Multilocus Sequence Typing Database Research Articles

Emergence of two novel sequence types (3366 and 3367) NDM-1- and OXA-48-co-producing K. pneumoniae in Italy.

The aim of this study was to analyze the alarming spread of NDM-1- and OXA-48-co-producing Klebsiella pneumoniae clinical isolates, collected between October 2016 and January 2018 in a neonatal intensive care unit of the University Hospital, Catania, Italy, through whole genome sequencing. All confirmed carbapenem-resistant K. pneumoniae (CRKp) isolates were characterized pheno- and geno-typically, as well as by whole genome sequencing (WGS). A total of 13 CRKp isolates were identified from 13 patients. Pulsed-field gel electrophoresis (PFGE) was performed, and the multilocus sequence typing (MLST) scheme used was based on the gene sequence as published on the MLST Pasteur website. Core genome MLST (cgMLST) was also performed. All isolates co-carried blaoxa-48 and blaNDM-1 genes located on different plasmids belonging to the IncM/L and IncA/C2 groups, respectively. The 13 strains had identical PFGE profiles. MLST and cgMLST showed that K. pneumoniae was dominated by CRKp ST101 and two novel STs (ST3666 and ST3367), identified after submission to the MLST database for ST assignment. All isolates shared the same virulence factors such as type 3 fimbriae, genes for yersiniabactin biosynthesis, yersiniabactin receptor, and iron ABC transporter. They carried the wzi137 variant associated with the K17 serotype. To the best of our knowledge, this is the first report of two novel STs, 3366 and 3367, NDM-OXA-48-co-producing K. pneumoniae clinical isolates, in Italy.

Dissecting the Heterogeneous Population Genetic Structure of Candida albicans: Limitations and Constraints of the Multilocus Sequence Typing Scheme

Candida albicans is a fungal opportunistic pathogen of significant public health importance mainly due to the recent emergence of strains with increased aggressiveness and antifungal resistance. Here, we aimed to describe the epidemiological profiles and approximate the population structure of C. albicans by analyzing the C. albicans multilocus sequence typing (MLST) database (Calb-MLST-DB), which contains the largest publically available dataset for this species. Based on 4,318 database isolates, we confirmed the ubiquitous nature of C. albicans including a group of diploid sequence types (DSTs) obtained from Healthy individuals exclusively (taken as an indicator of lack of association with illnesses in its host), until isolates established from Non-Healthy individuals (potentially associated with pathogenic processes) and other DSTs reported in both types (Healthy and Non-Healthy). The highest number of reported DSTs was related to blood, oral and vaginal swabs (32.4, 20.5, and 13.8%, respectively). High genetic diversity was observed in the seven housekeeping genes included in the MLST scheme, with a diverse population structure (154 clonal complexes, CCs; and a high number of singletons, n = 1,074). Phylogenetic reconstruction on the concatenated alignment of these housekeeping genes for all the reported DSTs (n = 3,483) was partially concordant with the CC assignment, however, an absence of bootstrap threshold supported nodes or p-distance, and the lack of association with the other epidemiological variables, evidenced the limitations of the MLST scheme. Marked genetic admixture signals were identified by STRUCTURE, with the majority being attributable to recombination events according to the RDP program results, although another type of exchange event cannot be ruled out. Our results reaffirm the genetic diversity inherent in the genes used for the MLST scheme, which are associated with the chromosomal remodeling already proposed for C. albicans. This was also corroborated with an internal validation at a micro geographical scale. Despite these results are biased due to the unavailability of considering the broad global spectrum of C. albicans isolates around the world. This suggests that the strategy used to population type this pathogen should be reevaluated to improve epidemiological monitoring of its health impact.

