Study of a Natural Mutant SHV-Type -Lactamase, SHV-104, from Klebsiella pneumoniae

Klebsiella pneumoniae ML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited by K. pneumoniae ML2011 was performed by Etest. The crude extract of the isolates contains four different -lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only the -lactamases with pI 7.3 and pI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomal -lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV-104 (pI 7.3) were encoded by bla gene located on a 50 kb highly conjugative plasmid. The SHV-104 -lactamase was produced in E. coli and purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype of K. pneumoniae ML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity

Emergence and dominance of CTX-M-15 extended spectrum beta-lactamase among Escherichia coli isolates from children

Of forty-seven extended-spectrum cephalosporin-resistant Escherichia coli isolates, collected from children at the Children’s Hospital in 2006 (Tunis, Tunisia), we analyzed 32 isolates that were genotypically different by enterobacterial repetitive intergenic consensus -polymerase chain reaction. For all isolates, the double-disk diffusion test revealed synergy between clavulanate and cefotaxime and/or ceftazidime, suggesting the production of extended-spectrum beta-lactamases. Polymerase chain reaction experiments, performed on plasmid DNA, and sequencing revealed the presence of blaTEM-1B (26 isolates, 81%), blaTEM-34(IRT-6) (3 isolates, 9%), blaSHV-12 (2 isolates, 6%), and blaCTX-M-15 (31 isolates, 97%). Further, the insertion sequence ISEcp1 was found upstream from the blaCTX-M-15 gene in 11 isolates. The bla genes were found alone or in various combinations in a single isolate. blaTEM-1B and blaCTX-M-15 genes were detected in 26 out of the 32 isolates. Three isolates harbored both blaTEM-34(IRT-6) and blaCTX-M-15. blaSHV-12 was identified either alone or with blaCTX-M-15 in a single isolate. Our investigation showed the dominance of CTX-M-type extended-spectrum beta-lactamases, with CTX-M-15 particularly common, and to our best knowledge, this is the first report of the coexistence of CTX-M-15 and IRT-6 in E. coli isolates from children in Tunisia.Fil: Réjiba, Samia. Université Tunis El-Manar; Túnez. Hôpital d’Enfants; TúnezFil: Mercuri, Paola Sandra. Universite de Liege; BélgicaFil: Power, Pablo. Universite de Liege; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Kechrid, Amel. Hôpital d’Enfants; Túne

Differential proteomic analysis of the response of Stenotrophomonas maltophilia to imipenem

This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of beta-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Two-dimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQA (R) differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in beta-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 beta-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the beta-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in beta-lactamase production

Detection and characterization of VIM-52, a new variant of VIM-1 from clinical isolate.

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-β-lactamases (MBLs), which inactivate virtually all β-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime as compared to VIM-1. These results were confirmed by steady state kinetic measurements, where VIM-52 yielded a lower activity towards ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221-241), which borders the active site. As Arg 224 and Ser 228 are both playing an important and interrelated role in enzymatic activity, stability and substrate specificity for the MBLs, targeted mutagenesis at both positions were performed and further confirmed their crucial role for substrate specificity

Characterization of the cattle serum antibody responses against TEM beta-lactamase and the nonimmunogenic Escherichia coli heat-stable enterotoxin (STaI)

In order to test the use of a subunit recombinant vaccine for its capacity to induce antibodies against the nonimmunogenic heat-stable enterotoxin STa from Escherichia coli and the TEM-1 beta-lactamase, cattle were immunized with a hybrid protein created by insertion of the STa sequence at position 197 of the TEM-1 beta-lactamase. Specific anti-STa IgG and IgG1 antibodies were detected at low levels, while no IgG2 antibodies were detected. In contrast, high levels of the different anti-TEM IgG subtypes were detected in cattle sera. In addition, beta-lactamase activity was inhibited by the sera. The presence of antibodies against STa and TEM-1 beta-lactamase was assessed in sera from 366 cattle taken from the field. No significant level of IgGs against the toxin or the TEM-1 was detected. A comparison of the antibody level between the immunized and the nonimmunized animals clearly demonstrated that STa was not able to induce a significant level of antibodies in the vaccinated animals. In contrast, a strong antibody response against TEM-1 beta-lactamase was demonstrated

1,2,4-Triazole-3-thione analogues with an arylakyl group at position 4 as metallo-β-lactamase inhibitors

International audienceMetallo-β-lactamases (MBLs) represent an increasingly serious threat to public health because of their increased prevalence worldwide in relevant opportunistic Gram-negative pathogens. MBLs efficiently inactivate widely used and most valuable β-lactam antibiotics, such as oxyiminocephalosporins (ceftriaxone, ceftazidime) and the last-resort carbapenems. To date, no MBL inhibitor has been approved for therapeutic applications. We are developing inhibitors characterized by a 1,2,4-triazole-3-thione scaffold as an original zinc ligand and few promising series were already reported. Here, we present the synthesis and evaluation of a new series of compounds characterized by the presence of an arylalkyl substituent at position 4 of the triazole ring. The alkyl link was mainly an ethylene, but a few compounds without alkyl or with an alkyl group of various lengths up to a butyl chain were also synthesized. Some compounds in both sub-series were micromolar to submicromolar inhibitors of tested VIM-type MBLs. A few of them were broad-spectrum inhibitors, as they showed significant inhibitory activity on NDM-1 and, to a lesser extent, IMP-1. Among these, several inhibitors were able to significantly reduce the meropenem MIC on VIM-1- and VIM-4- producing clinical isolates by up to 16-fold. In addition, ACE inhibition was absent or moderate and one promising compound did not show toxicity toward HeLa cells at concentrations up to 250 μM. This series represents a promising basis for further exploration. Finally, molecular modelling of representative compounds in complex with VIM-2 was performed to study their binding mode