Microbiology

OCCURRENCES AND GENOMIC CHARACTERIZATION OF FOODBORNE BACTERIAL PATHOGENS IN DIARRHEIC UNDER FIVE CHILDREN AND THEIR POTENTIAL EXPOSURE SOURCES IN HARAR CITY AND KERSA DISTRICT, EASTERN ETHIOPIA

Tue, 01 Oct 2024 00:00:00 GMT

OCCURRENCES AND GENOMIC CHARACTERIZATION OF FOODBORNE BACTERIAL PATHOGENS IN DIARRHEIC UNDER FIVE CHILDREN AND THEIR POTENTIAL EXPOSURE SOURCES IN HARAR CITY AND KERSA DISTRICT, EASTERN ETHIOPIA Dinaol Belina Kitila; Dr. Tesfaye Gobena; Dr. Meseret Chimdessa; Dr. Meseret Chimdessa; Dr. Yonas Hailu Diarrheagenic foodborne bacterial pathogens pose a significant global public health challenge, particularly in developing countries, such as Ethiopia. Among these foodborne pathogens (FBPs), nontyphoidal Salmonella (NTS), Escherichia coli, Shigella, and Campylobacter are major contributors to high childhood diarrhea rates. However, there is a lack of comprehensive understanding regarding the prevalence, genomic characteristics, and potential sources of foodborne bacterial pathogens contributing to diarrheal illnesses among children under five in Ethiopia. This knowledge gap hinders effective preventive measures and targeted interventions aimed at reducing the burden of diarrheal diseases associated with foodborne pathogens in vulnerable populations. Therefore, it is important to investigate the occurrence of FBPs and their genomic antimicrobial resistance (AMR) profiles and explore the relationships between strains isolated from children with diarrhea and their potential exposure sources in Harar town and Kersa district, Eastern Ethiopia. A health facility-based cross-sectional study was conducted from November 2021 to January 2023 to isolate and identify NTS, diarrheagenic E. coli (DEC), Shigella, and Campylobacter in stool and environmental samples. Stool samples were collected from children under five years (UFC) with diarrhea visiting healthcare facilities. Additionally, exposure source samples, which included caregivers and/or siblings, animals, and various food and environmental samples, were collected through case-based tracing. The bacterial pathogens were isolated and identified using plate culture and biochemical testing procedures. Genomic DNA was extracted, libraries for next-generation sequencing were prepared, and the samples were sequenced on a NextSeq 550 Illumina sequencer. Data analysis was conducted using statistical software and various bioinformatics tools, including SpeciesFinder for species determination, ResFinder and PointFinder for AMR profiling, multi- locus sequence typing (MLST) for sequence type (ST) profiling, and CSIPhylogeny for phylogenetic analysis. The overall prevalence of the four diarrheagenic pathogens—NTS, DEC, Shigella, and Campylobacter—was 24.8%, with occurrence rates of 27.9% in children, 24.1% in caretakers, and 23.1% in environmental samples. The study revealed an 8.5% coinfection rate and a 23.7% single pathogen infection rate among children with diarrhea. In caretakers, the bacterial coinfection rate was 6%. The prevalence in children with diarrhea was 6.1% for NTS, 10.3% for DEC, 7.3% for Shigella, and 8.4% for Campylobacter, with corresponding rates of 6%, 8.4%, 9.6%, xiii and 6% in caretakers, respectively. Notably, 4.5% and 3.9% of diarrhea cases in children from urban and rural settings, respectively, were attributed to bacterial coinfection. Among the 438 analyzed exposure source samples, the overall prevalence of the three aerobic pathogens—NTS, DEC, and Shigella—was 18.3%, with 3.9% co-occurrence and 14.4% single pathogen occurrence rates. The overall prevalence of Campylobacter in exposure sources was 5.5%, with 6% in Harar town and 5% in Kersa district. The highest prevalence of FBPs was observed in wastewater, at 40.9% for the three aerobic FBPs (40.9%; AOR = 3.3; 95% CI: 1.1–10.1), and 9.1% for Campylobacter. The detection of Campylobacter was 1.8 times more likely in household samples (8.7%; OR = 1.8; 95% CI: 0.7–4.5) than in samples from marketplaces, whereas the occurrence of the aerobic pathogens in samples collected from households was 6.5% (AOR = 0.4; 95% CI: 0.2–1.0). The food contamination rate was 17.9% (AOR = 1.2; 95% CI: 0.4–3.6) for the aerobic FBPs and 4.2% (OR = 0.4; 95% CI: 0.1–1.4) for Campylobacter, with no significant difference between the animal- sourced and nonanimal- sourced food categories. The occurrence rates of NTS, DEC, Shigella, and Campylobacter in the meat samples were 13.9%, 5.4%, 6.5%, and 3.2%, respectively. Notably, DEC and Shigella were detected in 3.7 (1/27) and 7.4% (2/27) of the cooked food