Genetic diversity and antimicrobial resistance? in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.     

MLST clustering of Campylobacter jejuni isolates from patients with gastroenteritis,reactive arthritis and Guillain–Barré syndrome

Aims: To determine the diversity and population structure of Campylobacter jejuni (C. jejuni) isolates from Danish patients and to examine the association between multilocus sequence typing types and different clinical symptoms including gastroenteritis (GI), Guillain–Barré syndrome (GBS) and reactive arthritis (RA). Methods and Results: Multilocus sequence typing (MLST) was used to characterize 122 isolates, including 18 from patients with RA and 8 from patients with GBS. The GI and RA isolates were collected in Denmark during 2002–2003 and the GBS isolates were obtained from other countries. In overall, 51 sequence types (STs) were identified within 18 clonal complexes (CCs). Of these three CCs, ST‐21, ST‐45 and ST‐22 clonal complexes accounted for 64 percent of all isolates. The GBS isolates in this study significantly grouped into the ST‐22 clonal complex, consistent with the PubMLST database isolates. There was no significant clustering of the RA isolates. Conclusions: Isolates from Denmark were found to be highly genetically diverse. GBS isolates grouped significantly with clonal complex ST‐22, but the absence of clustering of RA isolates indicated that the phylogenetic background for this sequela could not be reconstructed using variation in MLST loci. Possibly, putative RA‐associated genes may vary, by recombination or expression differences, independent of MLST loci. Significance and Impact of the Study: MLST typing of C. jejuni isolates from Danish patients with gastroenteritis confirmed that the diversity of clones in Denmark is comparable to that in other European countries. Furthermore, a verification of the grouping of GBS isolates compared to RA isolates provides information about evolution of the bacterial population resulting in this important sequela.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

The Bacillus cereus group: novel aspects of population structure and genome dynamics

AIMS: To provide new insights into the population and genomic structure of the Bacillus cereus group of bacteria. METHODS AND RESULTS: The genetic relatedness among B. cereus group strains was assessed by multilocus sequence typing (MLST) using an optimized scheme based on seven chromosomal housekeeping genes. A set of 48 strains from different clinical sources was included, and six clonal complexes containing several genetically similar isolates from unrelated patients were identified. Interestingly, several clonal groups contained strains that were isolated from similar human sources. Furthermore, comparative whole genome sequence analysis of 16 strains led to the discovery of novel ubiquitous genome features of the B. cereus group, such as atypical group II introns, IStrons, and hitherto uncharacterized repeated elements. CONCLUSIONS: The B. cereus group constitutes a coherent population unified by the presence of ubiquitous and specific genetic elements which do not show any pattern, either in their sequences or genomic locations, which allows to differentiate between the member species of the group. Nevertheless, the population is very dynamic, as particular lineages of clinical origin can evolve to form clonal complexes. At the genome level, the dynamic behaviour is indicated by the presence of numerous mobile and repeated elements. SIGNIFICANCE AND IMPACT OF THE STUDY: The B. cereus group of bacteria comprises species that are of medical and economic importance. The MLST data, along with the primers and protocols used, will be available in a public, web-accessible database (http://mlstoslo.uio.no).     

陕西省猕猴桃溃疡病病原菌分离鉴定及分型研究

明确陕西省猕猴桃主产区丁香假单胞菌猕猴桃致病变种（Pseudomonas syringae pv. actinidiae，Psa）的变型，从而有针对性地制定猕猴桃溃疡病的综合防治策略，采集陕西省猕猴桃主产区周至、眉县、灞桥、渭南、长安和鄠邑不同年份发病样本进行病原菌的分离，对病原菌进行初步鉴定后再应用特异性PCR进行分子鉴定，并进行回接实验。从鉴定出的Psa菌株中选出16株不同年份不同地区的代表性菌株应用7个管家基因进行MLSA分型分析。共分离鉴定出86株不同地区不同年份的Psa菌株，并通过回接实验验证了其致病性。MLSA分型结果表明16株代表性Psa菌株均属于Psa3，且Psa3与Psa6的遗传距离最近。陕西省猕猴主产区的Psa变型为Psa3，近十年未发生变异。研究结果为今后Psa的检测鉴定及防治研究提供参考。     

Evaluation of molecular assays for identification Campylobacter fetus species and subspecies and development of a C. fetus specific real-time PCR assay

Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus subspecies identification, but these methods are laborious and expensive. Several PCR assays for C. fetus subspecies identification have been described, but a reliable comparison of these assays is lacking.     

