日化产品中洋葱伯克霍尔德氏菌复合群(Bcc)的分类和神秘伯克霍尔德氏菌的耐药性研究

【目的】对从2020–2022年不同日化产品中分离的29株洋葱伯克霍尔德氏菌复合群(Burkholderia cepacia complex,Bcc)进行分类和分型,另将2020年前来源于日化产品中6株被鉴定为Burkholderia lata的菌株进行分类更正。探究神秘伯克霍尔德氏菌(Burkholderia aenigmatica)的耐药性。【方法】本文主要应用多位点分型研究方法(multilocus sequence typing,MLST),PCR扩增atpD、gltB、gyrB、recA、lepA、phaC和trp B 7个管家基因片段,将测序结果与MLST数据库中的数据比对分析,获得菌株各管家基因的编号和ST型(sequence type),对本检测中心分离自日化产品的Bcc进行分型;利用多位点序列分析(multilocus sequence analysis,MLSA),结合MLST中等位基因的核苷酸序列构建进化树,从而对Bcc进行系统发育分析和鉴定。利用最小抑菌浓度法(minimum inhibitory concentration,MIC)测定Bcc对常见防腐剂(1,...     

Biogeography of the ubiquitous marine bacterium Alteromonas macleodii determined by multilocus sequence analysis

Twenty‐three isolates of the widely distributed marine bacteria Alteromonas macleodii have been analysed by multilocus sequence analysis combined with phylogenetic and multivariate statistical analyses. The strains originated from the Pacific Ocean, Mediterranean Sea, English Channel, Black Sea and Thailand. Using the nucleotide sequences of nine loci for each of the 23 isolates, a robust identification was achieved of different clades within the single species. Strains generally clustered with the depth in the water column from which the isolate originated. Strains also showed more recombination with isolates from the same vicinity, suggesting that genetic exchange plays a role in diversification of planktonic marine prokaryotes. This study thus shows for the first time for a large set of isolates of a species of planktonic marine prokaryotes that multilocus sequence analysis overcomes the problems associated with the analysis of individual marker genes or presence of extensive recombination events. It can thus achieve intraspecific identification to the level of genotypes and, by comparison with relevant environmental data, ecotypes.     

Wolbachia 在花蓟马中的感染及其与寄主线粒体DNA多样性的关系

【目的】Wolbachia 是一种广泛存在于节肢动物中的胞内共生细菌,影响寄主的生物学特性。花蓟马 Frankliniella intonsa (Trybom)是重要的害虫,对农作物及园林植物造成危害。本研究旨在明确 Wolbachia 在花蓟马中的感染情况,并分析其与寄主线粒体DNA多样性的关系。【方法】采集中国境内26个花蓟马自然种群,运用多位点序列分型技术（multilocus sequence typing, MLST）对其体内 Wolbachia 感染率及株系进行分析;利用线粒体 COI 分子标记研究花蓟马的遗传分化及遗传多样性;通过比较感染和未感染 Wolbachia 个体 COI 数据,探究 Wolbachia 多样性与寄主线粒体DNA多样性之间的关系。【结果】花蓟马中 Wolbachia 的感染率为0%~60%,共检测到5种 Wolbachia 株系（wFint1,wFint2,wFint3,wFint4及wFint5）,均属于B大组且形成一个单系群。Wolbachia感染情况与这些花蓟马种群（除CC, GZ, TA和TY, N<5）的线粒体DNA多样性相关,表现为不感染 Wolbachia 的种群中线粒体DNA单倍型多样性（Hd）与核苷酸多样性（Pi）均高于感染 Wolbachia 的种群,且 Wolbachia 感染率与 Hd 呈显著负相关（ P <0.05）。AMOVA分析表明花蓟马线粒体DNA遗传分化与Wolbachia 感染情况有关。【结论】 Wolbachia 可能在侵染花蓟马种群后出现遗传分化;Wolbachia 感染与寄主线粒体DNA多样性有关。     

Multilocus sequence typing confirms synonymy but highlights differences between Candida albicans and Candida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.     

Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins

Eight strains of Bacillus cereus isolated from bacteremia and soft tissue infections were assigned to seven sequence types (STs) by multilocus sequence typing (MLST). Two strains from different locations had identical STs. The concatenated sequences of the seven STs were aligned with 65 concatenated sequences from reference STs and a neighbor-joining tree was constructed. Two strains were distantly related to all reference STs. Three strains were recovered in a clade that included Bacillus anthracis, B. cereus and rare Bacillus thuringiensis strains while the other three strains were assigned to two STs that were more closely affiliated to most of the B. thuringiensis STs. We conclude that invasive B. cereus strains do not form a single clone or clonal complex of highly virulent strains.     