samples, respectively. The water contamination rate of Shigella was 7.5% (3/40), and it was also the only FBP detected in drinking water in this study. A significantly higher prevalence of FBPs was detected in poultry than in other animal species, with the likelihood of Campylobacter contamination in the environment being 5.8 times higher in the presence of poultry (17.7%; OR = 5.8; CI: 1.1– 30.6) compared to shoats. The monthly occurrence patterns of the isolated FBPs revealed the highest frequency during the winter (dry) season in both children with diarrhea and their exposure sources, peaking at 73.9% in February 2022 in human stool samples. MLST analysis identified 13, 19, and 8 distinct STs for NTS, DEC, and Campylobacter, respectively. The most common STs were ST93 for NTS, ST13857 for DEC, and ST353 for Campylobacter. The Species determination analyses revealed low Campylobacter species diversity, with only C. jejuni (62.2%) and C. coli (37.8%) detected. The SeqSero tool identified 17 different Salmonella serotypes, with. Muenchen being the most frequently detected, accounting for 14% of the isolates. Among the sequenced NTS, DEC, and Campylobacter, 100% (14/14), 60% (15/25), and 84.6% (11/13) of the child case isolates, respectively, shared ST profiles with at least one isolate from an exposure source. Maximum likelihood phylogenetic analysis revealed that most strains isolated from children clustered with isolates from caregivers or environmental exposures (SNP < 30). Close genomic relatedness among case isolates and those from diverse sources was also observed in distinct clades (SNP < 10 for Campylobacter; SNP < 5 for NTS and DEC). PointFinder analysis revealed at least one antimicrobial resistance gene (ARG) in every NTS sample and 78% of the DEC isolates. The most prevalent ARGs included aac(6')-Iaa and aph(3’)-Ib, which predict xiv aminoglycoside resistance. Other commonly identified ARGs included sul2, aph(6)-Ib, blaTEM-1B, and tet(A). Notably, most of the identified β-lactam ARGs predicted that some NTS and DEC strains were resistant to more than three antibiotics, with blaCTX-M-15 found in 18% of DEC and 3.5% of NTS isolates, conferring resistance to nine antibiotics. DEC isolates from children revealed the highest ARG diversity, with genes such as aph(3'')- Ib, aph(6)-Id, sul2, and tet(A) detected across all sample types, although some ARGs were exclusive to specific sample types. Point mutations mediating AMR were detected in several genes, with mutations associated with nucleotide substitution being the most frequent. In conclusion, this study revealed a high prevalence of FBPs, with all four pathogens being detected in various exposure sources of children with diarrhea. There were also no significant differences (P > 0.05) in the occurrence of each pathogen between settings or between animal- sourced and nonanimal-sourced food, underscoring the importance of these pathogens in both Harar town and Kersa district. This study revealed diverse Salmonella serotypes and DEC strains but low Campylobacter species diversity. The majority of NTS, DEC, and Campylobacter case isolates presented similar ST profiles to those from potential exposure sources and clustered together, even including some isolates collected at different times within certain clades. This suggests the potential persistence and widespread distribution of specific pathogen strains from common sources. In some phylogenetic tree clusters, isolates from various sources clustered together and shared ST profiles, demonstrating the involvement of multiple vectors in the transmission of each pathogen. Furthermore, genotypic AMR profiling revealed widespread drug resistant NTS and DEC strains in Eastern Ethiopia. The study results underscore the need for enhanced food safety measures and improved environmental and personal hygiene practices among caregivers in both urban and rural settings to reduce the burden of FBP-associated diarrhea in children in the study area. As the first whole genome sequencing-based source-tracking study in Ethiopia, our findings highlight the importance of employing WGS in tracking the sources of FBPs and foodborne disease surveillance. Furthermore, one-health approach studies employing WGS and other genome-based diagnostic tools, such as metagenomic analysis and molecular clock analysis, are recommended to better determine the transmission dynamics of FBPs and the relative contributions of each source. Robust and sustained AMR surveillance is also recommended for monitoring the emergence and spread of resistant pathogens. 201p.

PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITIES OF RADISH (Raphanus sativus L.) TUBERS AND SEED CRUDE EXTRACTS AGAINST SELECTED HUMAN PATHOGENS

Sat, 01 Apr 2023 00:00:00 GMT

PHYTOCHEMICAL SCREENING AND ANTIMICROBIAL ACTIVITIES OF RADISH (Raphanus sativus L.) TUBERS AND SEED CRUDE EXTRACTS AGAINST SELECTED HUMAN PATHOGENS EMIRU TIBEBU; Anteneh Belayneh (PhD); Ameha Kebede (PhD) Traditional plants have been used for centuries to treat different human ailments. The phytochemical constituents of these plants contributed much to the reported antimicrobial activities. This study was carried out to determine the phytochemical composition of the crude extracts of the tuber and seeds of Raphanus sativus and evaluate their antimicrobial activities against Eschericia coli O157:H7, Staphylococcus aureus,Candida albicans and Aspergillus niger. Qualitative analysis of the major secondary metabolites was conducted following standard phytochemical screening procedures. The antimicrobial activities of the crude extracts were also evaluated against test organisms using the disc diffusion method at concentrations of 100, 125, and 150 mg/mL. The MIC, MBC, and the MFC of the crude extracts were assessed using the broth dilution method. The results indicated that the crude extracts of the tuber and seeds of Raphanus sativus demonstrated bioactive compounds such as tannins, saponins, steroids, terpenoids, and alkaloids. However, phlobatannins were absent in both plant parts. On the other hand, flavonoids were absent only from the aqueous and ethanol seed extracts. The ethanol extracts of the seeds at the concentration of 150 mg/m1 showed a significantly high zone of inhibition (15.16±0.28 mm) against S. aureus (P < 0.05). Among the test organisms, E. coli exhibited a significantly (P < 0.05) low inhibition zone (10.7±0.17mm) when tested using water extracts of the tuber (150 mg/ml). The ethanol extracts of the seeds (150 mg/ml) revealed the maximum antifungal activity (12.47±0.11mm) against A. niger. The tuber and seed crude extracts showed a statically significant inhibitory effects at p<0.05. The MIC (12.5 mg/ml) of the ethanol extracts of the tuber and seeds revealed its MBC at 25 mg/ml concentration against S. aureus. The ethanol extracts of tuber demonstrated the strongest antifungal activity with MIC of 20.83±7.21 mg/mL and MFC of 41.67±14.43 mg/mL against A. niger. On the basis of these results, it can be concluded that the parts of Raphanus sativus and the extraction solvents used have effects on the antimicrobial activities of the crude extracts. Thus, this suggests that Raphanus sativus tuber and seeds have the potential to be used as a source of alternative antimicrobial agents. 75

