Molecular Typing of Pathogenic Leptospira Serogroup Icterohaemorrhagiae Strains Circulating in China during the Past 50 Years

Background

Leptospirosis is one of the most important neglected tropical infectious diseases worldwide. Icterohaemorrhagiae has been throughout recent history, and still is, the predominant serogroup of this pathogen in China. However, very little in detail is known about the serovars or genotypes of this serogroup.

Methodology/Principal Findings

In this study, 120 epidemic strains from five geographically diverse regions in China collected over a 50 year period (1958~2008), and 8 international reference strains characterized by 16S rRNA sequencing and MLST analysis. 115, 11 and 2 strains were identified as L. interrogans, L. borgpetersenii, and L. kirschneri, respectively. 17 different STs were identified including 69 ST1 strains, 18 ST17, 18 ST128, 9 ST143 and 2 ST209. The remaining 12 strains belonged to 12 different STs. eBURST analysis demonstrated that, among the clonal complexes isolated (CCs), CC1 accounted for 73.3% (88/120) strains representing three STs: ST1, ST128 and ST98. ST1 was the most likely ancestral strain of this CC, followed by singleton CC17 (17/120) and CC143 (11/120). Further analysis of adding 116 serogroup Icterohaemorrhagiae strains in the MLST database and studies previously described using global eBURST analysis and MST dendrogram revealed relatively similar ST clustering patterns with five main CCs and 8 singletons among these 244 strains. CC17 was found to be the most prevalent clone of pathogenic Leptospira circulating worldwide. This is the first time, to our knowledge, that ST1 and ST17 strains were distributed among 4 distinct serovars, indicating a highly complicated relationship between serovars and STs.

Conclusions/Significance

Our studies demonstrated a high level of genetic diversity in the serogroup Icterohaemorrhagiae strains. Distinct from ST17 or ST37 circulating elsewhere, ST1 included in CC1, has over the past 50 years or so, proven to be the most prevalent ST of pathogenic leptospires isolated in China. Moreover, the complicated relationship between STs and serovars indicates an urgent need to develop an improved scheme for Leptospira serotyping.     

Extensive Genomic Diversity among Bovine-Adapted Staphylococcus aureus: Evidence for a Genomic Rearrangement within CC97

Staphylococcus aureus is an important pathogen associated with both human and veterinary disease and is a common cause of bovine mastitis. Genomic heterogeneity exists between S. aureus strains and has been implicated in the adaptation of specific strains to colonise particular mammalian hosts. Knowledge of the factors required for host specificity and virulence is important for understanding the pathogenesis and management of S. aureus mastitis. In this study, a panel of mastitis-associated S. aureus isolates (n = 126) was tested for resistance to antibiotics commonly used to treat mastitis. Over half of the isolates (52%) demonstrated resistance to penicillin and ampicillin but all were susceptible to the other antibiotics tested. S. aureus isolates were further examined for their clonal diversity by Multi-Locus Sequence Typing (MLST). In total, 18 different sequence types (STs) were identified and eBURST analysis demonstrated that the majority of isolates grouped into clonal complexes CC97, CC151 or sequence type (ST) 136. Analysis of the role of recombination events in determining S. aureus population structure determined that ST diversification through nucleotide substitutions were more likely to be due to recombination compared to point mutation, with regions of the genome possibly acting as recombination hotspots. DNA microarray analysis revealed a large number of differences amongst S. aureus STs in their variable genome content, including genes associated with capsule and biofilm formation and adhesion factors. Finally, evidence for a genomic arrangement was observed within isolates from CC97 with the ST71-like subgroup showing evidence of an IS431 insertion element having replaced approximately 30 kb of DNA including the ica operon and histidine biosynthesis genes, resulting in histidine auxotrophy. This genomic rearrangement may be responsible for the diversification of ST71 into an emerging bovine adapted subgroup.     

