Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Screening of mycoflora and ochratoxin A on common culinary herbs and spices in Kenya.

The study aimed to screen fungal diversity and ochratoxin A levels on culinary spice and herb samples sold in open-air markets and supermarkets in Nairobi County, Kenya. All herbs were grown in Kenya, while locally-produced and imported spices were purchased from both types of retail outlet. The results showed a high frequency of Aspergillus and Penicillium species contaminating the samples. The isolated species included Aspergillus ochraceous, Aspergillus nomiae, Aspergillus niger, Aspergillus flavus, Aspergillus ustus, Aspergillus terrus, Aspergillus nidulans, Aspergillus clavutus, Penicillium crustosum, Penicillium expansum, Penicillium brevicompactum, Penicillium glabrum, Penicillium thomii, Penicillium citrinum, Penicillium polonicum, and Cladosporium cladosporioides. Total fungal count on spice and herb samples collected from various sources varied between 6 and 7 CFU/mL. Of imported spices, garlic had the highest fungal diversity, while cardamom had the least. For spices from both open market and supermarket outlets, cloves had the highest fungal diversity, while white pepper had the least. For the herbs sampled from the open markets, basil was the most contaminated, while sage was the least. In supermarket samples, parsley, sage, and mint had the highest fungal diversity, and bay had the least. The results indicate the contamination of spices and herbs with OTA at high concentrations. The calibration curve was saturated at 40 µg/kg; with samples of garlic, cinnamon, red chili, basil, thyme, mint, sage, and parsley having levels above this. Of the spices, imported ginger had the highest OTA levels (28.7 µg/kg), while turmeric from the open market had the least, 2.14 µg/kg. For herb samples, parsley from the open market had the highest OTA levels at 29.4 µg/kg, while marjoram from the open market had the lowest at 6.35 µg/kg. The results demonstrate the presence of mycotoxigenic fungi and OTA contamination of marketed culinary herbs and spices beyond acceptable limits. Hence, there is a need for informed and sustainable mitigation strategies aimed at reducing human exposure in Kenya to OTA mycotoxicosis through dietary intake of spices and herbs.

Optimization of Growth Conditions for Chlorpyrifos-Degrading Bacteria in Farm Soils in Nakuru County, Kenya.

Chlorpyrifos (CP) is a chlorinated organophosphate pesticide. In Kenya, it is commonly used as an acaricide, particularly in dairy farming, leading to soil and water contamination. The study is aimed at isolating bacteria with CP-degrading potential and optimizing their growth conditions, including temperature, pH, and CP concentration. The enrichment culture technique was used, with minimal salt medium (MSM) supplemented with commercial grade CP. A multilevel factorial design was used to investigate the interactions of temperature, pH, and CP concentration. According to the findings, seven bacterial strains with potential to degrade CP were characterized and identified as Alcaligenes faecalis, Bacillus weihenstephanensis, Bacillus toyonensis, Alcaligenes sp. strain SCAU23, Pseudomonas sp. strain PB845W, Brevundimonas diminuta, and uncultured bacterium clone 99. Growth and biodegradation of bacteria differed significantly among the isolates across pH value, temperature, and concentrations (P ≤ 0.05). The optimum conditions for growth were pH 7, temperature of 25°C, and 25mg/l chlorpyrifos concentration, while optimum degradation conditions were pH 5, temp 25°C, and CP conc. 25mg/l. The Pearson correlation between optimum growth and degradation showed a weak positive relationship (R = 0.1144) for pH and strong positive relationship for temperature and concentration of chlorpyrifos. Other than pH, the study shows that there could be other cofactors facilitating the chlorpyrifos degradation process. The findings show that an efficient consortium, at 25°C and pH 5, can include Bacillus toyonensis 20SBZ2B and Alcaligenes sp. SCAU23 as they showed high optical density (OD) values under these conditions. These results indicate the potential for these bacteria to be employed in chlorpyrifos-contaminated ecosystem detoxification efforts upon manipulation of natural growth conditions. The findings of this study offer a potential foundation for future research into the reconstitution of a consortium. Based on the optimum conditions identified, the isolated bacterial strains could be further developed into a consortium to effectively degrade CP in both laboratory and field conditions. Dairy farmers can utilize the isolated strains and the consortia to decontaminate farm soils.

Evaluation of Agronomic Characteristics, Disease Incidence, Yield Performance, and Aflatoxin Accumulation among Six Peanut Varieties (Arachis hypogea L.) Grown in Kenya.

