Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 complex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak. Contributed equally to this work Supported by the National Key Technologies Research and Development Program (Grant No. 2005BA711A09) from the Ministry of Science and Technology of China     

腹泻仔猪大肠埃希菌分离株ST4214的全基因组测序分析

分析腹泻仔猪大肠埃希菌(E.coli)分离株血清型、毒力因子及耐药基因。

本课题组前期研究从腹泻仔猪粪便样本中分离到64株E.coli, 本研究取其中含有毒力因子最多的1株进行全基因组测序分析。

E.coli分离株基因组序列为5.5 Mb, 预测蛋白质编码序列数为5 743, 与已知致病菌E.coli O145的基因重合率较低, 为64.53%。经多位点序列分型分析, 确定分离株为E.coli ST4214, 血清型为O3∶H45。与病原菌O157∶H7、O145∶H28和O104∶H4的毒力基因比较表明: 4株菌共同含有的毒力基因为354个, E.coli ST4214含有64种独特的毒力基因、编码鞭毛蛋白、Ⅳ型分泌蛋白、Ⅱ型分泌蛋白、溶血素、菌毛、肠毒素、荚膜多糖相关蛋白质。耐药基因E.coli分离株含有氨基香豆素抗性基因、磺胺类耐药基因、β-内酰胺抗性基因、多粘菌素耐药基因、肽抗生素抗性基因和抗生素耐药基因外排泵等多重耐药基因。

腹泻仔猪E.coli ST4214分离株是一株新菌株, 具有较多毒力因子及耐药基因, 可能与仔猪腹泻发病相关。

     

天津地区气单胞菌分离株的鉴定与多位点序列分型

[目的]研究气单胞菌菌株分类情况,并分析其致病性.[方法]采集环境样品和鱼类标本,分离并鉴定气单胞菌菌株,并运用多位点序列分型(Multilocus sequence typing,MLST)方法进行分类研究,利用PCR和测序方法分析毒力基因Aera、Hly、Aha1、GCAT和Nuc的分布.[结果]通过对分离菌株的16S rRNA基因进行分析,确认属于4种不同气单胞菌的7个分离株.发现所有菌株至少有1种毒力基因阳性,其中3株具有4种毒力基因.药物敏感实验显示,6株分离株对3种或3种以上抗菌素具有多重耐药性.最后,对看家基因gyrB、groL、gltA、metG、ppsA和recA进行分析,与MLST数据库中的等位基因序列比对,发现7株分离株均为新的不同的序列型(Sequence type,ST).[结论]气单胞菌具有较高的遗传多样性.     

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ～65 kb. Complete sequence analysis of the ～65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ～65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.     

Screening for antimicrobial resistance genes and virulence factors via genome sequencing

Second-generation genome sequencing and alignment of the resulting reads to in silico genomes containing antimicrobial resistance and virulence factor genes were used to screen for undesirable genes in 28 strains which could be used in human nutrition. No virulence factor genes were detected, while several isolates contained antimicrobial resistance genes.     

Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.     

Molecular epidemiology and microbiological characteristics of Cryptococcus gattii VGII isolates from China

Cryptococcus gattii (C. gattii) is a fungal pathogen that once caused an outbreak of cryptococcosis on Vancouver Island, and had spread worldwide, while few data were available in China. In this study, seven clinical isolates of C. gattii VGII were collected from 19 hospitals, Multi-locus Sequence Typing (MLST) analysis and whole-genome sequencing (WGS) was performed, combined with published data for phylogenetic analysis. In addition, in vitro antifungal susceptibility testing, phenotypic analysis, and in vivo virulence studies were performed, subsequently, histopathological analysis of lung tissue was performed. C.gattii VGII infected patients were mainly immunocompetent male, and most of them had symptoms of central nervous system (CNS) involvement. MLST results showed that isolates from China exhibited high genetic diversity, and sequence type (ST) 7 was the major ST among the isolates. Some clinical isolates showed a close phylogenetic relationship with strains from Australia and South America. All clinical isolates did not show resistance to antifungal drugs. In addition, there was no correlation between virulence factors (temperature, melanin production, and capsule size) and virulence while in vivo experiments showed significant differences in virulence among strains. Lung fungal burden and damage to lung tissue correlated with virulence, and degree of damage to lung tissue in mice may highlight differences in virulence. Our work seeks to provide useful data for molecular epidemiology, antifungal susceptibility, and virulence differences of C. gattii VGII in China.     