Comparative genotypic and phenotypic characterisation of methicillin-resistant Staphylococcus aureus ST398 isolated from animals and humans

The high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) ST398 among pigs in certain European countries and North America and its occurrence in other animal species raises a question concerning the molecular mechanisms mediating the success of this lineage. In this study a panel of S. aureus strains belonging to sequence type (ST) 5 (n = 4), ST8 (n = 5), ST15 (n = 5), ST22 (n = 8), clonal complex (CC) 30 (n = 8), CC97 (n = 8), CC130 (n = 4), CC151 (n = 4) and ST398 (n = 18) were screened by DNA microarray and PCR for the carriage of virulence and antimicrobial resistance genes. Isolates belonging to the same sequence type/clonal complex (ST/CC) were found to share similar virulence gene profiles. The ST398 lineage displayed the lowest content of virulence genes, which consisted mainly of genes detected among the majority or all of the analysed lineages. All MRSA ST398 isolates lacked accessory virulence genes that were detected in other ST/CC. In contrast to virulence genotype, the antimicrobial resistance genes profiles varied between isolates belonging to the same ST/CC and profile similarities could be observed for isolates from different lineages. MRSA ST398 isolates in particular displayed significant diversity and high content of antimicrobial resistance genes. This was comparable with certain MRSA belonging to other sequence types particularly the equine MRSA ST8. The apparent lack of significant virulence genes among MRSA ST398 strains, demonstrates that the lineage features a unique genetic background but no ST398-specific virulence markers could be identified.     

Lack of genetic polymorphism among peregrine falcons Falco peregrinus of Fiji

We compared levels of genetic diversity and isolation among peregrine falcons Falco peregrinus from two South Pacific island complexes (Fiji and Vanuatu: F. p. nesiotes), relative to other island and mainland populations. Fragment data from 12 microsatellite loci and sequence information from the control region of the mitochondrial DNA indicated levels of genetic variation in the South Pacific populations were lower than other island and mainland populations. Indeed, diversity varied from extremely low (Vanuatu) to completely absent (Fiji). We find little support for a hypothesis that populations on Fiji or Vanuatu were colonized via Australia. The complete lack of polymorphism in peregrine falcons of Fiji is remarkable, and to our knowledge has not been observed in a natural avian population. This lack of polymorphism, and the inability to test for decrease in polymorphism using museum samples, precludes testing whether the lack of genetic diversity in the population on Fiji is due to a recent bottleneck, or sustained isolation over evolutionary time. Increased fertility in eggs of Fiji peregrines upon outbreeding with males from other areas is consistent with inbreeding depression within a population typified by heterozygote deficiency.     

Geographical and Temporal Structures of Legionella pneumophila Sequence Types in Comunitat Valenciana (Spain), 1998 to 2013

Legionella pneumophila is an accidental human pathogen associated with aerosol formation in water-related sources. High recombination rates make Legionella populations genetically diverse, and nearly 2,000 different sequence types (STs) have been described to date for this environmental pathogen. The spatial distribution of STs is extremely heterogeneous, with some variants being present worldwide and others being detected at only a local scale. Similarly, some STs have been associated with disease outbreaks, such as ST578 or ST23. Spain is among the European countries with the highest incidences of reported legionellosis cases, and specifically, Comunitat Valenciana (CV) is the second most affected area in the country. In this work, we aimed at studying the overall diversity of Legionella pneumophila populations found in the period from 1998 to 2013 in 79 localities encompassing 23 regions within CV. To do so, we performed sequence-based typing (SBT) on 1,088 L. pneumophila strains detected in the area from both environmental and clinical sources. A comparison with the genetic structuring detected in a global data set that included 20 European and 7 non-European countries was performed. Our results reveal a level of diversity in CV that can be considered representative of the diversity found in other countries worldwide.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