Asian isolates of Anaplasma phagocytophilum: Multilocus sequence typing

Anaplasma phagocytophilum is the bacterial agent of granulocytic anaplasmosis in humans and animals; it is widely distributed in Eurasia and North America and transmitted mainly by Ixodes ticks. Several approaches have been used to study genetic diversity in A. phagocytophilum, multilocus sequence typing (MLST) currently being the most reliable and comparable. The MLST method based on seven housekeeping loci, 2877 bp total length, has been used to create and maintain the MLST database available worldwide (https://pubmlst.org/aphagocytophilum/). Before this study, the database contained 150 sequence types (STs) and 418 isolates, 397 of them originating from Europe and 21 from the USA, with none from Asia. We typed 25 A. phagocytophilum isolated from Ixodes ticks collected in the Asian part of Russia and compared the results with the conventional 16S rRNA typing. Substantial variability in the primer binding sites was found, so we had to modify the original primers for six out of seven loci. None of the sequences obtained matched those from the database; 15 new STs and 39 new alleles were revealed. Russian isolates belonged to two clusters, cluster 1 (19 isolates) and 3 (6 isolates), in both of which they formed separate clades. For the first time, we found A. phagocytophilum isolates from Ixodes persulcatus and I. pavlovskyi to belong to cluster 3, previously containing only the strains from voles and shrews. Further research is needed to estimate the prevalence of two MLST clusters of A. phagocytophilum in ticks and vertebrate hosts in Asia.

Comprehensive subspecies identification of 175 nontuberculous mycobacteria species based on 7547 genomic profiles

ABSTRACT The prevalence of nontuberculous mycobacteria (NTM) pulmonary diseases has been increasing worldwide. NTM consist of approximately 200 species and distinguishing between them at the subspecies level is critical to treatment. In this study, we sequenced 63 NTM genomes, 27 of which were newly determined, by hybrid assembly using sequencers from Illumina and Oxford Nanopore Technologies (ONT). This analysis expanded the available genomic data to 175 NTM species and redefined their subgenus classification. We also developed a novel multi-locus sequence typing (MLST) database based on 184 genes from 7547 assemblies and an identification software, mlstverse, which can also be used for detecting other bacteria given a suitable MLST database. This method showed the highest sensitivity and specificity amongst conventional methods and demonstrated the capacity for rapid detection of NTM, 10 min of sequencing of the ONT MinION being sufficient. Application of this methodology could improve disease epidemiology and increase the cure rates of NTM diseases.

Molecular analysis of clinical Burkholderia pseudomallei isolates from southwestern coastal region of India, using multi-locus sequence typing.

BackgroundThe Gram-negative soil dwelling bacterium Burkholderia pseudomallei is the etiological agent of melioidosis. The disease is endemic in most parts of Southeast Asia and northern Australia. Over last few years, there has been an increase in number of melioidosis cases from India; however the disease epidemiology is less clearly understood. Multi-locus sequence typing (MLST) is a powerful genotypic method used to characterize the genetic diversity of B. Pseudomallei both within and across the geographic regions.MethodsIn this study, MLST analysis was performed on 64 B. pseudomallei clinical isolates. These isolates were obtained between 2008–2014 from southwestern coastal region of India. Broad population patterns of Indian B. pseudomallei isolates in context with isolates of Southeast Asia or global collection was determined using in silico phylogenetic tools.ResultsA total of 32 Sequence types (STs) were reported among these isolates of which 17 STs (53%) were found to be novel. ST1368 was found as group founder and the most predominant genotype (n = 11, 17%). Most of the B. pseudomallei isolates reported in this study (or other Indian isolates available in MLST database) clustered in one major group suggesting clonality in Indian isolates; however, there were a few outliers. When analyzed by measure of genetic differentiation (FST) and other phylogenetic tools (e.g. PHYLOViZ), Indian STs were found closer to Southeast Asian isolates than Australian isolates. The phylogenetic analysis further revealed that within Asian clade, Indian isolates grouped more closely with isolates from Sri Lanka, Vietnam, Bangladesh and Thailand.ConclusionsOverall, the results of this study suggest that the Indian B. pseudomallei isolates are closely related with lesser heterogeneity among them and cluster in one major group suggesting clonality of the isolates. However, it appears that there are a few outliers which are distantly related to the majority of Indian STs. Phylogenetic analysis suggest that Indian isolates are closely related to isolates from Southeast Asia, particularly from South Asia.

