Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts

To better understand the emergence and transmission of antibiotic-resistant Streptococcus agalactiae, we compared phenotypic and genotypic characteristics of 52 human and 83 bovine S. agalactiae isolates. Serotypes found among isolates from human hosts included V (48.1%), III (19.2%), Ia and Ib (13.5% each), and II (5.8%). Among isolates from bovine hosts, molecular serotypes III and II were predominant (53 and 14.5%, respectively). Four and 21 different ribotypes were found among human and bovine isolates, respectively. A combination of ribotyping and serotyping showed that two bovine isolates were indistinguishable from human isolates. Resistance to tetracycline and erythromycin was more common among human (84.6% and 26.9%, respectively) than bovine (14.5% and 3.6%, respectively) isolates. tetM was found in all tetracycline-resistant human isolates, while tetO was the predominant resistance gene among bovine isolates. tet genes were found among various ribotypes. ermB, ermTR, and mefA were detected among erythromycin-resistant human isolates, while ermB was the only erythromycin resistance determinant among isolates from bovine hosts. For isolates from human hosts, erythromycin resistance genes appeared to be associated with specific ribotypes. We conclude that (i) human and bovine S. agalactiae isolates represent distinct populations; (ii) human host-associated S. agalactiae subtypes may occasionally be transmitted to bovines; (iii) while emergence of erythromycin and tetracycline resistance appears to largely occur independently among human and bovine isolates, occasional cross-species transfer of resistant strains or transmission of resistance genes between human- and bovine-associated subtypes may occur; and (iv) dissemination of antibiotic-resistant S. agalactiae appears to include both clonal spread of resistant strains as well as horizontal gene transfer.     

Genetic Features of Streptococcus agalactiae Strains Causing Severe Neonatal Infections, as Revealed by Pulsed-Field Gel Electrophoresis and hylB Gene Analysis

A collection of 114 independent Streptococcus agalactiae strains, including 54 strains isolated from the cerebrospinal fluid (CSF) samples of neonates and 60 strains from asymptomatic patients, was characterized by pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI and by PCR analysis of the hylB gene. All strains were previously studied by multilocus enzyme electrophoresis (MLEE) (R. Quentin, H. Huet, F.-S. Wang, P. Geslin, A. Goudeau, and R. K. Selander, J. Clin. Microbiol. 33:2576-2581, 1995). Among these 114 strains, there were 92 PFGE patterns. Eleven genetic groups (A to K) were identified with 38% divergence. A more homogeneous group (PFGE group A) was defined, consisting of 73% of the strains previously identified as belonging to a particular MLEE phylogenetic group. A 162-kb fragment was identified as a marker of strains that invaded the central nervous system of neonates. It was detected in 69% of the PFGE patterns obtained with CSF isolates and in only 1.8% of the PFGE patterns obtained with carrier strains. The hylB gene encoding hyaluronate lyase was amplified for all strains in our collection. Ten of 15 isolates belonging to an MLEE subgroup, previously described as being likely to cause invasive infection, had an insertion in the hylB gene (IS1548).     

Genotyping of the capsule gene cluster (cps) in nontypeable group B streptococci reveals two major cps allelic variants of serotypes III and VII

Forty group B Streptococcus (GBS) isolates obtained from Europe and the United States previously reported to be nontypeable (NT) by capsule serotype determination were subjected to buoyant density gradient centrifugation. From nearly half of the isolates capsule-expressing variants could be selected. For characterization of the remaining NT-GBS isolates, the capsule operon (cps) was amplified by the long-fragment PCR technique and compared by restriction fragment length polymorphism (RFLP) analysis. The patterns from serotype reference isolates (n = 32) were first determined and used as a comparison matrix for the NT-GBS isolates. Using two restriction enzymes, SduI and AvaII, cluster analysis revealed a high degree of similarity within serotypes but less than 88% similarity between serotypes. However, serotypes III and VII were each split in two distant RFLP clusters, which were designated III(1) and III(2) and VII(1) and VII(2), respectively. Among the isolates that remained NT after repeated Percoll gradient selections, two insertional mutants were revealed. Both were found in blood isolates and harbored insertion sequence (IS) elements within cpsD: one harbored IS1548, and the other harbored IS861. All other NT-GBS isolates could, by cluster analysis, be referred to different serotypes by comparison to the RFLP reference matrix. In pulsed-field gel electrophoresis of SmaI-restricted chromosomal DNA, patterns from allelic type 1 and 2 isolates were essentially distributed in separate clusters in serotypes III and VII. A covariation with insertion sequence IS1548 in the hylB gene was suggested for serotype III, since allelic type III(1) harboring IS1548 in hylB, clustered separately. The variation in serotype VII was not dependent on the presence of IS1548, which was not detected at any position in the type VII chromosome.     

