Chromosomal 1-Lactamase Expression and Resistance to 3-LactamAntibiotics in Proteus vulgaris and Morganella morganii
YOUJUN YANG* AND DAVID M. LIVERMORE
Department of Medical Microbiology, The London Hospital Medical College,Turner Street, London El 2AD, United Kingdom
Received 29 February 1988/Accepted 20 June 1988
Indole-positive members of the Proteeae usually have inducible expression of chromosomal ,-lactamases.Mutants with stably derepressed P-lactamase expression occur in inducible populations at frequencies in therange of 10-6 to 10-8. The contribution of these P-lactamases to drug resistance was examined in Morganellamorganii and Proteus vulgaris. The M. morganii enzyme was a high-molecular-weight (49,000) class Icephalosporinase with low Vm.x rates for ampicillin, carbenicillin, and broad-spectrum cephalosporins. The P.vulgaris enzyme had a lower molecular weight (32,000) and high Vmax rates for ampicillin, cephaloridine,cefotaxime, and ceftriaxone. Imipenem and cefoxitin inactivated the P. vulgaris enzyme but were low-Vmax,low-Km substrates for that of M. morganii. Despite these differences, the two I-lactamases caused similarresistance profiles. Ampicillin and cephaloridine were strong inducers for both species, and ,I-lactamase-inducible strains and their stably derepressed mutants were resistant, whereas basal mutants (those withlow-level uninducible P-lactamase) were susceptible to these two compounds. Mezlocillin, cefotaxime,ceftriaxone, and (usually) carbenicillin were almost equally active against P-lactamase-inducible organisms andtheir basal mutants, but were less active against stably derepressed mutants. This behavior reflected theP-lactamase lability of these drugs, coupled with their weak inducer activity below the MIC. Carbenicillin wasa labile strong inducer for a single P. vulgaris strain, and inducible enzyme was protective against the drug inthis atypical organism. Cefoxitin and imipenem, both strong inducers below the MIC, were almost equallyactive against P-lactamase-inducible organisms and their basal and stably derepressed mutants.
Mutations which affect chromosomal class I ,-lactamaseexpression can cause resistance to many newer ,B-lactamantibiotics in Pseudomonas aeruginosa and Enterobactercloacae. Expression of class I enzymes usually is induciblein these species, and most newer penicillins and cephalospo-rins, although labile to hydrolysis, are poor inducers ofenzyme synthesis below the MIC (8, 9). Consequently, theyremain active as long as the enzyme remains inducible.Activity is lost against mutants (variously termed "consti-tutive," "macroconstitutive," or "stably derepressed") thatproduce ,-lactamase copiously without induction (8, 9, 16).Such mutants are segregated at high frequency by induciblepopulations (3, 23) and may be selected during cephalosporinor ureidopenicillin therapy, sometimes resulting in clinicalfailure (8, 15).Chromosomal ,-lactamase inducibility also is characteris-
tic of indole-positive members of the Proteeae (i.e., Proteusvulgaris, Morganella morganii, and Providencia rettgeri)(20), and Dworzack et al. (4) have reported selection of a,3-lactamase-derepressed M. morganii mutant during cefa-mandole therapy. In the present investigation, we examinedthe contribution of chromosomal P-lactamases to resistancein M. morganii and P. vulgaris under conditions in whichenzyme synthesis was bcth inducible and stably dere-pressed. These findings were correlated with drug hydrolysisassays performed with purified P-lactamases and with directassays of the P-lactamase-inducing power of various ,B-lactam antibiotics.
MATERIALS AND METHODSStrains. ,B-Lactamase-inducible, stably derepressed, and
basal wild-type strains were isolated from patients at The
* Corresponding author.
London Hospital between 1982 and 1986. Identification wasby classical biochemical tests or the API 20E system.1-Lactamase production characteristics of the organismsstudied in detail, and of mutants derived from them, arelisted in Table 1. Stably derepressed mutants were selectedfrom inducible strains on Diagnostic Sensitivity Test agar(Oxoid Ltd., Basingstoke, Hants, United Kingdom) contain-ing cefotaxime (1 or 4 ,ug/ml) by the procedure describedpreviously (24). 1-Lactamase basal mutants (those withlow-level uninducible 13-lactamase expression) were derivedfrom the stably derepressed organisms by mutagenesis withN-methyl-N'-nitro-N-nitrosoguanidine (2).
