A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Multilocus enzyme electrophoresis analysis of Echinostoma revolutum and Echinostoma malayanum(Trematoda:Echinostomatidae) isolated from Khon Kaen Province,Thailand

Objective:To explore the genetic variation and differentiation of 2 echinostomes from genus Echinostoma,i.e.Echinostoma revolutum(E.revolution) and Echinostoma malayanum(E. malayanum) from Khon Kaen Province,Thailand.Methods:These parasites were compared at 22 enzymes encoding a presumptive 30 loci by using multilocus enzyme electrophoresis (MEE) technique.Results:Twenty-two loci can be used as diagnostic markers to differentiate these 2 species.E.revolutum and E.malayanum had fixed genetic differences at 70%of loci, whereas both species had fixed genetic differences from the liver fluke,Opisthorchis viverrini at 91%of loci.Intraspecific variation within a population of E.revolutum was observed at 5 polymorphic loci.Conclusions:MEE is a powerful technique to investigate genetic variation and differentiation of E.revolutum and E.malayanum.

Multilocus Sequence Analysis of Root Nodule Bacteria Associated with <i>Lupinus</i>spp. and <i>Glycine max</i>

Lupinus is known to form endophytic associations with both nodulating and non-nodulating bacteria. In this study, multilocus sequence analysis (MLSA) was used to analyze phylogenetic relationships among root nodule bacteria associated with Lupinus and soybean. Out of 17 bacterial strains analyzed, 13 strains isolated from root nodules of Lupinus spp. were obtained from the National Rhizobium Germplasm Resource Collection, USDA. Additionally, two strains of root-nodule bacteria isolated each from native lupinus and domestic soybean were examined. Sequences of the 16S rRNA gene and three house-keeping genes (atpD, dnaK and glnII) were used. All the reference genes were retrieved from the existing complete genome sequences only. The clustering of 12 of the strains was consistent among single and concatenated gene trees, but not USDA strains 3044, 3048, 3504, 3715, and 3060. According to the concatenated phylogeny, we suggest that USDA 3040, 3042, 3044, 3048, 3051, 3060, 3504, 3709 and 3715 are Bradyrhizobium, USDA 3063 and 3717 are Mesorhizobium, USDA 3043 is Burkholderia and USDA 3057a is Microvirga. The two strains isolated from native lupines in this study are Burkholderia and Rhizobium, whereas the two from domestic soybean are Bradyrhizobium. This study emphasizes the robustness of MLSA, the diversity of bacterial species that are capable of nodulating lupine and the substantial capability of Burkholderia spp. to colonize lupine root nodules.

Serological and molecular capsular typing, antibiotic susceptibility and multilocus sequence typing of Streptococcuspneumoniae isolates from invasive and non-invasive infections

Background Streptococcus pneumoniae （S.pneumoniae） is a major causative agent of severe infections,including sepsis,pneumonia,meningitis,and otitis media,and has become a major public health concern.We report the pneumococcal serotype and sequence type （ST） distribution,and antimicrobial resistance of 39 S.pneumoniae strains from seven hospitals in China.Methods Blood/cerebrospinal fluid （CSF） and sputum isolates from patients were analyzed to determine S.pneumoniae serotypes by polymerase chain reaction （PCR） and the Neufeld Quellung reaction,the multilocus sequence types （MLST） by PCR and sequencing,and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.Results A total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates.Conventional serotyping by the Quellung reaction required 749 reactions.In contrast,PCR based typing needed only 106 PCR reactions.The most frequent serotypes from the blood/CSF isolates were 14 （38.1%）,19A （14.3%）,23F （9.5%）,and 18C （9.5%）.In the sputum isolates the most frequent serotypes were 19F （33.3%）,23F （16.7%）,19A （11.1%）,and 3 （11.1%）.The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%,respectively.Statistical analysis showed that patients ≤5 years old had a higher resistance to penicillin when they compared with the patients ≥65 years old （P=0.011）.Serotypes 14,19A and 19F were significantly associated with penicillin resistance （P 〈0.001）.ST320,ST271,and ST876 isolates showed high resistant rates to several antibiotics including penicillin （P=0.006）.All of the isolates of serotype 19A were resistant to both penicillin and erythromycin,and they were all multi-drug resistant （MDR） isolates.Conclusions The specificity and sensitivity of multiplexPCR are good,and this method represents a substantial savings of time and money,and can be widely used in the laboratory and clinical practice.Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant （MDR） rate in S.pneumoniae.A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance.Therefore,nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