Characterization of Neisseria meningitidis serogroup C by multilocus enzyme electrophoresis and ribosomal DNA restriction profiles (ribotyping).

We compared multilocus enzyme electrophoresis (MEE) and ribosomal DNA fingerprinting (ribotyping) for subtyping 44 strains of Neisseria meningitidis serogroup C that were isolated in Los Angeles County, California, between December 1985 and July 1986. The isolates were divided into six enzyme types (ETs) by MEE, but 36 of the isolates were clustered in one ET, 3. The same isolates were divided into 17 ribotypes by use of restriction endonucleases ClaI, EcoRI, and XhoI. Twenty of the 36 ET 3 isolates were divided into 17 ribotypes by use of restriction endonucleases ClaI, EcoRI, and XhoI. Twenty of the 36 ET 3 isolates were grouped in a single ribotype, J. The rate of infection with ribotype J strains was higher in the southern part of the study area than in the northern part. Isolates from each of eight pairs (each isolate pair was cultured from the same patient from the same or different sites) were found identical by MEE, but ribotyping revealed a difference in one pair. In this study, ribotyping showed a greater discriminating capacity than MEE for subtyping N. meningitidis serogroup C, but the epidemiologic relevance of this increased sensitivity needs further assessment.     

Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis

Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype 1/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE.     

Correlations between Molecular Subtyping and Serotyping of Listeria monocytogenes

To define relationships between Listeria monocytogenes genetic lineages, ribotypes, and serotypes, 235 L. monocytogenes isolates were characterized by serotyping and automated EcoRI ribotyping. Genetic lineage predicted the following serovar clusters: lineage I, comprising serotypes 1/2b, 3b, 3c, and 4b; lineage II, comprising serotypes 1/2a, 1/2c, and 3a; and lineage III, comprising serotypes 4a and 4c. Some EcoRI ribotypes contained multiple serotypes; a subset of these isolates was further differentiated with PvuII ribotyping. Of the 12 resultant EcoRI-PvuII combination types, only 4 contained multiple serotypes, demonstrating the potential of ribotyping for serotype prediction.     

Comparison of rapid,automated ribotyping and DNA macrorestriction analysis of Burkholderia pseudomallei

An automated ribotyping device (RiboPrinter) was used to determine the ribotypes of a collection of Burkholderia pseudomallei isolates. In a preliminary evaluation with the restriction enzymes BamHI and EcoRI, the protocol with EcoRI was more discriminating. The reproducibilities of the ribotypes obtained with EcoRI (EcoRI ribotypes) were determined by testing three levels of bacterial loads. The performance of the manufacturer's software was assessed by comparing the machine-optimized ribotypes with the type determined from the original gel image analyzed with Bionumerics software. The library of B. pseudomallei EcoRI ribotypes was then compared with the ribotypes obtained by DNA macrorestriction analysis of XbaI digests by pulsed-field gel electrophoresis. The typeability of B. pseudomallei by EcoRI ribotyping was 100%, and the discrimination index was 0.94. The slightly greater discrimination provided by DNA macrorestriction analysis (0.96) was achieved at the expense of a significantly longer processing time of 6 days, although the method was only half the cost of automated ribotyping. Typeability by macrorestriction analysis was lower (97%) unless a thiourea step was added to neutralize the action of Tris-dependent endonucleases. The digital record of B. pseudomallei isolates analyzed thus far provides a useful resource for future epidemiological studies and will help shorten the response time in the event of a further melioidosis outbreak or the deliberate release of B. pseudomallei as a biohazard.     

Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates.