ISOLATION AND CHARACTERIZATION OF AMYLASE PRODUCING FUNGI FROM BANANA PEEL AND PAPAYA RIND WASTES

Wed, 01 Nov 2023 00:00:00 GMT

ISOLATION AND CHARACTERIZATION OF AMYLASE PRODUCING FUNGI FROM BANANA PEEL AND PAPAYA RIND WASTES Nuria Mohammed; Mulugeta Desta (PhD); Zekeria Yusuf (PhD) Efficient biodegradation of starch and lignocellulosic waste materials by fungi or bacteria leads to a number of processes of great economic importance. The present study was undertaken to isolate and characterize amylase producing fungi from banana peel and papaya rind wastes. The screening for amylase fungal isolates was conducted based on zone of inhibition followed by morphological identification of fungal species. The amylase activity was determined for fungal colonies using dinitro salicylic acid method. Optimization of parameters for amylase activity was conducted using different pH, temperature, carbon and nitrogen source and incubation time. Important microbial organisms for amylase production like Aspergillus, Penicillium and Rhizopus were indicated from banana peel and Aspergillus, Penicillium, Rhizopus and Saccharomyces were isolated from papaya rind. The effect of pH on amylase activity of the fungal isolates indicated that the maximum enzyme activity (141.51 U/min) was recorded for Aspergillus papaya rind isolate (PI), followed by Rhizopus PI (124.73 U/min) at pH7. The minimum enzyme activity (96.97) was recorded by Rhizopus BI at the same pH7 indicating that the optimum pH for the amylase activity was at around pH7 (neutral). The effect of temperature on amylase activity of the fungal isolates indicated that the maximum enzyme activity (206.05U/min) was recorded for Aspergillus PI and Aspergillus BI (150.06U/min) at 500c.The maximum enzyme activity for Rhizopus PI (150.54 U/min) and for Rhizopus BI (121.82 U/min) at 40oc. The effect of incubation time on amylase activity of the fungal isolates indicated that the maximum enzyme activity (217.51 U/min) was recorded for Aspergillus PI, and Aspergillus BI (116.34 U/min) at 72 hours incubation time while the maximum enzyme activity for Rhizopus PI (154.26 U/min) and Rhizopus BI (112.30 U/min) during 96 hours incubation time. The effect of carbon supplement on amylase activity of the fungal isolate found that Aspergillus BI has recorded the highest enzyme activity (93.10 U/min) with glucose supplement and Rhizopus BI isolate has recorded maximum enzyme activity (78.87 U/min) with lactose supplement. The effect of carbon source on papaya rind isolate has presented maximum enzyme activity (60.67U/min) with glucose supplement for Aspergillus PI while Rhizopus PI has recorded maximum enzyme activity (62.77 U/min) with lactose supplement. The effect of Nitrogen source on amylase activity of the fungal isolate presented that Aspergillus BI (92.94 U/min) and Rhizopus BI (67.45 U/min). The papaya rind isolate has presented maximum enzyme activity (113.59U/min) for Aspergillus PI and (68.58 U/min) for Rhizopus PI has recorded with peptone supplement. It can be concluded that the Aspergillus spp isolates were found to be superior in amylase activity over other isolates, and the papaya rind fungal isolates superior in the amylase activity than the banana peel fungal isolates. In light of significant enzyme imports, despite the potential in waste materials like banana peels and papaya rinds, this study advocates large-scale production for practical and sustainable utilization. 65

THE EXISTING STATE OF MICROBIOLOGICAL MEAT SAFETY IN ETHIOPIAN ABATTOIRS AND BUTCHER SHOPS: PREVALENCE AND IDENTIFICATION OF SALMONELLA ENTERICA SEROTYPES, SAFETY INDICATORS, AND DEVELOPMENT OF MITIGATION TOOLS

