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In patients with cystic fibrosis (CF) lung damage secondary to chronic infection is the main cause of death. Treatment of lung disease to reduce the impact of infection, inflammation and subsequent lung injury is therefore of major importance. As Pseudomonas aeruginosa is the dominant pathogen in CF patients it has been the major target of all treatment strategies, possible antibiotic regimens and recommendations for years. More sophisticated antibiotic therapies introduced over the last decades have helped to improve the prognosis in cystic fibrosis, but then new multidrug-resistant pathogens emerged.

Delftia acidovorans is an aerobic, non-fermenting Gram-negative bacterium (NFGNB), found in soil, water and hospital environments. It is rarely clinically significant, most commonly affecting hospitalized or immunocompromised patients. The present study aimed to explore the genomic characteristics of a Bulgarian clinical D. acidovorans isolate (designated Dac759) in comparison to all strains of this species with available genomes in the NCBI Genome database (n = 34). Dac759 was obtained in 2021 from the sputum of a 65-year-old female immunocompetent outpatient with bronchitis. Species identification using MALDI-TOF mass spectrometry, antimicrobial susceptibility testing, whole-genome sequencing (WGS), and phylogenomic analysis were performed. The isolate demonstrated high-level resistance to colistin (16 mg L-1); resistance to gentamicin; reduced susceptibility to piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, ciprofloxacin, and levofloxacin; and susceptibility to imipenem, meropenem, amikacin, and tobramycin. The observed genome size (6.43 Mb) and GC content (66.76%) were comparable with the accessible data from sequenced D. acidovorans genomes. A limited number of resistance determinants were identified in the assembled genome as follows: blaOXA-459, emrE, oqxB, and mexCD-oprJ. The phylogenomic analysis indicated a high heterogenicity of the included D. acidovorans genomes. In conclusion, to the best of our knowledge, this is the first documented case of a clinically relevant D. acidovorans isolate in Bulgaria. Unlike the majority of reports in the literature, Dac759 affected a patient with no malignancies or other preexisting comorbidities. With this in mind, its genome sequence is a valuable resource for the fundamental study of uncommon bacterial pathogens of public health importance.

Extensively drug-resistant P. aeruginosa (XDR-PA) has been highlighted as a serious public health threat. The present study aimed to explore the genomic characteristics of two Vietnamese extended-spectrum β-lactamase-9 (VEB-9)-producing XDR-PA isolates from Bulgaria in comparison to all bla VEB-9 -positive strains with available genomes. The isolates designated Pae51 and Pae52 were obtained from tracheobronchial aspirates of intensive care unit (ICU) patients. Antimicrobial susceptibility testing, whole-genome sequencing, RT-qPCR, and phylogenomic analysis were performed. Pae51 and Pae52 were resistant to most antipseudomonal β-lactams including carbapenems, aminoglycosides, and fluoroquinolones but remained susceptible to colistin and cefiderocol. Numerous resistance determinants were detected: bla VEB-9 , bla PDC-3 , bla OXA-10 , bla OXA-50 , aac(6')-II , ant(2″)-Ia , ant(3″)-IIa , aph(3')-IIb , cprP , catB7 , dfrB2 , sul1 , fosA , and tet(A) . Both isolates carried complex integrons with bla VEB-9 and tet(A) embedded next to the conservative 3' end sequences. A variety of virulence factors were also identified, including the type III secretion system exotoxin U. Pae51 and Pae52 differed by only four SNPs and belonged to the high-risk clone ST357. To our knowledge, this is the first report of bla VEB-9 -positive XDR-PA isolates in Bulgaria presenting a detailed genomic analysis. The development of novel antimicrobial strategies for such pathogens should be an essential part of infection control stewardship practices in ICU wards.

