Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever

Bartonella quintana is a bacterium that causes a broad spectrum of diseases in humans including trench fever. Humans were previously considered to be the primary, if not the only, reservoir hosts for B. quintana. To identify the animal reservoir and extend our understanding of the ecological and evolutionary history of B. quintana, we examined blood samples from macaques and performed multilocus sequence typing (MLST) analysis. We demonstrated the prevalence of B. quintana infection was common in macaques from main primate centres in mainland China. Overall, 18.0% (59/328) of rhesus macaques and 12.7% (39/308) of cynomolgus macaques were found to be infected with B. quintana by blood culture and/or polymerase chain reaction. The infection was more frequently identified in juvenile and young monkeys compared with adult animals. In contrast with the relatively low level of sequence divergence of B. quintana reported in humans, our investigation revealed much higher genetic diversity in nonhuman primates. We identified 44 new nucleotide variable sites and 14 novel sequence types (STs) among the B. quintana isolates by MLST analysis. Some STs were found only in cynomolgus macaques, while some others were detected only in rhesus macaques, suggesting evidence of host‐cospeciation, which were further confirmed by phylogenetic analysis and Splits decomposition analysis. Our findings suggest that trench fever may primarily be a zoonotic disease with macaques as the natural hosts.     

Genetic diversity of Staphylococcus equorum isolates from Saeu-jeotgal evaluated by multilocus sequence typing

Staphylococcus equorum, the predominant bacterial species detected in Saeu-jeotgal, a Korean high-salt fermented seafood, is a candidate starter bacterium for Saeu-jeotgal fermentation. A multilocus sequence typing (MLST) scheme was developed to evaluate the genetic diversity and background of S. equorum strains isolated from Saeu-jeotgal. A total of 135 strains, including 117 isolates from Saeu-jeotgal, and others from Myeolchi-jeotgal, sausage, cheese and horse skin, were subjected to MLST, and the internal fragments of seven housekeeping genes, aroE, dnaJ, glpF, gmk, hsp60, mutS, and pta, were compared. This MLST scheme produced 45 sequence types (STs) and the eBURST algorithm clustered the STs into nine clonal groups and seven singletons. Clonal group 1, the major group, consisted of 30 isolates from cheese, Saeu-jeotgal and sausages, which were classified into 12 STs. The predominant ST, ST26, comprised 25 isolates and presented as a singleton. Most of the isolates from Myeolchi-jeotgal and sausages clustered on two different branches of a phylogenetic tree generated with a cluster analysis using the maximum likelihood algorithm. This MLST scheme established the genetic backgrounds of S. equorum strains isolated from different types of food. Among the housekeeping genes used for MLST, gmk had the fewest allele types and fairly low sequence identities (74.0–90.0 %) within the Staphylococcus species. Therefore, sequence analyses of the gmk gene and 16S rRNA gene can be used for the accurate and rapid identification of S. equorum.     

Multilocus Sequence Typing (MLST) for Characterization of Enterobacter cloacae

Enterobacter cloacae is an important emerging pathogen, which sometime causes respiratory infection, surgical site infection, urinary infection, sepsis, and outbreaks at neonatal units. We have developed a multilocus sequence typing (MLST) scheme utilizing seven housekeeping genes and evaluated the performance in 101 clinical isolates. The MLST scheme yielded 83 sequence types (ST) including 78 novel STs found in the clinical isolates. These findings supported the robustness of the MLST scheme developed in this study.     

Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group

In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.     

Classification of Bartonella Strains Associated with Straw-Colored Fruit Bats (Eidolon helvum) across Africa Using a Multi-locus Sequence Typing Platform