An Investigation of the Diversity of Strains of Enteroaggregative Escherichia coli Isolated from Cases Associated with a Large Multi-Pathogen Foodborne Outbreak in the UK

Following a large outbreak of foodborne gastrointestinal (GI) disease, a multiplex PCR approach was used retrospectively to investigate faecal specimens from 88 of the 413 reported cases. Gene targets from a range of bacterial GI pathogens were detected, including Salmonella species, Shigella species and Shiga toxin-producing Escherichia coli, with the majority (75%) of faecal specimens being PCR positive for aggR associated with the Enteroaggregative E. coli (EAEC) group. The 20 isolates of EAEC recovered from the outbreak specimens exhibited a range of serotypes, the most frequent being O104:H4 and O131:H27. None of the EAEC isolates had the Shiga toxin (stx) genes. Multilocus sequence typing and single nucleotide polymorphism analysis of the core genome confirmed the diverse phylogeny of the strains. The analysis also revealed a close phylogenetic relationship between the EAEC O104:H4 strains in this outbreak and the strain of E. coli O104:H4 associated with a large outbreak of haemolytic ureamic syndrome in Germany in 2011. Further analysis of the EAEC plasmids, encoding the key enteroaggregative virulence genes, showed diversity with respect to FIB/FII type, gene content and genomic architecture. Known EAEC virulence genes, such as aggR, aat and aap, were present in all but one of the strains. A variety of fimbrial genes were observed, including genes encoding all five known fimbrial types, AAF/1 to AAF/V. The AAI operon was present in its entirety in 15 of the EAEC strains, absent in three and present, but incomplete, in two isolates. EAEC is known to be a diverse pathotype and this study demonstrates that a high level of diversity in strains recovered from cases associated with a single outbreak. Although the EAEC in this study did not carry the stx genes, this outbreak provides further evidence of the pathogenic potential of the EAEC O104:H4 serotype.     

Virulence characteristic and MLST-agr genetic background of high-level mupirocin-resistant, MRSA isolates from Shanghai and Wenzhou, China

The emergence and prevalence of high-level mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MuH MRSA) is challenging the eradication of MRSA nasal carriage and the treatment of skin and soft tissue infections. To understand the potentially pathogenetic capacity and the genetic basis of MuH MRSA, it is important to have a detailed knowledge of the molecular traits of this organism. Fifty three MuH MRSA isolates were gathered from Shanghai (28 isolates) and Wenzhou (25 isolates) in China. These isolates, consisting of 27 different PFGE-SCCmec-spa patterns, were examined by PCR for 35 virulence genes and further typed using agr (accessory gene regulator) typing and MLST (multilocus sequence typing). All 53 strains were positive for the genes hlg/hlg variant and icaD, and negative for seb, sed, see, seh, eta, etb, hld, cap-5, and ACME-arcA. Compared with Wenzhou isolates, Shanghai isolates were more likely to carry seg (P?=?0.002) and several other genes which were not found in Wenzhou strains such as sec, sei, tst (P<0.001 each), and pvl (P?=?0.012), and less likely to contain sea (P<0.001), cna (P?=?0.031), and efb (P?=?0.045). MLST and agr typing showed that ST239-agr1, ST5-agr1, and ST239-agr2 were the common lineages in MuH MRSA isolates from these two different regions. Our results indicated that MuH MRSA strains from two different geographic regions of China have differences in distribution of some virulence genes, while their major MLST-agr genetic backgrounds were accordant.     

Biochemical characteristics and genetic diversity of <Emphasis Type="Italic">Vibrio</Emphasis> spp. and <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> strains isolated from the Lac of Bizerte (Tunisia)

This study characterized the phenotypic and genetic properties of Vibrio spp. and Aeromonas hydrophila strains isolated from seawater and mussels (Mytilus edulis and Crassostrea gigas) cultured in mollusc farm localized in the lac of Bizerte. The 37 strains (31 strains of V. alginolyticus, one strain of V. fluvialis, one strain of V. parahaemolyticus and four strains of A. hydrophila) typed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) showed a high polymorphism. Most of the isolates were resistant to at least two antimicrobial agents. All the tested strains were resistant to ampicillin. PCR was used to detect the presence of eight Vibrio cholerae virulence genes in the genome of the Vibrio spp. isolates. The results showed a wide dissemination of these genes in the genome of all Vibrio spp. isolates tested. Differentiation of these strains with the ERIC 2-PCR technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern.     