Multilocus sequence typing indicates diverse origins of invasive Candida tropicalis isolates in China

Background According to data from the China Hospital Invasive Fungal Surveillance Net （CHIF-NET） 2010, Candida tropica/is （C. tropica/is） is the third most common pathogen causing invasive candidiasis. Moreover, the majority of fluconazole-resistant C. tropicalis isolates were from a single hospital. Therefore, a molecular epidemiological survey is necessary to investigate the genetic relatedness of C. tropica/is isolates in China. Methods In this study, 48 C. tropicalis isolates causing invasive fungal infections from four tertiary hospitals in China were studied. All the isolates were identified by sequencing the internal transcribed spacer region. Antifungal susceptibility to triazoles, amphotericin B, and caspofungin was determined by the Clinical and Laboratory Standards Institute standard broth microdilution method. Multilocus sequence typing （MLST） was performed, and phylogenetic analysis was further performed by the eBURST and maximum parsimony （MP） methods to characterize the genetic relatedness of isolates. Results MLST discriminated 40 diploid sequence types （DSTs） among 48 isolates, including 36 novel DSTs, and the XYR1 gene showed the highest discriminatory power. The DSTs obtained from this study were compared with those of previously reported C. tropicalis isolates, and there was poor type alignment with regional strains. Nine groups and 11 singletons were identified by eBURST, whereas two groups and 10 subgroups were clustered by MP analysis. Generally, there were no obvious correlations between clonal clusters generated and the specimen source or hospital origin. Seven fiuconazole-resistant isolates were confirmed and assigned to three distinguishable branches. Conclusions The results suggested diverse origins of invasive C. tropicalis isolates in China. Although most invasive C. tropicalis strains in the mainland of China were clustered with previously characterized Asian isolates, major C. tropicalis clusters identified in this study were genetically distinct from those of other geographic regions.

Optimization of Bartonella henselae multilocus sequence typing scheme using single-nucleotide polymorphism analysis of SOLID sequence data

Background Multi-locus sequence typing （MLST） is widely used to explore the population structure of numerous bacterial pathogens. However, for genotypically-restricted pathogens, the sensitivity of MLST is limited by a paucity of variation within selected loci. For Bartonella henselae （B. henselae）, although the MLST scheme currently used has been proven useful in defining the overall population structure of the species, its reliability for the accurate delineation of closely-related sequence types, between which allelic variation is usually limited to, at most, one or two nucleotide polymorphisms. Exploitation of high-throughput sequencing data allows a more informed selection of MLST loci and thus, potentially, a means of enhancing the sensitivity of the schemes they comprise. Methods We carried out SOLID resequencing on 12 representative B. henselae isolates and explored these data using single nucleotide polymorphism （SNP） analysis. We determined the number and distribution of SNPs in the genes targeted by the established MLST scheme and modified the position of loci within these genes to capture as much genetic variation as possible. Results Using genome-wide SNP data, we found the distribution of SNPs within each open reading frame （ORF） of MLST loci, which were not represented by the established B. henselae MLST scheme. We then modified the position of loci in the MLST scheme to better reflect the polymorphism in the ORF as a whole. The use of amended loci in this scheme allowed previously indistinguishable ST1 strains to be differentiated. However, the diversity of B. henselae was still rare in China. Conclusions Our study demonstrates the use of SNP analysis to facilitate the selection of MLST loci to augment the currently-described scheme for B. henselae. And the diversity among B. henselae strains in China is markedly less than that observed in B. henselae populations elsewhere in the world.

