Application of single-strand conformation polymorphism and denaturing gradient gel electrophoresis for fla sequence typing of Campylobacter jejuni

Campylobacter jejuni is a frequent cause of bacterial gastroenteritis in humans all over the world. Several molecular typing methods are used to study the epidemiology of Campylobacter spp. infections. The aim of the present study was to investigate the application of single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) analysis as rapid primary subtyping methods for C. jejuni. A variable fragment from the 3' end of the flaA to the 3' end of the intergenic region, separating the flaA and flaB genes, was subjected to SSCP and DGGE analysis. A total of 48 clinical C. jejuni isolates, 49 C. jejuni strains isolated from poultry, 2 strains isolated from ducks and 1 strain isolated from a pheasant were assigned to 24 distinct SSCP patterns. Sequence analysis of the respective DNA fragments revealed that every different fla sequence type could be distinguished by SSCP. DGGE proved to be equally discriminatory. Both methods can be applied as primary subtyping methods, because pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) analysis further differentiated isolates belonging to the same fla sequence types.     

Comparison of automated ribotyping and pulsed-field gel electrophoresis for subtyping of Vibrio cholerae

Aims:  To compare the discriminatory power of an automated ribotyping method for Vibrio cholerae subtyping with the pulsed-field gel electrophoresis (PFGE), to evaluate the possibility of automated ribotyping in use of outbreak investigations and surveillance of cholera.
Methods and Results:  Eight-one epidemiologically unrelated isolates of V. cholerae , and 19 isolates from seven cholera outbreaks were used as the panels. When comparing the two methods using the epidemiologically unrelated isolates, automated ribotyping using Pvu II distinguished 38 different ribotypes with a D -value of 0·8956. When combined with serotyping, the D -value is 0·9466. However, PFGE with Not I and Sfi I digestions had higher D -values of 0·9951 and 0·9948, respectively. PFGE could cluster the isolates from each outbreak into the same pattern, and distinguish different patterns from different outbreaks, whereas automated ribotyping had lower discriminatory ability.
Conclusions:  The automated ribotyping has lower discriminatory ability compared to PFGE, and is limited to application in V. cholerae subtyping and outbreak investigation.
Significance and Impact of the Study:  The study evaluated the limitation in subtyping of automated ribotyping for V. cholerae , and raise the question of improvement for the automated ribotyping in subtyping.     

Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience

AIMS: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. METHODS AND RESULTS: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98-100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. CONCLUSIONS: Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. SIGNIFICANCE AND IMPACT OF THE STUDY: This work should facilitate progress towards inter-laboratory standardization of fla typing.     

High-resolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting.

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

Evaluation of randomly amplified polymorphic DNA and pulsed field gel electrophoresis techniques for molecular typing of Dermatophilus congolensis

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694-0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.     

Genotyping of Campylobacter jejuni strains from Danish broiler chickens by restriction fragment length polymorphism of the LPS gene cluster

AIMS: To apply and evaluate LG (LPS genes) genotyping, which is a genotyping method based on a cluster of genes involved in the synthesis of surface lipopolysaccharides (LPS) in Campylobacter species, for typing of Campylobacter jejuni isolates obtained from Danish broiler chickens. Furthermore, the LG genotyping method was used to study the genetic stability of four C. jejuni strains after gastrointestinal passage through experimentally infected chickens. METHODS AND RESULTS: In the present study, the LG genotyping method was modified with respect to the restriction enzymes used. To validate the method, 63 Penner serotype reference strains and 107 C. jejuni chicken isolates, representing the most common Penner serotypes of C. jejuni in Danish poultry, were selected for typing. The method was successfully used for typing all isolates and the LG genotype profiles were reproducible. There were no changes in the LG genotype of the C. jejuni strains obtained after experimental passage through chickens. CONCLUSIONS: All C. jejuni strains obtained from broiler chickens were typeable by the LG genotyping method. Application of the RsaI restriction enzyme improved the method in terms of ease and consistency of analyses and increase of discriminatory power. SIGNIFICANCE AND IMPACT OF THE STUDY: The LG genotyping method is a valuable tool for typing C. jejuni isolates obtained from poultry. However, the association between Penner serotyping based on passive haemagglutination of heat-stable antigens and LG genotyping was low when applied to poultry isolates. This is in contrast to previous studies on isolates of human origin that reported a high correlation between results obtained by the two typing methods (Shi et al. 2002).     

