The mechanisms of resistance to beta-lactam antibiotics in 325 isolates of Pseudomonas aeruginosa were examined. These isolates were selected because of their resistance to meropenem and imipenem (breakpoint, >4 mg/L), carbenicillin (>128 mg/L), ceftazidime (>8 mg/L), piperacillin and ticarcillin/clavulanate (>64 mg/L). The most frequent mechanism of resistance was beta-lactamase-independent, so called 'intrinsic resistance', which was found in 183 isolates and was probably due to impermeability and/or efflux mechanisms. beta-Lactamase-mediated resistance was demonstrated in 111 strains (11.1%). Derepression of Ambler Class C chromosomal beta-lactamase was detected in 64 isolates, most of which were resistant to ceftazidime and piperacillin but susceptible to meropenem, whereas secondary plasmid-encoded beta-lactamases were found in 34 isolates, all of them resistant to carboxypenicillins and ureidopenicillins and susceptible to carbapenems. Twelve strains showed more than one plasmid-encoded beta-lactamase plus derepression of chromosomal Class C enzyme. Resistance to carbapenems was independent of resistance to other beta-lactam antibiotics, indicating a different mechanism of resistance, probably due to the loss of the D2 porin. In total, 32 strains were resistant to carbapenems: 24 only to imipenem and eight to both imipenem and meropenem.

A clinical isolate of Klebsiella pneumoniae highly resistant to third- and fourth-generation cephalosporins, cephamycins and aminoglycosides, was isolated in 1991 from urine. Analysis of a crude extract showed the presence of three beta-lactamases with isoelectric points of 6.6, 7.5 and 8.2. The enzyme with pI 8.2 was transferred by conjugation into Escherichia coli K-12 J53 and was responsible for the resistance to third-generation cephalosporins and monobactams, but not to other antibiotics. Kinetic studies of partially purified beta-lactamase from the transconjugant strain confirmed that the enzyme was able to hydrolyse ceftazidime, cefotaxime and aztreonam but not cephamycins. Analysis of the transconjugant showed the presence of two small non-conjugative plasmids of 14 and 6 kb. A polymerase chain reaction was performed using primers specific for the bla(SHV) gene and a fragment of the expected size (about 961 bp) was obtained with both the K. pneumoniae clinical isolate and the transconjugant. Nucleotide sequence analysis of the fragment showed that it encoded the enzyme SHV-12, derived from SHV-5 (with Gln-35 to Leu). This is the first report of an SHV-12-like enzyme isolated in Italy.

From May 1996 to September 1997, 615 Pseudomonas aeruginosa strains isolated from patients in intensive care units collected from different Italian laboratories were studied. The susceptibility of piperacillin/tazobactam, in comparison with other antipseudomonal antibiotics, to their NCCLS breakpoints was evaluated: amikacin 79.6%, carbenicillin 67.0%, ceftazidime 73.4%, ciprofloxacin 55.8%, imipenem 64.1%, piperacillin 88.1%, piperacillin/tazobactam 92.4% and ticarcillin/clavulanic acid 69.0%. Seventy-three strains were selected because of their resistance to piperacillin and the mechanisms underlying such a resistance were investigated. Isoelectric focusing and hydrolysis assays revealed the presence of 15 plasmid-mediated β-lactamases. Chromosomal β-lactamase derepression was demonstrated in 34 isolates. The remaining 24 piperacillin-resistant strains did not produce β-lactamases and an ‘intrinsic mechanism’ of resistance was inferred. The piperacillin/tazobactam combination restored resistance in 25 piperacillin strains. Nine of these were derepressed for chromosomal β-lactamase, 8 showed impermeability and 8 showed plasmid enzymes.