Tue, 01 Mar 2022 00:00:00 GMT

THE EXISTING STATE OF MICROBIOLOGICAL MEAT SAFETY IN ETHIOPIAN ABATTOIRS AND BUTCHER SHOPS: PREVALENCE AND IDENTIFICATION OF SALMONELLA ENTERICA SEROTYPES, SAFETY INDICATORS, AND DEVELOPMENT OF MITIGATION TOOLS Andarge Zelalem; Yitagele Terefe (DVM, MSC, Assoc.); Ameha Kebede (Ph.D); Jessie L. Vipham (Ph.D); Kebede Abegaz (Ph.D) Foodborne disease is a major global public health concern. Animal source foods are the leading point of exposure for foodborne pathogens. Salmonella enterica is one of the main meat safety challenges due its ability to colonize and persist on facilities including meat contact surfaces. Meat handling practices with the lack of appropriate knowledge on food safety and workers’ behaviors at abattoirs and butcher shops attribute to contamination of meat and its risk of safety. Therefore, this study was intended to find out the existing microbial safety status of meat (Meta analysis vs. laboratory investigation); phenotypic antimicrobial resistances profiles of isolated pathogens, food safety knowledge, attitudes and hygienic practices (KAP) of workers in meat chain and to design appropriate meat safety challenge mitigation intervention. Consequently, a systematic literature review and meta-analysis was conducted from major electronic databases using indexing services including PubMed/MEDLINE, Google Scholar, Science Direct and WorldCat. The pooled prevalence estimate and antimicrobial resistance profiles of the bacterial pathogens of public health importance such as Samonella spp., E. coli O157:H7, L. monocytogens and Staphylococcus spp. were determined from meat chain in Ethiopia. The study protocol was registered on PROSPERO. A cross-sectional study design was also used to assess knowledge on food safety, attitudes, and hygienic practices of workers in abattoirs and butcher shop facilities. A food safety questionnaire was administered to 542 respondents selected from abattoirs (n=422) and butcher shops (n = 120). Additionally, various samples from abattoirs (n= 150 beef carcasses; 450 sub-swab samples) and butcher shops (n= 360) were collected for microbiological analysis. Detection and identification of S. enterica were carried out using Wellcolex™ color Salmonella rapid latex agglutination kit and finally the presence of invA gene in presumptive positive isolates were confirmed by Real-Time PCR. All Salmonella enterica serotypes were phenotypically evaluated for susceptibility to a panel of 13 antimicrobials using the disk diffusion method as described in Clinical and Laboratory Standards Institute guidelines. The hygiene indicator bacteria were detected and quantified after growth onto petrifilms. Lastly, training intervention using one-group pretest-posttest design was employed to enable them xxiv assess on process hygiene, workers’ knowledge in food safety and behaviors in Addis Ababa and Hawasssa Municipal Abattoirs. Eligible 114 pre-intervention and 108 post-intervention participants were engaged in the study. A total of 138 swab samples (69 pre-intervention and 69 post intervention) were collected from beef carcasses, equipment, environment and personnel to determine the levels of hygiene indicator bacteria of the generic E. coli (ECC), Coliform (CC), Total Coliform (TCC), enterobacteriaceae (EB) and Aerobic Plate Count (APC). The results of systematic review and meta-analysis indicated that a total of 27 original studies with 7828 meat samples were conducted in Ethiopia. When subjected to systematic review and meta-analysis, the pooled prevalence of L. monocytogenes, E. coli O157:H7, Salmonella spp., Staphylococcus spp. was 4%, 5%, 9%, and 21%, respectively. The sample sources showed a high Salmonella prevalence in abattoirs (6%), butcher shops (36%) and markets (11%). The E. coli O157:H7 prevalence in abattoirs, butcher shops and markets was 5, 6 and 8%, respectively. The bacterial isolates showed different