Novel plasmid-mediated beta-lactamase (TEM-10) conferring selective resistance to ceftazidime and aztreonam in clinical isolates of Klebsiella pneumoniae.

Two clinical isolates of Klebsiella pneumoniae from seriously ill patients in Chicago, Ill., have been identified as resistant to ceftazidime and aztreonam but susceptible to other cephalosporins. This unusual antibiogram was shown to be due to a novel plasmid-mediated beta-lactamase which readily hydrolyzed ceftazidime and aztreonam in addition to penicillins such as piperacillin and carbenicillin. This enzyme and its attendant resistance were transferred to Escherichia coli by conjugation on a 50-kilobase plasmid. Isoelectric focusing revealed a single beta-lactamase band with a molecular weight of 29,000 and an isoelectric point of 5.57 in the resistant isolates and transconjugants. The beta-lactamase inhibitors clavulanic acid and sulbactam restored beta-lactam susceptibility in the resistant isolates. Fifty percent inhibitory concentrations of clavulanic acid and sulbactam were 4.4 and 940 nM, respectively. DNA hybridization studies indicated that this enzyme, designated TEM-10, is related to well-established TEM-type beta-lactamases. However, the TEM-10 enzyme was inhibited by p-chloromercuribenzoate, in contrast to TEM-2 beta-lactamase. On the basis of substrate and inhibition profiles, the TEM-10 enzyme could be easily discriminated from TEM-5 and RHH-I beta-lactamases.     

Emergence of clinical isolates of Escherichia coli producing TEM-1 derivatives or an OXA-1 beta-lactamase conferring resistance to beta-lactamase inhibitors.

Sixteen Escherichia coli clinical isolates which were resistant to ampicillin and amoxicillin-clavulanate but susceptible to cephalothin were studied. Eight strains showed the presence of a beta-lactamase which comigrates with reference OXA-1 enzyme. The eight other strains produced different TEM-1 derivatives which had in common a higher Km for penicillins and a higher 50% inhibitory concentration for the beta-lactamase inhibitors. By oligotyping and sequencing of PCR products, it was shown that Ser (AGC) (TEM-30; also called TRI-1) in three strains and Cys (TGC) (TEM-31; also called TRI-2) in one strain were substituted for Arg-241 (CGC), that Leu (CTG) (TEM-33) and Val (GTG) (TEM-34) in one strain each were substituted for Met-67 (ATG), and that in other mutants the two latter substitutions occurred together with the substitution of Asp (GAT) (TEM-35 and TEM-36) for Asn-272 (AAT). Therefore, different sets of amino acid substitutions of TEM-1 can be found in clinical isolates and lead to resistance to beta-lactamase inhibitors.     

In vivo selection of a chromosomally encoded beta-lactamase variant conferring ceftazidime resistance in Klebsiella oxytoca

Klebsiella oxytoca clinical isolate A was recovered from the urine of a 55-year-old man with prostatic and urinary tract infections. This isolate displayed a beta-lactam resistance phenotype consistent with overproduction of a chromosomally encoded class A beta-lactamase and had decreased susceptibilities to all beta-lactams except ceftazidime, cephamycins, and carbapenems. Four weeks after treatment with an antibiotic regimen that included ceftazidime, K. oxytoca isolate B, which had a high level of resistance to ceftazidime, was isolated from the urine of the same patient. Isoelectric focusing analysis of the culture extracts of these isolates gave a pI of 5.4 for both isolates. Cloning experiments with the PCR products of the bla(OXY) gene resulted in two Escherichia coli DH10B recombinant clones with resistance phenotypes mirroring those of the parental isolates. Sequencing analysis revealed that the bla(OXY-2-5) gene from K. oxytoca B had a single nucleotide substitution compared to the sequence of the bla(OXY-2) gene from K. oxytoca A, leading to a proline-to-serine substitution at position 167, according to the numbering of Ambler. Biochemical analysis of purified OXY-2-5 showed that it had the ability to hydrolyze ceftazidime. This is the first report of in vivo selection of a K. oxytoca isolate that produced a chromosomally encoded beta-lactamase conferring resistance to ceftazidime.     

