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

Dynamic of SARS-CoV-2 spread in Bulgaria, 2020-2022

The COVID-19 pandemic is associated with high morbidity and significant mortality worldwide. The objective of this study was to track the circulation pattern of SARS-CoV-2 in Bulgaria over three consecutive years (2020-2022) and to analyze the involvement of SARS-CoV-2 in cases of co-infections. A total of 98 247 clinical samples were tested for SARS-CoV-2 using a Real-Time RT-PCR method and 25.2% of them were positive. The positive rate for SARS-CoV-2 was greater among hospitalized patients compared to outpatients (p<0.05). Approximately 48.3% of all SARS-CoV-2-positive cases were male and 51.7% were female (p<0.05). SARS-CoV-2 positivity was highest in the group of oldest adults (≥65 years) (average 40.6%), and lowest in the group of youngest children (0-5 years) (average 9.4%). Several peaks in the spread of SARS-CoV-2 infections were observed. Among the 1 463 SARS-CoV-2 positive clinical samples examined for the presence of other respiratory viruses, 109 (7.5%) cases of co-infections were found. The greatest variety of co-infections with SARS-CoV-2 and other respiratory viruses was detected during the Omicron wave. Surveillance of SARS-CoV-2 is important to continue in the future in order not to miss the emergence of new genetic variants with increased infectivity, virulence or immune escape

SARS-COV-2 GENOMIC SURVEILLANCE IN BULGARIA INDICATES DIVERSE DYNAMICS DRIVEN BY MULTIPLE INTRODUCTIONS OF DIFFERENT VIRAL VARIANTS IN 2022

Background. Evolution of the emerging SARS-CoV-2 variants raises concerns about the possibility of accelerated transmission,  disease severity, diagnostic challenges, and reduced vaccine effectiveness in the ever-evolving COVID-19 pandemic worldwide. Objectives for this study were to build a comprehensive national system for monitoring and genomic surveillance of SARS-CoV-2  and to identify the introduced virus variants in the country. Methods. We analyzed SARS-CoV-2 infections in 7948 representative clinical samples collected in medical institutions in different  geographical regions of the country in 2022. Whole-genome next-generation sequencing of SARS-CoV-2 was performed on samples  from randomly selected SARS-CoV-2-positive individuals by using a modified ARTIC v3-tailed amplicon method. A bioinformatic and  phylogenetic analyses of the obtained sequences was carried out. Results. Significant dynamics was observed in the spread of viral variants in 2022, which is characterized by the introduction and  spread of multiple SARS-CoV-2 variants. The phylogenomic analysis identified a high genetic heterogeneiety composed of a total of 152 different viral clades divided into 3 main supergroups: 114 (75.0%) of which were Omicron sub-variants, 35 (23.0%) Delta sub-variants, and 3 (2.0%) recombinant forms. Conclusion. Viral variants and their sub-clades with different potentials to impact disease severity were identified and the  information was immediately published for use by decision-makers and the scientific community. The global pandemic of COVID-19  has shown the importance of molecular biological surveillance, which is an indispensable element of the modern approach in the  fight against infectious diseases

Metagenomic Investigation of the Short-Term Temporal and Spatial Dynamics of the Bacterial Microbiome and the Resistome Downstream of a Wastewater Treatment Plant in the Iskar River in Bulgaria