Bartonellae are facultative intracellular bacteria and are highly adapted to their mammalian host cell niches. Straw-colored fruit bats (Eidolon helvum) are commonly infected with several bartonella strains. To elucidate the genetic diversity of these bartonella strains, we analyzed 79 bartonella isolates from straw-colored fruit bats in seven countries across Africa (Cameroon, Annobon island of Equatorial Guinea, Ghana, Kenya, Nigeria, Tanzania, and Uganda) using a multi-locus sequencing typing (MLST) approach based on nucleotide sequences of eight loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, ITS, and 16S rRNA). The analysis of each locus but ribC demonstrated clustering of the isolates into six genogroups (E1 – E5 and Ew), while ribC was absent in the isolates belonging to the genogroup Ew. In general, grouping of all isolates by each locus was mutually supportive; however, nuoG, gltA, and rpoB showed some incongruity with other loci in several strains, suggesting a possibility of recombination events, which were confirmed by network analyses and recombination/mutation rate ratio (r/m) estimations. The MLST scheme revealed 45 unique sequence types (ST1 – 45) among the analyzed bartonella isolates. Phylogenetic analysis of concatenated sequences supported the discrimination of six phylogenetic lineages (E1 – E5 and Ew) corresponding to separate and unique Bartonella species. One of the defined lineages, Ew, consisted of only two STs (ST1 and ST2), and comprised more than one-quarter of the analyzed isolates, while other lineages contained higher numbers of STs with a smaller number of isolates belonging to each lineage. The low number of allelic polymorphisms of isolates belonging to Ew suggests a more recent origin for this species. Our findings suggest that at least six Bartonella species are associated with straw-colored fruit bats, and that distinct STs can be found across the distribution of this bat species, including in populations of bats which are genetically distinct.     

Comparative Analysis of the Orphan CRISPR2 Locus in 242 Enterococcus faecalis Strains

Clustered, Regularly Interspaced Short Palindromic Repeats and their associated Cas proteins (CRISPR-Cas) provide prokaryotes with a mechanism for defense against mobile genetic elements (MGEs). A CRISPR locus is a molecular memory of MGE encounters. It contains an array of short sequences, called spacers, that generally have sequence identity to MGEs. Three different CRISPR loci have been identified among strains of the opportunistic pathogen Enterococcus faecalis. CRISPR1 and CRISPR3 are associated with the cas genes necessary for blocking MGEs, but these loci are present in only a subset of E. faecalis strains. The orphan CRISPR2 lacks cas genes and is ubiquitous in E. faecalis, although its spacer content varies from strain to strain. Because CRISPR2 is a variable locus occurring in all E. faecalis, comparative analysis of CRISPR2 sequences may provide information about the clonality of E. faecalis strains. We examined CRISPR2 sequences from 228 E. faecalis genomes in relationship to subspecies phylogenetic lineages (sequence types; STs) determined by multilocus sequence typing (MLST), and to a genome phylogeny generated for a representative 71 genomes. We found that specific CRISPR2 sequences are associated with specific STs and with specific branches on the genome tree. To explore possible applications of CRISPR2 analysis, we evaluated 14 E. faecalis bloodstream isolates using CRISPR2 analysis and MLST. CRISPR2 analysis identified two groups of clonal strains among the 14 isolates, an assessment that was confirmed by MLST. CRISPR2 analysis was also used to accurately predict the ST of a subset of isolates. We conclude that CRISPR2 analysis, while not a replacement for MLST, is an inexpensive method to assess clonality among E. faecalis isolates, and can be used in conjunction with MLST to identify recombination events occurring between STs.     

Multilocus Sequence Typing Supports the Hypothesis that Cow- and Human-Associated Salmonella Isolates Represent Distinct and Overlapping Populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.     

Epidemiological relationships of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> strains isolated from humans and chickens in South Korea

Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007–2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009–2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.     

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.     

Comparative study of different molecular methods for typing of Acinetobacter baumannii clinical isolates from University Hospitals

Acinetobacter baumannii, a rod-shape Gram-negative bacterium, is an opportunistic pathogen causing diseases in humans. This bacterium has been recognized as one of the leading causes of nosocomial infection which occurs in hospital or hospital-like setting. The antibiotic resistance of A. baumannii could result from the heavy use of antibiotics and has been recognized as a threat to human health. However, prevention against the disease caused by A. baumannii is difficult due to variable host susceptibility against their infections. We isolated 53 bacterial strains from four different university hospitals in South Korea and identified 34 out of the 53 isolates as A. baumannii, based on the nucleotide sequence of 16S rRNA and gpi genes. For the subtyping of the clinical isolates, we used enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and multilocus sequencing typing (MLST) and compared the results. The result of ERIC-PCR showed that there are 14 distinct DNA fingerprint patterns in the 34 A. baumannii clinical isolates. For MLST analysis of the isolates, we amplified and sequenced seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD) from each isolate. Each unique allelic profile at the concatenated nucleotide sequences of the seven genes was assigned as a sequence type (ST) and six different STs (ST92, ST105, ST138, ST169, ST262, and ST357) were detected in the 34 A. baumannii clinical isolates. Among the six STs, ST138 was the most ubiquitous in the A. baumannii clinical isolates. To examine the regional distribution of the isolates, STs were clustered into clonal complexes based on their similarity to a previously registered central genotype and the clustering was verified by network phylogenetic analysis.     

