1.Non-selective and selective enrichment media for the recovery of Clostridium difficile from chopped beef.
Chai C1, Lee KS1, Lee D1, Lee S1, Oh SW2. J Microbiol Methods. 2015 Feb;109:20-4. doi: 10.1016/j.mimet.2014.12.001. Epub 2014 Dec 9.
Clostridium difficile exists within the intestines of animals and in meat products. Enrichment of C. difficile in an appropriate medium is necessary for the detection of C. difficile in meat products. Non-selective media (brain heart infusion medium [TBHI] and cooked meat medium containing sodium taurocholate [TCM]) and selective media (cycloserine-cefoxitin-fructose medium [TCCFB] and C. difficile moxalactam-norfloxacin medium containing antibiotics and sodium taurocholate [TCDMN]) can be used to enrich C. difficile. This study aimed to evaluate non-selective and selective enrichment media for the recovery of C. difficile from beef specimens. The efficiency of the enrichment media was investigated on the basis of the recovery frequency of C. difficile from beef specimens inoculated with C. difficile. The beef specimens were inherently contaminated with bacteria (around 10(4)CFUg(-1)), and further inoculated with C. difficile (around 10(0)CFUg(-1)).
2.Analysis of the Structure and Function of FOX-4 Cephamycinase.
Lefurgy ST1, Malashkevich VN2, Aguilan JT2, Nieves E2, Mundorff EC1, Biju B1, Noel MA1, Toro R2, Baiwir D3, Papp-Wallace KM4, Almo SC2, Frere JM5, Bou G6, Bonomo RA7. Antimicrob Agents Chemother. 2015 Nov 2;60(2):717-28. doi: 10.1128/AAC.01887-15.
Class C β-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC β-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in β-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.
3.Covalent inhibition of New Delhi metallo-β-lactamase-1 (NDM-1) by cefaclor.
Thomas PW1, Cammarata M, Brodbelt JS, Fast W. Chembiochem. 2014 Nov 24;15(17):2541-8. doi: 10.1002/cbic.201402268. Epub 2014 Oct 10.
Covalent irreversible inhibitors can successfully treat antibiotic-resistant infections by targeting serine β-lactamases. However, this strategy is useless for New Delhi metallo-β-lactamase (NDM), which uses a non-covalent catalytic mechanism and lacks an active-site serine. Here, NDM-1 was irreversibly inactivated by three β-lactam substrates: cephalothin, moxalactam, and cefaclor, albeit at supratherapeutic doses (e.g., cefaclor KI =2.3 ± 0.1 mM; k(inact) =0.024 ± 0.001 min(-1)). Inactivation by cephalothin and moxalactam was mediated through Cys208. Inactivation by cefaclor proceeded through multiple pathways, in part mediated by Lys211. Use of a cefaclor metabolite enabled mass spectrometric identification of a +346.0735 Da covalent adduct on Lys211, and an inactivation mechanism is proposed. Lys211 was identified as a promising "handhold" for developing covalent NDM-1 inhibitors and serves as a conceptual example for creating covalent inhibitors for enzymes with non-covalent mechanisms.
4.IMP-51, a novel IMP-type metallo-β-lactamase with increased doripenem- and meropenem-hydrolyzing activities, in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.
Tada T1, Nhung PH2, Miyoshi-Akiyama T3, Shimada K1, Phuong DM4, Anh NQ4, Ohmagari N5, Kirikae T6. Antimicrob Agents Chemother. 2015 Nov;59(11):7090-3. doi: 10.1128/AAC.01611-15. Epub 2015 Aug 17.
A meropenem-resistant Pseudomonas aeruginosa isolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-β-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly). Escherichia coli expressing blaIMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing blaIMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.