Analysis of Recombination in Campylobacter jejuni from MLST Population Data

We analyze recombination in C. jejuni using MLST data from isolates taken from wild birds, cattle, wild rabbits, and water in a 100-km² study region in Cheshire, UK. We use a recent approximate likelihood method for inference, based on combining likelihood information from all pairs of segregating (polymorphic) sites in the data. We find substantial evidence for recombination, but only for recombination with short tract lengths, of around 225–750 bp. We estimate that the rate of recombination is of a similar magnitude to the rate of mutation.[Reviewing Editor: Dr. Magnus Nordborg]     

Genetic diversity of the Campylobacter genes coding immunodominant proteins

Three Campylobacter jejuni 72Dz/92 genes (cjaA (ompH1), cjaC (hisJ) and cjaD (omp18)) encoding immunodominant proteins are considered to be potential chicken vaccine candidates. The presence and conservation of cjaA, cjaC and cjaD genes among different Campylobacter clinical isolates were determined. The genes were detected in thirty Campylobacter strains using hybridization as well as Western blot analysis. However, PCR products of the predicted size were amplified only from ten out of thirty examined strains regardless of the employed primer pair. The nucleotide sequence of the C. jejuni 72Dz/92 genes was compared with the nucleotide sequences of their homologs cloned from other Campylobacter strains as well as with the whole genome sequence of C. jejuni NCTC 11168. The examined sequences revealed 0 to 16% divergence. Strain-dependent levels of divergence were observed. The polymorphism detected in cjaC was mainly within the 5' region of the gene, while the nucleotide substitutions in cjaA and cjaD are distributed uniformly along the whole genes. Most of the observed nucleotide substitutions occurred at the third base of the codons. This observation is consistent with the results of Western blot experiments.     

Cronobacter,the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis

Background

Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections

Results

Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains.

Conclusions

The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes ‘on the fly’, and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1121) contains supplementary material, which is available to authorized users.     

Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea

AIMS: To investigate the molecular epidemiological study of Staphylococcus aureus from staphylococcal food poisoning (SFP) incidents in South Korea. METHODS AND RESULTS: Three hundred and thirty-two strains isolated from ten provinces between June 1999 and January 2002 were characterized by staphylococcal enterotoxin genes, toxic shock syndrome toxin 1 (tst) gene, and exfoliative toxin genes. Toxin genotypes were sea-seh (n=197), sea (n=51), sea-seg-sei (n=14), seg-sei (n=10), seb (n=10), seb-sed-seg-sei-sej (n=3), sea-seg-seh-sei (n=1), sea-seb (n=1), sea-sec (n=1), seg-sei plus eta (n=4), and sea-seg-sei plus tst (n=40). Most of the strains could be classified into three clusters of pulsed-field gel electrophoresis (PFGE) types A and B with coagulase type VII and type E with coagulase type IV. Of the ten sequence types (ST), ST1, ST59, and ST30 were frequently showed by multilocus sequence typing. CONCLUSIONS: The strain belonging to PFGE pattern A with sea-seh gene, coagulase VII, and ST1 was the most epidemic clone of SFP incidents in Korea.     

Evolution of pathogenicity in the Bacillus cereus group

The Bacillus cereus group of bacteria comprises soil-dwelling saprophytes but on occasion these bacteria can cause a wide range of diseases in humans, including food poisoning, systemic infections and highly lethal forms of anthrax. While anthrax is almost invariably caused by strains from a single evolutionary lineage, Bacillus anthracis, variation in the virulence properties of strains from other lineages has not been fully addressed. Using multi-locus sequence data from 667 strains, we reconstructed the evolutionary history of the B. cereus group in terms of both clonal inheritance and recombination. The strains included 155 clinical isolates representing B. anthracis, and isolates from emetic and diarrhoeal food poisoning, septicaemia and related infections, wound, and lung infections. We confirmed the existence of three major clades and found that clinical isolates of B. cereus (with the exception of emetic toxin-producing strains) are evenly distributed between and within clades 1 and 2. B. anthracis in particular and emetic toxin-producing B. cereus show more clonal structure and are restricted to clade 1. Our characterization of the patterns of genetic exchange showed that there exist partial barriers to gene flow between the three clades. The pathogenic strains do not exhibit atypically high or low rates of recombination, consistent with the opportunistic nature of most pathogenic infections. However, there have been a large number of recent imports in clade 1 of strains from external origins, which is indicative of an on-going shift in gene-flow boundaries for this clade.