Presence of Coliforms in Water, Poultry Mouth and Rectal Swabs from Selected Smallholder Poultry Farming Projects of Capricorn District, South Africa

Poultry production provides by products such as meat and eggs which are regarded as affordable sources of proteins and other important nutrients needed by the human body (Mack et al., 2005). The South African poultry industry continues to dominate the agricultural sector, providing 63.1% of animal protein in 2019 (SAPA, 2020). However, a high incidence of bacterial pathogens in poultry products was reported (Olobatoke et al., 2014; Zhang et al., 2018). Muvhali et al., (2017) reported high morbidity and poultry mortalities following outbreaks of Salmonella enteretidis in South Africa. The contributing factors to this demise could be feed and water resources utilised by poultry farmers which are often of compromised quality as these are low input production systems with limited biosecurity measures (Mwale and Masika, 2011). Water sources for poultry are generally open and there is usually no treatment at the point of consumption (Walters et al., 2014). Faecal shedding by food-producing animals is the leading source of contamination of water and the environment, whereas intestinal carriage often leads to contamination of carcasses at slaughter (Abraham et al., 2014). These circumstances expose poultry to various contaminating pathogens since contaminated feed and water provides an excellent media for the growth of microorganisms (Asiegbu et al., 2016). The pathogens are transmitted through the food chain to humans and cause foodborne illnesses (Chapman, 2013). Campylobacter jejuni, Escherichia coli, Salmonella spp, Listeria monocytogenes and Shigella spp thrive in poor hygienic conditions. These bacterial species are the most commonly isolated pathogens in the poultry industry of South Africa, where they are implicated in high incidences of morbidity and mortality among chickens (Rossaint et al., 2015; Asiegbu et al., 2016). Shonhiwa et al., (2017) reported the presence of other pathogens such as Bacillus cereus, Staphylococcus aureus and Escherichia coli O157:H7 from farm products. Contamination of feeds and drinking water with pathogenic microorganisms may lead to low product yield (Nyamongo and Okioma, 2005). Rouger et al., (2017) reported that microbiological metabolic activities may lead to spoilage and production of toxins and these pose a serious health risk to the chicken consumers. Furthermore, bacterial pathogens are becoming drug resistant (Ayukekbong et al., 2017). Antibiotic resistance is of great public health concern because the antibiotic-resistant bacteria associated with the animals may be pathogenic to humans, easily transmitted to humans via food chains and widely disseminated in the environment via animal wastes. These may cause complicated, untreatable and prolonged infections in humans, leading to higher healthcare cost and sometimes death (href="#williams-nguyen_2016">Williams-Nguyen et al., 2016 and Ayukekbong et al., 2017). The objective of the study was to identify the bacterial species present in the water supplies, mouths and recta of chickens and determine their antimicrobial resistance profiles. The findings contribute to the literature on the zoonotic risks associated with the consumption of poultry products in the study area which to the best of our knowledge has not been extensively explored. Information on the microbiological quality of feeds and knowledge of common contaminants is important for improved monitoring and designing of quality control measures in the handling of important raw materials in poultry production such as water and feedstock.

The study was conducted in randomly selected smallholder poultry farms during the period of July to December 2020. The farms were all located in the Capricorn district, Limpopo Province, South Africa (51.4818°N latitude, 7.2162°E longitude) co-ordinates 29°00'4.68" (Statistics South Africa, 2015). Laboratory consumables used in this study were from SIGMA ALDRICH® Company, South Africa. Seventy-four (74) samples comprising of water, mouth and rectal swab samples were collected from the farms. Fifty millilitres of water samples were collected from the same bulk tank in duplicates and from the drinkers within each poultry house into 50ml sterile bottles. Mouth and rectal samples were collected from 10% of the randomly selected chickens. Collected samples were transported on ice to the University of Limpopo, Microbiology laboratory for analysis within three hours of collection.

Samples were cultured on the following selective media that were prepared according to the manufacturer’s instructions: MacConkey agar (SIGMA-ALDRICH®) was used for the isolation of Gram-negative enteric bacteria. Water samples were prepared, processed and analysed for bacterial contamination according to the ISO 6222:1999. Escherichia coli and other coliform bacteria were analysed according to ISO 9308-1:2000. To process the swab samples, each swab was rolled onto the total agar surface of MacConkey media and incubated according to the manufacturer’s instruction. The cotton bud of the swab was then cut and inoculated into the tubes containing the Rappaport Vassiliadis broth. The plates that had evidence of colony growth were observed, selected and counted. Enumeration was performed manually under white light on the media plates that contained total colony counts of 30-300. All the media cultures including the broth culture colonies were sub-cultured and Gram stained to check for purity and cellular characteristics of the isolates. The isolates were sub-cultured on MacConkey media for subsequent identification assay (Szakal, 2003).

