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A disk method for hippurate hydrolysis was compared with the ninhydrin tube method by using 140 genetically confirmed Campylobacter strains. Results were similar for 129 (92%) strains when the inoculum size for the disk method was standardized. Six strains (4.2%) showed variable results by each method. Our results conflict with those obtained in studies by others, who found the two methods to be dissimilar.

We used the scheme of Lior to biotype 140 genetically identified Campylobacter strains. Our results confirmed previous studies and extended Lior biotyping to show that nine C. jejuni subsp. doylei strains (100%) were one biotype and nine C. jejuni subsp. jejuni nalidixic acid-resistant strains (100%) were C. jejuni biotype I or II. All C. jejuni subsp. jejuni hippurate-negative strains studied and 6 of 35 C. lari strains (17%) were grouped with C. coli biotypes. These findings may be useful in epidemiologic investigations.

A rapid high-performance liquid chromatography method was developed to determine hippurate hydrolysis by Legionella spp. Benzoic acid, an end product of enzymatic activity, was directly detected by high-performance liquid chromatography after 1 and 24 h of incubation in 1% sodium hippurate. Because of its sensitivity, this procedure offers more precise identification of some Legionella spp.

Campylobacter jejuni (20 strains) and Campylobacter coli (12 strains) were assigned to four biovars for each species based on phenotypic tests that were easy to perform and interpret. The resulting biotyping schemes offer a greater degree of distinction among C. jejuni and C. coli strains than any of the other biotyping schemes previously described for these organisms.

Several biochemical test systems were studied for their potential usefulness for the examination of strains of Campylobacter species. Most (81%) of the C. jejuni strains hydrolyzed sodium hippurate, but strains of C. fetus, C. sputorum, and C. fecalis did not. Some (46%) of the C. jejuni strains and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains hydrolyzed DNA, but the C. fetus and C. sputorum subsp. mucosalis strains did not. Strains of all species of Campylobacter grew on charcoal-yeast extract agar, but 47% of the C. jejuni strains did not. Alkaline phosphatase activity was recorded for some strains of C. jejuni, but all other species were negative for this activity. Aryl sulfatase activity was detected in 7% of the C. jejuni, 15% of the C. fetus subsp. fetus, and all of the C. sputorum subsp. sputorum, C. sputorum subsp. bubulus, and C. fecalis strains, but it was not detected in the C. fetus subsp. venerealis and C. sputorum subsp. mucosalis strains. Most (93%) of the C. jejuni but none of the other Campylobacter strains contained lactobacillic acid when examined for cellular fatty acids. On the basis of results from three of these tests (hippurate hydrolysis, DNA hydrolysis, and growth on charcoal-yeast extract agar), clinical strains of C. jejuni were placed in eight biotypes.

The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni and C. coli strains. Glycine and benzoic acid are formed when hippurate is hydrolyzed by C. jejuni. The test used in most laboratories is one of several variations of the ninhydrin tube test described by Hwang and Ederer (M. Hwang and G. M. Ederer, J. Clin. Microbiol. 1:114-115, 1975) for detection of glycine. We evaluated three modifications of the Hwang and Ederer method and the gas-liquid chromatographic (GLC) method described by Kodaka et al. (H. Kodaka, G. L. Lombard, and V. R. Dowell, Jr., J. Clin. Microbiol. 16:962-964, 1982) for detecting benzoic acid. Campylobacter strains comprised 22 C. jejuni, 11 C. coli, and 8 C. laridis strains. The species identification of each strain was confirmed by DNA relatedness. All strains of C. jejuni were positive and all strains of C. coli and C. laridis were negative by the GLC method for detecting hippurate hydrolysis, whereas three strains of C. jejuni gave negative or variable results in the tube tests. The GLC method is more sensitive than the tube methods for detecting hippurate hydrolysis and should be used on cultures yielding variable or questionable test results.

Strains of Legionella pneumophila were shown to hydrolyze sodium hippurate in an overnight test system, but strains of L. bozemanii, L. micdadei, L. dumoffii, and some other organisms similar to the legionellae did not. Although only a small number of strains of legionellae other than L. pneumophila have been classified and tested, the results indicate that the hippurate hydrolysis test may prove useful for differentiating among Legionella species.