The present study aimed to explore the genomic characteristics of eight New Delhi metallo-β-lactamase-1 (NDM-1)-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Bulgarian tertiary hospital (2021-2023) in comparison to blaNDM-1-positive strains originating from the Balkans. Antimicrobial susceptibility testing, phenotypic assays for carbapenemase activity, PCR screening, whole-genome sequencing (WGS), and phylogenomic analysis were performed. Seven of the CRPA isolates investigated (Minimum inhibitory concentration values of imipenem and meropenem >32 mg L-1) were also resistant to piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, cefepime, ceftolozane-tazobactam, amikacin, tobramycin, ciprofloxacin, and levofloxacin, but were susceptible to colistin (0.5-2 mg L-1) and cefiderocol (0.25-1 mg L-1). The P. aeruginosa Pae57 isolate (designated Pae57) remained susceptible to aminoglycosides as well. WGS uncovered the co-existence of blaNDM-1 and blaGES-1. The isolates belonged to the ST654 high-risk clone, except for Pae57 (ST611). Alignment against reference sequences revealed the presence of a Tn21 transposon harboring bleMBL-blaNDM-1-ISAba125. It was similar to that found in the P. aeruginosa ST654 NDM1_1 strain (GCA_020404785.1) from Serbia. Phylogenomic analysis of our isolates indicated that seven of them (ST654) differed from each other in no more than 44 single-nucleotide polymorphisms (SNPs). Pae57 (ST611) was strikingly different (>21,700 SNPs) compared to all Balkan strains. In conclusion, to our knowledge this is the first report of blaNDM-1-positive P. aeruginosa ST611 isolation, which indicates the transmission dynamics of this determinant between high-risk and potentially high-risk P. aeruginosa clones. Obtained results unveil the dissemination of clonally related NDM-1-producing P. aeruginosa strains in the monitored hospital for approximately a 2-year period.

Cefiderocol (CFDC) is a first-in-class siderophore cephalosporin with potent activity against multidrug-resistant Gram-negative bacteria including carbapenem-resistant Acinetobacter baumannii. The present study aimed to explore the CFDC resistance mechanisms of an extensively drug-resistant A. baumannii isolate from Bulgaria. The A. baumannii Aba52 strain (designated Aba52) was obtained in 2018 from a blood sample of a critically ill patient. The methodology included antimicrobial susceptibility testing, whole-genome sequencing (WGS), reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), multilocus sequence typing, and phylogenomic analysis. The isolate demonstrated high-level resistance to CFDC (MIC = 64 mg L-1), resistance to carbapenems, aminoglycosides, fluoroquinolones, sulfamethoxazole-trimethoprim, and tigecycline, as well as susceptibility only to colistin. WGS-based resistome analysis revealed the existence of blaOXA-23, blaOXA-66 and blaADC-73. Seven non-conservative missense mutations affecting iron transport-related genes were detected: exbD4 (p.Ser61Pro), tonB2 (p.Ala268Val), bauA (p.Thr61Ala), ftsI (p.Ala515Val), piuA (p.Gly216Val), and feoB (p.Ser429Pro and p.Thr595Ala). A variety of virulence factors associated with adherence, biofilm formation, enzyme production, immune invasion, iron uptake, quorum sensing, and two-component regulatory systems were identified, suggesting a significant pathogenic potential of Aba52. The performed RT-qPCR analysis showed diminished (0.17) and absent expression of the pirA and piuA genes, respectively, encoding TonB-dependent siderophore receptors. Aba52 belonged to the widespread high-risk sequence type ST2 (Pasteur scheme). To the best of our knowledge, this is the first documented case of CFDC-resistant A. baumannii in Bulgaria even though, CFDC has never been applied in our country. The emerging resistance highlights the crucial need for nationwide surveillance targeting the implementation of novel antibiotics.

The present study aimed to explore the virulence characteristics in 221 Bulgarian nosocomial Stenotrophomonas maltophilia isolates (2011-2022) via screening for the presence of virulence genes, their mutational variability, and the corresponding enzyme activity. PCR amplification, enzymatic assays, whole-genome sequencing (WGS), and biofilm quantification on a polystyrene plate were performed. The incidence of virulence determinants was as follows: stmPr1 (encoding for the major extracellular protease StmPr1) 87.3%, stmPr2 (minor extracellular protease StmPr2) 99.1%, Smlt3773 locus (outer membrane esterase) 98.2%, plcN1 (non-hemolytic phospholipase C) 99.1%, and smf-1 (type-1 fimbriae, biofilm-related gene) 96.4%. The 1621-bp allele of stmPr1 was most frequently found (61.1%), followed by the combined allelic variant (17.6%), stmPr1-negative genotype (12.7%), and 868-bp allele (8.6%). Protease, esterase, and lecithinase activity was observed in 95%, 98.2%, and 17.2% of the isolates, respectively. The WGS-subjected isolates (n = 9) formed two groups. Five isolates possessed only the 1621-bp variant of stmPr1, higher biofilm formation ability (Optical Density at λ = 550 nm (OD550): 1.253-1.789), as well as a low number of mutations in the protease genes and smf-1. Three other isolates had only the 868-bp variant, weaker biofilm production (OD550: 0.788-1.108), and higher number of mutations within these genes. The only weak biofilm producer (OD550 = 0.177) had no stmPr1 alleles. In conclusion, the similar PCR detection rates did not allow differentiation of the isolates. In contrast, WGS permitted stmPr1 alleles-based differentiation. To the best of our knowledge, this is the first Bulgarian study presenting genotypic and phenotypic insights into virulence factors of S. maltophilia isolates.

