Multilocus Sequence Typing Database Research Articles

Genetic diversity and molecular epidemiology of Candida albicans from vulvovaginal candidiasis patients.

Candida albicans (C. albicans) is a common cause of vulvovaginal candidiasis (VVC). In this paper, the genetic diversity and molecular epidemiology of 173C. albicans strains were investigated by multilocus sequence typing (MLST). A total of 52 diploid sequence types (DSTs) were recognized, and 27 (51.9%) of which have not been reported in the MLST database. Genotyping was performed on the multiple isolates collected from patients with recurrent VVC (RVVC, referring to VVC which attacks more than 4 times in one year) in different acute infectious phases. The results showed that 59.1% (26/44) of the patients suffered a relapse, with DST 79 (65.4%) as the dominant genotype. The etiology of the remaining 40.9% (18/44) of patients was reinfection, and the main genotypes included DST 79 (33.3%), DST 124 (8.6%) and DST 1895 (8.6%). DST 79 (45%) and DST 1395 (7.5%) were the main isolates of VVC patients, while DST 79 (24.1%), DST 727 (6.9%), DST 732 (6.9%) and DST 1867 (6.9%) were the main types of healthy volunteers. The results of the genotypes between RVVC patients and other groups were statistically different. Furthermore, cluster analysis was carried out on 1468 isolates, among which 1337 were downloaded from the MLST database, 130 were divided into 8 Clades in the present study and the remaining one was taken as a singleton. 92.3% isolates from relapse patients, 58.3% isolates from re-infected patients, 77.5% isolates from VVC patients and 51.7% isolates from volunteers were distributed in Clade 1. The analysis of the genotypes of multiple isolates from RVVC patients further demonstrated that point mutation and loss of heterozygosity contributed to the microevolution of C. albicans.

Campylobacter vulpis sp. nov. isolated from wild red foxes

During a sampling of wild red foxes (Vulpes vulpes) for the detection of Epsilonproteobacteria, 14 strains were isolated from the caecal contents of 14 epidemiologically-unrelated animals. A genus-specific PCR indicated that the isolates belonged to the genus Campylobacter. Based on the results of a species-specific PCR, the isolates were initially identified as C. upsaliensis. However, multi-locus sequence typing (MLST) revealed that the isolates were significantly different from the C. upsaliensis present in the MLST database. A polyphasic study, including conventional biochemical and tolerance characteristics, morphology by transmission electron microscopy (TEM), MALDI-TOF analysis, and genetic comparisons based on partial 16S rDNA and atpA gene sequences, was undertaken. Finally, the complete genome sequence of the type strain 251/13T and the draft genome sequences of the other isolates were determined. Average nucleotide identity, average amino acid identity and in silico DNA-DNA hybridization analyses confirmed that the isolates represent a novel taxon for which the name Campylobacter vulpis sp. nov. is proposed, with isolate 251/13T (=CCUG 70587T = LMG 30110T) as the type strain. In order to allow a rapid discrimination of C. vulpis from the closely-related C. upsaliensis, a specific PCR test was designed, based on atpA gene sequences.

Overview of the distribution of Burkholderia pseudomallei sequence types and the Emergence of sequence type 1342 in Malaysia.

Burkholderia pseudomallei, a Gram-negative bacterial pathogen that causes melioidosis, is of public health importance in endemic areas including Malaysia. An investigation of the molecular epidemiology links of B.pseudomallei would contribute to better understanding of the clonal relationships, transmission dynamics and evolutionary change. Multilocus sequence typing (MLST) of 45 clinical B.pseudomallei isolates collected from sporadic melioidosis cases in Malaysia was performed. In addition, a total of 449 B.pseudomallei Malaysian strains submitted to the MLST database from 1964 until 2019 were included in the temporal analysis to determine the endemic sequence types (STs), emergence and re-emergence of ST(s). In addition, strain-specific distribution was evaluated using BURST tool. Genotyping of 45 clinical strains was resolved into 12 STs, and the majority were affiliated with ST46 (n=11) and ST1342 (n=7). Concomitantly, ST46 was the most prevalent ST in Malaysia, which was first reported in 1964. All the Malaysian B.pseudomallei strains were resolved into 76 different STs with 36 of them uniquely present only in Malaysia. ST1342 was most closely related to ST1034, in which both STs were unique to Malaysia and first isolated from soil samples in Pahang, a state in Malaysia. The present study revealed a high diversity of B.pseudomallei in Malaysia. Localized evolution giving rise to the emergence of new STs was observed, suggesting that host and environmental factors play a crucial role in the evolutionary changes in B.pseudomallei.

Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India.

Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of 208 clinical isolates of Cryptococcus from different parts of India, multilocus sequence typing (MLST) using ISHAM MLST consensus scheme for C. neoformans/C. gattii species complex was used. MLST analysis yielded a total of 10 Sequence Types (STs)-7 STs for C. neoformans and 3 for C. gattii species complex. The majority of isolates identified as C. neoformans belonged to molecular type VNI with predominant STs 31 and 93. Only 3 isolates of C. gattii species complex were obtained, belonging to ST58 and ST215 of VGI and ST69 of VGIV. Phylogenetic analysis revealed less diversity among the clinical Indian isolates compared to the global MLST database. No association between prevalent STs and HIV status, geographical origin or minimum inhibitory concentration (MIC) could be established.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA

BackgroundSeveral Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA.ResultsIn this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv.ConclusionsThe newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

Multilocus Sequence Typing Reveals Clonality of Fluconazole-Nonsusceptible Candida tropicalis: A Study From Wuhan to the Global.

Candida tropicalis is a globally distributed human pathogenic yeast, and its increasing resistance to azoles makes clinical treatment difficult. In this study, we investigated the clinical features, azole resistance and genetic relatedness of 87 C. tropicalis isolates from central China and combined with the global database to explore the relationship between genetic information and fluconazole susceptibility. Of the 55 diploid sequence types (DSTs) identified by multilocus sequence typing (MLST), 27 DSTs were new to the C. tropicalis MLST database. Fluconazole-nonsusceptible (FNS) isolates were genetically closely related. goeBURST analysis showed that DST225, DST376, DST506, and DST546 formed a distinct and unique FNS clonal complex (CC) in Wuhan. The local FNS CC belongs to the large FNS CC (CC2) in China, in which the putative founder DST225 has been reported from the environment. The three most prevalent types (DST506, DST525, and DST546) in Wuhan had high minimum inhibitory concentrations (MICs) for antifungal azoles, and the six possible nosocomial transmissions we captured were all FNS strains, most of which were from CC2. Unique FNS CCs have been found in Singapore (CC8) and India (CC17) and are close to China’s CC2 in the minimum spanning tree. There were no FNS CCs outside Asia. This study is the first to reveal a significant correlation between genetic information and fluconazole susceptibility worldwide and to trace geographical locations, which is of great value for molecular epidemiological surveillance and azole-resistance study of C. tropicalis globally.

Consensus Multilocus Sequence Typing Scheme for Pneumocystis jirovecii.

Pneumocystis jirovecii is an opportunistic human pathogenic fungus causing severe pneumonia mainly in immunocompromised hosts. Multilocus sequence typing (MLST) remains the gold standard for genotyping of this unculturable fungus. However, the lack of a consensus scheme impedes a global comparison, large scale population studies and the development of a global MLST database. To overcome this problem this study compared all genetic regions (19 loci) currently used in 31 different published Pneumocystis MLST schemes. The most diverse/commonly used eight loci, β-TUB, CYB, DHPS, ITS1, ITS1/2, mt26S and SOD, were further assess for their ability to be successfully amplified and sequenced, and for their discriminatory power. The most successful loci were tested to identify genetically related and unrelated cases. A new consensus MLST scheme consisting of four genetically independent loci: β-TUB, CYB, mt26S and SOD, is herein proposed for standardised P. jirovecii typing, successfully amplifying low and high fungal burden specimens, showing adequate discriminatory power, and correctly identifying suspected related and unrelated isolates. The new consensus MLST scheme, if accepted, will for the first time provide a powerful tool to investigate outbreak settings and undertake global epidemiological studies shedding light on the spread of this important human fungal pathogen.

