MICROBIOLO4ICAL REVIEWS, December 1981, p. 591-619 Vol. 45, No.4 0146-0749/81/040591-29$02.00/0 Antibiotic Resistance in Pathogenic and Producing Bacteria, with Special Reference to fl-Lactam Antibiotics HIROSHI OGAWARA Second Department of Biochemistry, Meiji College of Pharnacy, 35-23, Nozawa-1, Setagaya-ku, Tokyo 154, Japan INTRODUCIION ............................................................. 591 PRODUCTION OF 8-LACTAM ANTIBIOTICS ................................ 591 MODE OF ACIION OF .-LACTAM ANTIBIOTICS ........................... 593 RESISTANCE MECHANISMS IN PATHOGENIC BACTERIA .............. 597 RESISTANCE MECHANISMS IN STREPTOMYCES ........................... 604 CONCLUSION .............................................................. 610 LITERATURE CITED ......................................... 610 INTRODUCTION Antibiotic research is one of the most exciting fields in chemotherapy (237, 238). As physicians and users of antibiotics, we demand that anti- biotics be active against various pathogenic mi- croorganisms with no harmful action upon the host. It therefore is expected that the pathogenic microorganisms will be susceptible to these an- tibiotics. However, as a natural consequence of selection under the pressure of antibiotics, re- sistant microorganisms are becoming prevalent in the environment (125, 126). Under such cir- cumstances, resistant mautants are selected much more effectively and rapidly, because only re- sistant mutants can grow and the resistant trait can easily be transmitted to susceptible micro- organisms by conjugation and transduction, al- though the frequency of mutation remains un- changed. As manufacturers of antibiotics, we insist that they must be produced in the largest possible amounts without having deleterious ef- fects on the producing organisms. For this pur- pose the producer organisms must be resistant to their own metabolites, at least during the producing period (48, 84, 243). Therefore, for users as well as makers of antibiotics, we demand that microorganisms have conflicting properties: susceptibility and resistance to antibiotics. Turning to the genetic material governing re- sistance properties, it is noteworthy that most of such traits in pathogenic bacteria are carried on extrachromosomal genetic elements (124). Therefore, such properties can be easily trans- mitted to susceptible organisms. Also, produc- tion of and resistance to antibiotics in producing organisms can be controlled, in some cases, by extrachromosomal genetic elements, either di- rectly or indirectly (79, 80, 132, 156, 164). Thus, the extrachromosomal genetic elements also demonstrate two conflicting properties: the re- sistance trait in pathogenic and producing bac- teria and the production control trait in produc- ing organisms. In this review, I have attempted to describe antibiotic resistance mechanisms and their interrelation in pathogenic and producing bacteria, taking,-lactam antibiotics as an ex- ample. PRODUCTION OF f8-LACTAM ANTIBIOTICS ,8-Lactam antibiotics are distinct in that a fi- lactam antibiotic, penicillin, was the first anti- biotic discovered (58) and in that ,8-lactam an- tibiotics have been widely used for chemother- apy of infectious diseases in the 40 years since penicillin's rediscovery. This widespread use of ,B-lactam antibiotics stems from their intrinsic antibacterial activity together with a highly spe- cific inhibitory effect on the biosynthesis of pep- tidoglycan (212, 227, 228), a unique structure of the bacterial cell wall, and from their quite low toxicity to animals, including humans. Further- more, the discovery of 6-aminopenicillanic acid and 7-aminocephalosporanic acid (95, 115, 194) and their preparation by enzymic (penicillin amidase, EC 3.5.1.11) (10, 85) and chemical methods, followed by chemical modification of the side chains of penicillins and cephalosporins, have introduced tremendous numbers of semi- synthetic penicillins and cephalosporins into the chemotherapeutic field and opened a new era by giving new compounds effective against various species of bacteria resistant (due to production of ,B-lactamase [penicillinase, EC 3.5.2.6]) or not susceptible (e.g., Pseudomonas species; due to a permeability barrier or intrinsic resistance, but not due to production of 8l-lactamase) to ordi- nary penicillins. Improvement of chemothera- peutic agents with the aim of overcoming all pathogenic bacteria on the earth has been a persistent enthusiasm. However, the most im- portant cause of their wide application is surely 591
Transcript
MICROBIOLO4ICAL REVIEWS, December 1981, p. 591-619 Vol. 45, No.40146-0749/81/040591-29$02.00/0
Antibiotic Resistance in Pathogenic and Producing Bacteria,with Special Reference to fl-Lactam Antibiotics
HIROSHI OGAWARA
Second Department ofBiochemistry, Meiji College ofPharnacy, 35-23, Nozawa-1, Setagaya-ku, Tokyo 154,Japan
INTRODUCIION ............................................................. 591PRODUCTION OF 8-LACTAM ANTIBIOTICS ................................ 591MODE OF ACIION OF .-LACTAM ANTIBIOTICS ........................... 593RESISTANCE MECHANISMS IN PATHOGENIC BACTERIA .............. 597RESISTANCE MECHANISMS IN STREPTOMYCES ........................... 604CONCLUSION .............................................................. 610LITERATURE CITED ......................................... 610
INTRODUCTIONAntibiotic research is one of the most exciting
fields in chemotherapy (237, 238). As physiciansand users of antibiotics, we demand that anti-biotics be active against various pathogenic mi-croorganisms with no harmful action upon thehost. It therefore is expected that the pathogenicmicroorganisms will be susceptible to these an-tibiotics. However, as a natural consequence ofselection under the pressure of antibiotics, re-sistant microorganisms are becoming prevalentin the environment (125, 126). Under such cir-cumstances, resistant mautants are selected muchmore effectively and rapidly, because only re-sistant mutants can grow and the resistant traitcan easily be transmitted to susceptible micro-organisms by conjugation and transduction, al-though the frequency of mutation remains un-changed. As manufacturers of antibiotics, weinsist that they must be produced in the largestpossible amounts without having deleterious ef-fects on the producing organisms. For this pur-pose the producer organisms must be resistantto their own metabolites, at least during theproducing period (48, 84, 243). Therefore, forusers as well as makers of antibiotics, we demandthat microorganisms have conflicting properties:susceptibility and resistance to antibiotics.Turning to the genetic material governing re-
sistance properties, it is noteworthy that most ofsuch traits in pathogenic bacteria are carried onextrachromosomal genetic elements (124).Therefore, such properties can be easily trans-mitted to susceptible organisms. Also, produc-tion of and resistance to antibiotics in producingorganisms can be controlled, in some cases, byextrachromosomal genetic elements, either di-rectly or indirectly (79, 80, 132, 156, 164). Thus,the extrachromosomal genetic elements alsodemonstrate two conflicting properties: the re-sistance trait in pathogenic and producing bac-
teria and the production control trait in produc-ing organisms. In this review, I have attemptedto describe antibiotic resistance mechanisms andtheir interrelation in pathogenic and producingbacteria, taking,-lactam antibiotics as an ex-ample.
PRODUCTION OF f8-LACTAMANTIBIOTICS
,8-Lactam antibiotics are distinct in that a fi-lactam antibiotic, penicillin, was the first anti-biotic discovered (58) and in that ,8-lactam an-tibiotics have been widely used for chemother-apy of infectious diseases in the 40 years sincepenicillin's rediscovery. This widespread use of,B-lactam antibiotics stems from their intrinsicantibacterial activity together with a highly spe-cific inhibitory effect on the biosynthesis of pep-tidoglycan (212, 227, 228), a unique structure ofthe bacterial cell wall, and from their quite lowtoxicity to animals, including humans. Further-more, the discovery of 6-aminopenicillanic acidand 7-aminocephalosporanic acid (95, 115, 194)and their preparation by enzymic (penicillinamidase, EC 3.5.1.11) (10, 85) and chemicalmethods, followed by chemical modification ofthe side chains of penicillins and cephalosporins,have introduced tremendous numbers of semi-synthetic penicillins and cephalosporins into thechemotherapeutic field and opened a new era bygiving new compounds effective against variousspecies of bacteria resistant (due to productionof ,B-lactamase [penicillinase, EC 3.5.2.6]) or notsusceptible (e.g., Pseudomonas species; due to apermeability barrier or intrinsic resistance, butnot due to production of 8l-lactamase) to ordi-nary penicillins. Improvement of chemothera-peutic agents with the aim of overcoming allpathogenic bacteria on the earth has been apersistent enthusiasm. However, the most im-portant cause of their wide application is surely
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the low toxicity of penicillins and cephalosporinsto eucaryotic cells.Taking the naturally occurring /8-lactam com-
pounds into consideration, the detection of pro-ducers of these compounds has been limited,until recently (113), to eucaryotic microorga-nisms, such as Penicillium, Cephalosporium,and Aspergillus. These organisms have no pep-tidoglycan the biosynthesis of which is a targetof /1-lactam compounds, in their cell walls. Thesecompounds have no detrimental effect on theproducer organisms, even in extremely largeamounts, insofar as they and their productiondo not disturb the producing organisms' essen-tial metabolic pathways. In other words, theseB-lactams are xenotoxic (243) and pose no threatto the organisms that synthesize them. Thispicture of,-lactam producers changed com-pletely in 1971, when the Eli Lilly and Mercklaboratories (130, 211) reported 7a-methoxyce-phalosporins as metabolites of certain species ofStreptomyces, including S. clavuligerus, S. lip-manii, and S. griseus. Streptomyces organisms,in contrast to the eucaryotic producers describedabove, are procaryotic microorganisms andhence have peptidoglycan as an indispensablecell wall constituent. Thus, they must have a
way of protecting themselves from their owntoxic metabolites. Interestingly, all of these ,B-lactam compounds have a 8-(D-a-aminoadipyl)side chain, like cephalosporin C (produced byCephalosporium) and penicillin N (produced byCephalosporium and Streptomyces species; [I.M. Miller, E. 0. Stapley, and L. Chaiet, Bacte-riol. Proc., p. 32, 1962]) but unlike isopenicillinN (produced by Penicillium and Cephalospo-rium [101, 166]). Streptomyces and Cephalospo-rium thus share a common biosynthetic path-way to cephalosporins. The 0-acetyl group ofcephalosporin C, however, is often replaced bya carbamoyl or a substituted cinnamoyl group,and the introduction of a 7a-methoxy group intothe cephem ring gives compounds with highresistance to many /8-lactamases (43, 143) andalso a high antibacterial activity. Cefoxitin, amember of this group, is widely used againstgram-positive and gram-negative bacteria (165).Another new direction in f,-lactam research
originated from finding of new 18-lactam com-pounds in the cultured broth of Streptomycesspecies as a result of screening for,8-lactamaseinhibitors. Umezawa et al. (239) found two ,B-lactamase inhibitors (MC-696-SY2-A and MC-696-SY2-B) in the cultured broth of Strepto-myces fulvoviridis, one ofwhich is characterizedas a strong inhibitor with a low antibacterialactivity and the other of which is a compoundwith a potential for antibacterial activity and
MICROBIOL. REV.
low in f8-lactamase-inhibitory activity. At thetime of original publication (239), the structureswere not clear, but they were expected to havea /i-lactam ring from a competitive inhibition ofhydrolysis of benzylpenicillin byf,-lactamases.MC-696-SY2-A and MC-696-SY2-B were laterfound to have structures identical (116) withthose of MM-4550 and MM-13902, respectively,reported by Beecham researchers (19). Soonafter that, a related compound, clavulanic acid,was reported from the Beecham laboratories asa strong ,f-lactamase inhibitor with low antibac-terial activity (18, 181). This compound con-tained a ,8-lactam ring with no side chain fusedto an oxazolidine ring instead of the thiazolidinering found in penicillins. It is known that clavu-lanic acid has a synergistic effect, when usedwith penicillins (36, 121), against many resistantbacteria (92).
Anotherfl-lactam, thienamycin, isolated fromthe culture broth ofStreptomyces cattleya (249),had an extremely unstable fused f-lactam ringdifferent from that ofpenicillins and cephalospo-rins. This compound shows a wide-range of an-tibacterial activity against various bacteria, in-cluding Pseudomonas, Serratia, and the anaer-obic genus Bacteroides. In this respect, thiscompound is different from MM-4550 and cla-vulanic acid. PS-5, with a similar structure, alsohas a strong antibacterial and inhibitory activi-ties against various species of bacteria (163). Allof these ft-lactam compounds are produced asmixtures of related compounds, and sometimesby mixing fl-lactam antibiotics with cephamy-cin-group antibiotics. CP-45899, a synthetic /B-lactam with a similar structure, also was re-ported (8, 56). Fortunately, these compoundsshow broad antibacterial spectra. Therefore,these new types of compounds can be used clin-ically after chemical modifications to stablestructures, because the unstability of the ,B-lac-tam ring itself is not directly related to antibac-terial activity (for example, MM-4550 and cla-vulanic acid, which are quite unstable, showweak antibacterial activities). Hence, it is possi-ble to get a stable ,B-lactam with high antibac-terial activity. A new stabilized thienamycin de-rivative reported recently (96) can be cited as anexample.A third group of 8-lactams was isolated from
an actinomycete, Nocardia uniformis, by usingan Escherichia coli strain hypersusceptible tofi-lactam antibiotics (5, 94). This compound, no-cardicin A, is unusual in containing an unfused/1-lactam ring and showing antibacterial activityagainst gram-negative bacteria, including Pseu-domonas aeruginosa, with no activity againstStaphylococcus aureus. Similar compounds, sul-
RESISTANCE TO fl-LACTAM ANTIBIOTICS 593
fazecin and isoulfazecin, were found in the fer-mentation broth of Pseudomonas acidophila(86).
Accordingly, current research on,B-lactam an-tibiotics has been directed towards two prob-lems, that is, preparation and chemical modifi-cation of 6-aminopenicillanic acid and 7-amino-cephalosporanic acid as backbone structures iso-lated from eucaryotic microorganisms and prep-aration and modification of new types of /i-lac-tams, such as cephamycins, MC-696-SY2, cla-vulanic acid, thienamycin, PS-5, and nocardicinsisolated from procaryotic microorganisms (Ta-ble 1).
MODE OF ACTION OF B-LACTAMANTIBIOTICS
The bactericidal activities of penicillins andcephalosporins are believed to derive from inhi-bition of bacterial cell wall biosynthesis. Thiswas first shown in 1957, through observationsthat penicillin induced the formation of proto-plasts or spheroplasts in a hypertonic mediumand lysed growing susceptible bacteria in a usualmedium and that uridine nucleotides containingmuramic acid and pentapeptide accumulated inS. aureus and other bacteria inhibited by peni-cillins (110, 170). In 1965, Wise and Park (250)and Tipper and Strominger (228) showed inde-pendently that penicillins inhibited the trans-peptidation of peptidoglycan. In addition, Wiseand Park suggested that the key to this inhibi-tion was the structural imilarity between peni-cillins and the L-alanyl-y-D-glutamyl portion ofthe peptidoglycan, whereas Tipper and Strom-inger proposed that penicillin inhibited pepti-doglycan synthetase and killed growing suscep-tible bacteria through the conformational simi-larity between penicillins and the D-alanyl-D-alanine part of the peptidoglycan. The confor-mational similarity between D-alanyl-D-alanineand penicillins as well as their structural analogsamong intermediates in transition states of thenormal substrates in the biosynthetic reactionhas been supported by Lee (112) and Viruda-chalam and Rao (244). According to this model,penicillins acylate the transpeptidase in a man-ner similar to that of acylation by a D-alanineresidue in the peptidoglycan and allow the cellwall to be mechanically weakened to death as aresult of formation of poorly cross-linked pepti-doglycan which is eventually ruptured by a com-bination of mechanical and osmotic pressures.However, the recent experiments with various
bacteria have indicated that some features ofthis model should be modified and that themechanism is much more complicated. The
mode of action of 8-lactams should therefore bereconsidered.At least two features of the mechanism of
action of penicillins must be considered: inhibi-tory effect and lethal or lytic effect. The formeris elicited by the direct interaction of penicillinswith their biochemical targets, penicillin-bindingproteins (PBPs), and the latter is due to subse-quent reactions triggered by the former.As early as 1954 (54, 55), Eagle and co-workers
pointed out that most bacteria have not one butmultiple penicillin-binding sites in the cytoplas-mic membrane fraction (14). Furthermore, itwas shown that individual ,8-lactam compoundsacted on bacteria in selective manners, especiallyat their minimum inhibitory concentrations (61,62). In 1975, Spratt and Pardee showed visually,by gel electrophoresis in the presence of sodiumdodecyl sulfate and fluorography, that penicil-lins and cephalosporins bind selectively to someofthe components in the cytoplasmic membranefraction of E. coli (206, 210). Further biochemi-cal and genetic analyses by Spratt (207, 208) andothers (39, 119, 120, 216) clearly showed thateach penicillin-binding component performs itsspecific function and that the results obtainedwith E. coli can be applied to other gram-nega-tive bacteria including Neisseria gonorrhoeae(139). Similar patterns ofpenicillin-binding com-ponents in fluorography have been observed alsoin gram-positive bacteria, such as Bacillus sub-tilis (14, 98), Bacillus megaterium (24-26, 60),Bacillus licheniformis (25), Streptococcus fae-calis (37), S. aureus (103, 254) and Streptomycescacaoi (149), although the study of these specieshas not been developed as extensively as that ofgram-negative bacteria (Table 2). These topicswill be reviewed by B. G. Spratt (manuscript inpreparation), and therefore the details are omit-ted. Here, it is enough to say that some penicil-lins and cephalosporins may carry out their le-thal action through binding to other componentsthan those detected by fluorography with['4C]benzylpenicillin.Turning to lytic action, the following facts
indicate that autolysin plays an important rolein penicillin-induced bacterial lysis (173, 187,188): the inhibition of growth and the inhibitionof peptidoglycan biosynthesis by subinhibitoryconcentrations of penicillins are reversible;timely addition of penicillinase to a penicillin-inhibited culture restores growth, which depictsa curve indistinguishable from normal; and thebactericidal effect of penicillins in Streptococcusand E. coli can be antagonized by the presenceof antibiotics, such as chloramphenicol (179).This suggestion, that is, that bacterial cell lysisinduced by penicillins may be controlled by a
VOL. 45, 1981
TABLE 1. Main naturally occurring ,B-lactam antibioticsYr
a Sizes of PBPs, expressed as molecular weights x 10- (values in parentheses are approximate percentages).
factor not directly affected by penicillins, is sup-
ported by the following observations. Lederbergand Zinder (111) reported a phenomenon com-monly referred to as penicillin selection, in whichbacterial cells must be involved in active growthin order to be killed by penicillins. Activegrowth, rather than an interaction with the ac-cess of penicillins to their biochemical targets, isnecessary for the lytic step of penicillin action,because the same type of active growth is neededfor lysis induced by lysine starvation of an aux-otrophic mutant of S. faecalis (203) or by dia-
minopimelic acid deprivation of a diaminopi-melic acid-requiring mutant of E. coli (251).Furthermore, some inhibitors of cell wall syn-thesis cause lysis irrespective of their inhibitingsite. Thus, bacterial cell lysis implies an intrinsicfactor, such as autolysin, in addition to the directeffects of penicillins on their biochemical targets.A direct demonstration of the participation of
autolysin was accomplished by Tomasz and co-workers (17, 75-78, 234, 235). Upon penicillintreatment of Streptococcus pneumoniae mu-tants defective in autolytic enzymes they ob-
50 (70) 14, 98
26
25
103
37
257
81, 149
149
149
134a
62 494744
5552
134a
59
59
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served three phenomena: (i) growth of the mu-tants was inhibited by the same concentrationsof penicillin as those that induced lysis in thewild type, but the mutants were never lysed, andsuch cultures permitted regrowth when penicil-lin was destroyed with penicillinase; (ii) mutantstreated with penicillin at the miniimal inhibitoryconcentration were lysed after addition of wild-type autolysin to the growth medium, but cul-tures of mutants treated with autolysin alonedid not show any effect; and (iii) treatment withpenicillin caused release of a choline-containingmacromolecule with features similar to those oflipoteichoic acid.