Seventy-three isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, including 26 isolates from 10 hospital outbreaks, were typed by ribotyping with EcoRI and ClaI and by pulsed-field gel electrophoresis (PFGE) of genomic DNA after digestion with ApaI. Ribotyping with EcoRI distinguished 31 ribopatterns. Digestion with ClaI generated another eight ribotypes. PFGE, in contrast, identified 49 distinct patterns with seven variants. Both methods detected all outbreak-related isolates. By ribotyping, nine epidemiologically unrelated strains could not be differentiated from outbreak strains, in contrast to only one isolate not identified by PFGE. Thus, PFGE was more discriminating than ribotyping. However, ribotyping is known to generate banding patterns specific to each DNA group in the A. calcoaceticus-A. baumannii complex that may be used for taxonomic identification of the strains. PFGE was shown to lack this property. Both methods are therefore useful for strain differentiation in epidemiological studies of Acinetobacter isolates.     

Development of a ribotyping scheme forHaemophilus influenzae type b

Ribotyping and outer-membrane protein subtyping were used to characterise 283 consecutive isolates ofHaemophilus influenzae type b. These isolates were obtained primarily from patients with invasive disease in the UK and were received by the Public Health Laboratory Service Haemophilus Reference Laboratory prior to the implementation ofHaemophilus influenzae serotype b vaccine in the UK. A subtyping scheme using the ribotyping method is suggested. Twenty-two ribotypes are described, 14 of which were found amongst the 283 clinical isolates characterised in this study. In contrast, only four outer-membrane protein subtypes were found amongst the 283 isolates. The ribotyping profiles were further used to estimate the relatedness of isolates. The resulting dendrogram suggested a population genetic structure different from that previously described forHaemophilus influenzae type b using multi-locus enzyme electrophoresis. This study shows the value of ribotyping as a subtyping method for epidemiological studies ofHaemophilus influenzae type b. However, the further use of ribotyping for population genetic structure analysis ofHaemophilus influenzae type b may be misleading and therefore inappropriate.     

Extended multilocus variable-number tandem-repeat analysis of Clostridium difficile correlates exactly with ribotyping and enables identification of hospital transmission

PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.     

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.     

Multilocus sequence typing of Streptococcus uberis provides sensitive and epidemiologically relevant subtype information and reveals positive selection in the virulence gene pauA

Control of the bovine mastitis pathogen Streptococcus uberis requires sensitive and epidemiologically meaningful subtyping methods that can provide insight into this pathogen's epidemiology and evolution. Development of a multilocus sequence typing (MLST) scheme based on six housekeeping and virulence genes allowed differentiation of 40 sequence types among 50 S. uberis isolates from the United States (n = 30) and The Netherlands (n = 20). MLST was more discriminatory than EcoRI or PvuII ribotyping and provided subtype data with better epidemiological relevance, e.g., by discriminating isolates with identical ribotypes obtained from different farms. Phylogenetic analyses of MLST data revealed indications of reticulate evolution between genes, preventing construction of a core phylogeny based on concatenated DNA sequences. However, all individual gene phylogenies clearly identified a distinct pauA-negative subtaxon of S. uberis for which housekeeping alleles closely resembled those of Streptococcus parauberis. While the average GC content for five genes characterized was between 0.38 and 0.40, pauA showed a considerably lower GC content (0.34), suggesting acquisition through horizontal transfer. pauA also showed a higher nonsynonymous/synonymous rate ratio (dN/dS) (1.2) compared to the other genes sequenced (dN/dS < 0.12), indicating positive selection in this virulence gene. In conclusion, our data show that (i) MLST provides for highly discriminatory and epidemiologically relevant subtyping of S. uberis; (ii) S. uberis has a recombinatorial population structure; (iii) phylogenetic analysis of MLST data reveals an S. uberis subtaxon resembling S. parauberis; and (iv) horizontal gene transfer and positive selection contribute to evolution of certain S. uberis genes, such as the virulence gene pauA.     

Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophage-nontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates.