Multilocus sequence typing reveals high genetic diversity and epidemic population structure for the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the causative agent of enteric redmouth in fish and one of the major bacterial pathogens causing losses in salmonid aquaculture. Previously typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. In this work, we describe a multilocus sequence typing (MLST) scheme for Y.ruckeri based on the internal fragment sequence of six housekeeping genes. This MLST scheme was applied to 103 Y.ruckeri strains from diverse geographic areas and hosts as well as environmental sources. Sequences obtained from this work were deposited and are available in a public database (http://publmst.org/yruckeri/). Thirty different sequence types (ST) were identified, 21 of which were represented by a single isolate, evidencing high genetic diversity. ST2 comprised more than one-third of the isolates and was most frequently observed among isolates from trout. Two major clonal complexes (CC) were identified by eBURST analysis showing a common evolutionary origin for 94 isolates forming 21 STs into CC1 and for 6 isolates of 6 STs in the CC2. It was also possible to associate some unique ST with isolates from recent outbreaks in vaccinated salmonid fish.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Multilocus Sequence Typing Identifies Epidemic Clones of Flavobacterium psychrophilum in Nordic Countries

Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD), which affects a variety of freshwater-reared salmonid species. A large-scale study was performed to investigate the genetic diversity of F. psychrophilum in the four Nordic countries: Denmark, Finland, Norway, and Sweden. Multilocus sequence typing of 560 geographically and temporally disparate F. psychrophilum isolates collected from various sources between 1983 and 2012 revealed 81 different sequence types (STs) belonging to 12 clonal complexes (CCs) and 30 singleton STs. The largest CC, CC-ST10, which represented almost exclusively isolates from rainbow trout and included the most predominant genotype, ST2, comprised 65% of all isolates examined. In Norway, with a shorter history (<10 years) of BCWD in rainbow trout, ST2 was the only isolated CC-ST10 genotype, suggesting a recent introduction of an epidemic clone. The study identified five additional CCs shared between countries and five country-specific CCs, some with apparent host specificity. Almost 80% of the singleton STs were isolated from non-rainbow trout species or the environment. The present study reveals a simultaneous presence of genetically distinct CCs in the Nordic countries and points out specific F. psychrophilum STs posing a threat to the salmonid production. The study provides a significant contribution toward mapping the genetic diversity of F. psychrophilum globally and support for the existence of an epidemic population structure where recombination is a significant driver in F. psychrophilum evolution. Evidence indicating dissemination of a putatively virulent clonal complex (CC-ST10) with commercial movement of fish or fish products is strengthened.     

New Staphylococcus aureus genetic cluster associated with infectious osteomyelitis

Diverse genotyping methods, including multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), were used for genotyping Staphylococcus aureus in samples recovered from a clinical case of osteomyelitis. An unexpected genetic diversity of strains was determined, including four new sequence types (ST 1521, 1522, 1628 and 1629) belonging to the same genetic lineage, implying the appearance of a new subgroup derived from clonal complex CC121 isolated from that hospital. A close phylogenetic relationship among the STs was demonstrated, reflecting a possible diversifying evolution process. To our knowledge, there have no been previous reports of staphylococcal genetic variability observed within a single individual with such a high degree of variation. These findings emphasize the need for infection control measures to monitor the high genetic variability continuously occurring in this often dangerous infectious agent.     

Molecular Epidemiology of Clostridium difficile Infection in a Large Teaching Hospital in Thailand