Primary cultures of clinical material were screened for the presence of colonies suspected of being Streptococcus agalactiae (Lancefield group B). Sixty-three such cultures and 108 other isolates of beta-hemolytic streptococci (groups A, C, and G), encountered during the first 3 months of the investigation, were studied by Lancefield grouping, sodium hippurate hydrolysis, and a standardized CAMP test. All streptococci were inoculated perpendicularly to streaks of a beta-toxin-producing staphylococcus on sheep blood agar plates and incubated aerobically in a candle jar and anaerobically at 37 C. Plates were examined after 5 to 6 and 18 h of incubation. The production of a distinct "arrowhead" of hemolysis was indicative of a positive CAMP reaction. All group B streptococci produced a positive CAMP reaction in the candle jar or anaerobically, usually within 5 to 6 h, and aerobically after 18 h of incubation. All group A streptococci produced a positive reaction only under anaerobic conditions. Groups C and G streptococci were negative under all atmospheres. The CAMP reaction is a prompt and reliable procedure for the presumptive identification of group B streptococci when a candle jar atmosphere is used during incubation.

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

A gas-liquid chromatography technique which allows simultaneous detection of hippuric acid (N-benzoylglycine) hydrolysis and conversion of fumaric acid to succinic acid by microorganisms uses a new medium, hippurate-formate-fumarate broth, and a gas chromatograph equipped with a thermal conductivity detector. This technique gave more reproducible results than other tests used in the study for detecting hippurate hydrolysis and also gave consistent results in detecting succinic acid produced from utilization of fumaric acid.

A biotyping scheme using improved media and methods for the detection of hippurate hydrolysis, rapid H2S production, and DNA hydrolysis was applied to 1,826 cultures of Campylobacter jejuni, Campylobacter coli and "Campylobacter laridis" isolates from human and nonhuman sources. Four biotypes were identified among C. jejuni: 57.3% of the isolates belonged to biotype I; 36.0%, to biotype II; 4.0%, to biotype III; and 2.7%, to biotype IV. C. coli organisms were differentiated into biotype I (67.0% of the isolates) and biotype II (33.0%). All "C. laridis" isolates belonged to biotype I. The combination of the biotyping scheme with the serotyping of campylobacters provided additional epidemiological markers by further differentiating the serogroups by species and biotypes.

Investigations of the etiology of diarrhea in patients in South Australia and the Northern Territory showed that Campylobacter spp. other than Campylobacter jejuni and C. coli were common in children. Campylobacters which were hippurate positive, nitrate negative, and susceptible to cephalothin and polymyxins were shown to be closely related to C. jejuni by DNA studies. Thermotolerant catalase-negative campylobacters were also isolated. These were H2S negative and biochemically resembled the catalase-negative or weak strains found in dogs in Sweden. DNA studies showed these campylobacters to be distinct from C. sputorum subsp. sputorum and to form a homogeneous group distinct from the enteropathogenic catalase-positive campylobacters. Preliminary studies suggest that these campylobacters are related to the Swedish catalase-negative or weak strains.

An additional method for differentiating between Campylobacter fetus subsp. jejuni and C. fetus subsp. intestinalis is reported. Strains of C. fetus subsp. jejuni (18/20) were shown to hydrolyze hippurate in the 2-h rapid test, whereas strains of C. fetus subsp. intestinalis did not.

A rapid gas-liquid chromatographic procedure was developed to determine hippurate hydrolysis by microorganisms. Bacterial cells were inoculated into 0.4 ml of 1% sodium hippurate and incubated for 2 h at 37 degrees C. Cells were removed by centrifugation, and the benzoate released by enzyme activity was converted to methyl benzoate and analyzed by gas-liquid chromatography. This procedure is sensitive, and its specificity provides a high degree of reliability for organisms with weak hippuricase activity.

Glycine, one of the end products of hippurate hydrolysis by microorganisms, was detected by a rapid, specific technique utilizing two-dimensional thin-layer chromatography. A loopful of growth of each organism from its suitable agar medium was washed, suspended, and incubated with 0.1% sodium hippurate for 30 min at 37 degrees C. The supernatant of the incubated suspension from each organism was then dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. Glycine, a product of hippurate hydrolysis, was detected under UV light. This technique does not require prolonged incubation and was found to be more specific and reliable than the standard ninhydrin reaction. In addition, it is inexpensive and can be easily conducted in a clinical microbiological reference laboratory. By this method, 100% (22/22) of Campylobacter jejuni and 0% (0/9) of Campylobacter coli reference strains were positive. In addition, 100% (13/13) of group B streptococci, 100% (24/24) of group D streptococci, and 90% (18/20) of Gardenerella vaginalis clinical isolates were positive for hippurate hydrolysis. This method is useful for the identification to the species level of Campylobacter organisms and the biotyping of Gardnerella organisms and for the detection of hippurate hydrolysis by unknown microorganisms.

