RJHS 11(2).cdr Prevalence and antibiotic resistant Escherichia coli isolated from abattoir and aquaculture environment in Ebonyi State, South East Nigeria. 1,2 2 2 2 3 *Onuoha, S.C. , Okafor, C.O.O. , Eronmosele, B.O. , Ovia, K.N. , Nwosu, M.C. , 2 4 5 5 Onwere, C.C. , Ude, I.U. , Ezeme-Nwafor, A.C. , Ani, P. Abstract Background: The present study was carried out to evaluate the distribution and antibiotics profile of Escherichia coli from abattoir and aquaculture. Methods: Abattoir and aquaculture effluents were randomly collected from various parts of Abakaliki in Ebonyi State. Bacterial detection was conducted using cultural and biochemical analysis. Susceptibility of the E. coli to antimicrobials was investigated using the Kirby- Bauer disk diffusion method. 7 7 Results: The microbial load from abattoirs ranges from 0.26±0.11x10 to 4.08±0.11x10 cfu/ml and 7 7 aquacultures 0.40±0.04x10 to 4.06±2.74x10 cfu/ml differ significantly (P<0.05). Out of the total 44 E. coli isolates from abattoir, drainage shows the highest E. coli isolates (40.9 %) and waste water least (22.7 %), while of the 18 E. coli isolates from aquaculture, 55.6 % were from concrete pond, while 44.4 % were from earthen pond. The E. coli isolates showed reasonable susceptibility to cefeprime (62.5 %), followed by imipenem (50. 0 %). However, all the E. coli isolates were resistant to amoxicillin/clavulanic acid, cefixime, cefotaxime and tobramycin. E. coli MAR index range from 0.4 - 0.9. Conclusion: The high microbial load, antibiotic resistance and higher MAR index >2 is of public health concern and further demonstrates the need for adequate treatment and disposal of waste generated from abattoir and aquaculture. Keywords: E. coli, abattoir, aquaculture, antibiotics *Corresponding author Onuoha, S .C Email: sconuoha@yahoo.com 1 Department of Biotechnology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria. 2 Department of Microbiology, College of Sciences, Evangel University Akaeze, Nigeria. 3 Department of Biotechnology, College of Sciences, Evangel University Akaeze, Nigeria. 4 Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria. 5 Department of Microbiology, Caritas University, Amorji-Nike, Nigeria ORCID-NO: https://orcid.org/0000-0002-6076-3910 Received: July 4, 2022 Accepted: March 24, 2023 Published: June 30, 2023 Research Journal of Health Sciences subscribed to terms and conditions of Open Access publication. Articles are distributed under the terms of Creative Commons Licence (CC BY-NC-ND 4.0). (http://creativecommons.org/licences/by-nc-nd/4.0). http://dx.doi.org/10.4314/rejhs.v11i2.6 Original Article Research Journal of Health Sciences Res. J. Health Sci. Vol 11(2), June 2023 128 Prévalence et escherichiacoli résistant aux antibiotiques isolés de l'abattoir et de l'environnement aquacole dans l'état d'Ebonyi , au sud-est du Nigéria 1,2 2 2 2 3 *Onuoha, S.C. , Okafor, C.O.O. , Eronmosele, B.O. , Ovia, K.N. , Nwosu, M.C. , 2 4 5 5 Onwere, C.C. , Ude, I.U. , Ezeme-Nwafor, A.C. , Ani, P. Résumé Contexte général de l'étude: La présente étude a été réalisée pour évaluer la distribution et le profil d'antibiotiques d'Escherichia coli provenant d'abattoirs et d'aquaculture. Méthode de l'étude : Les effluents d'abattoir et d'aquaculture ont été collectés au hasard dans différentes parties d'Abakaliki dans l'état d'Ebonyi. La détection bactérienne a été effectuée à l'aide d'analyses culturales et biochimiques. La sensibilité d'E. coli aux antimicrobiens a été étudiée à l'aide de la méthode de diffusion sur disque de Kirby-Bauer. 