JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1987, p. 1747-17520095-1137/87/091747-06$02 .00/0Copyright (O 1987, American Society for Microbiology

Prevalence and Characterization of Hippurate-NegativeCampylobacterjejuni in King County, Washington

PAT A. TOTTEN,l* CHARLOTTE M. PATTON,2 FRED C. TENOVER,34 TIMOTHY J. BARRETT,`WALTER E. STAMM,5'6 ARNOLD G. STEIGERWALT,2 JAMES Y. LIN,5'67 KING K. HOLMES,5'6

AND DON J. BRENNER2

Departments of Microbiology,' Laboratory Medicine,3 Medicine,5 and Pathobiology,7 Uni'ersity of Washington, Seattle,Washington 98195; Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 303332; Harbori'iew

Medical Center, Seattle, Wushington 981046; and Seattle Veterans Administration Medical Center,Seattle, Washington 981084

Received 19 February 1987/Accepted 2 June 1987

A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrheaor from poultry in King County, Washington. Of these strains, 98 (52 hippurate-positive strains and all 46 ofthe hippurate-negative strains) were selected for further phenotypic characterization and genetic classification.Hippurate hydrolysis, the test typically used to differentiate Campylobacterjejuni and C. coli, did not alwayscorrelate with the genetic classification. All hippurate-positive strains were classified as C. jejuni. Of thehippurate-negative strains, 20% were C. jejuni, 78% were C. coli, and 2% were C. laridis. Assuming that theremaining hippurate-positive strains were all C. jejuni, then hippurate-negative C. jejuni represented a smallpercentage (9 of 556 or 1.6%) of C. jejuni strains but a significant percentage (9 of 46 or 20%) ofhippurate-negative strains. This finding suggests that hippurate hydrolysis should not be used as the solecriterion for differentiating thermophilic Campylobacter species, particularly when describing the disease statesassociated with these organisms.

Campylobacterjejuni is the most frequent bacterial causeof acute diarrheal disease among adults in the United Statesand is also a common cause of diarrhea in children (3). Itsisolation from human stools depends upon proper isolationconditions, which include reduced oxygen tension, incuba-tion at 42°C (thermophilic conditions), and growth on selec-tive media containing antimicrobial agents to inhibit normalintestinal flora. Although initial studies implicated C. jejunias the only Campylobacter species associated with diarrhea(2, 3), subsequent taxonomic studies showed that Campylo-bacter strains isolated by this method may belong to threeother species: C. coli, C. laridis, and C. fetus (5, 10, 11, 21,22). The association of these latter species with diarrhealdisease is not as firmly established as it is for C. jejuni.

C. jejuni and C. coli, the most common thermophilicspecies isolated from humans, are 25 to 58% related to eachother by DNA hybridization, but very few biochemical testsdifferentiate them (10, 18, 21). Hippurate hydrolysis, typi-cally performed by the rapid tube test with ninhydrin as an

indicator, was found to correlate closely with species differ-entiation in taxonomic studies (10, 21). Hippurate-negativeC. jejuni strains have been described (10, 21), but subse-quent studies (18) have indicated that C. jejuni strainsdetermined to be hippurate negative by the rapid tube testmay have detectable hippurate hydrolysis in gas-liquid chro-matography (GLC), indicating that few, if any, C. jejunistrains are determined to be hippurate negative by more

sensitive techniques.C. laridis has also been isolated from humans and is <20%

related to the other thermophilic Campylobacter species, butthere are few differential phenotypic tests for this species (1,

* Corresponding author.

22). Resistance to nalidixic acid is typically used to differ-entiate this species from C. jejuni and C. coli (1).

C. fetus subsp. fetus primarily causes systematic illness inhumans, is occasionally associated with gastrointestinal dis-ease, and is genetically (<35%) related to C. jejuni, C. coli,and C. laridis (5, 10, 21). Several phenotypic tests, primarilygrowth at 25°C, will biochemically separate thermotolerantC. feuts strains from strains of the other three species.Most large epidemiological studies analyzing the preva-

lence of the thermophilic Campylobacter species, C. jejuni,C. coli, and C. laridis, isolated from humans and theirassociation with disease have relied on phenotypic traits,particularly hippurate hydrolysis, to differentiate species (3,12-14). Whole-cell DNA relatedness, the test that mostaccurately differentiates species, was not done in thesestudies. Thus, the associations reported in these studiesdepend on the effectiveness of hippurate hydrolysis in iden-tifying thermophilic Campylobacter species.