Unveiling the Multilocus Sequence Typing (MLST) Schemes and Core Genome Phylogenies for Genotyping Chlamydia trachomatis.

Multilocus sequence typing (MLST) has become a useful tool for studying the genetic diversity of important public health pathogens, such as Chlamydia trachomatis (Ct). Four MLST schemes have been proposed for Ct (data available from Chlamydiales MLST databases). However, the lack of a sole standardized scheme represents the greatest limitation regarding typing this species. This study was thus aimed at evaluating the usefulness of the four MLST schemes available for Ct, describing each molecular marker's pattern and its contribution toward a description of intra-specific genetic diversity and population structure. The markers for each scheme, showed a variable power of dicrimination, exhibiting in some cases over estimation in the determination of Sequence Types (STs). However, individual analysis of each locus's typing efficiency and discrimination power led to identifying 8 markers as having a suitable pattern for intra-specific typing. analyzing the 8 candidate markers gave a combination of 3 of these loci as an optimal scheme for identifying a large amount of STs, maximizing discrimination power whilst maintaining suitable typing efficiency. One scheme was compared against core genome phylogenies, finding a higher typing resolution through the last approach. These results confirm once again that although complete genome data, in particular from core genome MLST (cgMLST) allow a high resolution clustering for Ct isolates. There are combinations of molecular markers that could generate equivalent results, with the advantage of representing an easy implementation strategy and lower costs leading to contribute to the monitoring and molecular epidemiology of Ct.

Virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China

ObjectivesWe aimed to report virulence-associated genes and molecular characteristics of non-O1/non-O139 Vibrio cholerae isolated from hepatitis B cirrhosis patients in China. MethodsPatient clinical data including course of disease, laboratory tests, antibiotic treatment and outcomes were collected. Antimicrobial susceptibility testing was performed and virulence-associated genes were detected by PCR. Genetic relatedness among non-O1/non-O139 V. cholerae strains was investigated by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). ResultsAll three strains in this study harbored pathogenicity related genes like rtxA, rtxC, toxR, hapA, hlyA and ompW whereas they lacked ctxA, ctxB, tcpA, ompU and zot genes. None of them showed resistance to any antibiotic detected. A new allele of gyrB was submitted to the MLST database and designated as 97. Two novel sequence types (ST518 and ST519) and ST271 were identified by multilocus sequence typing (MLST). PFGE indicated considerable diversity among three non-O1/non-O139 V. cholerae strains. ConclusionsThree sporadic cases highlight that non-O1/non-O139 V. cholerae can cause opportunistic invasiveness infection in cirrhosis patients. Pathogenicity may be related to virulence-associated genes. Timely detection and antibiotic therapy should be paid more attention to in clinic.

Genomic Sequencing of Bordetella pertussis for Epidemiology and Global Surveillance of Whooping Cough.

Bordetella pertussis causes whooping cough, a highly contagious respiratory disease that is reemerging in many world regions. The spread of antigen-deficient strains may threaten acellular vaccine efficacy. Dynamics of strain transmission are poorly defined because of shortcomings in current strain genotyping methods. Our objective was to develop a whole-genome genotyping strategy with sufficient resolution for local epidemiologic questions and sufficient reproducibility to enable international comparisons of clinical isolates. We defined a core genome multilocus sequence typing scheme comprising 2,038 loci and demonstrated its congruence with whole-genome single-nucleotide polymorphism variation. Most cases of intrafamilial groups of isolates or of multiple isolates recovered from the same patient were distinguished from temporally and geographically cocirculating isolates. However, epidemiologically unrelated isolates were sometimes nearly undistinguishable. We set up a publicly accessible core genome multilocus sequence typing database to enable global comparisons of B. pertussis isolates, opening the way for internationally coordinated surveillance.