Waste Water Treatment Plants (WWTP) aim to reduce contamination in effluent water; however, studies indicate antimicrobial resistance genes (ARGs) persist post-treatment, potentially leading to their spread from human populated areas into the environment. This study evaluated the impact of a large WWTP serving 125,000 people on the Iskar River in Bulgaria, by characterizing the spatial and short-term temporal dynamics in bacterial community dynamics and resistance profiles of the surface water. Pairs of samples were collected biweekly on four dates from two different locations, one about 800 m after the WWTP effluents and the other 10 km downstream. Taxonomic classification revealed the dominance of Pseudomonodota and Bacteriodota, notably the genera Flavobacterium, Aquirufa, Acidovorax, Polynucleobacter, and Limnohabitans. The taxonomic structure corresponded with both lentic and lotic freshwater habitats, with Flavobacterium exhibiting a significant decrease over the study period. Principal Coordinate Analysis revealed statistically significant differences in bacterial community composition between samples collected on different dates. Differential abundance analysis identified notable enrichment of Polynucleobacter and Limnohabitans. There were shifts within the enriched or depleted bacterial taxa between early and late sampling dates. High relative abundance of the genes erm(B), erm(F), mph(E), msr(E) (macrolides); tet(C), tet(O), tet(W), tet(Q) and tet(X) (tetracyclines); sul1 and sul2 (sulphonamides); and cfxA3, cfxA6 (beta-lactams) were detected, with trends of increased presence in the latest sampling dates and in the location closer to the WWTP. Of note, genes conferring resistance to carbapenems blaOXA-58 and blaIMP-33-like were identified. Co-occurrence analysis of ARGs and mobile genetic elements on putative plasmids showed few instances, and the estimated human health risk score (0.19) according to MetaCompare2.0 was low. In total, 29 metagenome-assembled genomes were recovered, with only a few harbouring ARGs. This study enhances our understanding of freshwater microbial community dynamics and antibiotic resistance profiles, highlighting the need for continued ARGs monitoring

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

Cross-Over Pathogenic Bacteria Detected in Infected Tomatoes (Solanum lycopersicum L.) and Peppers (Capsicum annuum L.) in Bulgaria

The ability of certain human pathogens to adapt to plants without losing their virulence toward people is a major concern today. Thus, the aim of the present work was the investigation of the presence of cross-over pathogenic bacteria in infected tomato and pepper plants. The objects of the study were 21 samples from seven different parts of the plants and three from tomato rhizosphere. In total, 26 strains were isolated, identified by MALDI-TOF, and phenotypically characterized. The PCR amplification of the rpoB gene was applied as an approach for the rapid detection of cross-over pathogens in plant samples. A great bacterial diversity was revealed from tomato samples as nine species were identified (Leclercia adecarboxylata, Pseudesherichia vulneris, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter bugandensis, Acinetobacter calcoaceticus, Pantoea agglomerans, Pantoea ananatis, and Pectobacterium carotovorum). Polymicrobial contaminations were observed in samples T2 (tomato flower) and T10 (tomato fruit). Five species were identified from pepper samples (P. agglomerans, L. adecarboxylata, Pseudomonas sp., Pseudomonas putida, and Enterococcus sp.). Antibiotic resistance patterns were assigned in accordance with EFSA recommendations. All isolates showed varying resistance to the tested antibiotics. The genetic basis for the phenotypic antibiotic resistance was not revealed. No genes for the virulence factors were found among the population. To our knowledge, this is the first overall investigation of tomato and pepper cross-over pathogenic bacterial populations in Bulgaria

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

Genomic Epidemiology and Lineage Dynamics of SARS-CoV-2 in Bulgaria: Insights from a Three-Year Pandemic Analysis

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought about significant challenges worldwide. In this study, we present a comprehensive analysis of the genomic epidemiology and lineage dynamics of SARS-CoV-2 in Bulgaria over a three-year period. Through extensive genomic sequencing and data analysis, we investigated the evolution of the virus, the emergence of variants of concern (VOCs), and their impact on the country’s pandemic trajectory. We also assessed the relationship between viral diversity and COVID-19 morbidity and mortality in Bulgaria. Our findings shed light on the temporal and spatial distribution of SARS-CoV-2 lineages and provide crucial insights into the dynamics of the pandemic in the country. The interplay between international travel and viral transmission plays a significant role in the emergence and dissemination of different SARS-CoV-2 variants. The observed proportions of exportation to various continents provide insights into the potential pathways through which these lineages spread globally. Understanding the genomic epidemiology of SARS-CoV-2 in Bulgaria is essential for formulating targeted public health strategies, enhancing vaccination efforts, and effectively managing future outbreaks

Whole genomes from bacteria collected at diagnostic units around the world 2020

Abstract The Two Weeks in the World research project has resulted in a dataset of 3087 clinically relevant bacterial genomes with pertaining metadata, collected from 59 diagnostic units in 35 countries around the world during 2020. A relational database is available with metadata and summary data from selected bioinformatic analysis, such as species prediction and identification of acquired resistance genes