7 7 Résultat de l'étude : La charge microbienne des abattoirs varie de 0,26±0,11x10 à 4,08±0,11x10 ufc /ml 7 7 et les aquacultures de 0,40±0,04x10 à 4,06±2,74x10 ufc /ml diffèrent significativement (P<0,05). Sur le total de 44 isolats d'E. coli provenant d'abattoir, le drainage montre les isolats d'E. coli les plus élevés (40,9 %) et les eaux usées le moins (22,7 %), tandis que sur les 18 isolats d'E. coli provenant de l'aquaculture, 55,6 % provenaient d'étangs en béton, tandis que 44,4 % provenaient d'un étang en terre. Les isolats d'E. coli ont montré une sensibilité raisonnable au céféprime (62,5 %), suivi de l'imipénem (50,0 %). Cependant, tous les isolats d'E. coli étaient résistants à l'amoxicilline/acide clavulanique , au céfixime, au céfotaxime et à la tobramycine. L'indice E. coli MAR varie de 0,4 à 0,9. Conclusion : La charge microbienne élevée, la résistance aux antibiotiques et l'indice MAR supérieur > 2 sont préoccupants pour la santé publique et démontrent en outre la nécessité d'un traitement et d'une élimination adéquats des déchets générés par les abattoirs et l'aquaculture. Mots-clés : E. coli, abattoir, aquaculture, antibiotiques Received: July 4, 2022 Accepted: March 24, 2023 Published: June 30, 2023 *Corresponding author Onuoha, S .C Email: sconuoha@yahoo.com 1 Department of Biotechnology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria. 2 Department of Microbiology, College of Sciences, Evangel University Akaeze, Nigeria. 3 Department of Biotechnology, College of Sciences, Evangel University Akaeze, Nigeria. 4 Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria. 5 Department of Microbiology, Caritas University, Amorji-Nike, Nigeria ORCID-NO: https://orcid.org/0000-0002-6076-3910 Research Journal of Health Sciences subscribed to terms and conditions of Open Access publication. Articles are distributed under the terms of Creative Commons Licence (CC BY-NC-ND 4.0). (http://creativecommons.org/licences/by-nc-nd/4.0). http://dx.doi.org/10.4314/rejhs.v11i2.6 Article Original Research Journal of Health Sciences Res. J. Health Sci. Vol 11(2), June 2023 129 INTRODUCTION Foodborne infections are widespread and growing public health problems in the world (1). Abattoir, otherwise known as slaughter house is a facility where animals are killed for consumption as food products (2). Like many other sewage types, effluents from the abattoir flow into water bodies such as ground water, streams, rivers, lakes and oceans thereby introducing enteric pathogens, excess nutrients and other contaminants into the water sources (3). Water pollution from abattoir effluents may cause substantial environmental and public health hazards especially in Nigeria, a country where abattoir effluents like most other wastewater are untreated before they are discharged (4, 5, 6, 7). The increasing demand for aquaculture products as a source of protein stimulates the propagation and expansion of aquaculture in many countries (8). Despite the high nutritional quality that links fish consumption to positive health effects in humans, the aquaculture system is tremendously vulnerable to pollution and run-offs from anthropogenic sources which contaminate fish products with microbiological hazards such as E. coli and Salmonella (9). Escherichia coli is among the most challenging Enterobacteriaceae group of bacterial meat contaminant worldwide (1). E. coli is a common inhabitant of human and animal intestinal tract. It is a Gram-negative facultative aerobic organism and the most common in the family Enterobacteriaceae (5). E. coli is the most common pathogen leading to uncomplicated cystitis, and also results in other extraintestinal illnesses, including pneumonia, bacteremia, and abdominal infections such as spontaneous bacterial peritonitis (9). The detection of pathogenic organisms as well as the incidence of proteolytic and lipolytic bacteria in a water body is suggestive of impending health hazards and public health concern. Micronutrients in abattoir wastewater sustain the