A universal rapid procedure to determine the DNA base composition (mol% guanine + cytosine) of Gram-positive bacteria is described. Cells of Gram-positive bacteria were lysed with achromopeptidase and the mol% G + C of their DNAs were determined by using high performance liquid chromatography. One ml of a Gram-positive bacterial suspension which matched MacFarland No. 3 standard turbidity was sufficient to determine the mol% G + C within 3 h.
The intestinal microflora was analysed together with short-chain fatty acids (SCFA) and bile acids in faeces from nine children with acute diarrhoeal disease in Lari, Kenya. Enteric pathogens such as enteroinvasive E. coli, enteropathogenic E. coli, Yersinia enterocolitica, rotavirus, Giardia lamblia and Entamoeba histolytica were isolated either singly or in combination from diarrhoeal faecal specimens. The most striking finding in these patients was a marked reduction of anaerobes. Analysis of the SCFA revealed a significantly higher quantity of the volatile fatty acids (VFA) such as acetic, propionic, and butyric acid in recovery period faeces in comparison to diarrhoeal faeces, although no significant difference was seen in the quantity of non-volatile fatty acids. On analysing bile acids in faeces, conjugated primary bile acids were detected from all cases in diarrhoea whereas the free form of secondary bile acids was seen only in recovery. The pH of recovery faecal specimens was significantly lower than that in diarrhoeal faecal specimens. There was a parallel between the decrease in number of anaerobes and fluctuation in the amount of SCFA, showing that the drastic reduction of VFA accompanying decrease of anaerobes during the diarrhoeal state, and the rise in pH thought to arise from these facts, result in an increase of water content.
To develop an effective multiplex PCR for simultaneous and rapid detection of Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus, the three most important Vibrio species that can cause devastating health hazards among human.Species-specific PCR primers were designed based on toxR gene for V. cholerae and V. parahaemolyticus, and vvhA gene for V. vulnificus. The multiplex PCR was validated with 488 Vibrio strains including 322 V. cholerae, 12 V. vulnificus, and 82 V. parahaemolyticus, 20 other Vibrio species and 17 other bacterial species associated with human diseases. It could detect the three target bacteria without any ambiguity even among closely related species. It showed good efficiency in detection of co-existing target species in the same sample. The detection limit of all the target species was ten cells per PCR tube.Specificity and sensitivity of the multiplex PCR is 100% each and sufficient for simultaneous detection of these potentially pathogenic Vibrio species in clinical and environmental samples.This simple, rapid and cost-effective method can be applicable in a prediction system to prevent disease outbreak by these Vibrio species and can be considered as an effective tool for both epidemiologist and ecologist.
Diarrheagenic Escherichia coli (DEC) is an important agent of endemic and epidemic diarrhea worldwide, particularly in developing countries. DEC cannot be differentiated from commensal E. coli on selective media, although there are a few exceptions. Most studies use the colony isolation method, which cannot detect low numbers of DEC, and therefore, these studies might underestimate the incidence of DEC. In the present study, we employed a colony sweep method with real-time PCR targeting virulence genes of 5 categories of DEC; this technique can detect very low numbers of DEC among hundreds of commensal E. coli. DEC was detected in 171 (55.9%) of 306 children with diarrhea in Kenya. The prevalence of DEC in Kenya was notably higher than that (30 in 143, 21.0%) in Indonesia. Occurrences of multiple DEC infection in Kenya were frequent (69 in 306, 23.2%), suggesting that the source of DEC infection may be related to grossly contaminated food and water. In contrast, only 9 (6.0%) of 150 healthy adults in Kenya carried DEC. Considering that healthy adults naturally harbor non-DEC, it is interesting how children exclude DEC but not non-DEC as they grow up. Several mechanisms, such as mucosal immunity and intestinal microbiota, might be involved in the exclusion of DEC.
Infectious diseases are the main causes of morbidity and mortality worldwide. There is increasing concern of indiscriminate use of antibiotics and incidences of multiple antibiotic resistances in human pathogens. The potential of higher plants as source of new drug leads has been demonstrated but is still under explored. In Africa and most developing countries, traditional medicine still forms the backbone of rural medicinal practice. Although a number of American and Asian countries medicinal herbs have been evaluated scientifically and their medicinal properties demonstrated. In Africa, attempts to evaluate medicinal plants in relation to their biological activities and medicinal usefulness are limited. The emergence of antibiotic resistance has led to increased use of herbal medicine as an alternative to combat various ailments. This study aimed at determining the antimicrobial activity of Vernoa hildebrandtii, Acacia stuhlmannii and Moringa oleifera leafy and root bark extracts using disc diffusion technique. Crude extracts were obtained from dried powder by single solvent maceration with ethanol and water. Bioassays were used to evaluate the bioactivity of the extracts against Escherichia coli and Staphylococcus aureas. Antimicrobial activity was determined by measuring the zones of growth inhibition in mm. Moringa aleifera root water extract was the most potent fraction with bioactivity arrange of between 6-32 mm followed by Acacia stuhlmannii root bark water extracts with bioactivity range between 12-31 mm and Moringa oleifera root bark with bioactivity range of between 6-29 mm. Vernoa hildebrandtii leave alcohol and Acacia stuhlmannii Taub root bark- alcohol extract had bioactivity range of between 9-28 and 5-28, respectively. However, Acacia stuhlmannii leaf extracts did not show any antimicrobial activities. The antimicrobial effects of the plant extracts were dose dependent. These findings validate what have been known about Moringa oleifera. They also demonstrate potential biochemical agents in Vernoa hildebrandtii and Acacia stuhlmannii extracts in the management of diarrheagenic microorganisms
In the intestinal flora of patients with diarrhea, a close correlation exists between a decrease in numbers of anaerobes and a reduction in levels of short-chain fatty acids. The drastic reduction of intestinal volatile fatty acids (VFAs) accompanying decreased counts of anaerobes during the diarrheal state and the increase in pH thought to arise from these changes result in increased fecal water content. Our data suggest that free bile acids and VFAs may be factors controlling intestinal bacterial populations in vivo, especially in enteric infections. Thus the role of nonspecific factors such as VFAs, which are among the bile acid metabolites produced by anaerobic intestinal bacteria, deserves emphasis in the evaluation of protective mechanisms provided by the intestinal flora against enteric infections.
