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TEM derivative-producing Enterobacter aerogenes strains: dissemination of a prevalent clone.

TEM-24 (CAZ-6) extended-spectrum beta-lactamase (ESBL) was detected in 1988 in Clermont-Ferrand, France, in Klebsiella pneumoniae (bla(TEM-24)) and Enterobacter aerogenes (bla(TEM-24b)), and since 1994, a TEM-24-producing E. aerogenes clonal strain has been observed elsewhere in the country. To determine if the spread of this clonal strain was restricted to TEM-24-producing E. aerogenes strains, 84 E. aerogenes strains (non-TEM/SHV-producing strains, TEM-1- or -2-producing strains, and different ESBL-producing strains), isolated from 1988 to 1999 in Clermont-Ferrand (n = 59) and in 11 other French hospitals in 1998 (n = 25), were studied. A clonal strain was found for TEM-24- but also for TEM-3- and TEM-1- or 2-producing isolates. This study shows that there is a clonal strain dependent on acquisition of the TEM-type enzyme (TEM-24 and other TEM types).

Bacterial Proteins↗

TEM-80, a novel inhibitor-resistant beta-lactamase in a clinical isolate of Enterobacter cloacae.

Enterobacter cloacae Ecl261 was isolated with Escherichia coli Ec257 from the urine of a patient living in a nursing home. Both isolates were resistant to ticarcillin (MICs, 1,024 microg/ml), without significant potentiation of its activity by 2 microg of clavulanate per ml (MICs, 512 microg/ml), and susceptible to naturally active cephalosporins. This inhibitor-resistant phenotype was conferred in both strains by similar conjugative plasmids of 40 kb (Ecl261) and 30 kb (Ec257), which also conveyed resistance to sulfonamides and trimethoprim. Clinical and transconjugant strains produced a beta-lactamase with a pI of 5.2 which belonged to the TEM family, as indicated by specific PCR amplification. Compared with TEM-1, this enzyme exhibited lower catalytic efficiencies (14- and 120-fold less for amoxicillin and ticarcillin, respectively), and higher concentrations of beta-lactamase inhibitors were required to yield a 50% reduction in benzylpenicillin hydrolysis (750-, 82-, and 50-fold higher concentrations for clavulanate, sulbactam, and tazobactam, respectively). Gene sequencing revealed four nucleotide differences with the nucleotide sequence of bla(TEM-1A). The first replacement (T32C), located in the promoter region, was described as being responsible for the increase in the level of beta-lactamase production. The three other changes led to amino acid substitutions that define a new inhibitor-resistant TEM (IRT) beta-lactamase, TEM-80 (alternate name, IRT-24). Two of them, Met69Leu and Asn276Asp, have previously been related to inhibitor resistance. The additional mutation, Ile127Val, was demonstrated by site-directed mutagenesis to have a very weak effect, at least alone, on the IRT phenotype. This is the first description of an IRT beta-lactamase in E. cloacae. The horizontal transfer of bla(TEM-80) may have occurred either from Ec257 to Ecl261 or in the reverse order.

Aged↗

Outbreak of infections caused by Enterobacter cloacae producing the integron-associated beta-lactamase IBC-1 in a neonatal intensive care unit of a Greek hospital.

Nineteen of 27 ceftazidime-resistant Enterobacter cloacae isolates from a neonatal intensive care unit in Thessaloniki, Greece, had genes coding for the novel extended-spectrum beta-lactamase IBC-1; 18 of those 19 harbored similar conjugative plasmids and belonged to two distinct genetic lineages. A synergy test with ceftazidime and imipenem enabled us to identify five unrelated bla(IBC-1)-carrying E. cloacae isolates from other wards of the hospital. It seems that this integron-associated gene is capable of dispersing both by clonal spread and by gene dissemination.

Anti-Bacterial Agents↗

Mutational replacement of Leu-293 in the class C Enterobacter cloacae P99 beta-lactamase confers increased MIC of cefepime.

The class C beta-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum beta-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 beta-lactamase conferring a higher MIC of cefepime (MIC, 8 microg/ml, compared with 0.5 microg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other beta-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 beta-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k(cat)/K(m)) of the Leu-293-Pro mutant beta-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 beta-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant beta-lactamase.

