Clostridium difficile is the aetiological agent for almost all cases of pseudo membranous colitis and 15-25% of antibiotic associated diarrhoea. In recent years, C. difficile associated disease (CDAD) has been increasing in frequency and severity due to the emergence of virulent strains. Severe cases of toxic mega colon may be associated with mortality rates of 24-38%. The prevalence of CDAD is global and the incidence varies considerably from place to place. In the initial stages of its discovery, C. difficile infection was regarded mainly as an outcome of antibiotic intake and not as a life threatening disease. Intervention by man has produced conditions making C. difficile a significant cause of morbidity and mortality. The recent outbreak of CDAD in Quebec has sent the alarm bells ringing. Apart from a threefold increase in the incidence of CDAD, clinicians have also reported a higher number of cases involving toxic mega colon, colectomy or death. Among all the risk factors, inclusive of the host and the environmental factors, antibiotics are the most important ones. Surgical patients comprise 55-75% of all patients with CDAD due to the fact that perioperative prophylaxis requires the use of antibiotics. However, other drugs such as immunosuppressants and proton pump inhibitors are also important risk factors. Thus CDAD is a growing nosocomial and public health challenge. Additionally, the recognition of community acquired CDAD signals the presence of several risk factors. In this review, the established and potential risk factors of CDAD, along with the epidemiology, diagnostic modalities, management and preventive measures of the disease have been elaborated.

Enterococcus, considered a normal commensal of intestinal tract, is fast emerging as a pathogen causing serious and life threatening hospital borne infections. This is attributed to acquisition of multi drug resistance and virulence factors of the organisms. The sequencing of Enterococcus faecalis has given a lot of insight into its genetic makeup. The E. faecalis strain V583, which has been sequenced, contains a total of 3182 open reading frames (ORFs) with 1760 of these showing similarity to known proteins and 221 of unknown functions. Strikingly unique to this genome is the fact that over 25% of the genome is made up of mobile and exogenously acquired DNA which includes a number of conjugative and composite transposons, a pathogenicity island, integrated plasmid genes and phage regions, and a high number of insertion sequence (IS) elements. This review addresses the genomic arrangement and the study of virulence factors that have occurred since the sequencing of the genome.

Purpose : The purpose of this study was to understand the molecular epidemiology of tuberculosis in Khorasan province of Iran was studied by spoligotyping 113 Mycobacterium tuberculosis isolates. The spoligotyping results were in comparison to the word Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Spoligotyping data from Iran has rarely been described and there is limited information on the major circulating clades of M. tuberculosis in Iran. Materials and Methods: Spoligotyping was performed on 113 M. tuberculosis isolates from Mashhad patients between November 2004 and September 2005. Results: The study found 57 spoligopatterns. 17 clusters and 32 true orphan genotype. The biggest cluster with 13 isolates had not been previously reported. The Beijing genotype was seen in eight (7.1%) isolates. Conclusions: Genotyping and Spoligotyping gives a unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis populations.

Purpose: To investigate the genetic diversity of toxigenic Clostridium strains isolated from soil, water, meat and its associated polluted sites of Southern India. Materials and Methods: A total of 27 identified isolates of six different toxigenic clostridial species including C. bifermentans , C. botulinum , C. chauvoei , C. ramosum , C. tetani and C. novyi were isolated and characterized by conventional DNA restriction digestion analysis (REA) and by whole-cell and excretory protein patterns on SDS-PAGE. Results: The DNA fragment size ranged from 35-160 kilobases and the protein bands 30-200 KDa, followed by numerical analyses and phylogenetic analyses. Whole-cell protein banding pattern were unique with strains of C. chauvoei , C. novyi and C. ramosum . All the strains were heterogeneous and distinct in restriction digestion pattern and excretory protein patterns. Conclusion: These analyses contribute to the understanding of prevalence of toxigenic clostridial species and phylogeny within the species and assist in development of improved diagnostics and therapeutics for the treatment of clostridial infections.

