Carbapenem resistance in a clinical isolate of Enterobacter aerogenes is associated with decreased expression of OmpF and OmpC porin analogs

We investigated the mechanism of imipenem resistance in Enterobacter aerogenes strain 810, a clinical isolate from the United States for which the imipenem MIC was 16 micro g/ml and the meropenem MIC was 8 micro g/ml. An imipenem-susceptible revertant, strain 810-REV, was obtained after multiple passages of the strain on nonselective media. For the revertant, the imipenem MIC was /=128 micro g/ml), cefoxitin (>/=32 micro g/ml), and cefotaxime (>/=64 micro g/ml) remained the same. The beta-lactamase and porin profiles of the parent, the revertant, and carbapenem-susceptible type strain E. aerogenes ATCC 13048 were determined. Strains 810 and 810-REV each produced two beta-lactamases with pIs of 8.2 and 5.4. The beta-lactamase activities of the parent and revertant were similar, even after induction with subinhibitory concentrations of imipenem. While 810-REV produced two major outer membrane proteins of 42 and 39 kDa that corresponded to Escherichia coli porins OmpC and OmpF, respectively, the parent strain appeared to produce similar quantities of the 39-kDa protein (OmpF) but decreased amounts of the 42-kDa protein (OmpC). When the parent strain was grown in the presence of imipenem, the 42-kDa protein was not detectable by gel electrophoresis. However, Western blot analysis of the outer membrane proteins of the parent and revertant with polyclonal antisera raised to the OmpC and OmpF analogs of Klebsiella pneumoniae (anti-OmpK36 and anti-OmpK35, respectively) showed that strain 810 expressed only the 42-kDa OmpC analog in the absence of imipenem (the 39-kDa protein was not recognized by the anti-OmpF antisera) and neither the OmpC nor the OmpF analog in the presence of imipenem. The OmpC analog is apparently down-regulated in the presence of imipenem; however, 810-REV expressed both OmpC and OmpF analogs. These data suggest that imipenem resistance in E. aerogenes 810 is primarily associated with the lack of expression of the analogs of the OmpC (42-kDa) and OmpF (39-kDa) outer membrane proteins, which also results in decreased susceptibility to meropenem and cefepime.     

Enhanced resistance to cefotaxime and imipenem associated with outer membrane protein alterations in Enterobacter aerogenes

Mutants exhibiting enhanced resistance to cefotaxime and imipenem were selected by plating a strain of Enterobacter aerogenes, which already produced chromosomal beta-lactamase constitutively, on to varying concentrations of different beta-lactam antibiotics. Frequencies of mutation varied from 10(-5) to 10(-8), depending upon the particular antibiotic and concentration used for selection. Only minor variations in beta-lactamase specific activities were observed and these could not be directly correlated with changes in resistance when compared with the original strain. In the majority of mutants, the selection of an enhanced level of resistance to cefotaxime was associated with a significant increase in resistance to imipenem, but no increase in resistance to the non-beta-lactam antibiotics tested was observed. Examination of outer membrane protein profiles revealed a number of complex changes in the mutants when directly compared to the original strain. In one mutant imipenem/cefotaxime resistance was directly associated with almost total loss of a 42K protein which was non-covalently associated with peptidoglycan and therefore possibly a porin protein.     

mar Operon involved in multidrug resistance of Enterobacter aerogenes

We determined the sequence of the entire marRAB operon in Enterobacter aerogenes. It is functionally and structurally analogous to the Escherichia coli operon. The overexpression of E. aerogenes MarA induces a multidrug resistance phenotype in a susceptible strain, demonstrated by a noticeable resistance to various antibiotics, a decrease in immunodetected porins, and active efflux of norfloxacin.     

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.     

产气肠杆菌质粒介导喹诺酮类耐药基因研究

产气肠杆菌广泛存在于自然界和健康人肠道中,是医院感染的重要病原菌,可引起泌尿道、呼吸道、伤口及血液等多种感染.喹诺酮类药物由于抗菌谱广、抗菌作用强,广泛用于临床抗感染治疗.     

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.     