Clostridium difficile infection (CDI) is a leading cause of healthcare-associated morbidity and mortality worldwide. In Thailand, CDI exhibits low recurrence and mortality and its molecular epidemiology is unknown. CDI surveillance was conducted in a tertiary facility (Siriraj Hospital, Bangkok). A total of 53 toxigenic C. difficile strains from Thai patients were analyzed by multi-locus sequence typing (MLST), PCR ribotyping, and pulse-field gel electrophoresis (PFGE). The mean age of the cohort was 64 years and 62.3% were female; 37.7% of patients were exposed to > two antibiotics prior to a diagnosis of CDI, with beta-lactams the most commonly used drug (56.3%). Metronidazole was used most commonly (77.5%; success rate 83.9%), and non-responders were treated with vancomycin (success rate 100%). None of the isolates carried binary toxin genes. Most isolates (98.2–100%) were susceptible to metronidazole, vancomycin, tigecycline and daptomycin. There were 11 sequence types (STs), 13 ribotypes (RTs) and four PFGE types. Six previously identified STs (ST12, ST13, ST14, ST33, ST41 and ST45) and five novel STs unique to Thailand (ST66, ST67, ST68, ST69 and ST70) were identified. PCR RTs UK 017 (ST45) (45.3%) and UK 014/020 (ST33) (24.5%) were the most common. High concordance was observed between the MLST and ribotyping results (p<0.001). C. difficile isolates from Thai patients were highly susceptible to standard antimicrobial agents. In conclusion, the five STs indicate the high genetic diversity and unique polymorphisms in Thailand. Moreover, the emergence of antimicrobial resistance to vancomycin warranted continuous surveillance to prevent further spread of the toxigenic C. difficile isolates.     

Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers,Hot Springs,and Potable Water Systems in China

Legionella pneumophila serogroup 1 causes Legionnaires'' disease. Water systems contaminated with Legionella are the implicated sources of Legionnaires'' disease. This study analyzed L. pneumophila serogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n = 96), hot springs (n = 42), and potable water systems (n = 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n = 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons among L. pneumophila strains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmental L. pneumophila serogroup 1 isolates, similar to its prevalence in Japan and South Korea.     

Salmonella enterica Diversity in Central Californian Coastal Waterways

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.     

Genetic Variability of Vancomycin-Resistant Enterococcus faecium and Enterococcus faecalis Isolates from Humans,Chickens, and Pigs in Malaysia

Vancomycin-resistant enterococci (VRE) have been reported to be present in humans, chickens, and pigs in Malaysia. In the present study, representative samples of VRE isolated from these populations were examined for similarities and differences by using the multilocus sequence typing (MLST) method. Housekeeping genes of Enterococcus faecium (n = 14) and Enterococcus faecalis (n = 11) isolates were sequenced and analyzed using the MLST databases eBURST and goeBURST. We found five sequence types (STs) of E. faecium and six STs of E. faecalis existing in Malaysia. Enterococcus faecium isolates belonging to ST203, ST17, ST55, ST79, and ST29 were identified, and E. faecium ST203 was the most common among humans. The MLST profiles of E. faecium from humans in this study were similar to the globally reported nosocomial-related strain lineage belonging to clonal complex 17 (CC17). Isolates from chickens and pigs have few similarities to those from humans, except for one isolate from a chicken, which was identified as ST203. E. faecalis isolates were more diverse and were identified as ST4, ST6, ST87, ST108, ST274, and ST244, which were grouped as specific to the three hosts. E. faecalis, belonging to the high-risk CC2 and CC87, were detected among isolates from humans. In conclusion, even though one isolate from a chicken was found clonal to that of humans, the MLST analysis of E. faecium and E. faecalis supports the findings of others who suggest VRE to be predominantly host specific and that clinically important strains are found mainly among humans. The infrequent detection of a human VRE clone in a chicken may in fact suggest a reverse transmission of VRE from humans to animals.     

Characterisation of Australian MRSA strains ST75- and ST883-MRSA-IV and analysis of their accessory gene regulator locus

Background

Community-acquired methicillin-resistant Staphylococcus aureus have become a major problem in Australia. These strains have now been isolated throughout Australia including remote Indigenous communities that have had minimal exposure to healthcare facilities. Some of these strains, belonging to sequence types ST75 and ST883, have previously been reported to harbour highly divergent alleles of the housekeeping genes used in multilocus sequence typing.