In this paper, we describe a rapid DNA hybridization testwhich can be used to classify thermophilic Campylobacterspecies without isolating the DNA of the test organism. Weused this test to differentiate thermophilic Campylobacterspecies isolated from a large epidemiological study in KingCounty, Washington. A significant number of strains whichwere identified as C. jejuni by DNA hybridization were

hippurate negative in the rapid tube hippurate hydrolysistest. The prevalence of hippurate-negative C. jejuni in poul-try and humans was determined, and the possibility thatthese strains hydrolyzed low levels of hippurate which couldbe detected by more sensitive techniques was investigated.

MATERIALS AND METHODS

Strains. Test strains were selected from a Food and DrugAdministration-sponsored study to examine the flow of

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1748 TOTTEN ET AL.

TEST STR AI NS

1 i2 3

5, 6 7 '

9̀ 10.,`.S i`1 2

__r-- r

à

C J'eluni C. co/i C /orlois

PROBE DNA

FIG. 1. Classification of thermophilic Cainpylobavter strains by the differential spot blot test. Test strains were treated on nitrocelluloseto lyse the cells and make their DNA available to hybridize with radiolabeled whole-cell DNA probes from reference strains. The degree ofreaction of test strains with each of the three probes was indicated by the darkness of the reaction on X-ray film overlaying each spot. Slots(and their interpretations) are as follows: 1 and 2, C. jejuni; 3. 5, 6, 7, 8, 9, 10, 11, and 12, C. coli; and 4, C. laridis.

Salmonella and Camnpylobacter species from food productsof animal origin to humans (7, 8, 13, 24). Poultry isolateswere obtained from poultry carcasses or surfaces in a localpoultry processing plant. Human isolates were obtainedfrom persons with diarrheal disease at the Group HealthCooperative of Puget Sound. Campvlobacter strains isolatedin this study had been tested for hippurate hydrolysis by therapid tube method as described by Harvey (9); all (46) of thehippurate-negative strains as well as 52 consecutive isolatesof hippurate-positive strains were selected for analysis.

Reference strains of C. jeJjun(i (NCTC 11351), C. (oli(NCTC 11366), and C. lridis (NCTC 11352) were main-tained in the laboratories of P. Totten and C. Patton.Reference C. jejuni strains which were weakly positive in therapid tube method (D1713, D127, D128, and D142) weresupplied by the Centers for Disease Control.Rapid screening by DNA hybridization. Thermophilic

Canmpylobacter isolates were screened for whole-cell DNArelatedness, without isolating the DNAs of the test organ-isms, by a procedure described for Camnpylobacîer-likeorganisms (25). The name of this test was changed fromtaxonomic spot blot (25) to differential spot blot (this paper)to better describe the ability of this test to differentiateknown Campl iobaiter species rather than to taxonomicallyclassify new species. Briefly, organisms were suspended inbroth to match the turbidity of a broth McFarland no. 3turbidity standard. Samples (10 t1) of each strain werespotted onto three identical nitrocellulose filters and treatedto lyse the cells and denature the DNA. C. jejilnii, C. o/li,and C. lnridis whole-cell DNAs were isolated from threereference strains and radiolabeled by nick translation with[cx-32P]dATP and _'-32P]dTTP (specific activity, 5 x 108cpm/>tg). Each of these radiolabeled probes was allowed tohybridize to one of three identical filters. Excess probe waswashed off, and the filters were exposed to X-ray film. Each10-,uI spot was estimated to contain roughly the sameamount of bacteria, so the darkness of the film overlayingeach spot was proportional to the degree of DNA-DNAhybridization between the probe DNA and each strain. Eachstrain was then classified by its reaction to those of thereference strains (C. jejuni NCTC 11351, C. (oli NCTC11366, and C. laridis NCTC 11352) spotted on the filters.Hippurate hydrolysis. Hippurate hydrolysis was evaluated

by three methods: rapid tube test, thin-layer chromatogra-phy (TLC), and GLC. Each method detected one of the twoend products of hippurate hydrolysis, benzoic acid or gly-cine.

