JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1977, p. 115-121Copyright (D 1977 American Society for Microbiology

Vol. 5, No. 2Printed in U.S.A.

Serotyping of Serratia marcescens: Evaluation of Le Minor'sH-Immobilization Test and Description of Three New

Flagellar H AntigensWALTER H. TRAUB* AND INGRID KLEBER

Institut far klinische Mikrobiologie und Infektionshygiene, Universitdit Erlangen-Niirnberg, 8520 Erlangen,West Germany

Received for publication 17 August 1976

The H-immobilization test of Le Minor for determining flagellar (H) antigenswas evaluated and compared with tube and slide H-agglutination tests. The testproved specific and easy to perform, and titration end points were clearlydiscernible. The degree of serological cross-reactivity between H antigen refer-ence strains of Serratia marcescens was low. Consequently, this test wasadopted for routine serological analysis of H antigens, using unabsorbed rabbitimmune anti-H sera. As a result of using this procedure, three new provisionalH antigens, designated H14, H15, and H16, are proposed.

Within the last 15 years, Serratia marces-cens, an opportunistic pathogenic microorga-nism, has assumed an ever-increasing role withrespect to outbreaks of nosocomial cross-infec-tions. From 1957 onward, Ewing, Davis, and co-workers successively defined the somatic (0)and flagellar (H) antigens of S. marcescens (1-3) for purposes oftaxonomy and epidemiologicalinvestigations within the clinical setting. Cur-rently, 15 0 and 13 H antigens of this microor-ganism are recognized (2). With appropriatemonospecific anti-O and anti-H rabbit immunesera, it was possible to serotype approximately95% of clinical isolates ofS. marcescens from avariety of clinical sources and geographic local-ities (2). Sedlak (12) examined 91 isolates of S.marcescens, which were assigned to 10 0 sero-groups. The value of serotyping as an epidemio-logical tool was also underscored by Wilfert etal. (22). Other investigators introduced bacteri-ocin and phage typing as additional means tofacilitate the epidemiological work-up of in-criminated nosocomially significant strains ofS. marcescens (4-6, 8, 10, 14, 15, 18, 20, 21);with these latter procedures, it was possible totype roughly 90% of the isolates.

Recently we encountered a multiple-drug-re-sistant strain of S. marcescens that, up untilAugust 1976, had caused infections in a total of19 patients from four different university hospi-tals at Erlangen. The nonconjugative strainproved to be of bacteriocin type 18 (19). How-ever, on several occasions, patients with long-standing S. marcescens infection due to thisparticular strain yielded phenotypic variants

* Send reprint requests to: Clinical Microbiology Labora-tory, VA Hospital, 4150 Clement St., San Franciscio,CA 94121.

(17) that had been bacteriocin type 4 or 9 butthat on repeated subcultures had reverted tobacteriocin type 18. Therefore, it was of interestto serotype this problem strain, including phe-notypic variants, as well as previously encoun-tered, nosocomially significant isolates of S.marcescens, some of which had proved to bebacteriocin nontypable (14). During the courseof the serological investigations, we wished tocompare a number of techniques, among theseLe Minor's H-immobilization test (unpublisheddata), to expedite and, if possible, to simplifythe serotyping procedures. Relevant data withregard to the serological analysis of flagellar(H) antigens of S. marcescens are presentedbelow, including the description of three new Hantigens.

MATERIALS AND METHODS

Bacteria. The 24 reference somatic (0) and flagel-lar (H) antigen strains of S. marcescens listed inTable 1 were a gift from Betty R. Davis, Enterobac-teriology Unit, Center for Disease Control, Atlanta,Ga. S. marcescens strain S326 n.t. (serotype013:H14), bacteriocin nontypable, had been isolatedin 1971 from a postmortem wound specimen of theindex case of a small outbreak of nosocomial cross-infections in the North Carolina Baptist Hospital,Winston-Salem. The group A bacteriocin (phagetail) control indicator strain S. marcescens Sli (sero-type 06:H15) had been isolated in the same institu-tion in 1969 (15, 16, 21); the source of this strain,however, is unknown. S. marcescens strain LF-Munchen (serotype 013:H16), a rapidly lactose-fer-menting isolate that also proved to be bacteriocinnontypable, was recovered in May 1976 from a tra-cheal aspirate of a newborn infant in the Children'sHospital, University of Munich, Munich, Germany.

Media. Brain heart infusion broth, tryptic soy115

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TABLE 1. Compilation of S. marcescens reference 0 and H antigen strains

S. marces-0 antigen no. cens strain Serotype H antigen no. S marcescens Serotype

no.