Analysis of DNA restriction fragment length polymorphisms of rRNA genes (ribotyping) was employed to assist in the epidemiologic investigation of the emergence and spread of ciprofloxacin-resistant Staphylococcus aureus at the Atlanta VA Medical Center because many isolates of interest were nontypeable by phages and harbored few plasmids useful as strain markers. Chromosomal DNAs of selected S. aureus isolates were digested initially with 20 different restriction enzymes. EcoRI appeared to give the best discrimination of hybridization banding patterns (ribotypes) and was used with all study isolates. Overall, 15 different ribotypes were seen among the 50 S. aureus isolates studied (7 ribotypes among 13 methicillin-susceptible S. aureus [MSSA] isolates and 9 ribotypes among 37 methicillin-resistant S. aureus [MRSA] isolates). Seven of eight ciprofloxacin-resistant MSSA (CR-MSSA) patient isolates had identical antibiograms, were nontypeable by phages, and had a single 22-MDa plasmid. Six of these seven CR-MSSA isolates had an identical ribotype pattern. Ribotyping distinguished this CR-MSSA strain or clone from MRSA and other MSSA isolates, including nontypeable isolates that contained a 22-MDa plasmid. Five ciprofloxacin-susceptible MSSA isolates studied had five ribotypes; one pattern was identical to the CR-MSSA clone. Twenty-three CR-MRSA isolates recovered from the Atlanta VA Medical Center had four different ribotypes. Ribotyping proved to be a useful molecular epidemiologic tool in the study of S. aureus because it differentiated isolates which were indistinguishable by more traditional methods. In addition, this technique demonstrated that at our institution, ciprofloxacin resistance emerged in multiple strains of MRSA, as opposed to primarily a single strain or clone of MSSA.     

Comparison of biotyping, ribotyping, and pulsed-field gel electrophoresis for investigation of a common-source outbreak of Burkholderia pickettii bacteremia.

Over a 3-month period, six immunocompromised patients developed one or more episodes of Burkholderia pickettii bacteremia and/or catheter infection. Vials of a commercially available, "sterile" saline for injection which had been used for flushing the patients' indwelling intravenous devices were implicated as the common source of the organisms. No further cases were diagnosed once the use of this saline was discontinued. Twenty-six isolates, including 9 outbreak-related strains from case patients and contaminated saline as well as 17 control strains, were tested comparatively by biotyping, ribotyping with EcoRI and HindIII, and pulsed-field gel electrophoresis (PFGE) with SpeI. Macrorestriction analysis revealed nine PFGE groups and was more discriminating than ribotyping (seven ribotypes) and biotyping (two biovars). Among the outbreak-related isolates, one B. pickettii type was found by the three typing methods. Furthermore, PFGE was useful for subdividing ribotypes and for distinguishing isolates involved in the outbreak from all epidemiologically unrelated strains.     

Comparison of three typing methods for Pseudomonas aeruginosa

Fifty-seven independent isolates of Pseudomonas aeruginosa from blood specimens were typed with 3 different methods: ribotyping, random amplified polymorphic DNA (RAPD) typing, and pyocin typing. Ribotyping was performed by probing the rRNA genes of genomic DNA that was digested separately with 4 different restriction enzymes. Digestion of DNA from 57 P. aeruginosa isolates with BamHI, ClaI, EcoRI, and PstI produced 4, 4, 6, and 7 patterns, respectively. As a result, ribotyping classified the 57 isolates into 22 types. Six new ribotypes that had not been described previously were found. One BamHI, 1 ClaI, 2 EcoRI, and 2 PstI patterns were novel. RAPD typing was performed with two different polymerase chain reaction (PCR) primers (RAPD1 and RAPD2). Both primers classified the 57 isolates into 15 RAPD types and produced identical patterns. The pyocin typing method classified the 57 isolates into 10 types. According to the results obtained in this study, the ribotyping has a discriminatory index of 0.865, RAPD, 0.785, and pyocin typing, 0.676, respectively. The ribotyping method was the most effective among the 3 methods compared for typing P. aeruginosa isolates.     

Subtype distribution of Haemophilus influenzae isolates from north India

A total of 120 Haemophilus influenzae isolates from blood, cerebrospinal fluid, sputum and throat swabs of patients and carriers in North India was characterised by biotyping, ribotyping and random amplification of polymorphic DNA (RAPD)-PCR. Of these, 77 isolates (64%) were serotype b; the other 43 (36%) were non-typable. Biotype I was the most predominant among the typable strains and biotype II among the non-typable strains. Ribotyping with restriction endonucleases HaeIII and EcoRI differentiated the isolates into three and six ribotypes, respectively. However, RAPD fingerprints generated by the application of arbitrary primers AP1 and AP2 provided a higher level of discrimination. RAPD typing revealed distinct polymorphism among the serologically typable isolates. This study is the first report that stratifies the subtypes of H. influenzae strains from India by molecular techniques.     