Methodology/Principal Findings

ST75-MRSA-IV and ST883-MRSA-IV isolates were characterised in detail. Morphological features as well as 16S sequences were identical to other S. aureus strains. Although a partial rnpB gene sequence was not identical to previously known S. aureus sequences, it was found to be more closely related to S. aureus than to other staphylococci. Isolates also were screened using diagnostic DNA microarrays. These isolates yielded hybridisation results atypical for S. aureus. Primer directed amplification assays failed to detect species markers (femA, katA, sbi, spa). However, arbitrarily primed amplification indicated the presence of unknown alleles of these genes. Isolates could not be assigned to capsule types 1, 5 or 8. The allelic group of the accessory gene regulator (agr) locus was not determinable. Sequencing of a region of agrB, agrC and agrD (approximately 2,100 bp) revealed a divergent sequence. However, this sequence is more related to S. aureus agr alleles I and IV than to agr sequences from other Staphylococcus species. The predicted auto-inducing peptide (AIP) sequence of ST75 was identical to that of agr group I, while the predicted AIP sequence of ST883 was identical to agr group IV.

Conclusions/Significance

The genetic properties of ST75/ST883-MRSA may be due to a series of evolutionary events in ancient insulated S. aureus strains including a convergent evolution leading to agr group I- or IV-like AIP sequences and a recent acquisition of SCCmec IV elements.     

Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

Population Structure of Clinical Vibrio parahaemolyticus from 17 Coastal Countries,Determined through Multilocus Sequence Analysis

Vibrio parahaemolyticus is a leading cause of food-borne gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of clinical strains from worldwide collections remains largely undescribed, and the recorded outbreaks of V. parahaemolyticus gastroenteritis highlight the need for the subtyping of this species. We present a broad phylogenetic analysis of 490 clinical V. parahaemolyticus isolates from 17 coastal countries through multilocus sequence analysis (MLST). The 490 tested isolates fell into 161 sequence types (STs). The eBURST algorithm revealed that the 161 clinically relevant STs belonged to 8 clonal complexes, 11 doublets, and 94 singletons, showing a high level of genetic diversity. CC3 was found to be a global epidemic clone of V. parahaemolyticus, and ST-3 was the only ST with an international distribution. recA was observed to be evolving more rapidly, exhibiting the highest degree of nucleotide diversity (0.028) and the largest number of polymorphic nucleotide sites (177). We also found that the high variability of recA was an important cause of differences between the results of the eBURST and ME tree analyses, suggesting that recA has a much greater influence on the apparent evolutionary classification of V. parahaemolyticus based on the current MLST scheme. In conclusion, it is evident that a high degree of genetic diversity within the V. parahaemolyticus population and multiple sequence types are contributing to the burden of disease around the world. MLST, with a fully extractable database, is a powerful system for analysis of the clonal relationships of strains at a global scale. With the addition of more strains, the pubMLST database will provide more detailed and accurate information, which will be conducive to our future research on the population structure of V. parahaemolyticus.     

Isolation by distance and vicariance drive genetic structure of a coral reef fish in the Pacific Ocean

We studied the genetic diversity of a coral reef fish species to investigate the origin of the differentiation. A total of 727 Acanthurus triostegus collected from 15 locations throughout the Pacific were analyzed for 20 polymorphic loci. The genetic structure showed limited internal disequilibrium within each population; 3.7% of the loci showed significant Hardy-Weinberg disequilibrium, mostly associated with Adh*, and we subsequently removed this locus from further analysis of geographic pattern. The genetic structure of A. triostegus throughout the tropical Pacific Ocean revealed a strong geographic pattern. Overall, there was significant population differentiation (multilocus F(ST) = 0.199), which was geographically structured according to bootstraps of neighbor-joining analysis on Nei's unbiased genetic distances and AMOVA analysis. The genetic structure revealed five geographic groups in the Pacific Ocean: western Pacific (Guam, Philippines, Palau, and Great Barrier Reef); central Pacific (Solomons, New Caledonia, and Fiji); and three groups made up of the eastern populations, namely Hawaiian Archipelago (north), Marquesas (equatorial), and southern French Polynesia (south) that incorporates Clipperton Island located in the northeastern Pacific. In addition, heterozygosity values were found to be geographically structured with higher values grouped within Polynesian and Clipperton populations, which exhibited lower population size. Finally, the genetic differentiation (F(ST)) was significantly correlated with geographic distance when populations from the Hawaiian and Marquesas archipelagos were separated from all the other locations. These results show that patterns of differentiation vary within the same species according to the spatial scale, with one group probably issued from vicariance, whereas the other followed a pattern of isolation by distance. The geographic pattern for A. triostegus emphasizes the diversity of the evolutionary processes that lead to the present genetic structure with some being more influential in certain areas or according to a particular spatial scale.     