Extremely Low Prevalence of Erythromycin-Resistant Streptococcus pyogenes Isolates and Their Molecular Characteristics by M Protein Gene and Multilocus Sequence Typing Methods

Background: Group A streptococci (GAS) are notorious bacteria causing a wide variety of clinical manifestations ranging from mild, acute streptococcal pharyngitis to chronic non-suppurative diseases and immunological sequelae. They are further complicated by the global rise on the emergence of macrolide resistance among these bacteria in which several M protein gene (emm) and sequence types are associated with invasive diseases. Objectives: The current study aimed at determining the erythromycin resistance patterns and molecular characteristics of GAS clinical strains by emm and multilocus sequence typing (MLST) methods. Methods: Thirty-five GAS clinical isolates were subjected to antibiotic susceptibility testing by disk diffusion method. The minimum inhibitory concentration (MIC) of erythromycin against GAS by E-test was determined. Clinical and laboratory standards institute (CLSI) guideline was used for the interpretation of results. Detection of ermA, ermB, and mefA genes by polymerase chain reaction (PCR) was performed and emm typing was done by amplification and sequencing of emm genes per standard protocol. Allele and sequence type (ST) of GAS were obtained using the S. pyogenes MLST database. Results: All the isolates were sensitive to erythromycin, penicillin, clindamycin, chloramphenicol, and vancomycin (100%). Resistance to tetracycline was 54.3%. The mefA gene was found in one erythromycin susceptible isolate. No other erythromycin resistance genes were detected in the isolates. Twenty different emm types were found and the most frequent emm types/subtypes detected were emm1, emm18.21, emm28.5, emm97.4, and emm102.2 (each 8.6%). However, no new emm type was detected. A total of 15 sequence types (STs), eight clonal clusters (CCs), and eight singletons were identified among 21 representative isolates. Three isolates exhibited CC1 (ST28/emm1). Conclusions: High susceptibility of GAS isolates against erythromycin could be due to low antibiotic selective pressure in Malaysian clinical settings. High diversity of emm and ST types revealed the heterogenic nature of the strains circulating in Malaysian hospitals. Continuous epidemiological monitoring by molecular typing methods is warranted to improve the management strategies of GAS infections in future.

Genotypic characterization of Haemophilus influenzae isolates from paediatric patients in Japan.

Purpose. β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae is frequently isolated from respiratory samples and is particularly problematic in Japan. The aim of this study was to characterize circulating isolates of H. influenzae genotypically by BLNAR-PCR and multilocus sequence typing (MLST), and to determine any associations between them.Methods. H. influenzae isolates (n=191) were collected from paediatric patients (1 month to 12 years old) between 2000 and 2011 for three types of infections: pneumonia (n=61), acute otitis media (AOM) (n=68) and meningitis (n=62). All were characterized for capsular type by agglutination tests, and for β-lactam resistance by real-time PCR. The sequence types (STs) determined by MLST were analysed using eburst v3.Results. Eighty-eight out of 191 (46.1 %) H. influenzae isolates were BLNAR by PCR; 37 of 61 (60.7 %) from pneumonia; 33 of 68 (48.5 %) from AOM and 18 of 62 (29.0 %) from meningitis cases. MLST identified 40 and 44 STs among isolates from pneumonia and AOM, respectively. BLNAR were found in singletons such as ST156 in pneumonia, and ST161 and ST396 in AOM. In contrast, eight STs were identified in meningitis, of which seven were genotypically closely related, while ST54 was the most frequent (62.9 %), unlike in the MLST database registrations, where ST6 predominated.Conclusion. Non-typeable H. influenzae (NTHi), mostly derived from pneumonia and AOM, were genetically diverse, in contrast to the predominance of H. influenzae type b (Hib) among meningitis cases. The associations between certain STs and β-lactam resistance among NTHi were confirmed.

Estimating the Intra-taxa Diversity, Population Genetic Structure, and Evolutionary Pathways of Cryptococcus neoformans and Cryptococcus gattii.