Purpose : Malassezia yeasts are globally distributed agents of pityriasis versicolor and are implicated in the pathogenesis of seborrhoeic and atopic dermatitis. The aim of this study is to identify the Malassezia species obtained from pityriasis versicolor patients, using morphological, biochemical, physiological as well as Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Materials and Methods: The identification of Malassezia species is performed according to microscopic features and physiological characteristics, including catalase reaction and Tween assimilation tests. The DNA is extracted from cultured Malassezia using the glass bead, phenol-chloroform method. The internal transcribed spacer 1(ITS1) region is amplified and there is restricted digestion of the PCR products with two enzymes Cfo I and Bst F5I. Results : The most commonly isolated species is M. globosa (47.6%). RFLP analysis of the PCR products of the ITS1 region is in complete agreement with those from the DNA sequences of the internal transcribed spacer (ITS) 1 region and the biochemical tests. Conclusion : Based on the findings of this study, it can be concluded that PCR-RFLP is a relatively simple and quick method, completely comparable to the routine methods used for Malassezia identification.

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for secretory diarrhoea. The production of heat labile enterotoxin (LT), by ETEC, is largely responsible for the pathogenesis of diarrhoea. In the present study we investigated the effect of stress factors such as temperature, pH, osmotic stress and nutritional limitation on the production of LT by ETEC using in-house GMI-ELISA. Four strains of E. coli consisting, one standard strain MTCC 723 and three clinical isolates were used in the study. Maximum amount of LT (OD 3.285) was produced at 37 ⁰ C followed by 40 ⁰ C (OD 3.305). Growth of E. coli in medium with pH 8.6 resulted in maximum amount of LT production (OD 3.489). LT was not detectable when bacteria were grown in medium with pH ≤7.2 and ≥ 9.2. Sodium chloride concentration of 0.2 M stimulated maximum amount of LT production. Maximum amount of LT was produced when the bacteria were grown in medium containing 2.5g/l of glucose. All the stress factors had a significant effect on the LT production by E. coli , though quantitative differences in the various strains were observed.

Background: The failure of empirical therapy is frequently observed, even in community-acquired urinary tract infections. We, therefore, conducted a prospective, clinic-based study in 2004-2005 to document anti-microbial resistance rates and correlate them with possible risk factors to assist empirical decision-making. Materials and Methods: Symptomatic patients with pyuria underwent urine culture. Isolates were identified using standard methods and anti-microbial resistance was determined by disk-diffusion. Ultrasonography was used to detect complicating factors. Patients were stratified by the presence of complicating factors and history of invasive procedures for comparison of resistance rates. Statistical Method Used: Chi-square or Fisher exact tests, as appropriate. Results: There were 156 E. coli isolates, of which 105 were community-acquired. Twenty-three community-acquired isolates were from patients with complicating factors while 82 were from patients without any. Fifty-one isolates were from patients who had recently undergone invasive procedures on the urinary tract. Thirty-two community-acquired isolates from reproductive-age women without apparent complicating factors had resistance rates of 50% or above against tetracyclines, Co-trimoxazole, aminopenicillins, Nalidixic acid, Ciprofloxacin and 1 ^st generation cephalosporins. Resistance rates were significantly higher among isolates from patients subjected to invasive procedures, except against Co-trimoxazole, tetracyclines and Amikacin. Conclusion: High rates of anti-microbial resistance in community-acquired uropathogens have made antimicrobial sensitivity testing necessary even in a rural, primary-care setting.

Purpose: In gram-negative bacteria, active efflux pumps that excrete drugs can confer resistance to antibiotics however, in Helicobacter pylori this role is not well established. The purpose of this study is to evaluate the role of active efflux in resistance of H. pylori isolates to antibiotics. Materials and Methods: Twelve multiple antibiotic resistant (MAR) isolates resistant to at least four antibiotics, including β-lactams, metronidazole, tetracycline, erythromycin, and ciprofloxacin; three resistant to only β-lactams, and two hyper-susceptible isolates, were obtained from screening of 96 clinical isolates of H. pylori . Their minimal inhibitory concentrations (MICs) for antibiotics and ethidium-bromide (EtBr) were compared in the presence- and absence of a proton-conductor, carbonyl cyanide-m chlorophenyl-hydrazone (CCCP) using agar-dilution and disc diffusion. Drug accumulation studies for EtBr and antibiotics were assessed in the presence and absence of CCCP using spectrofluorometry. Results: MIC of EtBr for eight MAR-isolates was decreased two- to four-folds in the presence of CCCP, of which five showed reduced MICs for β-lactam, metronidazole, tetracycline, and ciprofloxacin with CCCP. Accumulation of EtBr by the MAR-isolates was rapid and not dependant on the pattern of multiple resistance. Antibiotic accumulation assay confirmed the presence of energy-dependant efflux of β-lactam, metronidazole, tetracycline, and ciprofloxacin, but no erythromycin in five MAR isolates. Energy-dependant efflux of EtBr or antibiotics was not observed for four MAR-isolates, and three isolates were resistant only to β-lactams. Conclusion: Energy-dependant efflux plays a role in the resistance of H. pylori clinical isolates to structurally unrelated antibiotics in a broadly specific multidrug efflux manner. Difference in the efflux potential of MAR isolates may be related to the presence or absence of functional efflux-pumps in diverse H. pylori isolates.