If autolytic enzymes were involved in penicil-lin-induced bacterial lysis, how was the action ofautolysins controlled so as not to cause lysis ofnormally growing bacteria? When the cell wallinhibitors, such as penicillin, were added to suchcultures, how did these inhibitors perturb thecontrol mechanisms and cause lysis? The auto-lytic enzymes are known to have several impor-tant functions in cell wall assembly, surface en-largement, cell division (42), sporulation, andthe ability of cells to become competent fortransformation (229). The concept that autoly-sins are essential for enlargement of the cell wallin bacteria, which proposes that the phenomenaoccurring in cell wall can be processed througha balance between synthetic and degrading en-zymes, has been widely accepted. Inhibition ofthe enzymes involved in cell wall biosynthesisgive greater weight to the degrading enzyme,which results in cell lysis. However, if autolyticenzymes acted continuously during the course ofthe cellular growth cycle, the S. pneumoniaemutants defective in autolytic enzymes wouldnot be alive. Therefore, the exact roles of theautolytic enzymes (at least the amidase-typeenzyme in S. pneumoniae) have remained un-defined. In any case, the activities of the auto-lytic enzymes are subject to time-dependent,special, and spacial regulation. In other words,these enzymes act at a specific time in the cel-lular growth cycle, and there is a barrier betweenthe enzyme and the substrate, peptidoglycan(70).Peptidoglycan can also be protected from the
attack of autolytic enzymes by an endogenousinhibitor of these autolytic enzymes. Low con-centrations of five different lipoteichoic acidsisolated pure from Lactobacillus casei NIRD094, Lactobacillus fermentum NCTC 6991,Streptococcus lactis ATCC 9936, and S. faecalis39 and NCIB 8191 were reported to inhibit N-acetylmuramidases (lysozymes, EC 3.2.1.17) ofS. faecalis and Lactobacillus acidophilus andthe amidase of B. subtilis (32). However, since
the chemically deacylated lipoteichoic acids re-duced or completely abolished the inhibitoryactivity, the balance between acylated and de-acylated lipoteichoic acids would control auto-lytic activities in bacterial cells.
In S.pneumoniae, the autolytic enzyme seemsto be regulated by the presence of choline resi-dues in the cell wall teichoic acid. S.pneumoniaegrown on ethanolamine-containing medium con-tains autolytic amidase with an abnormally lowspecific activity (E-form), but this can be con-verted into the catalytically active (C-form) en-zyme by in vitro incubation with choline-con-taining cell wall (235). Another regulatory mech-anism in this microorganism operates throughthe action of teichoic acid phosphocholine ester-ase (74), which removes critical phosphocholineresidues from the cell wall or lipoteichoic acid.
In B. subtilis ATCC 6051, however, the activ-ity of N-acetylmuramyl-L-alanine amidase, re-sponsible for cell wall turnover and cell lysisduring energy deprivation, is modulated by amodifier protein (71, 72). It is noteworthy thatthe presence of the modifier protein allows therandom pattern of cleavage of peptidoglycan tochange into a sequential pattern, although themodifier protein alters the catalytic activity byonly a 2.5-fold stimulation. Here again, teichoicacid has a regulatory role on the autolytic en-zyme, without which the enzyme cannot bebound to the cell wall tightly and the functionalinteraction with the modifier protein cannot oc-cur (72). Therefore, the autolytic enzymes areunder regulatory control of the spacial, topol-ogical barrier and also specific inhibitors, suchas lipoteichoic acid, phospholipids (31, 33), andactivators or modifiers (233). There have beenrecent reviews on the autolytic enzymes (204,230, 231).
In summary, ,B-lactam compounds first attackthe specific PBPs in the membrane fraction.Depending on the PBP they attack, an apparentfeature of the injurious action may be discrimi-nated. ,B-Lactam compounds in somewhathigher concentrations induce cell lysis and deaththrough a breakdown of the barrier between theautolytic enzyme and the substrate, peptidogly-can. Recently, however, Shockman's group re-ported that penicillin inhibited ribonucleic acidand protein, as well as peptidoglycan, synthesisin Streptococcus mutans strains FA-1 and GS-5in a concentration-dependent manner (128).They explained this observation as follows: S.mutans possessed a mechanism by which inhi-bition of insoluble peptidoglycan assembly can"talk back" to the synthesis of ribonucleic acidand protein. Furthermore, they suggested thata highly phosphorylated guanosine derivative
MICROBIOL. REV.
RESISTANCE TO 8-LACTAM ANTIBIOTICS 597
(136) may be a messenger, because peptidogly-can synthesis is regulated by stringent control inE. coli (87, 88).
RESISTANCE MECHANISMS INPATHOGENIC BACTERIA
The mode of action of f-lactam compoundshas thus far been discussed as a basis of under-standing the resistance mechanisms of 8-lactamcompounds. Now we are in a position to considerthe resistance mechanisms. Davis and Maas (47)predicted the following possibilities as biochem-ical mechanisms ofdrug resistance: (1) providingthe cell with a replacement for the metabolicstep that is inhibited by the drug, a bypassmechanism; (2) production of a metabolite thatcan antagonize the inhibitory effect of the drug;(3) increasing the amount of the enzyme in-hibited by the drug; (4) decreasing the cell'smetabolic requirement for the reaction inhibitedby the drug; (5) detoxication or inactivation ofthe drug; (6) changing the target site; and (7)blocking the transport of the drug into the cell.Although these mechanisms actually function inresistance to other drugs-for example, resist-ance of S. aureus and other bacteria to sulfon-amide is due to the overproduction ofp-amino-benzoic acid, and that to 6-mercaptopurine and8-azaguanine is due to the deletion ofthe enzymethat converts them into biologically active nu-cleotides (1)-it is mainly mechanisms 5, 6, and7 (that is, detoxication or inactivation of thedrug, changes in the target site that reduce oreliminate the binding of the drug, and blockingthe transport of the drug into the cell by chang-ing the structural organization or function of thecytoplasmic and outer membranes [12, 44, 66])that are used by bacteria for antibiotic resist-ance.
In the case of /-lactam antibiotics, mecha-nisms 5, 6, and 7 also are frequently operative inboth clinically isolated and laboratory-isolatedresistant mutants. Spratt reported (209) that alaboratory-isolated E. coli mutant resistant tomecillinam, thienamycin, and clavulanic acidhad a decreased affinity of these 8)-lactams tothe target, PBP2. However, this type of resist-ance mechanism is uncommon and its clinicalsignificance remains obscure (20). Recently, To-masz and others (68, 174, 257) reported thatpenicillin resistance in South African S. pneu-moniae strains was accompanied by changes inPBP patterns when compared with those seenin wild-type susceptible laboratory strains. En-try of transforming deoxyribonucleic acid iso-lated from the resistant strains caused gradualacquisition of increasing levels of penicillin re-sistance in a susceptible strain, which was par-
alleled by gradual changes in the PBP patterns.A high level of resistance to penicillin was ac-quired only in a stepwise process, in accord withprevious laboratory findings. A similar observa-tion also was reported for acquisition of oxacillinresistance (256). A new mechanism, decrease ordeletion of the autolytic activity conjectured tobe the indirect target described above, can beadded to the list of mechanisms of resistance.One example is the tolerance phenomenon in S.pneumoniae (232). Although this may be oper-ative, it is not very widespread as a resistancemechanism in pathogenic bacteria at present(191, 247). However, since pathogenic bacteriatend to acquire any resistance mechanism at anycost in order to survive, this will become one ofthe main mechanisms at some time in the nearfuture.
Penicillin resistance due to the changes inpermeability and peptidoglycan structure hasbeen described in gonococci (65, 186). A bypassmechanism is operative in B. megaterium (60)and E. coli (221)-that is, replacement of thesensitive primary target by a second, less sensi-tive target or compensation mechanism-that isa new category and surmised to be a combina-tion of mechanisms 1, 4, and 6. A decrease in theamount of the lethal target, which is compen-sated by an increase of less sensitive penicillin-binding proteins, makes bacteria resistant. How-ever, this can be seen only in laboratory strainsand is not of clinical significance now.As for detoxication or inactivation of f8-lac-
tams, mechanism 5,8-lactam antibiotics are hy-drolyzed by three kinds of enzymes. One is pen-icillin amidase, which is isolated not only frommicroorganisms (240, 241), such as gram-nega-tive and gram-positive bacteria and fungi (As-pergillus, Cephalosporium, Penicillium), butalso from mammals (35). However, the amidase,its reaction being reversible (162), is not a seriouscause of penicillin resistance in pathogenic bac-teria, but is used for the preparation of semisyn-thetic penicillins and cephalosporins. Acetyles-terase is ranked second. This enzyme hydrolyzesthe acetyl group at C-3 on the dihydrothiazinering of cephalosporins, such as cephalosporin C,cephalothin, and cephaloglycin, and is isolatednot only from microorganisms, such as E. coliand S. aureus (138), but also from human serum(13). This is not very important in the resistanceof pathogenic bacteria to /i-lactam antibiotics,either. The third enzyme, f-lactamase catalyzesthe hydrolysis of the f,-lactam ring of penicillinsand cephalosporins (69). This reaction destroysone of the most important sites for their anti-bacterial activity, and it is irreversible. Thus, ,B-lactamase plays a significant role in resistance to
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,-lactam antibiotics in the clinical field, espe-cially when one considers the facts that the genefor 8B-lactamase in S. aureus, a gram-positivebacterium, is located mainly on a plasmid andcan be easily transferred to other susceptiblebacteria through bacteriophage and that ingram-negative bacteria it is also located mainlyon an R-plasmid and transferred by conjugation.#-Lactamase has one common property of cat-alyzing the hydrolysis of the ,B-lactam ring ofpenicillins and cephalosporins. In this respect, itis interesting that although,B-lactam antibioticshave various killing targets (PBP), they are hy-drolyzed to inactive compounds by only one
enzyme (Jl-lactamase). This forms a strking con-trast to many other antibiotics. However, thisenzyme is produced by a wide-range of differentprocaryotes with great variety in chemical, phys-ical, and enzymological properties (16), as is notin the case of other enzymes. Recently, it hasbeen reported (123) that eucaryotic microorga-
nisms (fungi) produce ,-lactamase; this is inter-esting from the standpoint of the evolution ofB-lactamase, but the clinical role is not clear.The roles of ,-lactamase in gram-positive bac-teria, especially in Bacillus and Streptomyces,are not completely clarified, whereas it is prob-able that ,B-lactamases in gram-positive bacteriaare a source of those in pathogenic bacteria. Of
interest, in this regard, are the facts that S.aureus isolated in 1937 produced ,B-lactamaseand that B. lichenifonnis in the root of a plantspecimen stored in the British Museum since1689 produced a 8l-lactamase similar to thatfrom B. licheniformis at present (176, 201). Thisindicates that these fi-lactamase genes existed inthese bacteria long before the first use of peni-cillin.
fi-Lactamases from gram-positive bacteria canbe classified into several groups on the basis oftheir substrate specificities (Table 3). Except forD-type /3-lactamase from S. aureus (189), the/3-lactamases from Bacillus and Staphylococcusare inducible enzymes. Almost all the enzymesare excreted extracellularly (27, 30), although itdepends on the physiological condition of bac-teria to some extent. These enzymes, from Ba-cillus cereus (46, 106), with the exception of 68-lactamase II have no cysteine residue, as is thecase with many other extracellular proteins.Complete amino acid sequences of the A-typeenzyme from S. aureus PC1 and of the enzymefrom B. licheniformis 746/C and 85% (233 resi-dues) of the sequence of ,B-lactamase I from B.cereus 756/H have been determined (2, 3, 223).Among the 233 residues of the B. cereus enzyme,139 residues (about 60%) have the same posi-tions in the B. licheniformis enzyme, and 93
TABLE 3. ,B-Lactamases in gram-positive bacteriaSubstrate specificity
K. for ben-,B-Lactamase Mol wt pI Benzyl- Ampi- Methi Cephzylpenic-uin Reference
penicil- cfllin ciiiin loridine Mlin
Bacillus cereusf8-LActamase I 28,000 9.25 100 194 4 1 6 x 10-5 224
9.59.7
f6-Lactamase II 22,500 8.7 100 47 120 18 3.3 x 10-3 224
B-type B-1actamase 29,600 100 1.5 12 x 10-6 183, 184
C-type B-lactamase 29,600 100 0.6 7.5 x 10-6 183,184
Mycobacterium smegmatis8 -lac- 100 >1 77 1.7 x 10-3 69tamase
Mypobacterium tuberculosis /?- 100 >1 187 2.5 x 10-3 69lactamse
MICROBIOL. REV.
RESISTANCE TO fi-LACTAM ANTIBIOTICS 599
residues (about 40%) have the same positions inthe S. aureus enzyme. From these facts,Thatcher (223) concluded that "the geneticevent leading to separation of the gene, whichgave rise to the S. aureus enzyme from theancestral ,-lactamase gene, must have occurredbefore divergence of the gene giving rise to thetwo Bacillus enzymes."
fi-Lactamase from B. cereus has quite dif-ferent properties from the two 8i-lactamasesmentioned above. This enzyme constitutes onlyabout 5% of a total ,B-lactamase activity in B.cereus 569/H. The molecular weight of the pro-tein part is smaller than those of many other f6-lactamases. However, it contains a carbohydratemoiety in its molecule, although it is not directlyconnected with the enzyme activity. In addition,it contains not only one cysteine residue but alsoone atom of zinc bound to the cysteine residue(45). Furthermore, it is stable under heat treat-ment (600C for 30 min). Thus, B. cereus fi-lac-tamase II has these unusual properties, unlikeother fi-lactamases. As for its substrate profile,it also is unusual among the f-lactamases ofgram-positive bacteria. Most ,8-lactamases fromgram-positive bacteria are penicillinase-type en-
zymes. On the contrary, 8-lactamase II hydro-lyzes cephalosporin C, cephalothin, and othercephalosporins quite well (105); in that, it issimilar to some f)-lactamases from gram-nega-
tive bacteria. As will be described below (Resist-ance Mechanisms in Streptomyces), some Strep-tomyces 8-lactamases hydrolyze cephalosporins(151). These Streptomyces fl-lactamases and B.cereus f6-lactamase II may be evolutionally re-
lated to each other. Although the amino acidsequence of fl-lactamase II has not yet beendetermined and the relationship with ,B-lacta-mase I in B. cereus is not clear, along with nearlynothing being known of the physiological signif-icance of f-lactamase in Bacillus, it is interest-ing that one strain has two f)-lactamases withdifferent properties.The genetic element is not yet clarified: it is
not known whether or not both of the 8-lacta-mase in B. cereus are mediated by chromosomalgenes. However, prolonged subcultivation of B.cereus 569/H gave f6-lactamase-negative mu-tants. A small proportion of the mutants re-
verted into a ,-lactamase-positive state whenthey were incubated at 45 or 350C in the pres-
ence of chloramphenicol. One conceivable rea-
son is that the expression of the /-lactamasegene is controlled by a thermolabile repressor,
but it also indicates that a transposable elementis involved in the expression of the ,/-lactamasegene (177).