Monoclonal antibodies to TEM-1 plasmid-mediated beta-lactamase

At least 28 plasmid-mediated beta-lactamases have been described in gram-negative bacteria. To assess the relationship among these enzymes, we produced and characterized 28 murine monoclonal antibodies to the TEM-1 plasmid-mediated beta-lactamase. Radial immunodiffusion identified 3 monoclonal antibodies as immunoglobulin M (IgM), 18 as subclass IgG1, 2 as IgG2a, and 5 as IgG2b. Using a newly described enzyme immunoassay, cross-reactivity of 16 of these monoclonal antibodies was tested against 24 plasmid-determined beta-lactamases. The 16 monoclonal antibodies cross-reacted with TEM-2 and TLE-1 and, to a certain extent, SHV-1. Different levels of cross-reactivity were also observed with OXA-3 (11 of 16), OXA-7 (8 of 16), OXA-1 (2 of 16), OXA-6 (2 of 16), and AER-1 (2 of 16). Six monoclonal antibodies demonstrated partial neutralization of beta-lactamase activity. This study suggests that common epitopes are shared by nine biochemically distinct plasmid-mediated beta-lactamases. On the basis of cross-reactivities with these monoclonal antibodies, we identified four epitopes on TEM-1, TEM-2, TLE-1, and SHV-1 beta-lactamases.     

Mutant TEM beta-lactamase producing resistance to ceftazidime, ampicillins, and beta-lactamase inhibitors

A derivative of the TEM-1 beta-lactamase producing clinically significant levels of resistance to ceftazidime and beta-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection. This complex mutant enzyme contained three amino acid substitutions including those of residues 104 and 276 that are known to produce extended-spectrum resistance and, correspondingly, resistance to beta-lactamase inhibitors. Although the Glu104Lys substitution by itself produced low levels of ceftazidime resistance, additional amino acid replacements in the enzyme with the triple mutation resulted in further enhancement of resistance to ceftazidime. Kinetic studies of the purified beta-lactamase enzyme with the triple mutation indicated enhancement of the catalytic efficiency for turnover (kcat/Km) of ceftazidime. The increases in the Ki values of both clavulanic acid and tazobactam for the enzyme with the triple mutation were consistent with the observed bacterial resistance to the reversibility of beta-lactam resistance with these inhibitors.     

Pharmacodynamics of ceftazidime plus the serine beta-lactamase inhibitor AM-112 against Escherichia coli containing TEM-1 and CTX-M-1 beta-lactamases

A strain of Escherichia coli containing TEM-1 and CTX-M-1 was tested in an in vitro pharmacokinetic model against ceftazidime with and without AM-112, a serine beta-lactamase inhibitor. Ceftazidime alone was less effective than ceftazidime plus AM-112, and a single dose was more effective than three fractionated doses.     

A new TEM-derived extended-spectrum beta-lactamase (TEM-91) with an R164C substitution at the omega-loop confers ceftazidime resistance

A new plasmid-mediated TEM-derived extended-spectrum beta-lactamase, TEM-91, was identified in a ceftazidime-resistant (MIC, >128 microg per ml) Escherichia coli strain isolated in 1996 in Japan. TEM-91 has three amino acid substitutions, R164C, M184T, and E240K, compared with TEM-1 penicillinase. The isoelectric point (pI), K(m), and k(cat) of TEM-91 for ceftazidime were 5.7, 179 microM, and 29.0 s(-1), respectively. The K(i) of clavulanic acid for ceftazidime hydrolysis was 30.3 nM.     

