Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping and serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens

Molecular characterization of Salmonella Enteritidis : comparison of an optimized multi-locus variable-number of tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis

Salmonella Enteritidis (SE) is a genetically homogenous serovar, which makes optimal subtype discrimination crucial for epidemiological research. This study describes the development and evaluation of an optimized multiple-locus variable number tandem-repeat assay (MLVA) for characterization of SE. The typeability and discriminatory power of this MLVA was determined on a selected collection of 60 SE isolates and compared with pulsed-field gel electrophoresis (PFGE) using restriction enzymes XbaI, NotI, or SfiI. In addition, the estimated Wallace coefficient (W) was calculated to assess the congruence of the typing methods. Selection of epidemiologically unrelated isolates and more related isolates (originating from layer farms) was also based on the given phage type (PT). When targeting six loci, MLVA generated 16 profiles, while PFGE produced 10, 9, and 16 pulsotypes using XbaI, NotI, and SfiI, respectively, for the entire strain collection. For the epidemiologically unrelated isolates, MLVA had the highest discriminatory power and showed good discrimination between isolates from different layer farms and among isolates from the same layer farm. MLVA performed together with PT showed higher discriminatory power compared to PFGE using one restriction enzyme together with PT. Results showed that combining PT with the optimized MLVA presented here provides a rapid typing tool with good discriminatory power for characterizing SE isolates of various origins and isolates originating from the same layer farm

Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37 degrees C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains

Development of Multi-Locus Variable Number Tandem Repeat Analysis for Outbreak Detection of Neisseria meningitidis

Neisseria meningitidis is a major cause of septicemia and meningitis worldwide. Traditional typing methods like pulsed-field gel electrophoresis (PFGE) for identifying outbreaks are subjective and time consuming. Multi-locus variable number tandem repeats analysis (MLVA) is an objective typing method amenable to automation that has been used to type other bacterial pathogens. This report describes the development of MLVA for outbreak detection of N. meningitidis. Tandem Repeats Finder software was used to identify variable number tandem repeats (VNTRs) from 3 sequenced N. meningitidis genomes. PCR amplification of identified VNTRs was performed on DNA from 7 serogroup representative isolates. PCR products were sequenced and repeats were manually counted. VNTR loci identified by this screen were evaluated on a collection of 46 outbreak and sporadic serogroup C isolates. Alleles at each locus were concatenated to define the MLVA type for each isolate. Minimum spanning tree (MST) analysis was performed to determine the genetic relationships among the isolates. The genetic distance was defined as the summed tandem repeat difference (STRD) between isolates MLVA types. Outbreak clusters were defined by a STRD less than or equal to 3. These data was compared to PFGE data to determine the utility of MLVA for outbreak detection. Twenty-one VNTR loci with variable copy numbers among the sequenced genomes were identified that met the established criteria of short repeat length and consensus sequence > 85%. Seven VNTR loci were reliably amplified among the 7 serogroups tested. These loci had repeat lengths between 4 and 20 nucleotides and exhibited between 10 and 26 alleles among 61 isolates belonging to 7 different serogroups. MST analysis with 7 loci differentiated serogroups, discriminated sporadic isolates and identified 7 out of 8 serogroup C outbreaks. In summary, MLVA with 5 VNTR loci distinguished N. meningitidis isolates from 7 different serogroups and sporadic isolates within each serogroup. In addition, MLVA identified 88% of PFGE-defined serogroup C outbreaks. Further investigation of these and other outbreak-associated isolates is necessary to define the optimal combination of VNTR loci and to evaluate MST analysis criteria in order to determine the utility of MLVA for N. meningitidis outbreak detection

Neutral genomic microevolution of a recently emerged pathogen, salmonella enterica serovar agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks

Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin.

A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the feces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being bla TEM, dfr, strB, tet(A), and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of bla TEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harboring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year

E. coli O157 on Scottish cattle farms: evidence of local spread and persistence using repeat cross-sectional data

<b>Background</b><p></p> Escherichia coli (E. coli) O157 is a virulent zoonotic strain of enterohaemorrhagic E. coli. In Scotland (1998-2008) the annual reported rate of human infection is 4.4 per 100,000 population which is consistently higher than other regions of the UK and abroad. Cattle are the primary reservoir. Thus understanding infection dynamics in cattle is paramount to reducing human infections.<p></p> A large database was created for farms sampled in two cross-sectional surveys carried out in Scotland (1998 - 2004). A statistical model was generated to identify risk factors for the presence of E. coli O157 on farms. Specific hypotheses were tested regarding the presence of E. coli O157 on local farms and the farms previous status. Pulsed-field gel electrophoresis (PFGE) profiles were further examined to ascertain whether local spread or persistence of strains could be inferred.<p></p> <b>Results</b><p></p> The presence of an E. coli O157 positive local farm (average distance: 5.96km) in the Highlands, North East and South West, farm size and the number of cattle moved onto the farm 8 weeks prior to sampling were significant risk factors for the presence of E. coli O157 on farms. Previous status of a farm was not a significant predictor of current status (p = 0.398). Farms within the same sampling cluster were significantly more likely to be the same PFGE type (p < 0.001), implicating spread of strains between local farms. Isolates with identical PFGE types were observed to persist across the two surveys, including 3 that were identified on the same farm, suggesting an environmental reservoir. PFGE types that were persistent were more likely to have been observed in human clinical infections in Scotland (p < 0.001) from the same time frame.<p></p> <b>Conclusions</b><p></p> The results of this study demonstrate the spread of E. coli O157 between local farms and highlight the potential link between persistent cattle strains and human clinical infections in Scotland. This novel insight into the epidemiology of Scottish E. coli O157 paves the way for future research into the mechanisms of transmission which should help with the design of control measures to reduce E. coli O157 from livestock-related sources

Characteristics of vancomycin-resistant enterococci (VRE) isolated from medical fields in Japan

学位記番号：医博甲169