Genotyping Diversity of Pseudomonas aeruginosa Isolates, Isolated from Baquba City

This research aims to assess the similarities and variances between MDR Pseudomonas aeruginosa clinicalisolates from different infections using MLST molecular typing technique in Baquba city. One hundredeighty clinical samples were collected from urinary tract, wounds and burns infections, included (110)urine, (50) wounds and (20) burns. Twenty isolates (11.11%) of Pseudomonas aeruginosa were diagnosedbased on microscopic, morphological, biochemical tests and confirmed by VITEK-2 system. Antibioticsusceptibility test for the twenty isolates was performed against six antibiotics using disk diffusion method,the results showed (100%) resistance for Cephalothin, Ceftazdim (60%), Colistin (35%), Amikacin (25%),Ciprofloxacin (20%), while (0%) resistance for Imipenem. The results also showed that 16 (80%) of isolateswere produced extended spectrum beta-lactamase (ES?L) and did not show their production for Metallobeta-lactamase (M?Ls). Biofilm was detected by Micro-Titer Plate method and the result showed that all theisolates (100%) were biofilm producers. Multi-locus sequence typing (MLST) was used because broadnessand ease of access of MLST database. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA,andtrpE) were sequenced and the results were compared with MLST database and allele profile for eachisolate was used to determine sequence types (STs).New Iraqi bacterial strain has been identified andrecorded in database of MLST (PubMLST) under the name (AS-85U).

MLST based serotype prediction for the accurate identification of non typhoidal Salmonella serovars.

Traditional serotyping based on the phenotypic variation of O- and H-antigen remains as the gold-standard for the identification and classification of Salmonella isolates for last 70years. Although this classification is a globally recognized nomenclature, huge diversity of Salmonella serotypes have made the serovar identification to be very complex. Seven gene multilocus sequence typing (MLST) on the other hand can provide serovar prediction as well as the evolutionary origin between the serovars. In this study non typhoidal Salmonella (NTS) strains (n = 45) isolated from clinical samples (blood, faeces and pus) were identified by traditional phenotypic serotyping and biochemical testing. All the tested Salmonella isolates were designated as serovar Typhimurium based on phenotyping. However, by MLST 60% (27/45) of the isolates were S. Typhimurium, 35.5% (16/45) were S. Agona (ST13), 2.2% (1/45) were S. Kentucky (ST198) and 2.2% (1/45) were S. Saintpaul (ST27). MLST analysis assigned S. Typhimurium isolates as ST36 (18/127), ST19 (7/27) and ST313 (2/27). Mismatches in serovar designation between MLST database and phenotypic serotyping can be due to the misinterpretation of phenotypic serotyping as the antigenic structures of S. Typhimurium, S. Agona differs by a surface antigen. MLST based phylogeny of study isolates showed clustering according to sequence types. Concordance between MLST based sequence type and phenotypic serotype is important to provide insights into genetic population structure of Salmonella.

Virulence Pattern and Genomic Diversity of Vibrio cholerae O1 and O139 Strains Isolated From Clinical and Environmental Sources in India.