Behaviour of TEM-1 beta-lactamase as a resistance mechanism to ampicillin, mezlocillin and azlocillin in Escherichia coli

Escherichia coli isolates which synthesised the extremely common 'TEM-1' plasmid mediated beta-lactamase were more resistant to the alpha-aminopenicillins, ampicillin, mezlocillin and azlocillin, than were strains which lacked this enzyme. However, many TEM-1+ isolates remained sensitive to therapeutic concentrations of mezlocillin (less than 64 mg/l), whereas virtually none was susceptible to such levels of ampicillin or azlocillin. Transconjugants of E. coli K12 into which we introduced various TEM-1 coding plasmids similarly acquired lower levels of resistance to mezlocillin than to ampicillin and azlocillin. Those which expressed relatively small amounts of enzyme remained sensitive to 64 mg/l of mezlocillin, whereas they were substantially resistant (MIC greater than 64 mg/l) to the other alpha-aminopenicillins. These data suggested that the enzyme afforded weaker protection against mezlocillin than against azlocillin and ampicillin and we attempted to relate this finding to its hydrolytic activity. Extracted TEM-1 beta-lactamase hydrolysed high concentrations of mezlocillin more rapidly than ampicillin and azlocillin; however, mezlocillin was calculated to be the weakest substrate at the low concentrations which are likely to be obtainable in the bacterial cell. These data may partly account for the residual activity of mezlocillin against enzyme producers, but target and permeability factors probably also contribute.     

Prediction of the evolution of ceftazidime resistance in extended-spectrum beta-lactamase CTX-M-9

A random mutagenesis technique was used to predict the evolutionary potential of beta-lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1- to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k(cat) value than CTX-M-9 against ceftazidime. The other two had 75- to 300-fold-lower Km values than CTX-M-9 against ceftazidime. The current emergence of CTX-M beta-lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.     

Experimental prediction of the evolution of ceftazidime resistance in the CTX-M-2 extended-spectrum beta-lactamase

We applied in vitro evolution to an Escherichia coli strain containing bla(CTX-M-2) and obtained 10 independent mutant bla(CTX-M-2) alleles that confer elevated resistance to ceftazidime (MIC > or = 32 microg/ml) but lost the ability to confer resistance to cefepime. All alleles had a Pro-to-Ser substitution at position 167.     

Molecular epidemiology of TEM-3 (CTX-1) beta-lactamase.

A total of 33 clinical isolates encoding TEM-3 (CTX-1) from four French hospitals were studied. The strains belonged to seven species, Klebsiella pneumoniae (n = 24), Escherichia coli (n = 3), Serratia marcescens (n = 2), Citrobacter freundii (n = 1), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 1), and Klebsiella oxytoca (n = 1). All the strains harbored an Inc7 or M self-transferable plasmid with a size of approximately 85 kilobases. The plasmids had closely related EcoRI, HincII, HindIII, and PvuII restriction endonuclease-generated patterns and conferred resistance to all beta-lactams, except cephamycins and imipenem; to tetracycline, because of the presence of the genes blatem-3 and tetC, respectively, as determined by hybridization with specific probes; and to sulfonamide. Depending on the presence or absence and level of expression of the genes aacA4, aadA, and dfrI and of insertion element IS15, four types of plasmids could be distinguished. Plasmid pCFF04, the prototype plasmid encoding TEM-3, was widespread and appeared, by Southern hybridization, as the progenitor of the other types of replicons. The plasmid epidemic responsible for dissemination of TEM-3 in clinical isolates of members of the family Enterobacteriaceae may have originated in S. marcescens since pCFF04 was first detected in this species.     

Laboratory mutants of OXA-10 beta-lactamase giving ceftazidime resistance in Pseudomonas aeruginosa

