Isolation of Klebsiella pneumoniae strains with altered susceptibility to carbapenems not carbapenemase mediated

The spread of enterobacteria producing extended-spectrum ß-lactamases (ESBLs) is sharply increasing in Italy, while the detection of isolates resistant to carbapenems is still sporadic. Isolates of Klebsiella pneumoniae resistant to all cephalosporins, aminoglycosides and fluoroquinolones have been isolated in Trieste since 2008. Because of the altered profile of resistance to carbapenems, these strains were reported as ESBL-negative and possible carbapenemases producer by the expert system, leaving tigecycline as the only therapeutic choice.The purpose of this study is the characterization of the mechanisms involved in resistance to carbapenems in these strains and the evaluation of a reliable and simple test for phenotypic confirmation of ESBL and/or carbapenemase production. 25 isolates of MDR K. pneumoniae were collected between October 2008 and May 2009, mainly from urinary samples of elderly patients hospitalized in medicine wards. Identification and susceptibility testing were performed using the Vitek 2 system.The double-disc (DD) test was used to check the production of ESBLs, while imipenem and imipenem-EDTA synergy test was used to detect the production of metallo-ßlactamase (MBL). Carbapenemase activity was tested by an hydrolysis assay and the production of MBLs was also investigated by PCR. The DD synergy test highlighted the possible production of ESBLs in 18 out of 22 strains, considered as negative by Vitek. All ESBLs producers tested positive for the blaCTX-M-15 allele. Only one isolate was resistant to carbapenems and resulted positive for production of MBL by the phenotypic test.The crude extract showed carbapenemase activity inhibited by EDTA; PCR test gave positive result for a blaVIM-type allele. PCR analysis performed on representative isolates, followed by sequencing, showed that coding sequence of ompk35 was not functional. Results of this study confirmed the emergence of ESBL-positive strains of K. pneumoniae that showed altered sensitivity to carbapenems in absence of carbapenemases. For a correct identification of the resistance mechanisms of these isolates we used phenotypic tests (modified DD test and synergy test for MBL) coupled with molecular techniques.The MDR strains isolated in Trieste are particularly difficult in terms of therapeutic management, especially in case of at-risk subjects.