Genomic epidemiology of Escherichia coli: antimicrobial resistance through a One Health lens in sympatric humans, livestock and peri-domestic wildlife in Nairobi, Kenya.

BACKGROUND: Livestock systems have been proposed as a reservoir for antimicrobial-resistant (AMR) bacteria and AMR genetic determinants that may infect or colonise humans, yet quantitative evidence regarding their epidemiological role remains lacking. Here, we used a combination of genomics, epidemiology and ecology to investigate patterns of AMR gene carriage in Escherichia coli, regarded as a sentinel organism. METHODS: We conducted a structured epidemiological survey of 99 households across Nairobi, Kenya, and whole genome sequenced E. coli isolates from 311 human, 606 livestock and 399 wildlife faecal samples. We used statistical models to investigate the prevalence of AMR carriage and characterise AMR gene diversity and structure of AMR genes in different host populations across the city. We also investigated household-level risk factors for the exchange of AMR genes between sympatric humans and livestock. RESULTS: We detected 56 unique acquired genes along with 13 point mutations present in variable proportions in human and animal isolates, known to confer resistance to nine antibiotic classes. We find that AMR gene community composition is not associated with host species, but AMR genes were frequently co-located, potentially enabling the acquisition and dispersal of multi-drug resistance in a single step. We find that whilst keeping livestock had no influence on human AMR gene carriage, the potential for AMR transmission across human-livestock interfaces is greatest when manure is poorly disposed of and in larger households. CONCLUSIONS: Findings of widespread carriage of AMR bacteria in human and animal populations, including in long-distance wildlife species, in community settings highlight the value of evidence-based surveillance to address antimicrobial resistance on a global scale. Our genomic analysis provided an in-depth understanding of AMR determinants at the interfaces of One Health sectors that will inform AMR prevention and control.

Draft Genome Sequence of an Enterotoxigenic Escherichia coli Strain Carrying Genes for Colonization Surface Antigen 13 and a Heat-Labile Toxin.

Here, we report the draft genome of ESEI_597, an enterotoxigenic Escherichia coli (ETEC) strain harboring genes encoding colonization surface antigen 13 (CS13) and a heat-labile toxin. The ESEI_597 strain was isolated from an 8-month-old child living in Korogocho, Kenya, in 2013.

Aflatoxin in cereals and groundnut from small holder farming households in Malawi.

Aflatoxin contamination in commonly consumed cereals and nuts may place children at higher risk of stunting and adults at risk of developing liver cancer. This study investigated knowledge on aflatoxins and the level of aflatoxin B1 contamination in commonly consumed cereals and nuts in Malawi. It also included an examination of the proportion of cereals and nuts contaminated above regulatory maximum limits. Aflatoxin contamination in samples was assessed using an enzyme-linked immunosorbent assay (ELISA) method. Less than half of all households knew that consumption of aflatoxin contaminated grain is associated with stunting and lowered immunity. Sorghum samples were the most contaminated and millet the least contaminated. Aflatoxin contamination was highest in southern Malawi and least in northern Malawi. Observed results indicate that this population is at risk of poor health due to lack of knowledge and aflatoxin exposure. Strategies to address contamination should therefore include a comprehensive education campaign to increase knowledge and promote accessible strategies.

Population genomics of Escherichia coli in livestock-keeping households across a rapidly developing urban landscape.

Quantitative evidence for the risk of zoonoses and the spread of antimicrobial resistance remains lacking. Here, as part of the UrbanZoo project, we sampled Escherichia coli from humans, livestock and peri-domestic wildlife in 99 households across Nairobi, Kenya, to investigate its distribution among host species in this rapidly developing urban landscape. We performed whole-genome sequencing of 1,338 E. coli isolates and found that the diversity and sharing patterns of E. coli were heavily structured by household and strongly shaped by host type. We also found evidence for inter-household and inter-host sharing and, importantly, between humans and animals, although this occurs much less frequently. Resistome similarity was differently distributed across host and household, consistent with being driven by shared exposure to antimicrobials. Our results indicate that a large, epidemiologically structured sampling framework combined with WGS is needed to uncover strain-sharing events among different host populations in complex environments and the major contributing pathways that could ultimately drive the emergence of zoonoses and the spread of antimicrobial resistance.