Random amplified polymorphic DNA (RAPD) profiling of Legionella pneumophila

Random amplified polymorphic DNA (RAPD) profiling of Legionella pneumophila by PCR can be used to provide a simple and efficient comparison of clinical and environmental isolates. RAPD profiling is quicker and cheaper to perform than restriction fragment length polymorphism (RFLP) typing, eliminating the need for blotting, hybridization and detection. For some isolates, RAPD profiling is more discriminatory than RFLP typing, being able to distinguish between isolates with identical RFLP types.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Comparison of Campylobacter jejuni PFGE and Penner subtypes in human infections and in water samples from the Taieri River catchment of New Zealand

AIMS: To determine the degree of overlap in strain types of Campylobacter jejuni isolated from clinical cases and water samples from the Taieri catchment in the South Island of New Zealand. METHODS AND RESULTS: Thermophilic Campylobacter were collected from human cases of infection, the main stem of the Taieri River and streams within distinct land-use types over a 1-year period. Campylobacter jejuni (187 isolates) and Campylobacter lari (four isolates) were identified using a multiplex polymerase chain reaction protocol. Isolates were typed by the Penner method and pulsed-field gel electrophoresis (PFGE) utilizing two restriction endonucleases. Several serotypes and PFGE types occurred in both water samples and clinical cases when the restriction profiles for each enzyme were considered separately. However, when PFGE profiles and serotyping were combined, there was no overlap between Camp. jejuni types from water and clinical cases. CONCLUSIONS: The results of this study indicate that recreational water in the Taieri catchment is not a major source of campylobacteriosis in the Dunedin area. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the risk of acquiring campylobacteriosis from surface waters in the Taieri catchment is considerably lower than previously predicted and highlights the necessity of using two endonucleases in PFGE typing.     

Characterization of Rhizobium 'hedysari' by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting

The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers. For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari'. As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used. By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh. 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position. The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible. In parallel, analysis of the same strains was performed by PCR amplification of their total DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR). By both AP and rep-PCR an identification of every particular strain was achieved. In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons. On the basis of the results presented here it can be concluded that AP and rcp-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.     

Genotypic Diversity among Campylobacter jejuni Isolates in a Commercial Broiler Flock

Analysis of nucleic acid polymorphism in the flagellin genes of Campylobacter jejuni was used to investigate genetic diversity among Campylobacter spp. in a commercial broiler flock. Three hundred single colonies of C. jejuni were isolated from fecal samples collected weekly for 3 weeks immediately before slaughter. Both the flaA and flaB genes were amplified by PCR, and the PCR product was digested with the restriction enzyme AluI. The fragments generated were then analyzed by agarose gel electrophoresis. Among the 300 recovered isolates, five different restriction fragment length polymorphism profiles were observed. Three of these profiles were dominant during the course of the study, and the other two profiles were detected at low frequency. Analysis of genetic variation in C. jejuni over the course of an experimental infection lasting 7 weeks indicated that there was no obvious drift in the flagellin gene type. These findings demonstrate that a range of bacterial genotypes can constitute the bacterial population within a commercial poultry flock, with the most likely sources of these types being multiple environmental exposure and/or genetic drift within the population. This degree of diversity must be considered in epidemiological analyses which utilize genetic typing methods that investigate Campylobacter contamination of any food source, including poultry, to ensure that the total gene pool for C. jejuni is evaluated.     

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.     

Improved group‐specific primers based on the full SILVA 16S rRNA gene reference database

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T‐RFLP) analysis, are well‐suited techniques for the examination of microbial community structures. The use of phylum‐ and class‐specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain‐specific primers. To date, several phylum‐ and class‐specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non‐target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T‐RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above‐mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.     

Development of a rapid strain differentiation method for methicillin-resistant Staphylococcus aureus isolated in Japan by detecting phage-derived open-reading frames

AIMS: To develop a rapid genotyping method for investigating outbreaks of methicillin-resistant strains of Staphylococcus aureus (MRSA) isolated in Japan. METHODS AND RESULTS: Isolates were genotyped by detecting the keeping pattern of 16 open-reading frames (ORFs), a process we call phage ORF typing (POT). Thirteen of the ORFs were selected from phage genomes and one from a genomic island SaGIm in the genome of strain Mu50. The other two ORFs, one from Tn554 and one from staphylococcal cassette chromosome mec (SCCmec) type II, were used as strain markers. Three hundred and sixty-eight isolates from five hospitals were classified into 133 types by POT, whereas they were classified into 139 types by pulsed-field gel electrophoresis (PFGE) subtyping. The discriminatory power of POT (D=0.989) was equal to that of PFGE subtyping (D=0.986). CONCLUSIONS: MRSA isolates collected in Japan can be genotyped by detecting the keeping pattern of phage-derived ORFs with a discriminatory power equal to that of PFGE subtyping. SIGNIFICANCE AND IMPACT OF THE STUDY: MRSA isolates can be genotyped rapidly by detecting phage-derived ORFs. As particular pandemic clones can be found in a specific region, a typing method localized to a pandemic clone may be effective for the rapid genotyping of MRSA during outbreaks.     