Vibrio cholerae is an autochthonous inhabitant of the aquatic environment. Several molecular methods have been used for typing V. cholerae strains, but there is no proper database for such scheme, including multilocus sequence typing (MLST) for V. cholerae O1 and O139 strains. We used 54 V. cholerae O1 and three O139 strains isolated from clinical and environmental sources and regions of India during the time period of 1975–2015 to determine the presence of virulence genes and production of biofilm. We devised a MLST scheme and developed a database for typing V. cholerae strains. Also, we performed pulsed-field gel electrophoresis to see the genomic diversity among them and compared it with MLST. We used the MEGA 7.0 software for the alignment and comparison of different nucleotide sequences. The advanced cluster analysis was performed to define complexes. All strains of V. cholerae, except five strains, showed variation in phenotypic characteristics but carried virulence-associated genes indicating they belonged to the El Tor/hybrid/O139 variants. MLST analysis showed 455 sequences types among V. cholerae strains, irrespective of sources and places of isolation. With these findings, we set up an MLST database on PubMLST.org using the BIGSdb software for V. cholerae O1 and O139 strains, which is available at https://pubmlst.org/vcholerae/ under the O1/O139 scheme. The pulsed-field gel electrophoresis (PFGE) fingerprint showed six fingerprint patterns namely E, F, G, H, I, and J clusters among 33 strains including strain N16961 carrying El Tor ctxB of which cluster J representing O139 strain was entirely different from other El Tor strains. Twenty strains carrying Haitian ctxB showed a fingerprint pattern classified as cluster A. Of the five strains, four carrying classical ctxB comprising two each of El Tor and O139 strains and one El Tor strain carrying Haitian ctxB clustered together under cluster B along with V. cholerae 569B showing pattern D. This study thus indicates that V. cholerae strains are undergoing continuous genetic changes leading to the emergence of new strains. The MLST scheme was found more appropriate compared to PFGE that can be used to determine the genomic diversity and population structure of V. cholerae.

Diversity of the diploid sequence type of Candida albicans clinical isolates from a tertiary-care hospital in Mwanza, Tanzania.

Geographical strain variations of Candida albicans causing different clinical conditions in susceptible individuals have been reported. In this study, the distribution of diploid sequence type of C. albicans was investigated in Mwanza, Tanzania. A total of 64 C. albicans were selected on the basis of their antifungal susceptibility patterns, followed by multilocus sequence typing (MLST) to establish the circulating sequence types (STs). Forty-eight MLST were obtained out of 64 isolates amounting to 75% population structure differences. Out of these STs, 27 (56.3%) were new diploid ST types. C. albicans isolates with new ST were more diverse than isolates with known STs (27/29, 93.1% vs. 21/35, 60%, p 0.002). In conclusion, C. albicans from clinical specimens were highly diverse, with more than half of the detected diploid ST not previously reported in the MLST database, thus confirming the genetic differences of C. albicans from different geographical regions.

Molecular characterisation of Trichomonas vaginalis isolates in Southwest Turkey with multilocus sequence typing and genetic structure analysis in relation to different countries

Trichomonas vaginalis, a flagellated protozoan parasite, is among the most common sexually transmitted pathogens in the world. The present study aimed to identify the genetic profiles of T. vaginalis in the southwest of Turkey with multilocus sequence typing (MLST) and to analyse the genetic structure of the parasite in a collection of isolates from different countries. The study included 27 T. vaginalis isolates from symptomatic females in Aydin, Turkey. Seven housekeeping genes of T. vaginalis were partially amplified and sequenced after genomic DNA extraction from in vitro cultures. The allele profiles and sequence types (STs) of the isolates were determined by using the MLST database (https://pubmlst.org/tvaginalis). The genetic structure and differentiation of the parasite were analysed in relation to findings from other countries by assembling the available MLST sequences. When referred to the database, a total of 22 STs, including 18 new STs were found; besides, there were two new allele types. The genetic analysis of MLST data demonstrated the presence of two main genetic structures: Type I and Type II. In addition, the neighbor-joining method also revealed that the isolates were clustered into two groups. The genetic types distributed almost equally in the Netherlands and the USA, however, the predominance of Type I was noted in Turkey and the UK. The genetic differentiation among four countries was significant (p < .05), the gene flow was relatively high between the Netherlands and the USA, in contrast to Turkey. Finally, genetic variations were originated within populations (93.8%) rather than among populations (6.2%). In conclusion, we studied the genetic diversity of T. vaginalis isolates with MLST in the southwest of Turkey and showed the origin of genetic differentiation of the parasite among different countries. The presentation of MLST profiles and genetic variance of T. vaginalis isolates will contribute to the development of new diagnostic and treatment options for the parasite.