Campylobacter spp. Recovered from the Upper Oconee River Watershed, Georgia in a 4-Year Study

Waterways should be considered in the migration routes of Campylobacter, and the genus has been isolated from several water sources. Inferences on migration routes can be made from tracking genetic types in populations found in specific habitats and testing how they are linked to other types. Water samples were taken over a 4-year period from waterways in the Upper Oconee River Watershed, Georgia, to recover isolates of thermophilic Campylobacter. The isolates were typed by multilocus sequence typing (MLST) and analyzed to determine the overall diversity of Campylobacter in that environment. Forty-seven independent isolates were recovered from 560 samples (8.4 %). Two (~4 %) isolates were Campylobacter coli, three (~6 %) isolates were putatively identified as Campylobacter lari, and the remaining 42 (~90 %) were Campylobacter jejuni. The C. jejuni and C. coli isolates were typed by the Oxford MLST scheme. Thirty sequence types (STs) were identified including 13 STs that were not found before in the MLST database, including 24 novel alleles. Of the 17 previously described STs, 10 have been isolated from humans, 6 from environmental water, and 6 from wild birds (five types from multiple sources). Seven sites had multiple positive samples, and on two occasions, the same ST was isolated at the same site. The most common type was STST61 with four isolates, and the most common clonal complex was CC179 with nine isolates. CC179 has been commonly associated with environmental water. Although some Campylobacter STs that were found in the Oconee River engage in widespread migration, most are tightly associated with or unique to environmental water sources.     

Population structure of Staphylococcus aureus from remote African Babongo Pygmies

Background

Pandemic community-acquired methicillin-resistant Staphylococcus aureus isolates (CA-MRSA) predominantly encode the Panton-Valentine leukocidin (PVL), which can be associated with severe infections. Reports from non-indigenous Sub-Saharan African populations revealed a high prevalence of PVL-positive isolates. The objective of our study was to investigate the S. aureus carriage among a remote indigenous African population and to determine the molecular characteristics of the isolates, particularly those that were PVL-positive.

Methodology/Principal Findings

Nasal S. aureus carriage and risk factors of colonization were systematically assessed in remote Gabonese Babongo Pygmies. Susceptibility to antibiotics, possession of toxin-encoding genes (i.e., PVL, enterotoxins, and exfoliative toxins), S. aureus protein A (spa) types and multi-locus sequence types (MLST) were determined for each isolate. The carriage rate was 33%. No MRSA was detected, 61.8% of the isolates were susceptible to penicillin. Genes encoding PVL (55.9%), enterotoxin B (20.6%), exfoliative toxin D (11.7%) and the epidermal cell differentiation inhibitor B (11.7%) were highly prevalent. Thirteen spa types were detected and were associated with 10 STs predominated by ST15, ST30, ST72, ST80, and ST88.

Conclusions

The high prevalence of PVL-positive isolates among Babongo Pygmies demands our attention as PVL can be associated with necrotinzing infection and may increase the risk of severe infections in remote Pygmy populations. Many S. aureus isolates from Babongo Pygmies and pandemic CA-MRSA-clones have a common genetic background. Surveillance is needed to control the development of resistance to antibiotic drugs and to assess the impact of the high prevalence of PVL in indigenous populations.