Evaluation of a simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli which is based on the O antigen.

A simplified procedure for serotyping Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens was developed and tested for its applicability as a routine typing method. The assay involves the sensitization of erythrocytes with an antigenic extract and performance of a slide agglutination assay with specific antisera. In order to simplify the typing system to a greater extent, the standard typing antisera were pooled into nine groups for C. jejuni and four groups for C. coli. The five antiserum samples allocated to each pool were selected so that pairs or groups of cross-reacting antisera were included in the same pool. When this system was tested with the serotype reference strains, it was found that, in most cases, a strain reacted in only one pool. The specific serotype of that strain could then be further defined by typing in each of the antisera belonging to that pool. To evaluate the specificity of the simplified method, 246 clinical isolates of C. jejuni and 57 clinical isolates of C. coli were typed at the same time by the standard passive hemagglutination assay and by the rapid slide agglutination system. Although both schemes effectively differentiated isolates and results from both schemes were generally very similar, differences were noted for a few isolates. On the basis of these findings, the simplified procedure may be recommended as an alternative means for serotyping these species for epidemiological purposes.

Antigenic shifts in serotype determinants of Campylobacter coli are accompanied by changes in the chromosomal DNA restriction endonuclease digestion pattern.

Changes in somatic (O) lipopolysaccharide (LPS) antigenic specificities of Campylobacter coli serostrains were observed after continuous laboratory subculture. Two serostrains (C. coli O34 and C. coli O48) lost O specificity and did not react with homologous or any of the available heterologous antisera. The C. coli serostrain for serogroup O5, after subculture, yielded a variant that had acquired a new specificity which was detectable with a heterologous antiserum. In a repeat experiment with the original isolate of the O5 strain, a second variant was obtained which had not only acquired the same new determinant but had, unlike the first variant, lost reactivity with the homologous antiserum. Immunoblot experiments with homologous and heterologous antisera indicated that changes in antigenic specificity were associated with the O side chains of the LPS molecules. Results of restriction endonuclease analysis of chromosomal DNA of the variants and their parents revealed minor differences in restriction patterns which suggested that C. coli is capable of undergoing genomic re-arrangements that lead to changes in LPS specificity and structure.

Application of serotyping and chromosomal restriction endonuclease digest analysis in investigating a laboratory-acquired case of Campylobacter jejuni enteritis.

A frequently passaged laboratory strain of Campylobacter jejuni was confirmed by serotyping on the basis of thermostable antigens and by bacterial chromosomal restriction endonuclease digests to be the causative agent of enteritis in a laboratory worker.

Investigation of a waterborne outbreak of Campylobacter jejuni enteritis with a serotyping scheme based on thermostable antigens.

Serotyping of 11 human and 2 water isolates of Campylobacter jejuni associated with a waterborne outbreak revealed two serotypes among the human isolates. One of these (serotype 58) was a new serotype and was added to the serotyping scheme. Serotypes were defined by using extracted thermostable antigens and passive hemagglutination titrations of both unabsorbed and cross-absorbed antisera. Two water isolates of the same serotype as six human isolates provided evidence to link a contaminated water supply to the outbreak.

Correlation of an expanded direct fluorescent-antibody system with an established passive hemagglutination system for serogrouping strains of Campylobacter jejuni and Campylobacter coli.

Rabbits were inoculated with whole, formalinized cells from eight passive hemagglutination reference strains of Campylobacter. Fluorescein isothiocyanate-labeled immunoglobulin G from these antisera defined seven new direct fluorescent-antibody serogroups of C. jejuni and one new serogroup of C. coli. This expanded the Campylobacter direct fluorescent antibody system to include 17 serogroups of C. jejuni, 3 serogroups of C. coli, and 2 serogroups of C. fetus. We then compared the passive hemagglutination method (57 serotypes) and the direct fluorescent-antibody method (20 serogroups) for typing strains of C. jejuni and C. coli. The data obtained by testing 101 strains by both methods revealed that the two test systems were measuring completely different sets of antigen complexes. The two serogrouping methods were complementary, and their combined use discriminated among strains more effectively than did either method individually.

