Identification of fecal contamination sources of groundwater in rural areas of Vhembe District Municipality, Limpopo Province, South Africa.

Groundwater is a valuable source of drinking water worldwide, recognized as an improved drinking water source. However, on-site sanitation systems may put groundwater at risk of fecal contamination. In the present study, two approaches were used to ascertain the sources of fecal contamination in groundwater used by communities of the Vhembe District Municipality. Overall, 87.5% of boreholes (n = 70) in the wet and 72.5% in the dry season were contaminated with Escherichia coli, and septic tank (n = 18) wastewaters displayed up to 104 cfu/mL E. coli. Host-specific Bacteroidales quantitative polymerase chain reaction (qPCR) assays established the presence of human (BacHum and HF183) and animal (Cytb, BacCan, and Pig-2Bac) genetic markers in groundwater from 15.7% of boreholes (wet) and 10% of boreholes (dry). No strong associations were founded between culturable E. coli counts and the presence/absence of marker genes for all the markers except for Cytb marker, which showed a weak significant correlation (r = 0.217; p = <0.01) between E. coli and the Cytb marker under dry seasonal conditions. Human markers and Cytb were present in the household septic tank wastewater samples. Significant differences in marker genes distribution in wastewater were observed using the Chi-squared test: HF183 (p = <0.001) and BacHum (p = <0.001). Overall, no association was recorded between markers in groundwater and in wastewater for 18 households' septic tanks. A combined culturable E. coli and host-specific Bacteroidales qPCR assays remain an appropriate approach for the identification of fecal contamination of groundwater. PRACTITIONER POINTS: Households primarily used private boreholes for drinking water, as a primary source. Most households used on-site sanitation systems, including ventilated improved pit latrines and flush toilets connected to septic tanks. Escherichia coli was detected in groundwater, and the sources of fecal contamination were humans and animals (pigs, dogs, and chickens). The presence of human and animal markers in groundwater suggests that humans and animals are liable for fecal contamination. Fecal contamination in drinking water sources poses a significant concern due to pathogenic microorganisms posing potential human health risks.

Synthesis, Characterization and In Vitro Antibacterial Evaluation of Pyrenacantha grandiflora Conjugated Silver Nanoparticles.

In the present study, silver nanoparticles (AgNPs) were synthesized using both the chemical and biological methods and conjugated with Pyrenacantha grandiflora extracts. These were then characterized and evaluated for antimicrobial activities against multi-drug resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumonia, and Escherichia coli. Nanoparticles were analyzed with UV-visible spectrophotometer, transmission electron microscopy (TEM), and energy dispersive X-ray analysis (EDX). Silver nanoparticles, P. grandiflora extracts, and the conjugates were also analyzed with Fourier transform infrared spectroscopy (FTIR). As a result, quasi-sphere-shaped AgNPs with sizes ranging from 5 to 33 nm and spherically shaped AgNPs with sizes ranging from 3 to 25 nm were formed from chemical and biological synthesis, respectively. A well diffusion assay showed that the activity of silver nanoparticles was most improved with acetone extract against all tested bacteria with diameters in the range of 19-24 mm. The lowest MIC value of 0.0063 mg/mL against MRSA was observed when biologically synthesized AgNPs were conjugated with acetone and water extracts. Chemically synthesized silver nanoparticles showed the lowest MIC value of 0.0063 mg/mL against E. coli when conjugated with acetone and methanol extracts. This study indicates that silver nanoparticles conjugated with P. gandiflora tubers extracts exhibit strong antibacterial activities against multi-drug resistant bacterial pathogens. Therefore, biosynthesized conjugates could be utilized as antimicrobial agents for effective disease management due to the synergistic antibacterial activity that was observed.

Functionalization and antimicrobial evaluation of ampicillin, penicillin and vancomycin with Pyrenacantha grandiflora Baill and silver nanoparticles.