Reference 0 antigens Reference H antigens01 866-57 01 :H5 Hi 863-57 03:H102 868-57 02:H1 H2 836-57 05:H203 863-57 03:HI H3 862-57 06:H304 864-57 04:H4 H4 864-57 04:H405 867-57 05:H1 H5 866-57 01 :H506 862-57 06:H3 H6 680-57 05:H607 843-57 07:H1O H7 814-57 04:H708 1604-57 08:H3 H8 877-57 06:H809 4534-60 09:Hll H9 1783-57 014:H9010 1287-59 010:H8 H10 2420-57 06:H10011 1914-63 011 :nonmotile H1l 827-57 05:H11012 6320-58 012 :H9 H12 874-57 014:H12013 3607-60 013:H4 H13 2436-57 05:H13014 4444-60 014:H12 H14 S326 n.t. 013:H14015 4523-60 015:H8 H15 Sli 06:H15

H16 LF Munchen 013:H16

broth (TSB) and agar (TSA), Difco agar, and Mac-Conkey agar with added crystal violet were pur-chased from Difco laboratories, Detroit, Mich. Themicroorganisms were maintained on TSA slants at4°C and in a mixture of 1 volume of brain heartinfusion broth plus 1 volume of heat-inactivatedbovine serum (Behringwerke AG, Marburg, Ger-many) at -65°C. Semisolid (ss)TSA U-tubes (10 mlof medium per tube) were prepared by addition of0.3% (wt/vol) Difco agar to TSB (= 0.3% semisolidssTSA); these tubes served for the determination ofmotility of all H antigen reference strains and allisolates to be typed. For the H-immobilization test(see below), TSB was rendered semisolid by theaddition of 0.4% (wt/vol) Difco agar to TSB (= 0.4%ssTSA).

Reagents. Analytical-grade formalin (35%) andcrystallized phenol were purchased from E. Merck,Darmstadt, Germany. A 0.6% solution of formalde-hyde and a 0.5% solution of phenol in aqueous 0.154M NaCl, respectively, were prepared according toEdwards and Ewing (2). Evipan-natrium (methyl-cyclohexenyl-methyl-barbituric acid, sodium salt)was a gift from Farbenfabriken Bayer AG, Leverku-sen, Germany. Phosphate-buffered saline (PBS), pH7.5, was prepared and autoclaved (121°C, 15 min)according to Schmidt (11).

Preparation of formalinized/phenolized S. mar-cescens H cells. In accordance with the technique ofB. R. Davis (personal communication) and that ofEdwards and Ewing (2), the S. marcescens strainswere successively passed through six 0.3% ssTSA U-tubes, after which 1 loopful of each motile strain wasinoculated in the late afternoon hours into 20 ml ofTSB; the cultures were incubated at 35°C overnight,and the next morning either 20 ml of 0.6% formalde-hyde or 20 ml of 0.5% phenolized saline was added.The cells were held at 35°C for an additional 24 h.Subcultures to plates of MacConkey agar with addedcrystal violet served to monitor effective killing of

the cells. Properly inactivated H cell suspensionsserved either as immunogens for rabbits or as agglu-tinogens for serological tests. The formalinized Hcells were stored at 4°C for no longer than 6 weeks.

Immunization of rabbits and processing of rabbitimmune anti-H sera. Based on Edwards and Ewing(2) and on the recommendations of B. R. Davis (per-sonal communication), rabbits (2.5 kg of bodyweight; Firma Willy Schmid, Holzgerlingen, Ger-many) received a series of five intravenous injec-tions of formalinized H cell suspensions, given at 5-day intervals; the volumes of the intravenous inoc-ula were 0.5, 1.0, 2.0, 4.0, and 4.0 ml, respectively.On day 7 after the last injection, the animals wereanesthetized by the intravenous inoculation of 1.0ml of Evipan-natrium, after which the animals wereexsanguinated aseptically by cardiac puncture. Therabbit blood was held at room temperature for 1 h,after which the blood clots were loosened, held at4°C for an additional 2 h, and separated by centrifu-gation at 2,500 x g (4°C) for 20 min. The sera weredispensed in 2-ml aliquots into sterile, screw-cappedvials and stored at -65°C. As required, sera wereheat-inactivated at 56°C for 30 min in a water bath.

