Characterization of an Unusual Strain of Proteusrettgeri Associated with an Outbreak ofNosocomial Urinary-Tract Infection
W. H. TRAUB, M. E. CRADDOCK, E. A. RAYMOND, M. FOX, AND C. E. McCALLDepartments of Pathology and Medicine, Bowman Gray School of Medicine,
Winston-Salem, North Carolina 27103
Received for publication 24 May 1971
An outbreak of nosocomial urinary-tract infection was caused by a strain ofProteus rettgeri that fermented lactose overnight and was resistant to all antimicro-bial drugs tested. The nonmotile isolates shared an 0 (somatic) antigen that differedfrom those of wild-type P. rettgeri. The organisms proved markedly serum-sensitive.In rats, the isolates elicited an acute interstitial nephritis with associated transientbacteriuria. Attempts to transfer the lac+ trait and drug-resistance markers torecipient strains of Escherichia coli K-12 failed; exposure of the isolates to acridineorange yielded small numbers of non-lactose-fermenting variants which, however,were still as drug-resistant as before. Epidemiological studies failed to uncover thesource of this unique strain and appeared to indicate exogenous spread of infection.
Between April 1969 and September 1970, atotal of 51 patients, all but 6 of whom were ofthe same surgical ward, contracted nosocomialurinary-tract infection due to an unusual, that is,promptly lactose-fermenting and multiple-drug-resistant, strain of Proteus rettgeri (31). Theorganism appeared to colonize the urinary tractof roughly two-thirds of the patients; the remain-der of the patients revealed moderate fever as theonly sign other than bacteriuria indicative ofurinary-tract infection. The majority of patientshad been hospitalized for extended periods oftime; many of them had indwelling catheters,and most of the patients had received variousantimicrobial drugs prior to acquisition of infec-tion with the organism. In this report, we describeseveral features of this unique strain of P. rett-geri.
MATERIALS AND METHODSBacteria. Seven isolates of "wild-type" P. rettgeri
from various clinical sources (designated P. rettgeriI to VII) and 15 isolates of the unusual strain of P.rettgeri were studied. Among the latter isolates, all ofwhich were from urinary-tract specimens, 12 fer-mented lactose overnight (designated LF P. rettgeri1 to 12) and 3 failed to ferment lactose (coded NLFP. retgeri 13 to 15). The organisms were identifiedaccording to the usual criteria (9, 33) and were main-tained on Brain Heart Infusion agar slants at 4 C.
Media. Nutrient broth, Brain Heart Infusion broth,Mueller Hinton broth, and tryptic soy broth werepurchased from Difco, as were Mueller Hinton agar,
MacConkey agar with added crystal violet, eosin-methylene blue agar, Casman's base (in 5% sheepblood-agar), and Brain Heart Infusion agar. Mac-Conkey agar without crystal violet was procuredfrom Colab Laboratories, Inc., Chicago Heights,Ill.
Antibiotics. Stock solutions of the antimicrobialdrugs sodium ampicillin (Bristol Laboratories, Syra-cuse, N.Y.), carbenicillin (Beecham Pharmaceuticals,Clifton, N.J.), cephalothin (Eli Lilly & Co., Indian-apolis, Ind.), chloramphenicol (Parke, Davis & Co.,Detroit, Mich.), gentamicin sulfate (Schering Corp.,Bloomfield, N.J.), kanamycin sulfate (Bristol Labora-tories), nalidixic acid (Sterling-Winthrop ResearchInstitute, Rensselaer, N.Y.), polymyxin B, andtetracycline hydrochloride (Pfizer Laboratories, NewYork, N.Y.) were prepared in distilled water (nal-idixic acid was dissolved in 0.1 N NaOH), ultra-membrane-filtered (0.2-um membrane filters; NalgeSybron Corp., Rochester, N.Y.) for sterilization, andfrozen and kept stored at -65 C.