Hospital-based evidence on cost-effectiveness of brucellosis diagnostic tests and treatment in Kenyan hospitals.

Hospitals in Kenya continue to use the Febrile Antigen Brucella Agglutination Test (FBAT) to diagnose brucellosis, despite reports showing its inadequacy. This study generated hospital-based evidence on the performance and cost-effectiveness of the FBAT, compared to the Rose Bengal Test (RBT).Twelve hospitals in western Kenya stored patient serum samples that were tested for brucellosis using the FBAT, and these were later re-tested using the RBT. Data on the running time and cost of the FBAT, and the treatment prescribed for brucellosis, were collected. The cost-effectiveness of the two tests, defined as the cost in US Dollars ($) per Disability Adjusted Life Year (DALY) averted, was determined, and a basic sensitivity analysis was run to identify the most influential parameters. Over a 6-month period, 180 patient serum samples that were tested with FBAT at the hospitals were later re-tested with RBT at the field laboratory. Of these 24 (13.3%) and 3 (1.7%) tested positive with FBAT and RBT, respectively. The agreement between the FBAT and RBT was slight (Kappa = 0.12). Treatment prescribed following FBAT positivity varied between hospitals, and only one hospital prescribed a standardized therapy regimen. The mean $/DALY averted when using the FBAT and RBT were $2,065 (95% CI $481-$6,736) and $304 (95% CI $126-$604), respectively. Brucellosis prevalence was the most influential parameter in the cost-effectiveness of both tests. Extrapolation to the national level suggested that an estimated $338,891 (95% CI $47,000-$1,149,000) per year is currently spent unnecessarily treating those falsely testing positive by FBAT. These findings highlight the potential for misdiagnosis using the FBAT. Furthermore, the RBT is cost-effective, and could be considered as the mainstay screening test for human brucellosis in this setting. Lastly, the treatment regimens must be harmonized to ensure the appropriate use of antibiotics for treatment.

The Genome of Caenorhabditis bovis.

The free-living nematode Caenorhabditis elegans is a key laboratory model for metazoan biology. C. elegans has also become a model for parasitic nematodes despite being only distantly related to most parasitic species. All of the ∼65 Caenorhabditis species currently in culture are free-living, with most having been isolated from decaying plant or fungal matter. Caenorhabditis bovis is a particularly unusual species that has been isolated several times from the inflamed ears of Zebu cattle in Eastern Africa, where it is associated with the disease bovine parasitic otitis. C. bovis is therefore of particular interest to researchers interested in the evolution of nematode parasitism. However, as C. bovis is not in laboratory culture, it remains little studied. Here, by sampling livestock markets and slaughterhouses in Western Kenya, we successfully reisolated C. bovis from the ear of adult female Zebu. We sequenced the genome of C. bovis using the Oxford Nanopore MinION platform in a nearby field laboratory and used the data to generate a chromosome-scale draft genome sequence. We exploited this draft genome sequence to reconstruct the phylogenetic relationships of C. bovis to other Caenorhabditis species and reveal the changes in genome size and content that have occurred during its evolution. We also identified expansions in several gene families that have been implicated in parasitism in other nematode species. The high-quality draft genome and our analyses thereof represent a significant advancement in our understanding of this unusual Caenorhabditis species.

Knowledge, Attitude and Practice of Malawian Farmers on Pre- and Post-Harvest Crop Management to Mitigate Aflatoxin Contamination in Groundnut, Maize and Sorghum-Implication for Behavioral Change.