Diseases contribute to attainment of less than 50% of the local groundnut potential yield in Kenya. This study aimed to evaluate the agronomic characteristics (flowering and germination), disease incidence, yield performance (biomass, harvest index, 100-pod, 100-seed, and total pod weight), and aflatoxin accumulation in six peanut varieties. A field experiment was conducted using four newly improved peanut varieties: CG9, CG7, CG12, and ICGV-SM 90704 (Nsinjiro), and two locally used varieties: Homabay local (control) and 12991, and in a randomized complete block design with three replications. The disease identification followed the International Crop Research Institute for the Semi-Arid Tropics (ICRISAT) rating scale and further isolation of fungal contaminants was conducted by a direct plating technique using potato dextrose agar. The aflatoxin levels in the peanuts were determined after harvesting using the ultrahigh performance liquid chromatography and fluorescence detection (UHPLC-FLD) technique. ICGV-SM 90704 showed the least average disease incidence of 1.31 ± 1.75%, (P < 0.05); the lowest total aflatoxin levels (1.82 ± 1.41 µg kg-1) with a range 0.00-0.85 µg kg-1 for total aflatoxins and a range 0.00-1.24 µg kg-1 for Aflatoxin B1. The locally used varieties (12991 and the control) revealed the highest disease incidence (5.41 ± 8.31% and 7.41 ± 1.88%), respectively. ICGV-SM 90704 was the best performing among all the six varieties with an average total pod weight (9.22 ± 1.19 kg), 100-pod weight (262.93 ± 10.8 g), and biomass of (27.21 ± 5.05 kg) per row. The 12991 variety and the control showed the least total pod weight (1.60 ± 0.28 and 1.50 ± 1.11 kg, respectively) (P = 0.0001). The newly improved varieties showed lower disease rates, low levels of aflatoxins, and higher yields than the locally used varieties.

Genetic Profiling of Aspergillus Isolates with Varying Aflatoxin Production Potential from Different Maize-Growing Regions of Kenya.

Highly toxigenic strains of Aspergillus flavus have been reported to frequently contaminate maize, causing fatal aflatoxin poisoning in Kenya. To gain insights into the environmental and genetic factors that influence toxigenicity, fungi (n = 218) that were culturally identified as A. flavus were isolated from maize grains samples (n = 120) from three regions of Kenya. The fungi were further characterized to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA which also revealed all of them to be A. flavus. A subset of 72 isolates representing ITS sequence-based phylogeny and the agroecological origin of maize samples was constituted for subsequent analysis. The analysis of partial calmodulin gene sequences showed that the subset consisted of A. flavus (87%) and Aspergillus minisclerotigenes (13%). No obvious association was detected between the presence of seven aflatoxin biosynthesis genes and fungal species or region. However, the presence of the aflD and aflS genes showed some association with aflatoxin production. The assessment of toxigenicity showed higher aflatoxin production potential in A. minisclerotigenes isolates. Given that A. minisclerotigenes were mainly observed in maize samples from Eastern Kenya, a known aflatoxin hotspot, we speculate that production of copious aflatoxin is an adaptative trait of this recently discovered species in the region.

Effects of Medicinal Plant Extracts and Photosensitization on Aflatoxin Producing Aspergillus flavus (Raper and Fennell).

This study was undertaken with an aim of exploring the effectiveness of medicinal plant extracts in the control of aflatoxin production. Antifungal properties, photosensitization, and phytochemical composition of aqueous and organic extracts of fruits from Solanum aculeastrum, bark from Syzygium cordatum, and leaves from Prunus africana, Ocimum lamiifolium, Lippia kituiensis, and Spinacia oleracea were tested. Spores from four-day-old cultures of previously identified toxigenic fungi, UONV017 and UONV003, were used. Disc diffusion and broth dilution methods were used to test the antifungal activity. The spores were suspended in 2 ml of each extract separately and treated with visible light (420 nm) for varying periods. Organic extracts displayed species and concentration dependent antifungal activity. Solanum aculeastrum had the highest zones of inhibition diameters in both strains: UONV017 (mean = 18.50 ± 0.71 mm) and UONV003 (mean = 11.92 ± 0.94 mm) at 600 mg/ml. Aqueous extracts had no antifungal activity because all diameters were below 8 mm. Solanum aculeastrum had the lowest minimum inhibitory concentration at 25 mg/ml against A. flavus UONV017. All the plant extracts in combination with light reduced the viability of fungal conidia compared with the controls without light, without extracts, and without both extracts and light. Six bioactive compounds were analyzed in the plant extracts. Medicinal plant extracts in this study can control conidia viability and hence with further development can control toxigenic fungal spread.