Several extended-spectrum beta-lactamases (ESBLs) belonging to molecular Class D have been described from Pseudomonas aeruginosa isolates collected in Turkey. Four of these, OXA-11, -14, -16 and -17, are derivatives of OXA-10 beta-lactamase. We tried to select similar mutants in vitro from OXA-10-producing transconjugants of P. aeruginosa, using a multistep method on ceftazidime-containing agars. Forty-four such mutants were obtained; all had increased resistance to ceftriaxone, cefsulodin, cefepime, cefpirome, latamoxef, aztreonam and, especially, ceftazidime whereas MICs of piperacillin, carbenicillin, cefotaxime, cefoperazone and carbapenems were little altered. Genes related to blaOXA-10 were sequenced from five mutants. One mutant enzyme had aspartate instead of glycine at position 157, and corresponded exactly to natural OXA-14 beta-lactamase. Another mutant strain appeared to have both OXA-14 and a new pI 6.2 enzyme, designated OXA-M102, with serine instead of alanine at position 124 and aspartate instead of glycine at position 157. This latter variant resembled natural OXA-16 enzyme, which has threonine at position 124 and aspartate at position 157. The remaining three mutant enzymes differed from any so far found in wild-type isolates. Two had leucine replacing tryptophan at position 154 (this enzyme was named OXA-M101) while the third (OXA-M103) had a pI of 7.6, and had lysine instead of asparagine at position 143. A different mutation at this position was previously found in OXA-11, a wild-type OXA-10 mutant. Thus, some of the ESBL mutants selected (OXA-14 and OXA-M102) correspond exactly or almost exactly to ESBLs found in wild-types, whereas others (OXA-M101 and OXA-M103) were totally new.     

An in-vitro and in-vivo comparison of the activity of beta-lactamase inhibitor combinations with imipenem and cephalosporins against Escherichia coli producing TEM-1 or TEM-2 beta-lactamase.

Reference strains of Escherichia coli (ampicillin-susceptible and -resistant ATCC strains, and known TEM-1 and TEM-2 beta-lactamase producers) were tested in vitro and in the in-vivo mouse thigh infection model against four beta-lactamase inhibitor compounds (BICs: amoxycillin/clavulanic acid, ampicillin/sulbactam, ticarcillin/clavulanic acid, and piperacillin/tazobactam), selected cephalosporins, and imipenem. The ATCC strains (ampicillin-susceptible and -resistant) were susceptible to the BICs in disc and MIC tests. Three or more logs of killing were observed at the NCCLS breakpoint concentrations. However, the TEM-1 and TEM-2 producers were resistant in disc tests to ampicillin/sulbactam and amoxycillin/clavulanic acid, and showed intermediate susceptibility to ticarcillin/clavulanic acid. MICs were at or near the breakpoint, but bactericidal activity was only noted at the probable breakpoint concentration of piperacillin/tazobactam. Cefoxitin, cefotaxime, cefpirome and imipenem, but not cephalothin, showed greater bactericidal activity and lower MICs for the TEM-producing strains than the BICs. The viable count of the TEM-1 producer was not reduced in the mouse thigh model by ampicillin/sulbactam or amoxycillin/clavulanic acid, but cefpirome and cefotaxime reduced the viable count by approximately three logs. There was a 50% mortality rate in mice receiving the two BICs. The ampicillin-susceptible ATCC strain of E. coli was killed to a similar degree by all agents tested. Overall, the BICs appeared inferior, in both in-vivo and in-vitro tests to selected cephalosporins and imipenem when tested against reference strains of E. coli producing TEM-1 or TEM-2 beta-lactamase. The large inoculum effect and poor bactericidal activity observed with the BICs suggest they could be less effective in certain clinical situations.     

Outbreak of ceftazidime resistance due to a novel extended-spectrum beta-lactamase in isolates from cancer patients.

Ceftazidime is widely used in the therapy of infectious complications in neutropenic patients. We studied an outbreak of ceftazidime-resistant gram-negative bacillary infections in pediatric cancer patients receiving empirical ceftazidime therapy for neutropenic fever. Fourteen isolates (12 Klebsiella pneumoniae and 2 Escherichia coli) from 13 patients were studied. Specimens were obtained from multiple clinical sites including blood, urine, throat, and lung. The organisms were resistant to ceftazidime, aztreonam, and penicillins but remained susceptible to cephamycins and imipenem. All resistant isolates produced a novel beta-lactamase (TEM-26) with a pI of approximately 5.58, which was transferred by transformation to E. coli on a 7.9-kb nonconjugative plasmid which cotransferred resistance to trimethoprim-sulfamethoxazole. This enzyme readily hydrolyzed ceftazidime, aztreonam, and penicillins in a spectrophotometric assay. DNA sequencing data suggest that TEM-26 is derived from TEM-1.     

