Effect of Lactobacillus acidophilus & epidermal growth factor on experimentally induced Clostridium difficile infection.

Background & objectives: Clostridium difficile-associated disease (CDAD) remains an important nosocomial ailment. Antimicrobial therapy used for CDAD gives inconsistent results. This experimental study was planned to investigate the beneficial effects of Lactobacillus acidophilus and epidermal growth factor (EGF) for CDAD management. Methods: Among 10 groups of BALB/c mice (6 in each), group 1 served as controls receiving no inoculum. Animals in groups 2-10 received C. difficile, those in groups 3, 6 and 9 received L. acidophilus and those in groups 4, 7 and 10 received EGF after C. difficile inoculation. Animals in groups 5-7 were pre-treated with ampicillin and those in groups 8-10 with lansoprazole prior to C. difficile. The animals were killed and investigated for colonisation by C. difficile and toxin production, myeloperoxidase (MPO) activity and histopathology. Results: Colonisation by C. difficile was found to be significantly different (P<0.001) in the various groups. C. difficile toxin titres and MPO activity were significantly lower in animals given L. acidophilus and EGF after ampicillin (groups 6 and 7) and lansoprazole (groups 9 and 10). The severity of acute inflammation was also significantly less (P<0.05) in caecal and colonic segments of animals in groups 6 and 7 compared to those in group 5. Although the severity of acute inflammation was less in the caecal and colonic segment of animals in groups 9 and 10, the reduction was not significant compared to group 8. Interpretation & conclusions: Our findings showed that the administration of L. acidophilus and EGF reduced the severity of C. difficile infection in the experimental animals.

Phenotypic and molecular characterization of clinically isolated Escherichia coli.

All diarrheagenic Escherichia coli carry at least one virulence-related property. Stool samples from 244 patients having acute or persistent diarrhea received after the exclusion of routine enteropathogens were investigated. Purely or predominantly isolated E. coli (n = 100) were subjected to serotyping, of which only 25 were typable. They belonged to 14 different O-serogroups comprising 5 O153, 4 O102, 3 O25, 2 each of O130 and O169, and 1 each of O1, O8, O15, O37, O86, O101, O127, O143, and O160. The typable E. coli isolates along with 5 other untypable isolates were investigated for molecular markers, such as intimin (eae), enterohemolysin (EhlyA), a-hemolysin, heat-labile enterotoxins (LT), heat-stable enterotoxins (STa), verotoxins (VT1 and VT2), invasivity (ial), enteroaggregative E. coli (EAEC) gene (EAGG), and enterotoxin (EAST). Two of the isolates (O153 and O86) were positive for enterohemolysin phenotypically and 5 for β-hemolysin both phenotypically and genotypically. Interestingly, 16.6% of the randomly isolated E. coli were O153, a serogroup common in cattle, and 10% belonged to EAEC pathotype of which two-thirds had the EAST gene, which is quite frequent in these strains. Additionally, there was one strain (O153) that was positive for EAST only. Between the two 0130:H6 strains isolated, one belonged to EAEC serogroup. None of the E. coli isolated were positive for verotoxins, eae, LT1, STa, and ial. Data obtained emphasize the need for additional research into the role of eae gene and other putative factors affecting the virulence of diarrheagenic E. coli in India.

Clostridium perfringens enterotoxin in antibiotic-associated diarrhea.

Clostridium perfringens type A is associated with 5-20% cases of antibiotic-associated diarrhea (AAD) even though Clostridium difficile is implicated in the most severe cases. Fecal specimens from one hundred hospitalized patients, who developed diarrhea regardless of antibiotic intake and who were negative for C. difficile toxin assay, were investigated for C. perfringens enterotoxin (CPE). Simultaneously, cultures were set up for other possible aetiological factors. Ten healthy controls were also similarly investigated. CPE was positive in 2/100 (2%) of the patients and the samples were also positive for the organism in culture. Other organisms isolated were non-toxigenic C. difficile (4%), staphylococci (6%), Candida (18%) and Klebsiella pneumoniae (1%). Stool samples from healthy controls grew mixed growth of no significance and CPE was negative in all of them. Detection of CPE is not part of routine laboratory investigation due to resource implication. Criteria for initiating investigations have to be therefore established by understanding the true burden of C. perfringens-associated AAD by further research.

Evaluation of an in-house rapid diagnostic method for detection of Salmonella enterica serovar Typhi in fecal specimens.

Salmonella enterica serovar Typhi is the etiological agent of typhoid fever. Laboratory diagnosis requires isolation and identification of the organism from the patient's blood or feces. Feces is the specimen most commonly submitted to laboratories. Detection of bacterial antigens is an important adjunct to laboratory diagnosis. We carried out an in-house diagnostic method by preparing test reagents comprising of latex beads coated with specific antisera to detect Vi, O9 and H-d antigens of S. typhi. Fecal specimens from one hundred patients with diarrhea and fever as well as from twenty healthy controls were incubated for enrichment in Selenite F broth for 6 hours or overnight. Latex agglutination tests to detect antigens of S. typhi were carried out on centrifuged broth supernatants. Parallel cultures on media selective for S. typhi were also set up. Nine of the supernatants were positive for two or more specific antigens and S. typhi grew in three of the corresponding cultures. None of the samples from 20 healthy controls were positive by either the diagnostic method or by culture. The result of the in-house diagnostic assay can be obtained overnight and may help in directing immediate antimicrobial therapy.