Phylogeography and diversification of Oriental weaverbirds(Ploceus spp.):A gradual increase of eurytopy

Weaverbirds are a speciose group of colorful passerines inhabiting the Old World Tropics.Nevertheless,the Oriental weaverbirds(Ploceus spp.),widespread across southern Asia,are much less diverse and restricted to a few ecological niches compared to their African counterpart.To investigate their phylogeography,we retrieved 101 samples of Baya Weaver(P.philippinus),Streaked Weaver(P.manyar),Black-Throated Weaver(P.benghalensis)and Asian Golden Weaver(P.hypoxanthus)along with GenBank sequences of Finn's Weaver(P.megarhynchus).We reconstructed the first molecular phylogeny based on a dataset consisting of both mitochondrial and nuclear genes,dating the most recent common ancestor of Oriental Ploceus to~11 mya.Subsequent speciation appears to have been a combination of divergence within the Indian subcontinent and dispersal across a barrier situated between the Indian subcontinent and the Indochinese region,which provided habitats with a varying degree of isolations and ultimately promoted divergences in allopatry.Two descendants of the earliest nodes,P.megarhynchus and P.hypoxanthus,are both rare and local,often found near large river systems,which perhaps reflects niche conservatism and a lack of adaptive potential.The three smaller species are all widespread,common and less habitat specific.The most recent divergence,between western and eastern P.philippinus populations,is supported by both phylogenetic and morphological evidence,pointing toward limited gene flow between them.However,a zone of intergradation may exist in Myanmar and Brahmaputra flood plains,thus preventing a recommendation for species level recognition without further study.

Molecular Characteristics of Neisseria meningitidis Isolated during an Outbreak in a Jail: Association with the Spread and Distribution of ST-4821 Complex Serogroup C Clone in China

Objective To characterize the meningococcal strains isolated from cases and close contacts with meningococcal disease associated with an outbreak in a jail in May 2010 by investigating the national distribution of hyperinvasive ST-4821 serogroup C clone associated with this outbreak. Methods The cases were described based on the clinical symptoms and laboratory results. Pharyngeal swabs were cultured for N. meningitidis from men in the jail. Meningococcal isolates were identified by serogrouping, pulsed-field gel electrophoresis （PFGE） and multilocus sequence typing （MLST）, respectively. Four hundred and sixteen serogroup C N. meningitidis strains were collected from 27 provinces between 2003 and 2010 for a nationwide survey and analyzed by PFGE and MLST. Results Three persons in a jail system were infected with invasive N. meningitidis serogroup C. All isolates tested had matching PFGE patterns and belonged to the multilocus sequence type （ST） 4821 clonal complex. All 47 N. meningitidis strains were identified from the pharyngeal swabs of 166 peoples in the jail, and 26 of them belonged to ST-4821 serogroup C clone, and 90.14% （375/416） serogroup C strains identified in the nationwide survey belonged to the ST-4821 complex. The ST-4821 serogroup C clone was spread nationwide, distributed in 24 provinces, especially in eastern provinces between 2003 and 2010. Conclusion Endemic transmission and carriage rate of ST-4821 serogroup C clone are high in this jail system. The ST-4821 serogroup C clone is spreading in China and nationwide distributed despite the existence of some effective vaccines.

Genetic and Antibiotic Resistance Characteristics ofCampylobacterjejuni Isolated from Diarrheal Patients,Poultry and Cattle in Shenzhen

Objective To investigate genetic and antibiotic resistance characteristics of Campylobacter jejuni(C. jejuni) isolated from Shenzhen. Methods Multilocs sequence typing and agar dilution methods were used to define the genotype and antibiotic resistance of C. jejuni, respectively. Results In total, 126 C. jejuni strains were isolated. The prevalence of C. jejuni was 5.3% in diarrheal patients. The prevalence in poultry meat(36.5%) was higher than that in cattle meat(1.1%). However, the prevalence in poultry cloacal swabs(27.0%) was lower than that in cattle stool(57.3%). Sixty-two sequence types were obtained, among which 27 of the STs and 10 alleles were previously unreported. The most frequently observed clonal complexes were ST-21(11.9%), ST-22(10.3%), and ST-403(7.1%). ST-21, ST-45, ST-354, ST-403, and ST-443 complexes overlapped between isolates from patients and cattle, whereas ST-45 and ST-574 complexes overlapped between isolates from patients and poultry. All C. jejuni were resistant to at least one antibiotic. The highest resistance rate was toward ciprofloxacin(89.7%), followed by tetracycline(74.6%), and nalidixic acid(69.0%). Conclusion This is the first report of the genotypes and antibiotic resistance of C. jejuni in Shenzhen. Overlapping clonal complexes were found between isolates from patients and cattle, and between patients and poultry.