Plasmid-Mediated Resistance to Expanded-Spectrum Cephalosporins among Enterobacter aerogenes Strains

Resistance to expanded-spectrum cephalosporins commonly develops in Enterobacter aerogenes during therapy due to selection of mutants producing high levels of the chromosomal Bush group 1 β-lactamase. Recently, resistant strains producing plasmid-mediated extended-spectrum β-lactamases (ESBLs) have been isolated as well. A study was designed to investigate ESBL production among 31 clinical isolates of E. aerogenes from Richmond, Va., with decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk potentiation test. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. β-Lactamases were investigated by an isoelectric focusing overlay technique which simultaneously determined isoelectric points (pIs) and substrate or inhibitor profiles. Decreased susceptibility to cefotaxime, ceftazidime, and aztreonam (MIC range, 1 to 64 μg/ml) was detected and associated with resistance to gentamicin and trimethoprim-sulfamethoxazole. All strains produced an inducible Bush group 1 β-lactamase (pI 8.3). Twenty-nine of the 31 isolates also produced an enzyme similar to SHV-4 (pI 7.8), while 1 isolate each produced an enzyme similar to SHV-3 (pI 6.9) and to SHV-5 (pI 8.2). The three different SHV-derived ESBLs were transferred by transconjugation to Escherichia coli C600N and amplified by PCR. Plasmid profiles of the clinical isolates showed a variety of different large plasmids. Because of the linkage of resistance to aminoglycosides and trimethoprim-sulfamethoxazole with ESBL production, it is possible that the usage of these drugs was responsible for selecting plasmid-mediated resistance to extended-spectrum cephalosporins in E. aerogenes. Furthermore, it is important that strains such as these be recognized, because they can be responsible for institutional spread of resistance genes.     

Rifampin resistance in Neisseria meningitidis due to alterations in membrane permeability.

Rifampin-resistant (Rifr) Neisseria meningitidis strains are known to have single point mutations in the central conserved regions of the rpoB gene. We have demonstrated two distinct resistance phenotypes in strains with identical mutations in this region, an intermediate level of resistance in Rifr clinical isolates and a high level of resistance in mutants selected in vitro. The possible role of membrane permeability in the latter was investigated by measuring MICs in the presence of Tween 80; values for high-level-resistance mutants were reduced to intermediate levels, whereas those for intermediate-level-resistance strains were unaffected. The highly resistant mutants were also found to have increased resistance to Triton X-100 and gentian violet. Sequencing of the meningococcal mtrR gene and its promoter region (which determine resistance to hydrophobic agents in Neisseria gonorrhoeae) from susceptible or intermediate strains and highly resistant mutants generated from them showed no mutation within this region. Two-dimensional gel electrophoresis of two parent and Rif mutant strains showed identical shifts in the pI of one protein, indicating that differences between the parent and the highly Rifr mutant are not confined to the rpoB gene. These results indicate that both permeability and rpoB mutations play a role in determining the resistance of N. meningitidis to rifampin.     

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.     

Imipenem resistance associated with the loss of a 40 kDa outer membrane protein in Enterobacter aerogenes.

An imipenem-resistant strain, Enterobacter aerogenes EA-Z, was isolated from a blood culture. Outer membrane protein (OMP) profiles indicated the loss of a 40 kDa OMP, decreased expression of 42 and 44 kDa OMPs, and increased expression of a 50 kDa OMP in strain EA-Z when compared with imipenem-susceptible clinical isolates of E. aerogenes. The OMP profile of EA-Z was similar to that of strain EA-SI16, an imipenem-resistant E. aerogenes second-step mutant selected on imipenem-containing media. A single-step imipenem-resistant mutant, EA-SI8, had lost expression of only the 40 kDa OMP. No new beta-lactamase could be detected by isoelectric focusing, and no increase in imipenem hydrolysis was seen when EA-Z was compared with imipenem-sensitive controls, even in the presence of added zinc. These data suggest that the 40 kDa OMP of E. aerogenes might be required for the normal diffusion of imipenem across the outer membrane.     

Ability of ceftibuten to induce the class-I beta-lactamases of Enterobacter cloacae, Serratia marcescens, and Enterobacter aerogenes

Ceftibuten, like cefotaxime, was observed to be a weak inducer of the class-I beta-lactamases of Enterobacter cloacae, Serratia marcescens, and Enterobacter aerogenes. In contrast, cefoxitin and imipenem induced these enzymes strongly at subinhibitory concentrations.     

Detection of ciprofloxacin resistance in gram-negative bacteria due to alterations in gyrA.