Antimicrobial drug susceptibility tests. The stand-ardized method of Bauer et al. (3) was employed fordisc susceptibility tests. For quantitative drug-sensi-tivity determinations, tube broth dilution tests (30)and the microtiter procedure (32) were performed inMueller Hinton broth with bacterial inocula adjustedto yield approximately 1.5 X 106 organisms/ml atzero time. The minimal inhibitory concentration(MIC) of a drug was defined as the lowest concentra-tion of drug that completely inhibited growth at 35 Cfor 16 to 18 hr, as judged by visual inspection. Theminimal bactericidal concentration (MBC) of anantimicrobial drug represented the lowest concentra-tion of drug that yielded no colonies from 3-mm
loopful samples streaked from clear tubes or wells toquarter sectors of 5% sheep blood-agar plates, whichwere incubated at 35 C for 18 hr. A strain of Escher-ichia coli of known antibiotic susceptibility (E. coliATCC 25922) served as a control for all drug-sensi-tivity tests performed.To screen for possible additive or synergistic effects
of various pairs of antimicrobial drugs, strips ofsterile Whatman no. 1 filter paper (5 by 0.5 cm) weresoaked in respective stock solutions and placed atright angles to one another on Mueller Hinton agarplates (100 by 15 mm) that had been streaked withthe isolates under study (26).
Serological tests. New Zealand White rabbits (tworabbits per isolate) were hyperimmunized with LFP. rettgeri isolates 2, 4, 6, 8, and 10, according to thetechnique of Roschka (20). The bacteria were grownon Brain Heart Infusion agar slants for 24 hr at 35 Cand removed with sterile 0.15 M saline. The suspen-sions were boiled for 2 hr and centrifuged at 2,000 Xg for 15 min. The sediments were suspended in 95%ethanol and incubated at 35 C for 4 hr. The suspen-sions were centrifuged, washed twice with acetone,and dried overnight at 35 C. The dry powders weretransferred to screw-capped test tubes and kept atroom temperature. For injection, the powders weresuspended in sterile 0.15 M saline at 4-day intervals.Blood was obtained through cardiac puncture on the5th day after the last injection. The collected rabbitblood specimens as well as blood specimens frompatients were processed as described previously (16).The sera were heat-inactivated at 56 C for 30 min,after which the respective pairs of sera were pooled,frozen, and kept stored at -65 C. Immediately priorto use, the sera were exposed to 56 C for 10 min.
For control purposes, rabbit hyperimmune serawere absorbed with homologous and heterologousLF P. rettgeri 0 antigen preparations according tothe technique of Kopeloff and Kopeloff (14).
For screening purposes, slide 0 agglutination testswere performed (9); tube 0 agglutination tests werecarried out and interpreted in accordance with thetechnique of Felix and Bensted (11). For indirect(passive) hemagglutination (IHA) tests, sheep redcells were sensitized according to the method ofNeter et al. (19); the microtiter modification of Leeet al. (15) served for titration of the rabbit hyper-immune sera.
Tests for susceptibility to the bactericidal activityof human serm. Tests to determine the susceptibilityof the organisms to the bactericidal activity of humanserum were performed and interpreted according topreviously published procedures (16, 34).
Attempts to characterize the lac+ trait and multiple-drug resistance of LF P. rettgeri isolates as episome-mediated. The method of Anderson and Lewis (2)was used in attempts to demonstrate bacterial conju-gation and transfer of resistance to carbenicillin,chloramphenicol, gentamicin, kanamycin, polymyxinB, or tetracycline from LF P. rettgeri isolates 1 and 7to recipient E. coli K-12 strains 1485 lac+F- andCS100 lac-F-.The technique of Watanabe and Fukasawa (35) was
used in attempts to "cure" LF P. rettgeri isolates.
The organisms were pregrown and diluted in NutrientBroth, pH 7.6, to yield 104, 103, and 102 organisms/mlat zero time; these were exposed to 100, 50, 25, 12.5,and 6.25 Ag of acridine orange (Fisher Scientific Co.,Raleigh, N.C.) per ml. The growth obtained was sub-cultured to MacConkey agar, and the plates wereexamined for the appearance of NLF colonies. Thesecolonies were subcultured, examined biochemicallyand serologically, and tested for antibiotic disc sus-ceptibility. Randomly chosen LF colonies likewisewere tested for antibiotic sensitivity.
Animal, pathogenicity studies. Female Holtzman(Sprague-Dawley) rats, which weighed 200 to 250 g,were inoculated in their left kidney (5). Groups offour rats each were infected with approximately7.5 X 106 organisms of LF P. rettgeri isolates 7 and9, and NLF isolates 13 and 15. Two rats received P.rettgeri III (positive controls), and two rats wereinoculated with sterile 0.15 M saline (negative con-trols).
Urine was aseptically aspirated from the exposedurinary bladders of the animals immediately afterlethal anesthesia (methoxyflurane). Urine sampleswere plated semiquantitatively on 5% sheep blood-agar and MacConkey agar (undiluted, one 3-mmloopful; 1:1,000 dilution, one 1:1,000 calibratedloopful). The kidneys of each animal were removedaseptically and processed as follows: one (longitudi-nal) half of each kidney was ground in 0.15 M salinein glass mortars. The homogenates were plated semi-quantitatively as above; all organisms isolated werefully identified. The remaining halves of the kidneyswere placed into buffered neutral Formalin for fixa-tion; after routine hematoxylin and eosin staining(17), sections were examined for microscopic evidenceof nephritis.
RESULTS
Identification. The LF and NLF P. rettgeriisolates under study grew readily on all mediaemployed. The isolates were characterized by anunmistakable odor, not unlike that of cheese-crackers. The isolates produced a metallic sheenon eosin-methylene blue agar. The isolates gavethe following biochemical reactions that wereconsistent with P. rettgeri: deamination of phen-ylalanine, hydrolysis of urea, utilization of citrate,late fermentation of inositol, lack of lysine andornithine decarboxylase, and lack of gas forma-tion during fermentation of glucose, with theexception of NLF isolates 13 to 15, which pro-duced less than 10% gas in Durham's tubes inglucose broth (Table 1). All isolates were non-motile. The NLF isolates thus differed from theirLF counterpart in that they lacked the lac+ traitand produced small amounts of gas in glucose.The wild-type P. rettgeri isolates yielded typicalreactions. The identity of LF P. rettgeri isolates1, 3, 5, 7, and 8 was confirmed as that ofpromptlylactose-fermenting P. rettgeri by W. H. Ewing of
Nitrate reductionH2S (Kligler iron agar).........Cytochrome oxidase............0-F test (Hugh-Leifson) ..
+
+
Acid only
+
+ (48 hr)
+
F (ermentation)
++
Acid, less than 10%7c, gas
+
+ (48 hr)
+
F
the Center for Disease Control, Atlanta, Ga.(personal communication).
Serological studies. All LF and NLF isolateswere agglutinated by the five rabbit anti-O hyper-immune sera when tested by the slide agglutina-tion technique. The titers of the hyperimmunesera ranged from 1: 2,048 to 1: 4,096 againsthomologous as well as against heterologous iso-lates as revealed by the tube 0 agglutination test.With the IHA procedure, the titers of the fiveantisera ranged from 1:5,120 to 1:40,960; again,the titers of the antisera against homologous iso-lates compared favorably with those againstheterologous isolates. Absorption of the rabbithyperimmune sera with heat-killed, ethanol- andacetone-extracted cells from homologous andheterologous isolates reduced the IHA titers toless than 1:80. None of the five rabbit hyper-immune sera agglutinated any of the seven wild-type P. rettgeri isolates examined. None of thesera obtained from five proven bacteriuric pa-tients yielded titers greater than 1 :32, as deter-mined with the IHA procedure.
Several of the LF P. rettgeri isolates were ex-
posed to fresh serum from homologous andheterologous patients as well as to fresh serum
from a presumably healthy adult donor. As shownin Table 2, the isolates proved markedly sensitiveto the bactericidal activity of fresh human serum.
Antibiotic susceptibility tests. It was found that
LF P. rettgeri isolates 1 to 12 were resistant toall antimicrobial drugs examined (Table 3)However, NLF P. rettgeri isolates 13 and 14proved susceptible to carbenicillin, gentamicinsulfate, kanamycin sulfate, nalidixic acid, andtriple sulfonamide. In contrast, NLF isolate 15was as resistant as the LF isolates.The LF isolates and NLF isolate 15 tolerated
greater than 100 ,ig of ampicillin, cephalothin,chloramphenicol, kanamycin, nalidixic acid, andtetracycline per ml; the isolates were inhibited by100 Mg of polymyxin B per ml, but were notinhibited by carbenicillin at concentrations up to500 ,g/ml. All LF isolates were resistant to gen-
tamicin, the concentrations of the drug requiredfor inhibition ranging from 25 to 50,ug/ml. Anexception was LF isolate no. 3, which provedborderline-resistant, in that this isolate was in-hibited by 12 ug of gentamicin per ml. This par-
ticular isolate yielded a zone of inhibition 11 mm
in diameter around gentamicin discs; the otherLF isolates yielded zones of inhibition rangingfrom 10 to 13 mm in diameter. J. A. Waitz of theSchering Corp., Bloomfield, N.J., likewise foundthat the gentamicin concentrations required toinhibit the LF isolates ranged from 30 to greaterthan 50 ,g/ml (personal communication). Alldrug combinations examined with the filter-stripmethod proved indifferent in activity.