Antibiotics. Mezlocillin was from Bayer Pharmaceuticals,Haywards Heath, Sussex, United Kingdom; ampicillin so-dium and carbenicillin disodium from Beecham ResearchLaboratories, Brockham Park, Surrey, United Kingdom;benzylpenicillin, cephaloridine and nitrocefin, from GlaxoGroup Research, Greenford, Middlesex, United Kingdom;cefotaxime, from Hoechst-Roussel, Somerville, N.J.; cefox-itin and imipenem, from Merck Sharp & Dohme Ltd.,Hoddesdon, Herts, United Kingdom; and ceftriaxone, fromRoche Products Ltd., Welwyn Garden City, Herts, UnitedKingdom.
Susceptibility tests. MICs of antibiotics were measured onDiagnostic Sensitivity Test agar. The inocula consisted of104 CFU per spot. Results were read after incubation for 18h at 37°C.
P-Lactamase induction assays. Induction assays were per-formed by exposure of logarithmic-phase cells to antibioticsin Iso-Sensitest broth (Oxoid) for 4 h, as described previ-ously (24). After induction, the cells were harvested andwashed by centrifugation at 5,000 x g and 30°C and thensonicated on ice. The 1-lactamase activity of the sonicextracts was assayed by spectrophotometry at 295 nm, with
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TABLE 1. ,-Lactamase inducibility mutants and their P-lactamase production characteristics
Enzyme activity (nmol of
Stra D p-Lactamase cephaloridine hydrolyzed/min perStrain Derivation inducibilityb mg of cell protein)c pINot induced Induced
V2 Clinical isolate I 16.8 326 8.2V2-con Cefotaxime-selected mutant of V2 SDR 1,010 373 8.2V2-def NTGd mutant of V2-con B 20.0 17.5 8.2
V3 Clinical isolate I 10.7 661 8.9V3-con Cefotaxime-selected mutant of V3 SDR 1,460 1,130 8.9V3-def NTG mutant of V3-con B 12.4 14.4 8.9
Vii Clinical isolate I 14.2 541 8.0Vll-con Cefotaxime-selected mutant of Vii SDR 2,360 540 8.0Vll-def NTG mutant of Vll-con B 5.0 6.0 8.0
V17 Clinical isolate I 16.1 310 8.6V17-con Cefotaxime-selected mutant of V17 SDR 1,550 557 8.6V17-def NTG mutant of V17-con B 6.6 6.4 8.6
Val Clinical isolate I 9.8 604 7.8Val-con Cefotaxime-selected mutant of Val SDR 8,600 280 7.8Val-def NTG mutant of Val-con B 10.4 7.12 7.8
Ml Clinical isolate I 20.25 1,424 6.6Mi-con Cefotaxime-selected mutant of Ml SDR 2,872 4,451 6.6Ml-def NTG mutant of Mi-con B 8.7 241 6.6
M3 Clinical isolate SDR 1,014 1,044 7.6M3-def NTG mutant of M3 B 56.2 23.2 7.6
M6 Clinical isolate I 30.1 1,000 7.6M6-con Cefotaxime-selected mutant of M6 , SDR 1,265 3,349 7.6M6-def NTG mutant of M6-con B 16.1 12.7 7.6
aV, P. vulgaris; M, M. morganifi.b B, Basal; I, inducible; SDR, stably derepressed.c Specific activity versus 10 mM cephaloridine. Induction was with 1 pLg of imipenem per ml (see text).NTG, N-Methyl-N'-nitro-N-nitrosoguanidine.
10 mM cephaloridine as the substrate in 0.1 M phosphatebuffer, pH 7.0. The light path was 1 mm. Enzyme yieldswere standardized against the protein concentration, whichwas measured by the method of Lowry et al. (10).
