Phenotypic study of resistance of beta-lactamase-inhibitor-resistant TEM enzymes which differ by naturally occurring variations and by site-directed substitution at Asp276.

At this time an amino acid substitution at position 276 in the TEM-1 enzyme is associated with an additional substitution at position 69 in natural beta-lactamase-inhibitor-resistant (IRT) beta-lactamases. The effect of the Asn276-->Asp substitution on resistance was assessed with the Asn276Asp variant, generated by site-directed mutagenesis. The mutant was resistant to beta-lactamase inhibitors, but the MICs of amoxicillin combined with clavulanic acid or tazobactam were strikingly different for E. coli strains producing the Asn276Asp variant and those producing naturally occurring IRTs with single or double substitutions. The inhibitory effects of clavulanic acid and tazobactam were the same in IRTs with substitutions at position 69 (IRT-5 and IRT-6). The effect of clavulanic acid on the MICs of amoxicillin for the Asn276Asp variant was greater than that of tazobactam. In IRTs with double substitutions, at positions 69 plus 276 (IRT-4, IRT-7, and IRT-8) or 69 plus 275 (IRT-14), tazobactam was a more potent inhibitor than clavulanic acid. The effect of the Asn276-->Asp substitution on the values of the kinetic constants and the concentration required to inhibit by 50% the hydrolysis of benzylpenicillin confirms that this single mutation is responsible for resistance to beta-lactamase inhibitors. Molecular modeling of the Asn276Asp mutant shows that Asp276 can form two salt bonds with Arg244 close to the penicillin-binding cavity. The addition of the Asp276 mutation to that preexisting at position 69 confers a higher selective advantage to bacteria, as shown by the reduction in beta-lactamase inhibitor efficiencies of the double variants. Therefore, the emergence of multiple mutations in TEM beta-lactamases by virtue of the use of beta-lactamase inhibitors increases selection pressure resulting in the convergent evolution of resistant strains.

Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

Molecular diversity and evolution of blaTEM genes encoding beta-lactamases resistant to clavulanic acid in clinical E. coli.

The molecular diversity of inhibitor-resistant TEM (IRT) enzymes was explored using a strategy which involved DNA amplification by polymerase chain reaction (PCR), analysis of restriction fragment length polymorphism (RFLP), and direct nucleotide sequencing. The study of plasmid-borne genes from 27 strains, resistant to amoxicillin and beta-lactamase-inhibitor combinations, identified mutations resulting in amino acid change at positions 69, 244, 275, and 276 known to be associated with the IRT phenotype and a mutation at nucleotide position 162 in the promoter region. These mutations were found to lie on two different gene sequences, described here as "TEM-1B like" and "TEM-2 like" restriction linkage groups. Further analysis, of nucleotide sequences of promoter and coding regions of the beta-lactamases, confirmed that a given mutation causing IRT phenotype could be associated with two different gene sequence frameworks and two different causal mutations could lie on identical gene sequence framework. These data argue in favor of convergent phenotypic evolution of IRT enzymes under the selective pressure imposed by the intensive clinical use of beta-lactam-beta-lactamase inhibitor combinations.

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to beta-lactamase inhibitors.

The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.

Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2).

Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.

Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.

Borrelia isolates associated with Lyme borreliosis were previously divided into 3 genospecies, B. burgdorferi sensu stricto, B. garinii and group VS461, on the basis of DNA homology. B. burgdorferi sensu stricto and B. garinii were identified by monoclonal antibodies (MAbs), H3TS and D6 respectively, but no MAbs were available to identify group VS461. Two MAbs were produced, I 17.3 and J 8.3 which reacted with OspB and OspA proteins, respectively, of strains belonging to group VS461, which should be named B. afzelii sp. nov. 24 strains were assigned to B. afzelii sp. nov., 11 of them being isolated from skin lesions, 6 from acrodermatitis chronica atrophicans (ACA) and 5 from erythema chronicum migrans (ECM). Although quite unknown in the USA, ACA has frequently been reported in northern Europe where B. afzelii sp. nov. is commonly isolated. This study documents the involvement of B. afzelii sp. nov. as a specific aetiological agent of ACA.

Pseudomonas cepacia: the sensitivity of nosocomial strains to new antibiotics.

Pseudomonas cepacia, considered a phytopathogenic organism for many years, has been shown recently to be widely distributed geographically. The hospital environment has become an important source of this organism but the resistance of Ps. cepacia to most antibiotics has made the treatment of infections a problem. One hundred per cent of the strains tested have proved to be sensitive to the sulphonamides and to novobiocin, 93.0% to the combination of trimethoprim and sulfamethoxazole (co-trimoxazole); 85.2% to minocycline; 77.8% to chloramphenicol and dibekacin and 44.4% to nalidixic acid. One hundred per cent of the strains exhibit resistance to ampicillin, cephalothin, cefamandole, cefoxitin, colistin, cefuroxime, tetracycline and cefazolin; 88.9% to amikacin, tobramycin and sisomycin; 85.2% to carbenicillin. The new beta-lactams, apalcillin, ceftazidime, N-formimidoyl-thienamycin, piperacillin, cefotaxime and azlocillin proved to be the most potent of the molecules tested, inhibiting 90% of the strains, at concentrations of 4, 8, 8, 8, 32 and 16 mg/l and 100% of the strains at 8, 16, 16, 32, 32 and 64 mg/l, respectively. In contrast to the usual sensitivity patterns of Pseudomonas spp, Ps. cepacia has been shown to be resistant to colistin, cefsulodin and the aminoglycosides. However, unlike Ps. aeruginosa, Ps. cepacia has been shown, by the dilution method, to be sensitive to co-trimoxazole, 92.3% of the strains being inhibited by 16 mg/l.

In vitro activity of ceftazidime and other beta-lactam antibiotics against nosocomial strains of Pseudomonas aeruginosa.

The in vitro sensitivity of 300 nosocomial strains of Pseudomonas aeruginosa to ceftazidime was compared with their sensitivities to eleven other beta-lactam antibiotics. Concentrations of 2 mg/l of ceftazidime were sufficient to inhibit all of the strains tested including the carbenicillin-resistant ones. Ceftazidime shows considerably greater activity against Ps. aeruginosa than the other beta-lactam antibiotics.