(i) Rapid tube test. A loopful of bacteria from a 24-h platewas suspended in 0.4 ml of 1% sodium hippurate, incubated

for 2 h at 37°C, and then overlaid with ninhydrin reagent asdescribed previously (9). The development of a deep purplecolor after 10 min indicated the presence of glycine and wasinterpreted as a positive hippurate reaction.

(ài) TLC. Glycine produced by the hydrolysis of hippuratewas detected by TLC as previously described (16). A loopfulof bacteria scraped from a 24-h plate was washed two times,suspended in 0.1% sodium hippurate, and incubated for 30min at 37°C. The suspensions were then centrifuged, and thesupernatants were reacted with dansyl chloride. Any glycinecontained in the supernatants was identified by its charac-teristic migration pattern on polyamine sheets subjected totwo-dimensional TLC.

(iii) GLC. The procedure for detecting hippurate hydroly-sis by GLC has been described previously (15). A standard-ized inoculum of each strain was grown overnight in hippu-rate-formate-fumarate broth. A sample of the bacterialculture was treated as described previously (15). Benzoicacid was detected by its characteristic elution profile.

Quantitative whole-cell DNA hybridization. DNA was iso-lated and purified as described previously (4). The percentDNA relatedness of test strains to reference strains of C.jejuni, C. coli, and C. laridis was determined by hybridiza-tion at optimal (50°C) and stringent (65°C) conditions, fol-lowed by assay of the degree of hybridization by the hy-droxyapatite method as previously described (4).

Phenotypic characterization. Auxotyping and plasmid anal-ysis were performed as described previously (23, 24). Iso-lates were serotyped by using antisera to heat-labile antigensas described by Lior et al. (17) and to heat-stable antigens asdescribed by Penner et al. (20). Serotyping results wereinterpreted as indicated in a previous study (19). Nalidixicacid resistance was determined by growth around a 30-p.gdisk as described previously (1).

RESULTS

Differential spot blot test. Isolates were classified intospecies by the differential spot blot test (Fig. 1). Of the 98thermophilic Canpylobactîer strains tested by this method,61 were classified as C. jejuni, 36 were C. coli, and 1 was C.laridis.

Quantitative whole-cell DNA hybridization. To confirm theclassification of the isolates by the differential spot blot test,we isolated whole-cell DNAs from 15 Cnpvlohacuter strainsand performed quantitative whole-cell DNA hybridization(Table 1). Reference strains of C. jejuni, C. coli, and C.luindis were hybridized to whole-cell DNAs isolated fromeight hippurate-negative C. jejuzli strains, two hippurate-

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TABLE 1. Classification of Camnpylobacier isolates by the differential spot blot test and quantitativewhole-celt DNA hybridization tests

% DNA relatedness with type strains as determined by quantitative whole-cell DNAhvbridization tests at indicated temp (éC:

Classification by Strain C. j/e/oni C. cofi C. laridisspot blot test NCTC 11351 NCTC 11366 NCTC 1135250 65 50 65 50 65

Test strainsC. jej.i

Hippurate negative' J10840001 90G 11160001 96Y11750001 87 44T00662201 86 50M01310301 88 78 38T01477211 84 85 54 33 26 8TO1491201 96M01572201 88 81 51 27 24 7

Hippurate positive S00008021 96S00009031 98

C. coli B11870001 62 84 80 7B11980001 70 42 84 80 27 8C01851221 60 42 82 77 32 14M01859211 70 38 100 93

C. lOiCdis M01687201 40 21 28 il 93 79

Reference strainsC. jejuni NCTC 11351 100 100 60 40 24 10C. coli NCTC 11366 56 100 100 23 7C. lridis NCTC 11352 29 28 5 100 100

One hippurate-negative strain was not included in the quantitative DNA study.

positive C. jejuni strains, four C. coli strains, and the sole C.laridis strain. In al cases, classification by the spot blot testwas confirmed by quantitative whole-cell DNA hybridiza-tion.