Reversal of clavulanate resistance conferred by a Ser-244 mutant of TEM-1 beta-lactamase as a result of a second mutation (Arg to Ser at position 164) that enhances activity against ceftazidime.

The mutation of Arg-244 to Ser (Arg-244-->Ser mutation) in the TEM-1 beta-lactamase has been shown to produce resistance to inactivation by clavulanate in the mutant enzyme and resistance to ampicillin plus clavulanate in a strain of Escherichia coli producing this enzyme. The Arg-164-->Ser mutation in the TEM-1 beta-lactamase (TEM-12 enzyme) is known to enhance the activity of the enzyme against ceftazidime, resulting in resistance to the drug in a strain producing the mutant enzyme (D. A. Weber, C. C. Sanders, J. S. Bakken, and J. P. Quinn, J. Infect. Dis. 162:460-465, 1990). The doubly mutated derivative of the TEM-1 enzyme (Ser-164/Ser-244) retains the characteristics of the Ser-164 mutant enzyme, i.e., enhanced activity against ceftazidime and sensitivity to inactivation by clavulanate. It also confers the same phenotype as the Ser-164 mutant enzyme, i.e., resistance to ceftazidime and ampicillin, with reversal of this resistance in the presence of clavulanate. Thus, the Arg-164-->Ser mutation in the TEM-1 beta-lactamase suppresses the effect of the Arg-244-->Ser mutation which, by itself, reduces the sensitivity of the enzyme to inactivation by clavulanate.     

Novel plasmid-mediated beta-lactamase (MIR-1) conferring resistance to oxyimino- and alpha-methoxy beta-lactams in clinical isolates of Klebsiella pneumoniae

Klebsiella pneumoniae isolates from 11 patients at the Miriam Hospital were identified as resistant to cefoxitin and ceftibuten as well as to aztreonam, cefotaxime, and ceftazidime. Resistance could be transferred by conjugation or transformation with plasmid DNA into Escherichia coli and was due to the production of a beta-lactamase with an isoelectric point of 8.4 named MIR-1. In E. coli, MIR-1 conferred resistance to aztreonam, cefotaxime, ceftazidime, ceftibuten, ceftriaxone, and such alpha-methoxy beta-lactams as cefmetazole, cefotetan, cefoxitin, and moxalactam. In vitro, MIR-1 hydrolyzed cephalothin and cephaloridine much more rapidly than it did penicillin G, ampicillin, or carbenicillin. Cefotaxime was hydrolyzed at 10% the rate of cephaloridine. Cefoxitin inactivation could only be detected by a microbiological test. The inhibition profile of MIR-1 was similar to that of chromosomally mediated class I beta-lactamases. Potassium clavulanate had little effect on cefoxitin or cefibuten resistance and was a poor inhibitor of MIR-1 activity. Cefoxitin or imipenem did not induce MIR-1. The gene determining MIR-1 was cloned on a 1.4-kb AccI-PstI fragment. Under stringent conditions, probes for TEM-1 and SHV-1 genes and the E. coli ampC gene failed to hybridize with the MIR-1 gene. However, a provisional sequence of 150 bp of the MIR-1 gene proved to be 90% identical to the sequence of ampC from Enterobacter cloacae but only 71% identical to that of E. coli, thus explaining the lack of hybridization to the E. coli ampC probe. Plasmid profiles of the 11 K. pneumoniae clinical isolates were not identical, but each contained a plasmid from 40 to 60 kb that hybridized with the cloned MIR-1 gene. Both transfer-proficient and transfer-deficient MIR-1 plasmids belonged to the N incompatibility group. Thus, the resistance of these K. pneumoniae strains was the result of plasmid acquisition of a class I beta-lactamase, a new resistance determinant that expands the kinds of beta-lactam resistance capable of spread by plasmid dissemination among clinical isolates.