A knowledge, attitude and practice (KAP) study was conducted in three districts of Malawi to test whether the training had resulted in increased knowledge and adoption of recommended pre- and post-harvest crop management practices, and their contribution to reducing aflatoxin contamination in groundnut, maize and sorghum. The study was conducted with 900 farmers at the baseline and 624 farmers at the end-line, while 726 and 696 harvested crop samples were collected for aflatoxin testing at the baseline and end-line, respectively. Results show that the knowledge and practice of pre- and post-harvest crop management for mitigating aflatoxin were inadequate among the farmers at the baseline but somewhat improved after the training as shown at the end-line. As a result, despite unfavorable weather, the mean aflatoxin contamination level in their grain samples decreased from 83.6 to 55.8 ppb (p < 0.001). However, it was also noted that increased knowledge did not significantly change farmers' attitude toward not consuming grade-outs because of economic incentive incompatibility, leaving potential for improving the practices further. This existing gap in the adoption of aflatoxin mitigation practices calls for approaches that take into account farmers' needs and incentives to attain sustainable behavioral change.

One Health in Action: Operational Aspects of an Integrated Surveillance System for Zoonoses in Western Kenya.

Surveillance of diseases in Kenya and elsewhere in East Africa is currently carried out by both human and animal health sectors. However, a recent evaluation highlighted the lack of integration between these sectors, leading to disease under-reporting and inefficiencies. This project aimed to develop an integrated and cost-effective surveillance and reporting system for 15 zoonotic diseases piloted in the counties of Bungoma, Busia, and Kakamega in western Kenya. Specifically, in this paper we describe the operational aspects of such a surveillance system. Interviews were carried out with key informants, and this was followed by field visits to identify sentinel sites and liaise with relevant stakeholders. Based on this information, a sampling strategy comprising 12 sentinel sites, 4 in each county, was developed. Each sentinel site comprised of a livestock market, 1-2 neighboring slaughter houses/slabs, and a hospital in the vicinity; each of the 12 sites, comprising 12 × 3 = 36 sampling locations, was visited every 4 weeks for 20 cycles. At each site, animal or patient sampling included a clinical examination and collection of blood, feces, and nasal swabs; in slaughtered animals, mesenteric lymph nodes, hydatid cysts, and flukes were also collected. At the end of each field visit, data on staff involved and challenges encountered were recorded, while biological samples were processed and tested for 15 zoonotic diseases in the field laboratory in Busia, Kenya. Public engagement sessions were held at each sentinel site to share preliminary results and provide feedback to both stakeholders and study participants. A livestock market visit lasted just over 3 h, and the most common challenge was the frequent refusals of animal owners to participate in the study. At the slaughterhouses, visits lasted just under 4 h, and challenges included poorly engaged meat inspectors or slaughter processes that were too quick for sampling. Finally, the hospital visits lasted around 4 h, and the most frequent challenges included low patients turn-out, frequent staff turn-over leading to poor institutional memory, and difficulty in obtaining patient stool samples. Our experiences have highlighted the importance of engaging with local stakeholders in the field, while also providing timely feedback through public engagement sessions, to ensure on-going compliance.

Publisher Correction: Genomic insights into the 2016-2017 cholera epidemic in Yemen.

In the HTML version of this Letter, the affiliations for authors Andrew S. Azman, Dhirendra Kumar and Thandavarayan Ramamurthy were inverted (the PDF and print versions of the Letter were correct); the affiliations have been corrected online.

Genomic insights into the 2016-2017 cholera epidemic in Yemen.

Yemen is currently experiencing, to our knowledge, the largest cholera epidemic in recent history. The first cases were declared in September 2016, and over 1.1 million cases and 2,300 deaths have since been reported1. Here we investigate the phylogenetic relationships, pathogenesis and determinants of antimicrobial resistance by sequencing the genomes of Vibrio cholerae isolates from the epidemic in Yemen and recent isolates from neighbouring regions. These 116 genomic sequences were placed within the phylogenetic context of a global collection of 1,087 isolates of the seventh pandemic V. cholerae serogroups O1 and O139 biotype El Tor2-4. We show that the isolates from Yemen that were collected during the two epidemiological waves of the epidemic1-the first between 28 September 2016 and 23 April 2017 (25,839 suspected cases) and the second beginning on 24 April 2017 (more than 1 million suspected cases)-are V. cholerae serotype Ogawa isolates from a single sublineage of the seventh pandemic V. cholerae O1 El Tor (7PET) lineage. Using genomic approaches, we link the epidemic in Yemen to global radiations of pandemic V. cholerae and show that this sublineage originated from South Asia and that it caused outbreaks in East Africa before appearing in Yemen. Furthermore, we show that the isolates from Yemen are susceptible to several antibiotics that are commonly used to treat cholera and to polymyxin B, resistance to which is used as a marker of the El Tor biotype.

