scholarly journals Cluster-specific gene markers enhance Shigella and enteroinvasive Escherichia coli in silico serotyping

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Xiaomei Zhang ◽  
Michael Payne ◽  
Thanh Nguyen ◽  
Sandeep Kaur ◽  
Ruiting Lan
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Type Species ◽  
Specific Gene ◽  
Read Mapping ◽  
Gene Markers ◽  
Genetic Characteristics ◽  
Content Type ◽  
Link Type ◽  
Invasion Mechanisms

Shigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. Differentiation of Shigella from EIEC is important for clinical diagnostic and epidemiological investigations. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli , making it difficult to find genetic markers to discriminate between Shigella and EIEC. In this study, we identified 10 Shigella clusters, seven EIEC clusters and 53 sporadic types of EIEC by examining over 17000 publicly available Shigella and EIEC genomes. We compared Shigella and EIEC accessory genomes to identify cluster-specific gene markers for the 17 clusters and 53 sporadic types. The cluster-specific gene markers showed 99.64% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella and EIEC serotype-specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired-end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70 and 99.74% cluster assignment accuracy for the assembled genomes and read mapping respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity of 99.40% for assembled genomes and 99.38% for read mapping for serotyping. The cluster-specific gene markers and our new serotyping tool, ShigEiFinder (installable package: https://github.com/LanLab/ShigEiFinder, online tool: https://mgtdb.unsw.edu.au/ShigEiFinder/), will be useful for epidemiological and diagnostic investigations.

scholarly journals Cluster-specific gene markers enhance Shigella and Enteroinvasive Escherichia coli in silico serotyping

2021 ◽  
Author(s):  
Xiaomei Zhang ◽  
Michael Payne ◽  
Thanh Nguyen ◽  
Sandeep Kaur ◽  
Ruiting Lan
Keyword(s):  
Escherichia Coli ◽  
Illumina Sequencing ◽  
In Silico ◽  
Specific Gene ◽  
Gene Markers ◽  
Genetic Characteristics ◽  
E Coli ◽  
Link Type ◽  
Invasion Mechanisms ◽  
Multiple Clusters

AbstractShigella and enteroinvasive Escherichia coli (EIEC) cause human bacillary dysentery with similar invasion mechanisms and share similar physiological, biochemical and genetic characteristics. The ability to differentiate Shigella and EIEC from each other is important for clinical diagnostic and epidemiologic investigations. The existing genetic signatures may not discriminate between Shigella and EIEC. However, phylogenetically, Shigella and EIEC strains are composed of multiple clusters and are different forms of E. coli. In this study, we identified 10 Shigella clusters, 7 EIEC clusters and 53 sporadic types of EIEC by examining over 17,000 publicly available Shigella/EIEC genomes. We compared Shigella and EIEC accessory genomes to identify the cluster-specific gene markers or marker sets for the 17 clusters and 53 sporadic types. The gene markers showed 99.63% accuracy and more than 97.02% specificity. In addition, we developed a freely available in silico serotyping pipeline named Shigella EIEC Cluster Enhanced Serotype Finder (ShigEiFinder) by incorporating the cluster-specific gene markers and established Shigella/EIEC serotype specific O antigen genes and modification genes into typing. ShigEiFinder can process either paired end Illumina sequencing reads or assembled genomes and almost perfectly differentiated Shigella from EIEC with 99.70% and 99.81% cluster assignment accuracy for the assembled genomes and mapped reads respectively. ShigEiFinder was able to serotype over 59 Shigella serotypes and 22 EIEC serotypes and provided a high specificity with 99.40% for assembled genomes and 99.38% for mapped reads for serotyping. The cluster markers and our new serotyping tool, ShigEiFinder (https://github.com/LanLab/ShigEiFinder), will be useful for epidemiologic and diagnostic investigations.Impact statementThe differentiation of Shigella strains from enteroinvasive E. coli (EIEC) is important for clinical diagnosis and public health epidemiologic investigations. The similarities between Shigella and EIEC strains make this differentiation very difficult as both share common ancestries within E. coli. However, Shigella and EIEC are phylogenetically separated into multiple clusters, making high resolution separation using cluster specific genomic markers possible. In this study, we identified 17 Shigella or EIEC clusters including five that were newly identified through examination of over 17,000 publicly available Shigella and EIEC genomes. We further identified an individual or a set of cluster-specific gene markers for each cluster using comparative genomic analysis. These markers can then be used to classify isolates into clusters and were used to develop an in silico pipeline, ShigEiFinder (https://github.com/LanLab/ShigEiFinder) for accurate differentiation, cluster typing and serotyping of Shigella and EIEC from Illumina sequencing reads or assembled genomes. This study will have broad application from understanding the evolution of Shigella/EIEC to diagnosis and epidemiology.Data summarySequencing data have been deposited at the National Center for Biotechnology Information under BioProject number PRJNA692536.RepositoriesRaw sequence data are available from NCBI under the BioProject number PRJNA692536.

