Detection of CMY-99, a novel acquired AmpC-Type β-lactamase, and VIM-1 in Proteus mirabilis isolates in Bulgaria.

During the last years, outbreaks and countrywide spread of isolates producing -lactamases of the AmpC type or carbapenemases have been reported (1, 2). We detected three Proteus mirabilis isolates with reduced susceptibility to cephalosporins and carbapenems at the Alexandrovska University Hospital in Sofia. They were isolated between November 2011 and January 2012 from the urine of three patients, all with a history of multiple hospitalizations, urologic procedures, and urinary bladder catheterizations (Table 1). Antimicrobial susceptibility testing (3) revealed resistance or reduced susceptibility to oxyimino-cephalosporins, cephamycins, and imipenem (Table 1). The isolates were resistant to all of the non-lactam antibiotics tested, except for fosfomycin, and P. mirabilis 60 additionally showed susceptibility to gentamicin and amikacin. The -lactam resistance determinants were not transferable in conjugation experiments with an Escherichia coli K-12: W3110 lac mutant strain resistant to rifampin using different selective agents, including ampicillin. A modified Hodge test and an imipenem-EDTA synergy test (4) were positive for all of the isolates, suggesting the production of a metallo-lactamase. The isolates were screened by PCR for the presence of the blaVIM, blaIMP, blaNDM, blaCTX-M, blaSHV, and blaTEM genes using primers described elsewhere (5, 6). Additionally CMY-2 groupspecific primers were used (CMY-2-K, GGTGCAAATCAAACA CAC; CMY-2-M, ACTGCAGCAACGACGGGC). All three isolates gave positive results with CMY-2 group, VIM, and TEM primers. Isolates 79 and 915 additionally were positive for blaSHV by PCR. Sequencing of the PCR products obtained with primers binding outside the coding regions (reference 6; for blaVIM, IntI1attI [TCTATGCCTCGGGCATCC] and VIM-R-A [HWGTTATG CCGCATCTG]) revealed the presence of blaTEM-1, blaSHV-12, and blaVIM-1 (Table 1). The CMY enzyme was identified as a new variant of the CMY-2 subgroup, CMY-99, which is closest to CMY-16,