Estimation of endotoxin in infected bile from patients with biliary diseases.

Diseases of the biliary tract can get complicated by infection. Endotoxin may theoretically be responsible for damage to the gall bladder due to its numerous pathophysiological effects. The aim of the present study was to detect and semi-quantitate the amount of endotoxin present in the bacteriologically positive bile samples and to correlate the endotoxin levels with the clinical profile of the patients. One hundred patients with gall bladder diseases and with infected bile constituted the population for investigation. The clinical profile included presence of fever, jaundice, abdominal pain and gall bladder stones. Endotoxin detection and semi-quantitation in the bile samples were carried out using the Limulus amoebocyte assay: Of 100 infected bile samples investigated, 9 samples (9%) were positive for endotoxin ranging from 1.9 EU/ml to 15 EU/ml. Four of them had Klebsiella pneumoniae, 2 had Acinetobacter anitratus and one each of the remaining 3 samples was positive for (i) Escherichia coli and Serratia marcescens (ii) Pseudomonas aeruginosa and (iii) Salmonella enteritidis. The stool sample of the patient with S. enteritidis in the bile also grew the same microorganism. Statistical analysis showed a significant increase in the presence ofjaundice (p<0.05) and abdominal pain (p<0.01) in the endotoxin positive patients compared to the endotoxin negative ones. Hitherto this is the first report that investigated the endotoxin levels in the bile of patients with gall bladder and biliary tract diseases, along with their biliary bacterial profile. Further research is warranted on the effects of endotoxin on gall stone formation.

Is Campylobacter involved in antibiotic associated diarrhoea?

Campylobacter jejuni is an important cause of acute bacterial diarrhoea. In developing countries like India, children gain immunity early during infancy. However, the incidence is higher in non-immune hosts. Antibiotic use destabilizes the gut flora and can inhibit the local immune responses, thereby compromising resistance to a variety of infections. It is not yet known whether antibiotic intake can also precipitate C. jejuni enteritis as the infectious dose is low and attack rates are high. We made a preliminary study to determine the prevalence of C. jejuni in hospitalized patients receiving antibiotics for various ailments. One hundred and thirty eight stool samples submitted for Clostridium difficile toxin assay were additionally cultured for C. jejuni in blood-free campylobacter selectivity agar. All suspected colonies were subjected to Gram staining, oxidase, catalase and nalidixic acid sensitivity tests. Confirmation of C. jejuni was done by the hippurate hydrolysis test. Of the 138 faecal samples investigated, 14 (10.1%) grew C. jejuni and 11 of them belonged to adults. Two of these 14 samples were also positive for C. difficile toxin. Though not as yet reported, C. jejuni may also be involved in antibiotic associated diarrhoea due to lowered immunity in the host. It may cause enteritis either by itself or in synergy with C. difficile infection.

Clostridium perfringens type A & antibiotic associated diarrhoea.

BACKGROUND & OBJECTIVE: Clostridium perfringens type A (CPA) isolates produce lethal necrotizing antigens and the heat resistant forms of the organism are associated with pathogenic outcome in humans. CPA has also been implicated in antibiotic associated diarrhoea (AAD). We therefore undertook this study to investigate the presence of CPA in stool samples of patients with AAD in a tertiary care setting in north India. METHODS: A total of 285 stool samples obtained from patients suspected for Clostridium difficile aetiology were examined for the presence of CPA antigens. Four sets of reagents (CP-I, CP-II, CP-III and CP- IV) comprising latex beads coated with polyvalent immune sera to 17 serotypes of heat resistant CPA were used in the study. Agglutination reaction was carried out using the reagents with the stool supernatants. RESULTS: Of the 285 stool samples tested, 25 (8.77%) were positive for at least one or more of the four polyvalent sets. Briefly, 48 per cent were positive for all the four sets, 12 per cent for 3 sets, 28 per cent for 2 sets and 12 per cent for only one set, indicating the prevalence of multiple serotypes of CPA. Twenty three (92%) of the 25 positive samples came from patients who were on antibiotics. C. difficile toxin was also present in 9 of 25 (36%) of the samples positive for CPA antigens. INTERPRETATION & CONCLUSION: In our setting, CPA could thus be associated with AAD either by itself or in synergy with C. difficile infection. Assessment of true burden of CPA associated AAD would be required to take appropriate steps for its control in our country.

The epidemiological and resistogram patterns of enteropathogenic and enterotoxigenic Escherichia coli isolated from diarrhoeal stools in a north Indian hospital.

We report our findings on the epidemiological and resistogram patterns of enteropathogenic Escherichia coli (E. coli) (EPEC) and enterotoxigenic E. coli (ETEC) in patients with diarrhoea in a north Indian hospital. A total of 153 diarrhoeic stool samples were cultured for E. coli. Pure or predominant growth obtained was biochemically identified, serogrouped and tested for antibiotic susceptibility. Among the E. coli that could be serogrouped, there were 7 EPEC and 11 ETEC isolates, most of which were multidrug resistant. Apart from this, there were 35 untypable and 4 rough strains of E. coli. Culturing stools and testing the antibiotic susceptibility for most categories of diarrhoeagenic E. coli should be performed in cases of persistent diarrhoea and during outbreaks. This will help to study its epidemiological pattern in a particular locale. Moreover, this information coupled with the clinical picture may be valuable in deciding the need for and type of antibiotic therapy.