A Critical Review of Aflatoxin Contamination of Peanuts in Malawi and Zambia: The Past, Present, and Future.

Peanut (Arachis hypogaea L.) is an important crop in Malawi and Zambia. The crop is valued for soil improvement in cereal-based cropping systems, for improving the livelihoods of farming households who consume it and also sell it for cash, and for earning foreign exchange when exported. Research and development efforts have resulted in an increase in both peanut production area and productivity. However, a key challenge that still needs to be solved in these countries is how to produce peanuts with acceptable levels of aflatoxin contamination. Data continues to show that aflatoxin continues to be a problem in both formal and informal trade. As a result, unlike 30 years ago, most of the peanut trade has now shifted to domestic and regional markets that do not restrict the sale of aflatoxin-contaminated peanuts. Impacts of aflatoxin contamination on health and also on the full cost burden of control are not well documented. Technologies are available for mitigating against aflatoxin contamination. The advantages, disadvantages, and gaps associated with these technologies are discussed. Considerable money and effort continues to be invested in Malawi and Zambia into mitigating aflatoxin contamination, but evidence of long-term success is limited. Based on past and current initiatives, the prospects of eliminating aflatoxin in the near future at the household level and in trade are not promising.

Aflatoxin B1 levels in groundnut products from local markets in Zambia.

In Zambia, groundnut products (milled groundnut powder, groundnut kernels) are mostly sold in under-regulated markets. Coupled with the lack of quality enforcement in such markets, consumers may be at risk to aflatoxin exposure. However, the level of aflatoxin contamination in these products is not known. Compared to groundnut kernels, milled groundnut powder obscures visual indicators of aflatoxin contamination in groundnuts such as moldiness, discoloration, insect damage or kernel damage. A survey was therefore conducted from 2012 to 2014, to estimate and compare aflatoxin levels in these products (n = 202), purchased from markets in important groundnut growing districts and in urban areas. Samples of whole groundnut kernels (n = 163) and milled groundnut powder (n = 39) were analysed for aflatoxin B1 (AFB1) by competitive enzyme-linked immunosorbent assay (cELISA). Results showed substantial AFB1 contamination levels in both types of groundnut products with maximum AFB1 levels of 11,100 µg/kg (groundnut kernels) and 3000 µg/kg (milled groundnut powder). However, paired t test analysis showed that AFB1 contamination levels in milled groundnut powder were not always significantly higher (P > 0.05) than those in groundnut kernels. Even for products from the same vendor, AFB1 levels were not consistently higher in milled groundnut powder than in whole groundnut kernels. This suggests that vendors do not systematically sort out whole groundnut kernels of visually poor quality for milling. However, the overall contamination levels of groundnut products with AFB1 were found to be alarmingly high in all years and locations. Therefore, solutions are needed to reduce aflatoxin levels in such under-regulated markets.

A Case for Regular Aflatoxin Monitoring in Peanut Butter in Sub-Saharan Africa: Lessons from a 3-Year Survey in Zambia.