scholarly journals ECTyper: in silico Escherichia coli serotype and species prediction from raw and assembled whole-genome sequence data

2021 ◽  
Vol 7 (12) ◽  
Author(s):  
Kyrylo Bessonov ◽  
Chad Laing ◽  
James Robertson ◽  
Irene Yong ◽  
Kim Ziebell ◽  
...  
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
In Silico ◽  
Type Species ◽  
Outbreak Detection ◽  
Health Concern ◽  
Whole Genome ◽  
Content Type ◽  
Link Type ◽  
O Antigens

Escherichia coli is a priority foodborne pathogen of public health concern and phenotypic serotyping provides critical information for surveillance and outbreak detection activities. Public health and food safety laboratories are increasingly adopting whole-genome sequencing (WGS) for characterizing pathogens, but it is imperative to maintain serotype designations in order to minimize disruptions to existing public health workflows. Multiple in silico tools have been developed for predicting serotypes from WGS data, including SRST2, SerotypeFinder and EToKi EBEis, but these tools were not designed with the specific requirements of diagnostic laboratories, which include: speciation, input data flexibility (fasta/fastq), quality control information and easily interpretable results. To address these specific requirements, we developed ECTyper (https://github.com/phac-nml/ecoli_serotyping) for performing both speciation within Escherichia and Shigella , and in silico serotype prediction. We compared the serotype prediction performance of each tool on a newly sequenced panel of 185 isolates with confirmed phenotypic serotype information. We found that all tools were highly concordant, with 92–97 % for O-antigens and 98–100 % for H-antigens, and ECTyper having the highest rate of concordance. We extended the benchmarking to a large panel of 6954 publicly available E. coli genomes to assess the performance of the tools on a more diverse dataset. On the public data, there was a considerable drop in concordance, with 75–91 % for O-antigens and 62–90 % for H-antigens, and ECTyper and SerotypeFinder being the most concordant. This study highlights that in silico predictions show high concordance with phenotypic serotyping results, but there are notable differences in tool performance. ECTyper provides highly accurate and sensitive in silico serotype predictions, in addition to speciation, and is designed to be easily incorporated into bioinformatic workflows.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽  
2021 ◽  
Vol 167 (3) ◽  
Author(s):  
Sathi Mallick ◽  
Shanti Kiran ◽  
Tapas Kumar Maiti ◽  
Anindya S. Ghosh
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Species ◽  
Pentose Phosphate ◽  
Bacterial Cells ◽  
Surface Adhesion ◽  
Content Type ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

scholarly journals Amedibacterium intestinale gen. nov., sp. nov., isolated from human faeces, and reclassification of Eubacterium dolichum Moore et al. 1976 (Approved Lists 1980) as Amedibacillus dolichus gen. nov., comb. nov

2020 ◽  
Vol 70 (6) ◽  
pp. 3656-3664 ◽  
Author(s):  
Nao Ikeyama ◽  
Atsushi Toyoda ◽  
Sho Morohoshi ◽  
Tadao Kunihiro ◽  
Takumi Murakami ◽  
...  
Keyword(s):  
In Silico ◽  
Type Species ◽  
Novel Species ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Content Type ◽  
Monophyletic Cluster ◽  
Link Type ◽  
Gene Sequence Analysis ◽  
Faecal Samples

Four strains (9CBEGH2T, 9BBH35, 6BBH38 and 6EGH11) of Gram-stain-positive, obligately anaerobic, rod-shaped bacteria were isolated from faecal samples from healthy Japanese humans. The results of 16S rRNA gene sequence analysis indicated that the four strains represented members of the family Erysipelotrichaceae and formed a monophyletic cluster with ‘ Absiella argi ’ strain N6H1-5 (99.4% sequence similarity) and Eubacterium sp. Marseille-P5640 (99.3 %). Eubacterium dolichum JCM 10413T (94.2 %) and Eubacterium tortuosum ATCC 25548T (93.7 %) were located near this monophyletic cluster. The isolates, 9CBEGH2T, ‘ A. argi ’ JCM 30884 and Eubacterium sp. Marseille-P5640 shared 98.7–99.1% average nucleotide identity (ANI) with each other. Moreover, the in silico DNA–DNA hybridization (DDH) values among three strains were 88.4–90.6%, indicating that these strains represent the same species. Strain 9CBEGH2T showed 21.5–24.1 % in silico DDH values with other related taxa. In addition, the ANI values between strain 9CBEGH2T and other related taxa ranged from 71.2 % to 73.5 %, indicating that this strain should be considered as representing a novel species on the basis of whole-genome relatedness. Therefore, we formally propose a novel name for ‘ A. argi ’ strains identified because the name ‘ A. argi ’ has been effectively, but not validly, published since 2017. On the basis of the collected data, strain 9CBEGH2T represents a novel species of a novel genus, for which the name Amedibacterium intestinale gen. nov., sp. nov. is proposed. The type strain of A. intestinale is 9CBEGH2T (=JCM 33778T=DSM 110575T).

