Multidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence in Enterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates of E. cloacae were used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). The acrA and tolC genes were deleted in strains EcDC64 and Jc194 to produce, respectively, Ec⌬acrA and Ec⌬tolC and Jc⌬acrA and Jc⌬tolC knockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance of E. cloacae to several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that the acrA and tolC genes both affect the fitness of E. cloacae, as fitness was clearly reduced in the acrA and tolC KO strains. The median CI values obtained in vitro and in vivo were, respectively, 0.42 and 0.3 for EcDC64/Ec⌬acrA, 0.24 and 0.38 for EcDC64/Ec⌬tolC, 0.15 and 0.11 for Jc194/Jc⌬acrA, and 0.38 and 0.39 for Jc194/Jc⌬tolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in both E. cloacae clinical strains when either the acrA or tolC gene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates of E. cloacae.

Acinetobacter baumannii is an opportunistic pathogen that has been associated with severe infections and outbreaks in hospitals. At present, very little is known about the biology of this bacterium, particularly as regards mechanisms of adaptation, persistence and virulence. To investigate the growth phase-dependent regulation of proteins in this microorganism, we analyzed the proteomic pattern of A. baumannii ATCC 17978 at different stages of in vitro growth. In this study, proteomics analyses were conducted using 2-DE and MALDI-TOF/TOF complemented by iTRAQ LC-MS/MS. Here we have identified 107 differentially expressed proteins. We highlight the induction of proteins associated with signaling, putative virulence factors and response to stress (including oxidative stress). We also present evidence that ROS (O(2)(-) and OH(-)) and RNI (ONOO(-)) accumulate during late stages of growth. Further assays demonstrated that stationary cells survive at high concentrations of H(2)O(2) (30 mM), the O(2)(-) donor menadione (500 muM) or the NO donor sodium nitroprusside (1 mM), and showed a higher survival rate against several bactericidal antibiotics. The growth phase-dependent changes observed in the A. baumannii proteome are discussed within a context of adaptive biological responses, including those related to ROS and RNI stress.

Enterobacter cloacae is an emerging clinical pathogen that may be responsible for nosocomial infections. Management of these infections is often difficult, owing to the high frequency of strains that are resistant to disinfectants and antimicrobial agents in the clinical setting. Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role as a mechanism of antimicrobial resistance in gram-negative pathogens. In the present study, we cloned and sequenced the genes encoding an AcrAcBTolC-like efflux pump from an E. cloacae clinical isolate (isolate EcDC64) showing a broad antibiotic resistance profile. Sequence analysis showed that the acrR, acrA, acrB, and tolC genes encode proteins that display 79.8%, 84%, 88%, and 82% amino acid identities with the respective homologues of Enterobacter aerogenes and are arranged in a similar pattern. Deletion of the acrA gene to yield an AcrA-deficient EcDC64 mutant (Ec⌬acrA) showed the involvement of AcrAB-TolC in multidrug resistance in E. cloacae. However, experiments with an efflux pump inhibitor suggested that additional efflux systems also play a role in antibiotic resistance. Investigation of several unrelated isolates of E. cloacae by PCR analysis revealed that the AcrAB system is apparently ubiquitous in this species.Multidrug efflux pumps, especially those belonging to the resistance-nodulation-division family, play a major role in establishing the "intrinsic or acquired" resistance of gram-negative bacteria to a wide range of toxic compounds, including antibiotics (20).A well-studied example is the AcrAB-TolC multidrug resistance (MDR) tripartite pump system of Escherichia coli, which confers resistance to a wide variety of lipophilic and amphiphilic compounds, including dyes, detergents, and antimicrobial agents such as ethidium bromide, crystal violet, sodium dodecyl sulfate, bile acids, tetracycline, chloramphenicol, fluoroquinolones, ␤-lactams, erythromycin, and fusidic acid (17, 18). The presence of similar systems has been reported for other members of Enterobacteriaceae (2,9,11,12,14,15,16,21,28).The species of Enterobacter that most commonly cause nosocomial infections are E. cloacae and E. aerogenes (24). The existence of an acrB-like gene (11) has previously been identified in E. cloacae, although none of the components of the efflux pump have been cloned.The objectives of the present study were (i) to characterize the genes encoding the AcrAB-TolC-like efflux pump of E. cloacae and (ii) to determine the involvement of this efflux pump in MDR in E. cloacae. The clinical strain used in the study was an MDR strain of E. cloacae (EcDC64) which overexpressed the AcrAB system and also overexpressed the chromosomal ampC gene and exhibited a drastic reduction of ompC gene expression.(These results were presented in part at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy [2a].) MATERIALS AND METHODSBacterial strains and growth media. The bacterial strains used in the study are listed i...