TABLE 2. Susceptibility of LF P. rettgeri isolates to the bactericidal activity offresh humani serum
Time after LF P. retteri 6 exposed LF P. rettgeri 5 LF P. rettgeri 5 exposed to T-serumexposuetoserum to fres serum of exposed to fresh serumexposure to serium patient no. 6 of patient no. 4
Fresh Heat-inactivated
0 2.6 X 103a 2.8 X 103 3.1 X 103 3. 1 X 10345 S.0 X 10° 5.0 X 100 1.0 X 101 4.6 X 103
a Numbers listed indicate number of colony-forming units per milliliter (survivors).
TABLE 3. Results of antibiotic-susceptibility tests
LF isolates 1-12; NLF isolate 15 NLF isolates 13 and 14 Control E. coli ATCC 25922
Antimicrobial drugZone size MIC Zone size MIC Zone size MIC(mm) (Jag/ml) (mm) (ug/mil) (mm) (jag/ml)
a Numbers in parentheses indicate drug content of discs.bThe discs measured 6 mm in diameter; therefore, readings of 6 mm indicate no zone of inhibition.c The isolates yielded zones of inhibition of varying diameter around gentamicin discs.d LF isolate no. 3 required 12,ug of gentamicin/ml for inhibition; the remainder of the isolates were
characterized by minimal inhibitory concentrations ranging from 25 to 50,g/ml.e Control Staphylococcus aureus ATCC 25923 yielded a zone of inhibition of 34 mm in diameter and
was inhibited by 0.2 ,ug of cephalothin/ml.f Denotes not determined.
orange. Attempts to transfer multiple-drugresistance from LF P. rettgeri isolates to recipi-ent cells of E. coli K-12 failed invariably. Aselected number of LF P. rettgeri isolates wereexposed to acridine orange at concentrationsranging from 100 to 6.25 ,g/ml (Table 4). Ex-ceedingly small numbers of NLF colonies wereobtained from LF isolate 1 after exposure to 50,ug of acridine orange per ml; these provedo-nitrophenyl-3-D-galactoside (ONPG)-negative,yet still had the characteristic odor, were positivefor urease and phenylalanine deaminase, were0-agglutinable, and produced less than 10% gasin glucose; they revealed the same disc antibio-gram as the parent strain. However, the soleNLF variant obtained from LF isolate 11 afterexposure to 25 ,ug of acridine orange per ml wasONPG-positive, but failed to ferment lactoseovernight; in all other respects, it was identical to
the previous NLF variants. All LF colonies exam-ined after exposure to acridine orange proved asmultiple-drug resistant as before.
Animal pathogenicity studies. Experiments withleft intrarenally inoculated rats were performedto learn more about the predilection of this par-ticular strain for the mammalian urinary tract.The control rats inoculated with 0.15 M saline,which were sacrificed on the 15th and 29th daysafter inoculation, yielded sterile and morpho-logically unremarkable kidneys. Of the two posi-tive control rats that had been inoculated withwild-type P. rettgeri m, one animal, which waskilled on day 15, revealed no renal lesions; how-ever, the homogenates of both kidneys yielded5 X 104 colonies of P. rettgeri m. The othercontrol animal, which was examined on day 29after injection, had a morphologically normalright kidney. The left kidney, however, appeared
extensively inflamed. Several irregularly shapedareas of the renal parenchyma, which were notconfined to the path of the inoculating needle,showed evidence of acute interstitial nephritis(13); the arteries, arterioles, and glomeruli werenot involved, nor was the renal pelvis affected.Those rats which had been inoculated with LFP. rettgeri isolates 7 and 9 revealed left renalmorphological changes, which did not appear assevere; however, tubular white cell casts wereprominent. The wedge-shaped areas of inflamma-tion had developed along the path of the inocu-lating needle and were sharply demarcated fromthe surrounding healthy parenchyma. The leftkidney of these animals remained bacterio-logically positive for 8 days; subsequent cultureswere negative. Those rats that had been injectedwith NLF P. rettgeri isolates 13 and 15 hadpositive urine cultures on the 15th day afterinoculation. One rat that had received NLF P.rettgeri 13 yielded greater than 105 organisms/mlof urine, and the homogenates of both kidneysgave colony counts in excess of 105 organisms/ml.The left kidney showed evidence of acute inter-stitial nephritis; in addition, the right kidney ofthis particular animal revealed inflammatoryinvolvement of the renal pelvis suggestive of con-tralateral ascending urinary-tract infection. Urineand kidney homogenate cultures were negativeon days 22 and 29 after injection.