多位点序列分型在食源性金黄色葡萄球菌分型中的应用研究

对食源性金黄色葡萄球菌进行多位点序列分型(MLST)分析,了解其基因型特征,并与流行病学资料进行对比分析。应用MLST方法对2012年石家庄市分离出的18株食源性金葡菌进行基因分型,并对该地区食源性金葡菌分子特性和流行病学特性进行分析。18株食源性金葡菌通过MLST分析得到10个ST序列型,其中ST5序列型最多,共5株;其次为ST464序列型,共3株;ST7型和ST15各2株;ST6型、ST9型、ST59型和ST2138型各1株,有2个菌株是2个新的ST型其ST码分别为287-1-1-8-1-1-1和10-14-8-6-278-3-2。本地区食源性金葡菌的ST型别丰富,主要流行克隆系为ST5和ST464,ST6、ST7、ST9、ST15、ST59和ST2138等克隆系也有分布。     

Comparison of the Diversilab® system with multi-locus sequence typing and pulsed-field gel electrophoresis for the characterization of Streptococcus agalactiae invasive strains

Streptococcus agalactiae (or group B streptococcus; GBS) is a leading cause of neonatal morbidity and mortality in the developed countries. Several epidemiological typing tools have been developed for GBS to investigate the association between genotype and disease and to assess genetic variations within genogroups. This study compared the semi-automated repetitive sequence-based PCR Diversilab® system (DL) with MLST and pulsed field gel electrophoresis (PFGE) for determining the relatedness of invasive GBS strains. We analysed 179 unrelated GBS strains isolated from adult (n = 108) and neonatal (n = 71) invasive infections. By MLST, strains were resolved into 6 clonal complexes (CCs) including 23 sequence-types (STs), and 4 unique STs, whereas DL differentiated these isolates into 12 rep-PCR clusters (rPCs) and 9 unique rep-PCR types. The discriminatory power of both methods was similar, with Simpson's diversity indexes of 71.9% and 70.6%, respectively. However, their clustering concordance was low with Wallace concordance coefficients inferior to 0.4. PFGE was performed on 31 isolates representative of the most relevant DLrPCs clustered within the 3 major MLST CCs (CC-17, CC-23 and CC-1). As already observed with MLST, the concordance of DL with PFGE was low for all three CCs (Wallace coefficient < 0.5), PFGE being more discriminative than rep-PCR. In summary, this work suggests that DL is less appropriate than MLST or PFGE to study GBS population genetic diversity.     

Campylobacter genotypes from poultry transportation crates indicate a source of contamination and transmission

Aims: Crates used to transport live poultry can be contaminated with Campylobacter, despite periodic sanitization, and are potential vectors for transmission between flocks. We investigated the microbial contamination of standard and silver ion containing crates in normal use and the genetic structure of associated Campylobacter populations. Methods and Results: Bacteria from crates were enumerated by appropriate culture techniques, and multilocus sequence typing (MLST) was used to determine the genetic structure of Campylobacters isolated from standard and silver ion containing crates. Compared to standard crates, counts of bacteria, including Campylobacter, were consistently lower on silver ion containing crates throughout the decontamination process. In total, 16 different sequence types were identified from 89 Campylobacter jejuni isolates from crates. These were attributed to putative source population (chicken, cattle, sheep, the environment, wild bird) using the population genetic model, structure . Most (89%) were attributed to chicken, with 22% attribution to live chicken and 78% to retail poultry meat. MLST revealed a progressive shift in allele frequencies through the crate decontamination process. Campylobacter on crates survived for at least 3 h after sanitization, a period of time equivalent to the journey from the processing plant to the majority of farms in the catchment, showing the potential for involvement of crates in transmission. Conclusions: Inclusion of a silver ion biocide in poultry transportation crates to levels demonstrating acceptable antibacterial activity in vitro reduces the level of bacterial contamination during normal crate use compared to standard crates. Molecular analysis of Campylobacter isolates indicated a change in genetic structure of the population with respect to the poultry‐processing plant sanitization practice. Significance and Impact of the Study: The application of a sustainable antimicrobial to components of poultry processing may contribute to reducing the levels of Campylobacter circulating in poultry.