Some antibiotics have lost their efficacy over common infections and this has led to the search for new antibiotics and chemically altering existing ones for a better control of infectious diseases. In the present study, Pyrenacantha grandiflora tubers extracts were conjugated with ampicillin, penicillin, vancomycin and silver nanoparticles and their antimicrobial activity was evaluated against Escherichia coli, Staphylococcus aureus and Klebsiella Pneumoniae. The reactions were confirmed by formation of new functional groups that were identified by Fourier transmission infrared spectroscopy (FTIR). Minimum inhibitory concentrations were determined using the microdilution assay. Minimum bactericidal concentrations and the fractional inhibition concentration index were also determined. FTIR analysis indicated different functional group associated with conjugation. The activity of ampicillin was improved when conjugated with silver nanoparticles against K. pneumonia and E. coli. Vancomycin showed improvement of activity when conjugated to silver nanoparticles against K. pneumonia. Penicillin was improved by acetone extracts and vancomycin showed to be more effective when conjugated with silver nanoparticles and water extracts. The conjugation of P. grandiflora with penicillin, ampicillin and vancomycin in the presence of silver nanoparticles improved their biological activities. Therefore, the conjugates are medicinally important and can be used to improve the activity of existing antibiotics.

Use of quantitative molecular diagnostic methods to investigate the effect of enteropathogen infections on linear growth in children in low-resource settings: longitudinal analysis of results from the MAL-ED cohort study.

BACKGROUND: Enteropathogen infections in early childhood not only cause diarrhoea but contribute to poor growth. We used molecular diagnostics to assess whether particular enteropathogens were associated with linear growth across seven low-resource settings. METHODS: We used quantitative PCR to detect 29 enteropathogens in diarrhoeal and non-diarrhoeal stools collected from children in the first 2 years of life obtained during the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) multisite cohort study. Length was measured monthly. We estimated associations between aetiology-specific diarrhoea and subclinical enteropathogen infection and quantity and attained length in 3 month intervals, at age 2 and 5 years, and used a longitudinal model to account for temporality and time-dependent confounding. FINDINGS: Among 1469 children who completed 2 year follow-up, 35 622 stool samples were tested and yielded valid results. Diarrhoeal episodes attributed to bacteria and parasites, but not viruses, were associated with small decreases in length after 3 months and at age 2 years. Substantial decrements in length at 2 years were associated with subclinical, non-diarrhoeal, infection with Shigella (length-for-age Z score [LAZ] reduction -0·14, 95% CI -0·27 to -0·01), enteroaggregative Escherichia coli (-0·21, -0·37 to -0·05), Campylobacter (-0·17, -0·32 to -0·01), and Giardia (-0·17, -0·30 to -0·05). Norovirus, Cryptosporidium, typical enteropathogenic E coli, and Enterocytozoon bieneusi were also associated with small decrements in LAZ. Shigella and E bieneusi were associated with the largest decreases in LAZ per log increase in quantity per g of stool (-0·13 LAZ, 95% CI -0·22 to -0·03 for Shigella; -0·14, -0·26 to -0·02 for E bieneusi). Based on these models, interventions that successfully decrease exposure to Shigella, enteroaggregative E coli, Campylobacter, and Giardia could increase mean length of children by 0·12-0·37 LAZ (0·4-1·2 cm) at the MAL-ED sites. INTERPRETATION: Subclinical infection and quantity of pathogens, particularly Shigella, enteroaggregative E coli, Campylobacter, and Giardia, had a substantial negative association with linear growth, which was sustained during the first 2 years of life, and in some cases, to 5 years. Successfully reducing exposure to certain pathogens might reduce global stunting. FUNDING: Bill & Melinda Gates Foundation.

Use of quantitative molecular diagnostic methods to assess the aetiology, burden, and clinical characteristics of diarrhoea in children in low-resource settings: a reanalysis of the MAL-ED cohort study.