prevalence of pathogenic and entropic organisms that constitute biohazards in water bodies. Antibiotic resistance is a pandemic that requires global health solutions (11). The emergence of antimicrobial resistance among bacterial pathogens demands a local understanding of the epidemiological situation. This information is needed both for clinical treatment decision-making purposes as well as for the revision of current care guidelines (12). Wastewaters are considered hotspots for antibiotic resistant bacteria and horizontal gene transfer among related and unrelated bacterial species (13). Abattoir effluents are potential carriers of resistant pathogenic bacteria and could be contributing to the global spread of these strains in the environments (14). As a consequence of the use of antibiotics in aquaculture, antibiotic resistance is induced in the surrounding bacteria in the column water, sediment, and fish-associated bacterial strains (15). Antibiotics are widely used in intensive fish farming, which in turn increases the emergence of antimicrobial-resistant bacteria in the aquatic environment (8). Pathogenic E. coli has become alarming and livestock acquired infection is now one of the leading cause infections globally. Antimicrobial resistance aggravates the already difficult treatment of bacterial infections. Due to many factors influencing antibiotic resistance, the correct choice of antimicrobial management remains debatable. Hence, this has necessitated the research in the distribution and antibiotics susceptibility profile of pathogenic E. coli from abattoir and aquaculture effluents in Abakaliki, Ebonyi State. MATERIALS AND METHODS Approval and consent for the study were obtained from the owners of the various abattoir and aquaculture farms. The study was conducted from March 2020 to June 2020 at the Microbiology Laboratory of Ebonyi State University Abakaliki, Nigeria. Sample Collection and Procession Fifty (50) samples were collected from a b a t t o i r w a s t e - w a t e r a n d a q u a c u l t u r e environment within Abakaliki Metropolis, Ebonyi State. The waste water from the abattoir were obtained from drainage, butcher table and wash water. All samples were collected aseptically using universal sterile container. Containers were filled leaving a top space of about 2.5cm. Samples were processed and incubated within 5 hours of sampling. Samples were transported in isothermal boxes with ice to the laboratory of Applied Microbiology Department, Ebonyi State University for Escherichia coli analyses. Isolation, Enumeration, and Identification of Escherichia coli The isolation, identification and enumeration of E. coli were carried out using standard microbiological/biochemical methods (16, 17). Antibiotics Sensitivity Testing Susceptibility patterns of the isolated E. Res. J. Health Sci. Vol 11(2), June 2023 130 Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. coli were tested against a wide range of antibiotics namely; Imipenem (10 µg), cefoxitin (30 µg), cefotaxime (30 µg), cefeprime (30 µg), meropenem (10 µg), tobramycin (10 µg) ceftazidime (30 µg) and amoxicillin clavulanic acid (30 µg) on Muller Hinton Agar (Oxoid, UK) using Kirby and Bauer disc diffusion methods of determining susceptibility (16). All the antibiotics disk were procured from Oxoid limited (Oxoid, UK). Multiple Antibiotic Resistance (MAR) Index MAR index was determined by following the procedure described by Ayandele et al. (18). MAR Index for an isolate = = Number of antibiotics to which isolate is resistant Total number of antibiotics against which isolate was tested Statistical Analysis The percentage frequency of occurrence of the E.coli isolated from abattoir and aquaculture environment was calculated using n 100 Frequency (%) = / x / N 1 Where n = Number of occurrence of bacteria species, N = Total number of bacteria isolated. E x p e r i m e n t a l d a t a w a s p r e s e n t e d a s mean±standard deviation, while one way ANOVA procedure was used to analyze statistical difference in the data generated. RESULTS The result from abattoir shows that samples from location designated AB had 3 significantly (P<0.05) high microbial load 7 (4.08±0.11x10 cfu/ml), followed by AB from 2 7 waste water (3.54±0.65x10 cfu/ml), while samples from AB waste water recorded the 2 7 l o w e s t m i c r o b i a l l o a d ( 0 . 2 6 ± 0 . 11 x 1 0 cfu/ml)(Table 1). No significant variation (p>0.05) was observed with microbial load from sample location designated AB from butchers 2 table, waste water and drainage (Table 1). No significance difference (p>0.05) was observed with microbial load of waste water and drainage from sample AB and AB , microbial load of 5 6 waste water and butcher table from sample AB , 6 microbial load of drainage and butcher table from sample AB and AB (Table 1).1 6 Result from aquaculture pond revealed a significant (p<0.05) difference in their microbial burden showing that PW earthen pond had 2 significantly (P<0.05) high microbial load 7 (4.06±2.74x10 cfu/ml), followed by PW earthen 1 7 pond (2.56±0.23 x10 cfu/ml), while samples from PW concrete ponds recorded the lowest 4 7 microbial load (0.40±0.04x10 cfu/ml) as shown in Table 2. No significant variation (p>0.05) was observed with microbial load from PW concrete 6 and earthen pond (Table 2). The result of the prevalence of E. coli showed that AB (33.3 %) had the highest 3 frequency of E. coli isolates from drainage, followed by AB (22.2 %), AB (16.2 %), while 4 2 AB (5.6 %) shows the least prevalence (Figure 1 1). AB (30.0 %) showed the highest frequency of 5 E. coli isolates from waste water, followed by AB (20.0 %), AB (20.0 %), while AB AB and 1 2 3, 4, AB (10.0 %) shows the least percentage 6 prevalence (Figure 1). There was also highest frequency of E. coli isolates from butcher table from AB (37.5 %), followed by AB (25.0 %), 3 4 while AB and AB (6.3 %) had the least 2 5 prevalence (figure 1). Also, PW (30.0 %) showed the highest 3 E. coli from concrete pond, followed by PW 2 (20.0 %), while others (PW , PW , PW and PW ) 1 4 5 6 recorded the least with 10.0 % prevalence respectively (figure 2). PW and PW (25.0 %) 1 3 had the highest E. coli from earthen pond, while PW , PW , PW and PW recorded the least with 2 4 5 6 12.5 % prevalence each (figure 2). T h e r e s u l t o f t h e a n t i b i o t i c s susceptibility of E. coli to the respective antibiotics revealed that the E. coli isolates s h o w e d h i g h e s t r e s i s t a n c e t o a m o x i c i l l i n / c l a v u l a n i c a c i d , c e f i x i m e ,cefotaxime and tobramycin at 100 %, , while imipenem recorded the least at 25.0%. Highest susceptibility was observed with cefeprime (62.5 %), followed by imipenem (50. 0 %) (Table 3). The result of the multiple antibiotics resistance index of the isolates ranges from 0.4 to 0.9 with the mean value of 0.6 (Table 4). The isolate from AB drainage recorded the highest 1 MARI of 0.9 while all the wastewater samples recorded MARI of 0.7 except the one collected from AB whose MAR index was 0.5. The result 2 of the multiple antibiotics resistance index of the E. coli isolates from aquaculture environment ranges from 0.5 to 0.7 (Table 4). DISCUSSION AND CONCLUSION The findings of the study demonstrated that Escherichia coli existed in high densities in samples in the study area with waste water from AB designated sampling showing the highest 3 7 microbial load 4.08±0.11x10 cfu/ml, while samples from AB waste water recording the 2 7 least microbial load 0.26±0.11x10 cfu/ml Res. J. Health Sci. Vol 11(2), June 2023 131 Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. (Table1). High bacterial load obtained is due to poor techniques of meat handling and non- hygienic practices used by the butchers. The variations of bacterial load observed in different sample locations were attributed to the hygiene standard in the processing and handling of meat. The result of the present study is in conformity with the findings of Joseph et al (19), Atlabachew et al (20) , Gufe et al. (14), Egwu et al. (21) , Ogunlade et al. (22) and Olawale et al. (23), who contended that the presence of bacteria in high densities obviously constitute a serious public health hazard as the presence of these microorganisms is associated with water borne diseases. The result from aquaculture samples shows that the sample point designated as PW 2 earthen pond shows the highest microbial load 7 (4.06±2.74x10 cfu/ml) and differs significantly at (P<0.05), it was immediately followed by PW 1 7 earthen pond (2.56±0.23 x10 cfu/ml), while samples from PW concrete ponds recorded the 4 7 lowest microbial load (0.40±0.04x10 cfu/ml) (Table 2). Higher microbial load obtained in earthen pond might be attributed to the use of animal manure in the fertilization of the pond. Pond water is known as an ideal culture medium for the proliferation of bacterial pathogens causing bacterial infection in fish and an important cause of food poisoning (24). High microbial load as obtained from our study corroborates work as reported by other authors (25, 26, 27). Untreated abattoir effluent constitutes potential reservoir for transmission of pathogenic strains of multiple antibiotic-resistant bacteria by pollution of surface and ground water sources (28). Antibiotics are widely used in intensive fish farming, which in turn increases the emergence of antimicrobial-resistant (AMR) bacteria in the aquatic environment (8). Antimicrobial r e s i s t a n c e t h r e a t e n s i n f e c t i o u s d i s e a s e management outcomes, especially in developing countries (29). E. coli isolates shows highest resistances to amoxicillin/clavulanic acid, cefixime, cefotaxime and tobramycin at 100 %, followed by meropenem and ceftazidime (Table 3). High resistance of antibiotics of human value as obtained in our study was previously reported by other authors both in Nigeria and elsewhere (Datok et al. (30) and Wu et al. 31). However, contrary report have been obtained from several authors (Mapanguy et al. 32, Aabed et al. 33, Montagnani et al. 34 and Isac et al 35) Multiple antibiotic resistance index (MARI) is an effective, valid, and cost-effective method that is used in source tracking of antibiotic resistant organisms (36). MARI is an important analysis to check antibiotic resistance and health risk factors (37). Organisms which have MAR indices of greater than ≥0.2 confirm the presence of multidrug-resistant genes originating from the environment where there is an abuse of these drugs and also that the plasmids contain one or more resistance genes, each encoding a single antibiotic resistance phenotype (38. 39). From the abattoir and aquaculture samples analyzed, E. coli MAR indices were greater than 0.2. It was an evidence that all the E. coli isolates originated from potentially dangerous sources where antibiotics are frequently used and possibly was introduced through fecal contamination. The result of the study agrees with work of Adinortey et al. (40); Afunwa et al., (37) and Kusunur et al. (41). The result from the study authenticated that there are high level of exposure to antibacterial agents in cat fish reared/sold within Abakaliki metropolis. The study implicated not just the presence highly pathogenic E. coli but also highly resistance pathogens in abattoir and aquaculture wastes. Hence wastewater from abattoir and aquaculture should be treated before discharge into soil and water bodies. Steps must be taken now in other to avoid a health crisis in the anticipated future. Funding Information The authors received no specific funding for this work. Conflict of Interest: The authors declare that they have no known competing Interest Acknowledgements: We wish to acknowledge the owners of the various fish ponds and abattoir workers for granting accesses to collect samples for the work. REFERENCES 1. 