Cefepime↗

The AcrAB-TolC efflux pump contributes to multidrug resistance in the nosocomial pathogen Enterobacter aerogenes.

We identified the genes encoding the AcrA-AcrB-TolC efflux pump in Enterobacter aerogenes and constructed acrAB and tolC mutants from a multidrug-resistant isolate. Both derivatives were more susceptible to antibiotics than the parental strain. Sequence analysis and complementation experiments revealed that the multidrug-resistant isolate is an acrR mutant.

Bacterial Outer Membrane Proteins↗

Prevalence of extended-spectrum beta-lactamase-producing Enterobacter cloacae in the Asia-Pacific region: results from the SENTRY Antimicrobial Surveillance Program, 1998 to 2001.

Enterobacter cloacae strains from hospitalized patients with a range of infections were collected by 17 laboratories in the Asia-Pacific region and South Africa. Isolates for which ceftriaxone MICs were above 1 microg/ml and/or ceftazidime MICs were above 2 microg/ml, as well as 46 strains for which ceftriaxone and/or ceftazidime MICs were at or below these values, were screened for levels of extended-spectrum beta-lactamase (ESBL) production through the use of broth microdilution for the detection of clavulanate enhancement of the activity of ceftriaxone, ceftazidime, and cefepime. Of the isolates examined, ceftriaxone and/or ceftazidime had elevated MICs for 44%, of which 36% were ESBL positive. ESBL-positive strains were commonly susceptible to piperacillin-tazobactam and more frequently resistant to several other antimicrobials studied. A cefepime MIC above 0.25 microg/ml had the highest sensitivity (100%) and specificity (74%) for predicting the presence of an ESBL.

Asia↗

Modification of outer membrane protein profile and evidence suggesting an active drug pump in Enterobacter aerogenes clinical strains.

Two clinical strains of Enterobacter aerogenes that exhibited phenotypes of multiresistance to beta-lactam antibiotics, fluoroquinolones, chloramphenicol, tetracycline, and kanamycin were investigated. Both strains showed a porin pattern different from that of a susceptible strain, with a drastic reduction in the amount of the major porin but with an apparently conserved normal structure (size and immunogenicity), together with overproduction of two known outer membrane proteins, OmpX and LamB. In addition, the full-length O-polysaccharide phenotype was replaced by a semirough Ra phenotype. Moreover, in one isolate the intracellular accumulation of chloramphenicol was increased in the presence of the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone, suggesting an energy-dependent efflux of chloramphenicol in this strain. The resistance strategies used by these isolates appear to be similar to that induced by stress in Escherichia coli cells.

Bacterial Outer Membrane Proteins↗

TEM-121, a novel complex mutant of TEM-type beta-lactamase from Enterobacter aerogenes.

Enterobacter aerogenes clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type beta-lactamase (TEM-121) differing by five amino acid substitutions from beta-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum beta-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant beta-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM beta-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.

Ceftazidime↗

Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-beta-lactamase encoded by a conjugative plasmid.

Resistance to carbapenems is an emerging problem among gram-negative hospital pathogens. A transferable plasmid encoding the VIM-4 metallo-beta-lactamase was detected in isolates of Klebsiella pneumoniae and Enterobacter cloacae obtained from a single patient under carbapenem therapy. Thus, enterobacteria appear to increasingly contribute to the spread of VIM-type enzymes.

Aged↗

Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate.

Enterobacter aerogenes resistant to cefepime (MIC, 32 microg/ml) was isolated from a patient treated with cefepime for an infection caused by a strain of E. aerogenes overproducing its AmpC beta-lactamase (MIC of cefepime, 0.5 microg/ml). The AmpC beta-lactamase of the resistant strain had an L-293-P amino acid substitution and a high k(cat)/K(m) ratio for cefepime. Both of these modifications were necessary for resistance to cefepime.

Amino Acid Sequence↗

Inhibitors of antibiotic efflux in resistant Enterobacter aerogenes and Klebsiella pneumoniae strains.