A multivariate partial least squares approach to joint association analysis for multiple correlated traits

Many complex traits are highly correlated rather than independent. By taking the correlation structure of multiple traits into account, joint association analyses can achieve both higher statistical power and more accurate estimation. To develop a statistical approach to joint association analysis that includes allele detection and genetic effect estimation, we combined multivariate partial least squares regression with variable selection strategies and selected the optimal model using the Bayesian Information Criterion(BIC). We then performed extensive simulations under varying heritabilities and sample sizes to compare the performance achieved using our method with those obtained by single-trait multilocus methods. Joint association analysis has measurable advantages over single-trait methods, as it exhibits superior gene detection power, especially for pleiotropic genes. Sample size, heritability,polymorphic information content(PIC), and magnitude of gene effects influence the statistical power, accuracy and precision of effect estimation by the joint association analysis.

Emerging Carbapenem-Resistant <i>Enterobacter cloacae</i>Producing OXA-48-, VIM- and IMP-Type-<i>β</i>-Lactamases in Eastern Cape Hospitals in South Africa

Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.

MLST analysis of genetic diversity of Bacillus coagulans strains to evaluate effects on constipation model

Bacillus coagulans can help ameliorate or prevent gastrointestinal diseases, but the genetic relationships among B. coagulans isolates are not well studied. Multilocus sequence typing analysis was conducted on 57 isolates of B. coagulans from 22 provinces or autonomous regions in China. B. coagulans isolates were highly diverse and a total of 33(sequence typings) STs were found. These isolates had a weak clonal population structure and strong indications of intraspecies recombination. The evolution direction of B. coagulans was not correlated with geography or isolation source. Fifteen strains were selected for further analysis based on proximity relationships from the phylogenetic tree. Five isolates(B. coagulans-1, B. coagulans-10, B. coagulans-39, B. coagulans-70 and B. coagulans-71) with good spore-forming ability relative to the rest of the isolates were evaluated for constipation relief. B. coagulans-39 significantly relieved constipation symptoms in mice by regulating intestinal flora, increasing the production of short-chain fatty acids and restoring the level of gastrointestinal regulatory peptides. Comparative genomic analysis showed the beneficial effects of B. coagulans-39 might be associated with specific functional genes that are involved in the utilization of various carbohydrates as primary substrates and short-chain fatty acid production.

Prevalence and fuoroquinolone resistance of Campylobacter spp.isolated from beef cattle in Japan

Beef is a source of human Campylobacter infections.Antimicrobial treatment is needed when patients are immuno‑compromised or have other comorbidities.Therefore,we investigated the prevalence and antimicrobial resistance of Campylobacter spp.in beef cattle in Japan.Rectal swab samples were collected from 164 beef cattle at an abattoir between March 2021 and August 2021,and Campylobacter spp.were isolated from 94(57.3%)cattle.C.jejuni and C.coli were isolated from 68 and 26 cattle,respectively.For Campylobacter jejuni,the resistant rates against ampicillin,tetracycline and ciprofoxacin were 20.6,75.0 and 64.7%,respectively.For C.coli,the resistant rates against ampicil‑lin,tetracycline and ciprofoxacin were 53.8,76.9 and 88.5%,respectively.No Campylobacter isolates were resistant to erythromycin.By multilocus sequence typing,C.jejuni and C.coli isolates were classifed into 22 and 2 sequence types(STs).The top three STs of C.jejuni were ST806(12 isolates),ST21(nine isolates),and ST459(eight isolates).The most frequent ST of C.coli was ST1068(23 isolates).The results suggest that Campylobacter spp.are prevalent in the gastrointestinal tract of beef cattle slaughtered at abattoirs.Furthermore,the administration of erythromycin is efec‑tive against human campylobacteriosis caused by beef consumption.Monitoring the prevalence and antimicrobial resistance of Campylobacter spp.in beef cattle could be useful for managing the risk of human campylobacteriosis.

