A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.

A clinical Pseudomonas aeruginosa strain, PAe391, was found to be resistant to a number of antibiotics including ticarcllin, piperacillin, cefsulodin and amikacin, and a disk diffusion assay showed evidence of pronounced synergy between imipenem and various β;-lactam antibiotics. Cloning and nucleotide sequence analysis revealed the dicistronic arrangement of an aac(6’)-lb variant and a novel bla OXA-type gene between the intl and qacEδ1 genes typical of integrons. In PAe391, this integron was apparently chromosome-borne. The β;-lactamase, named OXA-13, displayed nine amino acid changes with respect to OXA-10: I in position 10 of OXA-10 to T (I10T), G20S, D55N, N73S, T107S, Y174F, E229G, S245N and E259A. OXA-13 (plapp = 8.0) showed poor catalytic activity against penicillins as well as cephalosporins, but was efficient in hydrolysing some penicillinase-resistant β;-lactams, such as cefotaxime and aztreonam. It was efficiently inhibited by imipenem (K iapp = 11 nM), and formed a stable complex. While the K iapp value of meropenem was similar (16 nM), the corresponding complex was less stable.

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum ␤-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from bla TEM-1 by three mutations leading to the following amino acid substitutions: Glu 104 3Lys, Met 182 3Thr, and Gly 238 3Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated bla TEM-52 . The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (K i , 0.16 M) was 10-fold higher than that of TEM-3 (K i , 1.9 M). Allelic replacement analysis revealed that the combination of Lys 104 , Thr 182 , and Ser 238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.Klebsiella pneumoniae is an important pathogen that is usually susceptible to extended-spectrum cephalosporins. However, strains producing extended-spectrum ␤-lactamases (ESBLs) were described in the early 1980s, and, since that time, there has been an increase in the incidence of ceftazidime (CAZ)resistant Klebsiella strains responsible for nosocomial outbreaks (8,16,27). In most cases, ESBLs are plasmid-encoded enzymes providing resistance to oxyiminocephalosporins (CAZ, cefotaxime [CTX], ceftriaxone, cefpirome, and cefepime [FEP]) and to aztreonam (ATM) (16,24). Cephamycins (cefoxitin [FOX] and cefotetan [CTT]), moxalactam (MOX), and carbapenems are stable toward most ESBLs, and strains producing such enzymes remained susceptible to these molecules (7,24). The molecular basis of the extended spectrum often involves point mutations within plasmid-mediated ␤-lactamase genes resulting in either single or multiple amino acid substitutions in the corresponding enzymes (7,16,24). We describe here a novel TEM-type ESBL able to hydrolyze MOX. The enzyme is produced by a clinical isolate of K. pneumoniae.(Part of this work was presented at the 37th Interscience

The class A beta-lactamase PER-1, which displays 26% identity with the TEM-type extended-spectrum beta-lactamases (ESBLs), is characterized by a substrate profile similar to that conferred by these latter enzymes. The role of residues Ala164, His170, Ala171, Asn179, Arg220, Thr237 and Lys242, found in PER-1, was assessed by site-directed mutagenesis. Replacement of Ala164 by Arg yielded an enzyme with no detectable beta-lactamase activity. Two other mutants, N179D and A164R+N179D, were also inactive. Conversely, a mutant with the A171E substitution displayed a substrate profile very similar to that of the wild-type enzyme. Moreover, the replacement of Ala171 by Glu in the A164R enzyme yielded a double mutant which was active, suggesting that Glu171 could compensate for the deleterious effect of Arg164 in the A164R+A171E enzyme. A specific increase in kcat for cefotaxime was observed with H170N, whereas R220L and T237A displayed a specific decrease in activity towards the same drug and a general increase in affinity towards cephalosporins. Finally, the K242E mutant displayed a kinetic behaviour very similar to that of PER-1. Based on three-dimensional models generated by homology modelling and molecular dynamics, these results suggest novel structure-activity relationships in PER-1, when compared with those previously described for the TEM-type ESBLs.

A clinical isolate of Pseudomonas aeruginosa, PAe191, was found to be highly resistant to all anti-Pseudomonasβ-lactam antibiotics (except imipenem) and resistant also to aminoglycosides. It produced a β-lactamase (with an apparent pI of 7.6) which was not inhibited by clavulanic acid. Cloning and characterization of the β-lactamase gene showed that it coded for a novel extended-spectrum OXA-10 variant, called OXA-19, which differed from OXA-10 by nine amino acids and from OXA-13 by two, i.e., Asn in position 73 (Asn73) instead of Ser and Asp157 instead of Gly. Asparagine in position 157 is implicated in resistance to ceftazidime, while the amino acid in position 73, in this variant, seems to condition the level of resistance to penicillins. The oxa19gene was found to be inserted, in a typical integron structure, immediately downstream from anaac(6′)-Ib gene coding for an aminoglycoside acetyltransferase variant, which was called AAC(6′)-Ib9.