Fecal Shedding, Antimicrobial Resistance and In Vitro Biofilm formation on Simulated Gallstones by Salmonella Typhi Isolated from Typhoid Cases and Asymptomatic Carriers in Nairobi, Kenya.

Typhoid fever, caused by the human restricted pathogen Salmonella Typhi, remains a major global public health concern. Even after successful treatment, approximately 3-5% of patients with typhoid fail to clear the bacteria within one year and become chronic carriers. Most typhoid carriers have gallstones in their gallbladder, and biofilm formation on gallstones is highly correlated with chronic carriage. This study's goal was to identify asymptomatic typhoid carriers in an endemic setting in Kenya, and to compare acute versus chronic isolates. A cohort of typhoid fever patients identified through blood and/or stool culture, and their household contacts, were followed up after treatment to detect longitudinal S. Typhi stool shedding. An abdominal ultrasound scan was used to identify individuals with gallstones. A total of 32 index patients and 32 household contacts were successfully followed-up. Gallstones were detected in 4 cases and 1 household contact. The duration of S. Typhi shedding was significantly longer in individuals with gallstones compared to those without, P<0.001. Eighty-three (83) S. Typhi strains were tested for susceptibility to commonly used antimicrobials and examined by in vitro biofilm formation assays. Out of 37 infected individuals, 32.4% had infections caused by multidrug resistant (MDR) S. Typhi strains and only 18.9% were infected by susceptible strains. Non-MDR strains formed significantly better biofilms in vitro than the MDR strains (P<0.001). This study provides data on S. Typhi chronic carriage that will influence public health approaches aimed at reducing typhoid transmission and the burden of infection.

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.

Multispectral Sorting Based on Visibly High-Risk Kernels Sourced from Another Country Reduces Fumonisin and Toxigenic Fusarium on Maize Kernels.

Fusarium species infect maize crops leading to the production of fumonisin by their toxigenic members. Elimination of microbes is critical in mitigating further postharvest spoilage and toxin accumulation. The current study investigates the efficacy of a previously described multispectral sorting technique to analyze the reduction of fumonisin and toxigenic Fusarium species found contaminating maize kernels in Kenya. Maize samples (n = 99) were collected from six mycotoxin hotspot counties in Kenya (Embu, Meru, Tharaka Nithi, Machakos, Makueni, and Kitui County) and analyzed for aflatoxin and fumonisin using commercial ELISA kits. Aflatoxin levels in majority (91%) of the samples were below the 10 ng/g threshold set by the Kenya Bureau of Standards and therefore not studied further. The 23/99 samples that had >2,000 ng/g of fumonisin were selected for sorting. The sorter was calibrated using kernels sourced from Ghana to reject visibly high-risk kernels for fumonisin contamination using reflectance at nine distinct wavelengths (470-1,550 nm). Accepted and rejected streams were tested for fumonisin using ELISA, and the presence of toxigenic Fusarium using qPCR. After sorting, there was a significant (p < 0.001) reduction of fumonisin, by an average of 1.8 log ng/g (98%) and ranging between 0.14 and 2.7 log ng/g reduction (28-99.8%) with a median mass rejection rate of 1.9% (ranged 0% to 48%). The fumonisin rejection rate ranged between 0 and 99.8% with a median of 77%. There was also a significant reduction (p = 0.005) in the proportion of DNA represented by toxigenic Fusarium, from a mean of 30-1.4%. This study demonstrates the use of multispectral sorting as a potential postharvest intervention tool for the reduction of Fusarium species and preformed fumonisin. The spectral sorting approach of this study suggests that classification algorithms based on high-risk visual features associated with mycotoxin can be applied across different sources of maize to reduce fumonisin.

Efficacy of electron beam irradiation in reduction of mycotoxin-producing fungi, aflatoxin, and fumonisin, in naturally contaminated maize slurry.

Maize is a staple food in Kenya. However, maize is prone to fungal infestation, which may result in production of harmful aflatoxins and fumonisins. Electron beam (eBeam) food processing is a proven post-harvest technology, but published literature is rare on the ability of eBeam to reduce mycotoxins in naturally contaminated maize samples. This study evaluated the efficacy of eBeam doses in reducing viable fungal populations and the destruction of aflatoxins and fumonisins in naturally highly contaminated maize samples from eastern Kenya. Ninety-seven maize samples were analyzed for total aflatoxins and fumonisins using commercial ELISA kits. Then, 24 samples with >100 ng/g of total aflatoxins and >1000 ng/g of total fumonisins were chosen for eBeam toxin degradation studies. Prior to eBeam exposure studies, the samples were made into a slurry using sterile de-ionized water. These slurry samples were exposed to target doses of 5 kGy, 10 kGy, and 20 kGy, with 0 kGy (untreated) samples as controls. Samples were analyzed for total fungal load using culture methods, the quantity of total aflatoxins and fumonisins using ELISA, and the presence of Aspergillus and Fusarium spp. nucleic acids using qPCR for just control samples. There was a significant positive correlation in the control samples between total Aspergillus and aflatoxin levels (r = 0.54; p = 0.007) and total Fusarium and fumonisin levels (r = 0.68; p < 0.001). Exposure to eBeam doses 5 kGy and greater reduced fungal loads to below limits of detection by plating (<1.9 log(CFU/g)). There was also a significant (p = 0.03) average reduction of 0.3 log (ng/g) in aflatoxin at 20 kGy (range from -0.9 to 1.4 log (ng/g)). There was no significant reduction in fumonisin even at 20 kGy. eBeam doses below 20 kGy did not reduce mycotoxins. These results confirm the sensitivity of fungi to eBeam doses in a naturally contaminated maize slurry and that 20 kGy is effective at degrading some pre-formed aflatoxin in such maize preparations.

Correction to: A metagenomic study of the rumen virome in domestic caprids.

Unfortunately, the original article was online published with error in the results section. The error is correction by this erratum.

A metagenomic study of the rumen virome in domestic caprids.

This project sought to investigate the domestic caprid rumen virome by developing a robust viral DNA isolation and enrichment protocol (utilizing membrane filtration, ultra-centrifugation, overnight PEG treatment and nuclease treatment) and using RSD-PCR and high throughput sequencing (HTS) techniques. 3.53% of the reads obtained were analogous to those of viruses denoting Siphoviridae, Myoviridae, Podoviridae, Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseillevirus. Most of the sequenced reads from the rumen were similar to those of phages, which are critical in maintaining the rumen microbial populations under its carrying capacity. Though identified in the rumen, most of these viruses have been reported in other environments as well. Improvements in the viral DNA enrichment and isolation protocol are required to obtain data that are more representative of the rumen virome. The 102,130 unknown reads (92.31%) for the goat and 36,241 unknown reads (93.86%) for the sheep obtained may represent novel genomes that need further study.