Mamatha Ballal

Distribution of Candida Species in different clinical samples and their virulence: Biofilm formation, proteinase and phospholipase production: A study on hospitalized patients in Southern India

Introduction: Candida species are normal inhabitants of the skin and mucosa. The importance of epidemiological monitoring of yeasts involved in pathogenic processes is unquestionable due to the increase of these infections over the last decade; Materials and Methods: The clinical samples from the respiratory tract (sputum, bronchial wash, tracheal secretions), saliva, blood, urine, middle ear discharge, vitreous fluid, corneal ulcer, and plastic devices (endotracheal tube, catheter tip, suction tip) were collected and cultured. The species of Candida isolated were identified. Results: A total of 111 isolates of Candida species were recovered from 250 diverse clinical sources. C. albicans (39.64%) was the most isolated species, although the Candida non albicans species with 60.36% showed the major prevalence. In blood cultures, C. krusei (38.23%) and C. albicans (20.58%) were isolated frequently. C. albicans (63.27%) was the predominant species in mucosal surface. Urinary tract infections caused by yeasts were more frequent in hospitalized patients, C. krusei (50.0%) being commonly isolated, followed by C. albicans (25.0%). Discussion: Several virulence factors like, biofilm, proteinase, phospholipase, etc. contribute to the pathogenecity. Early detection of virulence factors by Candida is useful in clinical decision making. We therefore have aimed at demonstrating the formation of biofilm using the method proposed by Branchini et al, (1994). The proteinase produced by Candida was estimated as per the method of Staib et al, (1965). Phospholipase assay was carried out as per the method of Samaranayake et al, (2005). Conclusions: The data suggests that the capacity of Candida species to produce biofilm may be a reflection of the pathogenic potential of the isolates. C. krusei and C. tropicalis showed strong slime production. The non-Candida albicans produced more proteinase than C. albicans. C. albicans produced higher levels of phospholipase than non Candida albicans in this study.

Proteinase and phospholipase activity as virulence factors in Candida species isolated from blood

The number of nosocomial blood stream infections due to Candida species has increased over the past few decades. In order to establish an infection, opportunistic pathogens have to evade the immune system, survive, divide in the host environment, and spread to other tissues. Proteinase and phospholipase secretion has been implicated as potential virulence factors for some Candida species responsible for catheter related candidemia in intensive care unit (ICU) patients with indwelling devices. We therefore have aimed at demonstrating the secretion of proteinase and phospholipase enzymes as virulent factors by Candida species isolated from blood samples collected from ICUs, dialysis units and oncology units. One hundred and fourteen isolates of Candida species were obtained from the blood samples and the isolates include 37 Candida albicans, 7 Candida glabrata, 5 Candida guilliermondii, 3 Candida kefyr, 45 Candida krusei, 5 Candida parapsilosis, and 12 Candida tropicalis. Proteinase assay was performed by using the Staib et al method. Phospholipase assay was performed by using the method of Samaranayake et al. Precipitation zone (Pz value) was determined. The percentage of isolates which produced detectable amounts of proteinase is 74.56% and 44.73% of isolates produced detectable amounts of phospholipase. We believe that production of both phospholipase and proteinase enzimes could be an important virulence factor for several Candida species.

Epidemiological study of Vibrio cholerae using variable number of tandem repeats

By conventional genetic methods, including pulse-field gel electrophoresis and multilocus sequence typing, most pathogenic, cholera toxin-positive O1 and O139 isolates of Vibrio cholerae cannot be distinguished. We evaluated relationships among 173 V. cholerae isolates collected between 1992 and 2007 from different geographic areas in India by analyzing five variable number of tandem repeat (VNTR) loci. Each VNTR locus was highly variable, with between 5 and 19 alleles. eburst analysis revealed four large groups of genetically related isolates. Two groups contained genotypes of isolates with the O139 serogroup (which emerged for the first time in epidemic form in 1992), with the other two groups containing O1 strains. In subsequent analysis, it was possible to track the spread of specific genotypes across time and space. Our data highlight the utility of the methodology as an epidemiologic tool for assessing spread of isolates in both epidemic and endemic settings.

Rotavirus and enteric pathogens in infantile diarrhoea in Manipal, South India

The etiology of Rotavirus in acute diarrhoeal illness in children 0–5 years of age, admitted to the pediatric wards of Kasturba Medical College Hospital, Manipal was studied over a period of 5 years. Rotavirus in the faeces detected by Latex agglutination test accounted for 19.56% of the diarrhoea with maximum incidence (65%) in the 7–12 months of age group. Bacterial aetiological agents continued to play a significant role (69.6%) in diarrhoeal diseases.Enteroaggregative E. coli was common in the age group between 25–36 months, Shigellosis in 37–60 months andSalmonella typhimurium enteritis in 7–12 months of age. The other pathogens isolated werevibrio cholerae (4.98%), species of aeromonas (15.92%), along with cryptosporidium (6.47%) and Candida albicans (3.98%). In a control group consisting of 100 children without history of diarrhoea, 2 were positive for rotavirus, 3 for cryptosporidium and 12 forEscherichia coli.

Multidrug resistant enteroaggregative Escherichia coli diarrhoea in rural southern Indian population.