Molecular typing, and integron and associated gene cassette analyses in Acinetobacter baumannii strains isolated from clinical samples.

The present study aimed to investigate the association between drug resistance and class I, II and III integrons in Acinetobacter baumannii (ABA). Multilocus sequence typing (MLST) is a tool used to analyze the homology among house-keeping gene clusters in ABA and ABA prevalence and further provides a theoretical basis for hospitals to control ABA infections. A total of 96 clinical isolates of non-repeating ABA were harvested, including 74 carbapenem-resistant ABA (CRABA) and 22 non-CRABA strains, and used for bacterial identification and drug susceptibility analysis. Variable regions were sequenced and analyzed. Then, 7 pairs of housekeeping genes were amplified and sequenced via MLST and sequence alignment was performed against the Pub MLST database to determine sequence types (STs) strains and construct different genotypic evolutionary diagrams. The detection rate of CRABA class I integrons was 13.51% (10/74); no class II and III integrons were detected. However, class I, II and III integrons were not detected in non-CRABA strains. The variable regions of 9 of 10 class I integrons were amplified and 10 gene cassettes including aacC1, aac1, aadDA1, aadA1a, aacA4, dfrA17, aadA5, aadA1, aadA22 and aadA23 were associated with drug resistance. The 96 ABA strains were divided into 21 STs: 74 CRABA strains containing 9 STs, primarily ST208 and ST1145 and 22 non-CRABA strains containing 18 STs, primarily ST1145. Class I integrons are a critical factor underlying drug resistance in ABA. CRABA and non-CRABA strains differ significantly; the former primarily contained ST208 and ST1145, and the latter contained ST1145. Most STs were concentrated in intensive care units (ICUs) and the department of Neurology, with the patients from the ICUs being the most susceptible to bacterial infection. In the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital, ABA is potentially horizontally transmitted and MLST can be used for clinical ABA genotyping.

An Improved Approach to Identify Bacterial Pathogens to Human in Environmental Metagenome.

The identification of bacterial pathogens to humans is critical for environmental microbial risk assessment. However, current methods for identifying pathogens in environmental samples are limited in their ability to detect highly diverse bacterial communities and accurately differentiate pathogens from commensal bacteria. In the present study, we suggest an improved approach using a combination of identification results obtained from multiple databases, including the multilocus sequence typing (MLST) database, virulence factor database (VFDB), and pathosystems resource integration center (PATRIC) databases to resolve current challenges. By integrating the identification results from multiple databases, potential bacterial pathogens in metagenomes were identified and classified into eight different groups. Based on the distribution of genes in each group, we proposed an equation to calculate the metagenomic pathogen identification index (MPII) of each metagenome based on the weighted abundance of identified sequences in each database. We found that the accuracy of pathogen identification was improved by using combinations of multiple databases compared to that of individual databases. When the approach was applied to environmental metagenomes, metagenomes associated with activated sludge were estimated with higher MPII than other environments (i.e., drinking water, ocean water, ocean sediment, and freshwater sediment). The calculated MPII values were statistically distinguishable among different environments (p<0.05). These results demonstrate that the suggested approach allows more for more accurate identification of the pathogens associated with metagenomes.

New multilocus sequence typing primers to enable genotyping of AD hybrids within the Cryptococcus neoformans species complex.

We designed and tested new specific primers to amplify the two alleles of each of the seven MLST loci in C. neoformans species complex hybrids. The sequences obtained from hybrids can be compared with those present in the Cryptococcus global MLST database for future molecular epidemiology studies.