A 3-year comprehensive analysis of aflatoxin contamination in peanut butter was conducted in Zambia, sub-Saharan Africa. The study analyzed 954 containers of 24 local and imported peanut butter brands collected from shops in Chipata, Mambwe, Petauke, Katete, and Nyimba districts and also in Lusaka from 2012 to 2014. For analysis, a sample included six containers of a single brand, from the same processing batch number and the same shop. Each container was quantitatively analyzed for aflatoxin B1 (AFB1) in six replicates by using competitive enzyme-linked immunosorbent assay; thus, aflatoxin contamination level of a given sample was derived from an average of 36 test values. Results showed that 73% of the brands tested in 2012 were contaminated with AFB1 levels >20 µg/kg and ranged up to 130 µg/kg. In 2013, 80% of the brands were contaminated with AFB1 levels >20 µg/kg and ranged up to 10,740 µg/kg. Compared with brand data from 2012 and 2013, fewer brands in 2014, i.e., 53%, had aflatoxin B1 levels >20 µg/kg and ranged up to 1,000 µg/kg. Of the eight brands tested repeatedly across the 3-year period, none consistently averaged ≤20 µg/kg. Our survey clearly demonstrates the regular occurrence of high levels of AF B1 in peanut butter in Zambia. Considering that some of the brands tested originated from neighboring countries such as Malawi, Zimbabwe, and South Africa, the current findings provide a sub-Saharan regional perspective regarding the safety of peanut butter.

Knowledge, attitude, and practices concerning presence of molds in foods among members of the general public in Malawi.

This study assessed knowledge, attitude, and practices (KAP) of the public in Malawi on issues related to molds in foodstuffs. A survey involving structured questionnaire was conducted with 805 respondents from seven districts of Malawi. Descriptive statistics, t tests, and analyses of variance were calculated. A majority of respondents recognized that molds were dangerous to human health (88%); however, about 50% of respondents were not informed that mold toxins are thermally stable and that they are not destroyable by normal cooking processes. About 33% of the respondents asserted that they buy moldy maize, while approximately 20% of respondents reported that they consume moldy fruits having discarded moldy fraction. There were significant differences in knowledge scores among different demographic groups. Females had significantly (p < 0.05) lower knowledge scores on issues related to molds in foods. Additionally, the respondent's location had a significant effect. However, respondent's education had subtle effect on knowledge score and the overall population's knowledge score was generally low (3.55 ± 1.32 score out of 9). Results of the study underline the need to raise public's knowledge about health risks associated with spoilage molds in food and prevention and management options.

A putative, novel coli surface antigen 8B (CS8B) of enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) strains harbor multiple fimbriae and pili to mediate host colonization, including the type IVb pilus, colonization factor antigen III (CFA/III). Not all colonization factors are well characterized or known in toxin positive ETEC isolates, which may have an impact identifying ETEC isolates based on molecular screening of these biomarkers. We describe a novel coli surface antigen (CS) 8 subtype B (CS8B), a family of CFA/III pilus, in a toxin producing ETEC isolate from a Kenyan collection. In highlighting the existence of this putative CS, we provide the sequence and specific primers, which can be used alongside other ETEC primers previously described.

Uncommon occurrence ratios of aflatoxin B1, B 2, G 1, and G 2 in maize and groundnuts from Malawi.

We report an unusual aflatoxin profile in maize and groundnuts from Malawi, with aflatoxin G1 found routinely at equal or even higher levels than aflatoxin B1. Aflatoxin B1 (AFB1) ratio in a contaminated sample is generally greater than 50% of total aflatoxin (sum of aflatoxin B1, B2, G1, and G2). In Malawi, the aflatoxin occurrence ratios were determined by examining LC-MS/MS and HPLC fluorescence detection (FLD) data of 156 naturally contaminated raw maize and 80 groundnut samples collected in 2011 and 2012. Results showed that natural aflatoxin occurrence ratio differed. In 47% of the samples, the concentration of AFG1 was higher than that of AFB1. The mean concentration percentages of AFB1/AFB2/AFG1/AFG2 in reference to total aflatoxins were found to be 47:5:43:5%, respectively. The AFG1 and AFB1 50/50 trend was observed in maize and groundnuts and was consistent for samples collected in both years. If the AFB1 measurement was used to check compliance of total aflatoxin regulatory limit set at 10, 20, 100, and 200 µg/kg with an assumption that AFB1≥50% of the total aflatoxin content, 8, 13, 24, and 26% false negative rates would have occurred respectively. It is therefore important for legislation to consider total aflatoxins rather than AFB1 alone.

