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We report the first confirmed cases of NDM-1-producing Klebsiella pneumoniae infections in two hospitals in Bulgaria. The isolates were diverse in terms of plasmid and co-resistance gene content. K. pneumoniae PR2682, causing sepsis in patient with polytrauma due to traffic accident, harbored bla NDM-1, bla CMY-4 , bla CTX-M-15 , bla SHV-1 , bla TEM-1b , qnrB, and aac(6')-Ib. bla NDM-1 was transferable by conjugation and located on an IncA/C plasmid of 176-kb, which also carried bla CMY-4 , bla CTX-M-15 , bla TEM-1b , and qnrB. K. pneumoniae PR2830, causing urinary tract infection in prostate cancer patient, harbored bla NDM-1, bla SHV-1 , bla TEM-1 , and aac(6')-Ib. bla NDM-1 was carried on an 86-kb IncA/C plasmid transferable by conjugation together with bla TEM-1 , and aac(6')-Ib. Multilocus sequence typing indicated that the two isolates belonged to sequence type ST11. The emergence of NDM-1-producing K. pneumoniae indicates that bla NDM-1 -mediated resistance is already disseminated among Enterobacteriaceae in Bulgaria. Our results further confirm the role of the Balkans as a secondary reservoir where NDM-encoding genes originate., (Copyright © 2016 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We report the identification of OXA-48-producing Klebsiella pneumoniae, causing peritonitis in a cancer patient admitted to the Oncology Hospital in Sofia. The isolate had reduced susceptibility to carbapenems but remained susceptible to extended-spectrum cephalosporins. PCR and sequencing confirmed the presence of blaOXA-48 gene flanked by two intact copies of IS1999 on truncated ΔTn1999.1. This transposon was located on unusual non-typeable 29-kb plasmid that could be transferred only by transformation. Multilocus sequence typing (MLST) indicated the presence of the sequence type ST530.This is the first documented infection due to OXA-48-producing Enterobacteriaceae strain in Bulgaria.

This report describes the first identification of OXA-24 carbapenemase-producing Acinetobacter baumannii isolates from Bulgaria. According to national surveillance data A. baumannii along with Pseudomonas aeruginosa are the most troublesome microorganisms in hospital environment with high rates of acquired carbapenem resistance. In the present study real-time multiplex PCR was performed to identify the most common carbapenemase genes in 15 non-duplicate carbapenem-resistant A. baumannii isolates collected in 2012. The results showed lack of KPC, GES, VIM, IMP-type enzymes. Four A. baumannii isolates tested positive by PCR for the acquired OXA-24 together with the intrinsic OXA-51 carbapenemase. OXA-24 and OXA-23 were determined as co-existent in one isolate. Two isolates were identified with OXA-23 in addition to the OXA-51 carbapenemase.

A panel of 29 multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from seven hospitals as part of a country-wide surveillance of antimicrobial resistance in Bulgarian hospitals was studied. Molecular typing through multiple-locus variable number tandem-repeat analysis (MLVA6) yielded 23 different profiles. Phenotypic and genotypic tests for the detection of acquired carbapenemases yielded negative results in all cases. In contrast, 76% of the isolates produced other acquired β-lactamases, including extended-spectrum β-lactamases (ESBLs). Namely, 6 of the isolates (21%) produced a VEB-1 ESBL; 14 (48%) produced an OXA-10-type enzyme (7 OXA-10 and 7 OXA-10 ESBL variants, including 2 OXA-17 [A218G], 2 OXA-74 [C197T, A218G], and 3 OXA-142 [A218G, G470A]); 8 (28%) an OXA-2-type enzyme (all OXA-2); and 1 (3%) a PSE-1 carbenicillinase. Further analysis through multilocus sequence typing (MLST) revealed that the six VEB-1-producing strains, recovered from four hospitals, belonged to ST111 or ST244 international high-risk clones. Additionally, nearly all of the isolates (97%) lacked OprD production, explaining carbapenem resistance. Overexpression of AmpC was documented in 5 (17%) of the isolates, including most of the MDR isolates not producing any acquired β-lactamase. Particularly noteworthy was the very high prevalence of MexXY-OprM overexpression, documented in 72% of the isolates, whereas the prevalence of MexAB-OprM overexpression was lower (21%). In summary, while the production of metallo-β-lactamases is uncommon among P. aeruginosa isolates from Bulgarian hospitals, MDR profiles frequently result from the production of ESBLs combined with the lack of production of the carbapenem porin OprD and the overexpression of the MexXY-OprM efflux pump.

1,310 Enterobacteriaceae, 242 Pseudomonas aeruginosa and 97 Acinetobacter baumannii clinical isolates collected at a cancer hospital in Bulgaria were screened for the presence of 16S rRNA methylases. The armA methylase gene was identified in 20 (1.5%) Enterobacteriaceae (7 Klebsiella pneumoniae, 3 Escherichia coli, 3 Serratia marcescens, 3 Citrobacter freundii , 3 Enterobacter cloacae and 1 Klebsiella oxytoca). ArmA-mediated aminoglycoside resistance was transferable by conjugation and carried by closely related IncL/M plasmids which also carried ant3"9, dfrXII, sul1, bla(TEM-1), and bla(CTX-M-3) genes encoding resistance to streptomycin-spectinomycin, trimethoprim, sulfonamides, and ss-lactams, respectively. Most of the isolates were genetically different according to PFGE but shared similar restriction patterns of the armA -encoding plasmids. Our findings highlight the strong association of armA and bla(CTX-M-3) extended-spectrum ss-lactamase genes across various species in the family Enterobacteriaceae . The spread of multiresistant isolates expressing 16S rRNA methylases and ESBLs is a worrisome development requiring continuous monitoring.