Eleven strains of Campylobacter from earlier fluorescent-antibody studies were examined by DNA hybridization to determine their species. Three of the strains hydrolyzed sodium hippurate, and eight did not. Four of the hippurate-negative strains were in Campylobacter jejuni serogroups, and the remaining strains were in both C. jejuni and Campylobacter coli serogroups. DNA relatedness to type strains of C. jejuni and C. coli indicated that all three of the hippurate-positive strains and two of the hippurate-negative strains were C. jejuni. The six remaining hippurate-negative strains were C. coli. Two of the hippurate-negative strains in C. jejuni serogroups were C. jejuni, and two were C. coli. Three of the strains in serogroups of both species were C. jejuni, and four were C. coli. These studies confirm that a few strains of C. jejuni are hippurate negative and show that identical or highly related antigens are found in C. coli and C. jejuni.

A rapid test of hippurate hydrolysis and a test of tolerance to triphenyltetrazolium chloride (TTC) were studied in 315 strains of Campylobacter fetus subsp. jejuni to determine their usefulness for biotyping this organism and for distinguishing it from C. fetus subsp. intestinalis. Of the 315 strains tested, 84% hydrolyzed hippurate and 97% were resistant to TTC. Ability to hydrolyze hippurate was seen in 99% of 155 human isolates, 75% of 60 avian isolates, 100% of 41 cattle and dog isolates, 84% of 31 zoo mammal isolates, and none of 28 hog isolates. Resistance to 400 micrograms of TTC per ml was seen in 97% of the human isolates, 95% of the avian isolates, and 100% of the mammalian isolates (other than human). In no case did any of the 315 isolates of C. fetus subsp. jejuni show both lack of ability to hydrolyze hippurate and sensitivity to TTC. In contrast, all 18 strains of C. fetus subsp. intestinalis failed to hydrolyze hippurate and were sensitive to TTC. These two tests may be useful to distinguish between C. fetus subsp. jejuni and subsp. intestinalis and also to biotype strains of C. fetus subsp. jejuni.

The enhanced rapid identification method (RIM; Austin Biological Laboratories), a micromethod for the identification of Gardnerella vaginalis, is based on starch and raffinose fermentation and hippurate hydrolysis. We tested 105 clinical isolates of G. vaginalis with both the RIM and standard biochemical tests. The RIM agreed with the standard biochemical methods for 96 (91.4%) of the strains; nine isolates which were hippurate hydrolysis positive by standard biochemical tests were hippurate hydrolysis negative in the RIM. RIM may serve as a useful adjunct to Gram stain and colony morphology for the identification of G. vaginalis.

The rapid hippurate hydrolysis test was evaluated with the conventional test, using 17 group A streptococci, 9 non-enterococcal group D streptococci, 108 enterococcal group D streptococci, and 2 strains of Listeria monocytogenes. There was complete correlation between the rapid and conventional tests with all organisms except enterococcal group D. The rapid hippurate hydrolysis method was more sensitive with the enterococci; 95.4% were positive with the rapid method, and 9.3% were positive with the conventional method. Thin-layer chromatography (TLC) was performed on all isolates to determine if the end product of hydrolysis, glycine, was indeed present. The TLC results were in agreement with the rapid and conventional methods for group A streptococci, nonenterococcal group D streptococci, and L. monocytogenes. TLC results were in total agreement with the rapid hippurate hydrolysis test for the enterococcal group D isolates, thus verifying the accuracy of this more sensitive test. Trace amounts of glycine were found in the substrate, indicating the need for including an uninoculated substrate control as well as stock strains of group A and B beta-hemolytic streptococci (negative and positive controls, respectively) each time the rapid hippurate hydrolysis test is performed.

A disk method for detecting hippurate hydrolysis by Campylobacter spp. was evaluated and compared with the conventional tube test used at the Centers for Disease Control (CDC) (G.K. Morris and C.M. Patton, p. 302-308, in E.H. Lennette, A. Balows, W.J. Hausler, Jr., and H.J. Shadomy, ed., Manual of Clinical Microbiology, 4th ed., 1985) and high-performance liquid chromatography (HPLC). A total of 118 Campylobacter strains were tested. Eighty-seven strains (74%) were hippurate positive by the HPLC method, and the remaining 31 (26%) were found to be hippurate negative. By using HPLC as the reference technique, the CDC method showed a sensitivity of 80% and a specificity of 81%; the disk method showed a sensitivity of 93% and a specificity of 94%. The disk method can be performed with a small inoculum of bacteria, did not present problems of interpretation, and showed better results than the CDC method (P = 0.015).