In Enterobacter aerogenes and Klebsiella pneumoniae, efflux provides efficient extrusion of antibiotics and contributes to the multidrug resistance phenotype. One of the alkoxyquinoline derivatives studied here, 2,8-dimethyl-4-(2'-pyrrolidinoethyl)-oxyquinoline, restores noticeable drug susceptibility to resistant clinical strains. Analyses of energy-dependent chloramphenicol efflux indicate that this compound inhibits the efflux pump mechanism and improves the activity of structurally unrelated antibiotics on multidrug-resistant E. aerogenes and K. pneumoniae isolates.

Anti-Bacterial Agents↗

Synergistic activities of moxifloxacin combined with piperacillin-tazobactam or cefepime against Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii clinical isolates.

The bactericidal activity of moxifloxacin alone and in combination with cefepime or piperacillin-tazobactam against clinical isolates of Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii was evaluated by using time-kill methods and antimicrobial concentrations of one-half and one times the MIC. Synergy was observed in 58 to 88% of the strains and resulted in bactericidal activity against 60 to 100% of the strains. Combinations including moxifloxacin demonstrated enhanced bactericidal activity compared with that of either agent tested alone.

Acinetobacter Infections↗

Omp35, a new Enterobacter aerogenes porin involved in selective susceptibility to cephalosporins.

In Enterobacter aerogenes, beta-lactam resistance often involves a decrease in outer membrane permeability induced by modifications of porin synthesis. In ATCC 15038 strain, we observed a different pattern of porin production associated with a variable antibiotic susceptibility. We purified Omp35, which is expressed under conditions of low osmolality and analyzed its pore-forming properties in artificial membranes. This porin was found to be an OmpF-like protein with high conductance values. It showed a noticeably higher conductance compared to Omp36 and a specific location of WNYT residues in the L3 loop. The importance of the constriction region in the porin function suggests that this organization is involved in the level of susceptibility to negative large cephalosporins such as ceftriaxone by bacteria producing the Omp35 porin subfamily.

Amino Acid Sequence↗

The AcrAB-TolC pump is involved in macrolide resistance but not in telithromycin efflux in Enterobacter aerogenes and Escherichia coli.

The role of the AcrAB-TolC pump in macrolide and ketolide susceptibility in Escherichia coli and Enterobacter aerogenes was studied. Efflux pump inhibitor restored erythromycin, clarithromycin, and telithromycin susceptibilities to multidrug-resistant isolates. No modification of telithromycin accumulation was detected in E. aerogenes acrAB or tolC derivatives compared to that in the parental strain. Two independent efflux pumps, inhibited by phenylalanine arginine beta-naphthylamide, expel macrolides and telithromycin in E. aerogenes.

Anti-Bacterial Agents↗

Molecular analysis of the simultaneous production of two SHV-type extended-spectrum beta-lactamases in a clinical isolate of Enterobacter cloacae by using single-nucleotide polymorphism genotyping.

Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of bla(SHV) gene products gave a "double peak" at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different bla(SHV) genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.

Enterobacter cloacae↗

Successive emergence of Enterobacter aerogenes strains resistant to imipenem and colistin in a patient.

Enterobacter aerogenes is an agent of hospital-acquired infection that exhibits a remarkable resistance to beta-lactam antibiotics during therapy. Five successive isolates of E. aerogenes infecting a patient and exhibiting a multiresistance phenotype to beta-lactam antibiotics and fluoroquinolones were investigated. Among these clinical strains, four presented resistant phenotypes during successive imipenem and colistin treatments. The involved resistance mechanisms exhibited by the successive isolates were associated with alterations of the outer membrane that caused a porin decrease and lipopolysaccharide modifications.

Anti-Bacterial Agents↗

Properties of silver sulfadiazine-resistant Enterobacter cloacae.

Two silver sulfadiazine-resistant isolates of Enterobacter cloacae obtained in a burns unit where the drug was in use were studied. These strains were resistant to elevated levels of the drug, and they were cross-resistant to silver benzoate, but not to silver nitrate. Growth of the strains in nutritionally poor defined media sensitized them to the inhibitory action of the drug. Exposure of the bacteria to penicillins rendered them susceptible to silver sulfadiazine. The resistant bacteria harbored episomes for resistance to carbenicillin and kanamycin; however, resistance to silver sulfadiazine could not be transferred by these episomes. Twenty-three strains of E. cloacae isolated in a general hospital were sensitive to much lower levels of the drug (</=50 mug/ml).

Cell Wall↗