antimicrobial resistance profiles against the tested drugs. About 25% of Salmonella spp. and 10% of E. coli 157:H7 isolates were found to be resistant to ampicillin and ceftriaxone, respectively. The results of this study also revealed that 63.3% and 55.8% of the respondents from abattoirs and butcher shops, respectively, demonstrated poor meat safety knowledge. About 46% of the respondents demonstrated good attitude towards meat safety in abattoirs. The respondents from abattoirs (61%) and butcher shops (65%) were classified as having good practices towards meat safety. Logistic regression analysis showed that abattoir workers with primary school education (AOR = 28.93, 95% CI = 16.14 to 51.85) and informal education (AOR = 39.95, 95% CI = 9.19 to 173.70) were more likely to have poor meat safety knowledge (P < 0.0001). The respondents with primary school education (AOR = 13.28, 95% CI = 1.728 to 101.97, P = 0.013) and informal education (AOR = 74.86, 95% CI = 9.05 to 619.22, P < 0.0001) were more likely to have poor attitude toward food safety. Respondents with informal education (AOR = 7.88, 95% CI = 2.56 to 24.25), contract employee status (AOR = 10.86, 95% CI = 5.09 to 23.17), < 2 years of work experience (AOR = 2.50, 95% CI = 1.132 to 5.51, P = 0.023) and poor knowledge (AOR = 3.07, 95% CI = 1.59 to 5.95, P = 0.001) were more likely to have poor food safety practice. The prevalence S. enterica on beef carcasses was 22.7% (95% CI, 16.0 - 30.0) in abattoirs and 16.7% (95% CI, 8.3 – 26.7) in butcher shops. Further study in abattoirs indicated that the prevalence of S. enterica on hides was 13.3% (95% CI, 8.0 - 18.7), pre-evisceration was 12.0% (95% CI, 7.3 - 17.3), and post-evisceration was 4.7% xxv (95% CI, 1.3 - 8.0). The prevalence of S. enterica was significantly lower in post evisceration samples compared to hides (P = 0.009) and pre-evisceration (P = 0.022). A total of five Salmonella enterica serotypes including Salmonella enterica serotype anatum, Salmonella enterica serotype hadar, Salmonella enterica serotype dublin, Salmonella enterica serotype typhimurium and Salmonella enterica serotype choleraesuis were identified from abattoirs and butcher shops in which Salmonella enterica serotype anatum was the most dominant. The prevalence of S. enterica serotype in abattoirs was the highest during wet season (P = 0.011). No significant difference was detected in the prevalence of S. enterica between abattoirs (P = 0.346), and among butcher shop location (P = 0.806). In abattoirs, the overall mean log CFU/cm2 ± SD of generic E. coli, coliform and total coliform counts were 4.55±0.99, 4.91±1.13 and 4.98±1.09, respectively. In butcher shop facilities, the overall mean ± SD (log CFU/cm2 ) of ECC, CC, TCC, EB and APC were 4.31 ± 1.15, 4.61 ± 1.33, 4.77 ± 1.32, 4.59  1.38, and 5.87  1.5. In abattoirs, about 20% of S. enterica were phenotypically multi drug resistant. In butcher shops, (80%) of S. enterica isolates were susceptible to the panel of 11 out of 13 antimicrobials tested. The participant’s food safety knowledge scores on hygienic practices (P = 0.009), pathogens and its associated illness (P = 0.004) significantly increased after intervention. The contamination level of generic E. coli (P = 0.034) and Enterobacteriaceae (P = 0.046) were significantly decreased after intervention. This study revealed a high meat contamination level of indicator bacteria organisms suggesting the existence of poor hygienic practices along the meat supply chain continuum. The presence of S. enterica serotypes on beef with multidrug resistance to commonly used antibiotics may show a public health care challenge. Therefore, integrated intervention approach, standard practical operation protocol for abattoirs and butcher shops, enforcement of food safety standards, swift food safety inspection, refresher trainings and strict meat safety monitoring are recommended in Ethiopian meat supply chain 230