,B-Lactamase extraction and purification. The ,B-lactamase-derepressed clinical isolate M. morganii M3 and the labora-tory mutants P. vulgaris V3-con and Val-con (Table 1) werechosen as sources of P-lactamases for biochemical study.Cultures were grown overnight in Iso-Sensitest broth at37°C, with continuous agitation, and then diluted 10-fold into20-liter amounts of prewarmed (37°C) identical broth. Incu-bation was continued for 4 h under the same conditions.Subsequently, the cells were harvested by centrifugation for15 min at 5,000 x g, washed in 0.1 M phosphate buffer, pH7.0, and suspended at 200 times their original density in thesame buffer.P-Lactamases were released from the cell suspensions of
M. morganii M3 and P. vulgaris V3-con by six to sevencycles of alternate freezing and thawing and from P. vulgarisVal-con by sonication. Cell debris was removed from thesonic extracts by centrifugation at 100,000 x g for 30 min.The supematants were retained, and the ,B-lactamases werepurified by chromatography. Enzyme from M. morganii wassubjected first to cation-exchange chromatography on car-boxymethyl-Sephadex C-50 (Pharmacia Fine Chemicals,Uppsala, Sweden) in 10 mM phosphate buffer, pH 6.5.Elution was with the same buffer containing a linear (0 to 0.5
M) NaCl gradient. p-Lactamase-containing eluent fractionswere dialyzed against 20 mM Tris hydrochloride, pH 8.8,and then rechromatographed on DEAE-Sephadex A-50.Elution was with 20 mM Tris hydrochloride buffer, pH 8.8,containing a linear (0 to 0.5 M) NaCl gradient. The ,-lactamase-rich eluent fractions were pooled and dialyzedagainst distilled water, then lyophilized, and then redis-solved in 10 mM phosphate buffer, pH 7.0, and subjected togel filtration on Sephadex G-100 Superfine.The ,-lactamases from both P. vulgaris strains were
chromatographed first on carboxymethyl-Sephadex C-50and then on QAE-Sephadex A-50. Equilibration bufferswere 10 mM phosphate, pH 7.5, and 10 mM diethanolaminehydrochloride, pH 10.0, respectively, for enzyme from V3-con and 10 mM phosphate, pH 6.5, and 10 mM diethanol-amine hydrochloride, pH 8.8, respectively, for enzyme fromVal-con. Elution was with the equilibration buffer contain-ing a linear (0 to 0.5 or 0 to 1.0 M) NaCl gradient. Enzymefrom P. vulgaris Val-con was further purified by gel filtra-tion on Sephadex G-100 Superfine in 10 mM phosphatebuffer, pH 7.0. The molecular weights and purity of the,-lactamase preparations were examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis as described pre-viously (9).
Hydrolysis assays. Hydrolysis of P-lactams was examinedby UV spectrophotometric assays in 1-cm or 1-mm light pathcuvettes at 37°C (13, 22). Antibiotic solutions were prepared
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TABLE 2. MICs of 13-lactam antibiotics for wild types and mutants of P. vulgaris and M. morganji
in 0.1 M phosphate buffer, pH 7.0. The wavelengths selectedfor the assays were as follows: ampicillin, benzylpenicillin,carbenicillin, and mezlocillin, 235 nm; cefotaxime, 255 nm;ceftriaxone, 257 nm, cefoxitin, 260 nm; cephaloridine, 295nm; and imipenem, 297 nm. Kinetic parameters (Vmax andKin) were derived by linear regression analysis of Hanes (slvagainst s) or Lineweaver-Burk (1/v against lls) plots of initialvelocity (v) data obtained at 8 to 10 different substrate (s)concentrations.
Isoelectric focusing. Isoelectric focusing was performed bythe method of Matthew et al. (12).