b␤-Lactamases and penicillin-binding proteins (PBPs) have evolved from a common ancestor. ␤-Lactamases are enzymes that degrade ␤-lactam antibiotics, whereas PBPs are involved in the synthesis and processing of peptidoglycan, which forms an elastic network in the bacterial cell wall. This study analyzed the interaction between ␤-lactamases and peptidoglycan and the impact on fitness and biofilm production. A representative set of all classes of ␤-lactamases was cloned in the expression vector pBGS18 under the control of the CTX-M promoter and expressed in Escherichia coli MG1655. The peptidoglycan composition of all clones was evaluated, and quantitative changes were found in E. coli strains expressing OXA-24, OXA-10-like, and SFO-1 (with its upstream regulator AmpR) ␤-lactamases; the level of cross-linked muropeptides decreased, and their average length increased. These changes were associated with a statistically significant fitness cost, which was demonstrated in both in vitro and in vivo experiments. The observed changes in peptidoglycan may be explained by the presence of residual DD-endopeptidase activity in these ␤-lactamases, which may result in hydrolysis of the peptide cross bridge. The biological cost associated with these changes provides important data regarding the interaction between ␤-lactamases and the metabolism of peptidoglycan and may provide an explanation for the epidemiology of these ␤-lactamases in Enterobacteriaceae.

Overall, the results showed that the two residues located in the L3 loop are sufficient to confer the substrate specificity of each enzyme, thus highlighting the importance of the L3 loop of the active site in the evolution of VIM-type metallo-beta-lactamases.

Arg-276 in CTX-M-type beta-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid.

The CTX-M ␤-lactamases are an increasingly prevalent group of extended-spectrum ␤-lactamases (ESBL). Point mutations in CTX-M ␤-lactamases are considered critical for enhanced hydrolysis of cefotaxime. In order to clarify the structural determinants of the activity against cefotaxime in CTX-M ␤-lactamases, screening for random mutations was carried out to search for decreased activity against cefotaxime, with the CTX-M-1 gene as a model. Thirteen single mutants with a considerable reduction in cefotaxime MICs were selected for biochemical and stability studies. The 13 mutated genes of the CTX-M-1 ␤-lactamase were expressed, and the proteins were purified for kinetic studies against cephalothin and cefotaxime (as the main antibiotics). Some of the positions, such as Val103Asp, Asn104Asp, Asn106Lys, and Pro107Ser, are located in the 103 VNYN 106 loop, which had been described as important in cefotaxime hydrolysis, although this has not been experimentally confirmed. There are four mutations located close to catalytic residues-Thr71Ile, Met135Ile, Arg164His, and Asn244Asp-that may affect the positioning of these residues. We show here that some distant mutations, such as Ala219Val, are critical for cefotaxime hydrolysis and highlight the role of this loop at the top of the active site. Other distant substitutions, such as Val80Ala, Arg191, Ala247Ser, and Val260Leu, are in hydrophobic cores and may affect the dynamics and flexibility of the enzyme. We describe here, in conclusion, new residues involved in cefotaxime hydrolysis in CTX-M ␤-lactamases, five of which are in positions distant from the catalytic center.

Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases.