Beta-lactamases represent the main mechanism of bacterial resistance to beta-lactam antibiotics. The recent emergence of bacterial strains producing inhibitor-resistant TEM (IRT) enzymes could be related to the frequent use of beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam in hospitals and in general practice. The IRT beta-lactamases differ from the parental enzymes TEM-1 or TEM-2 by one, two or three amino acid substitutions at different locations. This paper reviews the phenotypic, genetic and biochemical characteristics of IRT beta-lactamases in an attempt to shed light on the pressures that have contributed to their emergence.

Approximately 10% (89 isolates) of Klebsiella pneumoniae isolated in 1985 from patients in intensive care units in Clermont-Ferrand exhibited a complex resistance phenotype towards antibiotics. They were resistant to amino-, carboxy- and ureidopenicillins, aminoglycosides (except gentamicin), chloramphenicol, sulphonamides, tetracyclines and, most importantly, to cephalosporins (except cefoxitin and latamoxef) and to aztreonam. The metabolic profile of fifty isolates was identical and seven were selected for further study. All the resistance characters in these isolates were transferable to Escherichia coli by conjugation and were lost en bloc after treatment with ethidium bromide. Agarose gel electrophoresis of crude lysates of the wild types and their transconjugants indicated that the multiple resistances were mediated by a 95kb plasmid, pCF04. The seven isolates selected for study and their corresponding transconjugants, constitutively produced a plasmid-mediated beta-lactamase with a pI of 6.3 that was much more active against third-generation cephalosporins than against cephalothin. The substrate profile and the isoelectric-focusing behaviour of this enzyme differed from those of other known plasmid-mediated beta-lactamases, and the enzyme was designated CTX-1. A chromosomally-encoded SHV-1 (PIT-2) penicillinase (pI 7.7) was also present in the seven K. pneumoniae isolates but did not transfer. Resistance to aminoglycosides in the K. pneumoniae isolates was due to synthesis of a 6'-aminoglycoside acetyltransferase type IV. Our data indicate an epidemic of antibiotic multiply-resistant strains of K. pneumoniae producing a new beta-lactamase.

Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla CTX-M genes encoding ␤-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla CTX-M-9 gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-Mencoding gene, designated bla CTX

To estimate the diversity of extended-spectrum ␤-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains (Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae, E. aerogenes, and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as a bla CTX-M gene. E. coli HB101 transconjugants and the E. coli DH5␣ transformant harboring a recombinant plasmid produced a CTX-M ␤-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes. The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

From January 1988 to August 1989, 267 non-repetitive strains of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBla) derived from TEM (CTX-1/TEM-3, CAZ-6) or SHV (CAZ-5) were isolated from 219 colonized or infected patients. ESBlas were characterized by analytical isoelectric focusing. Biotypes, resistance phenotypes and plasmid patterns were determined in order to differentiate the isolates in each species. Among the 116 CTX-1-producing Enterobacteriaceae, 48 strains were differentiated: 27 from 74 Klebsiella pneumoniae isolates, seven from 22 Enterobacter aerogenes isolates, and 14 from a combined total of seven K. oxytoca, five Serratia marcescens, six Escherichia coli, 1 Enterobacter cloacae and 1 Citrobacter freundii. CAZ-5 has been isolated since January 1988 in 16 different strains among 101 K. pneumoniae isolates. CAZ-6 was first identified in K. pneumoniae (January 1988). Among the 48 Enterobacteriaceae producing CAZ-6, 12 strains were differentiated: four from 39 E. aerogenes isolates, three from four K. pneumoniae, and five from a combined total of two S. marcescens, two E. coli and one E. cloacae. During this outbreak, CTX-1 was found to be encoded by 85 kb (Inc 7/M) or greater than or equal to 150 kb (Inc 6/C) plasmids. CAZ-6 was always encoded by an 85 kb (Inc 7/M) plasmid and CAZ-5 by a greater than 150 kb plasmid. These results show that strain epidemics and plasmid dissemination occurred mainly in K. pneumoniae and E. aerogenes for CTX-1, in E. aerogenes for CAZ-6, and in K. pneumoniae for CAZ-5. They also suggest that the bla(tem) gene (CTX-1) has spread between different plasmids present in the same ecosystem.