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India

Aims: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)‐PCR. Methods and Results: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0·95, while ERIC‐PCR showed a DI of 0·94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. Conclusions: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC‐PCR generates useful information in the case of V. parahaemolyticus associated with seafood. Significance and Impact of the Study: The study demonstrates the usefulness of nucleic acid and protein‐based studies in understanding the relationship between various isolates from seafood.     

New restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus

In this study, we isolated and tested restriction fragment length polymorphism (RFLP) markers for Aspergillus fumigatus based on PCR products amplified by the random amplified polymorphic DNA (RAPD) primer R108. Four DNA fragments, Afd, Af5, Af4, and Af4A, were amplified. Fragments Afd and Af5 were 85% and 88% identical at the DNA level to part of the Afut1 retrotransposon from A. fumigatus. Fragment Af4A is a duplication of fragment Af4 and both showed similarity at the amino acid level with endonucleases from other fungal retrotransposons. We used both RAPD with primer R108 and RFLP assays with Afut1, Afd, and Af4A, to determine the genetic relatedness of clinical isolates of A. fumigatus isolated sequentially from four patients colonized with A. fumigatus. The combination of these different methods suggested that the isolates infecting the four patients were not identical.     

Evaluation of a commercial diagnostic PCR for the identification of Campylobacter jejuni and Campylobacter coli

AIMS: DuPont Qualicon recently developed a new PCR assay for the identification of Campylobacter jejuni and Campylobacter coli. We evaluated the selectivity and utility of this assay compared with a PCR method already in use in our laboratory. METHODS AND RESULTS: A group of 133 Campylobacter isolates from poultry carcass rinse samples were screened using the commercial PCR and standard PCR. Identical results were found for 89.5% (119/133) of the isolates. However, 10.5% (14/133) gave conflicting results suggesting mixed cultures. These 14 strains were retested by both PCR methods. Of these, 78.6% (11/14) showed identical results for both PCR methods after retesting; the results for the remaining 21.4% (3/14) again indicated mixed cultures. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: The new multiplex PCR is a rapid and accurate alternative to more conventional PCR methods. The persistence of mixed Campylobacter cultures noted in this study suggests certain strains may be very difficult to isolate clonally by standard culture methods.     

Typings of Campylobacter jejuni isolated from patients of 2 outbreak cases by genotypic and phenotypic methods

We compared Campylobacter jejuni strains isolated from the patient stools associated with two food-borne diarrheal outbreak cases by the serotypic methods (Lior and Penner systems) and the genotypic methods (restriction fragment length polymorphism (RFLP) of flaA gene and pulsed-field gel electrophoresis (PFGE)). Fla-RFLP was based on the digestion of 410 bp DNA fragment by MboI restriction enzyme amplified from a 5' portion of C. jejuni flaA gene. Six distinctive fla-RFLP patterns were identified by examining 29 serotype reference strains and 58 strains isolated from the patients infected with C. jejuni independently. In the first outbreak case, 4 isolates were shown to be the same patterns each other by the fla-RFLP and PFGE, and by the Lior serotyping, except the Penner system that serotyped into 2 distinct types. On the other hand, in the second case, out of 10 isolates, 5 isolates were identical by the both genotypic and the both serotypic methods, and 4 isolates were not differentiated by the fla-RFLP and Penner system, but were separated into 4 types by PFGE in a little difference. The rest isolate was completely different from the other isolates by the all of methods used now. The findings suggest that the second case occurred by the infection of at least 3 different strains of C. jejuni.     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Phenon cluster analysis as a method to investigate epidemiological relatedness between sources of Campylobacter jejuni

AIMS: To develop a method for assessing the relative epidemiological significance of possible infection sources for human campylobacteriosis. METHODS AND RESULTS: Using fluorescent amplified fragment length polymorphism (AFLP), 243 apparently epidemiologically unrelated Campylobacter jejuni isolates were genotyped (77 human, 46 cattle, 49 pet and 71 poultry isolates). In total 136 different phena were identified, of which 48 were clusters grouping at least two isolates. Isolates from different sources were frequently clustered together, underlining the high degree of source mixing and the lack of host specificity of C. jejuni. The phena were classified into different phenon types according to the sources of the isolates they contained. The occurrence of these phenon types was analysed using an area-proportional Euler diagram to describe epidemiological relatedness among C. jejuni isolates. Group separation statistics revealed that 43% of analysed human isolates expressed maximum similarity to other human isolates, 9% to cattle isolates, 21% to pet isolates and 27% to poultry isolates; these results were in accordance with the pattern observed in the phenon cluster analysis. CONCLUSIONS: Based on the grouping of strains into molecular similarity clusters, ecological patterns between sources can be investigated. Significance and IMPACT OF THE STUDY: This approach is a new methodological contribution to establish the relative epidemiological significance of concurrent infection sources.