A colorimetric test for the determination of hippurate hydrolysis was developed. Brain heart infusion broth made with 1% sodium hippurate served as the test medium. Hydrolysis was determined by the addition of two chemical developers, M (rhodamine B) and A (uranium acetate). A dark pink color indicated hydrolysis; no color change indicated no hydrolysis. The method was efficacious in either rapid or overnight incubation. One hundred twenty-five strains of group B, 44 strains of group A, 15 strains of group C, and 10 strains of group G Streptococcus were tested. By using the Lancefield method as the standard, there was 100% agreement with both the colorimetric and ferric chloride tests for hippurate hydrolysis, and 96% agreement with the CAMP test.

Rabbits were inoculated with whole, formalinized Campylobacter jejuni, C. coli, and C. fetus cells; the C. jejuni and C. coli immunogens were identified by their DNA relatedness at the species level to the type strains of C. jejuni or C. coli. The designation C. coli was not used among the other C. jejuni strains; they were classified as hippurate-positive or hippurate-negative C. jejuni. Immunoglobulin G was isolated from the antisera and labeled with fluorescein isothiocyanate. These conjugates defined 10 serogroups of C. jejuni, 2 serogroups of C. coli, and 2 serogroups of C. fetus. Of the 316 strains of C. jejuni tested, 258 (82%) were groupable; 173 were single-serogroup strains, and 85 were multiple-serogroup strains. Of the 226 strains in C. jejuni serogroups, 223 (98.7%) were hippurate positive; of the 27 strains in C. coli serogroups, 26 (96.3%) were hippurate negative. Five strains were equally reactive in immunofluorescent staining with a conjugate for a C. jejuni serogroup and a conjugate for a C. coli serogroup. A total of 58 strains of C. jejuni were ungroupable: 33 (13%) of the 259 hippurate-positive strains and 25 (44%) of the 57 hippurate-negative strains. All 121 strains of C. fetus tested were groupable as A, B, or A:B. The 14 conjugates used to define serogroups of C. jejuni, C. coli, and C. fetus reacted with the flagella but not the cells of other Campylobacter species and were negative on 256 other bacteria from 21 genera.

Streptococci from clinical isolates were studied for their ability to produce pigment in stab cultures in Columbia agar. Serological grouping of these organisms was done by counterimmunoelectrophoresis using Burroughs-Wellcome antisera. In this group of isolates, 66 of the 68 organisms grouped as B by serological testing produced pigment in the Columbia agar stab cultures. Pigment was not produced by any of the other 36 streptococci studied (11 group A, 9 group C, 4 group D, and 12 nongroupable). The use of the Columbia agar stab culture is recommended as a rapid and simple test for recognition of group B streptococci. The counterimmunoelectrophoresis test is also suggested as a convenient, rapid, and sensitive method for grouping the streptococci.

A rapid test to detect the hydrolysis of sodium hippurate by beta-hemolytic streptococci within 2 h was developed. All group B streptococci tested were positive using this method and all other groups were negative.

A gas-liquid chromatographic method which requires no chloroform extraction of the split products has been investigated for the detection of hippurate hydrolysis by Campylobacter spp. This technique gave better reproducibility than other tests also used in this study and allows the routine use of the gas-liquid chromatographic method for identification of Campylobacter isolates.

A total of 163 strains, including 106 strains of Legionella pneumophila, 28 strains of Tatlockia micdadei, and 29 strains of other legionellae (including members of the proposed genus Fluoribacter), were studied. Ten tests which together could distinguish the genera previously proposed were identified. These tests included catalase-peroxidase, gelatinase, hippurate hydrolysis, starch hydrolysis, medium browning, acetoin production, oxidase, medium fluorescence, colony fluorescence, and the bromcresol purple spot test. T. micdadei strains were strongly catalase positive and bromcresol purple spot test positive and produced acetoin but otherwise were usually inert in the other tests. L. pneumophila and Fluoribacter species could usually be distinguished by strength of catalase activity, blue-white colony fluorescence (if present), and differences in frequency of hippurate hydrolysis, starch hydrolysis, yellow-green medium fluorescence, and, to a lesser extent, oxidase activity. With a simple algorithm and computer program, the overall accuracy was 98.8%.