Comparison of Crop Rotation for Verticillium Wilt Management and Effect on Pythium Species in Conventional and Organic Strawberry Production.

The effects of broccoli and lettuce rotations on population densities of Verticillium dahliae and Pythium spp. in soil and on strawberry (Fragaria × ananassa) growth, yield, and Verticillium wilt were evaluated in conventional and organic production systems in California for 2 years. Under both management systems, strawberry was planted after two successive crops of broccoli or lettuce. The control treatment in the conventional field was strawberry planted in soils fumigated with methyl bromide + chloropicrin. Preplant densities of V. dahliae and Pythium sp. did not differ in these fields. At the end of the second broccoli crop, V. dahliae densities in conventional plots had declined by 44% in both years. In contrast, after the second broccoli crop, densities in organic fields decreased 47% in 2000 and 25% in 2001. In general, there were no differences in V. dahliae inoculum densities in organic and conventional plots following lettuce rotations. After the second vegetable production cycle, population densities of V. dahliae in broccoli rotated organic (24 CFU/g of soil in 2000 and 27 CFU/g of soil in 2001) or conventional (23 CFU/g of soil in 2000 and 19 CFU/g of soil in 2001) fields were significantly lower than those in lettuce rotated organic (40 CFU/g of soil in 2000 and 42 CFU/g of soil in 2001) or conventional (39 CFU/g of soil in 2000 and 35 CFU/g of soil in 2001) fields. However, crop rotation treatments had no consistent effect on the inoculum densities of Pythium spp. Canopy diameters of strawberry plants grown in rotation with broccoli were not different from those in fumigated control plots, whereas those from lettuce plots were 10% smaller. Strawberry plant nutrient analysis showed that fertilizer inputs into organic or conventional production were not responsible for the observed differences in plant size. Increases in strawberry yields were not consistent between years. Verticillium wilt incidence on strawberry was 12 to 24% lower in fields rotated with broccoli compared with fields rotated with lettuce. Wilt severity on strawberry was 22 to 36% lower in fields rotated with broccoli compared with those rotated with lettuce. The strategy of using broccoli rotation coupled with postharvest incorporation of broccoli residue continues to show promise as a tool in the management of Verticillium wilt in both conventional and organic strawberry production systems.

Effect of Incorporation of Brassica spp. Residues on Population Densities of Soilborne Microorganisms and on Damping-off and Fusarium Wilt of Watermelon.

Incorporating Brassica spp. residue to reduce populations of soilborne fungi and manage damping-off and Fusarium wilt of watermelon (Citrullus lanatus var. lanatus) was studied in two field experiments. Treatments included incorporating flowering Brassica napus cv. Dwarf Essex canola or B. juncea cv. Cutlass mustard and laying black polyethylene mulch at incorporation or 1 month after incorporation, methyl bromide, and a nontreated control. In both years, glucosinolates were identified and quantified in the shoots and roots of the flowering plants. In both years, the total concentration of glucosinolates incorporated per square meter was significantly higher for B. juncea than for B. napus. Isothiocyanates were inconsistently detected in the amended soils and none were detected more than 12 days postincorporation. After incorporation in 2004 and 2005, amended plots had higher populations of Fusarium oxysporum and Pythium spp. than the methyl bromide treatment, and in some treatments, populations were higher than in the control. Fluorescent Pseudomonas spp. were not suppressed in amended soils, and their populations were significantly higher in some amended treatments than those in methyl bromide-treated soils or nontreated control soils. Incidence of damping-off and severity of Fusarium wilt on seedless watermelon cv. Tri-X 313, which is susceptible to Fusarium wilt, were not consistently lower in brassica-amended soils or methyl bromide-treated plots than in nontreated control plots. Therefore, under spring conditions and methods used in this study, neither biofumigation nor methyl bromide fumigation in coastal South Carolina was an effective disease management tool for two soilborne pathogens of watermelon.