In another gram-positive bacterium, S. au-
reus, a large proportion of penicillin-resistantstrains are indebted to ,B-lactamase for theirresistance, with the exception of some intrinsi-cally resistant or tolerant strains (methicillin-resistant) (191). Furthermore, in contrast to B.cereus, all four types of 8-lactamases are con-trolled by genes on a PC-plasmid (53, 109, 140,142). The amino acid sequence of A-type ,8-lac-tamase has been determined. It is of interest, asdescribed above, that 40% of the amino acidsequence is homologous with that of a B. cereusenzyme, considering that one is mediated byplasmid, whereas the other is mediated by chro-mosomal deoxyribonucleic acid.Regarding the transfer of a fi-lactamase gene,
Asheshov performed the following experimentwith S. aureus strain ps-80, a naturally occur-ring, penicillinase-producing strain with the pen-icilhinase gene on the chromosome (6). Thestrain was verified to have a metal ion (Hg andCd) resistance marker on a plasmid throughtreatment at high temperature and the effect ofultraviolet light on the transduction. The co-transduction frequency of this strain for bothmetal resistance and penicillin resistancemarkers in the original isolate was less than 10'-.After a few years, the cotransduction frequencyfor these resistance markers was found to in-crease up to 10'-. In addition, although the effectof ultraviolet light on the transduction phenom-ena of the penicillin resistance marker indicatedthat the gene controlling penicillin resistancewas located on the chromosome, the curve ofcotransduction frequency as a function of theultraviolet light dose was typical for transduc-tion ofan extrachromosomal gene. Furthermore,by position effect analysis (7), Asheshov andDykes concluded that the penicilhinase gene,originally located on the chromosome, was du-plicated, with one copy being retained on thechromosome and the second being incorporatedinto the plasmid, and that both of the genesexpressed their trait simultaneously. Environ-mental change can thus cause such a duplicationof the penicillinase gene. This phenomenon is ofgreat interest not only regarding the evolutionof a gene on the plasmid which controls a largeproportion of the biosynthesis of f8-lactamasesin S. aureus, but also regarding the epidemiolog-ical aspects of fi-lactamases. A similar phenom-enon, consistent with the replication model oftransposable elements proposed by Grindley andSherratt (63) and Shapiro (202), has been ob-served with IS4 in gram-negative bacteria (97).Up to this time, among plasmids containing
the 8-lactamase gene, precise physical geneticmaps of plasmids pI258, pI524, and pI6187 ofincompatibility group I and pII147 of group II
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600 OGAWARA
have been established (127, 141, 142). Whereasp1258 and pI524 control the biosynthesis of A-type- ,8-lactamase, the gene on pII147 mediatesthat of C-type ft-lactamase. Despite differencesin f-lactamase produced and that in incompati-bility group, the arrangements of the genes sofar clarified are not that much different (142).Among these plasmids, pI524, the first plasmididentified in S. aureus and the first example ofa fB-lactamase plasmid, contains a 2.2-kilobasesegment located close to the ,f-lactamase locuswhich can undergo a site-specific, rec-independ-ent, and reversible inversion. Such short inver-tible segments have also been reported in genesconcerned with phase variation of flagellum an-tigen in Salmonella (255) and adsorbabilitychanges of bacteriophage Mu to the host (21,242). It is of interest in this regard that invertedrepeat sequences on transposon Tn5 carryingneomycin and kanamycin resistance markershave different binding abilities for ribonucleicacid polymerase (ribonucleic acid nucleotidyl-transferase), being different in degree of neo-mycin resistance and function in the transposi-tion process, although they give identical restric-tion endonuclease cleavage patterns (190). Tak-ing these facts into consideration, it is probablethat changes in the direction of the invertiblesegment on pI524 cause different phenotypes. Inany case, it is surely true that the bla region onpI524 is a hot spot of genetic rearrangement.
Integration of the f)-lactamase gene on thechromosome into plasmid is discussed above (6).Much has been reported elsewhere (93, 141, 172,199) regarding the integration of the plasmid(PC-plasmid) deoxyribonucleic acid containingthe ,B-lactamase gene into the chromosome.p1258 inc-i and pI254 repA18, carrying an eryth-romycin resistance marker together with the fl-lactamase gene, are integrated into the chro-mosome near the attachment site of 4d1 pro-phage (att4ll) and also near the chromosomallocation ofthe ,8-lactamase determinant ofstrainPs53, which may be also a transposable element.As described above, a large proportion of the
,8-lactamases in gram-positive bacteria actmainly as penicillinases, especially as enzymescatalyzing the hydrolysis of benzylpenicillin.The production of these f8-lactamases is medi-ated by both chromosomal and plasmid genes.However, as in the case with S. aureus, the geneon the plasmid can be derived from that on thechromosome. Thus, it can be assumed with easethat rearrangement ofthe fl-lactamase gene mayoccur not only between chromosomes and plas-mids but also within chromosomes and plasmidsat fairly high frequencies. However, cephalospo-rin resistance due to the production of fl-lacta-
mase is seldom seen in gram-positive bacteria,indicating that, although gram-positive bacteriahave less contact with cephalosporins thangram-negative bacteria, ,B-lactamases hydrolyz-ing penicillins and those hydrolyzing cephalo-sporins may be fairly different in their origins.This indication is supported by the facts that ingram-negative bacteria most ofthe fi-lactamaseshydrolyzing cephalosporins are controlled by agene on the chromosome and inducible and thata strain of Citrobacter freundii contains severalsoluble proteins bound with cephalosporins spe-cifically (146). The latter fact also indicates thatthe construction modes of the amino acid resi-dues involved in the binding of penicillins andthe binding of cephalosporins are somewhat dif-ferent from each other. In other words, at leastsome proteins can differentiate penicillins andcephalosporins by their binding abilities. Thus,it can be said that, in contrast to the case ofpenicillin-hydrolyzing fl-lactamases, the gene forcephalosporin-hydrolyzing,-lactamases is notsufficiently evolved to have the ability to trans-pose itself now.
In many Streptomyces strains there is no lin-ear relationship between fl-lactamase produc-tion and the minximum inhibitory concentrationof benzylpenicillin (145). This also can be seenin the case of B. licheniformis strains 749/C and6346/C; although they produce large amounts of/B-lactamase, they are highly susceptible to ben-zylpenicillin, the minimum inhibitory concentra-tion being 0.16 ,ug/ml (175). Bacillus anthracisV1B-189 is highly susceptible to benzylpenicillin,but the strain actively produces ,8-lactamase si-multaneously (180). In addition, an S. aureusstrain susceptible to 0.06 ,ug of benzylpenicillinper ml and 0.56 jig of methicillin per ml producedB-lactamase at 1 U/(1.3 x 109) cells (193). Sazand co-workers have proposed (167, 168, 192,197) thatf,-lactamase plays an important, butunknown, role in spore formation on the basis offindings that some synthetic circular peptidesinduce f,-lactamase production in B. cereus 569or S. aureus S-1 at a lower concentration thanthat induced by benzylpenicillin; that a largeamount of circular peptides are accumulated,especially at the time of spore formation, in B.cereus; and that peptidoglycans with molecularweights of 8,000 to 12,000 isolated from B. cereusinduce /i-lactamase production at 1/20 to 1/30of the concentration of benzylpenicillin requiredto produce the same effect. It is known that /8-lactamase production in B. subtilis increases atthe time of spore formation. When the ,B-lacta-mase gene from S. aureus was transformed, B.subtilis produced two kinds of 8-lactamases, anoriginal /?-lactamase and another from S. aureus,
MICROBIOL. REV.
RESISTANCE TO fi-LACTAM ANTIBIOTICS 601
and at the same time the biosynthesis of pepti-doglycan was impaired to some extent (T. Ya-makawa, personal communication). These find-ings, when evaluated in combination, revealsome assumable relationships between ,8-lacta-mase production and spore or peptidoglycan for-mation, at least in Bacillus.Turning to the active site(s) of ,B-lactamase,
there has been no substantial analysis differen-tiating amino acid residues involved in the bind-ing of the substrate, in the reaction directly, and,in some cases, in the maintenance of the activeconformation of the enzyme. Up to now, aminoand carboxyl groups and cysteinyl, histidyl,seryl, tryptophanyl, and tyrosyl residues havebeen proposed for such moieties. However, therehas been no conclusive evidence for the aminoacid residue in the active site.As for the histidine residue, Ogawara and
Umezawa (158, 159), using site-specific reagents,such as penicillin isocyanates, and from the pHdependence of enzyme inactivation, suggestedthat an imidazole group was involved in theactivities of,-lactamases from E. coli (153) andB. cereus. In this case a part of the reactionproduct was surmised to migrate to a nearbyamino group (147). However, f-lactamases fromB. cereus and E. coli were weakly inhibited byiodoacetic acid, diethyl pyrocarbonate, and bro-moacetone, so-called histidine-specific reagents,even at high concentrations (160). Waley indi-cated that a histidine residue with a pK. of 5.5was involved in the active site of B. cereus ,8-lactamase when it was assessed from the pHdependence of kcat/Km and inactivation byWoodward reagent (222); later, he changed thisassignment to a carboxyl group (245). Scott iso-lated a peptide containing an essential histidineresidue after modification of an E. coli ,8-lacta-mase with 0.25 M iodoacetic acid (200). How-ever, the histidine residue is not conserved inthe corresponding position of the f8-lactamasefrom S. aureus (2). Thus, although a histidineresidue is involved in the enzyme activities ofmany ,B-lactamases, it can be replaced by otheramino acid residues in some cases. In,-lacta-mase II of B. cereus, a histidine residue wasreported to be the second zinc ligand. The firstligand is a cysteine residue (9).From a reaction analysis with tetranitrome-
thane and iodine, a tyrosine residue was reportedto be essential for the activity of a fi-lactamasefrom B. lichenifonnis (38). However, fi-lacta-mases from B. cereus and E. coli were not in-activated by these reagents even at high concen-trations or only partially inactivated even afterprolonged treatment at high concentrations(200). This result is similar to the cases with a
histidine residue. These results indicate thateven though histidine and tyrosine residues areessential for the activities off8-lactamases, theseresidues are buried inside the enzyme moleculesand inaccessible to ordinary chemical reagents.For inactivation to occur, the enzymes mustchange their conformation by the action ofchemical reagents at high concentrations, dena-turation reagents, or surfactants. However, sub-strates or their analogs induce these active-siteresidues to the correct positions, and the enzymeperforms the catalytic reaction quite easily (28,161). This concept is supported by the findingthat inactivation of B. cereus f,-lactamase byiodine is highly stimulated by the presence ofurea or urea and methicillin (67).Although a tryptophan residue does not exist
in S. aureus fl-lactamases, it is preferentiallymodified in B. cereus ,B-lactamase I by treatmentwith N-bromosuccinimide, a finding verified byintrinsic fluorescence titration. This reactionconsists of two steps, with partial inactivation ofthe enzyme activity as the first step. The enzymeat this stage has one less tryptophan residuethan the original enzyme, a different Kmn, anddifferent behavior towards an affinity column(160). Thus, a tryptophan residue may have animportant role in maintenance of the active con-formation of the B. cereus f,-lactamase (161).This may also be seen in the case of the S.cacaoi enzyme (154).As for a cysteine residue, its clearest function
is to bind Zn2+ ion in B. cereus ,8-lactamase II(107). It is known thatfl-lactamases from gram-negative bacteria generally inhibited by p-chlo-romercuribenzoate catalyze the hydrolysis ofcloxacillin and that those from gram-negativebacteria generally inhibited by cloxacillin arenot inhibited byp-chloromercuribenzoate (185).However, since B. cereus ,8-lactamase I, havingno cysteine residue in its molecule, is partiallyinactivated by p-chloromercuribenzoate (223),the inactivation by this reagent may be due toa conformational change of the enzyme causedby its bulky group. But at the same time, it canbe said that a cysteine residue is involved in themaintenance of the active conformation in thesense that the enzyme can differentiate a sub-strate, cloxacillin, depending on whether the en-zyme has a cysteine residue or not (161).
Recently, Pratt and Loosemore (178) andKnott-Hunziker and co-workers (99, 100) re-ported independently the inactivation of B. cer-eus,8-lactamase I by 6,8-bromopenicillanic acid.This affinity label modifies the serine-44 residueof ,B-lactamase I specifically. Although it inacti-vates an E. coli fi-lactamase, it does not inhibitB. cereus 8i-lactamase II. Related to this, it is
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602 OGAWARA
interesting that several D-alanine carboxypepti-dases, which have been shown to have weak ,8-lactamase activities, have a serine residue intheir active site (248, 253), and that other D-alanine carboxypeptidases are metalloenzymes(49, 50), in that they are related to fB-lactamaseII. However, diisopropylfluorophosphate, a spe-cific chemical reagent for serine enzymes, cannotinactivate B. cereus ,8-lactamase I (29), an Aero-bacter cloaceae (Enterobacter cloacae) fl-lac-tamase (90), and a fi-lactamase from S. cacaoi(154). This situation is similar to those of theother amino acid residues described above. Inaddition, these /i-lactamases are unusual serineenzymes.
Patil and Day (171) reported that a carboxylgroup was involved in the active site of the ,B-lactamase of B. cereus 569/H from an inactiva-tion experiment with products formed by reac-tions of nitrous acid with 6-aminopenicillanicacid and ampicillin. Waley (245) reported theinvolvement of an amino acid residue with a pKaof 5.5 from the pH dependence of k,cft/Km of B.cereus,B-lactamase I and designated it as a car-boxyl group. Recently, Durkin et al. (51) reex-amined this reaction and found that the inacti-vation was maximal at pH 4 to 5, but reversibleat pH 7.0. In any case, however, such a grouphas not yet been identified.As described above, amino acid residues di-
rectly implicated in the enzymatic activities of,f-lactamases have not yet been determined con-clusively. Pain and Virden (169) and Thatcher(222) reported that the differences in free energyamong a native active form, a partially unfoldedform, and a completely unfolded form of a S.aureus f?-lactamase are far less than those ofother enzymes, such as ribonuclease and lyso-zyme. This conformational flexibility of /8-lacta-mases reflects the varied properties of ,B-lacta-mases, not seen in other enzymes. Moreover,this is one of the reasons for the discrepancybetween the results of the reactivity towardsspecific chemical reagents and the active-siteamino acid residues. To clarify these points fur-ther, much more useful affinity labels, especiallyphotoaffinity labels, should be developed (157).As for 83-lactamases from gram-negative bac-
teria, attention has been focused upon theseenzymes since the introduction of broad-spec-trum penicillins and cephalosporins in the early1960s (185, 217). 8l-Lactamases from gram-neg-ative bacteria have many properties distinctfrom those from gram-positive bacteria, withseveral exceptions. Almost all of the /i-lacta-mases are cell bound, whether they are mediatedby R-factors or chromosomes. At one time, itwas proposed that the enzymes controlled by R-
factors were periplasmic and those controlled bychromosomes were cell bound (135). However,this is not true at the present time (205). A ,B-lactamase from Bacteroides fragilis is not cellbound (219). Another distinct point is that manyf8-lactamases from gram-negative bacteria areproduced constitutively, in contrast to thosefrom gram-positive bacteria. Many new ,B-lac-tamases from gram-negative bacteria are foundevery year. However, these f,-lactamases can beclassified into several groups on the basis oftheirsubstrate specificity, isoelectric points, andwhether they are controlled by R-factors orchromosomes (91). Whereas the enzymes con-trolled by R-plasmids can be divided into twogroups, those mediated by deoxyribonucleic acidon the chromosomes are distributed amongmany groups (185, 217). Matthew and Harris(122) have claimed thatf,-lactamases can beused as criteria for bacterial taxonomy, becausemost gram-negative bacteria produce genus-,species-, and strain-specific fl-lactamases, evenif in small amounts, the biosyntheses of whichare controlled by chromosomal genes. However,it is reported (15) that an identicalf,-lactamaseis produced under either R-plasmid or chromo-somal control. In addition, since these geneticelements can be transferred as components oftransposable elements, the genus-, species-, andstrain-specific properties of f8-lactamases de-scribed above will gradually disappear. Thus, itis now a time of mixing up or chaos for thesegenes.
Despite these facts, chromosomally controlled/3-lactamases from gram-negative bacteria canbe roughly classified into three classes on thebasis of their substrate specificities (Table 4). Agreat majority of chromosomally mediated ,B-lactamases catalyze the hydrolysis of susceptiblecephalosporins 5 to 10 times faster than they dothat of benzylpenicillin. These enzymes are in-hibited by cloxacilhin and carbenicillin and re-sistant to inhibition by p-chloromercuriben-zoate. This class of ,B-lactamases is subdividedinto two types on the basis of inducibility. Theinducible ,B-lactamases are produced by a wide-range of bacterial genera, such as Citrobacter,Enterobacter, indole-positive Proteus, Provi-dencia, Pseudomonas, and Serratia, with theirproperties being species specific. In contrast,large proportions of E. coli strains produce thecephalosporin-hydrolyzing enzyme constitu-tively, as do Shigella and Salmonella. Only asmall number ofgram-negative bacteria producechromosomally mediated class 2 /3-lactamases,which are differentiated by their predQminantactivity against penicillins and lack of activityagainst cephalosporins. Although these enzymes
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RESISTANCE TO #l-LACTAM ANTIBIOTICS 603
TABLE 4. f8-Lactamases in gram-negative bacteria
I. Chromosomally mediated f?-lactamasesClass 1. The great majority of chromosomally mediated /8-lactamases belong to this class, they hydrolyze
susceptible cephalosporins 5 to 10 times as rapidly as they do benzylpenicillin; inhibited by cloxacillin orcarbenicillin but resistant to inhibition by p-chloromercuribenzoate
Subclass 1. Inducible, which is characteristic of this class of f8-lactamases; found in a wide range of genera,notably, Citrobacter, Enterobacter, indole-positive Proteus, Providencia, Pseudomonas, and Serratia
Subclass 2. Constitutive, most Escherichia coli fi-lactamases belong to this class; also found in Acineto-bacter, Bacteroides fragilis, ShigeUa, and SalnoneUa
Class 2. These f-lactamases hydrolyze penicillins, but hydrolyze cephalosporins only slightly; similar to R-factor-mediated ,6-lactmases
Class 3. These $-lactamases show a broad range of substrate specificity; found in many strains of Klebsiella,Bacteroides, and Neisseria, resistant to inhibition byp-chloromercuribenzoate and relatively resistant toinhibition by cloxacillin
II. R-factor-mediated ,-lactamases (most are constitutive)Class 1. TEM-type 8-lactamases; comparatively able to hydrolyze benzylpenicillin, ampicillin, and cephalori-
dine; found also in Neisseria gonorrhoeae and Haemophilus influenzae; divided into TEM I and TEMII by their isoelectric points, but most belong to TEM I
Clms 2. Oxacillin-hydrolyzing fi-lactamases, lower turnover numbers; resistant to inhibition by p-chloromer-curibenzoate and cloxacillin, but susceptible to chloride ion
Subclass 1. High activity against methicillin; mol wt about 24,000Subclass 2. Low activity against methicillin; mol wt over 40,000; consist of two subunits, bind blue dextran
Class 3. Other fi-lactamases; hydrolyze carbenicillin and cephalosporins
have not been elaborately studied, a imilaritybetween their substrate specificities and those ofR-factor-mediated f-lactamass should at leastbe pointed out and are described hereunder.The most characteristic ,B-lactamases of class
3 are produced by KlebsieUa and catalyze thehydrolyses of both penicillins and cephalospo-rins at similar rates. The enzymes of this class,as shown in those of class 2, are found rarely andare reistant to inhibition by p-chloromercuri-benzoate. Furthermore, they are relatively re-sistant to cloxacillin.
