The conserved aspartate in motif III of b family AdoMet-dependent DNA methyltransferase is important for methylation

S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) are involved in diverse cellularfunctions. These enzymes show little sequence conservation but have a conserved structural fold. The DNAMTases have characteristic motifs that are involved in AdoMet binding, DNA target recognition and catalysis.Motif III of these MTases have a highly conserved acidic residue, often an aspartate, whose functionalsignificance is not clear. Here, we report a mutational study of the residue in the b family MTase of the Type IIIrestriction-modification enzyme EcoP15I. Replacement of this residue by alanine affects its methylationactivity. We propose that this residue contributes to the affinity of the enzyme for AdoMet. Analysis of thestructures of DNA, RNA and protein MTases reveal that the acidic residue is conserved in all of them, andinteracts with N6 of the adenine moiety of AdoMet. Interestingly, in the SET-domain protein lysine MTases,which have a fold different from other AdoMet-dependent MTases, N6 of the adenine moiety is hydrogenbonded to the main chain carbonyl group of the histidine residue of the highly conserved motif III. Our studyreveals the evolutionary conservation of a carbonyl group in DNA, RNA and protein AdoMet-dependentMTases for specific interaction by hydrogen bond with AdoMet, despite the lack of overall sequenceconservation

Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot infections.

AIMS: Diabetic foot lesions are a major medical, social, and economic problem and are the leading cause of hospitalization for patients with diabetes, worldwide. ESBL-producing bacteria may not be detectable by routine disc diffusion susceptibility test, leading to inappropriate use of antibiotics and treatment failure. There is not much information on ESBL-producing organisms causing diabetic foot infection. An attempt was therefore made to study the ESBL-producing Escherichia coli and Klebsiella pneumoniae in diabetic foot patients with type 2 diabetes mellitus. MATERIALS AND METHODS: A total of 134 isolates of E. coli and K. pneumoniae were obtained from tissue, pus swab, and wound swab samples from diabetic foot ulcers submitted for routine microbiological analysis during the period January to December 2005 from patients with diabetic foot infections who had type 2 diabetes mellitus, attending S. L. Raheja Hospital. The above isolates were tested for antimicrobial susceptibility by disc diffusion technique according to clinical and laboratory standards institute (CLSI) guidelines. The screening for ESBL production was done by phenotypic confirmatory test using ceftazidime disc in the presence and absence of clavulanic acid as recommended by CLSI. RESULTS: Among the 134 isolates, 54 (40.29%) were E. coli and 80 (59.70%) were K. pneumoniae; among which, ESBL production was detected in 31 (23.13%) isolates. Of these 31, 15 (48.38%) were E. coli and 16 (51.61%) were K. pneumoniae. All the ESBL-producing isolates were found to be 100% sensitive to carbapenem (imipenem and meropenem). Mortality was found to be 3.22%, the cause of death being septicemia leading to multiple organ failure. CONCLUSIONS: The prevalence of ESBLs among members of Enterobacteriaceae constitutes a serious threat to the current beta-lactam therapy, leading to treatment failure and consequent escalation of costs. There is an urgent need to emphasize rational use of drugs to minimize the misuse of available antimicrobials.

Incidence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in diabetes and cancer patients.

Metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa strains have been reported to be an important cause of nosocomial infections. There is not enough information from India regarding their prevalence in diabetic and cancer patients. The present study was undertaken over a period of one year from January to December 2006 to study the incidence of MBL P. aeruginosa and the clinical outcome in diabetes and cancer patients admitted to S.L. Raheja Hospital, Mumbai. Two hundred and thirty isolates of P. aeruginosa were obtained from different samples of patients. These isolates were subjected to susceptibility testing to anti-pseudomonal drugs as per CLSI guidelines. They were further screened for the production of MBL by disc potentiation testing using EDTA-impregnated imipenem and meropenem discs. Of the 230 isolates of P. aeruginosa, 60 (26%) isolates were found resistant to carbapenems (both imipenem and meropenem) and 33 (14.3%) were found to be MBL producers. Of the 33 MBL-producing isolates, 24 (72.7%) were diabetic patients, six (18.1%) were cancer patients and three (9%) patients had both diabetes and cancer. Five (15.1%) patients responded to the combination therapy of colistin, piperacillin with tazobactam and amikacin, while 28 (84.8%) patients responded to the combination therapy of amikacin, piperacillin with tazobactam and gatifloxacin. Thus, the rapid dissemination of MBL producers is worrisome and necessitates the implementation of not just surveillance studies but also proper and judicious selection of antibiotics, especially carbapenems.

Incidence of metallo beta lactamase producing Pseudomonas aeruginosa in ICU patients.

BACKGROUND & OBJECTIVE: Metallo beta lactamase (MBL) producing Pseudomonas aeruginosa have been reported to be important cause of nosocomial infections. The appearance of MBL genes and their spread among bacterial pathogens is a matter of concern with regard to the future of antimicrobial therapy. The present study was undertaken to determine the incidence of MBL producing P. aeruginosa in patients with diabetes and cancer admitted to the intensive care unit of a tertiary care hospital in western India and to assess the clinical outcome after antimicrobial treatment. METHODS: A total of 240 isolates of P. aeruginosa from various specimens between January and December 2005 were subjected to susceptibility testing against various antibiotics by disc diffusion test as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Imipenem and meropenem resistant isolates were selected for the detection of MBL production by disc potentiation test. Enhancement of inhibition zone around imipenem and meropenem discs impregnated with EDTA as compared to those without EDTA confirmed MBL production. RESULTS: Of the 240 P. aeruginosa isolates, 60 (25%) were found to be carbapenem resistant and 50 (20.8%) were found to be MBL producers. Of the 50 MBL producing isolates, 38 (76%) were from diabetes patients and 12 (24%) from cancer patients. Overall, 36 per cent patients responded to gatifloxacin, 42 per cent responded to piperacillin/tazobactam while 14 per cent responded to combination of gatifloxacin and piperacillin/tazobactum. Due to this nosocomial pathogen, the average hospital stay was 32 days and was associated with 20 per cent mortality due to septicaemia. INTERPRETATION & CONCLUSION: Our findings showed that there is a need to do surveillance to detect MBL producers, judiciously use carbapenems to prevent their spread and use effective antibiotics, such as gatifloxacin and piperacillin-tazobactum, after sensitivity testing for treatment.