scholarly journals d-Serine induces distinct transcriptomes in diverse Escherichia coli pathotypes

Microbiology ◽  
2021 ◽  
Vol 167 (10) ◽  
Author(s):  
James P. R. Connolly ◽  
Natasha C. A. Turner ◽  
Jennifer C. Hallam ◽  
Patricia T. Rimbi ◽  
Tom Flett ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Species ◽  
Pathogenic Bacteria ◽  
Neonatal Meningitis ◽  
Published Data ◽  
Toxic Metabolite ◽  
Transcriptional Responses ◽  
Content Type ◽  
E Coli ◽  
Link Type

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. d-Serine (d-ser) is a toxic metabolite present in highly variable concentrations at different colonization sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes – enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) – to d-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC, and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA – a genetic locus required for detoxification and hence normal growth in the presence of d-ser. Specific transcriptional responses were induced in strains accumulating d-ser (WT EHEC and UPEC/NMEC mutants lacking the d-ser-responsive transcriptional activator DsdC), corroborating the notion that d-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that d-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity.

Roseomonas oleicola sp. nov., isolated from an oil production mixture in Yumen Oilfield, and emended description of Roseomonas frigidaquae

2021 ◽  
Vol 71 (10) ◽  
Author(s):  
Danni Wu ◽  
Hongcan Liu ◽  
Yuguang Zhou ◽  
Xiaolei Wu ◽  
Yong Nie ◽  
...  
Keyword(s):  
Type Species ◽  
Genomic Analysis ◽  
Oil Production ◽  
Gene Families ◽  
Specific Gene ◽  
Rrna Gene ◽  
Strictly Aerobic ◽  
Content Type ◽  
Link Type ◽  
Type Strains

A pink, ovoid-shaped, Gram-stain-negative, strictly aerobic and motile bacterial strain, designated ROY-5-3T, was isolated from an oil production mixture from Yumen Oilfield in PR China. The strain grew at 4–42 °C (optimum, 30 °C), at pH 5–10 (optimum, 7) and with 0–5 % (w/v) NaCl (optimum, 0%). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that ROY-5-3T belongs to the genus Roseomonas and shared the highest pairwise similarities with Roseomonas frigidaquae CW67T (98.1%), Roseomonas selenitidurans BU-1T (97.8%), Roseomonas tokyonensis K-20T (97.7%) and Roseomonas stagni HS-69T (97.3%). The average nucleotide identity and digital DNA–DNA hybridization values between ROY-5-3T and other related type strains of Roseomonas species were less than 84.08 and 28.60 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate ROY-5-3T shared 3265 core gene families with the four closely related type strains in Roseomonas , and the number of strain-specific gene families was 513. The major fatty acids were identified as summed feature 8 (C18 : 1 ω6c/C18 : 1 ω7c), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c) and C16 : 0. Strain ROY-5-3T contained Q-10 as the main ubiquinone and the genomic DNA G+C content was 69.8 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. Based on the phylogenetic, morphological, physiological, chemotaxonomic and genome analyses, strain ROY-5-3T represents a novel species of the genus Roseomonas for which the name Roseomonas oleicola sp. nov. is proposed. The type strain is ROY-5-3T (=CGMCC 1.13459T =KCTC 82484T).

scholarly journals bla OXA-48-like genome architecture among carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the Netherlands

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Antoni P. A. Hendrickx ◽  
Fabian Landman ◽  
Angela de Haan ◽  
Sandra Witteveen ◽  
Marga G. van Santen-Verheuvel ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
The Netherlands ◽  
Resistance Genes ◽  
Type Species ◽  
Antibiotic Resistance Genes ◽  
Gene Content ◽  
Content Type ◽  
E Coli ◽  
Link Type

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48-like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48-like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48-like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48-like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48-like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48-like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48, bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48-like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48-like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

scholarly journals Lipopolysaccharide core type diversity in the Escherichia coli species in association with phylogeny, virulence gene repertoire and distribution of type VI secretion systems

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Sébastien O. Leclercq ◽  
Maxime Branger ◽  
David G. E. Smith ◽  
Pierre Germon
Keyword(s):  
Escherichia Coli ◽  
Type Species ◽  
Virulence Gene ◽  
Uneven Distribution ◽  
Gene Repertoire ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Wide Range ◽  
K 12

