DEVELOPMENT AND PILOT VALIDATION OF A NOVEL PCR-BASED REPLICON TYPING SCHEME FOR PLASMID FAMILIES ASSOCIATED WITH ANTIBIOTIC RESISTANCE IN PSEUDOMONAS SPP.

Background. Pseudomonas species are ubiquitous environmental Gram-negative bacteria increasingly associated with difficult to treat healthcare-associated infections. Along with their substantial intrinsic antimicrobial resistance, the ability to acquire additional resistance and pathogenicity determinants contributes to increased morbidity and mortality. Plasmids represent the major vehicles of gene transfer among hospital strains. Accumulation and dissemination of resistance genes through horizontal gene transfer is exceptionally problematic since it leads to the emergence of multi-resistant and stable phenotypes highlighting the importance of novel tools for studying plasmid epidemiology. Materials and Methods. In this study we introduce a novel PCR-based replicon typing (PBRT) scheme for differentiation of various Pseudomonas spp. plasmid families requiring only two multiplex PCR (mPCR) assays. mPCR 1 is composed of previously published primer sets for IncP-1, IncP-7, IncP-9, IncQ, A/C, N, W, IncU. Primers for multiplex PCR 2 were designed after an in-depth in-silico bioinformatic analysis of the repA gene of more than 50 reference IncP-2, IncP-6, IncP-10, pKLC102-like and pMOS94-like plasmids some of which studied for the first time as a group. Results. The scheme was tested on a set of 90 previously genotyped multi-resistant clinical Pseudomonas spp. isolates. The detection rate of the target plasmid families was low in our strain collection. Replicons were registered in only 3/90 isolates from the IncP-7 (n=1), IncP-10 (n=1), and pMOS94-like (n=1) families.  This pilot study demonstrates a novel PBRT scheme applicable to Pseudomonas spp. targeting plasmids of incompatibility groups known to harbour genes associated with antibiotic resistance

Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis

Background: Infections due to antibiotic-resistant bacteria are threatening modern health care. However, estimating their incidence, complications, and attributable mortality is challenging. We aimed to estimate the burden of infections caused by antibiotic-resistant bacteria of public health concern in countries of the EU and European Economic Area (EEA) in 2015, measured in number of cases, attributable deaths, and disability-adjusted life-years (DALYs). Methods: We estimated the incidence of infections with 16 antibiotic resistance–bacterium combinations from European Antimicrobial Resistance Surveillance Network (EARS-Net) 2015 data that was country-corrected for population coverage. We multiplied the number of bloodstream infections (BSIs) by a conversion factor derived from the European Centre for Disease Prevention and Control point prevalence survey of health-care-associated infections in European acute care hospitals in 2011–12 to estimate the number of non-BSIs. We developed disease outcome models for five types of infection on the basis of systematic reviews of the literature. Findings: From EARS-Net data collected between Jan 1, 2015, and Dec 31, 2015, we estimated 671 689 (95% uncertainty interval [UI] 583 148–763 966) infections with antibiotic-resistant bacteria, of which 63·5% (426 277 of 671 689) were associated with health care. These infections accounted for an estimated 33 110 (28 480–38 430) attributable deaths and 874 541 (768 837–989 068) DALYs. The burden for the EU and EEA was highest in infants (aged <1 year) and people aged 65 years or older, had increased since 2007, and was highest in Italy and Greece. Interpretation: Our results present the health burden of five types of infection with antibiotic-resistant bacteria expressed, for the first time, in DALYs. The estimated burden of infections with antibiotic-resistant bacteria in the EU and EEA is substantial compared with that of other infectious diseases, and has increased since 2007. Our burden estimates provide useful information for public health decision-makers prioritising interventions for infectious diseases

High quality RNA extraction from Pseudomonas aeruginosa and other bacteria with a novel rapid and cost-effective method