BACKGROUND: Optimum management of childhood diarrhoea in low-resource settings has been hampered by insufficient data on aetiology, burden, and associated clinical characteristics. We used quantitative diagnostic methods to reassess and refine estimates of diarrhoea aetiology from the Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study. METHODS: We re-analysed stool specimens from the multisite MAL-ED cohort study of children aged 0-2 years done at eight locations (Dhaka, Bangladesh; Vellore, India; Bhaktapur, Nepal; Naushero Feroze, Pakistan; Venda, South Africa; Haydom, Tanzania; Fortaleza, Brazil; and Loreto, Peru), which included active surveillance for diarrhoea and routine non-diarrhoeal stool collection. We used quantitative PCR to test for 29 enteropathogens, calculated population-level pathogen-specific attributable burdens, derived stringent quantitative cutoffs to identify aetiology for individual episodes, and created aetiology prediction scores using clinical characteristics. FINDINGS: We analysed 6625 diarrhoeal and 30 968 non-diarrhoeal surveillance stools from 1715 children. Overall, 64·9% of diarrhoea episodes (95% CI 62·6-71·2) could be attributed to an aetiology by quantitative PCR compared with 32·8% (30·8-38·7) using the original study microbiology. Viral diarrhoea (36·4% of overall incidence, 95% CI 33·6-39·5) was more common than bacterial (25·0%, 23·4-28·4) and parasitic diarrhoea (3·5%, 3·0-5·2). Ten pathogens accounted for 95·7% of attributable diarrhoea: Shigella (26·1 attributable episodes per 100 child-years, 95% CI 23·8-29·9), sapovirus (22·8, 18·9-27·5), rotavirus (20·7, 18·8-23·0), adenovirus 40/41 (19·0, 16·8-23·0), enterotoxigenic Escherichia coli (18·8, 16·5-23·8), norovirus (15·4, 13·5-20·1), astrovirus (15·0, 12·0-19·5), Campylobacter jejuni or C coli (12·1, 8·5-17·2), Cryptosporidium (5·8, 4·3-8·3), and typical enteropathogenic E coli (5·4, 2·8-9·3). 86·2% of the attributable incidence for Shigella was non-dysenteric. A prediction score for shigellosis was more accurate (sensitivity 50·4% [95% CI 46·7-54·1], specificity 84·0% [83·0-84·9]) than current guidelines, which recommend treatment only of bloody diarrhoea to cover Shigella (sensitivity 14·5% [95% CI 12·1-17·3], specificity 96·5% [96·0-97·0]). INTERPRETATION: Quantitative molecular diagnostics improved estimates of pathogen-specific burdens of childhood diarrhoea in the community setting. Viral causes predominated, including a substantial burden of sapovirus; however, Shigella had the highest overall burden with a high incidence in the second year of life. These data could improve the management of diarrhoea in these low-resource settings. FUNDING: Bill & Melinda Gates Foundation.

Evaluation of Antimicrobial Activities of Extract from Pyrenacantha grandiflora Baill. (Icacinaceae).

BACKGROUND AND OBJECTIVE: Microbial drug resistance is a growing health problem. This has led to search for new antimicrobial compound and plants are considered as one of the most promising sources for new antimicrobials discovery. Pyrenacantha grandiflora (P. grandiflora) Baill is used for the treatment and management of diarrhea, gastrointestinal related infections, dysentery, inflammation and tooth pain by traditional healers in the Venda region. The goal of the present study was to evaluate the antimicrobial activity of P. grandiflora tubers using different extraction solvents against 15 bacterial and 11 fungal strains. MATERIALS AND METHODS: Plant extracts were obtained using 5 solvents separately, boiled water, cold water, methanol, dichloromethane, chloroform and ethyl acetate. Hole plate assay was used for initial evaluation of antimicrobial properties of plant materials. Minimum inhibitory concentrations (MIC) of the most active plant extracts were determined by the broth microdilution method. One-way ANOVA was used for data analysis. RESULTS: The hole plate assay revealed that the highest antibacterial activity was against Micrococcus kristinae with ethyl acetate extract and no extract was active against Candida and Fusarium species using this method. The MIC of the extracts was determined and all the extracts showed antimicrobial activity against all tested strains ranging from 0.06-7.5 mg mL-1. Some extract appeared to be fungicidal and hot water extract were more active against Cryptococcus neoformans with the MFC value of 0.06 mg mL-1. Methanol extract was also active against most test strains including Candida tropicalis with the minimum fungicidal concentration value of 3.75 mg mL-1. CONCLUSION: Pyrenacantha grandiflora contains substances that make it active against bacterial and fungal pathogens. This is the first time the antimicrobial and antifungal activities of P. grandiflora have been demonstrated scientifically. Extraction with hot water as done by the traditional healers showed activity thereby justifying the traditional use of this plant.