Subtyping of Salmonella enterica serotype enteritidis strains by manual and automated PstI-SphI ribotyping

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). A recently developed method for ribotyping of this organism, which uses a mixture of PstI and SphI (PS) for restriction of DNA (PS ribotyping), has proved useful for further subtyping of a number of PTs of this organism, including PT 4. However, it has not been extensively tested with PT 8. In the present study the PS ribotyping method was used to investigate outbreaks of both S. enterica serotype Enteritidis PT 4 and PT 8 and provided subtyping data that were consistent with information obtained from epidemiologic investigations. The method proved to be more discriminatory than phage typing and pulsed-field gel electrophoresis (PFGE) combined and was useful for investigating a pseudo-outbreak involving isolates that had identical PTs and PFGE types but that could not be linked epidemiologically. Several PS ribotypes were found within the cluster of isolates indistinguishable by other subtyping methods, confirming the epidemiologic findings. Although the PS ribotyping method proved to have a superior discriminatory ability in resolving clusters, it did not have high enough throughput for use in outbreak investigations. This method has therefore been adapted for use in automated ribotyping with a RiboPrinter, and the results were compared with those obtained by manual ribotyping. Both methods produce equivalent results and are useful for obtaining epidemiologically relevant subtyping data for S. enterica serotype Enteritidis, including PT 8 strains not extensively tested previously.     

Differentiation of Shigella flexneri strains by rRNA gene restriction patterns.

We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.     

Practical evaluation of molecular subtyping and phage typing in outbreaks of infection due to Salmonella enterica serotype typhimurium

Identification and control of food-poisoning outbreaks due to salmonellosis depend on prompt microbiological diagnosis and subtyping to identify the causative strain. In Australia, Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) is responsible for 40-70% of cases of human salmonellosis. Phage typing is the usual method of subtyping S. typhimurium, but on its own, has limitations. We compared it with three molecular subtyping methods using 100 isolates of S. typhimurium, representing four different phage types (PT 1, 9, 126 and 135) and comprising 74 isolates from three presumed outbreaks, 25 isolates from sporadic cases of salmonellosis and S. typhimurium ATCC 10428 (phage type 126). The isolates were divided into 11 subtypes by IS200 restriction fragment length polymorphism (RFLP) typing, four each by ribotyping and pulsed-field gel electrophoresis (PFGE) and 17 distinct strains using a combination of phage and molecular typing. Isolates from two presumed outbreaks were resolved into multiple strains, possibly explaining the failure to identify a common source for either during the original investigations. IS200 RFLP analysis was the most discriminatory and reproducible typing method. Several strains were identifiable within and shared between phage types 1, 9 and 126. Phage and IS200 RFLP typing together, would provide improved definition of S. typhimurium outbreaks.     

Value of plasmid profiling, ribotyping, and detection of IS200 for tracing avian isolates of Salmonella typhimurium and S. enteritidis.

Seventy selected strains of Salmonella typhimurium and S. enteritidis isolated from related poultry flocks in three independent geographical areas were characterized by phenotypic and genotypic methods to compare the usefulness of the methods in epidemiological studies. The 56 S. typhimurium isolates were poorly discriminated by their biotypes, resistance patterns, and plasmid profiles. Nine different ribotypes were obtained after DNA digestion by BglII, PvuII, and SmaI. Seven IS200 types, characterized by six to nine copies of IS200 on the chromosome, were detected after digestion of genomic DNA by PstI. These studies resulted in the definition of 15 clonal lineages distributed in three clusters. The 14 S. enteritidis strains were not discriminated either by ribotyping or by detection of IS200 (IS200 typing), but were separated on the basis of antibiotic resistance and plasmid profiling. The stability of the insertion sequence type was confirmed by inoculation of an S. typhimurium strain to axenic chickens reared for 15 weeks in sterile isolators.