Pathogenomic Analysis of a Novel Extensively Drug-Resistant Citrobacter freundii Isolate Carrying a blaNDM-1 Carbapenemase in South Africa.

Pathogenomic analysis was performed on a novel carbapenem-resistant Citrobacter freundii isolate (H2730R) from a rectal swab of an adult male patient admitted to a tertiary hospital, Durban, South Africa. H2730R was identified using selective media and API 20e kit. Confirmatory identification and antibiotic susceptibility testing were performed using the VITEK II. H2730R was whole-genome sequenced on the Illumina MiSeq platform. H2730R was resistant to all tested antibiotics except tigecycline and was defined as ST498 by the C. freundii multilocus sequence typing (MLST) database. The estimated pathogenic potential predicted a higher probability (Pscore ≈ 0.875), supporting H2730R as a human pathogen. H2730R harbored 25 putative acquired resistance genes, 4 plasmid replicons, 4 intact prophages, a class 1 integron (IntI1), 2 predominant insertion sequences (IS3 and IS5), numerous efflux genes, and virulome. BLASTn analysis of the blaNDM-1 encoding contig (00022) and its flanking sequences revealed the blaNDM-1 was located on a plasmid similar to the multireplicon p18-43_01 plasmid reported for the spread of carbapenem resistance in South Africa. Phylogenomic analysis showed clustering of H2730R with CF003/CF004 strains in the same clade, suggesting a possible association between C. freundii strains/clones. Acquiring the p18-43_01 plasmid containing blaNDM-1, the diversity, and complex resistome, virulome, and mobilome of this pathogen makes its incidence very worrying regarding mobilized resistance. This study presents the background genomic information for future surveillance and tracking of the spread of carbapenem-resistant Enterobacteriaceae in South Africa.

MLST-based genetic relatedness of Campylobacter jejuni isolated from chickens and humans in Poland.

Campylobacter jejuni infection is one of the most frequently reported foodborne bacterial diseases worldwide. The main transmission route of these microorganisms to humans is consumption of contaminated food, especially of chicken origin. The aim of this study was to analyze the genetic relatedness of C. jejuni from chicken sources (feces, carcasses, and meat) and from humans with diarrhea as well as to subtype the isolates to gain better insight into their population structure present in Poland. C. jejuni were genotyped using multilocus sequence typing (MLST) and sequence types (STs) were assigned in the MLST database. Among 602 isolates tested, a total of 121 different STs, including 70 (57.9%) unique to the isolates' origin, and 32 STs that were not present in the MLST database were identified. The most prevalent STs were ST464 and ST257, with 58 (9.6%) and 52 (8.6%) C. jejuni isolates, respectively. Isolates with some STs (464, 6411, 257, 50) were shown to be common in chickens, whereas others (e.g. ST21 and ST572) were more often identified among human C. jejuni. It was shown that of 47 human sequence types, 26 STs (106 isolates), 23 STs (102 isolates), and 29 STs (100 isolates) were also identified in chicken feces, meat, and carcasses, respectively. These results, together with the high and similar proportional similarity indexes (PSI) calculated for C. jejuni isolated from patients and chickens, may suggest that human campylobacteriosis was associated with contaminated chicken meat or meat products or other kinds of food cross-contaminated with campylobacters of chicken origin. The frequency of various sequence types identified in the present study generally reflects of the prevalence of STs in other countries which may suggest that C. jejuni with some STs have a global distribution, while other genotypes may be more restricted to certain countries.

Molecular and epidemiologyical analysis of a Campylobacter jejuni outbreak in China, 2018.

