Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.     

A Bacillus thuringiensis strain producing a polyglutamate capsule resembling that of Bacillus anthracis

Bacillus thuringiensis serovar Monterrey strain BGSC 4AJ1 produced a microscopically visible capsule that reacted with a fluorescent antibody specific for the poly-gamma-d-glutamic acid (PGA) capsule of Bacillus anthracis. PGA capsule biosynthesis genes with 75%, 81%, 72%, 65% and 63% similarity, respectively, to those of the B. anthracis capBCADE cluster were present on a plasmid (pAJ1-1). Strain BGSC 4AJ1, together with five strains of Bacillus cereus that hybridized to a PGA cap gene probe, were analyzed phylogenetically using six housekeeping genes of a B. cereus multilocus sequence typing scheme. Bacillus thuringiensis BGSC 4AJ1 shared four identical alleles with B. anthracis and was the second most closely related to this bacterium of the 674 isolates in the multilocus sequence typing database. The other cap+ strains were distributed among various lineages of Clade 1 of the B. cereus group.     

Differential in vitro infectious abilities of two common Japan-specific sequence-type (ST) clones of disease-associated ST-2032 and carrier-associated ST-2046 Neisseria meningitidis strains in human endothelial and epithelial cell lines

The Japan-specific sequence type (ST) clones, as well as several major epidemic-prone clones such as ST-32, have been identified previously among Neisseria meningitidis isolates in Japan. In this study, the infectious properties of various ST clones, including the two common Japan-specific ones, were examined and compared by in vitro infection assays using human endothelial and epithelial cell lines. The known invasive clones, as well as the Japan-specific ST-2032 strains that were frequently isolated from patients, exhibited high infectious abilities in adherence and invasion. In contrast, the Japan-specific ST-2046 and ST-198 strains, both of which were frequently isolated from carriers in Japan, were less efficient in adherence and invasion. The expression of the bacterial surface molecules such as pilin, Opc, Opa and PilC, and the lipooligosaccharide structure, did not differ between disease-associated and carrier-associated isolates. These results suggest that in vitro infection assays may discriminate between disease-associated (patient-dominant) and carrier-associated (carrier-dominant) meningococcal ST clones. The ST-2032 clone showed the highest infectious activity in vitro, suggesting that it may possess some unidentified factors necessary for the infectious ability that were not present in the ST-2046 clone with the lowest infectious ability.     

Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico

Aims: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Methods and Results: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed‐field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 μg ml^?1) and trimethoprim (MIC of >32 μg ml^?1); few were resistant to clindamycin (MIC₉₀ 4 μg ml^?1), erythromycin (MIC₉₀ 8 μg ml^?1) and tetracycline (MIC₉₀ 8 μg ml^?1); but none was resistant to azithromycin (MIC₉₀ 4 μg ml^?1), ciprofloxacin (MIC₉₀ 1 μg ml^?1) or gentamycin (MIC₉₀ 4 μg ml^?1). Most strains restricted with SmaI, but a combination of SmaI–KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST‐2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco‐2 cells, medium cytotoxicity with INT‐407 and Hep‐2 cells and high cytotoxicity with CHO cells. Conclusion: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. Significance and Impact of the Study: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.     

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     

Strengthening the laboratory diagnosis of pathogenic Corynebacterium species in the Vaccine era

Over the last three decades, successful implementation of the diphtheria vaccination in the developed and developing countries has reduced the infections caused by the toxigenic strains of Corynebacterium diphtheriae, but a concomitant increase in the invasive infections due to the nontoxigenic strains was seen. In addition, the recent reports on the emergence of nontoxigenic toxin gene‐bearing strains, having the potential to revert back to toxigenic form poses a significant threat to human beings. Besides infections caused by C. diphtheriae, the emergence of the respiratory, cutaneous and invasive infections by related pathogenic Corynebacterium species like C. ulcerans and C. pseudotuberculosis, complicate the diagnosis and management of infection. These observations together with the widespread prevalence of diphtheria in the vaccine era, necessitates the strengthening of the epidemiological surveillance and laboratory diagnosis of the pathogen. This review provides the overview of the advantages and limitations of different molecular methods and the role of MALDI‐TOF in the laboratory diagnosis of Diphtheria. The contribution of next generation sequencing technology and different genotyping techniques in understanding the pathogenicity, transmission dynamics and epidemiology of the C. diphtheriae is discussed.     

