Detection of mutations in gyrB using denaturing high performance liquid chromatography (DHPLC) among Salmonella enterica serovar Typhi and Paratyphi A

Background:- Fluoroquinolone resistance is mediated by mutations in the quinolone-resistance determining region (QRDR) of the topoisomerase genes. Denaturing high performance liquid chromatography (DHPLC) was evaluated for detection of clinically important mutations in gyrB among Salmonella. Method:- S. Typhi and S. ParatyphiA characterised for mutation in QRDR of gyrA, parC and parE were studied for mutation in gyrB by DHPLC and validated by sequencing. Result:- The DHPLC analysis was able to resolve the test mutant from isolates with wild type gyrB and distinguished mutants from other mutant by peak profile and shift in retention time. Three sequence variants were detected at codon 464, and a novel mutation Ser→Thr was also detected. gyrB mutation was associated with non classical quinolone resistance (NALS-CIPDS) in 34 isolates of S. Typhi only and was distinct from classical quinolone resistance associated with gyrA mutations (NALR-CIPDS). Conclusion: DHPLC is effective for the detection of mutation and can reduce the need forsequencing to detect clinically significant gyrB mutations.

Antimicrobial Susceptibility Patterns of Salmonella enterica Serotype Typhi in Eastern Nepal

The aim of the present study was to evaluate antimicrobial susceptibility patterns with special reference to multidrug resistance, susceptibility to ciprofloxacin, and bacteriophage typing of Salmonella enterica serotype Typhi isolated from blood sent for culture in a tertiary-care teaching hospital in eastern Nepal during January 2000\u2013December 2004. In total, 132 strains of S. enterica Typhi, isolated from 2,568 blood culture samples collected from cases of suspected enteric fever, were tested for susceptibility to commonly-used antimicrobials by the disc-diffusion method. There were 35 multidrug-resistant strains. None of the isolates were resistant to ciprofloxacin.Of 52 isolates tested for minimum inhibitory concentration (MIC) of ciprofloxacin, 36 (69.23%) showed reduced susceptibility (MIC 650.25 mg/L). Of 112 strains tested for nalidixicacid susceptibility,86(76%) were resistant. Strains with reduced susceptibility to ciprofloxacin and resistance to nalidixic acid could be correlated. The commonest phage type was E1. Nalidixic acid susceptibility could be a useful screening test for the detection of decreased susceptibility of S. Typhi to ciprofloxacin, a drug which is commonly used even for minor ailments in this area

Ciprofloxacin-Resistant Neisseria meningitidis, Delhi, India

Decreased susceptibility of Neisseria meningitidis isolates to ciprofloxacin emerged from an outbreak in Delhi, India. Results of antimicrobial susceptibility testing of the meningococcal isolates to ciprofloxacin and further sequencing of DNA gyrase A quinolone-resistance–determining region confirmed the emergence of ciprofloxacin resistance in the outbreak

Clinically and microbiologically derived azithromycin susceptibility breakpoints for Salmonella enterica serovars Typhi and Paratyphi A.

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤ 16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P 16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A

Rapid Characterization of Mycobacterium tuberculosis Complex isolated from Clinical Samples by SD TB Ag MPT 64 kits

Purpose: The present study aimed at evaluation of SD TB Ag MPT 64 Rapid kit for confirmation of isolates of Mycobacterium tuberculosis complex (MTBC) grown from clinical samples.Material & Methodology: The present study included a total of 105 mycobacterial growths recovered from various pulmonary and extra-pulmonary clinical specimens in a tertiary care center. Culture was performed using Lowenstein Jensen’s media and BacT ALERT 3D automation system. The growths were subjected to both Accuprobe culture identification (Genprobe, San Deigo, CA) and SD TB Ag MPT 64 rapid kit (Standard Diagnostics, INC, Korea). SD TB Ag MPT 64 rapid kit was evaluated in the present study using Accuprobe as gold standard.Results: The sensitivity and specificity of SD TB Ag MPT 64 rapid kit was 92.3% and 100% respectively.Conclusion: SD TB Ag MPT 64 rapid kit is easy to perform, rapid and cheap test which can be used as a screening tool for rapid confirmation of the MTBC isolates in a routine tertiary care setting

Original Article Use of the cefepime-clavulanate ESBL Etest for detection of extendedspectrum beta-lactamases in AmpC co-producing bacteria

Background: Extended-spectrum beta-lactamases (ESBLs) may not always be detected in routine susceptibility tests. This study reports the performance of the cefepime-clavulanate ESBL Etest for the detection of ESBLs in Enterobacteriaceae, including those producing AmpC enzyme. Methodology: Consecutive non-duplicate isolates of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolated from bloodstream infections from January to June 2008 were tested for ESBL by both the standard CLSI double-disk diffusion method using ceftazidime and cefotaxime disks and Etests using ceftazidime/ceftazidime-clavulanate, cefotaxime/cefotaxime-clavulanate and cefepime/cefepime-clavulanate gradients. Isolates were also tested for the presence of transferable AmpC beta-lactamase by AmpC disk test and the efficacies of the different Etests in detecting ESBL production were compared. Results: A total of 113 bacterial isolates (61 K. pneumoniae, 50 E. coli, and 2 P. mirabilis) were recovered. Respectively, 42 (37.2%) and 55 (48.7%) isolates were positive for ESBL by the ceftazidime-clavulanate and cefotaxime-clavulanate combined disk tests. The cefepime/cefepime-clavulanate Etest strip detected the maximum number of isolates (70/113, 61.9 %) as ESBL-positive compared to the ceftazidime/ceftazidime-clavulanate and cefotaxime/cefotaxime-clavulanate strips, which detected 57 (50.4%) isolates each as ESBLpositive. All three ESBL Etest strips were equally effective in detecting ESBL in the isolates that were AmpC negative. In the 66 (58.4%) isolates that co-produced AmpC in addition to the ESBL enzymes, cefepime/cefepime-clavulanate Etest strip detected ESBL in an additiona