Phenotypic and genetic diversity among intestinal spirochaetes (genus Brachyspira) in free-living wild mallards (Anas platyrhynchos) sampled in southern Sweden

Brachyspira spp. are anaerobic intestinal spirochaetes that colonize vertebrates. Some species cause enteric diseases in pigs, chickens and possibly in humans, whereas others display a commensual relationship with their hosts. The aims were to investigate the prevalence among colonized free-living wild mallards (Anas platyrhynchos) of three enteropathogenic Brachyspira spp., and to describe the biodiversity of Brachyspira spp. isolates. Isolates from 150 birds were screened by PCR for 3 pathogenic Brachyspira spp., and 35 isolates from 20 mallards, 4 pigs and 1 chicken were subjected to phenotypic tests, 9 diagnostic PCRs, sequencing of the 16S rRNA and NADH oxidase (nox) genes, phylogenetic analysis and nox gene restriction enzyme analysis in silico. Of the 150 birds, 47%, 33% and 11% were positive by PCR for Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira hyodysenteriae, respectively. Thirty-one characterized isolates were provisionally identified as B. intermedia, Brachyspira alvinipulli, "Brachyspira pulli", or B. pilosicoli, whereas 4 were of indeterminate species affiliation. Many isolates were phylogenetically related to isolates from livestock. Isolates identified by PCR as B. pilosicoli displayed particularly high biodiversity. Up to five different Brachyspira genotypes were found from the same bird. Sequencing of amplicons from isolates that displayed ambiguous results as judged from PCR and phenotyping showed that several diagnostic PCRs were non-specific. Nox gene restriction enzyme analysis in silico correctly identified 2 of 34 characterized isolates. A culture technique based on filtration that produced uncontaminated spirochaete isolates was described. The results show that mallard intestines support a high degree of biodiversity among Brachyspira spp.     

Amplified Fragment Length Polymorphism and Pulsed Field Gel Electrophoresis for Subspecies Differentiation ofSerpulina pilosicoli

Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due to the low workload and high capacity and sensitivity of AFLP, this technique is recommended for future studies of S. pilosicoli. S. pilosicoli isolates were genetically diverse, and generally, identical subtypes were only identified in cultures from the same herd.     

Identification and genetic fingerprinting of Brachyspira species

Six Brachyspira type and reference strains, and 14 well characterized porcine field isolates representing all recognised porcine Brachyspira spp. were compared by different molecular methods. Sequence analysis of the 16S rRNA and the nox genes, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) were used in the study. In addition the isolates were analysed by five species-specific PCR systems. The topologies of the dendrograms obtained from each of the four typing systems were different. The B. pilosicoli isolates formed monophyletic clusters in all dendrograms, but with different sister lines. All five porcine Brachyspira species formed monophyletic clusters in the nox gene-based dendrogram only. All five PCR systems accurately identified their targets, except for the nox gene-based B. intermedia-specific system, by which it was not possible to identify one of the presumed B. intermedia isolates, and the other B. intermedia-specific system, based on the 23S rRNA gene, gave a positive reaction for one B. innocens isolate. In an extended study, 46 additional isolates and the original eight isolates with the phenotypes of B. hyodysenteriae or B. intermedia were compared by PFGE and PCR. The PFGE results indicated a high genetic diversity of isolates with the phenotype of B. intermedia. Thirty-three of 34 tested isolates could be identified by one or both of the two B. intermedia-specific PCR systems used, however, only 19 of the 34 isolates were positive in both systems.     

Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Characterization of Staphylococcus aureus Isolates from Buffalo, Bovine, Ovine, and Caprine Milk Samples Collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Comparison of culture and biochemical tests with PCR for detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli

Traditional culture and biochemical tests (CBT) were compared with PCR for sensitivity and detection of Brachyspira hyodysenteriae and Brachyspira pilosicoli in seeded faeces and clinical samples from diarrhoeic pigs. A duplex PCR system was developed based on primers detecting the tlyA-gene of B. hyodysenteriae and the 16S rRNA-gene of B. pilosicoli. Sensitivities for the PCR system were determined on seeded faeces, using DNA that had been recovered from primary cultures or extracted directly from faeces. Compared to CBT, PCR applied to DNA extracted directly from faeces lowered the sensitivity by a factor of 1000 to 10,000. B. hyodysenteriae and B. pilosicoli detection was compared for CBT and PCR using 200 clinical samples. CBT detected more B. hyodysenteriae isolates in the clinical samples than PCR, but fewer B. pilosicoli positive samples. An atypical strongly haemolytic isolate was detected only by CBT.     

Persistence of a Salmonella enterica serotype typhimurium clone in Danish pig production units and farmhouse environment studied by pulsed field gel electrophoresis (PFGE)

The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996-1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella.     

Molecular analysis of environmental and human isolates of Salmonella typhi.

Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.     

