The gastrointestinal tract is an important reservoir of extended spectrum beta-lactamase (ESBL)/carbapenemase-producing Enterobacterales isolates. This study included patients from two Bulgarian hospitals. Overall, 98 ESBL producers (including 68 Escherichia coli and 20 Klebsiella pneumoniae isolates) were detected among 99 hospitalized patients, 212 patients at admission, and 92 hospital staff in 42.4%, 24.5%, and 4%, respectively. We observed bla_CTX-M-15 in 47% of isolates, bla_CTX-M-3 in 39% and bla_CTX-M-14 in 11%. Three bla_CTX-M-15 positive isolates were also bla_KPC-2 positive. High transferability was detected for bla_CTX-M-3 carrying plasmids (55%) with L/M and I1 replicon plasmids, followed by CTX-M-14 (36.4%) and CTX-M-15 (27.9%) with IncF plasmids. BlaKPC-2 was carried by FIIAs plasmids. Epidemiology typing revealed 8 K. pneumoniae ST types—ST15(8/20), ST17(4/20), ST37(2/20) and 9 E. coli ST types—ST131 (30.9%, 21/68), ST38 (8/68), ST95(7/68) and ST316(7/68). All ST131 isolates but one was from the highly virulent epidemic clone O25bST131. This is the first report in Bulgaria about ESBL/carbapenemase faecal carriage. We observed high ESBL/carbapenemases prevalence. A predominant number of isolates were members of highly epidemic and virulent PanEuropean clones ST15 K. pneumoniae and O25bST131 E. coli. High antibiotics usage during the COVID pandemic will worsen the situation. Routine screenings and strict infection control measures should be widely implemented.

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. bla_NDM-5 was the only NDM variant, and it was always combined with bla_OXA-232. The coexistence of bla_OXA-232 and bla_NDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All bla_NDM-5- and bla_OXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed bla_OXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for bla_NDM-5- and bla_OXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for bla_OXA-232- or bla_KPC-2-positive isolates. All bla_OXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.

To solve the problem with pan-drug resistant and extensively drug-resistant Gram-negative microbes, newly approved drugs such as ceftazidime/avibactam, cefiderocol, plazomicin, meropenem/vaborbactam, and eravacycline have been introduced in practice. The aim of the present study was to collect carbapenemase-producing clinical Enterobacterales isolates, to characterize their carbapenemase genes and clonal relatedness, and to detect their susceptibility to commonly used antimicrobials and the above-mentioned newly approved antibiotics. Sixty-four carbapenemase producers were collected in a period of one year from four Bulgarian hospitals, mainly including Klebsiella pneumoniae (89% of the isolates) and also single Proteus mirabilis, Providencia stuartii and Citrobacter freundii isolates. The main genotype was bla_NDM-1 (in 61%), followed by bla_KPC-2 (23%), bla_VIM-1 (7.8%) and bla_OXA-48 (7.8%). Many isolates showed the presence of ESBL (bla_CTX-M-15/-3 in 76.6%) and AmpC (bla_CMY-4 in 37.5% or bla_CMY-99 in 7.8% of isolates). The most common MLST type was K. pneumoniae ST11 (57.8%), followed by ST340 (12.5%), ST258 (6.3%) and ST101 (6.3%). The isolates were highly resistant to standard-group antibiotics, except they were susceptible to tigecycline (83.1%), colistin (79.7%), fosfomycin (32.8%), and aminoglycosides (20.3–35.9%). Among the newly approved compounds, plazomicin (90.6%) and eravacycline (76.3%) showed the best activity. Susceptibility to ceftazidime/avibactam and meropenem/vaborbactam was 34.4% and 27.6%, respectively. For cefiderocol, a large discrepancy was observed between the percentages of susceptible isolates according to EUCAST susceptibility breakpoints (37.5%) and those of CLSI (71.8%), detected by the disk diffusion method. This study is the first report to show patterns of susceptibility to five newly approved antibiotics among molecularly characterized isolates in Bulgaria. The data may contribute to both the improvement of treatment of individual patients and the choice of infection control strategy and antibiotic policy.

The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017–2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-β-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 β-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)—ST11, ST258, and ST15 and five main Escherichia coli clones—ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented.

The aim of the present work was to genetically characterise cefotaxime-resistant enterobacteria isolated from community carriers in Bulgaria. In total, 717 faecal samples from children and adults in five medical centres in Sofia, Pleven and Burgas were examined. Antimicrobial susceptibility was evaluated by the disk diffusion method. bla_ESBL or plasmidic AmpC (pAmpC) genes were detected by PCR and sequencing. MLST and ERIC-PCR were used to detect clonal relatedness. Among the faecal samples, 140 cefotaxime-resistant enterobacteria were found. The most frequently detected species was Escherichia coli (77.9%, 109/140 samples), followed by Klebsiella pneumoniae (7.9%, 11/140). Among the isolates, bla_CTX-M-15 (37.1%) was predominant, followed by bla_CTX-M-3 (19.2%), bla_CTX-M-14 (10%), and bla_CTX-M-27 (4.3 %). Genes encoding pAmpC were observed in 11.4% (bla_DHA-1, 16/140) and in 1.4% (bla_CMY-2, 2/140). The frequency of ESBL and pAmpC producers among the subjects was 14.6% and 2.5%, respectively. No carbapenem-resistant isolates were found. Four main clonal complexes (CC131, CC10, CC38, and CC155) were detected among E. coli isolates. The most common type was ST131, phylogroup B2 (16.5%). The increased frequency of ESBL- and pAmpC-producing enterobacteria in the community is a prerequisite for treatment failures of the associated infections and a good background for further studies.

The aim of the present study was to reveal the characteristics of an Enterobacter cloacae complex isolate producing DHA-1 AmpC enzyme, recovered from a patient hospitalized in Saint Marina Hospital – Varna. Material and methods: Susceptibility testing, conjugation experiments, isoelectric focusing, PCR and sequencing were carrying out. Results: From 176 Enterobacter spp. isolates only one isolate was positive for blaDHA. The sequencing revealed the presence of blaDHA-1 and blaCTX-M-3. The antimicrobial susceptibility testing showed higher resistance rates to almost all beta-lactams (ceftazidime, cefotaxime, cefepime, amoxicillinclavulanic acid, piperacillin/tazobactam), tobramycin, gentamycin, trimethoprim/sulphomethoxazole and quinolones (ciprofloxacin and levofloxacin). The isolate was susceptible to imipenem, meropenem and amikacin. The isoelectric focusing showed a band at pI 5.4 without ceftazidime and cefotaxime activity; a band at pI 7.8 with cefoxitin activity and another - with pI 8.4 with cefotaxime activity. Conjugation experiments were successful only for blaCTX-M-3 carrying determinants. Conclusions: To the best of our knowledge this is the first report of DHA-1 producing isolate in Bulgaria. The emergence of DHA-1 producing E. cloacae complex demonstrates the possibility for further dissemination of the gene encoding this enzyme. Infectious control measures are needed for the prevention of this phenomenon.