Campylobacter spp. is the most common gastrointestinal pathogen worldwide with a very low reported incidence in China. In April 2018, one 36 cases of diarrhea outbreak occurred in a high school in Beijing after a trip to another province in Southern China. The investigation for the enteric pathogen infection was conducted to identify the cause. Eighteen stool specimens from 11 diarrheal patients and 3 close contacts were collected and tested for 16 enteric bacterial and viral pathogens using real-time PCR methods. Multilocus Sequence Typing (MLST), pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing were applied to C. jejuni isolates in this outbreak in order to characterize the incident. Ten (90.9%) of 11 stool specimens from diarrheal patients were positive for Campylobacter jejuni, confirmed as the cause of outbreak. Eight C. jejuni strains were obtained and produced 2 sequence types (STs) and 3 PFGE patterns. Six of them had the same ST (ST2274) and PFGE pattern (SMA001). The dominant outbreak strain with an identical subtyping profile was clustered with most chicken isolates from national MLST and PulseNet Campylobacter database. The entire 8 isolates were multi-drug resistant, with the dominant resistance pattern of nalidixic acid, tetracycline and ciprofloxacin combined, except for 2 isolates resistant to florfenicol. Molecular typing confirmed that most of the cases belonged to a clonal cluster supporting the hypothesis of a common source; however, the source was not identified. This was the first recognized Campylobacteriosis outbreak in China among 20 years.

Identifying geographic origins of the Escherichia coli isolates from food by a method based on single-nucleotide polymorphisms

BackgroundE.coli is an important foodborne pathogen. Rapid and robust tracking of the source of E. coli is the key step to control foodborne infections. ResultsIn this study, a genotyping and tracing method based on highly discriminatory single nucleotide polymorphisms (SNPs) was developed to investigate the geographical origin of E. coli in food. A highly informative set of 12 SNPs was derived from 4 housekeeping genes in E. coli multilocus sequence typing (MLST) database. A collection of 253 E. coli isolates from food in 12 countries and regions were screened, resulting in a total of 61 profiles, 35 geographically specific SNP profiles were revealed and further verified by blind sample test. Also, the evolutionary relationship of 61 SNP profiles with different geographical origins was established by the enhanced analysis Based Upon Related Sequence Types (eBURST) analysis, which provided evidence that strains of different geographical origins owned the same ancestor strain. ConclusionsOur study established a powerful method based on a set of 12 SNPs for identifying geographical origins. The blind sample analysis proved that this SNPs panel had a high traceability of E. coli in food. Furthermore, this method based on SNPs combined with eBURST analysis revealed the potential evolutionary relationship between E.coli strains of different geographical origins.

Genetic diversity of pathogenic leptospires from wild, domestic and captive host species in Portugal.

Leptospirosis is a neglected zoonotic disease of worldwide distribution with a significant veterinary and public health impact. It is caused by pathogenic bacteria of the genus Leptospira. The availability of effective tools to accurately identify and type leptospires is of utmost importance for the diagnosis of the disease and for assessing its epidemiology. Several multi-locus sequence typing (MLST) approaches were described for the typing of worldwide isolates of Leptospira but an extensive agreement towards the adoption of a unique consensus scheme for this agent is still lacking. Most genotyped strains originate from Asian and South American countries, with a minority originating from Europe (being most countries represented only by one or a few isolates). The knowledge of the diversity of circulating leptospires is the key to understanding the disease transmission and its zoonotic implications. In this study, we revisited the taxonomy of several isolates of pathogenic Leptospira obtained from domestic, wild and captive animals in Portugal, between 1990 and 2012. A selection of these isolates was genotyped using two previously published MLST schemes. A total of seven distinct sequence types (STs) were detected among the Portuguese isolates with two STs representing L.borgpetersenii (ST149 and ST152), two STs representing L.kirschneri (ST117 and ST100) and three STs representing L.interrogans (ST17, ST24 and ST140). Global widespread (and maybe more virulent) Leptospira genotypes seem to circulate in Portugal, particularly the L.interrogans ST17 isolates which are associated with several outbreaks of leptospirosis among humans and animals in different regions of the world. This study contributes to the enrichment of the global MLST databases with a new set of allele and sequence type information also providing novel data on circulating Leptospira serovars in Portugal.