Members of the Cryptococcus complex, includes Cryptococcus neoformans (most common fungal infection of the brain) and Cryptococcus gattii (high-impact emerging pathogen worldwide). Currently, the fungal multilocus sequence typing database (Fungal MLST Database) constitutes a valuable data repository of the genes used for molecular typing of these pathogens. We analyzed the data available in the Fungal MLST Database for seven housekeeping genes, with the aim to evaluate its contribution in the description of intra-taxa diversity, population genetic structure, and evolutionary patterns. Although the Fungal MLST Database has a greater number of reports for C. neoformans (n = 487) than for C. gattii (n = 344), similar results were obtained for both species in terms of allelic diversity. Phylogenetic reconstructions revealed grouping by molecular type in both species and allowed us to propose differences in evolutionary patterns (gradualism in the case of C. neoformans and punctuated evolution in the case of C. gattii). In addition, C. neoformans showed a population genetic structure consisting of 37 clonal complexes (CCs; CC1 being predominant), high crosslinking [without sequence type (ST) grouping by molecular type], marked divergence events in phylogenetic analysis, and few introgression events (mainly between VNI and VNIV). By contrast, C. gattii showed 50 CCs (with greater homogeneity in ST number by CC) and clustering by molecular type with marked crosslinking events in phylogenetic networks being less evident. Understanding relationships at the molecular level for species of the Cryptococcus complex, based on the sequences of the housekeeping genes, provides information for describing the evolutionary history of these emerging pathogens.

Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep.

Whole genome sequence of an MCR-1-carrying, extended-spectrum β-lactamase (ESBL)-producing Escherichia coli ST746 isolate recovered from a community-acquired urinary tract infection.

Colistin is regarded as one of the last-resort antimicrobials for severe infections. Isolates carrying the plasmid-borne mobile colistin resistance gene mcr-1 were rarely reported in community-acquired infections. Here we report the draft genome sequence of an MCR-1-carrying, extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolate from community-acquired urinary tract infection. Antimicrobial susceptibility testing (AST) was performed by the broth microdilution method. Transferability of the mcr-bearing plasmid was determined by filter mating using E. coli EC600 as recipient strain. Multilocus sequence typing (MLST) was undertaken using the E. coli MLST database. The draft genome sequence of isolate LX13 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SOAPdenovo. Acquired antimicrobial resistance genes were identified using ResFinder 2.1. AST showed that LX13 was resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefazolin, cefepime and polymyxins. MLST showed that isolate LX13 belongs to ST746. The MCR-1-producing plasmid was conjugative and conferred increased resistance to colistin the transconjugant. The draft genome of E. coli LX13 was 4914035bp in size. In silico analysis revealed the presence of eight putative acquired resistance genes, including blaCTX-M-14, blaTEM-1B, aadA5, mcr-1, dfrA17, sul2, tet34 and tetA. plasmidSPAdes revealed that the mcr-1 gene was harboured by a plasmid of replicon type IncI2. This study highlights the potential risk of spread of MCR-1-carrying, ESBL-producing E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant E. coli.

The Applied Development of a Tiered Multilocus Sequence Typing (MLST) Scheme for Dichelobacter nodosus.

Dichelobacter nodosus (D. nodosus) is the causative pathogen of ovine footrot, a disease that has a significant welfare and financial impact on the global sheep industry. Previous studies into the phylogenetics of D. nodosus have focused on Australia and Scandinavia, meaning the current diversity in the United Kingdom (U.K.) population and its relationship globally, is poorly understood. Numerous epidemiological methods are available for bacterial typing; however, few account for whole genome diversity or provide the opportunity for future application of new computational techniques. Multilocus sequence typing (MLST) measures nucleotide variations within several loci with slow accumulation of variation to enable the designation of allele numbers to determine a sequence type. The usage of whole genome sequence data enables the application of MLST, but also core and whole genome MLST for higher levels of strain discrimination with a negligible increase in experimental cost. An MLST database was developed alongside a seven loci scheme using publically available whole genome data from the sequence read archive. Sequence type designation and strain discrimination was compared to previously published data to ensure reproducibility. Multiple D. nodosus isolates from U.K. farms were directly compared to populations from other countries. The U.K. isolates define new clades within the global population of D. nodosus and predominantly consist of serogroups A, B and H, however serogroups C, D, E, and I were also found. The scheme is publically available at https://pubmlst.org/dnodosus/.