Molecular Diversity of <i>Staphylococcus aureus</i>from the Nares of Hospital Personnel, HIV-Positive and Diabetes Mellitus Patients in Yaounde Cameroon

Nasal carriage of Staphylococcus aureus has been identified as a risk factor for the development of staphylococcal infections caused by endogenous colonizing strains. Information on the genotypic diversity of Staphylococcus aureus is relevant for managing epidemiological and clinical challenges resulting from the evolutionary differences of this bacterium. The objective of this study was to determine and compare the molecular diversity of Staphylococcus aureus isolates from three high-risk populations in Yaounde, Cameroon. Molecular analysis confirmed that 95% of 100 tested isolates were S. aureus. The mecA and Panton Valentine-Leukocidin (PVL) genes (lukS/F-PV) were detected in 37% (35/95) and 43% (41/95) of isolates respectively and 18% (17/95) of the isolates harboured both the mecA and lukS/F-PV genes. A mixed distribution of both methicillin sensitive S. aureus (MSSA)/PVL and methicillin resistant S. aureus (MRSA)/PVL strains were detected within the study population. Community associated MRSA accounted for 94% (33/35) of the isolates, further classified into allotypes SCCmec type IV 54% (19/35) and SCCmec type V 40% (14/35), while two isolates were hospital associated SCCmec type II strains. A majority of the isolates harboured a single aggressive gene regulator allele agr type I. Pulsed Field Gel Electrophoresis (PFGE) generated 18 pulsotypes that grouped isolates irrespective of the study population. Multilocus Sequence Typing (MLST) of 12 selected isolates was assigned to six pandemic clonal complexes (CC): CC5 (ST5), CC8 [ST8, (n = 3)], CC15 (ST 15), CC25 (ST 25), CC72 [ST72 (n = 2)] and CC121 [ST 121 (n = 2)] and three atypical sequence types ST 508, ST 699 (CC45) and ST 1289 (CC 88). The study population represents an important reservoir for MRSA, MRSA-PVL and MSSA-PVL which could serve as focal point for further dissemination bringing about significant clinical and epidemiological implications. The predominance of SCCmec IV and agr types in this setting warrants further investigation. Isolates were genetically diverse with MLST indicating that pandemic ST8 was predominant. Detection of atypical STs has provided an insight into the necessity for constant monitoring.

Molecular Epidemiology of Nontuberculous Mycobacteria in a German CF Center and Clinical Course of NTM Positive Patients

Goal of this study was to analyse the clinical course of cystic fibrosis (CF) patients with nontuberculous mycobacteria (NTM) in their respiratory secretions and to investigate the molecular epidemiology of the most prevalent NTM species by multilocus sequence analysis (MLSA). The respiratory specimen and the clinical parameters forced expiratory volume in one second (FEV1), body-mass-index (BMI), erythrocyte sedimentation rate (ESR) 1 h and immunoglobulin G (IgG) of 357 CF patients, 0 - 52.4 years, mean FEV1 2009 81.5% pred were analysed between 1998 and 2010. In 13 patients NTM were detected. 12 of 13 patients carried M. abscessus, for one patient the NTM species was not characterized. 4 patients carried a second NTM species (M. avium, M. chelonae (2x), M. intracellulare). 6 patients exhibited a significant decline in FEV1, however changes in BMI, IgG and ESR were discordant. Molecular genotyping of M. abscessus isolates revealed a unique MLSA pattern in 6 patients. 2 patients harboured identical strains, and one patient a closely related strain. Whether the presence of identical strains is attributed to the acquisition of NTM clones from common environmental sources or to patient-to-patient transmission cannot be definitely clarified. Although cross-in- fection of the three patients with identical/closely related strains in the present cohort is highly unlikely, we recommend strict hygiene measures for all CF patients harbouring NTM.