Serological tests. (i) Tube H-agglutination tests.Tube H-agglutination tests were performed accord-ing to the technique of Edwards and Ewing (2). TheH antigens (formalinized H cells) had been preparedas described above. Heat-inactivated rabbit immuneanti-H sera were diluted 1:10, 1:20, and so on, until1:10,240, in PBS, pH 7.5. Small Widal tubes (11 by80 mm) received 0.5 ml of serum dilution plus 0.5 mlofH antigen; control tubes were inoculated with 0.5ml of PBS plus 0.5 ml of H antigen. The tubes wereincubated in a water bath for 2 h at 50°C, after whichthey were examined for flocculent, loose aggluti-nates. Titers were recorded as the reciprocal of thehighest dilution of serum that had yielded macro-scopic evidence of H-agglutination.

(ii) Slide H-agglutination tests. Slide H-aggluti-

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H ANTIGENS OF S. MARCESCENS 117

nation tests were performed with the same serum

dilutions in PBS (pH 7.5) and the same H anti-gens (formalized H cells) as in tube H-aggluti-nation tests. One drop (0.05 ml) ofserum dilution (orcontrol PBS) plus 1 drop ofH antigen were combinedon one-quarter sections of ordinary microscope glassslides. The slides were gently tilted back and forthmanually for a total of 3 min, after which they wereread and interpreted (9). As a rule, the terminal andone or two subterminal reactive serum dilutionsresulted in faintly visible agglutination, which hadto be read with a x3 magnifying lens against an

obliquely illuminated dark background. Titers weredefined as above.

(iii) Tube 0-agglutination tests. Tube 0-aggluti-nation tests were carried out in accordance with thetechnique of Felix and Bensted (7), except for thefollowing minor modifications. For the preparationof 0 cells, each S. marcescens isolate was subcul-tured to a chocolate agar plate, which was incubatedovernight at 35°C. On the next morning, 10 smooth-appearing colonies of each isolate were picked andtransferred into 4 ml of TSB; after incubation at35°C for 5 h, two large (14 cm in diameter) TSAplates were inoculated with 0.2 ml of TSB growth,respectively. The plates were incubated at 35°Covernight. The next morning, the growths were har-vested with a total of 20 ml of sterile 0.154 M NaCl.The cells were centrifuged at 12,062 x g (10,000 rpm)and 4°C for 10 min (Sorvall RC-5 centrifuge, SS34rotor), after which the supernatant fluids were dis-carded. The cell sediments were resuspended in 20ml of 0. 154 M NaCl and boiled in a water bath for 30min. The cells were washed once more in 0.154 MNaCl, resuspended in 20 ml of saline, and subse-quently stored at 4°C for no longer than 3 months.The turbidity of the 0 cell suspensions correspondedapproximately to that of McFarland barium sulfatestandard no. 10 (3 x 109 colony-forming units/ml).The 0-agglutination tests were performed by adding0.5 ml ofdiluted (1:2 in PBS) stock 0 cells to 0.5 ml ofa rabbit immune serum dilution in small Widaltubes. The master serum dilutions were 1:0, 1:10,1:20, and so on, until 1:1,280, in PBS, pH 7.5. Controltubes received 0.5 ml of PBS plus 0.5 ml of 0 cells.The tubes were incubated in a water bath at 37°C for2 h, after which the racks were transferred and heldat 40C overnight (ca. 16 to 18 h). The tubes were thenheld at room temperature for an additional 2 h, afterwhich they were inspected macroscopically for gran-ules and aggregates floating in the clear fluid,which resisted very gentle shaking (9). The serumtiters were recorded as the reciprocal values of thehighest dilutions of sera that yielded 0-agglutina-tion.

(iv) Slide 0-agglutination tests. Comparativetests consisted ofadding 0.05 ml ofdiluted (1:2) stockO cells (boiled for 30 min; see above) to 0.05 ml ofserum dilution (or PBS for control purposes) onstandard microscope glass slides that had been di-vided into four sections (9). For the determination ofthe serogroup (O antigen) of clinical isolates of S.marcescens, undiluted stock 0 cells were used. Theslides were gently tilted back and forth manually for

2 min at room temperature, after which they wereread with the naked eye and recorded. Again, serumtiters were defined as the reciprocal values of thehighest serum dilutions that resulted in macroscop-cially evident 0-agglutination.