Multilocus sequence types of clinical Burkholderia pseudomallei isolates from peninsular Malaysia and their associations with disease outcomes

BackgroundPrevious studies on the Burkholderia pseudomallei genetic diversity among clinical isolates from melioidosis-endemic areas have identified genetic factors contributing to differential virulence. Although it has been ruled out in Australian and Thai B. pseudomallei populations, it remains unclear whether B. pseudomallei sequence types (STs) correlate with disease in Malaysian patients with melioidosis.MethodsIn this study, multi-locus sequence typing (MLST) was performed on clinical B. pseudomallei isolates collected from Kelantan state of Malaysia, patients’ clinical data were reviewed and then genotype-risk correlations were investigated.ResultsGenotyping of 83 B. pseudomallei isolates revealed 32 different STs, of which 13(40%) were novel. The frequencies of the STs among the 83 isolates ranged from 1 to 12 observations, and ST54, ST371 and ST289 were predominant. All non-novel STs reported in this study have also been identified in other Asian countries. Based on the MLST data analysis, the phylogenetic tree showed clustering of the STs with each other, as well as with the STs from Southeast Asia and China. No evidence for associations between any of B. pseudomallei STs and clinical melioidosis presentation was detected. In addition, the bacterial genotype clusters in relation with each clinical outcome were statistically insignificant, and no risk estimate was reported. This study has expanded the data for B. pseudomallei on MLST database map and provided insights into the molecular epidemiology of melioidosis in Peninsular Malaysia.ConclusionThis study concurs with previous reports concluding that infecting strain type plays no role in determining disease presentation.

Genetic characterization of Listeria monocytogenes from ruminant listeriosis from different geographical regions in the U.S.

Listeria monocytogenes infections are an important disease of ruminants worldwide, causing encephalitis, septicemia, and abortions. Ruminant listeriosis can also pose a food safety risk due to the potential for L. monocytogenes to enter the food supply via the farm environment. Data on the genetic diversity of L. monocytogenes from ruminant clinical cases in the United States is limited. Our goal was to assess the genetic diversity of clinical listeriosis isolates from ruminants in the Upper Great Plains states, a population not well-studied, and compare this population to isolates from ruminants in New York State. Multi-locus sequence typing (MLST) was used to classify and compare the genetic diversity of the isolates from the two regions. Loci sequences were compared to all known sequence types using the Pasteur Institute L. monocytogenes MLST database. Four novel sequence types (ST) were identified among the Upper Great Plains isolates, and four new STs were classified in the New York collection. Five STs were found to be common across the 2 geographical regions; ST 1, 7, 191, and 204. Strains of ST 7 were most frequently isolated (7/46 isolates). Strains of ST 91 were all associated with fetal infections from the Upper Great Plains. Our results demonstrate that while there are some subtypes commonly found between the two geographic regions, there are also subtypes distinct to each region.

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China.

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2) was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2) and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs) were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS)-single-nucleotide polymorphism (SNP)-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

Prevalence and genetic diversity of human diarrheagenic Escherichia coli isolates by multilocus sequence typing

