First detection of a cefiderocol-resistant and extensively drug-resistant Acinetobacter baumannii clinical isolate in Bulgaria.

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.

Genotypic and phenotypic insights into virulence factors of nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria (2011-2022).

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.

Risk factors for gut colonization with vancomycin-resistant enterococci among Bulgarian critically ill patients.

Vancomycin-resistant enterococci (VRЕ) are recognized as important hospital pathogens which have become common in patients admitted to the intensive care units (ICUs). The purpose of this study was to evaluate the incidence of and the risk factors for colonization with VRE among ICU patients. A total of 91 patients who had duration of hospitalization more than 48 h and without infection caused by VRE or/and other microorganisms in the ICU at University Hospital, Pleven were screened for colonization with VRE. The following data were collected: demographic characteristics, clinical information and antimicrobials use. The statistical analysis was performed using SPSS version 27.0. Colonization with VRE was established in 22 patients and one was carrying two enterococcal species. A total of 23 VRE were isolated. The univariate analysis showed that the postoperative critical cares (p < 0.001), cardiovascular diseases (p = 0.009) and the presence of an endotracheal tube (p = 0.003) were risk factors for colonization with VRE. Also, the postoperative critical cares (p = 0.021) and cardiovascular diseases (p = 0.018) were confirmed as independent risk factor for VRE acquisition by multivariate analysis. The prevalence of VRE colonization among the ICU patients was relatively high (24.2%). Risk factors for acquisition of intestinal VRE were the postoperative cares, cardiovascular diseases and the presence of an endotracheal tube.

Molecular Epidemiology of Bulgarian Clinically Significant Staphylococcus aureus Isolates.

Severe infections due to highly virulent and resistant Staphylococcus aureus pose a serious health threat in Bulgaria and worldwide. The purpose of this study was to explore the clonal spread of recent clinically significant methicillin-susceptible S. aureus (MSSA) isolates from inpatients and outpatients treated in three university hospitals in Sofia, Bulgaria, during the period 2016-2020 and evaluate the relationship between their molecular epidemiology, virulence profiling, and antimicrobial resistance. A total of 85 isolates (invasive and noninvasive) were studied using RAPD analysis. Ten major clusters (A-K) were identified. The first major cluster A (31.8%) was found to be predominant during 2016 and 2017 and was widespread in two hospitals, unlike its case in the following years, when it was found to be replaced by newer cluster groups. All MSSA members of the second most common cluster F (11.8%) were recovered from the Military Medical Academy, mainly during 2018-2020, and were determined to be susceptible to all other groups of antimicrobials, except for penicillins without inhibitors because they harboured the blaZ gene. The newer cluster I, with 9.4% of the isolates absent in 2016-2017, showed significantly higher virulence and macrolide resistance (42.9%) due to ermB and ermC. All the isolated MSSA in groups F and I were nosocomial and mostly invasive. In conclusion, this 5-year study demonstrates the molecular epidemiology of MSSA infections in three Bulgarian hospitals. Findings can be helpful for the understanding of staphylococcal infection distribution in hospital settings and their prevention.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Acinetobacter baumannii Isolates from Bulgarian Intensive Care Unit Patients.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is designated as an urgent public health threat, both due to its remarkable multidrug resistance and propensity for clonal spread. This study aimed to explore the phenotypic and molecular characteristics of antimicrobial resistance in CRAB isolates (n = 73) from intensive care unit (ICU) patients in two university hospitals in Bulgaria (2018-2019). The methodology included antimicrobial susceptibility testing, PCR, whole-genome sequencing (WGS), and phylogenomic analysis. The resistance rates were as follows: imipenem, 100%; meropenem, 100%; amikacin, 98.6%; gentamicin, 89%; tobramycin, 86.3%; levofloxacin, 100%; trimethoprim-sulfamethoxazole, 75.3%; tigecycline, 86.3%; colistin, 0%; and ampicillin-sulbactam, 13.7%. All isolates harbored blaOXA-51-like genes. The frequencies of distribution of other antimicrobial resistance genes (ARGs) were: blaOXA-23-like, 98.6%; blaOXA-24/40-like, 2.7%; armA, 86.3%; and sul1, 75.3%. The WGS of selected extensively drug-resistant A. baumannii (XDR-AB) isolates (n = 3) revealed the presence of OXA-23 and OXA-66 carbapenem-hydrolyzing class D ß-lactamases in all isolates, and OXA-72 carbapenemase in one of them. Various insertion sequencies, such as ISAba24, ISAba31, ISAba125, ISVsa3, IS17, and IS6100, were also detected, providing increased ability for horizontal transfer of ARGs. The isolates belonged to the widespread high-risk sequence types ST2 (n = 2) and ST636 (n = 1) (Pasteur scheme). Our results show the presence of XDR-AB isolates, carrying a variety of ARGs, in Bulgarian ICU settings, which highlights the crucial need for nationwide surveillance, especially in the conditions of extensive antibiotic usage during COVID-19.

Whole-Genome Sequencing-Based Resistome Analysis of Nosocomial Multidrug-Resistant Non-Fermenting Gram-Negative Pathogens from the Balkans.

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.

Analysis of biofilm formation in nosocomial Stenotrophomonas maltophilia isolates collected in Bulgaria: An 11-year study (2011-2022).

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.

Human ELISA Detects anti-SARS-CoV-2 Antibodies in Cats: Seroprevalence and Risk Factors for Virus Spread in Domestic and Stray Cats in Bulgaria.

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.

First detection of a colistin-resistant Klebsiella aerogenes isolate from a critically ill patient with septic shock in Bulgaria.

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 ß-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.

Molecular epidemiology, virulence and antimicrobial resistance of Bulgarian methicillin resistant Staphylococcus aureus isolates.

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.

WGS-based characterization of the potentially beneficial Enterococcus faecium EFD from a beehive.

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.

Carbapenem-resistant Acinetobacter baumannii: Current status of the problem in four Bulgarian university hospitals (2014-2016).

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 blaOXA-51-like genes were found in all of the isolates. The majority of the A. baumannii isolates harboured either blaOXA-23-like associated with the upstream-located ISAba1 (26.1%) or blaOXA-40/24-like (46.7%), 45 isolates (19.9%) harboured both genes, and 1 isolate harboured blaOXA-58-like surrounded by ISAba3C upstream and ISAba3 downstream. The blaOXA-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.

Stenotrophomonas maltophilia - a low-grade pathogen with numerous virulence factors.

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.