R-factor-mediated ,B-lactamases from gram-negative bacteria are produced in large amountsconstitutively. They act mainly as penicillinases,although a few enzymes catalyze the hydrolysisof cephalosporins, such as cephaloridine. On thebasis of their substrate specificity, they aregrouped into three classes. TEM-type ,B-lacta-mases belong to class 1 and hydrolyze benzyl-penicillin, ampicillin, and cephaloridine at com-parable rates. Their different isoelectric pointsmake it possible to distnguish them as TEM-1and TEM-2 enzymes. However, almost all of the,B-lactas isolated as the R-factor-mediatedenzymes are TEM-1 enzymes, and their geneticelement is easily transferred to various gram-negative bacteria as a transposable element. Re-cently, N. gonorrhoeae and H. influenzae havebeen reported to produce the enzymes of thistype (218, 219). The nucleotide sequence (214,215) and the amino acid sequence, including thesignal sequence, have been determined for thisclass of /B-lactamases (4). Class 2 ,B-lactamasesare characterized by hydrolysis of "penicilhinase-
resistant penicillin," oxacillin, and low turnovernumber. They are found rarely, but they havethe peculiar feature of being resistant to inhibi-tion by p-chloromercuribenzoate and cloxacillinand sensitive to inhibition by chloride ion (252).The enzymes of this group can be divided intotwo subclasses: subclass 1 includes those withhigh activity against methicillin and molecularweights of about 24,000, and subclass 2 includesthose with low activity against methicillin andmolecular weights of over 40,000, consisting oftwo subunits, and exhibiting significant bindingto blue dextran (40, 41).As thus far described, it is clear that the ,B-
lactamase enzymes have diverse properties. Re-lated to this, inactivation by clavulanic acidshould be mentioned here. Incubation of clavu-lanic acid with the f8-lactamase from E. coliRTEM leads to irreversible inactivation of theenzyme, forming three species of inactivated en-zymes on polyacrylamide isoelectric focusinggels (23, 57). Irreversible inhibition was observedwith B. cereus /-lactamase I (34, 52, 99, 100, 114,178). This,B-lactam also inhibited both extracel-lular and cell-bound ,B-lactamases from four S.aureus strains. However, even with excess cla-vulanic acid, these enzymes gradually restoredtheir enzymatic activity, the time course ofwhich was coincident with the slow disappear-ance and then the complete loss of clavulanicacid from the reaction mixture (22, 182). Inaddition, "cephalosporinases" in general are notinactivated by clavulanic acid. This indicatesthat /1-lactamases have more than two bindingsites and behave in a different fashion towards
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604 OGAWARA
clavulanic acid. Similar differentiation amongfi-lactamases was observed with a semisynthetic,B-lactamase inhibitor, CP-45,899 (108). Further-more, it was reported that the affinities, ex-pressed as Ki values, off,-lactamase inhibitorsfor various /t-lactamases range over 5 orders ofmagnitude (64). These facts support the notionthat /-lactamases at the present time are con-vergently evolved from many different proteinsinto one common property: hydrolysis of the ,B-lactam ring (148, 154).On one hand, the biosynthesis of 8-lactamases
in gram-negative bacteria is controlled by ge-netic elements on R-plasmids. These enzymesbehave mainly as penicillinases. The genetic de-terminants for these enzymes are transferredeasily, as components of R-plasmids containingtransposable elements, from enteric bacteria,such as E. coli, Shigella, Salmonella, and Citro-bacter, to Enterobacter, Klebsiella, Proteus,Pseudomonas, and Serratia and further to pe-culiar bacteria, such as Yersinia enterocolitica,H. influenzae, and N. gonorrhoeae. On the otherhand, almost all of the gram-negative bacteriaproduce species-specific fi-lactamases as induc-ible enzymes, though in small amounts. Al-though these enzymes existed as latent and si-lent proteins at a time when their apparentfunction should not have been necessary, theyhave been awakened from this dormant condi-tion to afford protection against many newlydeveloped,8-lactam compounds, especially ceph-alosporins. Since it has been shown that someR-plasmid-controlled ,8-lactamases are identicalwith those controlled by chromosomal deoxyri-bonucleic acid (15), genetic determinants forsuch species-specific, chromosomally mediated,B-lactamases must be transferred to other gram-negative or gram-positive bacteria sometime inthe near future. Thus, we shudder at the pros-pect of such species-specific fi-lactamases pre-vailing in nature.
In summary, fl-lactamase plays a significantrole at present in causing resistance of patho-genic bacteria to various /8-lactam antibiotics.One reason for this is that hydrolysis of the f8-lactam ring is the most efficient way for patho-genic bacteria to protect themselves against ex-ogenous ,B-lactam antibiotics. Even though theseenzymes are complicated in their chemical,physicochemical, and enzymological properties,they will become far more complicated year byyear, because many pathogenic bacteria producespecies- or strain-specific f8-lactamases, as onecan see in gram-negative bacteria. Besides theseenzymes, the pathogenic bacteria will protectthemselves from fi-lactam antibiotics by chang-ing the target sites and their properties and
autolytic enzyme systems and by the mecha-nisms listed above.
RESISTANCE MECHANISMS INSTREPTOMYCES
Streptomyces is one of the three main micro-organisms in soil, along with bacteria and fungi,and is known as a producer of tremendous num-bers of antibiotics, including penicillins andcephalosporins (129, 237, 238). Since it producessuch various kinds of antibiotics, it should havemeans for protecting itself against these auto-toxic substances (48, 243). This is true also forfl-lactam compounds. In pathogenic bacteria, asdescribed above, the,B-lactamases, modificationsof target proteins and of autolytic enzymes, andpermeability barriers shown as tolerance phe-nomena can be cited as such means. In Strep-tomyces, however, tolerance mechanisms havenot been observed. First, fl-lactamase will beconsidered.As described above, ,8-lactamases are distrib-
uted among wide range of procaryotic cells andhave far more varied properties in chemical,physical, and enzymological senses than manyother enzymes, except that they have the onecommon property of catalyzing the hydrolysis ofthe fl-lactam ring of penicillins and cephalospo-rins to produce inactive compounds (27, 30, 185,217). As a natural consequence of hydrolysis ofthe /8-lactam ring, an essential structure for theantibacterial activity, of these compounds thephysiological role of,-lactamase in bacterialresistance to penicillins and cephalosporins, es-pecially in gram-negative bacteria, is extremelyimportant. However, at least in Streptomycesand Bacillus, the levels of ,B-lactamases pro-duced, especially penicillinases, are not relatedto the degrees of resistance to /8-lactam com-pounds. For example, three-quarters of Strep-tomyces strains isolated in 1975 (145) and three-quarters of Streptomyces strains isolated about30 years ago (151) produced ,8-lactamase consti-tutively and extracellularly without connectionwith their resistance against benzylpenicillin. Insome cases, these Streptomyces strains contin-ued to produce 83-lactamase after over 200 h ofcultivation. The frequency of producing strainsshould have been higher, because nonproducerstrains, such as Streptomyces strains S-35 and S-44, at these primary screenings were later foundto produce 8i-lactamase in the membrane frac-tion. Thus, most Streptomyces strains produceor have the potential to produce /3-lactamase atsome time in their life cycle. Although many ,/-lactamases from gram-positive bacteria can beinduced by the addition of penicillin and pepti-doglycan (167, 168, 192), the production of those
MICROBIOL. REV.
RESISTANCE TO /?-LACTAM ANTIBIOTICS 605
from Streptomyces cannot be influenced. Thus,Streptomyces,B-lactamases are constitutive en-zymes. Such enzymes are also produced bymany non-Streptomyces Actinomycetales (198).Among the f-lactamase-producing strains,Streptomyces lavendulae, Streptomyces diasta-tochromogenes, and Streptomyces albus werethe highest producing strains under the condi-tions encountered, and the amounts producedwere comparable to those produced by B. cereus569/H and B. licheniformis 749/C, althoughthey depended on the composition of the me-
dium (132). On the basis of their substrate spec-ificities, isoelectric points and molecular weights,these f8-lactamases are divided into five classes(H. Ogawara, in H. J. Kutzner, G. Pulverer, andK. P. Schaal, ed., Actinomycete Biology, inpress) (Table 5). The class 1 enzymes, which are
basic, selectively hydrolyze penicillins, and theirmolecular weights are below 25,000. The class 2enzymes cannot be differentiated from the class1 enzymes by substrate specificity, althoughthese enzymes are neutral or acidic proteins.The class 3 enzymes are peculiar in that theyhydrolyze cephazolin fairly well. The ,B-lacta-mase from Streptomyces coelicolor S-6 (class 4)is similar to fB-lactamases in the class 3 in manypoints, but this enzyme cannot hydrolyze cloxa-cillin and methicillin at all. In substrate specific-ity and molecular weight, the enzymes of class1 and S. coelicolor S-6 are similar to the TEM-type enzymes from gram-negative bacteria, andthe class 4 and TEM-type enzymes are alsosimilar in that they are acidic proteins. The class5 enzyme, an acidic protein, hydrolyzes cloxacil-lin, methicillin, and carbenicillin as well as ben-zylpenicillin; its molecular weight is 34,000, alittle greater than those of other Streptomycesfl-lactamases. Apart from this preliminary clas-sification, the ideal classification should be basedon the structures ofthe active sites for substratesand antibodies and closeness in the evolutionarytree. Taking a particular property, such as bind-ing to blue dextran, for example, the enzymeswith such a property are distributed over class1 (Streptomyces cellulosae S-127) and class 2(Streptomyces phaeochromogenes S-70, Strep-tomyces fradiae Y-59, and Streptomyces sp.strain 7394). Thus, the Streptomyces f8-lacta-mases cannot satisfactorily be classified basedon a few properties. Rather, it can be said thatBi-lactamases in Streptomyces are species spe-cific, as in the case of chromosomally mediatedenzymes in gram-negative bacteria. Thus, theproperties of the Streptomyces -lactamases aremuch more complicated than previously as-sumed (145).As stated above, the ,8-lactamase of S. cellu-
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VOL. 45, 1981
606 OGAWARA
losae S-127 is peculiar in that it binds stronglyto blue dextran. This property was used to purifythe enzyme to apparent homogeneity, with arecovery of 25% (148). The final preparation hada specific activity of 1,130 U/mg of protein.Thompson et al. (226) have proposed that theprotein in general, which forms a complex withblue dextran being dissociable by salt, has asupersecondary structure, called a dinucleotidefold. The S. cellulosae S-127 ,B-lactamase is noexception to this rule. Thus, fluorescence titra-tion revealed that the enzyme interactedstrongly with oxidized nicotinamide adenine di-nucleotide (NAD) phosphate, but not with oxi-dized NAD or adenosine 5'-triphosphate ATP.Furthermore, when the enzyme solution wasapplied to a column of oxidized NAD-phos-phate-agarose, the enzyme was adsorbed andeluted only with 0.5 M NaCl, whereas the en-zyme was passed through or eluted completelywith less than 0.1 M NaCl, from a column ofSepharose 4B. These results indicate that the.8-lactamase interacts strongly with oxidizedNAD-phosphate and has a dinucleotide fold.However, it is very interesting in this connectionthat the ,B-lactamase shows a small but definitivedecrease of its enzymatic activity in the presenceof oxidized NAD-phosphate. This suggests thatoxidized NAD-phosphate acts as a negative al-losteric effector in this fi-lactamase, even thoughits exact physiological role is yet unknown. Be-cause some other,-lactamases from Strepto-myces interact strongly with blue dextran (151,152), these enzymes should also interact withsome dinucleotides, even if their properties, suchas molecular weights and isoelectric points, arequite different from each other.The finding that the f8-lactamase has a dinu-
cleotide fold is quite interesting also from thepoint of the view of the evolution or source off-lactamases in pathogenic bacteria. As previ-ously mentioned, the properties of f8-lactamasesare quite diverse. Consequently, it is probablethat the f,-lactamases at the present time havebeen evolved convergently from many differentproteins into one common property: hydrolysisof the ,B-lactam ring. If one accepts the idea thatthe enzymes inactivating antibiotics in patho-genic bacteria may be derived from antibiotic-producing Streptomyces (11, 137, 246), these di-vergent properties can be easily understood. Thereason is that the Streptomyces must protectitself at any cost from f,-lactam compoundswhich are its own metabolites. If this were notthe case it would kill itself by allowing these ,B-lactam compounds to bind with its own targets,penicillin-binding proteins (83, 149, 150). Theconcept that ,B-lactamases now come from many
origins is supported by the fact that the S. cel-lulosae 8-lactamase has a rather weak affinityfor oxidized NAD-phosphate and benzylpenicil-lin (148). Thus, the enzyme might have beenrelated in the past to a dehydrogenase or anoxygenase which used oxidized NAD-phosphateas a cofactor. Turing to fl-lactamases fromgram-negative bacteria, some R-factor-mediated,B-lactamases which hydrolyze oxacillin havebeen reported to bind blue dextran (40). Amongthe enzymes hydrolyzing oxacillin, the propertyof binding blue dextran is only manifested inthose having a molecular weight of over 40,000and comprising two subunits (41). However, theinteraction of these enzymes with dinucleotidesand the mechanism of interaction between thedimer enzyme and blue dextran have not beenanalyzed. Furthermore, whereas the f)-lacta-mases from gram-negative bacteria are inhibitedby sodium chloride at low concentrations, the S.cellulosae enzyme is not inhibited even at highconcentration. In any case, however, the rela-tionship of these enzymes at the molecular levelis quite interesting from the evolutional point ofview.
In contrast, in respect to hydrolyzing a "pen-icillinase-resistant" penicillin, such as oxacillin,the ,-lactamase from S. cacaoi S-352, belongingto class 5 in the above classification, is similar tothose of gram-negative bacteria. Both S. cacaoiS-352 ,B-lactamase and the 8-lactamases ofgram-negative bacteria are resistant to the in-hibition by p-chloromercuribenzoate and cloxa-cillin. However, the enzymes from gram-nega-tive bacteria are susceptible to inhibition bysodium chloride (40, 252), whereas the enzymeactivity from S. cacaoi is not sensitive to sodiumchloride (154). Moreover, the turnover numberwas calculated to be 2.84 x 104 mol of benzyl-penicillin hydrolyzed per mol of enzyme per minfor the latter enzyme, which is comparable tothat for S. aureus and TEM-type enzymes butmuch more than that for cloxacillin- and oxacil-lin-hydrolyzing enzymes from gram-negativebacteria (27). In any case, these types ofenzymesare relatively rare among R-factor-mediated andStreptomyces,B-lactamases (145,220). Sykes andSmith explained this fact by their lower sub-strate turnover rate as compared with those ofthe commoner, TEM-type ,8-lactamases (220).However, this explanation is not applicable tothe Streptomyces ,8-lactamases. Although the S.cacaoi f8-lactamase is similar to those of gram-negative bacteria in that none can hydrolyzecephalospoins and 7-methoxycephalosporins,the relative rates of hydrolysis of methicillin andcloxacillin to benzylpencillin for the S. cacaoienzyme and those from gram-negative bacteria
MICROBIOL. REV.
RESISTANCE TO 13-LACTAM ANTIBIOTICS 607
are quite different. This indicates that the ar-rangement of the functional groups involved inthe catalytic or binding reaction is somehowdifferent from enzyme to enzyme, whereas theessential features are similar.Another characteristic of S. cacaoi ,B-lacta-
mase is that the mechanism of hydrolysis ofbenzylpenicillin is quite different from that ofcloxacillin. The Km values for benzylpenicillin inthis enzyme sharply increased with decreasingpH. From this curve, the involvement of anamino acid residue with pKI of between 6.5 and7.0 in the binding or catalytic reaction of theenzyme was suggested. In this, the enzyme isquite different from the B. cereus ,8-lactamase,whose Km is independent ofpH (245). The curveof Km versus pH for cloxacillin as a substratewas completely different from that for benzyl-penicillin. The different behavior towards eachsubstrate also was observed in V,,-versus-pHcurves. In addition, Km values for benzylpenicil-lin altered with temperature within a smallrange, whereas those for cloxacillin changed farmore. These results suggest that the mechanismof hydrolysis of cloxacillin by this ,8-lactamase isdifferent from that of benzylpenicillin. The dif-ferent activation energies for the hydrolysis re-actions of benzylpenicilhin and cloxacillin furthersupport this notion. 83-Lactamases inhibited bymethicillin and cloxacillin are known to responddifferently, in a conformational sense, to thesepenicillins than to benzylpenicillin or other or-dinary penicillins (28, 169, 195). However, it is ofinterest that such a differential capacity hasbeen retained in a S. cacaoi f,-lactamase, a clox-acillin-hydrolyzing enzyme.