Escherichia coli is a very versatile species for which diversity has been explored from various perspectives highlighting, for example, phylogenetic groupings and pathovars, as well as a wide range of O serotypes. The highly variable O-antigen, the most external part of the lipopolysaccharide (LPS) component of the outer membrane of E. coli , is linked to the innermost lipid A through the core region of LPS of which five different structures, denominated K-12, R1, R2, R3 and R4, have been characterized so far. The aim of the present study was to analyse the prevalence of these LPS core types in the E. coli species and explore their distribution in the different E. coli phylogenetic groups and in relationship with the virulence gene repertoire. Results indicated an uneven distribution of core types between the different phylogroups, with phylogroup A strains being the most diverse in terms of LPS core types, while phylogroups B1, D and E strains were dominated by the R3 type, and phylogroups B2 and C strains were dominated by the R1 type. Strains carrying the LEE virulence operon were mostly of the R3 type whatever the phylogroup while, within phylogroup B2, strains carrying a K-12 core all belonged to the complex STc131, one of the major clones of extraintestinal pathogenic E. coli (ExPEC) strains. The origin of this uneven distribution is discussed but remains to be fully explained, as well as the consequences of carrying a specific core type on the wider aspects of bacterial phenotype.

scholarly journals Phylum barrier and Escherichia coli intra-species phylogeny drive the acquisition of antibiotic-resistance genes

2021 ◽  
Vol 7 (8) ◽  
Author(s):  
Marie Petitjean ◽  
Bénédicte Condamine ◽  
Charles Burdet ◽  
Erick Denamur ◽  
Etienne Ruppé
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Type Species ◽  
Antibiotic Resistance Genes ◽  
Resistance Pattern ◽  
Content Type ◽  
E Coli ◽  
Link Type ◽  
Specific Distribution

Escherichia coli is a ubiquitous bacterium that has been widely exposed to antibiotics over the last 70 years. It has adapted by acquiring different antibiotic-resistance genes (ARGs), the census of which we aim to characterize here. To do so, we analysed 70 301 E. coli genomes obtained from the EnteroBase database and detected 1 027 651 ARGs using the AMRFinder, Mustard and ResfinderFG ARG databases. We observed a strong phylogroup and clonal lineage specific distribution of some ARGs, supporting the argument for epistasis between ARGs and the strain genetic background. However, each phylogroup had ARGs conferring a similar antibiotic class resistance pattern, indicating phenotypic adaptive convergence. The G+C content or the type of ARG was not associated with the frequency of the ARG in the database. In addition, we identified ARGs from anaerobic, non- Proteobacteria bacteria in four genomes of E. coli , supporting the hypothesis that the transfer between anaerobic bacteria and E. coli can spontaneously occur but remains exceptional. In conclusion, we showed that phylum barrier and intra-species phylogenetic history are major drivers of the acquisition of a resistome in E. coli .

scholarly journals Genetic diversity and epidemiology of accessory gene regulator loci in Clostridioides difficile

2020 ◽  
Vol 2 (7) ◽  
Author(s):  
Yuta Okada ◽  
Shu Okugawa ◽  
Mahoko Ikeda ◽  
Tatsuya Kobayashi ◽  
Ryoichi Saito ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
In Silico ◽  
Type Species ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Accessory Gene ◽  
Content Type ◽  
Link Type ◽  
Accessory Gene Regulator ◽  
Clostridioides Difficile

Quorum sensing is known to regulate bacterial virulence, and the accessory gene regulator (agr) loci is one of the genetic loci responsible for its regulation. Recent reports examining Clostridioides difficile show that two agr loci, agr1 and agr2, regulate toxin production, but the diversity of agr loci and their epidemiology is unknown. In our study, in silico analysis was performed to research genetic diversity of agr, and C. difficile isolates from clinical samples underwent multilocus sequence typing (MLST) and PCR analysis of agr loci. To reveal the distribution of agr among different strains, phylogenetic analysis was also performed. In our in silico analysis, two different subtypes, named agr2R and agr2M, were found in agr2, which were previously reported. PCR analysis of 133 C . difficile isolates showed that 131 strains had agr1, 61 strains had agr2R, and 26 strains had agr2M; agr2R was mainly found in clade 1 or clade 2 organisms, whereas agr2M was only found in clade 4. With rare exception, agr1-negative sequence types (STs) belonged to clade C-Ⅰ and C-Ⅲ, and one clade 4 strain had agr2R. Our study revealed subtypes of agr2 not previously recognized, and the distribution of several agr loci in C. difficile . These findings provide a foundation for further functional and clinical research of the agr loci.

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