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Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. Res. J. Health Sci. Vol 11(2), June 2023 135 Table 1 Ó Microbial load (x107 cfu/ml) from abattoir samples Sample code Waste Water Drainage Butcher Table AB1 0.76±0.02a 1.86±0.28b 1.29±0.01b AB2 3.54±0.65a 1.31±0.27b 0.26±0.11c AB3 4.08±0.11a 1.60±0.66b 3.18±1.44c AB4 0.40±0.33a 2.20±0.28b 1.04±0.05c AB5 1.19±0.65a 1.32±0.04a 2.22±0.85b AB6 1.19±0.37a 1.50±0.98a 1.92±0.33a Key: AB= Abattoir, Values were mean ± standard deviation (SD), Values with no common superscripts within the same row are significantly different at p < 0.05, Values with same superscripts within same row are not significantly different (P > 0.05). Table 2Ó Microbial load (x107cfu/ml) from aquaculture environment Sample Code Concrete Pond Earthen Pond PW1 0.66±0.19 a 2.56±0.23b PW2 1.02±0.02a 4.06±2.74b PW3 1.50±0.25a 0.70±0.14b PW4 0.40±0.04a 1.50±0.09b PW5 1.92±0.03a 2.22±0.88b PW6 1.86±0.03a 1.29±0.01a Key: PW= Pond water, Values were mean ± standard deviation (SD), Values with no common superscripts within the same row are significantly different at p < 0.05, Values with same superscripts within same row are not significantly different (P > 0.05). Table 3: Antibiotics sensitivity pattern of the E. coli isolates Antibiotics Resistance (%) Intermediate (%) Susceptibility (%) IPM 2(25.0) 2(25.0) 4(50.0) AMC 8(100) 0(0) 0(0) CXM 8(100) 0(0) 0(0) CTX 8(100) 0(0) 0(0) TOB 8(100) 0(0) 0(0) FEP 3(37.5) 0(0) 5(62.5) MEM 6(75.0) 0(0) 2(25.0) CAZ 6(75.0) 0(0) 2(25.0) Key: IPM = Imipenem, CTX= Cefotaxime, CXM= Cefixime, FEP = Cefeprime, MEM = Meropenem, TOB = Tobramycin, CAZ = Ceftazidime, AMC= Amoxicillin/clavulanic acid. Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. Table 4: Multiple Antibiotics Resistance Index (MARI) of the E. coli Isolates Sample Sample Code MARI ANTIBIOTICS Drainage AB1 0.9 IPM, AMC, CTX, TOB, FEP, MEM, CAZ AB2 0.6 AMC, CTX, TOB, MEM, CAZ AB3 0.4 AMC, CTX, TOB AB4 0.6 AMC, CTX, TOB, MEM, CAZ AB5 0.5 AMC, CTX, TOB, MEM AB6 0.7 AMC, CTX, TOB, FEP, MEM, CAZ Waste Water AB1 0.7 AMC, CTX, TOB, FEP, MEM, CAZ AB2 0.5 AMC, CTX, TOB, CAZ AB3 0.7 AMC, CTX, TOB, FEP, MEM, CAZ AB4 0.7 AMC, CTX, TOB, FEP, MEM, CAZ AB5 0.7 AMC, CTX, TOB, FEP, MEM, CAZ AB6 0.7 AMC, CTX, TOB, FEP, MEM, CAZ Butchers Table AB1 0.5 AMC, CTX, TOB, CAZ AB2 0.5 AMC, CTX, TOB, MEM AB3 0.6 AMC, CTX, TOB, MEM, CAZ AB4 0.6 AMC, CTX, TOB, MEM, CAZ AB5 0.6 AMC, CTX, TOB, MEM, CAZ AB6 0.6 AMC, CTX, TOB, MEM, CAZ Concrete pond PW1 0.5 AMC, CTX, TOB, MEM PW2 0.6 AMC, CTX, TOB, MEM, CAZ PW3 0.6 AMC, CTX, TOB, MEM, CAZ PW4 0.6 AMC, CTX, TOB, FEP, CAZ PW5 0.7 AMC CTX, TOB, FEP, MEM, CAZ PW6 0.7 AMC, CTX, TOB, FEP, MEM, CAZ Earthen pond PW1 0.5 AMC, CTX, TOB, CAZ PW2 0.6 AMC, CTX, TOB, MEM, CAZ PW3 0.5 AMC, CTX, TOB, CAZ PW4 0.6 AMC, CTX, TOB, FEP, CAZ PW5 0.6 AMC, CTX, TOB, MEM, CAZ PW6 0.7 AMC, CTX, TOB, FEP, MEM, CAZ Mean 0.6 Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. Res. J. Health Sci. Vol 11(2), June 2023 136 Figure 1: Prevalence of E. coli across the different abattoir sample sources Figure 2: Prevalence of E. coli across the different aquaculture sample sources Antibiotic resistant Escherichia coli in abattoir and aquaculture environment Onuoha et al. Res. J. Health Sci. Vol 11(2), June 2023 137