Hippurate hydrolysis. To compare the ability of TLC andGLC to detect low levels of hippurate hydrolysis, we testedfour strains of C. jejuni from the Centers for Disease Controlwhich gave equivocal results in the rapid tube test. Althoughone strain (D1713) was positive in GLC when it was firstisolated, it was subsequently negative the three additionaltimes it was tested. This strain was also negative in TLC.The other three strains (D127, D128, and D142) were posi-

tive in both TLC and GLC. Thus, both GLC and TLC were

capable of detecting hippurate hydrolysis when it was weakor undetectable in the rapid tube test.The 52 hippurate-positive and 46 hippurate-negative

strains analyzed by the rapid tube method were also ana-

lyzed for hippurate hydrolysis by TLC. In all cases theresults of the hippurate testing by TLC and the rapid tubemethod agreed (Table 2). A subset of these strains was

TABLE 2. Comparison of three hippurate hydrolysis methodswith the differential spot blot test for identification

of 98 Cainpylohacter isolates

No. ofstrainstested

Identity in

spot blot test

No. positive/no. tested by:

Rapidtube test

TLC

further tested by GLC (two hippurate-positive and ninehippurate-negative C. jejuni, three C. coli, and one C.laidis). Again, al GLC results were in agreement with theresults of hippurate testing by TLC and the rapid tubemethod. Thus, no end products of hippurate hydrolysiscould be detected by any of the three methods in nine C.jejuni strains, indicating that they were truly hippurate-negative C. jejuni.

Correlation of hippurate hydrolysis and species differentia-tion. We found that all 52 hippurate-positive Campylobacterstrains were C. jejuni in differential spot blots. However, ofthe 46 hippurate-negative Catnpylobacter strains, 9 (20%)were C. jejuniii, 36 (78%) were C. coli, and 1 (2%) was C.laridis (Table 2).

Isolation source and phenotypic characterization of hippu-rate-negative C. jejuni strains. The hippurate-negative C.jejuni strains were isolated from both poultry and humans.Serotyping, auxotyping, and plasmid content were used todetermine the diversity of hippurate-negative C. jejunistrains (Table 3). The nine strains were classified as fourPenner serotypes and four Lior serotypes. Two of theisolates contained plasmids of different sizes, and one had a

distinctive auxotype. When transient antigens were ex-

cluded, five of the nine hippurate-negative C. jejuni strainsreacted with both C. coli (serotype 5-) and C. jejuni(serotype 5 ) antisera in the Penner system. The Lior systemcan not be used to differentiate species. However, there was

a predominance of certain serotypes in the hippurate-negative C. jejuni strains; six reacted wjth Penner serotype5-, and six reacted with Lior serotype 9.Only one strain was resistant to nalidixic acid, consistent

with its genetic classification as C. laiudis.Correlation of isolation source and species differentiation.

52 C. jejuni 52/52 52/52 2/29 C. j'j/uni 0/9 (/9 0/9

36 C. co/i 0/36 0/36 0/31 C. laiudis (/1 (/1 0/1

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TABLE 3. Isolation source and characteristics of hippurate-negative C. jejuni strains

Serotype(s) in indicated system: PlasmidsStrain Source

Penner" Lior (kilobases)" Auxotypel'J10780001 Human 1 33 45 0J10840001 Human 5-, 5+ 9 0 0G11160001 Human 10, 17 (4, 6, 7, 25) 16 0 0Y11750001 Human 5- (25, 6) 9 0 0T00662201 Poultry 5- 5+ (25, 6, 7) 9 0 0M01310301 Poultry 5-, 5+ (25, 6) 9 0 0T01477211 Poultry 5-, 5+ 9 0 0T01491201 Poultry 21, 9 20 45 ILV-, Met'M01572201 Poultry 5-, 5+ (56) 9 18 0

O, No auxotrophic requirements or no plasmids isolated.b Antigens in parentheses are sometimes seen as transient antigens in our laboratory; see reference 19.ILV-, Requires isoleucine, leucine, and valine for growth; Met-, requires methionine for growth.