In some Streptomyces strains, the trait of 8-lactamase production is spontaneously lost athigh frequency or by treatment with mutagenicagents, such as acriflavine and ethidium bromide(118, 156). In S. lavendulae such a mutationalpattern presents several unusual features: (i)partial loss of 8l-lactamase production, accom-panied by auxotrophy to arginine or arginino-succinate and loss of ability to form aerial my-celia and spores; (ii) development of acid pH andlow saturation density of growth in liquid cul-ture; (iii) a decrease in antibiotic production; (iv)an increase in susceptibility to benzylpenicillin;(v) a decrease in production of pigment; and (vi)reversion of mutants to the prototroph (argininenonrequiring), restoring the ability to form aerialmycelia and spores, but not leading to recoveryof fl-lactamase activity (132). One of the possiblereasons for this is that the structural gene forfB-lactamase transposes near or into the argini-nosuccinate synthetase gene on the chromosomeand elicits the multiple effects. Another possi-
bility is that the transposable element is a com-mon regulatory gene necessary for secondarymetabolism. Considering that the time course of,8-lactamase production in Streptomyces in gen-eral follows that of usual secondary metabolitesand that no extrachromosomal genetic elementhas been detected in either the parent or themutant strains, the latter possibility is morereasonable (134; M. M. Nakano and H. Ogawara,in J. E. Zajic and P. End [ed.], Proceedings ofthe Sixth International Fermentation Sympo-sium, in press). This is confirmed by the follow-ing experiments. Repression of ,B-lactamase pro-duction in mutants with multiple mutationscould be recovered by changing the nitrogensource of the medium. When polypeptone (hy-drolysate of casein), polypeptone S (hydrolysateof soybean meal) or Casamino Acids were usedas a nitrogen source, ,8-lactamase production wasrepressed. However, when casein, soybean meal,or serum albumin was used as a nitrogen source,the /3-lactamase production increased to the par-ent's level. In other words, retarded utilizationof nitrogen source was essential for the highproduction of ,B-lactamase in mutant strains,demonstrating multiple effects. In addition, con-sumption of a carbon-source, such as glycerol,was also dependent on the nitrogen source,which was in parallel with the production of ,8-lactamase. Of interest, in this connection, is thefact that glutamine synthetase activity in theparent strain is high throughout cultivation,whereas that in the mutants is extremely low(M. M. Nakano and H. Ogawara, unpublisheddata). Thus, the unknown genetic element,which is associated with the argininosuccinatesynthetase gene on the chromosome, is relatedto nitrogen and carbon catabolite repression(104, 117) in mutants of S. lavendulae.
In contrast, no clear interrelation was ob-served in Streptomyces kasugaensis between ar-ginine auxotrophy and the repression of fl-lac-tamase production, although mutants requiringarginine were also obtained at high frequency;however, a close correlation was observed be-tween arginine auxotrophy and the loss of pig-ment and spore formation (134; Nakano andOgawara, in press). From the parent strain withplasmids one could seldom obtain an arginine-auxotrophic mutant. However, arginine-nonre-quiring mutants with no plasmids could be ob-tained spontaneously. Such mutants gave rise toarginine-requiring mutants, in which no plasmidwas detected, at a rate of over 10%. This indi-cates that once the plasmid is integrated intothe chromosome, part of it or some other ele-ment transposes easily to the arginine gene onthe chromosome. Transposition of a plasmid to
VOL. 45, 1981
608 OGAWARA
the arginine region on the chromosome was sup-ported by the isolation of revertants which didnot require arginine any more and had the orig-inal plasmids (134). Southem blotting of thehydridized deoxyribonucleic acids suggests thatsome sequences similar to insertion sequencesmay be present in these parent and mutantstrains (Nakano and Ogawara, unpublisheddata). However, the nature of the genetic ele-ments involved in the biosynthesis of 8-lacta-mases in Streptomyces strains remains to beclarified (131, 133).Streptomyces produce various kinds of 8i-lac-
tamases, and their properties are thought to bespecies specific, as in the cases with gram-nega-tive bacteria. How, then, do the fi-lactamasesplay their roles in resistance to ,B-lactam com-pounds? Or does the enzyme have some otherphysiological role? As stated above, most Strep-tomyces strains have a potential to produce f8-lactamase, and the amount produced in somestrains is comparable to those in B. cereus 569/H and B. licheniformis 749/C. The fact thatStreptomyces strains produce fi-lactamase con-stitutively with high frequency and in a largeamount means that they have an effective meansof self-protection against ,B-lactam compounds,autotoxic substances which should kill otherbacteria in the natural environment (73). How-ever, there being no definite relationship be-tween the minimum inhibitory concentration ofbenzylpenicillin, a natural,-lactam, and theamounts of 8-lactamase produced in manyStreptomyces strains (144, 149), the productionof,-lactamase over a certain amount should notbe necessary for self-protection, although theminimum amount is needed. On the other hand,when minimum inhibitory concentration forbenzylpenicillin is examined in some producersof small amounts of 18-lactamase that were ob-tained by treatment with mutagenic reagents,the minimum inhibitory concentrations werelower than those in the parent strain (118). Thisindicates that, in some strains, /8-lactamase con-tributes to some extent to resistance to ,8-lactamcompounds. However, when Streptomyces be-came able to biosynthesize ,8-lactam compoundsat some time in the past, ,B-lactamase was not asufficient defense against endogenous metabo-lites, for reasons described below. Nevertheless,since /1-lactamase presents the most efficientway to destroy exogenous, toxic compounds, itcan easily be postulated that the trait is trans-mitted to the pathogenic bacteria and thereexpresses its character.Let us now turn our attention to the PBPs in
Streptomyces. Examining PBPs by autoradiog-raphy and fluorography (134a, 149, 150), at least
five PBPs could be detected in S. cacaoi (a ,B-lactamase producer, but a /1-lactam nonpro-ducer), Streptomyces felleus and S. lavendulae(,B-lactam nonproducers), and fl-lactamase-non-producing mutants of Streptomyces E750-3. Incontrast, two to five PBPs at most could bedetected in the membrane fraction of Strepto-myces olivaceus (an MC-696-SY2 producer)(116, 239) and Streptomyces clavuligerus (a cla-vulanic acid and cephamycin producer) (18, 130,181). However, the possibility that the PBPs inBl-lactam producers are already saturated withthe ,B-lactams produced by the organisms them-selves, such as cephamycins and clavulanic acid,was denied by experiments with hydroxylamine(149) (it is possible, however, that hydroxyla-mine does not cleave the chemical bond in thecomplexes as it does in other bacteria [248]).The finding that fewer PBPs were detected in S.olivaceus and S. clavuligerus and other ,B-lac-tam producers (134a) may be due to the follow-ing causes. (i) The number of PBPs is affectedby the growth of the organisms. Even thoughPBPs are essential for the survival of the orga-nisms, different PBPs may have different func-tions at different times. It is known that in E.coli there are at least seven PBPs in membrane-bound forms, but they function differently atvarious growth stages in this species, such aselongation, morphogenesis, and formation ofsepta (61, 221). Thus, the quantity and qualityof PBPs can change in the course of the growthcycle. In fact, the fluorographic patterns ofPBPsin the early logarithmic phase are slightly differ-ent from those in other growth phases in S.clavuligerus, S. olivaceus, and some otherStreptomyces strains producing ,B-lactams. How-ever, in other strains, especially f)-lactam non-producers, no such difference has so far beenobserved (134a, 149). (ii) It can be said that somePBPs are not essential for the organisms. How-ever, even if this is so, if each PBP functionsdifferently, miniimum numbers of PBPs shouldexist. Two PBPs are surmised to be fewer thanthe minimum number. Almost all of the bacte-ria studied, including ,B-lactam-nonproducingStreptomyces strains, have five PBPs or more intheir membrane fractions. Moreover, the PBPbands in S. cacaoi correspond well to those inB. subtilis (83, 150). (iii) The best conceivablereason is that the /8-lactams produced or theproperty ofproducing ,8-lactams itself affects theproperties or production of PBP. Streptomycesstrains which produce ,B-lactams may becomeresistant not only to their own ,B-lactam metab-olites but also to all relatedf,-lactams and un-related compounds. This possibility can be sup-ported by the facts that S. olivaceus and S.
MICROBIOL. REV.
RESISTANCE TO f8-LACTAM ANTIBIOTICS
clavuligerus, ,B-lactam-producing species, aremore resistant to benzylpenicillin than are non-producing species, even though S. cacaoi pro-duces fi-lactamase constitutively (149), and that,under the same conditions, fluorographic bandsof PBPs in S. olivaceus and S. clavuligerus aregenerally fainter than those in S. cacaoi. Inaddition, all of the PBPs detected by fluorogra-phy in S. clavuligerus have lower affinities to[14C]benzylpenmilhin than do those found in S.cacaoi (81). However, production of fl-lactamcompounds does not affect all PBPs to the sameextent; rather, it affects each PBP in a differentmanner and to a different degree, and the levelmay depend on the strain, the growth conditions,the growth phase, and so on. For example, al-though S. clavuligerus produced more kinds ofPBPs in the culture supernatant and the solublefraction inside the cells than in the membrane,S. olivaceus showed no or only a few kinds ofPBPs in the culture supematant and the solublefraction (149). Furthermore, for S. cacaoi and S.clavuligerus, the heat stabilities of the bindingof [14C]benzylpenicillin to PBP are quite differ-ent and so are the curves of 14C release from the['4C]benzylpenicilhin-PBP complex (81). Thesedifferences are quite definite, considering that S.cacaoi, Streptomyces E750-3, S. olivaceus, andS. clavuligerus belong to the same genus andthat PBP patterns in E. coli, P. aeruginosa,Salmonella typhimurium, and Proteus are sim-ilar to one another, although they belong todifferent genera (59). This is also true with PBPpatterns in B. subtilis, B. licheniformis, and B.megaterium. The main reason for these differ-ences is considered to be the production of fi-lactam compounds or the natures of producing,8-lactams themselves.
Semisynthetic B-lactam compounds, mecilli-nam and methicillin (82), selectively and pref-erentially bind to specific PBPs in S. cacaoi,whereas they do not bind to any PBPs in S.olivaceus and S. clavuligerus. In contrast, a
natural ,f-lactam, clavulanic acid, binds to PBPin S. cacaoi only at a very high concentration,whereas no binding of clavulanic acid to PBP inS. olivaceus and S. clavuligerus is observed.Similar phenomena can be seen with anothernatural product, PS-5 (150). Thus, in fi-lactam-producing strains, fewer PBPs can be detectedin fluorographic patterns, with affinity to variousfl-lactam compounds being extremely low.Through these mechanisms, Streptomycesstrains become resistant to fl-lactam compounds,self metabolites. There has been no report ofisolation of strains which can produce high levelsof thienamycin and,PS-5, but one could obtainstrains which can produce high levels of cepha-
mycins. To produce a great deal of 8l-lactams,such strains change their morphology and some-times kill themselves. This indicates that thereis a limitation on the ability of Streptomyces tobecome resistant to ,B-lactam compounds bychanging the essential PBPs and that the bind-ing amount necessary for such a change is farless than that for any other antibiotics. Changingthe PBPs to become resistant to B-lactamsmakes Streptomyces fail to perforn functionsessential for their existence.Furthermore, PBPs in Streptmyces in general
have low affinities to benzylpenicillin. AlthoughPBP 3 (Mr = 64,000) has the highest affinity tobenzylpenicillin among the PBPs in S. cacaoi,10 jig/ml is needed to saturate the PBP 3. All ofthe other PBPs cannot be saturated at a concen-tration of 50 jig/ml. PBP-2 (Mr = 91,000), aprobable lethal target of benzylpenicillin, hasthe lowest affinity to benzylpenicillin (150). Thislow affinity is more remarkable with PBPs in S.clavuligerus. The high concentration needed forsaturation in Streptomyces in general forms astiking contrast to PBPs in E. coli (8 ,ug/ml atmost [207]) and in B. subtilis (less than 0.15 ,g/ml [98]). These facts reflect on the high mini-mum inhibitory concentrations of benzylpenicil-lin for Streptomyces in general, although theyare gram-positive bacteria. In other words,Streptomyces strains are resistant to 8B-lactamcompounds by possessing PBPs with low affini-ties for f,-lactams. Another mechanism of resist-ance to ,-lactams in Streptomyces is a permea-bility barrier (H. Ogawara and H. Nakazawa,unpublished data).As thus far described, it seems that, in contrast
to pathogenic bacteria, the main means of re-sistance to,8f-lactam compounds in Streptomycesis the low affinity of PBP (killing targets) for fi-lactam compounds; ,-lactamase, which playsthe main role in the resistance of pathogenicbacteria, performs only a minor function. Thereasons for this may be as follows. (i) ,B-Lactamsare biosynthesized in autotoxic forms inside thecells, in contrast to aminoglycoside antibiotics(213). When they are released to the outside, theessential PBPs for existence should be resistantto fi-lactams, because they are in contact witheach other in this process. (ii) f8-Lactamase can-not hydrolyze ,8-lactams in the permeation proc-ess, because in some strains of Streptomyces, I)-lactamase is readily released into the culturemedium immediately after its biosynthesis with-out keeping its enzymatic activity in the mem-brane and the soluble fraction (154), although itis uncertain whether this can also apply to otherStreptomyces strains. Rapid excretion into theculture medium presents a striking contrast to
609VOL. 45, 1981
610 OGAWARA
the cases in B. licheniformis (89, 196) and S.typhimurium (102). In addition, some fi-lacta-mases can hydrolyze ,B-lactams from Strepto-myces, whereas some cannot. (iii) The times ofmaximum production are different for /?-lacta-mase and /)-lactam (unpublished data).
Conversely, to achieve production of largeamounts of ,B-lactam compounds, the followingconditions are necessary: Streptomyces shouldnot produce ,B-lactamase simultaneously, ,B-lac-tams should be biosynthesized inside the cells innontoxic forms (including a complex with a f-
lactamase-like compound) and converted to au-totoxic forms after they are released into themedium, and PBPs should have low affinity forfi-lactams. To satisfy these conditions, specialcircumstances, such as temperature, pH, growthspeed, and so on, are necessary. They may bepoints of demarcation between life and death forthe producer organisms. It is possible that toachieve resistance to their own metabolites, thefB-lactam producers have already changed theirPBPs to an extent beyond which the PBPs canperform no essential function. This is now oneof the main subjects of investigation in the fieldof fi-lactam antibiotics.
CONCLUSION
The introduction of various antibiotics, suchas penicillin, streptomycin, tetracycline, chlor-amphenicol, and kanamycin, made it possible toexpect that diseases due to infection by patho-genic bacteria could be eliminated completelyfrom the earth in a few years. Contrary to thatexpectation, however, the number of pathogenicbacteria resistant to high levels of these anti-biotics has increased far more rapidly than be-fore. Thus, we have been compelled to exploreantibiotics effective not only against these re-
sistant bacteria but also against bacteria previ-ously thought not to be serious threats. In otherwords, we must work harder.As described in this review, a consideration of
drug resistance should take into account at leasttwo features: the resistance of pathogenic bac-teria and that of producing bacteria. However,only limited mechanisms are concerned. In thecase of f3-lactam antibiotics, the main mecha-nisms are change in the target, hydrolyzing en-zymes, ,8-lactamases, and the permeability bar-rier. Whereas,8-lactamases cause drug resistancein pathogenic bacteria, a change in the target(decrease in affinity of the target or the struc-tural change) is the main cause in the producingbacteria, Streptomyces. To achieve resistance tohigh levels of ,-lactams by changing the targetsor the affinity of the targets, bacteria must ac-quire such traits gradually. In other words, mul-tiple mutations are required. Thus, it is unlikely
MICROBIOL. REV.
that such traits are carried on extrachromosomalelements. Because the targets are essential com-ponents of the bacteria and even if the resistantphenomena are controlled by regulatory ele-ments, some of their genes, if not all, should beon the chromosome (120, 206, 216).
In contrast, the genetic element(s) controllingfl-lactamase is either on the chromosome or onextrachromosomal elements in the pathogenicbacteria. This also is true for some strains ofStreptomyces (132, 156). It is interesting, in thisconnection, that many penicillinase-type /?-lac-tamases similar to those in pathogenic bacteriaare detected in Streptomyces (155). ,B-Lacta-mases in Streptomyces are much more compli-cated than those in other bacteria. Probably,they are derived not only from enzymes impli-cated in the biosynthesis of peptidoglycan (254)or spores but also from other enzymes, such asdehydrogenases (148), peptidases, or esterases.It is understandable that the genetic elementscontrolling ,B-lactamases in Streptomyces aretransferred to the pathogenic bacteria throughbacteriophage (131) and plasmids (133), because,in some strains at least, their biosyntheses arecontrolled by such factors as transposable ele-ments (118, 132, 156). However, cephalosporin-ase-type ,B-lactamases are specific for gram-neg-ative bacteria. Possibly, they come from otherorigins.Microorganisms can create new traits (new
,8-lactamases, new PBPs, and new factors), aim-ing to the full extent at their survival, by recom-bination of the existing genes. We therefore haveto struggle continuously with the two opposedproblems: development of new ,B-lactams andtheir resistance in the producing and pathogenicmicroorganisms.
ACKNOWLEDGMENTSI thank Hamao Umezawa of the Institute of Micro-
bial Chemistry for his hearty encouragement through-out the work performed in my laboratory.The work carried out in my laboratory is supported
in part by the Institute of Microbial Chemistry and bygrants-in-aid from the Ministry of Education, Scienceand Culture of Japan.
LITERATURE CITED1. Albert, A. 1973. Selective toxicity. Chapman &
Hall, Ltd., London.2. Ambler, R. P. 1975. The amino acid sequence of
3. Ambler, R. P., and R. J. Meadway. 1969.Chemical structure of bacterial penicillinases.Nature (London) 222:24-26.
4. Ambler, R. P., and G. K. Scott 1978. Thepartial amino acid sequence of penicillinasecoded by Escherichia coli plasmid R6K. Proc.Natl. Acad. Sci. U.S.A. 75:3732-3736.
RESISTANCE TO fi-LACTAM ANTIBIOTICS 611
5. Aoki, H., H. Sakai, M. Kohsaka T. Konomi,J. Hosoda, Y. Kubochi, E. Iguchi, and H.Imanaka. 1976. Nocardicin A, a new mono-cyclic f-lactam antibiotic. I. Discovery, isola-tion and characterization. J. Antibiot. 29:492-500.