Variable within- and between-herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle

bla(CTX-M) beta-lactamases confer resistance to critically important cephalosporin drugs. Recovered from both hospital- and community-acquired infections, bla(CTX-M) was first reported in U.S. livestock in 2010. It has been hypothesized that veterinary use of cephalosporins in livestock populations may lead to the dissemination of beta-lactamase-encoding genes. Therefore, our objectives were to estimate the frequency and distribution of coliform bacteria harboring bla(CTX-M) in the fecal flora of Ohio dairy cattle populations. In addition, we characterized the CTX-M alleles carried by the isolates, their plasmidic contexts, and the genetic diversity of the bacterial isolates themselves. We also evaluated the association between ceftiofur use and the likelihood of recovering cephalosporinase-producing bacteria. Thirty fresh fecal samples and owner-reported ceftiofur use data were collected from each of 25 Ohio dairy farms. Fecal samples (n = 747) yielded 70 bla(CTX-M)-positive Escherichia coli isolates from 5/25 herds, 715 bla(CMY-2) E. coli isolates from 25/25 herds, and 274 Salmonella spp. from 20/25 herds. The within-herd prevalence among bla(CTX-M)-positive herds ranged from 3.3 to 100% of samples. Multiple pulsed-field gel electrophoresis (PFGE) patterns, plasmid replicon types, and CTX-M genes were detected. Plasmids with CTX-M-1, -15, and -14 alleles were clonal by restriction fragment length polymorphism (RFLP) within herds, and specific plasmid incompatibility group markers were consistently associated with each bla(CTX-M) allele. PFGE of total bacterial DNA showed similar within-herd clustering, with the exception of one herd, which revealed at least 6 different PFGE signatures. We were unable to detect an association between owner-reported ceftiofur use and the probability of recovering E. coli carrying bla(CTX-M) or bla(CMY-2).     

Multiple-locus variable-number tandem repeat analysis of Salmonella enterica subsp. enterica serovar Typhimurium: comparison of isolates from pigs, poultry and cases of human gastroenteritis

AIMS: Pulsed-field gel electrophoresis (PFGE) and variable number tandem repeat (VNTR) profiles of 195 epidemiologically unrelated Salmonella Typhimurium strains isolated in 1997-2004 from pigs were analysed and the results compared to establish the discriminatory ability of each method. In order to investigate the epidemiology of S. Typhimurium from different populations, the VNTR profiles from pigs were compared with those obtained from 190 S. Typhimurium strains isolated from poultry and 186 strains isolated from human cases of gastroenteritis. METHODS AND RESULTS: A total of 195 strains of S. Typhimurium were tested by PFGE and VNTR. For PFGE, the restriction enzyme XbaI was used, and for VNTR, the number of repeats at five loci (STTR 9, 5, 6, 10pl and 3) were counted and assigned an allele number based on an established VNTR scheme. The results obtained showed improved discrimination of VNTR when compared with PFGE with 34 PFGE profiles identified compared with 96 different VNTR profiles for the pig isolates and 56 different VNTR types within the most common PFGE type. Within the three different populations, VNTR showed distinct subpopulations of VNTR type related not only to source, but also demonstrated common VNTR types within samples obtained from humans, poultry and pigs, especially in strains of phage type DT104. CONCLUSIONS: VNTR has taken the discrimination to a further level than that obtained through PFGE, and demonstrated an overlap in the genetic diversity of isolates tested across the three different populations, confirming previous suggestions that animals have an involvement in the dissemination of S. Typhimurium through the food chain. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Typhimurium remains an important concern as a food-borne zoonotic agent. The VNTR strategy described provides an accurate method of tracing strain dissemination, and adds a further level of discrimination to the PFGE type, providing potential benefits to epidemiological studies and the possibility of deciphering source attribution of cases.     

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.     

Campylobacter hyointestinalis subsp. hyointestinalis, a common Campylobacter species in reindeer

AIMS: To study the prevalence of Campylobacter spp. in the faecal material of reindeer, and to identify the isolates by means of a polyphasic approach. In addition, to study the genetic diversity of Camp. hyointestinalis subsp. hyointestinalis reindeer isolates by pulsed-field gel electrophoresis (PFGE). METHODS AND RESULTS: The material, collected during the slaughter period in autumn 1998, comprised 399 faecal contents from the reindeer (Rangifer tarandus), a semi-domesticated, meat-producing ruminant of northern Finland. These samples came from 16 herds in the areas of eight reindeer slaughterhouses. Samples were cultured by methods suitable for isolation of fastidious Campylobacter species. Of all samples, 6% (24/399) were Campylobacter-positive. Phenotypic characteristics, SDS-PAGE protein patterns, dot blot DNA-DNA hybridization, 23S rDNA restriction fragment polymorphism analysis and PFGE identified the isolates as Camp. hyointestinalis subsp. hyointestinalis. CONCLUSIONS: Campylobacter hyointestinalis subsp. hyointestinalis was the only Campylobacter species isolated from reindeer in this study. The isolates showed high genomic diversity in PFGE with the restriction enzymes SmaI and KpnI. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE analysis is a useful subtyping method for epidemiological studies. Contaminated reindeer meat can be a source for human infections.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates ( n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant ( P > 0.05). There was a significant difference ( P > 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates (n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant (P greater than 0.05). There was a significant difference (P less than 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.     