RESULTS
j8-Lactamase production by wild types and mutants. Directinduction assays with imipenem (1 ,ug/ml) demonstratedP-lactamase inducibility in 27 of 28 P. vulgaris isolatesexamined and in 8 of 11 M. morganii isolates. The inductionratios (P-lactamase specific activity in the presence of in-ducer divided by P-lactamase specific activity in the absenceof inducer) varied from 5 to 170. Two M. morganii isolatesproduced large amounts of chromnosomal ,-lactamase withor without induction. Single 1-lactamase "basal" P. vulgarisand M. morganii isolates had low enzyme activity regardlessof the presence of inducer.The P-lactamases induced in P, vulgaris strains showed a
scatter of pls from 7.7 to 9.2; enzyme from the P-lactamasebasal strain had a pl of 9.2. Eight M. morganii strains(including the enzyme basal isolate) produced ,-lactamasesfocusing at pl 7.6; three produced enzymes focusing at pI6.6. ,-Lactamase production data for the strains studied indetail, and for their stably derepressed and basal mutants,are listed in Table 1. None of the organisms listed producedadditional plasmid-type P-lactamases: such enzymes werepresent in only 1 of the 39 isolates in the complete collection.
,B-Lactamase-inducible P. vulgaris and M. morganii iso-lates segregated stably derepressed mutants at frequencies inthe range of 10-6 to 10-8. P-Lactamase derepression in these
mutants usually was total. The P. vulgaris strains alsosegregated, at similar frequency, mutants with enzyme in-ducibility identical to that of their parents but with signifi-cantly increased resistance to carbenicillin, mezlocillin,cefotaxime, and ceftriaxone, but not imipenem. These or-ganisms were not studied in detail but may represent pertme-ability mutants. Analogous mutants were not segregated byM. morganii strains at detectable frequency (>10-9).
Antibiotic susceptibility. The MICs of P-lactams.for thewild types and mutants are listed in Table 2. P-Lactamase-inducible strains of both species typically were susceptibleto all P-lactams tested except ampicillin and cephaloridite.Atypically, P. vulgaris Val was resistant to carbenicillin(MIC, 128 ,ug/ml).The stably derepressed mutants, whether selected in the
laboratory or isolated from clinical mnaterial, remained resis-tant to ampicillin and cephaloridine and also showed greatlydecreased susceptibility to all other antibiotics tested, ex-cept cefoxitin and imipenem, compared with inducible or-ganisms. For most derepressed mutants the MICs of carben-icillin and mezlocillin were increased 4- to 64-fold, and MICsof cefotaxime and ceftriaxone were raised 32- to 128-foldcompared with the MICs for the inducible strains. Never-theless, MICs of cefotaxime and ceftriaxone often remainedin the clinically achievable range. Stable derepression ofP-lactamase synthesis caused only a twofold increase incarbenicillin MIC for strain Val.The ,B-lactamase basal mutants mostly were susceptible,
or moderately so, to all antibiotics tested, itncluding ampicil-lin and cephaloridine. With one exception, MICs of carben-icillin, mezlocillin, cefotaxime, and ceftriaxone for the basalmutants were similar to, or slightly lower than, those fortheir inducible parent strains. The exception was for the P.vulgaris Val series; the basal mutant (Val-def) was consid-erably more susceptible to carbenicillin than was the corre-sponding inducible strain (Val).MICs of cefoxitin and imipenem, unlike those of the other
,-lactams, remained almost constant within each mutant
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0
0
v2C
0
0-aEC.00
1 41 1 1 1o '1 /4 1/2 1 2 XMIC
o ' 10 1z,/ 1/2 1 2 XMIC
FIG. 1. Induction of P-lactamase in M. morganii M3 (a), P.vulgaris V3 (b), and P. vulgaris Val (c) by ampicillin (0), carbeni-cillin (O-O), cefotaxime (l), cefoxitin (0- -0), imipenem (A),and mezlocillin (A). Induction ratios correspond to enzyme activityper milligram of cell protein of induced cells divided by that inuninduced cells. The behavior of ceftriaxone (not shown) wasvirtually identical to that of cefotaxime.
series, regardless of the P-lactamase expression. MICs ofnalidixic acid, chloramphenicol, tetracycline, and gentami-cin likewise were constant within each mutant series (datanot shown), suggesting that modification of the permeabilitycharacteristics had not occurred during the mutation proce-dures.