As mentioned previously, strains in this study were a subsetof those from a larger study in which 593 strains wereisolated from humans and poultry. We included in this studyall (n = 46) hippurate-negative strains but only 52 of the 547hippurate-positive strains. Assuming that all 547 hippurate-positive strains were C. jejuni, then only 1.6% (9 of 556) ofC. jejuni strains were hippurate negative. The hippurate-negative C. jejuni strains, however, constituted a substantialproportion of all hippurate-negative thermophilic Campylo-bacter species. Of the 17 strains of hippurate-negative cam-pylobacters isolated from humans with diarrhea, 4 (24%)were C. jejuni and 13 (76%) were C. coli. Of 29 hippurate-negative strains isolated from poultry, 5 (17%) were C.jejuni, 23 (79%) were C. coli, and 1 (3%) was C. laridis.

DISCUSSION

In clinical studies, C. jejuni and C. coli, the two mostcommon thermophilic Campylobacter species isolated fromhumans, are routinely differentiated by a rapid tube test forhippurate hydrolysis. This differentiation is based on taxo-nomic studies which showed that 100% of C. coli strains arehippurate negative and that 95% of C. jejuni strains arehippurate positive (10, 21). Subsequent studies (18) showedthat the few C. jejuni strains which are hippurate negative inthe rapid tube test may be hippurate positive in the moresensitive GLC test and suggest that few, if any, C. jejunistrains are truly hippurate negative.

In this study, we classified 98 thermophilic Campylobacterstrains by a rapid DNA hybridization test, the differentialspot blot test, and compared the results with those obtainedby the rapid tube test for hippurate hydrolysis. We foundthat hippurate-positive strains were indeed C. jejuni, but ofthe hippurate-negative strains, 20% were C.jejuni, 78% wereC. coli, and 2% were C. laridis. The classification of thehippurate-negative C. jejuni strains was confirmed by aquantitative whole-cell DNA hybridization test. Specieshave been genetically defined as groups of strains that are70% or more related at criteria optimal for DNA reas-sociation (50°C incubation temperature for Campylobacterspecies) and at least 55% related at stringent criteria forDNA reassociation (65°C for Campylobacter species) (4).The strains designated C. jejuni on the basis of DNAhybridization clearly fulfilled this definition, showing 84% ormore relatedness to the C. jejuni type strain, as comparedwith 54% or less relatedness to the C. coli type strain, in50°C reactions and 78% or more relatedness to the C. jejunitype strain, as compared with 33% or less relatedness to the

C. coli type strain, in 65°C reactions. Two of the strainsdesignated C. coli were 70% related to the C. jejuni typestrain in 50°C reactions, but relatedness dropped to 42% orless in 65°C reactions. These strains and the other C. colistrains tested showed 82% or more relatedness to the C. colitype strain at 50°C and 77% or more relatedness at 65°C.There is no doubt that they should be classified as C. coli. Atotal of 24% of hippurate-negative thermophilic Campylo-bacter strains isolated from humans and 17% isolated frompoultry were actually C. jejuni. If we assume that 100% ofthe remaining hippurate-positive isolates in this study wereC. jejuni, then 1.6% (9 of 556) of the C. jejuni strains in thisstudy were hippurate negative. Thus, hippurate-negative C.jejuni strains represented a small percentage (1.6%) of C.jejuni strains but a significant portion (20%) of hippurate-negative strains in this study.Because previous studies have shown that C. jejuni strains

which are negative for hippurate hydrolysis in the rapid tubetest may be positive in more sensitive methods (18), wetested our strains for end products of hippurate hydrolysisby GLC and TLC. Initial studies with four strains of C.jejuniwhich were weakly reactive in the rapid tube test confirmedthe sensitivity of GLC and TLC. While one of these strainswas initially positive in GLC, it was later negative in bothGLC and TLC. Perhaps through repeated subculturing thisstrain lost its ability to hydrolyze hippurate. The other threestrains were positive in GLC and TLC, even though theywere equivocal in the rapid tube test. We analyzed all nineC. jejuni strains from our study which were hippuratenegative in the rapid tube test and found they were alsohippurate negative in GLC and TLC. Thus, the nine strainsof hippurate-negative C. jejuni isolated in this study appearto be truly hippurate negative.The high prevalence of hippurate-negative C. jejuni strains