Epidemiological studies. Epidemiological stud-ies failed to detect the source of this particularstrain of P. rettgeri. As to the reservoirs of thisorganism, it was found that the utility room, usedfor emptying and cleansing of bedpans, wasgrossly contaminated (sink, floor, bedpans, bed-pan hopper, and air). Ward air samples werenegative, as were the unused catheter kits tested.None of the ward personnel, including physi-cians, carried this organism; all stool, perineal,and hand skin cultures examined were negative.Experimentally, the organism was transferred tothe hands of the epidemiology surveillance nurseafter brief manipulation of the catheter tubing
and collection bag of a bacteriuric patient; how-ever, simple soap-washing (Dial soap) renderedthe hands free from this organism. Only 4 of 12examined patients with bacteriuria due to thisorganism yielded positive stool cultures.
DISCUSSIONThis unique strain of P. rettgeri had a number
of traits compatible with E. coli, such as promptfermentation of lactose, production of a metallicsheen on eosin-methylene blue agar, and, in thecase of the three NFL variants, production ofgas in glucose. However, the majority of the bio-chemical reactions of this organism were foundto be consistent with those of P. rettgeri, e.g.,hydrolysis of urea, deamination of phenylala-nine, utilization of citrate, fermentation of inosi-tol, and lack of lysine and ornithine decarboxyl-ases. Furthermore, several of the isolates of thisparticular strain proved susceptible to the species-specific activity of bacteriocins elaborated bywild-type P. rettgeri isolates III and VI, afterinduction with mitomycin C (M. E. Craddockand W. H. Traub, Experientia, in press). Thus,we choose to designate this organism a promptlylactose-fermenting strain of P. rettgeri (21, 24,25, 28, 29).
Unfortunately, our attempts aimed at demon-stration of transfer of the lac+ trait and anti-biotic-resistance markers from the isolates torecipient E. coli failed; the small numbers ofNLF variants obtained after exposure of LF iso-lates to acridine orange are not indicative of"cure" of this trait, since these variants mighthave arisen through spontaneous or acridineorange-induced mutation. Thus, the data ob-tained do not allow one to state with any degreeof certainty whether these traits are chromosomalor episomal in nature (25).The isolates of this unusual strain proved
pathogenic for the urinary tract of rats, as re-vealed by the resultant interstitial nephritis withassociated transient bacteriuria. Only in oneanimal was there evidence of right renal pelvic
involvement, a finding suggestive of ascendingurinary-tract infection.
Althouth this unique strain carried epidemio-logical markers that facilitated its isolation, ourstudies failed to disclose the source of this or-ganism. Nor were we able to define precisely themode of acquisition of urinary-tract infectionduring this outbreak. The epidemiological datasuggested exogenous spread of infection, that is,transmission through contaminated bed utensils,and, possibly, through contaminated hands ofward personnel (1, 4, 6-8, 10, 12, 18, 22, 23, 27).On the basis of indirect evidence, this strain of
P. rettgeri was interpreted to colonize the urinarytract of most of the patients involved. First, theisolates gave rise to asymptomatic bacteriuria inthe majority of patients; only a third of thepatients had low-grade fever as the only addi-tional sign of urinary-tract infection. Second,none of the patient sera tested had titers greaterthan 1: 32 as determined with the IHA technique.
ACKNOWLEDGMENT
We thank the Schering Corp., Bloomfield, N.J., for financialsupport.
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