Two plasmids containing the cloned Escherichia coli wild-type gyrA gene were used to transform ciprofloxacin-resistant Gram-negative clinical isolates to screen for DNA gyrase A-mediated quinolone resistance. The results show that the technique is simple and applicable to a wide range of Gram-negative species including E. coli, Enterobacter cloacae, Klebsiella aerogenes, Morganella morganii, Proteus mirabilis, Pseudomonas aeruginosa, Campylobacter jejuni and Neisseria gonorrhoeae. The use of an arithmetical MIC series of dilutions (as opposed to standard geometrical ones) was found to be essential during screening for the detection of altered gyrase A. The observations were consistent with the suggestion that DNA gyrase is highly conserved among different species of bacteria and that gyrase A-mediated resistance can occur in all.     

阴沟肠杆菌临床感染分布特征及耐药性分析

目的了解川北地区阴沟肠杆菌的临床分布特征及耐药情况。方法回顾性分析临床分离59株阴沟肠杆菌的临床感染分布情况,琼脂稀释法进行药敏试验,改良三维试验检测产超广谱β-内酰胺酶(ESBLs)和产AmpCβ-内酰胺酶(AmpC),敏感、中介、耐药的判定采用美国临床实验室标准化协会(CLSI)2006年公布的标准。结果 59株阴沟肠杆菌在痰液和尿液标本、重症监护病房和呼吸科检出率高,且全部菌株除对亚胺培南耐药率极低外(3.39%),对其他11种抗菌药物的耐药率均大于20%;59株细菌中,检出产ESBLs和AmpC共计34株,单产ESBLs、同时产AmpC和ESBLs、单产AmpC菌株检出率分别为30.5%、8.47%、18.65%,ESBLs阳性菌株的检出率为38.97%,且ESBLs阳性菌株对除亚胺培南之外的其他11种抗菌药物显示了极高耐药率(44.45%~100%)。结论川北地区阴沟肠杆菌以引起危重患者的呼吸道和泌尿道感染为主,阴沟肠杆菌具有较高的ESBLs检出率,且多重耐药严重。     

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.     

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.     

Pseudomonas pseudomallei resistance to beta-lactam antibiotics due to alterations in the chromosomally encoded beta-lactamase.

Pseudomonas pseudomallei, the causative agent of melioidosis, is generally susceptible to some of the newer extended-spectrum cephalosporins or to combinations of a beta-lactam and clavulanic acid, a beta-lactamase inhibitor. Resistance to these agents may, however, emerge during treatment. We report on alterations in the chromosomal beta-lactamase associated with the development of resistance. Three resistance patterns resulted from three different mechanisms in the strains investigated. Derepression of the chromosomal enzyme resulted in a general increase in the MICs of all of the beta-lactams tested. The second mechanism observed was an insensitivity to inhibition of the beta-lactamase by clavulanic acid. In this case, the level of susceptibility to beta-lactams as independent entities remained unchanged. The final "resistance" pattern occurred in a patient treated with ceftazidime and resulted in a beta-lactamase that was capable of hydrolyzing this antibiotic at detectable levels, but with reduced efficacy against other beta-lactams. The net result was a strain that was generally susceptible to all of the beta-lactams tested except ceftazidime. In all cases, the level of susceptibility to antibiotics other than beta-lactams remained unchanged. Such variability found within one genus over a relatively short time course suggests that treatment of infections caused by this organism should be carefully monitored to detect susceptibility alterations to the chosen therapy.     

Prevalence of outer membrane porin alteration in beta-lactam-antibiotic-resistant Enterobacter aerogenes.

We evaluated the prevalence of impermeability as a mechanism associated with resistance against beta-lactam antibiotics in members of the family Enterobacteriaceae. During a 1-year period, 80 strains were selected from 3,110 routinely isolated strains according to their noticeable cross-resistance pattern to cephalosporins. They were tested for (i) outer membrane nonspecific porins involved in the entry of small hydrophilic molecules; (ii) the MICs of cefepime, cefotaxime, imipenem, and moxalactam; and (iii) beta-lactamase production. Immunological investigations using specific probes showed that 23 of 80 strains presented an alteration of the porin content, most of them expressing an additional resistance mechanism. The prevalence of this porin-deficient phenotype is especially high in Enterobacter aerogenes and concerns 6.4% of the clinical isolates.