Identification of genes and genomic islands correlated with high pathogenicity in Streptococcus suis using whole genome tiling microarrays

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and sepsis in both pigs and humans. Infections in humans have been sporadic worldwide but two severe outbreaks occurred in China in recent years, while infections in pigs are a major problem in the swine industry. Some S. suis strains are more pathogenic than others with 2 sequence types (ST), ST1 and ST7, being well recognized as highly pathogenic. We analyzed 31 isolates from 23 serotypes and 25 STs by NimbleGen tiling microarray using the genome of a high pathogenicity (HP) ST1 strain, GZ1, as reference and a new algorithm to detect gene content difference. The number of genes absent in a strain ranged from 49 to 225 with a total of 632 genes absent in at least one strain, while 1346 genes were found to be invariably present in all strains as the core genome of S. suis, accounting for 68% of the GZ1 genome. The majority of genes are located in chromosomal blocks with two or more contiguous genes. Sixty two blocks are absent in two or more strains and defined as regions of difference (RDs), among which 26 are putative genomic islands (GIs). Clustering and statistical analyses revealed that 8 RDs including 6 putative GIs and 21 genes within these RDs are significantly associated with HP. Three RDs encode known virulence related factors including the extracellular factor, the capsular polysaccharide and a SrtF pilus. The strains were divided into 5 groups based on population genetic analysis of multilocus sequence typing data and the distribution of the RDs among the groups revealed gain and loss of RDs in different groups. Our study elucidated the gene content diversity of S. suis and identified genes that potentially promote HP.     

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

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

Genetic Affinities within a Large Global Collection of Pathogenic Leptospira: Implications for Strain Identification and Molecular Epidemiology

Leptospirosis is an important zoonosis with widespread human health implications. The non-availability of accurate identification methods for the individualization of different Leptospira for outbreak investigations poses bountiful problems in the disease control arena. We harnessed fluorescent amplified fragment length polymorphism analysis (FAFLP) for Leptospira and investigated its utility in establishing genetic relationships among 271 isolates in the context of species level assignments of our global collection of isolates and strains obtained from a diverse array of hosts. In addition, this method was compared to an in-house multilocus sequence typing (MLST) method based on polymorphisms in three housekeeping genes, the rrs locus and two envelope proteins. Phylogenetic relationships were deduced based on bifurcating Neighbor-joining trees as well as median joining network analyses integrating both the FAFLP data and MLST based haplotypes. The phylogenetic relationships were also reproduced through Bayesian analysis of the multilocus sequence polymorphisms. We found FAFLP to be an important method for outbreak investigation and for clustering of isolates based on their geographical descent rather than by genome species types. The FAFLP method was, however, not able to convey much taxonomical utility sufficient to replace the highly tedious serotyping procedures in vogue. MLST, on the other hand, was found to be highly robust and efficient in identifying ancestral relationships and segregating the outbreak associated strains or otherwise according to their genome species status and, therefore, could unambiguously be applied for investigating phylogenetics of Leptospira in the context of taxonomy as well as gene flow. For instance, MLST was more efficient, as compared to FAFLP method, in clustering strains from the Andaman island of India, with their counterparts from mainland India and Sri Lanka, implying that such strains share genetic relationships and that leptospiral strains might be frequently circulating between the islands and the mainland.     

Genotypic Diversity and Virulence Characteristics of Clinical and Environmental Vibrio vulnificus Isolates from the Baltic Sea Region

The genetic diversity of Vibrio vulnificus isolates from clinical and environmental sources originating from the Baltic Sea region was evaluated by multilocus sequence typing (MLST), and possible relationships between MLST clusters, potential genotypic and phenotypic traits associated with pathogenicity, and source of isolation were investigated. The studied traits included genotyping of polymorphic loci (16S rRNA, vcg, and pilF), presence/absence of potential virulence genes, including nanA, nab, and genes of pathogenicity regions, metabolic features, hemolytic activity, resistance to human serum, and cytotoxicity to human intestinal cells. MLST generated 35 (27 new) sequence types and divided the 53 isolates (including four reference strains) into two main clusters, with cluster I containing biotype 1 and 2 isolates of mainly environmental origin and cluster II containing biotype 1 isolates of mainly clinical origin. Cluster II isolates were further subdivided into two branches. Branch IIB included isolates from recent cases of wound infections that were acquired at the German Baltic Sea coastline between 2010 and 2011 and isolates from seawater samples of the same regions isolated between 1994 and 2010. Comparing the MLST data with the results of genotyping and phenotyping showed that strains of MLST cluster II possess a number of additional pathogenicity-associated traits compared to cluster I strains. Rapid microbiological methods such as matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry combined with typing of selected virulence-associated traits (e.g., serum resistance, mannitol fermentation, nanA, and pathogenicity region XII) could be used for risk assessment purposes regarding V. vulnificus strains isolated from the Baltic Sea region.     

Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China

A total of 108 S. aureus isolates from 16 major hospitals located in 14 different provinces in China were characterized for the profiles of 18 staphylococcal enterotoxin (SE) genes, 3 exfoliatin genes (eta, etb and etd), and the toxic shock syndrome toxin gene (tsst) by PCR. The genomic diversity of each isolate was also evaluated by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and accessory gene regulator (agr) typing. Of these strains, 90.7% (98/108) harbored toxin genes, in which tsst was the most prevalent toxin gene (48.1%), followed by sea (44.4%), sek (42.6%) and seq (40.7%). The see and etb genes were not found in any of the isolates tested. Because of high-frequency transfer of toxin gene-containing mobile genetic elements between S. aureus strains, a total of 47 different toxin gene combinations were detected, including a complete egc cluster in 19 isolates, co-occurrence of sea, sek and seq in 38 strains, and sec and sel together in 11 strains. Genetic typing by PFGE grouped all the strains into 25 clusters based on 80% similarity. MLST revealed 25 sequence types (ST) which were assigned into 16 clonal complexes (CCs) including 2 new singletons. Among these, 11 new and 6 known STs were first reported in the S. aureus strains from China. Overall, the genotyping results showed high genetic diversity of the strains regardless of their geographical distributions, and no strong correlation between genetic background and toxin genotypes of the strains. For genotyping S. aureus, PFGE appears to be more discriminatory than MLST. However, toxin gene typing combined with PFGE or MLST could increase the discriminatory power of genotyping S. aureus strains.     

Clonal analysis and virulent traits of pathogenic extraintestinal Escherichia coli isolates from swine in China

ABSTRACT: BACKGROUND: Extraintestinal pathogenic Escherichia coli (ExPEC) can cause a variety of infections outside the gastrointestinal tract in humans and animals. Infections due to swine ExPECs have been occurring with increasing frequency in China. These ExPECs may now be considered a new food-borne pathogen that causes cross-infections between humans and pigs. Knowledge of the clonal structure and virulence genes is needed as a framework to improve the understanding of phylogenetic traits of porcine ExPECs. RESULTS: Multilocus sequence typing (MLST) data showed that the isolates investigated in this study could be placed into four main clonal complexes, designated as CC10, CC1687, CC88 and CC58. Strains within CC10 were classified as phylogroup A, and these accounted for most of our porcine ExPEC isolates. Isolates in the CC1687 clonal complex, formed by new sequence types (STs), was classified as phylogroup D, with CC88 isolates considered as B2 and CC58 isolates as B1. Porcine ExPECs in these four clonal complexes demonstrated significantly different virulence gene patterns. A few porcine ExPECs were indentified in phylogroup B2, the phylogroup in which human ExPECs mainly exist. However some STs in the four clonal groups of porcine ExPECs were reported to cause extraintestinal infections in human, based on data in the MLST database. CONCLUSION: Porcine ExPECs have different virulence gene patterns for different clonal complexes. However, these strains are mostly fell in phylogenentic phylogroup A, B1 and D, which is different from human ExPECs that concentrate in phylogroup B2. Our findings provide a better understanding relating to the clonal structure of ExPECs in diseased pigs and indicate a need to re-evaluate their contribution to human ExPEC diseases.     

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.     

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.     

Genetic Characterization of Type A Enterotoxigenic Clostridium perfringens Strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.     

Molecular epidemiology of clinically high-risk Pseudomonas aeruginosa strains: Practical overview

In recent years, numerous outbreaks of multidrug-resistant Pseudomonas aeruginosa have been reported across the world. Once an outbreak occurs, besides routinely testing isolates for susceptibility to antimicrobials, it is required to check their virulence genotypes and clonality profiles. Replacing pulsed-field gel electrophoresis DNA fingerprinting are faster, easier-to-use, and less expensive polymerase chain reaction (PCR)-based methods for characterizing hospital isolates. P. aeruginosa possesses a mosaic genome structure and a highly conserved core genome displaying low sequence diversity and a highly variable accessory genome that communicates with other Pseudomonas species via horizontal gene transfer. Multiple-locus variable-number tandem-repeat analysis and multilocus sequence typing methods allow for phylogenetic analysis of isolates by PCR amplification of target genes with the support of Internet-based services. The target genes located in the core genome regions usually contain low-frequency mutations, allowing the resulting phylogenetic trees to infer evolutionary processes. The multiplex PCR-based open reading frame typing (POT) method, integron PCR, and exoenzyme genotyping can determine a genotype by PCR amplifying a specific insertion gene in the accessory genome region using a single or a multiple primer set. Thus, analyzing P. aeruginosa isolates for their clonality, virulence factors, and resistance characteristics is achievable by combining the clonality evaluation of the core genome based on multiple-locus targeting methods with other methods that can identify specific virulence and antimicrobial genes. Software packages such as eBURST, R, and Dendroscope, which are powerful tools for phylogenetic analyses, enable researchers and clinicians to visualize clonality associations in clinical isolates.