Since 2007, Kenya has experienced an increase in cholera outbreaks characterized by a high fatality rate. In this study, we characterized 81 Vibrio cholerae isolates from diarrhoeal stool samples in Nyanza, a cholera-endemic lake region of Kenya, for virulence properties, clonality and antibiotic susceptibility. Eighty of these isolates were V. cholerae O1 El Tor variants carrying the classical ctxB gene sequence, while one isolate was V. cholerae non-O1/O139. All of the El Tor variants were of clonal origin, as revealed by PFGE, and were susceptible to ampicillin, tetracycline, ciprofloxacin, fosfomycin, kanamycin and norfloxacin. However, the isolates showed resistance to sulfamethoxazole/trimethoprim and streptomycin, and intermediate resistance to nalidixic acid, chloramphenicol and imipenem. The non-O1/O139 isolate carried the cholix toxin II gene (chxA II) and was susceptible to all antimicrobials tested except ampicillin. We propose that an El Tor variant clone caused the Nyanza cholera outbreak of 2007-2008.
Abstract Diarrheal diseases are major causes of morbidity and mortality among children in developing countries. We have analyzed the causative agents of diarrhea in children under five years of age who resided in rural environments but attended a hospital in Malindi, a coastal town in Kenya. Bacterial diarrhea was found in 239 (27.7%) of 862 patients with diarrhea. Diarrheagenic Escherichia coli , including enteropathogenic, enterotoxigenic, and enterohaemorrhagic strains, was isolated from 119 (13.8%) patients, followed by Salmonella spp. (63 cases, 7.3%) and Shigella spp. (56 cases, 6.5%). Intestinal parasites were found in 109 (12.6%) of the patients. Entamoeba histolytica and Giardia lamblia were found in 67 (7.8%) and 42 (4.9%) of the cases, respectively. Rotavirus was found in 69 (16.1%) of 428 cases, a part of the 862 cases. Significant differences in age distribution were seen in diarrheal cases due to Campylobacter spp., G. lamblia , and rotavirus. No significant seasonal incidence of specific pathogens was found, but the number of diarrheal patients was significantly correlated to rainfall. Drinking water was contaminated with bacteria at concentrations ranging from 10 3 to 10 6 CFU/ml in 98% of the households and by coliform bacteria at concentrations of 10 2 to 10 5 CFU/ml in 72% of the households. These results suggest that the main routes of infection may be contaminated drinking water and fecal‐oral transmission of enteric pathogens. Consequently, we propose that the enhancement of hygienic practice through health education is a feasible control measure of diarrhea in the study area.
Over the last decade, cholera outbreaks in parts of Kenya have become common. Although a number of recent studies describe the epidemiology of cholera in Kenya, there is paucity of information concerning the diversity and occurrence of mobile genetic elements in Vibrio cholerae strains implicated in these outbreaks. A total of 65 Vibrio cholerae O1 El Tor serotype Inaba isolated between 1994 and 2007 from various outbreaks in Kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements (ICEs), conjugative plasmids and for their genotypic relatedness. All the strains were haemolytic on 5% sheep blood and positive for the Vibrio cholerae El Tor-specific haemolysin toxin gene (hylA) by PCR. They all contained strB, sulII, floR and the dfrA1 genes encoding resistance to streptomycin, sulfamethoxazole, chloramphenicol and trimethoprim respectively. These genes, together with an ICE belonging to the SXT/R391 family were transferable to the rifampicin-resistant E. coli C600 en bloc. All the strains were negative for integron class 1, 2 and 3 and for transposase gene of transposon Tn7 but were positive for integron class 4 and the trpM gene of transposon Tn21. No plasmids were isolated from any of the 65 strains. All the strains were also positive for all V. cholera El Tor pathogenic genes except the NAG- specific heat-stable toxin (st) gene. None of the strains were positive for virulence genes associated with the V. cholerae classical biotype. All the strains were positive for El Tor-specific CTXphi bacteriophage rstrR repressor gene (CTXETΦ) but negative for the Classical, Calcutta, and the Environmental repressor types. Pulse Field Gel Electrophoresis (PFGE) showed that regardless of the year of isolation, all the strains bearing the SXT element were clonally related. This study demonstrates that the V. cholerae O1 strains carrying an SXT/R391-like element implicated in recent cholera outbreaks in Kenya has not changed significantly between 1994 and 2007 and are clonally related.
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