Serotyping of Campylobacter jejuni and Campylobacter coli on the basis of thermostable antigens.

Hippurate hydrolysis tests performed on the serotype reference strains of the serotyping scheme based on thermostable antigens under development for Campylobacter jejuni showed that 42 strains were Campylobacter jejuni and 17 were Campylobacter coli. Moreover, only four (0.2%) of 2025 hippurate positive Campylobacter jejuni isolates reacted in Campylobacter coli antisera and 12 (4.3%) of the 282 Campylobacter coli reacted in Campylobacter jejuni antisera. Evidently each species has its own array of antigenic specificities. Separate schemes for serotyping Campylobacter jejuni and Campylobacter coli are advocated.

Occurrence of plasmid DNA in serologically defined strains of Campylobacter jejuni and Campylobacter coli.

Forty Campylobacter jejuni and 17 Campylobacter coli strains that constitute the set of reference strains for our serotyping scheme were each examined for the presence of plasmid DNA. Agarose gel electrophoresis of alkaline-extracted DNA showed the occurrence of 29 bands in 11 C. jejuni strains and 40 bands in C. coli strains. Plasmids ranged in size from 1.6 to 70 megadaltons. Most strains that carried plasmids had between 2 and 6 of them; however, one strain had 14 plasmids, and two strains contained only 1 plasmid each. Repeated electrophoresis demonstrated that all plasmid profiles were stable. A different plasmid profile was seen for each of the 19 plasmid-carrying strains, but it was clear that plasmids of the same or similar molecular weight could be found in different strains. On the basis of these findings, we are persuaded that plasmid profiles determined by a rapid procedure for DNA extraction will play a significant role in resolving complexities among strains that are difficult to serotype and could be useful in epidemiological studies in which the implicated isolates are plasmid bearers.

The serotype and biotype distribution of clinical isolates of Campylobacter jejuni and Campylobacter coli over a three-year period.

Two hundred eighty-five isolates of Campylobacter jejuni-Campylobacter coli from children with gastroenteritis at The Hospital for Sick Children (Toronto, Canada) over a three-year period were biotyped by the hippurate hydrolysis test and serotyped on the basis of thermostable, soluble antigens by the passive hemagglutination technique. Hippurate-negative strains (C. coli) were only 3.2% of the isolates. Ninety-seven percent of the isolates were serotypable with 55 antisera. About half of the strains belonged to one of four serotypes (2, 4, 3, or 1); about three-quarters belonged to one of 10 serotypes. Serotype 2 was consistently the commonest serotype in each of the three years of the study, accounting for 15%-20% of all isolates tested.

Serotyping of Campylobacter jejuni isolated from sporadic cases and outbreaks in British Columbia.

Campylobacter jejuni from sporadic cases and outbreaks of gastroenteritis were serotyped on the basis of heat-extracted soluble thermostable antigens identified with the use of the passive hemagglutination technique. A total of 168 isolates were separated into 45 different types. The largest proportion of the isolates fell into three serotypes, each with 11 to 12.5% of the total number. Three less frequently occurring serotypes each included approximately 5%, and the remaining 50% of the isolates were distributed among 39 other serotypes. In most cases, serotyping demonstrated that epidemiologically linked isolates were of the same serotype, but the outbreak strains could belong either to frequently or to infrequently isolated serotypes. The high correlation between clinical findings and serotyping results confirmed the applicability of the serotyping scheme in epidemiological investigations of C. jejuni infections.

Species differences in susceptibilities of Proteeae spp. to six cephalosporins and three aminoglycosides.

Six cephalosporins and three aminoglycosides were examined for activity against 1,693 isolates belonging to six species of Proteeae. The most notable species-specific differences included the marked susceptibility of Providencia alcalifaciens and Proteus mirabilis to cephalothin, the resistance of Proteus vulgaris to cefamandole, and the resistance of Providencia stuartii to gentamicin and tobramycin. The third-generation cephalosporins cefotaxime and moxalactam were substantially more inhibitory than were cefoperazone, cefamandole, and cefoxitin. P. stuartii, generally the most resistant species, was, however, markedly susceptible to moxalactam and cefotaxime.

