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

Aminoglycoside Resistance Gene ant(4′)-IIb of Pseudomonas aeruginosa BM4492, a Clinical Isolate from Bulgaria

The ant(4′)-IIb gene of Pseudomonas aeruginosa BM4492, which encodes an aminoglycoside 4′-O-adenylyltransferase, was identified as a coding sequence of 756 bp corresponding to a protein with a calculated mass of 27,219 Da. Analysis of the deduced sequence indicated that the protein was related to aminoglycoside 4′-O-adenylyltransferases IIa and Ia found in P. aeruginosa and gram-positive bacteria, respectively. The enzyme conferred resistance to amikacin and tobramycin but not to dibekacin, gentamicin, or netilmicin. The ant(4′)-IIb gene had a chromosomal location in five of six clinical isolates of P. aeruginosa tested and was plasmid borne in the remaining strain. The ant(4′)-IIb gene was detected by PCR in some clinical strains of P. aeruginosa from the same hospital but not in members of other bacterial genera

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

Consumption of penicillins in the community, European Union/European Economic Area, 1997-2017

Data on consumption of penicillins in the community were collected from 30 EU/European Economic Area (EEA) countries over two decades. This article reviews temporal trends, seasonal variation, presence of change-points and changes in the composition of the main subgroups of penicillins.For the period 1997-2017, data on consumption of penicillins, i.e. β-lactam antibacterials, penicillins (ATC group J01C), in the community aggregated at the level of the active substance, were collected using the WHO ATC/DDD methodology (ATC/DDD index 2019). Consumption was expressed in DDD per 1000 inhabitants per day and in packages per 1000 inhabitants per day. Consumption of penicillins was analysed based on ATC-4 subgroups, and presented as trends, seasonal variation, presence of change-points and compositional changes.In 2017, consumption of penicillins in the community expressed in DDD per 1000 inhabitants per day varied by a factor of 4.9 between countries with the highest (Spain) and the lowest (the Netherlands) consumption. An increase in consumption of penicillins, which was not statistically significant, was observed between 1997 and 2003 and up to 2010. A decrease, which was not statistically significant, was observed from 2010 onwards. Proportional consumption of combinations of penicillins, including β-lactamase inhibitors (J01CR) increased during 1997-2017, which coincided with a decrease in the proportional consumption of extended-spectrum penicillins (J01CA) and narrow-spectrum penicillins (J01CE).Considerable variation in the patterns of consumption of penicillins was observed between EU/EEA countries. The consumption of penicillins in the EU/EEA community did not change significantly over time, while the proportional consumption of combinations of penicillins increased

Consumption of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in the community, European Union/European Economic Area, 1997-2017

Data on consumption of tetracyclines, sulphonamides and trimethoprim, and other antibacterials were collected from 30 EU/European Economic Area (EEA) countries over two decades. This article reviews temporal trends, seasonal variation, presence of change-points and changes in the composition of main subgroups of tetracyclines, sulphonamides and trimethoprim and other antibacterials.For the period 1997-2017, data on consumption of tetracyclines (ATC group J01A), sulphonamides and trimethoprim (ATC group J01E), and other antibacterials (ATC group J01X) in the community and aggregated at the level of the active substance, were collected using the WHO ATC/DDD methodology (ATC/DDD index 2019). Consumption was expressed in DDD per 1000 inhabitants per day and in packages per 1000 inhabitants per day. Consumption of tetracyclines, sulphonamides and trimethoprim, and other antibacterials was analysed based on ATC-4 subgroups and presented as trends, seasonal variation, presence of change-points and compositional changes.In 2017, consumption of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in the community expressed in DDD per 1000 inhabitants per day varied considerably between countries. Between 1997 and 2017, consumption of tetracyclines did not change significantly, while its seasonal variation significantly decreased over time. Consumption of sulphonamides and trimethoprim significantly decreased until 2006, and its seasonal variation significantly decreased over time. The consumption of other antibacterials showed no significant change over time or in seasonal variation.Consumption and composition of tetracyclines, sulphonamides and trimethoprim, and other antibacterials showed wide variations between EU/EEA countries and over time. This represents an opportunity to further reduce consumption of these groups in some countries and improve the quality of their prescription

Consumption of macrolides, lincosamides and streptogramins in the community, European Union/European Economic Area, 1997-2017

Data on the consumption of macrolides, lincosamides and streptogramins (MLS) in the community were collected from 30 EU/European Economic Area (EEA) countries over two decades. This article reviews temporal trends, seasonal variation, presence of change-points and changes in composition of the main subgroups of MLS.For the period 1997-2017, data on consumption of MLS, i.e. ATC group J01F, in the community and aggregated at the level of the active substance, were collected using the WHO ATC/DDD methodology (ATC/DDD index 2019). Consumption was expressed in DDD per 1000 inhabitants per day and in packages per 1000 inhabitants per day. Consumption of MLS was analysed and presented as trends, seasonal variation, presence of change-points and compositional changes, using a classification based on mean plasma elimination half-life for macrolides.In 2017, consumption of MLS in the community expressed in DDD per 1000 inhabitants per day varied by a factor of 13 between countries with the highest (Greece) and the lowest (Sweden) consumption. Consumption of MLS did not change significantly up to 2003, after which it significantly increased up to 2007. No significant change was observed after 2007. Consumption of MLS showed high seasonal variation. The proportional consumption of long-acting macrolides significantly increased over time compared with that of intermediate-acting macrolides, and proportional consumption of the latter increased compared with that of short-acting macrolides.Consumption of MLS did not change significantly over time during 2007-2017, while the proportional consumption of long-acting macrolides increased. Seasonal variation remained high, which suggests that MLS are still prescribed inappropriately in many countries