Clonal Analysis of Meningococci during a 26 Year Period Prior to the Introduction of Meningococcal Serogroup C Vaccines

Meningococcal disease remains a public health burden in the UK and elsewhere. Invasive Neisseria meningitidis, isolated in Scotland between 1972 and 1998, were characterised retrospectively to examine the serogroup and clonal structure of the circulating population. 2607 isolates causing invasive disease were available for serogroup and MLST analysis whilst 2517 were available for multilocus sequence typing (MLST) analysis only. Serogroup distribution changed from year to year but serogroups B and C were dominant throughout. Serogroup B was dominant throughout the 1970s and early 1980s until serogroup C became dominant during the mid-1980s. The increase in serogroup C was not associated with one particular sequence type (ST) but was associated with a number of STs, including ST-8, ST-11, ST-206 and ST-334. This is in contrast to the increase in serogroup C disease seen in the 1990s that was due to expansion of the ST-11 clonal complex. While there was considerable diversity among the isolates (309 different STs among the 2607 isolates), a large proportion of isolates (59.9%) were associated with only 10 STs. These data highlight meningococcal diversity over time and the need for ongoing surveillance during the introduction of new meningococcal vaccines.     

Bartonella spp. Bacteremia in Blood Donors from Campinas,Brazil

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.     

Application of Streptococcus uberis Multilocus Sequence Typing: Analysis of the Population Structure Detected among Environmental and Bovine Isolates from New Zealand and the United Kingdom

We recently developed a multilocus sequence typing (MLST) scheme to differentiate S. uberis isolates and facilitate an understanding of the population biology of this pathogen. The scheme was initially used to study a collection of 160 bovine milk isolates from the United Kingdom and showed that the majority of isolates were from one clonal complex (designated the ST-5 complex). Here we describe the MLST analysis of a collection of New Zealand isolates. These were obtained from diverse sources, including bovine milk, other bovine anatomical sites, and environmental sources. The complete allelic profiles of 253 isolates were determined. The collection was highly diverse and included 131 different sequence types (STs). The New Zealand and United Kingdom populations were distinct, since none of the 131 STs were represented within the previously studied collection of 160 United Kingdom S. uberis isolates. However, seven of the STs were members of the ST-5 clonal complex, the major complex within the United Kingdom collection. Two new clonal complexes were identified: ST-143 and ST-86. All three major complexes were isolated from milk, other bovine sites, and the environment. Carriage of the hasA gene, which is necessary for capsule formation, correlated with clonal complex and isolation from clinical cases of mastitis.     

Using multilocus sequence data to define the pneumococcus

We investigated the genetic relationships between serotypeable pneumococci and nonserotypeable presumptive pneumococci using multilocus sequence typing (MLST) and partial sequencing of the pneumolysin gene (ply). Among 121 nonserotypeable presumptive pneumococci from Finland, we identified isolates of three classes: those with sequence types (STs) identical to those of serotypeable pneumococci, suggesting authentic pneumococci in which capsular expression had been downregulated or lost; isolates that clustered among serotypeable pneumococci on a tree based on the concatenated sequences of the MLST loci but which had STs that differed from those of serotypeable pneumococci in the MLST database; and a more diverse collection of isolates that did not cluster with serotypeable pneumococci. The latter isolates typically had sequences at all seven MLST loci that were 5 to 10% divergent from those of authentic pneumococci and also had distinct and divergent ply alleles. These isolates are proposed to be distinct from pneumococci but cannot be resolved from them by optochin susceptibility, bile solubility, or the presence of the ply gene. Complete resolution of pneumococci from the related but distinct population is problematic, as recombination between them was evident, and a few isolates of each population possessed alleles at one or occasionally more MLST loci from the other population. However, a tree based on the concatenated sequences of the MLST loci in most cases unambiguously distinguished whether a nonserotypeable isolate was or was not a pneumococcus, and the sequence of the ply gene fragment was found to be useful to resolve difficult cases.     