Non-fermenting Gram-negative bacilli (NFGNB), such as Pseudomonas aeruginosa and Acinetobacter baumannii , are among the major opportunistic pathogens involved in the global antibiotic resistance epidemic. They are designated as urgent/serious threats by the Centers for Disease Control and Prevention and are part of the World Health Organization's list of critical priority pathogens. Also, Stenotrophomonas maltophilia is increasingly recognized as an emerging cause for healthcare-associated infections in intensive care units, life-threatening diseases in immunocompromised patients, and severe pulmonary infections in cystic fibrosis and COVID-19 individuals. The last annual report of the ECDC showed drastic differences in the proportions of NFGNB with resistance towards key antibiotics in different European Union/European Economic Area countries. The data for the Balkans are of particular concern, indicating more than 80% and 30% of invasive Acinetobacter spp. and P. aeruginosa isolates, respectively, to be carbapenem-resistant. Moreover, multidrug-resistant and extensively drug-resistant S. maltophilia from the region have been recently reported. The current situation in the Balkans includes a migrant crisis and reshaping of the Schengen Area border. This results in collision of diverse human populations subjected to different protocols for antimicrobial stewardship and infection control. The present review article summarizes the findings of whole-genome sequencing-based resistome analyses of nosocomial multidrug-resistant NFGNBs in the Balkan countries.

The present study aimed to explore the genotypic and phenotypic characteristics of biofilm formation in Bulgarian nosocomial Stenotrophomonas maltophilia isolates (n = 221) during the period 2011-2022, by screening for the presence of biofilm-associated genes (BAG) (spgM, rmlA and rpfF), their mutational variability, and assessment of the adherent growth on a polystyrene surface. The methodology included: PCR amplification, whole-genome sequencing (WGS) and crystal violet microtiter plate assay for biofilm quantification. The overall incidence of BAG was: spgM 98.6%, rmlA 86%, and rpfF 66.5%. The most prevalent genotype was spgM+/rmlA+/rpfF+ (56.1%), followed by spgM+/rmlA+/rpfF- (28.5%), and spgM+/rmlA-/rpfF+ (9.5%), with their significant predominance in lower respiratory tract isolates compared to those with other origin (P < 0.001). All strains examined were characterized as strong biofilm producers (OD550 from 0.224 ± 0.049 to 2.065 ± 0.023) with a single exception that showed a weak biofilm-forming ability (0.177 ± 0.024). No significant differences were observed in the biofilm formation according to the isolation source, as well as among COVID-19 and non-COVID-19 isolates (1.256 ± 0.028 vs. 1.348 ± 0.128, respectively). Also, no correlation was found between the biofilm amounts and the corresponding genotypes. WGS showed that the rmlA accumulated a larger number of variants (0.0086 per base) compared to the other BAG, suggesting no critical role of its product to the biofilm formation. Additionally, two of the isolates were found to harbour class 1 integrons (7-kb and 2.6-kb sized, respectively) containing sul1 in their 3' conservative ends, which confers sulfonamide resistance. To the best of our knowledge, this is the first study on S. maltophilia biofilm formation in Bulgaria, which also identifies novel sequence types (ST819, ST820 and ST826). It demonstrates the complex nature of this adaptive mechanism in the multifactorial pathogenesis of biofilm-associated infections.