Inducer power of I8-lactams. The inducer power of -lactams was examined for strains V2, V3, Val, Ml, and M6.Results for strains V3, Val, and Ml are shown in Fig. la-c.Results for strain V2 resembled those for V3, and results forstrain M6 resembled those for Ml. At low concentrationsimipenem was the strongest inducer for all strains, giving 30-to 80-fold induction below the MIC. Cefoxitin and ampicillinalso were strong inducers below the MIC. Carbenicillin,mezlocillin, cefotaxime, and ceftriaxone were poor inducersbelow the MIC for most strains except P. vulgaris Val, forwhich carbenicillin caused up to 60- to 70-fold inductionbelow the MIC (Fig. lc).
Hydrolysis of (8-lactam antibiotics by purified ,-lactamases.The P-lactamases purified from P. vulgaris V3-con andVal-con gave single bands on sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. The molecular weight ofboth enzymes was 32,000 + 1,000. Enzyme purified from M.morganii M3 gave a major band with a molecular weight of49,000, but remained contaminated with a larger protein.
Kinetic parameters for ,B-lactam hydrolysis are shown inTable 3. Both of the P. vulgaris enzymes behaved similarly,but very differently from that from M. morganii M3. The P.vulgaris enzymes had substantial Vmax rates for ampicillin
0 U
I 0 /1
I
0 1I o 1/4 1/2 1 2 XMIC
and carbenicillin, with the former exceeding the Vmax forbenzylpenicillin. Vmax rates for cefotaxime and ceftriaxonealso were high, although affinity for these drugs was low(high Kms). The M. morganii enzyme, on the other hand,had a Vmax for ampicillin of under 5% of the Vmax forbenzylpenicillin, and Vmax rates for carbenicillin, cefotax-ime, and ceftriaxone were less than 1% of the Vnmax forbenzylpenicillin. The Km values for the last three drugs werelow, however, elevating the Vmax/Km ratios.Hydrolyses of cefoxitin and imipenem by the M. morganii
enzyme, and the hydrolysis of cefoxitin by the V3-conenzyme, were slow but obeyed Michaelis-Menten kinetics.Hydrolyses of imipenem by both of the P. vulgaris enzymesand of cefoxitin by the Val-con enzyme, however, werebiphasic. Initially, drug hydrolysis was rapid, but the ratedecelined more rapidly than was explicable by substratedepletion, halving before 20% of the antibiotic had beendestroyed. After 80 to 100 s, the hydrolysis rate was 20 to25% of its initial value and thereafter remained almostconstant (Fig. 2). If further substrate was added midwaythrough a hydrolysis assay, the initial fast phase of reactionwas not repeated. The fast phase was repeated, however, if
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V-LACTAMASES OF PROTEEAE 1389
TABLE 3. Hydrolysis of P-lactam antibiotics by 13-lactamases from strains V3-con, Val-con, and M3
a Relative to benzylpenicillin at 37°C and pH 7.0.
further substrate was added after all substrate initially pro-vided had been hydrolyzed. Addition of further enzymemidway through a hydrolysis assay gave a repetition of thefast phase (Fig. 2).Few substantial differences were observed between the
kinetic properties of the P. vulgaris V3-con and Val-conenzymes: the latter did, however, have a lower Km forcarbenicillin. Turnover numbers (kcat) of benzylpenicillinwere 1,160 and 950 per min for the V3-con and Val-conenzymes, respectively.
DISCUSSION
P. vulgaris, M. morganii, and Providencia rettgeri arecollectively termed the indole-positive Proteeae and arenoted for their resistance to many older ,B-lactam antibiotics.P. vulgaris and M. morganii are isolated more frequentlyfrom clinical material than is Providencia rettgeri, and weexamined the contribution of chromosomal 1-lactamases toresistance in the former two species. As has been remarked
0.05-
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EcO-a
t-
Scx
Ea0C0
CL
0.03-
0.02-
0.01-
0-
0 20 40 60 80 100 120
Time (minutes)
FIG. 2. Hydrolysis of imipenem by ,B-lactamase from P. vulgarisV3. At time zero, enzyme (2.2 nmol) and drug (100 nmol) weremixed in a 1-ml volume. The decline in optical density was moni-tored at 297 nm and 37°C in a 1-cm-light path cuvette. At 60 min,either (a) the imipenem concentration was readjusted to its originallevel or (b) a further 1.1 nmol of enzyme was added.