Purpose: Bloodstream infection remains a major cause of morbidity and mortality in patients undergoing treatment for cancer. Severe infections due to Gram-negative bacilli & staphylococci are common in cancer patients. Altered gut flora because of frequent antibiotic administration and damage of epithelial surfaces contribute to the development of infection. To access the use of new potent antibiotics against bloodstream infection in cancer patients and to determine the cross resistance of Gram-negative bacterial strains. Materials and Methods: We studied the bacterial spectrum & antimicrobial susceptibility pattern of cephalosporins, fluoroquinolones, carbapenems and aminoglycosides against Gram-negative bacterial strains in cancer patients. The susceptibility was determined by broth dilution method according to National Committee for Clinical Laboratory Standards (NCCLS) now called Clinical Laboratory Standards Institute (CLSI) during study period (July 2006 to Jan 2007). Results: A total of 60 Gram-negative bacterial blood cultures were examined. Among these, Pseudomonas aeruginosa was the most common (38%). The Minimum Inhibitory Concentration at which 50% (MIC ₅₀ ) and 90% (MIC ₉₀ ) of Enterobacteriaceae and P. aeruginosa inhibited were found. Resistance in P.aeruginosa against cefepime, meropenem, ciprofloxacin, ceftriaxone, tobramycin, cefoperazone and imipenem was 60%, 13%, 80%, 67%, 40%, 90% and 10% respectively while for Enterobacteriaceae 80%, 20%, 88%, 72%, 20%, 90% and four per cent resistance was observed. Meropenem was found to be the most effective antimicrobial against Gram-negative bacteria. Conclusion: High resistance observed in this study warrants the needs of surveillance of resistant pattern of antimicrobial agents. Due to increased level of drug resistance, carbapenem would be a prudent choice in high- risk cases.

Hantaviruses cause hemorrhagic fever with renal syndrome in Europe and Asia. There are about 20 documented hantavirus species and newer species are being described worldwide, especially in non-rodent reservoirs, i.e shrews. Focus reduction neutralization test is the classical serotyping technique for hantavirus. However, this study employs a previously established serotyping ELISA, to retrospectively analyze known hantavirus IgG reactive samples for infecting serotypes. The result suggests presence of Thailand virus- like and Hantaan virus -like strains in India.

We have analyzed the suitability and potential of Oral Fluid (OF) to substitute serum in estimating measles IgG antibodies, during community surveys, by comparing the Optical Density (OD) of measles IgG antibodies in OF and serum of 100 apparently asymptomatic children. IgG antibody status was determined using commercially available - Measles IgG Capture ELISA. Sensitivity 89.5%, specificity 90.6% Concordance of 89%, coefficient of correlation r is equal to 0.97 (Karl Pearson's) and rho is equal to 0.86 (Spearman's), was found between OD value of OF and serum. The study emphasizes the potential of OF to surrogate serum in estimating Measles IgG antibody among children. The OF collection is advantageous over blood as it is painless. It is suitable for non-technical staff, easy to transport and less bio-hazardous.

Serological and molecular characterization of Leptospiral isolates helps us to identify serovar, which is useful, for epidemiological study. Serological characterization is tedious and requires a panel of monoclonal antibodies and expertise to read the results. This study is a preliminary work to evaluate the usefulness of Denaturing Gradient Gel Electrophoresis (DGGE) to identify serovars of leptospira. The V3 region of most conserved 16S rDNA of five pathogenic leptospiral serovars and one saprophytic serovar was characterized. DGGE method was employed to separate the amplified V3 region based on the nucleotide sequence. On DGGE, amplified V3 region of leptospiral serovars, under study, showed bands at different positions indicating DGGE as the effective method of characterization in the future. DNA sequencing of V3 region of the three serovars showed great difference in nucleotide sequence supporting the results of DGGE.