BackgroundThe population structure of human diarrheagenic Escherichia coli (DEC) isolates derived from worldwide collections remains undefined. MethodsA total of 1196 clinical isolates were obtained from a multilocus sequence typing (MLST) database. Genetic diversity analysis, MLST analysis, and phylogenetic analysis combined with different pathotypes were performed through a variety of calculation software applications. ResultsAll isolates were categorized as one of 579 different sequence types (STs). The eBURST algorithm resolved these 579 STs into 27 clonal complexes (CCs), 37 concatemers, and 210 singletons, revealing a high level of genetic diversity in the population structure of DEC. CC10 was the most prevalent CC, comprising 276 (23.08%, 276/1196) isolates with 85 (14.68%, 85/579) STs widely distributed in 20 countries. The population structure of five common pathotypes was highly diversified, and isolates with the same ST or CC were heterogeneous for different pathotypes. Sequence variations were more abundant in fumC and gyrB than in the other five genes, and these exhibited the highest degree of nucleotide diversity (0.03886 and 0.03075, respectively) and the greatest number of polymorphic nucleotide sites (137 and 139, respectively). The dN/dS ratios of seven analyzed loci varied from 0.0083 (recA) to 0.0434 (purA), and the ratio for the concatenated sequence was 0.2518, revealing the effects of purifying selection on housekeeping genes during the evolutionary process. Significant allele linkage disequilibrium was detected when the standardized index of association (ISA) was calculated both for the entire collection of isolates (0.3174, p<0.001) and for the 579 STs (0.1475, p<0.001). ConclusionsThis study facilitated a comprehensive understanding of the genetic diversity of human DEC distributed across the global population. The results provide genetic evidence that will allow us to uncover the microevolutionary relationships among different pathogenic isolates of DEC.

Antimicrobial susceptibility, virulence potential and sequence types associated with Arcobacter strains recovered from human faeces.

The genus Arcobacter includes bacteria that are considered emergent pathogens because they can produce infections in humans and animals. The most common symptoms are bloody and non-bloody persistent diarrhea but cases with abdominal cramps without diarrhea or asymptomatic cases have also been described as well as cases with bacteremia. The objective was to characterize Arcobacter clinical strains isolated from the faeces of patients from three Spanish hospitals. We have characterized 28 clinical strains (27 of A. butzleri and one of A. cryaerophilus) isolated from faeces, analysing their epidemiological relationship using the multilocus sequence typing (MLST) approach and screening them for their antibiotic susceptibility and for the presence of virulence genes.Results/Key findings. Typing results showed that only one of the 28 identified sequence types (i.e. ST 2) was already present in the MLST database. The other 27 STs constituted new records because they included new alleles for five of the seven genes or new combinations of known alleles of the seven genes. All strains were positive for the ciaB virulence gene and sensitive to tetracycline. However, 7.4 % of the A. butzleri and A. cryaerophilus strains showed resistance to ciprofloxacin. The fact that epidemiological unrelated strains show the same ST indicates that other techniques with higher resolution should be developed to effectively recognize the infection source. Resistance to ciprofloxacin, one of the antibiotics recommended for the treatment of Arcobacter intestinal infections, demonstrated in 10.7 % of the strains, indicates the importance of selecting the most appropriate effective treatment.

First Identification of Taylorella equigenitalis From Genital Tracts of Thoroughbred Horses From the Inland Area of South Korea by Multilocus Sequence Typing

The bacterium, Taylorella equigenitalis, is responsible for the disease contagious equine metritis (CEM), a highly contagious venereal disease of horses. There have been substantial economic losses reported in various equine industries across the world as a result of CEM. So far, there had been no reported cases of T. equigenitalis in the inland area of South Korea. This study was performed to determine the prevalence and the genotype of T. equigenitalis in the inland area of South Korea. In this study, 1 of 38 Thoroughbred horses was found positive for T. equigenitalis using bacterial culture. Multilocus sequence typing (MLST) and construction of a neighbor-joining tree based on the Taylorella spp. MLST database (http://pubmlst.org/taylorella) indicated that the inland South Korean T. equigenitalis strain in this study showed a distinct genotype and no epidemiologic relationship with other regional strains suggesting that the inland South Korean T. equigenitalis strain is unique. In order to prevent serious repercussions to the South Korean equine industry, a full epidemiologic investigation and comprehensive treatment regimen are needed.