Three episodes of nosocomial urinary tract infections caused by one O-serotype of Providencia stuartii.

The results of O-serotyping Providencia stuartii isolates in a general hospital showed that 43 isolates were the same serotype (063) and were from 15 patients located in 1 or 2 adjacent wards. On retrospective examination it was found that the series of infections occurred during a 9-month period and could be separated into 3 episodes, involving 7, 6 and 2 patients. All patients who acquired the Providencia stuartii 063 strain were catheterized. The introduction of the strain into the hospital was attributed to a patient catheterized before admission from an institution in which the 063 strain had been identified previously. Antibiotic irrigation was not successful in eliminating Providencia stuartii from the urine and the procedure may be a predisposing factor in preferentially selecting strains of this species.

Transferable urease activity in Providencia stuartii.

Six urea-positive Providencia stuartii strains were tested for transmissible urease determinants. Two strains, when implanted with "helper" conjugative plasmids, were found to be capable of transferring urease genes to Escherichia coli or urea-negative P. stuartii. Recombination of the urease genes with the helper plasmid in P. stuartii was noted in one case. One of the urea-positive P. staurtii strains was found to harbor a conjugative plasmid which mediated both urease activity and ampicillin resistance. This large plasmid (molecular weight, approximately 140 x 10(6)) was transmissible to and stably maintained in E. coli strains. The demonstration of transmissible genes for urease activity in P. stuartii is significant in that it accounts for previous problems associated with classifying urea-positive strains of this species.

Passive hemagglutination technique for serotyping Campylobacter fetus subsp. jejuni on the basis of soluble heat-stable antigens.

Antigenic materials were extracted from Campylobacter fetus subsp. jejuni strains by heating bacterial suspensions in saline at 100 degrees C and by exposure to ethylenediaminetetraacetic acid. The antigens were heat stable at 100 degrees C, capable of sensitizing sheep erythrocytes for agglutination in antisera, and able to elicit production of specific antibody in rabbits; they occurred with different immunological specificities in 23 strains. Antisera against the 23 strains could be used for discriminating among isolates of the species when the passive hemagglutination technique was used for serotyping. Three serotypes were more common than others among a collection of human isolates.

Separate O-grouping schemes for serotyping clinical isolates of Proteus vulgaris and Proteus mirabilis.

Antisera were prepared against type strains of the original scheme of B. Perch (Acta Pathol. Microbiol. Scand. 25:703-714, 1948) and against newly defined types to produce separate schemes for O-grouping Proteus vulgaris and Proteus mirabilis. In assessing the schemes for their effectiveness it was found that 82% of 208 P. vulgaris isolates and 88% of 194 P. mirabilis isolates from two hospitals were typable. Only 3.4% of the P. vulgaris isolates agglutinated in P. mirabilis antisera, and 1.5% of the P. mirabilis agglutinated in P. vulgaris antisera, indicating that separation of the schemes would be more advantageous in routine typing. P. mirabilis of groups O3, O6, O10, O29, and O30 were most frequently isolated. Of the P. vulgaris isolates, 25% belonged to newly defined O-groups, and one of these was the largest with 14% of all isolates of this species. The application of serotyping using separate schemes for each species was advocated in epidemiological studies.

O-serotyping Providencia alcalifaciens.

The O-serotyping scheme for Providencia was tested on Providencia alcalifaciens isolates collected mostly from two hospitals. The specificites of the somatic (O) antigens of P. alcalifaciens were found to be different from those of Providencia stuartii, and separation of the Providencia typing scheme to allow separate typing of each species led to more efficient typing. All but 4 of 86 isolates were typable. Eighteen serotypes occurred among 53 typable isolates obtained from a pediatric hospital, and 11 occurred among 19 isolates from a general hospital. Thirty-two percent of the isolates from the pediatric hospital belonged to serotype O3, the most frequently isolated and most widely distributed type. The use of the serotyping scheme for P. alcalifaciens is advocated for further studies to examine strains of the species for enteropathogenic types.

