Evaluation of a modified double-disc synergy test for detection of extended spectrum beta-lactamases in AMPC beta-lactamase-producing proteus mirabilis.

The detection of extended-spectrum beta-lactamases (ESBLs) in gram-negative bacteria that produce AmpC beta-lactamases is problematic. In the present study, the performance of modified double-disc synergy test (MDDST) that employs a combination of cefepime and piperacillin-tazobactam for the detection of Proteus mirabilis producing extended spectrum and AmpC beta-lactamases was evaluated and compared with double-disc synergy test (DDST) and NCCLS phenotypic disc confirmatory test (NCCLS-PDCT). A total of 90 clinical isolates of P. mirabilis , which met the CLSI (Clinical and Laboratory Standards Institute) screening criteria that these had broth microdilution (BMD) MIC of > or =2 mg/mL for at least one extended spectrum cephalosporin [ceftazidime (CAZ), cefotaxime (CTX) and cefpodoxime], were selected for the study. MDDST detected ESBLs in 40/90 of the isolates, whereas DDST detected ESBLs in only 25 isolates. NCCLS-PDCT could detect ESBLs in 39 isolates using CAZ and CAZ + clavulanic acid (CLA) combination, whereas CTX and CTX + CLA combination could detect only 37 isolates as ESBL positive. As many as 34/40 ESBL positive isolates were confirmed to be AmpC beta-lactamase positive by the modified three-dimensional test (MTDT). MDDST and NCCLS-PDCT could detect ESBLs in all the 34 AmpC positive isolates, whereas DDST could detect ESBLs in only 19 isolates. The study demonstrated that MDDST is superior to DDST and as sensitive as NCCLS-PDCT. However, MDDST seems to have enhanced potential for the detection of ESBLs in AmpC beta-lactamase-producing P. mirabilis .

Molecular epidemiology of clinical isolates of ampc producing Klebsiella pneumoniae.

PURPOSE: AmpC producing K. pneumoniae have been increasingly reported from India but epidemiological studies are lacking. In the present study, molecular epidemiology of extended-spectrum AmpC beta-lactamases (ESACs) producing clinical isolates of K. pneumoniae prevalent in our hospital was studied. METHODS: Fifty-one non-repeat, consecutive, clinical isolates of K. pneumoniae producing AmpC enzymes, were subjected to whole cell protein profile analysis (SDS-PAGE) and ribotyping. The antimicrobial susceptibility was determined using standard disk diffusion technique. The isolates showing decreased susceptibility to cefoxitin (< 18 mm) or cefotetan (< 16 mm) were subjected to modified three- dimensional test for detection of AmpC enzyme. RESULTS: Six different types of protein profiles were observed. Ribotyping could further discriminate between the strains that were clustered by protein fingerprinting. Twelve different ribo-patterns were identified. Ribotyping was found to have a better Discriminatory Index (0.98) than that of SDS-PAGE (0.78). Of the 26 isolates that showed decreased susceptibility to cefoxitin and/or cefotetan 13 isolates were found to harbour AmpC enzyme. CONCLUSIONS: The study demonstrated the usefulness of SDS-PAGE whole cell protein profile analysis and ribotyping to identify the clonality of the ESACs isolates, the latter having a higher discriminatory power. The presence of ESACs isolates in the community as well as in hospital settings emphasizes the need for regular monitoring of antimicrobial resistance.

Molecular typing of Salmonella enterica serotype Worthington isolates from infantile diarrhoea.

BACKGROUND AND OBJECTIVES: Salmonella Worthington has been known to be a causative agent for childhood diarrhoea. There is a paucity of information on the molecular relatedness of the strains isolated in various hospitals in India. The present study was carried out to attempt molecular typing of a cluster of Salmonella Worthington isolates obtained from cases of infantile diarrhoea during a six month period, from a tertiary care paediatric hospital in Delhi, India. METHODS: Nine isolates of S. Worthington obtained from faecal samples of infants suffering from diarrhoea during October 2001 to March 2002, were identified by the conventional biochemical methods and by serotyping. The antimicrobial susceptibility was determined by the disk diffusion method. Molecular typing was done by ribotyping. RESULTS: Eight patients were admitted to 3 different wards of the hospital and one was an outpatient. Four patients including the first patient visited the hospital with diarrhoea as the presenting symptom while five developed diarrhoea after admission. Stool microscopy showed no specific findings. Salmonella Worthington was isolated from stool cultures of these patients. Repeated cultures of the common drinking water source of the hospital and the milk supplied to children from central kitchen were negative for known pathogens. All S. Worthington isolates were resistant to all the beta-lactams tested including third generation cephalosporins. Eight isolates were sensitive to furazolidone and 6 to ciprofloxacin. Molecular characterization by ribotyping revealed four different clones. INTERPRETATION AND CONCLUSION: As four different ribotypes of the isolated Salmonella Worthington isolates were identified, it was clear that there was no single source of infection.

Liver abscess caused by Edwardsiella tarda biogroup 1 and identification of its epidemiological triad by ribotyping.

Two clinical isolates and an environmental isolate of Edwardsiella tarda biogroup 1 (ETB1), recovered from liver pus, the stool specimen and from the pond water of the village of the patient, diagnosed to have liver abscess, were found to be identical by protein fingerprinting and ribotyping. It can be construed that the pond water served as the source of infection. The epidemiological triad of the agent (ETB1), host (the patient) and environment (pond water) was thus established. This is the first report in which the triad for extraintestinal Edwardsiellosis caused by ETB1 has been identified. This also constitutes the first report of typing of ETB1 strains by SDS-PAGE and ribotyping.

Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae & evaluation of available phenotypic techniques for detection of extended spectrum beta-lactamases.

BACKGROUND & OBJECTIVES: Identifying organisms that harbour extended spectrum beta lactamases (ESBLs) is a major challenge for a diagnostic clinical microbiology laboratory. Wide variety of ESBLs produced and lack of a sensitive phenotypic method for their detection make the detection of ESBLs difficult and is responsible for under-recognition. The present study was undertaken to evaluate phenotypic characteristics, initial screening tests and established confirmatory phenotypic methods for detection of ESBLs Klebsiella pneumoniae isolates prevalent in a hospital in north India. METHODS: One hundred, non-repeat clinical isolates of K. pneumoniae collected over a period of six months were included in the study. Susceptibilities of the isolates to 20 different antimicrobial agents were determined. Agar dilution and broth dilution methods were used to determine minimum inhibitory concentrations (MICs) of ceftazidime (CAZ) and cefotaxime (CTX). CAZ and CTX were used with and without clavulanic acid to detect ESBL harbouring isolates. Using agar dilution and broth dilution, MIC reduction of two and three doubling dilutions were evaluated as a criterion for ESBL harbouring isolates. Standard double disk synergy test (DDST) with disks placed at 30 mm and modified DDST with disks placed at 16 mm center-to-center distance, using at least two different third generation cephalosporins and combined disk method were also performed to detect ESBL harbouring isolates. RESULTS: Multi-drug resistance (resistance to three or more antimicrobials of different classes) was found among 94 per cent of the isolates. Pooling the results of all the three confirmatory techniques MIC reduction of >3 doubling dilutions using broth dilution method (using CTX and CAZ), combined disk method [(using CTX, ceftriaxone [(CRO), CAZ and aztreonam)] and standard DDST (using CTX, CRO, CAZ and aztreonam), revealed as many as 87 per cent of the isolates as ESBL producers. CTX had greater sensitivity in identifying isolates that harboured ESBLs. Modified DDST using CTX was as sensitive method as broth dilution method and combined disk method in detecting ESBL harbouring isolates. MIC reduction technique using agar dilution method and standard DDST had lowest overall sensitivity in detecting ESBLs. INTERPRETATION & CONCLUSION: Modified DDST using at least two different third generation cephalosporins was considered to be the best technique for detection of ESBL producing K. pneumoniae at our hospital. MIC reduction test with >2 doubling dilution reduction in MICs was found to be a better criterion than the presently recommended >3 doubling dilution reduction. For screening of potential ESBL producers, MIC determination using agar dilution was as good as that using broth dilution method. However, while performing MIC reduction test agar dilution method was found highly unreliable for detection of ESBL harbouring isolates.

Development of a computer software for analysis of SDS-PAGE protein fingerprints of bacterial isolates.

Protein fingerprinting is a widely used technique in epidemiological studies for typing bacterial strains. This study reports the development of a computer based gel analysis system. The system has the capability to analyse SDS-PAGE whole-cell protein profiles using digital image processing techniques. The software incorporates spatial and frequency domain operators for image enhancement, support for geometric correction of images and new algorithms for identification of strain tracks and protein bands. The system also provides facilities for correcting imaging defects for inter-gel comparison, similarity analysis, clustering and pictorial representation of results as a dendrogram. The software is highly interactive, user-friendly and can produce accurate results for differentiation of bacterial strains with minimal overhead of time.

Evaluation of efficacy of standard haemodialysis and verapamil added peritoneal dialysis.

The study was carried out on 30 adult subjects of acute renal failure who were randomly divided into two groups of 15 patients each. Group A patients were subjected to standard haemodialysis (HD) for 4 hours using holofibre dialyser and group B patients received 36 hours of peritoneal dialysis (PD) where Verapamil was added intraperitonealy in the dose of 10 mg/ L/cycle in the clearance periods III, V, VII, IX and XI (Total dose 150 mg). The 36 hours of PD consisted of 36 cycles of one hour duration each and these were divided into 12 clearance periods (CP) of 3 cycles each. Following both the forms of dialytic treatments, there was significant improvement in the signs of uraemia with fall in the blood urea and serum creatinine levels. The peritoneal clearances, percentage fall of urea and creatinine, and protein loss were similar in the two groups (p > 0.5). However, ultrafiltration was significantly higher in the group B. No untoward effects were noticed in group B however group A patients had episodes of hypotension (5)] disequilibrium (6) and cardiac arrhythmias (1). It can be concluded that both clinically and biochemically, 36 hours of Verapamil added PD and 4 hours of hemodialysis are comparable and therefore, peritoneal dialysis may be used more frequently in acute renal failure specially in situations where trained dialysis staff is not available and patients are not haemodynamically stable.

Clindamycin in the treatment of anaerobic lung abscess.

A case of anaerobic lung abscess who had treatment failure after 4 weeks of supervised parenteral penicillin and oral metronidazole is described. Anaerobic pathogens resistant to one or the other of the above drugs were isolated. The patient had a striking clinical response to subsequent therapy with oral clindamycin. Failure of therapy should alert physicians to the possibility of infection with resistant anaerobic pathogens and in such situations, clindamycin is considered as an effective alternative.

Chronic anaerobic pneumonitis.

A case of chronic anaerobic pneumonitis, without any predisposing factors, nor the classical features often associated with it, which masqueraded as pulmonary tuberculosis is described. Therapy with metronidazole resulted in striking improvement.