(v) H-immobilization test of Le Minor. Followingthe suggestion of B. R. Davis to evaluate L. LeMinor's flagellar immobilization test for S. marces-cens (unpublished data), and after having obtainedDr. Le Minor's consent (L. Le Minor, personal com-munication), the following protocol was adopted forthe routine performance of the test subsequent to aseries ofpreliminary experiments. Each isolate ofS.marcescens to be typed was passed once through a0.3% ssTSA U-tube (check for motility). Dependingon the number of isolates or sera to be examined,appropriate numbers of metal-capped tubes (13 by100 mm) received 3 ml of 0.4% ssTSA (the height ofthe medium column measured about 3.4 cm); afterautoclaving (121°C, 15 min), the tubes were trans-ferred to a water bath and held at 56°C for about 1 h.Heat-inactivated, unabsorbed rabbit immune anti-H sera were diluted in PBS, pH 7.5, as follows: 1:0,1:4, 1:8, and so on, until 1:512. Then, 0.05 ml of eachserum dilution was delivered aseptically with 0.1- or0.2-ml pipettes to the appropriately labeled tubes,which had been removed from the 56°C water bathimmediately prior to addition of serum. Motilitycontrol tubes were used as such. The tubes wereshaken gently on a laboratory shaker to ensure evendistribution of the antibodies. The tubes were thentransferred to and held at 4°C for 20 min; this timewas sufficient for adequate gelation to occur. Aftersolidification, the tubes, including control tubes,were inoculated with S. marcescens candidatestrains to a depth ofno more than 2 mm. The needleswere flamed between each tube. The tubes wereincubated at 35°C overnight, after which they wereread and interpreted. It should be stressed that afterthe serum dilutions had been added to the ssTSAtubes, the sera had been further diluted 60-fold;thus, the final serum dilutions were 1:60, 1:240,1:480, and so on, until 1:30,720. The titer of sera wasdefined as the highest dilution permitting the motileisolate (relative to the control tube) to migratedownward from the point of inoculation for one-halfof the total height of semisolid medium (ca. 1.7 cm).This end point empirically was found to correlatewell with H-slide agglutination titers. H antigendeterminations of clinical isolates of S. marcescenswere performed as follows. After one U-tube pas-sage, the motile isolates were screened with a 1:4dilution (final dilution, 1:240) of each rabbit immuneanti-H serum, respectively. Those sera that had im-mobilized the isolate were then titrated in a manneranalogous to the above-mentioned procedure.

Bacteriocin typing. Isolates were typed as basedon susceptibility to one or more of 10 selected groupA (phage tail) bacteriocins of S. marcescens (13, 21).

RESULTSA series ofpreliminary experiments served to

determine a number of potential variables with

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118 TRAUB AND KLEBER

regard to the three H-typing procedures used,i.e., the tube and slide H-agglutination and theH-immobilization tests. During the preparationofH cells ofS. marcescens, it was observed thatphenolized saline (0.25% final concentration ofphenol) failed to kill the majority of strains,whereas formalinized saline (0.3% final concen-tration of formaldehyde) reliably rendered allS. marcescens strains nonviable. Therefore,immunogenic and agglutinogenic H cells of S.marcescens were prepared by formalinizationexclusively. It should be added, though, thatphenolized and formalinized H cells yieldedidentical titers in slide H-agglutination tests. Itwas noted that formalinized H cells that hadbeen stored at 4°C for up to 6 weeks still provedadequately agglutinogenic, whereas storage for8 weeks or longer at 4°C resulted in weaklyagglutinogenic cells. Therefore, formalinized Hcell suspensions were discarded after storage inexcess of 6 weeks. In this context, 30-min-boiledO cells of the majority of S. marcescens strainsbecame slightly rough after storage at 4°C formore than 3 months, as evidenced by traceauto-agglutinability in PBS, pH 7.5. Thus, 30-min-boiled stock 0 cells were discarded prior tothe 3-month expiration date. Formalinized Hcell suspensions of selected S. marcescensstrains that had been concentrated 10- and 20-fold (Sorvall RC-5 centrifuge, SS34 rotor; 12,062x g, 10 min, 4°C) yielded macroscopically veryclearly discernible titration end points in slideH-agglutination tests, whereas cells that hadbeen concentrated up to fivefold required theaid of a magnifying lens for the terminal andone or two subterminal serum dilutions. Forexample, rabbit immune serum no. 115, whichhad been made against S. marcescens strainCDC 05:H2, had a titer of 1:5,120 against stan-dard (1 x) and 5 x concentrated homologous for-malinized H2 cells and a titer of 1:2,560 againstlOx and 20x concentrated H2 cells, respec-tively. Boiling of formalinized or live cells ofselected S. marcescens reference CDC H anti-gen strains for 30 min abolished H-agglutina-bility. The tube and slide 0- and H-agglutina-tion tests yielded comparable titers with regardto rabbit immune and patient sera; titers dif-fered by no more than one dilution step. Forexample, patient G.S., a female patient with achronically infected hip endoprosthesis of over 1year's duration due to the multiple-drug-resist-ant strain ofS. marcescens (bacteriocin type 18;serotype 06/014:H12), had anti-H antibody ti-ters of 1:1,280 by both tests immediately prior toinstitution of curative amikacin chemotherapy;likewise, her 0 titer against antigen 014 was