Application of O-serotyping in a study of Providencia rettgeri (Proteus rettgeri) isolated from human and nonhuman sources.

A somatic (O) antigen serotyping scheme for Providencia rettgeri (Proteus rettgeri) was modified to exclude O-type strains recently reclassified as urea-positive Providencia stuartii and was extended to include new serotypes to provide for serotyping on the basis of 93 O-antigens. Isolates from two hospitals, five public health laboratories, and nonhuman sources (polluted water and frogs) were serotyped. The 112 isolates collected from a large general hospital over a 99-month period were distributed among 42 O-serotypes. No serotype showed significant predominance that would suggest the occurrence of human strains that are more prone than others to cause human infections, but in an institution experiencing cross-infection, 11 of the 22 (50%) isolates belonged to one serotype. The 54 isolates from the five public health laboratories belonged to 33 serotypes, 15 of which were found also among hospital isolates. All but 5 of 99 frog isolates were typable, and the 94 typable isolates were separated into 25 serotypes. Each of the four isolates from polluted water samples was of a different serotype. Sixteen of the serotypes found in frogs and three found in water were also identified among human isolates.

O antigen grouping of Morganella morganii (Proteus morganii) by slide agglutination.

Antisera were prepared against Morganella morganii (Proteus morganii) type strains from the scheme described by Rauss et al. (Acta Microbiol. Acad. Sci. Hung. 22:315--321, 1975) and Raus and Vörös (Acta Microbiol. Acad. Sci. Hung. 6:233--248, 1959; Acta Microbiol. Acad. Sci. Hung. 14:195--198, 1967). The specificities of the somatic (O) antigens were studied using the passive hemagglutination and slide agglutination techniques. Previously unreported interstrain relations were observed, and O groups were provisionally defined on the basis of related strains. Of 143 isolates, collected mostly from two hospitals, 96% could be placed in one or another of the O groups. Forty-eight percent belonged to O group 1 and most of these were of two serotypes O1ab, 2 (20%) and O1ad, 2 (12%).

O serotyping of Providencia stuartii isolates collected from twelve hospitals.

A collection of 829 isolates of Providencia stuartii, mostly from urological specimens of patients in 12 hospitals, were O serotyped. Hospitals varied in serotype distribution, but most isolates (97%) fell into one or another of 14 O types of P. stuartii. One type (O63) was found in 10 hospitals, and six types (O4, O17, O25, O52, O55, O56) were found in 5 or more hospitals. These seven types were more common than others and included 753 (91%) of the isolates. Only four isolates agglutinated in Providencia alcalifaciens antisera and, for increased efficiency in serotyping, it is recommended that separate schemes be employed for P. stuartii and P. alcalifaciens. Strains endemic in different hospitals may differ in serotype and give rise to nosocomial infections that are clinically recognizable when infections occur in obvious clusters. Nosocomial infections occurring in low frequency among patients not located close to each other in the hospital may be detected with the aid of serotyping.

Variation in urease activity of endemic hospital strains of Proteus rettgeri and Providencia stuartii.

Both urease-positive and urease-negative Proteeae isolated from cross-infected patients in the same hospitals and, in three cases, from the same patients were examined for their biochemical reactions and somatic (O-) antigens. All isolates gave the same reactions in 17 biochemical tests and possessed O-antigens characteristic of Providenic O-type strains 4 or 17. Study of the isolates indicated that endemic strains are capable of undergoing variation in urease activity. In the current classification urease-positive and urease-negative strains are classified as Proteus rettgeri and Providencia stuartii, respectively. The observed variation in urease activity of nosocomial isolates of Proteeae suggests that taxonomy should be modified so that all such strains would be accommodated in a single group.