previously (17, 20), the chromosomal 3-lactamases of M.morganii and P. vulgaris differ radically. The M. morganiienzyme had low Vmax rates for carbenicillin, cefotaxime,and ceftriaxone, but also exhibited high affinity for thesedrugs (low K,). The Vmax for ampicillin was low relative tothat for benzylpenicillin. In all of these respects, and in itshigh molecular weight (49,000), the M. morganii enzymeresembled the class I 3-lactamases typical of Enterobactercloacae (Ta enzyme), Escherichia coli (Tb enzyme), andPseudomonas aeruginosa (Id enzyme), which are closelyrelated to one another (6). By contrast, the P. vulgarisenzymes had high Vmax rates for carbenicillin, cefotaxime,and ceftriaxone, but low affinity (high Km) for the last twodrugs. The Vmax for ampicillin exceeded that for benzylpen-icillin, and the molecular weight (32,000) of the enzyme wasconsiderably below that of a typical class I ,-lactamase.Although the P. vulgaris 1-lactamase often is listed as a classI type (type Ic) (14, 20), the differences noted above togetherwith its cefuroximase activity (11) and its susceptibility toinhibition by clavulanate (1) distinguish it from more typicalmembers of the class. Neither the M. morganii nor the P.vulgaris 1-lactamase had significant activity against imipe-nem. The drug was a low-Km, ultralow-Vmax substrate forthe M. morganii P-lactamase, in which hydrolysis obeyedMichaelis-Menten kinetics, and caused a partial inactivationof the P. vulgaris enzymes. The ability of imipenem toinactivate P. vulgaris P-lactamase has been noted previouslyby Sawai and Tsukamoto (18) and Hashizume et al. (5), whodiscussed possible mechanisms for this behavior.
Despite their considerable differences, the M. morganiiand P. vulgaris enzymes caused similar resistance profiles.Ampicillin was a labile strong inducer for both species,lacking activity against both inducible and stably dere-pressed organisms, but being active (or moderately so)against their basal mutants. Mezlocillin, cefotaxime, andceftriaxone, although labile to both M. morganii and P.vulgaris enzymes (Table 3), remained almost as activeagainst the inducible strains as against their 3-lactamasebasal mutants. Stably derepressed mutants were consider-ably less sensitive. The failure of inducibly expressed P-lactamase to protect against these antibiotics correlated withtheir feeble inducer activity below the MIC (Fig. la to c).Carbenicillin also was a labile weak inducer for most of thestrains, being similarly active against r-lactamase-inducibleand basal organisms but less so against their derepressedmutants. P. vulgaris Val and its mutants were an exceptionto this pattern. Direct assays revealed that subinhibitory
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concentrations of carbenicillin induced P-lactamase stronglyin P. vulgaris Val. Consequently, strain Val was almost asresistant to carbenicillin as its derepressed mutant Val-con,whereas the basal mutant (Val-def) was considerably moresusceptible. The reasons for this behavior by the Val seriesremain obscure, and the behavior itself seems atypical of thespecies. Screening of the other 26 P-lactamase-inducible P.vulgaris isolates revealed that none was resistant to 16 ,ug ormore of carbenicillin per ml, except for a single strain thatwas broadly insusceptible to all ,-lactams while remainingP-lactamase inducible. This last organism may have had animpermeability based resistance.
In general, the P. vulgaris enzyme, when stably dere-pressed, gave greater protection against carbenicillin thandid the M. morganii enzyme, an observation which agreedwith the greater hydrolytic activity (whether defined as Vmaxor VmaxlKm) of the former enzyme. The failure of the P.vulgaris enzyme to give high-level resistance to cefotaximeand ceftriaxone, despite high Vmax rates, probably related toits low affinity for these drugs (high Kin), which would renderhydrolysis inefficient at low drug concentrations (7, 21).