Shigella is an important cause of acute invasive diarrhea in children and others. Antimicrobial susceptibility of Shigella spp. isolated from diarrhoeal/ dysenteric patients in Bangalore was studied in our hospital from January 2002 to December 2007. One hundred and thirty-four isolates were identified as Shigella species. S. flexneri, S. sonnei , S. boydii and S. dysenteriae were accounted respectively for 64.9%, 21.6%, 8.2% and 3.7% of the total number of Shigella isolated. Of these 56 (41.8%) were from children (0 to 14 years) and 78 (58.2%) were from adults and elderly patients. Over 70% of Shigella isolates were resistant to two or more drugs including Ampicillin and Co-trimoxazole. During 2002 to 2007, resistance to Ampicillin had increased from 46.7% to 68%. For Co-trimoxazole, though the resistance had gradually decreased from 100% to 72%, but still the resistance is high. Chloramphenicol resistance showed sudden decline from 73.3% to 25% from 2002 to 2003, but gradually has reached 48%. Nalidixic acid resistance was more than 70%. All isolates were sensitive to Ciprofloxacin during the period 2002 to 2004, but over the years the resistance pattern gradually increased up to 48%. Ceftriaxone had shown no resistance. The results of the study revealed the endemicity of Shigellosis with S. flexneri as the predominant serogroup. Children were at a higher risk of severe shigellosis. The results also suggest that Ampicillin, Co-trimoxazole, Chloramphenicol, Nalidixic acid and Ciprofloxacin should not be used empirically as the first line drugs in the treatment of Shigellosis. Periodic analysis and reporting of antibiotic susceptibility is an important measure to guide antibiotic treatment.

HIV-induced immunosuppression paves the way for several infections, tuberculosis being very common in our country. Female genital tuberculosis (FGTB), presenting as menstrual irregularities, is a diagnostic challenge in an adolescent female when these may be considered normal. The present case is of a young female who presented with menstrual irregularities, diagnosed subsequently as a case of genital tuberculosis. Microbiological relapse after anti-tubercular treatment of six months caused suspicion of a co-existing immunodeficiency and investigations revealed HIV co-infection; thus emphasizing the need of HIV testing in all patients of tuberculosis for timely diagnosis and treatment support thereafter.

Tubercular brain abscess are uncommon and tubercular cerebellar abscess are rarely reported. Most of these cases occur in immunocompromised patients. We report a case of multiple cerebellar abscesses in a 55-year-old HIV seronegative non-diabetic female, who complained of headache, neck pain and unsteadiness of gait since two months. She had been on treatment for pulmonary tuberculosis, diagnosed earlier. Diagnosis was made by CT scan of brain and confirmed by bacteriological examination of drained pus obtained by suboccipital craniotomy. The patient showed signs of recovery.

Salmonella enterica serotype Dublin, a bovine adapted serotype, is the commonest cause of salmonellosis in cattle. Salmonellosis in animals always presents a potential zoonotic threat. Infected cattles serves as a source of infection to humans. We present here Salmonella Dublin Bacteraemia in an elderly patient, with all the clinical details, due to the rarity of its occurrence. He was treated successfully with ciprofloxacin and his follow up period was uneventful.

Primary cutaneous aspergillosis is a rare entity, usually caused by A. fumigatus and A. flavus . Here, we present such a case, manifested by ulceration due to A. niger, which remained undiagnosed for a prolonged period. The immunological status was intact, although the patient had associated severe fungal infection. Recurrence of the lesion occurred despite repeated anti-fungal therapies. Anti fungal testing was done based on the broth dilution (M-38A, NCCLS, USA) method. The culture isolate was found to be sensitive to fluconazole and amphotericin B. Continuation of antifungal therapy improved the symptoms, reducing the size of the lesion.

Staphylococcus aureus and some other bacteria are known to cause botryomycosis. These bacteria are known to produce yellowish-white soft grains. Only Actinomadura pelletieri is known to produce red grains. We report here a case of an intensely inflammatory type of botryomycosis. A 35-year-old male labourer presented with swelling, redness and multiple sinuses on his foot, of eight months duration. The purulent discharge contained bright red coloured grains, 0.5 to 1 mm in size, which were round to oval in shape. Gram-positive cocci were demonstrated in crushed granules and tissue sections. Culture yielded pure and heavy growth of Staphylococcus aureus . He responded very well to cefazolin. There is no other report of such red grain botryomycosis due to Staphylococcus aureus , available in literature. This is first case report of its kind in world literature.