1:80 as measured with the tube 0- and the slide0-agglutination procedures, respectively. Itshould be stressed that the slide H-agglutina-tion test was not suitable for checkerboard ti-trations of rabbit immune anti-H sera, as nu-merous cross-reactions at low serum dilutionsbecame evident, confirming the experiences ofEdwards and Ewing (2). Furthermore, CDC S.marcescens reference strain 04:H7 gave rise to"flaky" growth in TSB, which persisted afteraddition of formaldehyde, rendering the inter-pretation of titration end points by both tubeand slide H-agglutination tests extremely diffi-cult; with the H-immobilization tests, however,clear-cut end points were obtained.

Finally, Dr. Le Minor's H-immobilizationtest was evaluated in a further series of prelim-inary experiments. This test was found to yieldspecific and reproducible results. Isolates of S.marcescens required only one passage through0.3% ssTSA U-tubes prior to performance of thetest. Rabbit immune anti-H sera with titers ashigh as 1:320 against the homologous 0 antigenof the immunogenic S. marcescens strain (Ta-ble 2) failed to inhibit the motility of the teststrains in 0.4% ssTSA. Properly immunizedrabbits developed immobilizing titers of-1:3,840 (Table 3), and, furthermore, the titersobtained with the H-immobilization test corre-lated well with the results of the slide H-agglu-tination tests; titers differed no more than four-fold (Table 3). In addition, the degree of serolog-ical cross-reactivity detected among the 13 S.marcescens H antigen reference strains waslow (Table 4); the same was true for S. marces-cens isolates S326 n.t. (bacteriocin nontypable),Sli (formerly bacteriocin type 1, now type 16),and LF-Munchen (bacteriocin nontypable),whose flagella were found to represent new Hantigens, designated H14, H15, and H16, re-spectively (Table 4). The only major cross-reac-tion observed was the reciprocal one between S.marcescens strains CDC 06:H8 and CDC014:H9 (Table 4). For instance, our clinical S.marcescens isolate no. S86 (bacteriocin type14), which proved to be of serogroup 013,yielded the following typing results with the H-immobilization test. Rabbit immune serum no.126, anti-CDC 06:H8, gave a titer of 1:1,920,whereas serum no. 127, anti-CDC 014:H9, im-mobilized the strain up to a dilution of 1:15,360,an eightfold difference, which was interpretedas significant; therefore, the H antigen of thisisolate was recorded as H9. Most important, theresults obtained with the checkerboard titra-tions of the rabbit immune anti-H sera permit-ted the use of unabsorbed sera (Table 4). As a

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H ANTIGENS OF S. MARCESCENS 119

consequence, the H-typing procedure for clini-cal isolates of S. marcescens evolved into arelatively simple task. As stated in Materialsand Methods, the isolates were passed firstthrough one U-tube of 0.3% ssTSA (check formotility). Next, the isolates were exposed torabbit anti-H immune sera diluted 1:4 (finaldilution, 1:240) in 0.4% ssTSA. Finally, onlythose sera that had immobilized the isolates inthe screen test were titrated fully againt thecandidate strains.

DISCUSSIONIn our hands, Le Minor's H-immobilization

test proved the simplest procedure for the sero-logical analysis of flagellar antigens of S. mar-

cescens; this test yielded clearly discernible endpoints. Furthermore, the degree of serologicalcross-reactivity between certain H antigens ofS. marcescens was similar to the results Ed-wards and Ewing (2) had obtained with tube H-agglutination tests and to those obtained by B.R. Davis (personal communication) with com-mercially available rabbit immune sera. Theresults from the checkerboard titrations of all16 anti-H sera led to a simplified approach forthe routine H-typing of clinical isolates of S.marcescens with the aid of unabsorbed anti-H

TABLE 2. Titration of rabbit immune anti-H sera

against homologous 0 cells of S. marcescens

Rabbit Titers ob-immune H antigen reference tainedserum stranmologous 0

no. cellsa

122 03:H1 320116 05:H2 160118 06:H3 40120 04:H4 160123 O1:H5 160124 05:H6 20125 04:H7 160126 06:H8 320127 014:H9 40128 06:H1O 320129 05:H11 40130 014:H12 160131 05:H13 80

961 S326 n.t., 013:H14 160177 Sli, 06:H15 160180 LF-Munchen, 013:H16 80

a Titrations were carried out by using the slide 0-agglutination test (30-min-boiled 0 cells). Titerslisted are the reciprocal values of the highest serumdilutions that yielded macroscopically evident 0-

agglutination.

