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During the period March 2003 – May 2004 at the Laboratory of Clinical Microbiology “Redaelli” LTCRF in Milan, Italy, a total of 529 E. coli, obtained from inpatients of 3 different Long Term Care Rehabilitation Facilities (LTCRFs) in Northern Italy, were processed and 77 ESßLs producers (14.5%) were identified by Vitek System. The results were confirmed by double-disk synergy test with tazobactam (TZP). 61/77 isolates were characterized by higher levels of resistance to cefotaxime (CTX) than to ceftazidime (CAZ). (ß-lactamase production was investigated by analytical isoelectric focusing (IEF) coupled with a bioassay and showed multiple (ß-lactamase bands including one enzyme with pI 8.4 that, in a bioassay, was more active on CTX,ATM than on CAZ. The presence of (ß-lactamase genes was investigated by colony blot hybridization and by PCR amplification of blaTEM, blaSHV and blaCTX-M alleles. 43/61 isolates produced both TEM-1 and CTX-M-type enzymes, 14/61 expressed only CTX-M-type while in 4 cases were found blaCTX-M, blaTEM and blaSHV genes.The remainders (16/77), characterized by high levels of resistance to both CTX and CAZ, produced TEM-1 and SHV-5 enzymes (1/16) and TEM type ESßLs (15/16). Conjugation experiments, performed in liquid medium, confermed that the ESßLs determinants were transferable. Pulsed-field gel electrophoresis profiles of genomic DNA, digested with NotI, were analysed and revealed clonal heterogeneity. Our work confirms the emergence of CTX-M-type enzymes and their spread in Northern Italy also in longterm care and rehabilitation facilities that may be an important reservoir of ES?L producing E. coli.
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