6. Asheshov, E. H. 1969. The genetics of penicillin-ase production in Staphylococcus aureus strainps80. J. Gen. MicrobioL 59:289-301.
7. Asheshov, E. H., and K. G. H. Dykes. 1968.Regulation of the synthesis of penicillinase indiploids of Staphylococcus aureus. Biochem.Biophys. Res. Commun. 30:213-218.
8. Aswapokee, N., and H. C. Neu. 1978. A sulfone,-lactam compound which acts as a ,B-lacta-mase inhibitor. J. Antibiot. 31:1238-1244.
9. Baldwin, G. S., A. Galdes, H. A. 0. Hill, B. E.Smith, S. G. Waley, and E. P. Abraham.1978. Hstidine residues as zinc ligands in ,B-lactamase II. Biochem. J. 175:441-447.
10. Batchelor, F. R., F. P. Doyle, J. H. C. Nayler,and G. N. Rolinson. 1959. Synthesis of peni-cillin: 6-APA in penicillin fermentation. Nature(London) 183:257-258.
11. Benveniste, R., and J. Davies. 1973. Amino-glycoside antibiotic-inactivating enzymes in ac-tinomycetes similar to those present in clinicalisolates of antibiotic-resistant bacteria. Proc.Natl. Acad. Sci. U.SA. 70:2276-2280.
12. Benveniste, R., and J. Davies. 1973. Mecha-nims ofantibiotic resistance in bacteria. Annu.Rev. Biochem. 42:471-506.
13. Binderup, F., W. 0. Godtfedsen, and K. Ro-holt. 1971. Orally active cephalosporin esters.J. Antibiot. 24:767-773.
14. Blumberg, P. M., and J. L Strominger. 1972.Five penicillin-binding components occur inBacillus subtilis membranes. J. BioL Chem.247:8107-8113.
15. Bobrowskd, MK M., M. Matthew, P. T. Barth,N. Datta, N. J. Grinter, A. E. Jacob, P.Kontomichalou, J. W. Dale, and J. T.Smith. 1976. Plasmid-determined P-lactamaseindisnguishable from the chromosomal ,8-lac-tamase of Escherichia coli. J. Bacteriol. 125:149-157.
16. Bondi, A., Jr., and C. C. Dietz. 1944. Produc-tion ofpenicillinase by bacteria. Proc. Soc. Exp.BioL Med. 56:132-134.
17. Briles, E. B., and A. Tomas 1973. Pneumo-coccal Forseman antigen. A choline-containinglipoteichoic acid. J. BioL Chem. 248:6394-6397.
18. Brown, A. G., D. Butterworth, M. Cole, G.Hanscomb, J. D. Hood, and C. Reading1976. Naturally-occurring 16-lactamase inhibi-tors with antibacterial activity. J. Antibiot. 29:668-669.
19. Brown, A. G., D. F. Corbett, A. J. Eglington,and T. T. Howarth. 1977. Structure of oli-vanic acid derivatives MM4550 and MM13902;two new fused B-lactams isolated from Strep-tomyces olivaceus. J. Chem. Soc. D 1977:523-525.
20. Buchanan, C. E., and J. L. Strominger. 1976.
Altered penicillin-binding components in pen-iciflin-reistant mutants of Bacillus subtilis.Proc. NatL Acad. Sci. U.S.A. 73:1816-1820.
21. Bu}hari, A. L, and L Ambrosio. 1978. Theinvertible segment of bacteriophage Mu DNAdetermines the adsorption properties of Muparticles. Nature (London) 271:575-577.
22. Cartwright, S. J., and A. F. W. Coulson. 1979.A semi-synthetic penicillinase inactivator. Na-ture (London) 278:360-361.
23. Charnas, R. L, J. Fisher, and J. R. Knowles.1978. Chemical studies on the inactivation ofEscherichia coli RTEM 8-lactamase by cla-vulanic acid. Biochemistry 17:2185-2189.
24. Chase, H. A. 1980. Purification offour penicillin-binding proteins from Bacillus megaterium. J.Gen. MicrobioL 117:211-224.
25. Chase, H. A., P. E. Reynolds, and J. R. Ward.1978. Purification and characterization of thepenicillin-binding protein that is the lethal tar-get of penicillin in Bacillus megaterium andBatillus licheniformis. Protein exchange andcomplex stability. Eur. J. Biochem. 88:275-285.
26. Chase, H. A., S. T. Shepherd, and P. E. Rey-nolds. 1977. Studies on the penicillin-bindingcomponents of Bacillus megaterium. FEBSLett. 76:199-203.
27. Citri, N. 1971. Penicillin and other #-lacta-mases, p. 23-46. In P. D. Boyer (ed.), Theenzymes, voL 4, 3rd ed. Academic Press, Inc.,New York.
28. Citri, N. 1973. Conformational adaptability inenzymes. Adv. Enzymol. Relat. Areas Mol.Biol. 37:397-648.
29. Citri, N., N. Garber, and M. Sela. 1960. Theeffect of urea and guanidine hydrochloride onactivity and optical rotation of penicllinas. J.BioL Chem. 235:3454-3459.
30. Citri, N., and AL R. Poflock. 1966. The bio-chemistry and function of f6-lactamase (peni-ciflinase). Adv. EnzymoL Relat. Areas Mol.Biol. 28:237-323.
31. Cleveland, R. F., L Daneo-Moore, A. J.Wicken, and G. D. Shockman. 1976. Effectof lipoteichoic acid and lipids on lysis of intactcells of Streptococcus faecalis. J. Bacteriol.127:1582-1584.
32. Cleveland, R. F., J. V. Holtje, A. J. Wicken,A. Tomasz, L Daneo-Moore, and G. D.Shockman. 1975. Inhibition of bacterial walllysins by lipoteichoic acids and related com-pounds. Biochem. Biophys. Res. Commun. 67:1128-1135.
33. Cleveland, R. F., A. J. Wicken, L Daneo-Moore, and G. D. Shockman. 1976. Inhibi-tion of wall autolysis in Streptococcus faecalisby lipoteichoic acid and lipids. J. Bacteriol.126:192-197.
34. Cohen, S. A., and RI F. Pratt 1980. Inactiva-tion of Bacilus cereus fl-lactamase I by 6,B-bromopenicillanic acid: mechanism. Biochem-istry 19:3996-4003.
35. Cole, M. 1964. Properties of the penicillin dea-cylase enzyme of Escherichia coli. Nature
VOL. 45, 1981
612 OGAWARA
(London) 203:519-520.36. Cole, M. 1979. Inhibitors of antibiotic-inactivat-
ing enzymes, p. 99-135. In J. D. Wlliams (ed.),Antibiotic interactions. Academic Press, Lon-don.
37. Coyette, J., J. M. Ghuysen, and R. Fontana.1978. Solubilization and isolation of the mem-brane-bound DD-carboxypeptidase of Strepto-coccus faecalis ATCC9790. Properties of thepurified enzyme. Eur. J. Biochem. 88:297-305.
38. Csanyi, V., L. Ferenez, andL. Mile. 1971. Chem-ical nature of the inactivation of Bacillus cer-eus penicilinas by iodine. Biochim. Biophys.Acta 236:619-627.
39. Curtis, N. A. C., D. Orr, G. W. Ross, and M.G. Boulton. 1979. Competition of 8i-lactamantibiotics for the penicillin-binding proteins ofPseudomonas aeruginosa, Enterobacter cloa-cae, Klebsiella aerogenes, Proteus rettgeri,and Escherichia coli: comparison with anti-bacterial activity and effects upon bacterialmorphology. Antimicrob. Agents Chemother.16:325-328.
40. Dale, J. W., and J. T. Smith. 1974. R-factor-mediated fl-lactamases that hydrolyze oxacil-lin: evidence for two distinct groups. J. Bacte-riol. 119:351-356.
41. Dale, J. W., and J. T. Smith. 1976. The dimericnature of an R-factor mediated ,-lactamase.Biochem. Biophys. Res. Commun. 68:1000-1005.
42. Daneo-Moore, L., and G. D. Shockman. 1977.The bacterial cell surface in growth and divi-sion, p. 597-715. In G. Poste and G. L. Nicolson(ed.), The synthesis, assembly and turnover ofcell surface components. North-Holland Pub-lishing Co., Amsterdam.
43. Daoust, D. R., H. R. Onishi, H. Wallick, D.Hendli, and E. 0. Stapley. 1973. Cepha-mycin, a new family of ,B-lactam antibiotics:antibacterial activity and resistance to ,B-lac-tamase degradation. Antimicrob. AgentsChemother. 3:254-261.
44. Davies, J., and D. I. Smith. 1978. Plasmid-determined resistance to antimicrobial agents.Annu. Rev. Microbiol. 32:469-518.
45. Davies, R. B., and E. P. Abraham. 1974. Metalcofactor requirements of ,B-lactamase II. Bio-chem. J. 143:129-135.
46. Davies, R. B., E. P. Abraham, and J. Melling.1974. Separation, purification and properties of,B-lactamase I and f8-lactamase II from Bacilluscereus 569/H/9. Biochem. J. 3:115-127.
47. Davis, B. D., and W. K. Maas. 1952. Analysisof the biochemical mechanism of drug resist-ance in certain bacterial mutants. Proc. Natl.Acad. Sci. U.S.A. 38:775-785.
48. Demain, A. L. 1974. How do antibiotic-produc-ing microorganisms avoid suicide? Ann. N.Y.Acad. Sci. 235:601-612.
49. Dideberg, O., P. Charleir, L. Dupont, M. Ver-meire, J. M. Frere, and J. M. Ghuysen.1980. The 4.5 A resolution structure analysis ofthe exocellular DD-carboxypeptidase of Strep-tomyces albus G. FEBS Lett. 117:212-214.
50. Dideberg, O., B. Joris, J. M. Frere, J. M.Ghuysen, G. Weber, R. Robaya, J. M. Bel-brouck, and I. Ruelandts. 1980. The exocel-lular DD-carboxypeptidase of Streptomyces al-bus G: a metallo (Zn2+) enzyme. FEBS Lett.117:215-218.
51. Durkin, J. P., G. I. Dimitrienko, and T. Vis-wanatha. 1977. Reversibility ofthe ampicillin-and nitrite-induced inactivation of,B-lactamaseI. Can. J. Biochem. 55:453-457.
52. Durkin, J. P., and T. Viswanatha. 1978. Cla-vulanic acid inhibition of ,B-lactamase I fromBacillus cereus 569/H. J. Antibiot. 31:1162-1169.
53. Dyke, K. G. H., and M. H. Richmond. 1967.Occurrence of various types of penicillinaseplasmid among 'hospital' staphylococci. J. Clin.Pathol. 20:75-79.
54. Eagle, H. 1954. The binding of penicillin in re-lation to its cytotoxic action. II. The reactivitywith penicillin of resistant variants of strepto-cocci pneumococci, and staphylococci. J. Exp.Med. 100:103-115.
55. Eagle, H., M. Levy, and R. Fleischman. 1954.The multiple mechanisms of penicillin resist-ance. J. Bacteriol. 68:610-616.
56. English, A. R., J. A. Retsema, A. E. Girard,J. E. Lynch, and W. E. Barth. 1978. CP-45,899, a beta-lactamase inhibitor that extendsthe antibacterial spectrum of beta-lactams: in-itial bacteriological characterization. Antimi-crob. Agents Chemother. 14:414-419.
57. Fisher, J., R. L Charnas, and J. R. Knowles.1978. Kinetic studies on the inactivation ofEscherichia coli RTEM fl-lactamase by cla-vulanic acid. Biochemistry 17:2180-2184.
58. Fleming, A. 1929. On the antibacterial action ofcultures of a penicillium, with special referenceto their use in the isolation of B. influenzae.Br. J. Exp. Pathol. 10:226-236.
59. Georgopapadakou, N. H., and F. Y. Liu. 1980.Penicillin-binding proteins in bacteria. Anti-microb. Agents Chemother. 18:148-157.
60. Giles, A. F., and P. E. Reynolds. 1979. Bacillusmegaterium resistance to cloxacillin accompa-nied by a compensatory change in penicillin-binding proteins. Nature (London) 280:167-168.
61. Greenwood, D., and F. O'Grady. 1973. Thetwo sites of penicillin action in Escherichiacoli. J. Infect. Dis. 128:791-794.
62. Greenwood, D., and F. O'Grady. 1975. Lysisenhancement: a novel form of interaction be-tween ,B-lactam antibiotics. J. Med. Microbiol.8:205-208.
63. Grindley, N. D. F., and D. J. Sherratt. 1979.Sequence analysis at IS1 insertion sites: modelsfor transposition. Cold Spring Harbor Symp.Quant. Biol. 43:1257-1261.
64. Guay, R., R. Letarte, J. C. Pechere, and B.Roy. 1980. Comparative efficiency of inhibitorson ,B-lactamases produced by gram-negativebacteria. IRCS (Int. Res. Commun. Syst.) Med.Sci. Libr. Compend. 8:209.
65. Guymon, L. F., D. L. Walstad, and P. F.
MICROBIOL. REV.
RESISTANCE TO ,B-LACTAM ANTIBIOTICS 613
Sparling. 1978. Cell envelope alterations inantibiotic-sensitive and -resistant strains ofNeisseria gonorrhoeae. J. Bacteriol. 136:391-401.
66. Hahn, F. E. 1976. Antimutagens and the preven-tion of chromosomal mutations to drug resist-ance. Antibiot. Chemother. (Basel) 20:112-132.
67. Hajdu, J., L. Ferencz, L. Mile, and V. Csanyi.1971. Effect of urea on the two iodine inacti-vation reactions of Bacillus cereus penicillin-ase. Acta Microbiol. Acad. Sci. Hung. 18:17-21.
68. Hakenbeck, R., M. Tarpay, and A. Tomasz.1980. Multiple changes of penicillin-bindingproteins in penicillin-resistant clinical isolatesof Streptococcus pneumoniae. Antimicrob.Agents Chemother. 17:364-371.
69. Hamilton-Miller, J. M. T., and J. T. Smith.1979. Beta-lactamases. Academic Press, Lon-don.
70. Hartmann, R., S. B. Bock-Henning, and U.Schwarz. 1974. Murein hydrolases in the en-velope of Escherichia coli. Properties in situand solubilization from the envelope. Eur. J.Biochem. 41:203-208.
71. Herbold, D. R., and L Glaser. 1975. Bacillussubtilis N-acetylmuramic acid L-alanine ami-dase. J. Biol. Chem. 250:1676-1682.
72. Herbold, D. R., and L. Glaser. 1975. Interactionof N-acetylmuramic acid L-alanine amidasewith cell wall polymers. J. Biol. Chem. 250:7231-7238.
73. Hill, P. 1972. The production of penicillins insoils and seeds by Penicillium chrysogenumand the role of penicillin 8i-lactamase in theecology of soil Bacillus. J. Gen. Microbiol. 70:243-252.
74. Holtie, J. V., and A. Tomasz. 1974. Teichoicacid phosphorylcholine esterase. A novel en-zyme activity in pneumococcus. J. Biol. Chem.249:7032-7034.
75. Holtje, J. V., and A. Tomasz. 1975. Lipotei-choic acid: a specific inhibitor of autolysin ac-tivity in pneumococcus. Proc. Natl. Acad. Sci.U.S.A. 72:1690-1694.
76. Holtje, J. V., and A. Tomasz. 1975. Biologicaleffects of lipoteichoic acids. J. Bacteriol. 124:1023-1027.
77. Holtje, J. V., and A. Tomasz. 1975. Specificrecognition of choline residues in the cell wallteichoic acid by the N-acetylmuramyl-L-ala-nine amidase of pneumococcus. J. Biol. Chem.250:6072-6076.
78. Holtje, J. V., and A. Tomasz. 1976. Purificationof the pneumococcal N-acetylmuramyl-L-ana-line amidase to biochemical homogeneity. J.Biol. Chem. 251:4199-4207.
79. Hopwood, D. A. 1978. Extrachromosomally de-termined antibiotic production. Annu. Rev.Microbiol. 32:373-392.
80. Hopwood, D. A., and M. J. Merrick. 1977.Genetics of antibiotic production. Bacteriol.Rev. 41:595-635.
81. Horikawa, S., H. Nakazawa, and H. Oga-
wara. 1980. Penicillin-binding proteins inStreptomyces cacaoi and Streptomyces clavu-ligerus. Kinetics of [14C]benzylpenicillin bind-ing, temperature sensitivity and release of[14C]benzylpenicillin from the complex. J. An-tibiot. 33:1363-1368.
82. Horikawa, S., and H. Ogawara 1978. Mem-brane-bound penicillin-binding proteins ofEscherichia coli. Comparison of a strain carry-ing an R-factor and the parent strain. J. Anti-biot. 31:1283-1291.
83. Horikawa, S., and H. Ogawara. 1980. Penicil-lin-binding proteins in Bacillus subtilis. Theeffects on penicillin-binding proteins and theantibacterial activities of fi-lactams. J. Anti-biot. 33:614-619.
84. Hotta, K., and Y. Okami. 1976. Productivityand resistance of antibiotic producer. HakkoTo Kogyo 34:488-499. (In Japanese.)
85. Huber, F. M., R. R. Chauvette, and B. G.Jackson. 1972. Preparative methods for 7-aminocephalosporanic acid and 6-aminopeni-cillanic acid, p. 27-73. In E. H. Flynn (ed.),Cephalosporins and penicillins, chemistry andbiology. Academic Press, Inc., New York.
86. Imada, A., K. Kitano, K. Kintaka, M. Muroi,and M. Asai. 1981. Sulfazecin and isosulfaze-cin, novel ft-lactam antibiotics of bacterial ori-gin. Nature (London) 289:590-591.
87. Ishiguro, E. E., and W. D. Ramey. 1976. Strin-gent control of peptidoglycan biosynthesis inEscherichia coli K-12. J. Bacteriol. 127:1119-1126.