Multilocus sequence typing for phylogenetic view and <Emphasis Type="Italic">vip</Emphasis> gene diversity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains of the Assam soil of North East India

An agriculturally important insecticidal bacterium, Bacillus thuringiensis have been isolated from the soil samples of various part of Assam including the Kaziranga National Park. Previously, the isolates were characterized based on morphology, 16S rDNA sequencing, and the presence of the various classes’ crystal protein gene(s). In the present study, the phylogenetic analysis of a few selected isolates was performed by an unambiguous and quick method called the multiple locus sequence typing (MLST). A known B. thuringiensis strain kurstaki 4D4 have been used as a reference strain for MLST. A total of four the MLST locus of housekeeping genes, recF, sucC, gdpD and yhfL were selected. A total of 14 unique sequence types (STs) was identified. A total number of alleles identified for the locus gdpD and sucC was 12, followed by locus yhfL was 11, however, only 6 alleles were detected for the locus recF. The phylogenetic analysis using MEGA 7.0.26 showed three major lineages. Approximately, 87% of the isolates belonged to the STs corresponding to B. thuringiensis, whereas two isolates, BA07 and BA39, were clustered to B. cereus. The isolates were also screened for the diversity of vegetative insecticidal protein (vip) genes. In all, 8 isolates showed the presence of vip1, followed by 7 isolates having vip2 and 6 isolates for vip3 genes. The expression of Vip3A proteins was analyzed by western blot analyses and expression of the Vip3A protein was observed in the isolate BA20. Thus, the phylogenetic relationship and diversity of Bt isolates from Assam soil was established based on MLST, in addition, found isolates having vip genes, which could be used for crop improvement.     

Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates

BackgroundGiardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains.Methodology/Principal findingsTwo G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411).Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction.Conclusions/SignificanceTyping of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.     

Clonal spread of carbapenem resistant Acinetobacter baumannii ST92 in a Chinese Hospital during a 6-year period

The carbapenem resistance rate of Acinetobacter baumannii in our hospital has increased steadily since 2004. The molecular epidemiology of carbapenem resistant A. baumannii (CRAB) clinical isolates was characterized by multilocus sequence typing (MLST) and rep-PCR in parallel, with pan-drug susceptible A. baumannii (PSAB) used as control. MLST was performed to determine the sequence types (STs), and eBURST algorithm was used to analyze their relatedness. Carbapenem resistance related genes (oxa-23, oxa-24, oxa-51, oxa-58, imp, vim, and adeB) were screened using Polymerase Chain Reaction (PCR) method. 23 STs were identified in the 65 included isolates, with ST92 being the predominant clone. PSAB clustered into more singletons than CRAB. The positivity of oxa-23 and adeB correlated with high level carbapenem resistance (MIC_IPM>32 mg/L, MIC_MEM >32 mg/L) of CRAB ST92 isolates in 2009, which was different from the resistance pattern (MIC_IPM≤4 mg/L, 8 mg/L ≤MIC_MEM≤16 mg/L) of CRAB ST92 isolates in 2004. These observations suggest that clonal spread of CRAB ST92 isolates longitudinally is the possible reason for carbapenem resistance rate increase and correlate with high level carbapenem resistance in our hospital.     

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.     

Antimicrobial Susceptibility Profiles and Molecular Typing of Campylobacter jejuni and Campylobacter coli Isolates from Ducks in South Korea

Campylobacter is a food-borne zoonotic pathogen that causes human gastroenteritis worldwide. Campylobacter bacteria are commensal in the intestines of many food production animals, including ducks and chickens. The objective of the study was to determine the prevalence of Campylobacter species in domestic ducks, and the agar dilution method was used to determine resistance of the isolates to eight antibiotics. In addition, multilocus sequence typing (MLST) was performed to determine the sequence types (STs) of selected Campylobacter isolates. Between May and September 2012, 58 duck farms were analyzed, and 56 (96.6%) were positive for Campylobacter. Among the isolates, 82.1% were Campylobacter jejuni, 16.1% were C. coli, and one was unidentified by PCR. Of the 46 C. jejuni isolates, 87.0%, 10.9%, and 21.7% were resistant to ciprofloxacin, erythromycin, and azithromycin, respectively. Among the C. coli isolates, all 9 strains were resistant to ampicillin, and 77.8% and 33.3% were resistant to ciprofloxacin and azithromycin, respectively. The majority of the Campylobacter isolates were classified as multidrug resistant. Twenty-eight STs were identified, including 20 STs for C. jejuni and 8 STs for C. coli. The most common clonal complexes in C. jejuni were the ST-21 complex and the ST-45 complex, while the ST-828 complex predominated in C. coli. The majority of isolates were of STs noted in ducks and humans from earlier studies, along with seven STs previously associated only with human disease. These STs overlapped between duck and human isolates, indicating that Campylobacter isolates from ducks should be considered potential sources of human infection.