AbstractHigh-quality mRNA extraction is essential for gene expression assays. In this study, we developed a rapid method (20 min) named FACTS for the extraction of intact RNA from Pseudomonas aeruginosa and compared its performance to established rapid techniques like RNAsnap and CiAR. The RNA integrity, yield, purity and presence of residual genomic DNA were adopted as assessment criteria. Multiple assays for RNA integrity were applied, including the Agilent 2200 TapeStation, QIAxcel capillary electrophoresis, and the newly available Qubit RNA Integrity and Quality (IQ) Assay Kit. The RNA purity and DNA/RNA yield were assessed by spectrophotometry and fluorimetry, respectively. Following Dnase treatment, two-step RT-qPCR for the expression of the rpoD reference gene was performed to evaluate the performance of each method. In terms of RNA integrity, FACTS showed the highest RNA integrity, while in terms of purity, CiAR scored best. RNAsnap resulted in a substantial amount of residual DNA. Pilot experiments for RNA extraction with FACTS from other Gram-negative and Gram-positive bacteria revealed promising results. FACTS is a novel RNA extraction method for rapid highly effective extraction of high-quality RNA from P. aeruginosa and can be used as a cost-efficient alternative to other methods in gene expression studies

Genomic Characterization of IMP-Producing <i>Pseudomonas aeruginosa</i> in Bulgaria Reveals the Emergence of IMP-100, a Novel Plasmid-Mediated Variant Coexisting with a Chromosomal VIM-4

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections represent a major public health concern and require comprehensive understanding of their genetic makeup. This study investigated the first occurrence of imipenemase (IMP)-carrying P. aeruginosa strains from Bulgaria. Whole genome sequencing identified a novel plasmid-mediated IMP-100 allele located in a a novel In4886 integron embedded in a putative Tn7700 transposon. Two other closely related chromosomal IMP variants, IMP-13 and IMP-84, were also detected. The IMP-producers were resistant to last-line drugs including cefiderocol (CFDC) (two out of three) and susceptible to colistin. The IMP-13/84 cassettes were situated in a In320 integron inserted in a Tn5051-like transposon as previously reported. Lastly, the p4782-IMP plasmid rendered the PA01 transformant resistant to CFDC, suggesting a transferable CFDC resistance. A variety of virulence factors associated with adhesion, antiphagocytosis, iron uptake, and quorum sensing, as well as secretion systems, toxins, and proteases, were confirmed, suggesting significant pathogenic potential consistent with the observed strong biofilm formation. The emergence of IMP-producing MDR P. aeruginosa is alarming as it remains unsusceptible even to last-generation drugs like CFDC. Newly detected IMP-100 was even located in a CFDC-resistant XDR strain

Improvement and Validation of a Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA8+) for <i>Klebsiella pneumoniae</i>, <i>Klebsiella variicola</i>, and <i>Klebsiella quasipneumoniae</i>

The genotyping of the multidrug-resistant Klebsiella pneumoniae species complex is essential to identify outbreaks and to track their source and spread. The aim of this study was to improve and extend the typeability, availability, cost and time efficiency of an existing multi-locus VNTR analysis (MLVA). A modified scheme (MLVA8+) was adopted and validated for strain-level differentiation of the three Klebsiella species involved in human pathology. A diverse set of 465 K. pneumoniae clinical isolates from 22 hospitals and 3 outpatient laboratories in Bulgaria were studied, where 315 were carbapenem-resistant. The MLVA8+ typeability was significantly improved and the typing data were validated against 158 isolates which were previously typed by WGS. The MLVA8+ results were highly concordant with the classic 7-locus MLST and the novel K. variicola MLST, but had greater congruency coefficients (adjusted Wallace). A major advantage was the differentiation of the hybrid cluster ST258 into its corresponding clades. Furthermore, the applicability of MLVA8+ was demonstrated by conducting a retrospective investigation of the intra-hospital spread of blaKPC-, blaNDM- and blaOXA-48-like producers. The MLVA8+ has improved utility and extended typing scope to K. variicola and K. quasipneumoniae, while its cost and time-to-result were reduced

Втора научна сесия за преподаватели и студенти 3-4 Октомври 2013, Варна

Варненски медицински форум (Varna Medical Forum) V. 2, Suppl 3(2013