BLUPmrMLM:A Fast mrMLM Algorithm in Genome-wide Association Studies

Multilocus genome-wide association study has become the state-of-the-art tool for dissecting the genetic architecture of complex and multiomic traits.However,most existing multilocus methods require relatively long computational time when analyzing large datasets.To address this issue,in this study,we proposed a fast mrMLM method,namely,best linear unbiased prediction multilocus random-SNP-effect mixed linear model(BLUPmrMLM).First,genome-wide single-marker scanning in mrMLM was replaced by vectorized Wald tests based on the best linear unbiased prediction(BLUP)values of marker effects and their variances in BLUPmrMLM.Then,adaptive best subset selection(ABESS)was used to identify potentially associated markers on each chromosome to reduce computational time when estimating marker effects via empirical Bayes.Finally,shared memory and parallel computing schemes were used to reduce the computational time.In simulation studies,BLUPmrMLM outperformed GEMMA,EMMAX,mrMLM,and FarmCPU as well as the control method(BLUPmrMLM with ABESS removed),in terms of computational time,power,accuracy for estimating quantitative trait nucleotide positions and effects,false positive rate,false discovery rate,false negative rate,and F1 score.In the reanalysis of two large rice datasets,BLUPmrMLM significantly reduced the computational time and identified more previously reported genes,compared with the aforementioned methods.This study provides an excellent multilocus model method for the analysis of large-scale and multiomic datasets.The software mrMLM v5.1 is available at BioCode(https://ngdc.cncb.ac.cn/biocode/tool/BT007388)or GitHub(https://github.com/YuanmingZhang65/mrMLM).

Public health implications of Yersinia enterocolitica investigation:an ecological modeling and molecular epidemiology study

Background Yersinia enterocolitica has been sporadically recovered from animals,foods,and human clinical samples in various regions of Ningxia,China.However,the ecological and molecular characteristics of Y.enterocolitica,as well as public health concerns about infection in the Ningxia Hui Autonomous Region,remain unclear.This study aims to analyze the ecological and molecular epidemiological characteristics of Y.enterocolitis in order to inform the public health intervention strategies for the contains of related diseases.Methods A total of 270 samples were collected for isolation[animals(n=208),food(n=49),and patients(n=13)],then suspect colonies were isolated and identified by the API20E biochemical identification system,serological tests,biotyping tests,and 16S rRNA-PCR.Then,we used an ecological epidemiological approach combined with machine learning algorithms(general linear model,random forest model,and eXtreme Gradient Boosting)to explore the associations between ecological factors and the pathogenicity of Y.enterocolitis.Furthermore,average nucleotide identity(ANI)estimation,single nucleotide polymorphism(SNP),and core gene multilocus sequence typing(cgMLST)were applied to characterize the molecular profile of isolates based on whole genome sequencing.The statistical test used single-factor analysis,Chi-square tests,t-tests/ANOVA-tests,Wilcoxon rank-sum tests,and Kruskal–Wallis tests.Results A total of 270 isolates of Yersinia were identified from poultry and livestock(n=191),food(n=49),diarrhoea patients(n=13),rats(n=15),and hamsters(n=2).The detection rates of samples from different hosts were statistically different(χ^(2)=22.636,P<0.001).According to the relatedness clustering results,270 isolates were divided into 12 species,and Y.enterocolitica(n=187)is a predominated species.Pathogenic isolates made up 52.4%(98/187),while non-pathogenic isolates made up 47.6%(89/187).Temperature and precipitation were strongly associated with the pathogenicity of the isolates(P<0.001).The random forest(RF)prediction model showed the best performance.The prediction result shows a high risk of pathogenicity Y.enterocolitica was located in the northern,northwestern,and southern of the Ningxia Hui Autonomous Region.The Y.enterocolitica isolates were classified into 54 sequence types(STs)and 125 cgMLST types(CTs),with 4/O:3 being the dominant bioserotype in Ningxia.The dominant STs and dominant CTs of pathogenic isolates in Ningxia were ST429 and HC100_2571,respectively.Conclusions The data indicated geographical variations in the distribution of STs and CTs of Y.enterocolitica isolates in Ningxia.Our work offered the first evidence that the pathogenicity of isolates was directly related to fluctuations in temperature and precipitation of the environment.CgMLST typing strategies showed that the isolates were transmitted to the population via pigs and food.Therefore,strengthening health surveillance on pig farms in high-risk areas and focusing on testing food of pig origin are optional strategies to prevent disease outbreaks.

