High Prevalence of the aac(6′)-Ib-cr Gene and Its Dissemination among Enterobacteriaceae Isolates by CTX-M-15 Plasmids in Bulgaria

Since 1998, three mechanisms of plasmid-mediated quinolone resistance (PMQR) have been reported: Qnr-mediated topoisomerase protection (6), enzymatic modification of ciprofloxacin and norfloxacin by the aminoglycoside acetyltransferase AAC(6′)-Ib-cr (10), and active efflux due to QepA (8). PMQR genes confer low-level quinolone resistance and are frequently cotransmitted with extended-spectrum β-lactamase (ESBL) genes (9). We report the prevalence of aac(6′)-Ib-cr and its association with qnr genes in ESBL-producing Enterobacteriaceae isolates in a Bulgarian hospital. A total of 163 ESBL-producing enterobacteria (4.6% of 3,516 consecutive isolates) were recovered among 10 species at increasing overall prevalence rates between 2000 and 2005 (Table ​(Table1). 1). These increases reflected the increasing rates of ESBL production in Escherichia coli (from 1.2% in 2000 to 10.0% in 2005), whereas similar rates of ∼7% in Klebsiella pneumoniae and the irregular appearance of ESBL production in other species have been observed during the study period. As shown in Table ​Table1,1, using primers 5′-ACTGAGCATGACCTTGCGATGC-3′ and 5′-TTAGGCATCACTGCGTGTTCG-3′, aac(6′)-Ib was detected in 99 (60.7%) of the ESBL producers distributed among nine species. Of these, 52 (52.5%) were found to carry the cr variant by sequencing, including 2 Citrobacter freundii isolates; one isolate each of Enterobacter aerogenes, Morganella morganii, and K. pneumoniae (all from 2005); and 47 E. coli isolates recovered since 2002, increasing from 0% to 67% during that period. For seven C. freundii isolates, including the two aac(6′)-Ib-cr-positive isolates, PCR for qnrA, qnrB, and qnrS (11, 12) yielded amplicons only for qnrB, identified as qnrB10 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"DQ631414","term_id":"110226166","term_text":"DQ631414"}}DQ631414), qnrB13 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"EU273755","term_id":"161579477","term_text":"EU273755"}}EU273755), and qnrB18 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AM919399","term_id":"161338643","term_text":"AM919399"}}AM919399) by using sequencing (1). The overall prevalence of qnrB (7/163; 4.3%) was sevenfold lower than that of aac(6′)-Ib-cr (52/163, 31.9%), and the coexpression of QnrB and AAC(6′)-Ib-cr occurred only in two (1.4% of all) isolates. TABLE 1. Distribution of aac(6′)-Ib-cr and qnrB genes among 163 ESBL-producing enterobacterial isolates at the National Oncology Center, Sofia, Bulgaria, from 2000 to 2005 and the respective ESBL prevalence rates The 52 aac(6′)-Ib-cr-positive isolates were characterized by antibiotic susceptibility testing and bla content determination as previously described (3, 13). Forty-three isolates were resistant to ciprofloxacin, 41 to gentamicin, and 25 to trimethoprim-sulfamethoxazole. All isolates were resistant to tobramycin and exhibited reduced susceptibility to amikacin, but worrisomely, 39 (75%) of the isolates were classified as amikacin susceptible according to CLSI breakpoints. Fifty isolates had both blaCTX-M-15 and blaOXA-1. Of these, 2 isolates also carried blaSHV-12 and 32 carried blaTEM-1. The remaining two aac(6′)-Ib-cr-positive isolates expressed ESBLs not of the TEM, SHV, CTX-M, VEB, PER, or GES type, associated with the TEM-1 enzyme. Thirty different XbaI-pulsed-field gel electrophoresis patterns were observed among the 47 E. coli isolates (data not shown). These findings suggest that the high prevalence of aac(6′)-Ib-cr was not solely due to the spread of a specific E. coli clone. Transferability of AAC(6′)-Ib-cr determinant in broth and on filters was examined using rifampin-resistant recipient E. coli ML4909 (F− galK2 galT22 hsdR metB1 relA supE44 Rifr) (4). Transconjugants were selected on bromothymol blue lactose agar containing rifampin (200 μg/ml) and kanamycin (25 μg/ml). Conjugative transfer of aac(6′)-Ib-cr was achieved for 42 of the 52 isolates, including one isolate each of K. pneumoniae and M. morganii, two C. freundii isolates, and 38 E. coli isolates. aac(6′)-Ib-cr was mostly cotransferred with blaCTX-M-15 and blaOXA-1, variably with blaTEM-1, but not with blaSHV-12 and qnrB. This is the first report of qnrB and aac(6′)-Ib-cr in clinical Enterobacteriaceae isolates from a Bulgarian hospital. The aac(6′)-Ib and its cr variant were highly prevalent in ESBL-producing E. coli. CTX-M-15 plasmid-mediated dissemination of aac(6′)-Ib-cr among Enterobacteriaceae isolates was particularly observed, as has been found in other countries (2, 5). In this work, qnrB had a low prevalence and was not cotransferred with the aac(6′)-Ib-cr gene. This result supports previous findings suggesting that aac(6′)-Ib-cr might already be widespread and substantially more prevalent than qnr genes (7, 10). Most of the isolates carrying the aac(6′)-Ib-cr variant were resistant to ciprofloxacin, probably reflecting its ability to promote higher-level quinolone resistance mutations (10).