Antimicrobial susceptibility and pulsed-field gel electrophoresis of Shigella sonnei strains in Malaysia (1997-2000)

AIMS: Antimicrobial resistance of Shigella sonnei from Malaysia was determined and subtyping was carried by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these strains. METHODS AND RESULTS: A total of 62 isolates of S. sonnei from sporadic cases of shigellosis in different parts of Malaysia were studied by antimicrobial susceptibility test and PFGE. Approximately 35.5% of the strains showed resistance to three or more antimicrobial agents. Eight resistant phenotypes, i.e. RI to RVIII, was defined. Resistant phenotype RV and RVIII only appeared in year 2000. PFGE analysis with NotI and XbaI restriction showed that a great heterogeneity existed at the DNA level among Malaysian S. sonnei isolates. Fifty-eight NotI and 61 XbaI-PFGE profiles were observed in 63 S. sonnei isolates, including ATCC 11060 isolate. Drug sensitive isolates displayed very different profiles from drug-resistant isolates, with a few exceptions. Isolates of resistant phenotype RVI (SXTr.TETr.STRr) showed a greater similarity among each other compared with isolates of resistant phenotype RI and drug-sensitive isolates. CONCLUSION: Multi-drug-resistant S. sonnei were circulated in different parts of Malaysia and the emergence of new resistant phenotype was observed. Wide genetic variations among Malaysian S. sonnei were observed and the drug-sensitive strains could be differentiated from drug-resistant strains by PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study verifies the usefulness of PFGE in characterizing and comparing strains of S. sonnei. Minor variations among S. sonnei isolates could be detected by PFGE.     

Intraspecific genotypic characterization of Lactobacillus rhamnosus strains intended for probiotic use and isolates of human origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

Occurrence and strain diversity of thermophilic campylobacters in cattle of different age groups in dairy herds

AIMS: To investigate the occurrence and numbers of thermophilic campylobacters excreted by cattle in dairy herds, and to assess the strain diversity within herds. METHODS AND RESULTS: Faecal samples from 15 animals at each of 24 cattle farms were cultured quantitatively for thermophilic campylobacters and 23% of animals and 83% of farms were positive for Campylobacter jejuni. Young animals had a higher prevalence and higher faecal concentration than older animals. Serotyping and pulsed field gel electrophoresis (PFGE) of isolates showed that the most common serotypes were 2, 4-complex and 11. Serotype 2 was especially prevalent among calves (68% of the positive calves). In eight of the 20 positive herds, all isolates had the same sero- and PFGE type while, in the other herds, two to five different types were isolated. CONCLUSIONS: Significant differences were found between age groups in relation to the prevalence and numbers of excreted campylobacters, serotype distribution and strain diversity. The relatively few different strains in each herd indicate that transmission between animals is common. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence on cattle farms of the human pathogen C. jejuni and the wide distribution of serotype 2, the most common serotype among Danish patients, indicate that cattle might be an important reservoir for human infections. The ability of this serotype to colonize calves in high numbers further indicates that serotype 2 strains may have an advantage over other serotypes.     

Intraspecific Genotypic Characterization of Lactobacillus rhamnosus Strains Intended for Probiotic Use and Isolates of Human Origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

Intraspecies diversity of Coxiella burnetii as revealed by com1 and mucZ sequence comparison

Coxiella burnetii is classified within the γ subgroup of the Proteobacteria. All strains tested to date have an identical 16S rRNA sequence but 20 different genotypes have been determined by pulsed field gel electrophoresis (PFGE). In this study, intraspecies genetic diversity was investigated by sequence comparison of 715 bp of the Com1 encoding gene (com1) and 774 bp of the MucZ encoding gene (mucZ) in 37 strains isolated from animals and humans with acute or chronic Q fever in Europe, North America and Africa. Five and four groups were established from sequence analysis of com1 and mucZ, respectively. Neither relation of the defined groups to geographical distribution of the isolates was noted nor relation to disease form (acute/chronic). The same isolates were grouped together regardless of the gene being investigated. Comparison of the five proposed groups to previous groups, yielded after digestion by NotI PFGE, allowed for an intermediate classification of C. burnetii isolates between those obtained by using 16S rDNA (one group) and PFGE (20 groups).     

Pulsed-field gel electrophoresis typing of Hafnia alvei isolated from chub-packed and retail ground beef

Thirty-eight isolates of Hafnia alvei were characterized by biochemical profiles, ribotyping and pulsed-field gel electrophoresis (PFGE) patterns. The isolates were recovered from chub-packed (19 isolates) or retail (nine isolates) ground beef, or were obtained from culture repositories (10 isolates). Biochemical profiling differentiated the 38 isolates into five groups and a commercial ribotyping method recognized 11 groups, whereas PFGE differentiated the same 38 isolates into 19 groups. These data substantiate that PFGE is a highly discriminatory tool for establishing the relatedness among Hafnia alvei strains.