Preliminary molecular epidemiology of the Staphylococcus aureus in lower respiratory tract infections： a multicenter study in China

Background Staphylococcus aureus （S. aureus） remains as an important microbial pathogen resulting in community and nosocomial acquired infections with significant morbidity and mortality. Few reports for S. aureus in lower respiratory tract infections （LRTIs） have been documented. The aim of this study was to explore the molecular epidemiology of S. aureus in LRTIs in China.Methods A multicenter study of the molecular epidemiology of S. aureus in LRTIs was conducted in 21 hospitals in Beijing, Shanghai and twelve other provinces from November 2007 to February 2009. All the collected S. aureus strains were classified as minimum inhibitory concentration （MIC）, mecA gene, virulence genes Panton-Valentine Leukocidin （PVL） and y-hemolysin （hlg）, staphylococcal cassette chromosome mec （SCCmec） type, agr type, and Multilocus Sequence Typinq （MLST）.Results Totally, nine methicillin-sensitive S. aureus （MSSA） and 29 methicillin-resistant S. aureus （MRSA） strains were isolated after culture from a total of 2829 sputums or bronchoalveolar lavages. The majority of MRSA strains （22/29） had a MIC value of 〉512 μg/ml for cefoxitin. The mecA gene acting as the conservative gene was carried by all MRSA strains. PVL genes were detected in only one S. aureus strain （2.63%, 1/38）. The hlg gene was detected in almost the all S. aureus （100% in MSSA and 96.56% in MRSA strains）. About 75.86% of MRSA strains carried SCCmec Ⅲ. Agr type 1 was predominant （78.95%） among the identified three agr types （agr types 1,2, and 3）. Totally, ten sequence type （ST） of S. aureus strains were detected. A new sequence type （ST1445） was found besides confirming ST239 as the major sequence type （60.53%）. A dendrogram generated from our own MLST database showed all the bootstrap values 〈50%. Conclusion Our preliminary epidemiology data show SCCmec Ⅲ, ST239 and agr type 1 of S. aureus as the predominant strains in LRTIs in Mainland of China.

A first meningococcal meningitis case caused by serogroup X Neisseria meningitidis strains in China

Neisseria meningitidis is the leading cause of bacterial meningitis and classified into 13serogroups based on the immunological reactivity of the capsular polysaccharide.1 Serogroups A, B, C, W135 and Y are the most common causes of meningitis.2 Among them, serogroup A and C are the major causes of epidemics in Africa and Asia.2 Most of the epidemic outbreaks of meningococcal meningitis are caused by serogroup A Neisseria meningitidis strain from the 1950s to the 1980s in China.3 During the years 2003 and 2005, a new sequence type （ST-4821） of serogroup C was identified in the Anhui and 11 other provinces of China.4

Five Colletotrichum species are responsible for mango anthracnose in northeastern Brazil

Colletotrichum species are the most important and widespread form of decay affecting mango fruit worldwide.In this study,Colletotrichum species associated with fruit anthracnose isolated from mango in northeastern Brazil were subject to molecular and morphological analyses.The partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene of 143 Colletotrichum isolates was amplified,as an initial measure of genetic diversity.A subset of 47 isolates,selected to represent the range of genetic diversity and geographic origin,were further sequenced using the partial actin,β-tubulin,calmodulin,glutamine synthetase genes and rDNA-ITS region.The multilocus sequence analysis,together with a critical examination of the phenotypic characters,revealed four previously described species(Colletotrichum asianum,Colletotrichum fructicola,Colletotrichum tropicale and Colletotrichum karstii)and one new species.The new species is introduced as Colletotrichum dianesei and formally described,illustrated and compared with similar taxa.Only C.asianum and C.karstii have previously been reported from mango,while the other species represent the first report associated with the mango fruits worldwide.All species are reported for the first time associated with the mango fruits in Brazil.