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The primary goal of synthetic biology is to make the process of engineering biological systems easier. One of the biggest steps in this direction was the development of the BioBrick registry. It is open and contains the largest collection of standard and well characterized genetic parts worldwide. Here, we present an optimized procedure for preparation of chemical competent cells that can be used with different BioBrick parts from this registry. It is based on transformation and storage solution (TSS) procedure with some additional modifications. Our improvements include the addition of a heat shock step, the use of a different type of polyethylene glycol and the omission of the pH adjustment step. We intensively optimized all transformation conditions for the widely used general cloning strain E. coli DH5alfa. Our modifications resulted in a very simple, quick and cost efficient protocol that gave reliable and reproducible results with the BioBrick distribution kits. The cells were tested in a variety of applications like plasmid transformations, BioBrick assembly procedures and Aqua cloning of complex genetic constructs. [ABSTRACT FROM AUTHOR]

Two novel plasmid cloning vectors, pBlue and pYellow, were constructed by substituting the LacZ' gene fragment of pUC19 with amilCP and amilGFP chromoprotein coding sequences obtained from the coral Acropora millepora. Both sequences were codonoptimized for expression in Escherihia coli and, when expressed, produced blue and yellow coloring, respectively. The substitution of the LacZ' gene fragment eliminates the need for adding X-Gal to the growth medium while keeping the same mode for positive transformants selection as the one used by the ancestral vector. As a proof of the concept, a 644 bp long fragment from the dia-1 gene in D. melanogaster was cloned in our new vectors pBlue and pYellow using standard restriction and ligation techniques. Positive transformants were selected using the white color of the colonies as an indicator for a successful cloning event. All colonies that contained empty vectors developed the corresponding blue or yellow coloring in the process of cultivation, allowing their visual identification. [ABSTRACT FROM AUTHOR]

Linezolid is an oxazolidinone antibiotic and is considered a last-resort treatment option for serious infections caused by problematic Gram-positive pathogens, including vancomycin-resistant enterococci. The present study aimed to explore the linezolid resistance mechanisms and genomic characteristics of two vancomycin-susceptible Enterococcus faecalis isolates from Bulgaria. The strains designated Efs2503-bg (inpatient from Pleven) and Efs966-bg (outpatient from Varna) were recovered from wounds in 2018 and 2023, respectively. Antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, and phylogenomic analysis based on 332 linezolid-resistant E. faecalis genomes were performed. Efs2503-bg was high-level resistant to linezolid (MIC > 256 mg/L) and displayed the G2576T mutation affecting three of the four 23S rDNA loci. Efs966-bg (MIC = 8 mg/L) carried a plasmid-located optrA determinant surrounded by fexA and ermA . No mutations in the genes encoding for ribosomal proteins L3, L4, and L22 were detected. The isolates belonged to the sequence types ST6 (Efs2503-bg) and ST1102 (Efs966-bg). Phylogenomic analysis revealed that Efs2503-bg and Efs966-bg are genetically distinct, with a difference of 12,051 single-nucleotide polymorphisms. To our knowledge, this is the first report of linezolid-resistant enterococci in Bulgaria. Although the global incidence of linezolid-resistant enterococci is still low, their emergence is alarming and poses a growing clinical threat to public health.

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.

As belonging to one of the most isolated continents on our planet, the microbial composition of different environments in Antarctica could hold a plethora of undiscovered species with the potential for biotechnological applications. This manuscript delineates our discoveries after an expedition to the Bulgarian Antarctic Base "St. Kliment Ohridski" situated on Livingston Island, Antarctica. Amplicon-based metagenomics targeting the 16S rRNA genes and ITS2 region were employed to assess the metagenomes of the bacterial, fungal, and archaeal communities across diverse sites within and proximal to the research station. The predominant bacterial assemblages identified included Oxyphotobacteria , Bacteroidia , Gammaprotobacteria , and Alphaprotobacteria . A substantial proportion of cyanobacteria reads were attributed to a singular uncultured taxon within the family Leptolyngbyaceae . The bacterial profile of a lagoon near the base exhibited indications of penguin activity, characterized by a higher abundance of Clostridia , similar to lithotelm samples from Hannah Pt. Although most fungal reads in the samples could not be identified at the species level, noteworthy genera, namely Betamyces and Tetracladium , were identified. Archaeal abundance was negligible, with prevalent groups including Woesearchaeales , Nitrosarchaeum , Candidatus Nitrosopumilus , and Marine Group II.

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.

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 bla OXA-51-like genes. The frequencies of distribution of other antimicrobial resistance genes (ARGs) were: bla OXA-23-like , 98.6%; bla OXA-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 β-lactamases in all isolates, and OXA-72 carbapenemase in one of them. Various insertion sequencies, such as IS Aba24 , IS Aba31 , IS Aba125 , IS Vsa3 , IS 17 , and IS 6100 , 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.

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.

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.

Paenibacillus profundus YoMME was isolated from the anodic biofilm of a sediment microbial fuel cell and recognized as one of the few exoelectrogenic Gram-positive bacteria, capable of transferring electrons extracellularly. Here, we report its draft genome sequence. The genome project is deposited at DDBJ/ENA/GenBank under the accession number JAJNBZ000000000.

Information about the fungal composition of bee bread, and the fermentation processes to which the fungi contribute significantly, is rather scarce or fragmentary. In this study, we performed an NGS-based metagenomics snapshot picture study of the fungal composition of bee bread in four locations in Bulgaria during the most active honeybee foraging period at the end of June 2020. The sampling locations were chosen to differ significantly in climatic conditions, landscape, and anthropogenic pressure, and the Illumina 2 × 250 paired-end reads platform was used for amplicon metagenomics study of the ITS2 region. We found that some of the already reported canonical beneficial core fungal species were present within the studied samples. However, some fungal genera such as Monilinia , Sclerotinia , Golovinomyces , Toxicocladosporium , Pseudopithomyces , Podosphaera and Septoriella were reported for the first time among the dominant genera for a honeybee related product. Anthropogenic pressure negatively influences the fungal composition of the bee bread in two different ways-urban/industrial pressure affects the presence of pathogenic species, while agricultural pressure is reflected in a decrease of the ratio of the beneficial fungi.

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.