88. Ishiguro, E. E., and W. D. Ramey. 1978. In-volvement of the relA gene product and feed-back inhibition in the regulation of UDP-N-acetylmuramyl-peptide synthesis in Esche-richia coli. J. Bacteriol. 136:766-774.
89. Izui, K., J. B. K. Nielsen, M. P. Caulfield, andJ. 0. Lampen. 1980. Large exopenicillinase,initial extracellular form detected in culturesof Bacillus lichenifornis. Biochemistry 19:1882-1886.
90. Jack, G. W. 1971. The purification and someproperties of a ,B-lactamase sensitive to inhibi-tion by p-chloromercuribenzoate. Biochim.Biophys. Acta 250:428-436.
91. Jack, G. W., and M. H. Richmond. 1970. Acomparative study of eight distinct 8i-lacta-mases synthesized by gram-negative bacteria.J. Gen. Microbiol. 61:43-61.
92. Jackson, R. T., L. F. Harris, and R. H. Alford.1978. Sodium clavulanate potentiation of ceph-alosporin activity against clinical isolates ofcephalothin-resistant Klebsiella pneumoniae.Antimicrob. Agents Chemother. 14:118-125.
93. Johnston, L. H., and K. G. H. Dyke. 1974.Staphylococcal penicillinase plasmids: studieson the reversion of a temperature-sensitivereplication mutant to temperature stability. J.Gen. Microbiol. 82:309-317.
94. Kamiya, T. 1977. Studies of the new monocyclicB8-lactam antibiotics, nocardicins, p. 281-294.In J. Elks (ed.), Recent advances in the chem-istry of fl-lactam antibiotics. The Chemical So-
VOL. 45, 1981
614 OGAWARA
ciety, London.95. Kato, K. 1953. Occurrence of penicillin-nucleus
in culture broths. J. Antibiot. Ser. A 6:130-139.96. Kesado, T., T. Hashizume, and Y. Asahi.
1980. Antibacterial activities ofa new stabilizedthienamycin, N-formimidoyl thienamycin, incomparison with other antibiotics. Antimicrob.Agents Chemother. 17:912-917.
97. Klaer, R., D. Pfeifer, and P. Starlinger. 1980.IS4 is still found its chromosomal site aftertranscription to galT. Mol. Gen. Genet. 178:281-284.
98. Kleppe, G., and J. L. Strominger. 1979. Stud-ies of the high molecular weight penicillin-binding proteins of Bacillus subtilis. J. Biol.Chem. 254:4856-4862.
99. Knott-Hunziker, V., B. S. Orlek, P. G.Sammes, and S. G. Waley. 1979. 61i-Bromo-peniciIlanic acid inactivates.,-lactamase I. Bio-chem. J. 177:365-367.
100. Knott-Hunziker, V., S. G. Waley, B. S. Orlek,and P. G. Sammes. 1979. Penicilinase activesites: labelling of serine-44 in ,B-lactamase I by6,B-bromopenicillanic acid. FEBS Lett. 99:59-61.
101. Konomi, T., S. Herchen, J. E. Baldwin, M.Yoshida, N. A. Hunt, and A. L. Demain.1979. Cell-free conversion of 8-(L-a-aminoadi-pyl)-L-cysteinyl-D-valine into an antibioticwith the properties of isopenicillin N in Ceph-alosporium acremonium. Biochem. J. 184:427-430.
102. Koshland, D., and D. Botatein. 1980. Secretionof 16-lactamase requires the carboxyl end of theprotein. Cell 20:749-760.
103. Kozarich, J. W., and J. L. Strominger. 1978.A membrane enzyme from Staphylococcus au-reus which catalyzes transpeptidase, carboxy-peptidase, and penicillinase activities. J. Biol.Chem. 253:1272-1278.
104. Kustu, S., D. Burton, E. Garcia, L McCarter,and N. McFarland. 1979. Nitrogen control inSalmonella: regulation by the glnR and glnFgene products. Proc. Natl. Acad. Sci. U.S.A.76:4576-4580.
105. Kuwabara, S., and E. P. Abraham. 1967. Someproperties of two extracellular ,-lactsmasesfrom Bacillus cereus 569/H. Biochem. J. 103:27c-30c.
106. Kuwabara, S., E. P. Adams, and E. P. Abra-ham. 1970. The composition of fi-lactamase Iand fi-lactamase II from Bacillus cereus 569/H. Biochem. J. 118:475-480.
107. Kuwabara, S., and P. H. Uloyd. 1970. Proteinand carbohydrate moieties of a preparation of,B-lactamase II. Biochem. J. 124:215-220.
108. Labia, R., V. Lelievre, and J. Peduzzi. 1980.Inhibition kinetics of three R-factor-mediatedf,-lactamases by a new ,B-lactam sulfone (CP45899). Biochim. Biophys. Acta 611:351-357.
109. Lacey, R. W. 1975. Antibiotic resistance plas-mids of Staphylococcus aureus and their clin-ical importance. Bacteriol. Rev. 39:1-32.
110. Lederberg, J. 1957. Mechanism of action ofpenicillin. J. Bacteriol. 73:144.
111. Lederberg, J., and N. Zinder. 1948. Concentra-tion of biochemical mutants of bacteria withpenicillin. J. Am. Chem. Soc. 70:42674268.
112. Lee, B. 1971. Conformation of penicillin as atransition state analog of the substrate of pep-tidoglycan transpeptidase. J. Mol. Biol. 61:463-469.
113. Lemke, P. A., and D. R. Brannon. 1972. Mi-crobial synthesis of cephalosporin and penicil-lin compounds, p. 370-437. In E. H. Flynn (ed.),Cephalosporins and penicillins, chemistry andbiology. Academic Press, Inc., New York.
114. Loosemore, M. J., S. A. Cohen, and R. T.Pratt 1980. Inactivation of Bacillus cereus,B-lactamase I by 6,8-bromopenicillanic acid: ki-netics. Biochemistry 19:3990-3995.
115. Lund, F., and L. Tybring. 1972. 6/i-Amidino-penicillanic acids. A new group of antibiotics.Nature (London) New Biol. 236:135-137.
116. Maeda, K., S. Takahashi, M. Sezaki, K. Iim-uma, H. Naganawa, S. Kondo, M. Ohno,and H. Umezawa. 1977. Isolation and struc-ture of a f8-lactamase inhibitor from Strepto-myces. J. Antibiot. 30:770-772.
117. Martin, J. F., and A. L Demain. 1980. Controlof antibiotic biosynthesis. Microbiol. Rev. 44:230-251.
118. Matsubara-Nakano, M., Y. Kataoka, and H.Ogawara. 1980. Unstable mutation of fi-lac-tamase production in Streptomyces lavendu-lae. Antimicrob. Agents Chemother. 17:124-128.
119. Matsuhashi, M., T. Kamiryo, P. M. Blum-berg, P. Linnett, E. Willoughby, and J. LStrominger. 1974. Mechanism of action anddevelopment of resistance to a new amidinopenicillin. J. Bacteriol. 117:578-587.
120. Matsuhashi, M., Y. Takagaki, L. N. Maru-yama, S. Tamaki, Y. Nishimura, H. Suzuki,U. Ogino, and Y. Hirota. 1977. Mutants ofEscherichia coli lacking in highly penicillin-sensitive D-alanine carboxypeptidase. Proc.Natl. Acad. Sci. U.S.A. 74:2976-2979.
121. Matsuura, M., H. Nakazawa, T. Hashimoto,and S. Mitauhashi. 1980. Combined antibac-terial activity of amoxicillin with clavulanicacid against ampicillin-resistant strains. An-timicrob. Agents Chemother. 17:908-911.
122. Matthew, M., and A. M. Harris. 1976. Identi-fication of al-lactamases by analytical isoelec-tric focusing: correlation with bacterial taxon-omy. J. Gen. Microbiol. 94:55-67.
123. Mehta, R. J., and C. H. Nash. 1978. ,B-Lacta-mase activity in yeast. J. Antibiot. 31:239-240.
124. Mltsuhashi, S. 1977. R factor. Drug resistanceplasmid. University of Tokyo Press, Tokyo.
125. Mitsuhashi, S. 1979. Microbial drug resistance,vol. 2. Japan Scientific Societies Press, Tokyo.
126. Mitsuhashi, S., and H. Hashimoto. 1975. Mi-crobial drug resistance. University of TokyoPress, Tokyo.
127. Murphy, E., and R. P. Novick 1979. Physicalmapping of Staphylococcus aureus penicillin-ase plasmid pI 524: characterization of an in-vertible region. Mol. Gen. Genetics 175:19-30.
MICROBIOL. REV.
RESISTANCE TO 8i-LACTAM ANTIBIOTICS 615
128. Mychajlonka, M., T. D. McDowell, and G. D.Shockman. 1980. Inhibition of peptidoglycan,ribonucleic acid, and protein synthesis in tol-erant strains of Streptococcus mutans. Anti-microb. Agents Chemother. 17:572-582.
129. Nagarajan, R. 1972. ,B-Lactam antibiotics fromStreptomyces. p. 636-61. In E. H. Flynn (ed.),Cephalosporins and penicillins, chemistry andbiology. Academic Press, Inc., New York.
130. Nagarajan, R., L. D. Boeck, M. Gorman, R.L Hamill. C. E. Higgens, M. M. Hoehn, W.H. Stark, and J. G. Whitney. 1971. fi-Lactamantibiotics from Streptomyces. J. Am. Chem.Soc. 93:2308-2310.
131. Nakano, M. M., H. Ishihara, and H. Ogawara.1981. Characterization of temperate bacterio-phage Ba isolated from Streptomyces laven-dulae. J. Gen. Microbiol. 122:289-293.
132. Nakano, M. M., and H. Ogawara. 1980. Mul-tiple effects induced by unstable mutation inStreptomyces lavendulae. J. Antibiot. 33:420-425.
133. Nakano, M. M., K. Ozawa, and H. Ogawara.1980. Isolation and characterization of a plas-mid pSL 1 from Streptomyces lavendulae.FEMS (Fed. Eur. Microbiol. Soc.) Microbiol.Lett. 9:111-113.
134. Nakano, M. M., K. Ozawa, and H. Ogawara.1980. Possible involvement of a plasmid in ar-ginine auxotrophic mutation of Streptomyceskasugaensis. J. Bacteriol. 3:1501-1503.
134a.Nakazawa, H., S. Horikawa, and H. Oga-wara. 1981. Penicillin-binding proteins in,Streptomyces strains. J. Antibiot. 34:1070-1072.
135. Neu, H. C. 1968. The surface localization ofpenicilinases in Escherichia coli and Salmo-nella typhimurium. Biochem. Biophys. Res.Commun. 32:258-263.
136. Nierlich, D. P. 1978. Regulation of bacterialgrowth, RNA and protein synthesis. Annu.Rev. Microbiol. 32:393-432.
137. Nimi, O., G. Ito, S. Sueda, and R. Nomi. 1971.Phosphorylation of streptomycin at C6-OH ofstreptidine moiety by an intracellular enzymeofStreptomycesgriseus. Agric. Biol. Chem. 35:848855.
138. Nishida, M., Y. Yotota, M. Okui, Y. Mine, andT. Matsubara. 1968. Studies on microbial deg-radation of cephalosporin C derivatives. I. Therole of 18-lactamase and acylesterase in theenzymatic degradation of cephalosporins. J.Antibiot. 21:165-169.
139. Nolan, R. D., and J. F. Hildebrandt. 1979.Comparison of the penicillin-binding proteinsof different strains of Neisseria gonorrhoeae.Antimicrob. Agents Chemother. 16:336-340.
140. Novick, R. P. 1967. Penicilhinase plasmids ofStaphylococcus aureus. Fed. Proc. Fed. Am.Soc. Exp. Biol. 26:29-38.
141. Novick, R. P., L. Edelman, M. D. Schwesin-ger, A. D. Gruss, E. C. Swanson, and P. A.Pattee. 1979. Genetic translocation in Staph-ylococcus aureus. Proc. Natl. Acad. Sci. U.S.A.76:400-404.
142. Novick, R. P., E. Murphy, T. J. Gryczan, E.Baron, and L. Edelman. 1979. Penicillinaseplasmids of Staphylococcus aureus: restric-tion-deletion maps. Plasmid 2:109-129.
143. O'Callaghan, C. H. 1979. Description and clas-sification of the newer cephalosporins and theirrelationships with the established compounds.J. Antimicrob. Chemother. 5:635-671.
144. Ogawara, H. 1975. Penicillin-inactivating en-zyme, ,B-lactamase, with special reference tothe active site, the role of the existence and themolecular evolution. Tampakushitsu KakusanKoso 20:1214-1227. (In Japanese.)
145. Ogawara, H. 1975. Production and property ofbeta-lactamases in Streptomyces. Antimicrob.Agents Chemother. 8:402-408.
146. Ogawara, H. 1976. A specific cephalosporin-binding protein of Citrobacter freundii.Biochim. Biophys. Acta 420:155-164.
147. Ogawara, H. 1977. Penicillin isocyanates for fl-lactamase. Methods Enzymol. 46:531-537.
148. Ogawara, H., and S. Horikawa. 1979. Purifi-cation of 1-lactamase from Streptomyces cel-lulosae by affinity chromatography on BlueSepharose. J. Antibiot. 32:1328-1335.
149. Ogawara, H., and S. Horikawa. 1980. Penicil-lin-binding proteins of Streptomyces cacaoi,Streptomyces olivaceus, and Streptomyces cla-vuligerus. Antimicrob. Agents Chemother. 17:1-7.
150. Ogawara, H., and S. Horikawa. 1980. Penicil-lin-binding proteins in Streptomyces cacaoi.The effects on penicillin-binding proteins andthe antibacterial activities of ,-lactams. J. An-tibiot. 33:620-624.
151. Ogawara, H., S. Horikawa, S. Shimada-Mi-yoshi, and K. Yasuzawa. 1978. Productionand property of beta-lactamases in Strepto-myces: comparison of the strains isolated newlyand thirty years ago. Antimicrob. AgentsChemother. 13:865-870.
152. Ogawara, H., S. Horikawa, S. Shimada-Mi-yoshi, and K. Yasuzawa. 1979. ,B-Lactamasesin Streptomyces, p. 331-333. In S. Mitsuhashi(ed.), Microbial drug resistance, vol. 2. JapanScientific Societies Press, Tokyo.
153. Ogawara, H., K. Maeda, and H. Umezawa.1972. A f6-lactamase of Escherichia coli.Biochim. Biophys. Acta 289:203-211.
154. Ogawara, H., A. Mantoku, and S. Shinada.1981. ,B-Lactamase from Streptomyces cacaoi.Purification and properties. J. Biol. Chem. 256:2649-2655.
155. Ogawara, H., T. Minagawa, and H. Nishi-zaki. 1978. Property of the ,B-lactamase fromStreptomyces E750-3. J. Antibiot. 31:923-925.
156. Ogawara, H., and S. Nozaki. 1977. Effect ofacriflavine on the production of ,8-lactamase inStreptomyces. J. Antibiot. 30:337-339.
157. Ogawara, H., and S. Shimada. 1980. Affinitylabeling off,-lactamases. Tampakushitsu Kak-usan Koso Suppl. 22:185-189. (In Japanese.)
158. Ogawara, H., and H. Umezawa. 1973. Affinitylabeling of an Escherichia coli f8-lactamase.Biochim. Biophys. Acta 327:481-489.
VOL. 45, 1981
616 OGAWARA
159. Ogawara, H., and H. Umezawa. 1974. Inacti-vation of 16-lactamase by some site-specific re-agents. J. Antibiot. 27:567-569.
160. Ogawara, H., and H. Umezawa. 1975. Bacilluscereus ,B-lactamase. Reaction with N-bromo-succinimide and the properties of the product.Biochim. Biophys. Acta 391:435-447.
161. Ogawara, H., and H. Umezawa. 1975. Activesites and conformation of/8-lactamases, p. 375-389. In S. Mitauhashi and H. Hashimoto (ed.),Microbial drug resistance. University of TokyoPress, Tokyo.
162. Okachi, R. 1979. Enzymatic synthesis of penicil-lins and cephalosporins. Nippon Nogei KagakuKaishi 53:R169-R177. (In Japanese.)
163. Okamura, K., S. Hirata, Y. Okumura, Y. Fu-kagawa, Y. Shimauchi, K. Kouno, T. Ish-ikura, and J. Lein. 1978. PS-5, a new ,B-lactamantibiotic from Streptomyces. J. Antibiot. 31:480-482.
164. Okanishi, M. 1979. Plasmids and antibiotic syn-thesis in streptomycetes, P. 134-140. In 0. K.Sebek and A. I. Laskin (ed.), Genetics of indus-trial microorganisms. American Society forMicrobiology, Washington, D.C.
165. Onishi, H. R., D. R. Daoust, S. B. Zimmer-man, D. Hendlin, and E. 0. Stapley. 1974.Cefoxitin, a semisynthetic cephamycin anti-biotic: resistance to beta-lactamase inactiva-tion. Antimicrob. Agents Chemother. 5:38-48.
166. O'Sullivan, J., R. C. Bleaney, J. A. Huddles-ton, and E. P. Abraham. 1979. Incorporationof 3H from 8-(L-a-amino[4,5-3H]adipyl)-L-cyS-teinyl-D-[4,4-3H]valine into isopenicillin N.Biochem. J. 184:421-426.
167. Ozer, J. H., D. L Lowery, and A. K. Saz. 1970.Depression off,-lactamase (penicillinase) inBacillus cereus by peptidoglycan. J. Bacteriol.102:52-3.
168. Ozer, J. H., and A. K. Saz. 1970. Possible in-volvement of,B-lactamase in sporulation in Ba-cillus cereus. J. Bacteriol. 102:64-71.
169. Pain, R. H., and R. Virden. 1979. The structuraland conformational basis of beta-lkctamase ac-tivity, p. 141-180. In J. M. T. Hamilton-Millerand J. T. Smith (ed.), Beta-lactamases. Aca-demic Press, London.