isolated in this study contrasts with the correlation ofhippurate hydrolysis and DNA hybridization in identifyingspecies as reported in previous taxonomic studies. Harveyand Greenwood (10) found that 94% (16 of 17) of C. jejunistrains were hippurate positive and that 100% (21 of 21) of C.coli strains were hippurate negative. Roop et al. (21) re-ported that 95% (20 of 21) of C. jejuni strains were hippuratepositive and that 100% (12 of 12) of C. coli strains werehippurate negative. Analyzed differently, these findings alsoindicated that only 4.5% (1 of 22) (10) and 7.7% (1 of 13) (21)of hippurate-negative strains were C. jejuni. Perhaps thedifferences in prevalence in these studies and ours reflectdifferences in the geographic distribution of hippurate-negative C. jejuni. Alternatively, these differences could be

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due to sample selection. In the taxonomic studies, Campylo-bacter species were selected from a wide variety of animalsand from humans. In our study, Camiipvlobac(ter strains wereselected only from poultry and humans.

In addition to hippurate hydrolysis, serotyping has alsobeen used to differentiate thermophilic Camiipyloba(cter spe-cies. Of the nine hippurate-negative C. jejuni strains in thisstudy, five reacted in the Penner system with antisera to bothC. jejuni (serotype 5 -) and C. (oli (serotype 5-). The twoserotype 5 antisera are known to cross-react (20); however,the hippurate-negative C. jejuni strains showed higher titerswith the C. coli serotype 5- antisera (from three to ninedilutions higher; data not shown), indicating that serotypingwould not have been useful in classifying these strains orstrain Y11750001, which reacted only with C. caoli serotype5- antisera. Others have encountered similar inconsistenciesin the association of species with serotype (6). Penner et al.(20) found that serotyping correlated with hippurate hydrol-ysis and that only 16 (0.7%) of 2,238 strains had conflictinghippurate and serotyping reactions. Again, differences be-tween that study and ours could be due to sample selectionor geographic distribution. Of the nine hippurate-negative C.jejuni strains tested, six were of Penner serotype 5- and sixwere of Lior serotype 9. perhaps indicating a nonrandomassociation of these serotypes with hippurate-negative C.jejuni.

Hippurate-negative C. jejuni strains were also analyzed bythe Lior serotyping scheme, plasmid content, and auxotyp-ing. The strains fell into four groups by the Penner serotyp-ing system, four groups by the Lior serotyping system, twoauxotypes, and three plasmid patterns, indicating that thesestrains represented a diverse group of isolates. Thus, theprevalence of hippurate-negative C. jejuni strains in theSeattle area is not due to the spread of a single clone.Because we have shown that hippurate-negative C. jejuni

strains do exist, it may be necessary to reevaluate theassociation of hippurate-negative thermophilic Carnpylobac-ter species with disease. In taxonomic studies with geneti-cally defined species, anecdotal instances of C. c oli isolatedfrom humans have been reported (10, 21). However, moststudies analyzing the association of thermophilic Campylo-bacter species with disease have relied on phenotypic tests,particularly hippurate hydrolysis, to distinguish species (3,12-14). To our knowledge, this is the first study in whichhippurate hydrolysis was correlated with genetic classifica-tion for large numbers of strains in a defined population. Inaddition, no other study has looked at the prevalence ofgenetically defined C. jejuni and C. coli in human disease. Inour study, both species were isolated from humans withdiarrhea. When more data are accumulated, it will be inter-esting to look at the association of the isolation of C. jejuniand C. coli with different symptoms. Additional studies inwhich genetic means are used to classify Campylobacterspecies will be necessary to correlate these species withother factors, such as isolation source, virulence properties,and association with disease.While this manuscript was in preparation, a new GLC

procedure was developed at the Centers for Disease Controlto determine hippurate hydrolysis (26). Four C. coli strainswere found weakly positive by this technique; however,none of the nine hippurate-negative C. jejuni strains used inthis study were evaluated with this new technique.

ACKNOWLEDGNIENTSWe thank Jennifer Wezenberg. Kate Bruch, and Sue Williams for

technical assistance. Kirk C. S. Chen and George K. Morris for

helpful advice. and Charles Nolan for providing the strains used inthis study.

This research was supported by the Medical Research Service ofthe Veterans Administration and by Public Health Service grant AI17805 from the National Institutes of Health.

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