TABLE 3. Titers ofanti-H sera compared by slide H-agglutination and H-immobilization tests

Rabbit Titers obtained with:immune H antigen

reference Slide H- H-immobi-serum strain no. agglutina- lizationno. ~~~~~tion test test

122 03:H1 30,720a 30,720b116 05:H2 15,360 3,840118 06:H3 7,680 7,680120 04:H4 15,360 7,680123 O1:H5 30,720 30,720124 05:H6 15,360 30,720125 04:H7 30,720 30,720126 06:H8 15,360 15,360127 014:H9 15,360 15,360128 06:H10 30,720 30,720129 05:H11 30,720 7,680130 014:H12 15,360 7,680131 05:H13 7,680 3,840

a Titers listed are the reciprocal values of thehighest dilutions of sera that yielded macroscopi-cally visible H-agglutination; the serum master di-lutions had been modified to give final dilutionsidentical to those of the H-immobilization test.

b Titers shown are the reciprocal values of thehighest dilutions of sera that prevented inocula ofmotile S. marcescens strains from migrating beyondone-half the length of the semisolid medium column.

sera, a considerable saving in effort and ex-pense. In addition, with the antecedent screen-ing procedure, i.e., the use of 1:4 diluted (1:240final dilution) immune serum, the number ofminor cross-reactions was reduced (Table 4),and consequently the number of required fullserum titrations could be limited.As for the three new H antigens ofS. marces-

cens described in the present study, undoubt-edly there exist additional H antigens of thismicroorganism. Therefore, the H antigenscheme presented here should be considered atentative one.Our experiences with serogrouping (determi-

nation of 0 antigens) of clinical S. marcescensisolates, particularly with respect to the majorand extremely bothersome cross-reactions ofantigens 06 and 014, will be the subject of aseparate communication.

ACKNOWLEDGMENTSWe should like to express our sincere thanks to Betty R.

Davis of the Enterobacteriology Unit, Center for DiseaseControl, Atlanta, Ga., for her gift of the 24 currently usedCDC H and 0 antigen reference strains of S. marcescens,and likewise to Leon Le Minor of the Institut Pasteur,Paris, France, for his kind permission to use his as yetunpublished H-immobilization test.

This study was supported in part by a grant from theDeutsche Forschungsgemeinschaft (Tr 136/1).

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VOL. 5, 1977

LITERATURE CITED

1. Davis, B. R., W. H. Ewing, and R. W. Reavis. 1957. Thebiochemical reactions given by members of the Serra-tia group. Int. Bull. Bacteriol. Nomencl. Taxon.7:151-160.

2. Edwards, P. R., and W. H. Ewing. 1972. Identificationof Enterobacteriaceae, 3rd ed., p. 308-317. BurgessPublishing Co., Minneapolis.

3. Ewing, W. H., B. R. Davis, and R. W. Reavis. 1959.Studies on the Serratia group. Communicable Dis-ease Center Publication, U. S. Department of Health,Education, and Welfare, Atlanta.

4. Farmer, J. J., III. 1972. Epidemiological differentiationof Serratia marcescens: typing by bacteriocin produc-tion. Appl. Microbiol. 23:218-225.

5. Farmer, J. J., III. 1972. Epidemiological differentiationofSerratia marcescens: typing by bacteriocin sensitiv-ity. Appl. Microbiol. 23:226-231.

6. Farmer, J. J., III., F. W. Hickman, and J. V. Sikes.1975. Automation of Salmonella typhi phage typing.Lancet ii:787-790.

7. Felix, A., and H. Bensted. 1954. Proposed standardagglutinating sera for typhoid and paratyphoid A andB fevers. Bull. W.H.O. 10:919-926.

8. Hamilton, R. L., and W. J. Brown. 1972. Bacteriophagetyping of clinically isolated Serratia marcescens.Appl. Microbiol. 24:899-906.

9. Kauffmann, F. 1975. Classification of bacteria, p. 31-38. Munksgaard, Copenhagen.

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