The aim of this study was to verify whether the human DR-ELISA for the detection of anti-SARS-CoV-2 antibodies can be applied in cats, and to assess the risk factors that determine the spread of the virus among the cat population in Bulgaria. The study included 92 serum samples collected from 68 domestic and 24 stray cats aged from 3 months to 20 years of age in the period of January-June 2021. The samples originated from three regions in Bulgaria and from three places of inhabitance. DR-ELISA based on peroxidase-labeled SARS-CoV-2 N protein was employed to detect IgA, IgG and IgM antibodies in the samples. Subsequently, the results were compared with a commercially available multi-species ELISA kit. There was high seroprevalence (83.33%) in stray cats and 41.18% in domestic cats, confirmed by the human and veterinary ELISA kit. The positive cases in the regional cities were 42.86%, in small towns 50% and in villages 78.26%. Cats under 7 years had a five times higher risk than those over 7 years ( p = 0.001). The risk was seven times higher for stray cats than for domestic cats ( p = 0.001). In addition, the results indicate that the risk was the highest for cats in villages ( p = 0.006) compared to cats in other places of inhabitance. This study demonstrates that human DR-ELISA may be successfully applied to monitor the circulation of SARS-CoV-2 in cats and other susceptible species. Cats might serve as sentinel animals for tracking the virus in nature and in inhabited areas (strays) and to discover asymptomatic cases in humans/owners.

Colistin is considered as the last-line antibiotic for the treatment of infections caused by extensively drug-resistant Gram-negative pathogens belonging to the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group. The present study aimed to explore the colistin resistance mechanisms of a Klebsiella aerogenes (formerly Enterobacter aerogenes) isolate (Kae1177-1bg) obtained from a Bulgarian critically ill patient with septic shock in 2020. Antimicrobial susceptibility testing and whole-genome sequencing using DNA nanoball technology were performed. The resulting read pairs were used for draft genome assembly, MLST analysis and mutation screening in the pmrA/B, phoP/Q, and mgrB genes. Kae1177-1bg demonstrated high-level resistance to colistin, resistance to 3rd generation cephalosporins and susceptibility to all other antibiotics tested. In our strain a CMY-2-type class C cephalosporinase was the only β-lactamase identified. No mobile colistin resistance (mcr) genes were detected. A total of three missense variants in the genes for the two-component PmrA/PmrB system were identified. Two of them were located in the pmrB (pR57K and pN275K) and one in the pmrA gene (pL162M). The pN275K variant emerged as the most likely cause for colistin resistance because it affected a highly conservative position and was the only nonconservative amino acid substitution. In conclusion, to the best of our knowledge, this is the first documented clinical case of a high-level colistin-resistant K. aerogenes in Bulgaria and the first identification of the nonconservative amino acid substitution pN275K worldwide. Colistin-resistant Gram-negative pathogens of ESKAPE group are serious threat to public health and should be subjected to infection control stewardship practices.

Background: Severe infections of virulent methicillin-resistant Staphylococcus aureus (MRSA) are a serious health problem. The present study aimed to investigate clonal spread, virulence and antimicrobial resistance rates of Bulgarian MRSA isolates in 2016-2020., Methods: Molecular identification and mecA gene detection were performed with PCR. Clonal relatedness was evaluated by RAPD PCR and MLST. MRSA epidemiology, virulence and resistance patterns were investigated by PCR., Results: All 27 isolates were identified as S. aureus and were mecA positive, and all were susceptible to linezolid, tigecycline and vancomycin. The toxin genes hlg (in 92.6% of isolates), seb (77.8%), sei (77.8%), seh (59.3%), sej (55.6%), and seg (48.1%), were frequently found among the isolates. Epidemiological typing by RAPD identified 4 clones (16 isolates) and 11 were with a unique profile. MLST analysis of the same MRSA isolates showed five MLST clonal complexes and 11 ST types, including CC5 (33.3%) (ST5, ST221, ST4776), CC8 (22.2%) (ST8, ST239, ST72), CC15 (ST582), CC22 (14.8%) (ST217, ST5417), CC30 (ST30) CC398 (ST398), and CC59 (ST59). The isolates from CC5 showed higher virulence potential and almost all were macrolide resistant (ermB or ermC positive). CC8 isolates showed higher level of resistance., Conclusion: To the best of our knowledge, this study is the first describing the clonal spreading of Bulgarian MRSA and the association with their virulence and resistance determinants. Monitoring of MRSA epidemiology, resistance and virulence profile can lead to better prevention and faster therapeutic choice in cases of severe infections.