Enteroaggregative Escherichia coli (EAEC) has been implicated in acute and persistent diarrhoea in children, adults, and in HIV/AIDS patients. Enteroaggregative Escherichia coli strains isolated from different parts of the world show a low to high level resistance to antimicrobial agents. The aim of the present study was to determine antimicrobial susceptibility in vitro among 64 EAEC strains isolated from children and adults with diarrhoea. 270 E. coli strains were isolated from children and adults. A total of 64 were identified as EAEC by multiplex PCR targeting 2 specific genes, AggR and EAST, from July 2006 to July 2007. Susceptibility to various antibiotics was checked using the Kirby Bauer disk diffusion method. Disk diffusion testing for 11 commonly used antimicrobial agents showed EAEC resistant to trimethoprim sulfamethoxazole, ampicillin and nalidixic acid. 75% of strains isolated showed multidrug resistance, i.e. resistance to >3 antimicrobial agents. Most of the isolates showing multidrug resistance were from children below 5 y of age. An increase in resistance of EAEC strains to quinolones was observed in the study. Our study indicated that EAEC resistance in southern India is much higher than that reported from other parts of country. Our study also showed that monitoring sensitivity to antibiotics commonly used in acute and persistent diarrhoea is necessary for optimum selection of effective antibiotics and elimination of antibiotics with little therapeutic value. Further clinical epidemiological and laboratory studies are needed to clarify these issues.

Molecular epidemiology of diarrhoeagenic Escherichia coli associated with sporadic cases and outbreaks of diarrhoea between 2000 and 2001 in India.

Diarrhoeal infection caused by Escherichia coli is common in India with occasional outbreaks. However, association of different pathotypes of diarrhoeagenic E. coli(DEC) with the disease and its phenotypic and genotypic characteristics are not fully demonstrated. In this study, E. coli strains from sporadic cases and outbreaks of diarrhoea during 2000–2001 were confirmed as DEC by polymerase chain reaction (PCR) targeting the specific virulence genes. DEC represented by enterotoxigenic E. coli(ETEC), enteropathogenic E. coli(EPEC) and enteroaggregative E. coli(EAggEC) were mostly belonged to O serogroups 25, 86a, 114 and 146. The gene astA was frequently detected among ETEC and EAggEC than EPEC. After initial screening of 200 DEC strains with serology and antibiotic susceptibility test, 32 strains representing ETEC, EPEC, and EAggEC isolated from different areas of India were included in the pulsed-field gel electrophoresis (PFGE) analysis. Using the PFGE results, the hierarchical representation of different linkage levels between the DEC strains were determined by unweighed pair-group arithmetic mean (UPGAMA) method. Except for few strains, clonotyping by PFGE revealed no correlation between pathotypes and serogroups as well as the place of isolation of the DEC strains. The prevailing clonal diversity among the different categories of DEC strains suggests that the pathotypes of DEC belonged to diverse clones.

Slime production a virulence marker in Pseudomonas aeruginosa strains isolated from clinical and environmental specimens: a comparative study of two methods.

Detection of slime in Pseudomonas aeruginosa can be useful in understanding the virulence of this organism. Here, comparative studies of two phenotypic methods using the tube method and the spectrophotometric method for slime production from 100 clinically and 21 environmentally significant isolates of P. aeruginosa were performed. A total of 68 isolates were positive by either of the tests whereas only 34 were positive by both the tests. The tube method detected slime significantly in more number of isolates than the spectrophotometric method. The tube test was found to be superior to the spectrophotometric method in ease of performance, interpretation and sensitivity. Among the clinical isolates, systemic isolates produce less slime compared to wound, respiratory and urinary isolates. Isolates from the hospital environment produced more slime indicating that this virulence marker helps the organism to survive for longer periods and cause nosocomial infections.

Identification of enteroaggregative Escherichia coli in infants with acute diarrhea based on biofilm production in Manipal, south India

BACKGROUND Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes persistent diarrhea among infants, both in developing and industrialized countries. The EAEC strains adhere to epithelial cell surface, to the glass substratum and to each other in a distinctive stacked brick-formation. Thus, gold standard for identification of EAEC remains the HEp-2 cell adherence test, which is time consuming and requires specialized facilities. AIM To evaluate the usefulness of quantitative biofilm assay to screen for EAEC from children with acute diarrhea. MATERIALS AND METHODS A total of 100 E. coli strains were collected from acute diarrheal cases from December 2005 to November 2006. The strains were screened for biofilm production using microtiter plate method. The biofilm in the microtiter plate was visualized after staining with crystal violet and was quantified using enzyme immunosorbent assay plate reader. The Aggregative plasmid and Heat stable toxin genes were evaluated by a multiplex polymerase chain reaction. The strains were identified as EAEC with an optical density at 570 nm (OD570)>0.2. RESULTS Of the total 100 Escherichia coli strains, 28 were positive by Polymerase Chain Reaction for two genes, AggR and EAST. Of the 28 PCR-positive strains screened for biofilm, 25 (89.2%) showed positive results by microtiter plate method. CONCLUSION The quantitative biofilm assay using microtiter plate is convenient and economical and can be used as a screening method to screen E. coli isolates from acute diarrheal cases. The best use of this test is to screen large number of isolates quickly, and if positive this can be confirmed by multiplex PCR for AggR and EAST genes. This assay may contribute to demonstrating the true incidence of EAEC with and without AggR among clinically isolated E. coli strains, which can cause acute diarrhea.

Chromobacterium violaceum diarrhea

This is the first case ofChromobacterium violaceum diarrhoea from coastal Karmataka reported in a 2 year 10 months old girl. Stool culture yieldedChromobacterium violaceum and was sensitive to ampicillin, gentamicin, chloramphenicol, erythromycin and septran. Patient completely recovered with ampicillin and gentamicin.

Prevalence of rubella virus in suspected cases of congenital infections

This study includes a total of 342 infants suspected of having congenital infections from January 1991–December 1993. Serum samples of these infants were tested for rubella specific IgM antibodies by μ ELISA. Of the total 342 infants, 52 (15.2%) were found to be positive for IgM antibodies to rubella virus. The commonest clinical presentation in infants with IgM antibodies to rubella virus was bilateral congenital cataract and hepatosplenomegaly.