170. Park, J. T., and J. L Strominger. 1957. Modeof action of penicillin. Biochemical basis forthe mechanism of action of penicillin and forits selective toxicity. Science 125:99-101.
171. Patil, G. V., and R. A. Day. 1973. Involvementofa carboxyl group in the active site ofBacilluscereus 569/H penicillinase (Bt-lactamase I).Biochim. Biophys. Acta 293:490-496.
172. Pattee, P. A., N. E. Thompson, D. Haubrick,and R. P. Novick. 1977. Chromosomal maplocation of integrated plasmids and related ele-ments in Staphylococcus aureus. Plasmid 1:38-51.
173. Pennington, J. H., and F. O'Grady. 1967. Re-versal of the action of penicillin. Nature (Lon-don) 213:34-35.
174. Percheson, P. B., and L. E. Bryan. 1980. Pen-icillin-binding components of penicillin-suscep-
tible and -resistant strains of Streptococcuspneumoniae. Antimicrob. Agents Chemother.18:390-396.
175. Pollock, M. R. 1965. Purification and propertiesof penicillinases from two strains of Bacilluslicheniformis: a chemical, physicochemical andphysiological comparison. Biochem. J. 94:666-675.
176. Pollock, M. R. 1967. Origin and function of pen-icillinase: a problem in biochemical evolution.Br. Med. J. 4:71-77.
177. Pollock, M. R., and J. Fleming. 1969. Heritablemass conversion of a mutant penicillinase-neg-ative culture of Bacillus cereus to a positivefully de-repressed state. J. Gen. Microbiol. 59:303-316.
178. Pratt, R. F., and M. J. Loosemore. 1978. 6,8-bromopenicillinic acid, a potent ,B-lactamaseinhibitor. Proc. Natl. Acad. Sci. U.S.A. 75:4145-4149.
179. Prestidge, L. S., and A. Pardee. 1957. Induc-tion of bacterial lysis by penicillin. J. Bacteriol.74:48-59.
180. Ratney, R. S. 1965. The chemistry of the cellwalls of Bacillus anthracis: the effect of peni-cillin. Biochim. Biophys. Acta 101:1-5.
181. Reading, C., and M. Cole. 1977. Clavulanic acid:a beta-lactamase-inhibiting beta-lactam fromStreptomyces clavuligerus. Antimicrob.Agents Chemother. 11:852-857.
182. Reading, C., and P. Hepburn. 1979. Inhibitionof staphylococcal ,B-lactamase by clavulanicacid. Biochem. J. 179:67-76.
183. Richmond, M. H. 1965. Wild-type variants ofexopenicillinase from Staphylococcus aureus.Biochem. J. 94:584-593.
184. Richmond, M. H. 1975. ,8-Lactamase (Staphy-lococcus aureus). Methods Enzymol. 43:664-672.
185. Richmond, M. H., and R. B. Sykes. 1973. TheB-lactamases of gram-negative bacteria andtheir possible physiological role. Adv. Microb.Physiol. 9:31-88.
186. Rodriguez, W. J., and A. K. Saz. 1978. Differ-ential binding of penicillin by membrane frac-tions from penicillin-susceptible and -resistantgonococci. Antimicrob. Agents Chemother. 13:589-597.
187. Rogers, H. J. 1967. The inhibition of mucopep-tide synthesis by benzylpenicillin in relation toirreversible fixation of the antibiotic by staph-ylococci. Biochem. J. 103:90-102.
188. Rogers, H. J. 1967. Killing of staphylococci bypenicillins. Nature (London) 213:31-33.
189. Rosdahl, V. T. 1973. Naturally occurring consti-tutive,8-lactamase of novel serotype in Staph-ylococcus aureus. J. Gen. Microbiol. 77:229-231.
190. Rothstein, S. J., R. A. Jorgensen, K. Postle,and W. S. Reznikoff. 1980. The inverted re-peats of Tn5 are functionally different. Cell 19:795-05.
191. Sabath, L. D., N. Wheeler, M. Laverdiere, D.Blazevic, and B. J. Wilkinson. 1977. A newtype of penicillin resistance of Staphylococcus
MICROBIOL. REV.
RESISTANCE TO ,B-LACTAM ANTIBIOTICS 617
aureus. Lancet i:443-446.
192. Sachithanandam, S., D. L Lowery, and A. K.Saz. 1974. Endogenous, spontaneous formationof beta-lactamase in Staphylococcus aureus.Antimicrob. Agents Chemother. 6:763-769.
193. Sachithanandam, S., D. L. Lowery, and A. K.Saz. 1978. Isolation of beta-lactamase from apenicillin-susceptible strain of Staphylococcusaureus. Antimicrob. Agents Chemother. 13:289-292.
194. Sakaguchi, K., and S. Murao. 1950. A prelim-inary report on a new enzyme, penicillin-ami-dase. Nippon Nogei Kakagu Kaishi 23:411.
195. Samuni, A., and N. Citri. 1979. How specific isthe effect of penicillins on the conformation ofpenicllinase? An experimental model. Mol.Pharmacol. 16:250-255.
196. Sargent, M. G., B. K. Ghosh, and J. 0. Lam-pen. 1969. Characteristics of penicillinase se-
cretion by growing cells and protoplasts ofBacillus lichenifornis. J. Bacteriol. 97:820-826.
197. Saz, A. K. 1970. An introspective view of peni-cillinase. J. Cell. Physiol. 76:397-404.
198. Schwartz, J. L., and S. P. Schwartz. 1979.Production of ,B-lactamase by non-Strepto-myces Actinomycetales. Antimicrob. AgentsChemother. 15:123-125.
199. Schwesinger, M. D., and R. P. Novick. 1975.Prophage-dependent plasmid integration inStaphylococcus aureus. J. Bacteriol. 123:724-738.
200. Scott, G. K. 1973. Structure and mechanism offB-lactamase from Escherichia coli. Biochem.Soc. Trans. 1:159-162.
201. Segalove, M. 1947. The effect of penicillin ongrowth and toxin production by enterotoxicstaphylococci. J. Infect. Dis. 81:228-243.
202. Shapiro, J. A. 1979. Molecular model for thetransposition and replication of bacteriophageMu and other transposable elements. Proc.Natl. Acad. Sci. U.S.A. 76:1933-1937.
203. Shockman, G. D., M. J. Conover, J. J. Kolb,P. M. Phillips, L. S. Riley, and G. Toennies.1961. Lysis of Streptococcus faecalis. J. Bac-teriol. 81:36-43.
204. Shockman, G. D., L. Daneo-Moore, J. B. Cor-nett, and M. Mychajlonka. 1979. Does peni-cillin kill bacteria? Rev. Infect. Dis. 1:787-796.
205. Smith, J. T., and J. M. Wyatt. 1974. RelationofR factor and chromosomal ,8-lactamase withthe periplasmic space. J. Bacteriol. 117:931-939.
206. Spratt, B. G. 1975. Distinct penicillin bindingproteins involved in the diversion, elongation,and shape of Escherichia coli K12. Proc. Natl.Acad. Sci. U.S.A. 72:2999-3003.
207. Spratt, B. G. 1977. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur.J. Biochem. 72:341-352.
208. Spratt, B. G. 1977. Companson of the bindingproperties of two 6/1-amidinopenicillanic acidderivatives that differ in their physiologicaleffects on Escherichia coli. Antimicrob.Agents Chemother. 11:161-166.
209. Spratt, B. G. 1978. Escherichia coli resistanceto 16-lactam antibiotics through a decrease inthe affinity of a target for lethality. Nature(London) 274:713-715.
210. Spratt, B. G., and A. B. Pardee. 1975. Penicil-lin-binding proteins and cell shape in E. coli.Nature (London) 254:516-517.
211. Stapley, E. O., M. Jackson, S. Hernandez, S.B. Zimmerman, S. A. Currie, S. Mochales,J. M. Mata, H. B. Woodruff, and D. Hend-Un. 1972. Cephamycins, a new family of /-lactam antibiotics. I. Production by actinomy-cetes, including Streptomyces lactamduranssp. n. Antimicrob. Agents Chemother. 2:122-131.
212. Strominger, J. L 1977. How penicillin kills bac-teria: a short history, p. 177-181. In D. Schles-singer (ed.), Microbiology-1977. American So-ciety for Microbiology, Washington, D.C.
213. Sugiyama, M., H. Kobayashi, 0. Nimi, andR. Nomi. 1980. Susceptibility of protein syn-thesis to streptomycin in streptomycin-produc-ing Streptomyces griseus. FEBS Lett. 110:250-252.
214. Sutcliffe, J. G. 1978. Nucleotide sequence of theampicillin resistance gene of Escherichia coliplasmid pBR 322. Proc. Natl. Acad. Sci. U.S.A.75:3737-3741.
215. Sutcliffe, J. G. 1979. Complete nucleotide se-quence of the Escherichia coli plasmid pBR322. Cold Spring Harbor Symp. Quant. Biol.43:77-90.
216. Suzuki, H., Y. Nishimura, and Y. Hirota.1978. On the process of cellular division inEscherichia coli: a series of mutants on E. colialtered in the penicillin-binding proteins. Proc.Natl. Acad. Sci. U.S.A. 75:664-668.
217. Sykes, R. B., and M. Matthew. 1976. The ,B-lactamases of gram-negative bacteria and theirrole in resistance to ,8-lactam antibiotics. J.Antimicrob. Chemother. 2:115-157.
218. Sykes, R. B., M. Matthew, and C. H.O'Callaghan. 1975. R-factor mediated ,B-lac-tamase production by Haemophilus influ-enzae. J. Med. Microbiol. 8:437-441.
219. Sykes, R. B., and A. Percival. 1978. Studies ongonococcal 8i-lactamases, p. 68-74. In G. F.Brooks, E. C. Gotschlich, K. K. Holmes, W. D.Sawyer, and F. E. Young (ed.), Immunobiologyof Neisseria gonorrhoeae. American Societyfor Microbiology, Washington, D.C.
220. Sykes, R. B., and J. T. Smith. 1979. Biochem-ical aspects of beta-lactamases from gram-neg-ative organisms, p. 369-401. In J. M. T. Ham-ilton-Miller and J. T. Smith (ed.), Beta-lacta-mases. Academic Press, London.
221. Tamaki, S., S. Nakajima, and M. Matsuhashi.1977. Thermosensitive mutation in Esche-richia coli simultaneously causing defects inpenicillin-binding protein-lBs and in enzymeactivity for peptidoglycan synthesis in vitro.Proc. Natl. Acad. Sci. U.S.A. 74:5472-5476.
222. Thatcher, D. R. 1975. Progress on penicillinase.Nature (London) 255:526.
223. Thatcher. D. R. 1975. The partial amino acid
VOL. 45, 1981
618 OGAWARA
sequence of the extracellular 8-lactamase I ofBacillus cereus 569/H. Biochem. J. 147:313-326.
224. Thatcher, D. R. 1975. ,B-Lactamase (Bacilluscereus). Methods Enzymol. 43:640-652.
225. Thatcher, D. R. 1975. f8-Lactamase (Bacilluslicheniformis). Methods Enzymol. 43:653-664.
226. Thompson, S. T., K. H. Cass, and E. Stell-wagen. 1975. Blue-dextran Sepharose: an af-finity column for the dinucleotide fold in pro-tein. Proc. Natl. Acad. Sci. U.S.A. 72:669-672.
227. Tipper, D. J. 1979. Mode of action of 8i-lactamantibiotics. Rev. Infect. Dis. 1:39-53.
228. Tipper, D. J., and J. L. Strominger. 1965.Mechanism of action of penicillins: a proposalbased on their structural similarity to acyl-D-alanyl-D-alanine. Proc. Natl. Acad. Sci. U.S.A.54:1133-1141.
229. Tomasz, A. 1973. Cell surface structures and theabsorption of DNA molecules during transfor-mation in bacteria, p. 321-355. In L. Leive (ed.),Bacterial membrane and walls. Marcel Dekker,Inc., New York.
230. Tomasz, A. 1974. The role of autolysins in celldeath. Ann. N.Y. Acad. Sci. 235:439-448.
231. Tomasz, A. 1979. The mechanism of the irre-versible antimicrobial effects of penicillins: howthe fi-lactam antibiotics kill and lyse bacteria.Annu. Rev. Microbiol. 33:113-137.
232. Tomasz, A., A. Albino, and E. Zanati. 1970.Multiple antibiotic resistance in a bacteriumwith suppressed autolytic system. Nature(London) 227:138-140.
233. Tomasz, A., and J. V. Holtje. 1977. Mureinhydrolases and the lytic and killing action ofpenicillin, p. 209-215. In D. Schlessinger (ed.),Microbiology-1977. American Society for Mi-crobiology, Washington, D.C.
234. Tomasz, A., and S. Waks. 1975. Mechanism ofaction of penicillin-triggering of the pneumo-coccal autolytic enzyme by inhibitors of cellwall synthesis. Proc. Natl. Acad. Sci. U.S.A.72:4162-4166.
235. Tomasz, A., and S. Waks. 1975. Enzyme re-placement in a bacterium: phenotypic correc-tion by the experimental introduction of thewild type enzyme into a live enzyme-defectivemutant pneumococcus. Biochem. Biophys.Res. Commun. 65:1311-1319.
236. Tomasz, A., and M. Westphal. 1971. Abnormalautolytic enzyme in a pneumococcus with al-tered teichoic acid composition. Proc. Natl.Acad. Sci. U.S.A. 68:2627-2630.
237. Umezawa, H. 1967. Index of antibiotics fromActinomycetes. University of Tokyo Press,Tokyo.
238. Umezawa, H. 1978. Index of antibiotics fromActinomycetes, vol. 2. Japan Scientific Socie-ties Press, Tokyo.
239. Umezawa, H., S. Mitsuhashi, M. Hamada, S.lyobe, S. Takahashi, R. Utahara, T. Osato,S. Yamazaki, H. Ogawara, and K. Maeda.1973. The fi-lactamase inhibitors produced bya Streptomyces. J. Antibiot. 26:51-54.
MICROBIOL. REV.
240. Vandamme, E. J. 1977. Enzymes involved inB-lactam antibiotic biosynthesis. Adv. Appl.Microbiol. 21:89-123.
241. Vandamme, E. J., and J. P. Voets. 1974. Mi-crobial penicillin acylases. Adv. Appl. Micro-biol. 17:311-369.
242. van de Putte, P., S. Cramer, and M. Giphart-Gassler. 1980. Invertible DNA determineshost specificity of bacteriophage Mu. Nature(London) 286:218-222.
243. Vining, L. C. 1979. Antibiotic tolerance in pro-ducer organisms. Adv. Appl. Microbiol. 25:147-168.
244. Virudachalam, R., and V. S. R. Rao. 1977.Theoretical studies on fi-lactam antibiotics. I.Conformational similarity of penicillins andcephalosporins to X-D-alanyl-D-alanine andcorrelation of their structure with activity. Int.J. Pept. Protein Res. 10:51-59.
245. Waley, S. G. 1975. The pH-dependence andgroup modification of,8-lactamase I. Biochem.J. 149:547-551.
246. Walker, M. S., and J. B. Walker. 1970. Strep-tomycin biosynthesis and metabolism. Enzy-matic phosphorylation of dihydrostreptobios-amine moieties of dihydrostreptomycin-(strep-tidino)phosphate and dihydrostreptomycin byStreptomyces extracts. J. Biol. Chem. 245:6683-6689.
247. Watanakunakorn, C. 1978. Antibiotic-tolerantStaphylococcus aureus. J. Antimicrob. Chem-other. 4:561-568.
248. Waxman, D. J., and J. L. Strominger. 1979.Cleavage of a COOH-terminal hydrophobic re-gion from D-alanine carboxypeptidase, a peni-cillin-sensitive bacterial membrane enzyme.Characterization of active, water-soluble frag-ments. J. Biol. Chem. 254:4863-4875.
249. Weaver, S. S., G. P. Bodey, and B. M. Le-Blanc. 1979. Thienamycin: new beta-lactamantibiotic with potent broad-spectrum activity.Antimicrob. Agents Chemother. 15:518-521.
250. Wise, E. M., and J. T. Park. 1965. Penicillin: itsbasic site of action as an inhibitor of a peptidecross-linking reaction in cell wall mucopeptidesynthesis. Proc. Natl. Acad. Sci. U.S.A. 54:75-81.
251. Work, E. 1961. The mucopeptides of bacterialcell walls. A review. J. Gen. Microbiol. 25:167-189.
252. Yamagishi, S., K. O'Hara, R. Sawai, and S.Mitsuhashi. 1969. The purification and prop-erties of penicillin 8)-lactamases mediated bytransmissible R-factors in Escherichia coli. J.Biochem. 66:11-20.
253. Yocum, R. R., D. J. Waxman, J. R. Rasmus-sen, and J. L. Strominger. 1979. Mechanismof penicillin action: penicillin and substratebind covalently to the same active site serinein two bacterial D-alanine carboxypeptidases.Proc. Natl. Acad. Sci. U.S.A. 76:2730-2734.
254. Yocum, R. R., D. J. Waxman, and J. L.Strominger. 1980. Interaction of penicillinwith its receptors in bacterial membranes.
RESISTANCE TO,-LACTAM ANTIBIOTICS 619
Trends Biochem. Sci. 5:97-101.255. Zieg, J., M. Silverman, M. Hilmen, and M.
Simon. 1977. Recombinational switch for geneexpression. Science 196:170-172.
256. Zighelboim, S., and A. Tomasz. 1979. Stepwiseacquisition of resistance to oxacilhin in Strep-tococcuspneumoniae, p. 290-292. In D. Schles-
singer (ed.), Microbiology-1979. American So-ciety for Microbiology, Washington, D.C.
257. Zighelboim, S., and A. Tomasz. 1980. Penicil-lin-binding proteins of multiply antibiotic-re-sistant South African strains of Streptococcuspneumoniae. Antimicrob. Agents Chemother.17:434-442.