Nowadays, due to their potential application as probiotics for humans or animals, many beneficial lactic acid bacteria have been isolated from different natural environments. These include members of the genus Enterococcus - quite specific due to their ambiguous nature, varying from pathogens to probiotics. In our work we present a whole-genome sequencing (WGS)-based approach for assessing the potential of bacteriocin-producing Enterococcus isolates from beehives to serve as natural preserving agents against bacterial infections associated with honeybees. Potential Enterococcus spp. isolates from pollen granules were tested with the well diffusion assay for bacteriocin activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (AFB). Two of them gave positive results and were determined at species level by 16S rRNA genes sequencing. They were then subjected to WGS using the Illumina HiSeq platform. The resulting raw data reads were processed and further analyzed by using only freely available web-based tools (the Shovill pipeline, QUAST, BAGEL4, ResFinder, VirulenceFinder and PlasmidFinder). The analysis revealed that both of them represent clonally identical isolates of the same strain. This specific strain was named Enterococcus faecium EFD, and was genotyped by the MLST-2.0 Server. Five bacteriocin genes were found in the assembled genome, providing a possible explanation for the antimicrobial properties of the isolate. The protein nature of the inhibitory agent/s was confirmed by treatment with proteinase K. No resistance determinants for clinically important antibiotics and functional virulence factor genes were detected. The bioinformatic analyses of the draft genome sequence suggest that E. faecium EFD is not pathogenic.The observation that E. faecium EFD was present within more than one of the beehives in the apiary proposes the idea that E. faecium EFD is there as a part of the normal beehive microbiota. This finding, in combination with its antibacterial activity against P. larvae, highlights this novel isolate as a potential natural preserving agent against AFB. Furthermore, the WGS-based approach reported here proved to be very cost- and time- efficient, for screening the applicability of new pro- and prebiotic Enterococcus strains as beehive protection agents.

Stenotrophomonas maltophilia is an increasingly prevalent opportunistic pathogen responsible for a wide range of nosocomial infections in intensive care unit patients, life-threatening diseases in immunocompromised haematology-oncology patients and chronic pulmonary infections in individuals with cystic fibrosis. Therapy of these infections is problematic due to the remarkable intrinsic antimicrobial resistance of the species and to acquired resistance to multiple antimicrobial agents. As this organism is a low-grade pathogen, the pathogenesis of S. maltophilia infections involves numerous virulence factors as well as the ability of bacterial cells to form biofilms on abiotic surfaces and host tissues. The present review summarizes the literature data regarding extracellular and cell-associated virulence factors of S. maltophilia (some of which have still not been studied in detail) and considers the basic characteristics of biofilm formation. Many virulence features such as extracellular enzymes, bacterial motility and biofilm formation are finely controlled by quorum sensing (QS) that enable the bacteria to express these virulence factors in a coordinated, cell-density-dependent manner and overwhelm the host defence mechanisms. Manipulating the QS regulatory system is a promising approach for development of new strategies for control of S. maltophilia infections.

Objectives: A total of 226 carbapenem-resistant Acinetobacter baumannii (CRAB) isolates was collected during 2014-2016 from inpatients (age range 5-88 years) in four Bulgarian university hospitals (H1-H4) to assess their antimicrobial susceptibility and to explore carbapenem resistance mechanisms as well as the molecular epidemiology of the isolates., Methods: Antimicrobial susceptibility testing, multiplex PCR, DNA sequencing and electrotransformation experiments were performed. Epidemiological typing by random amplification of polymorphic DNA (RAPD)-PCR was also performed., Results: The resistance rates were as follows: imipenem, 90.7%; meropenem, 98.2%; doripenem, 100%; amikacin, 92.9%; gentamicin, 87.2%; tobramycin, 55.8%; levofloxacin, 98.2%; trimethoprim/sulfamethoxazole, 86.3%; tigecycline, 22.1%; colistin, 0%; and ampicillin/sulbactam, 41.6%. Intrinsic bla OXA-51-like genes were found in all of the isolates. The majority of the A. baumannii isolates harboured either bla OXA-23-like associated with the upstream-located ISAba1 (26.1%) or bla OXA-40/24-like (46.7%), 45 isolates (19.9%) harboured both genes, and 1 isolate harboured bla OXA-58-like surrounded by ISAba3C upstream and ISAba3 downstream. The bla OXA-58 gene was transferable by electroporation indicating its plasmid location. Epidemiological typing revealed the dissemination of nosocomial CRAB with high clonal relatedness (70% similarity threshold) belonging to six, four, three and two clusters in H1, H2, H3, and H4 hospitals, respectively., Conclusions: The A. baumannii isolates studied were problematic nosocomial pathogens. Their multidrug resistance greatly limits therapeutic options. The persistence of endemic clones comprised of OXA carbapenemase-producing multidrug-resistant A. baumannii in the monitored hospitals over a period of ca. 3 years is of concern and requires continuous detailed investigations in the future., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)