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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 blaVEB-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: blaVEB-9, blaPDC-3, blaOXA-10, blaOXA-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 blaVEB-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 blaVEB-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. [ABSTRACT FROM AUTHOR]

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: bla OXA-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. [ABSTRACT FROM AUTHOR]

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 bla NDM-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 bla NDM-1 and bla GES-1. The isolates belonged to the ST654 high-risk clone, except for Pae57 (ST611). Alignment against reference sequences revealed the presence of a Tn 21 transposon harboring ble MBL– bla NDM-1–IS Aba125. 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 bla NDM-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. [ABSTRACT FROM AUTHOR]

In this research, we report the isolation of Enterococcus durans EDD2, which showed strong inhibitory activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (AFB). The strain was found in freshly collected pollen granules from four beehives in Dushantzi village (Pirdop region, Bulgaria), and was determined by polyphase approach including molecular, biochemical, and physiological tests. Whole-genome sequencing by the Illumina HiSeq NGS platform with 2 x 150 bases paired-end reads revealed the presence of two contigs containing genetic determinants for Enterocin L50A/L50B and Enterocin P -like bacteriocins, the first ones being carried out by the chromosome, while the second one being present on a putative plasmid. E. durans EDD2 was also subjected to genetic and microbiological tests for several pathogenicity factors, and none was found. These results, combined with the fact that the strain was present in four, but not all of the 15 beehives in the apiary, suggest that it is not a result from some human or animal fecal or other types of contamination in the adjacent fields, but rather it is maintained within the bees microbiomes and was transferred to the pollen granules by the insects' saliva. It was proven that E. durans EDD2 possesses a strong probiotic inhibitory activity against P. larvae due to the production of bacteriocins. [ABSTRACT FROM AUTHOR]

The Bulgarian 'mehovo sirene' cheese is a type of regional artisanal cheese prepared from raw ewe milk in a skin bag relying on spontaneous fermentation. A metagenomics study revealed an unusual microbiome comprising beneficial bacteria such as Staphylococcus equorum, Lactococcus lactis, Streptococcus thermophilus, Lactobacillus delbrueckii, Lactobacillus kefiranofaciens, Lactococcus raffinolactis, Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Loigolactobacillus coryniformis. Our findings suggest that 'mehovo sirene' could have some functional food properties and is a source of beneficial microorganisms. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]