Purpose. Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing.
Methodology. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson’s correlation coefficient.
Results. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here.
Conclusion. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.
Purpose. Solid-organ transplant recipients may display high rates of colonization and/or infection by multidrug-resistant bacteria. We analysed and compared the phenotypic and genotypic diversity of carbapenem-resistant (CR) strains of Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii isolated from patients in the Solid Organ Transplantation department of our hospital.
Methodology. Between March 2012 and August 2013, 56 CR strains from various biological fluids underwent antimicrobial susceptibility testing with VITEK 2, molecular analysis by PCR amplification and genotypic analysis with pulsed-field gel electrophoresis (PFGE). They were clustered according to antimicrobial drug susceptibility and genotypic profiles. Diversity analyses were performed by calculating Simpson’s diversity index and applying computed rarefaction curves.
Results/Key findings. Among K. pneumoniae, KP-producers predominated (57.1 %). VIM and OXA-23 carbapenemases prevailed among P. aeruginosa and A. baumannii (89.4 and 88.9 %, respectively). KPC-producing K. pneumoniae and OXA-23 A. baumannii were assigned in single PFGE pulsotypes. VIM-producing P. aeruginosa generated multiple pulsotypes. CR K. pneumoniae strains displayed phenotypic diversity in tigecycline, colistin (CS), amikacin (AMK), gentamicin (GEN) and co-trimoxazole (SXT) (16 clusters); P. aeruginosa displayed phenotypic diversity in cefepime (FEP), ceftazidime, aztreonam, piperacillin, piperacillin–tazobactam, AMK, GEN and CS (9 clusters); and A. baumannii displayed phenotypic diversity in AMK, GEN, SXT, FEP, tobramycin and rifampicin (8 clusters). The Simpson diversity indices for the interpretative phenotype and PFGE analysis were 0.89 and 0.6, respectively, for K. pneumoniae strains (P<0.001); 0.77 and 0.6 for P. aeruginosa (P=0.22); and 0.86 and 0.19 for A. baumannii (P=0.004).
Conclusion. The presence of different antimicrobial susceptibility profiles does not preclude the possibility that two CR K. pneumoniae or A. baumannii isolates are clonally related.
Purpose. To colonize and cause infection in the host, pathogens must be well equipped to respond to and survive in several hostile conditions. TolC, an outer membrane channel component used by multidrug efflux pumps and type I secretion systems, is considered to be largely involved in bacterial physiology and virulence. In this study, we attempted to investigate the possible roles of TolC2, a homologue of TolC, in the pathogenesis of Actinobacillus pleuropneumoniae.
Methodology. The cell viability was investigated under stress conditions (oxidative, thermal, acid and osmotic). Virulence was assessed by lethal intraperitoneal injection of mice. The underlying mechanisms of the attenuation were further explored by serum bactericidal, in vivo phagocytosis and organ burden assays.
Results/Key findings. The deletion of tolC2 caused increased sensitivity to oxidative, thermal and acid challenges, indicating a critical role of TolC2 in A. pleuropneumoniae survival under stress conditions. The intraperitoneal injection of mice showed that the ΔtolC2 mutant caused significantly decreased mortality, suggesting the involvement of TolC2 in the virulence of A. pleuropneumoniae. In the serum-killing assays, the ΔtolC2 mutant showed significantly reduced survival ability when exposed to fresh serum. By the in vivo phagocytosis assays, we found that the loss of tolC2 rendered the mutant susceptible to phagocytosis by macrophages. Finally, the organ burden assays revealed decreased colonization of ΔtolC2 in lungs, indicating a higher bacterial clearance rate in mice in the absence of TolC2.
Conclusion. Our findings demonstrate that TolC2 contributes to the virulence of A. pleuropneumoniae by helping survival and maximal colonization in the host.
Several adenoviruses are known to cause severe disease in veterinary species. Recent evidence suggests that canine adenovirus type 1 (CAV-1) persists in the tissues of healthy red foxes (Vulpes vulpes), which may be a source of infection for susceptible species. It was hypothesized that mustelids native to the UK, including pine martens (Martes martes) and Eurasian otters (Lutra lutra), may also be persistently infected with adenoviruses. Based on high-throughput sequencing and additional Sanger sequencing, a novel Aviadenovirus, tentatively named marten adenovirus type 1 (MAdV-1), was detected in pine marten tissues. The detection of an Aviadenovirus in mammalian tissue has not been reported previously. Two mastadenoviruses, tentatively designated marten adenovirus type 2 (MAdV-2) and lutrine adenovirus type 1 (LAdV-1), were also detected in tissues of pine martens and Eurasian otters, respectively. Apparently healthy free-ranging animals may be infected with uncharacterized adenoviruses with possible implications for translocation of wildlife.
Homeless individuals face an elevated risk of methicillin-resistant Staphylococcus aureus (MRSA) infection. Identifying the prevalence and risk factors for MRSA nasal colonization may reduce infection risk. A cross-sectional study was conducted at a health clinic for homeless persons in Boston, MA, USA (n=194). In-person interviews and nasal swab specimens were collected. MRSA isolates were genotyped using pulse-field gel electrophoresis (PFGE) and assessed for antibiotic susceptibility. The prevalence of MRSA nasal colonization was 8.3 %. Seventy-five percent of isolates reflected clonal similarity to USA300. USA100 (18.8 %) and USA500 (6.3 %) were also recovered. Resistance to erythromycin (81.3 %), levofloxacin (31.3 %) and clindamycin (23.1 %) was identified. Recent inpatient status, endocarditis, haemodialysis, heavy drinking, not showering daily and transience were positively associated with MRSA nasal colonization. Carriage of community-acquired MRSA strains predominated in this population, although nosocomial strains co-circulate. Attention to behavioural and hygiene-related risk factors, not typically included in MRSA prevention efforts, may reduce risk.
Extra-corporeal membrane oxygenation (ECMO) is a promising life-saving technique for critically ill patients. Bacterial infection is a frequent complication, and Escherichia coli the predominant causative pathogen, but little is known about the characteristics of E. coli strains in these infections. We therefore conducted a retrospective study of 33 E. coli strains responsible for 33 ECMO-related infections, in 30 subjects. Antimicrobial susceptibility, phylotyping, O-typing, clonal relatedness determination and the screening for four virulence factor genes were conducted. Polymicrobial infections were evidenced in 61.6 % of episodes, irrespective of E. coli characteristics. Extra-intestinal pathogenic strains represented the large majority (69.7 %) of all E. coli isolates. Their advantageous genetic background may explain their predominance in this context. The potential for targeted digestive decontamination should be investigated in these patients for whom infectious complications are a heavy burden.