Molecular typing and occurrence of beta‐lactam resistance genes of Shigella sonnei strains isolated from 1983 to 2014 in the São Paulo state of Brazil

Shigella sonnei , which has generally been associated with dysentery in developed countries, has recently been emerging in developing countries. Specifically, in Brazil few published studies have that molecularly characterized this species. The aims of this study were to analyze the efficacy of typing using multiple‐locus variable‐number tandem‐repeat analysis (MLVA), study the phylogeny by multi‐locus sequence typing (MLST) and assess the presence of some beta‐lactam resistance genes in S. sonnei strains isolated from human diarrhoeic faeces in the São Paulo State in Brazil between 1983 and 2014. Seventy‐two such S. sonnei strains were typed by MLVA and grouped into two clusters. The discrimination index of MLVA was found to be 0.996. Twenty strains were typed by MLST as ST152. In addition, the bla _TEM gene was detected in eight (72.7%) of the 11 S. sonnei strains that had previously been shown to be resistant to β‐lactams. However, bla _{CTX‐M‐1group}, bla _{CTX‐M‐9group} and bla _SHV genes were not found. MLVA results suggested the existence of two prevalent subtypes in the S. sonnei strains studied, confirming previous results. Moreover, MLVA efficiently discriminated monomorphic S. sonnei species. Because the S. sonnei strains studied belonged to clonal complex 152 and all isolates were typed as ST152, MLST is not a suitable method for studying the population structure of S. sonnei . Although, the rates of β‐lactam resistance were not high in the present study, the frequency of bla _TEM may represent a risk for patients receiving antimicrobial treatment. Taken together, the results provide better molecular characterization of this globally clinically important pathogen.

     

Tropical Drosophila pandora carry Wolbachia infections causing cytoplasmic incompatibility or male killing

Wolbachia infections have been described in several Drosophila species, but relatively few have been assessed for phenotypic effects. Cytoplasmic incompatibility (CI) is the most common phenotypic effect that has been detected, while some infections cause male killing or feminization, and many Wolbachia infections have few host effects. Here, we describe two new infections in a recently described species, Drosophila pandora, one of which causes near‐complete CI and near‐perfect maternal transmission (the “CI” strain). The other infection is a male killer (the “MK” strain), which we confirm by observing reinitiation of male production following tetracycline treatment. No incompatibility was detected in crosses between CI strain males and MK strain females, and rare MK males do not cause CI. Molecular analyses indicate that the CI and MK infections are distantly related and the CI infection is closely related to the wRi infection of Drosophila simulans. Two population surveys indicate that all individuals are infected with Wolbachia, but the MK infection is uncommon. Given patterns of incompatibility among the strains, the infection dynamics is expected to be governed by the relative fitness of the females, suggesting that the CI infection should have a higher fitness. This was evidenced by changes in infection frequencies and sex ratios in population cages initiated at different starting frequencies of the infections.     

Wolbachia infection in natural populations of Dictyophara europaea,an alternative vector of grapevine Flavescence dorée phytoplasma: effects and interactions

The European lantern fly, Dictyophara europaea, is an alternative vector of the Flavescence dorée phytoplasma (FDp) disease of grapevine in European vineyards, enabling infection initiation from wild reservoir compartment (Clematis vitalba). Heretofore recorded rate of D. europaea FDp‐infection has been very low (3%), making it less epidemiologically significant than would be expected based on reservoir plant infection rate (30%). In this study we present findings on a heavily FDp‐infected D. europaea population (>60%), on the natural Wolbachia infection of populations with low FDp‐infection rates (DeWo+) and on Wolbachia absence in highly FDp‐infected population (DeWo?). We examine several possible causes underlying the differences in vector infection rates: (a) population genetic characteristics of D. europaea and correlation with Wolbachia strain wEur natural infections, (b) Wolbachia effects on fitness components of DeWo+ laboratory colony and (c) rate of reservoir plant FDp‐infection and differences in FDp genotypes harboured by low and highly infected vector populations. The vector genetic diversity level was found to be lower in DeWo+ than in uninfected individuals and to exhibit a different evolution of fixed haplotypes. All DeWo+ populations were infected with the same strain of wEur. The FDp was found to be genetically diversified (five genotypes) but had no relation to infection rates. We did not find evidence of fitness upgrades with regard to Wolbachia infection status. Although more experimentation is needed, it seems that Wolbachia confers protection against FDp or is in competition with FDp according to the observed correlations: low FDp‐infected vector populations are infected with Wolbachia and vice versa.     

Typing Chlamydia trachomatis: from egg yolk to nanotechnology

A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations and pathogenicity. Serotyping using the major outer membrane protein (MOMP) has been the mainstay of epidemiological work for several decades. However, the development of nucleic acid amplification techniques (NAAT) and easy access to sequencing have shifted the focus from MOMP serotypes to omp1 genotypes. However, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1 . This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems at hand to further simplify and automate the high-resolution genotyping method based on NAAT and sequencing of various targets in the C. trachomatis genome. Thereby, a high throughput can be achieved and more epidemiological information can be obtained. However, it is important to realize that culture of C. trachomatis may still be needed to detect and characterize new variants of C. trachomatis .