Neither the M. morganii nor the P. vulgaris P-lactamasescould protect significantly against imipenem or cefoxitin,even when stably derepressed. Insofar as both drugs couldinactivate the P. vulgaris P-lactamase, the failure of thisenzyme to protect is unsurprising. The failure of the M.morganii enzyme to protect against cefoxitin is more note-worthy. Regardless of whether Vmax or Vmax/Km was takenas a measure of lability, cefoxitin appeared almost as labileas cefotaxime and ceftriaxone to the M. morganii enzymeand was significantly more labile than carbenicillin. Yet theM. morganii enzyme provided considerably less protectionagainst cefoxitin than against cefotaxime and ceftriaxoneand generally rather less than against carbenicillin. Thefailure of inducible and/or stably derepressed M. morganjiP-lactamase to protect the organism against cefoxitin isevident also from the results of others (3, 19) and is incontrast to the function of other class I enzymes, such asthose of E. cloacae and Pseudomonas aeruginosa. Thereason for cefoxitin susceptibility in P-lactamase-inducibleor -derepressed M. morganii is unclear, but it may be thatthe drug is an unusually good permeant of this species, suchthat it readily overloads the P-lactamase.The levels of resistance to new penicillins and cephalo-
sporins seen in stably derepressed Proteeae mutants weremuch lower than those observed in stably derepressed E.cloacae and Pseudomonas aeruginosa, for which MICs ofcefotaxime and ceftriaxone often exceed 256 ,ug/ml (3, 8, 16).The lower resistance of the Proteeae mutants probablyrelates to their less dramatic enzyme hyperproduction.Crude sonic extracts of totally derepressed M. morganjistrains had a specific activity against cephaloridine of 1 to 3,umol of drug hydrolyzed/min per mg of cell protein. Analo-gous ranges for E. cloacae were from 16 to 32 Fmol ofcephaloridine hydrolyzed per min per mg of protein (24).Other contributory factors to the residual susceptibility ofderepressed M. morganii potentially include high penicillin-binding protein sensitivity and high permeability. Althoughindole-positive Proteeae commonly appear on lists of ,B-lactamase-inducible species that are prone to segregatederepressed mutants, there would appear to be much lessreason for concern than with Pseudomonas aeruginosa andE. cloacae in view of this lower resistance of mutants of theProteeae.
ACKNOWLEDGMENTWe are grateful to Merck Sharp & Dohme (United Kingdom) for
financial support.
LITERATURE CITED
1. Aspiotis, A., W. Cullmann, W. Dick, and M. Steiglitz. 1986.Inducible ,B-lactamases are principally responsible for the natu-rally occurring resistance towards P-lactam antibiotics in Pro-teus vulgaris. Chemotherapy (Basel) 32:236-246.
2. Curtis, N. A. C., C. Brown, M. Boxall, and M. G. Boulton. 1978.Modified peptidoglycan transpeptidase activity in a carbenicil-lin-resistant mutant of Pseudomonas aeruginosa 18s. Antimi-crob. Agents Chemother. 14:246-251.
3. Curtis, N. A. C., R. L. Eisenstadt, C. Rudd, and A. J. White.1986. Inducible Type I P-lactamases of Gram-negative bacteriaand resistance to P-lactam antibiotics. J. Antimicrob. Chemo-ther. 17:51-61.
4. Dworzack, D. L., M. P. Pugsley, C. C. Sanders, and E. A.Horowitz. 1987. Emergence of resistance in Gram-negativebacteria during therapy with expended-spectrum cephalospo-rins. Eur. J. Clin. Microbiol. 6:456-459.
5. Hashizume, T., A. Yamaguchi, T. Hirata, and T. Sawai. 1984.Kinetic studies on the inhibition of Proteus vulgaris 1-lactamaseby imipenem. Antimicrob. Agents Chemother. 25:149-151.
6. Joris, B., J. Dusart, J.-M. Frere, J. Van Beeumen, E. L.Emanuel, S. Petursson, J. Gagnon, and S. G. Waley. 1984. Theactive site of the P99 ,B-lactamase from Enterobacter cloacae.Biochem. J. 223:271-274.
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