The bacterial isolates were identified with MALDI-TOF MS through a modified bio- typing protocol that was provided by the manufacturer (Bruker Daltonics, Maldi Biotyper). A rapid, on-plate method that requires less time and reagents for its performance was adopted in this study (Matsuda et al., 2012; Rodriguez-Sanchez et al., 2014).  Antibiotic sensitivity assays for the isolated bacteria were performed using nine different antibiotics of varying strengths presented on discs (MASTIDISCSTM) following the Kirby Bauer disk diffusion method (Bauer et al., 1966). The antibiotics discs used include streptomycin (S₁₀), ampicillin (AP₂₅), penicillin (PG₁₀), tetracycline (T₃₀), nalidixic acid (NA₃₀), erythromycin (E₁₅), neomycin (NE₃₀), ciprofloxacin (CIP₅) and gentamycin (GM₁₀). The results were interpreted as susceptible, intermediate, or resistant according to the zone diameter interpretative standards suggested by the Clinical and Laboratory Standards Institute (CLSI, 2001). The multiple antibiotic resistance (MAR) index of each strain was also detected using the equation provided by Singh et al., (2010) as follows:
 
 

Statistical Package for Social Sciences (IBM SPSS, 2019) version 26.0 was used for data analysis. Descriptive statistics such as percentages were computed for bacterial percentage resistance to antibiotics and the average number of colonies counted from water samples. 

The current study adopted MALDI-TOF MS technique using the simplified on plate method for microbial analysis of the samples. MALDI-TOF MS results revealed that all sampled farms were contaminated with the targeted microorganisms (Escherichia coli and Klebsiella spp) at varying levels (Table 1). Escherichia coli was found to be in abundance and positive to 88% of the sampled farms. Klebsiella spp tested positive in 38% of the farms. Bacterial contaminations from culture source (mouth, rectum and water) showed similar trend to farm contamination where E. coli tested positive to all culture source. Mouth swabs tested highly positive followed by rectum swabs and water resources were the least contaminated. Great variation in resistance to antibiotics tested was observed with respect to the water, poultry mouth and rectal swabs. The highest resistance occurred in rectal samples from farm code RAS with 96% resistance (Table 2) and the lowest resistance was observed in water samples from farm codes RAM and MAU with 44% resistance (Table 3).







Coliform bacteria are often referred to as “indicator organisms” because they indicate the potential presence of disease-causing bacteria in water. The presence of coliform bacteria in water indicates that a contamination pathway exists between a source of bacteria (surface water, septic system, animal waste, etc.) and the water supply. Our study revealed 18% occurrence of E. coli in collected samples. The detection level of E. coli reported in the presented study is higher compared to 13% reported by Strongmberg et al., (2017). The current study reported an occurrence of 13.3% of E. coli in poultry rectal swabs, however, Nyabundi et al., (2017) reported an occurrence rate of 3.6% in poultry rectal swabs. Obeng et al., (2012) reported a 10% occurrence rate of E. coli isolates from faeces of commercial egg layers. Varying isolation methods, classification methods, geographic locations and management practices are the possible reasons for the differences in frequency of occurrence between these studies. In addition, direct transmission of the bacteria from humans as well as differences in contamination levels of poultry feeds may be used to justify these observations.

Differences in E. coli contamination were observed between the farms. These differences may be linked to either environmental factors or the equipment being used in the farms (Voidarou et al., 2011). The type of bacteria and their loads depend on the initial bacterial contamination and proliferation of the bacterial pathogens might be aggravated by poor hygiene (Brightwell et al., 2007). Ribot et al. (2006) reported that E. coli is distributed among poultry of all ages. Escherichia coli in poultry production facilities may be introduced by air or faecal contamination or by any other contaminated substances to the water resources. On the other hand, the presence of Klebsiella spp. could be attributed to the exposure of the poultry drinkers and harvested rain to the unsanitary environment which allowed such organisms to proliferate and contaminate the water resources (Tzouvelekis et al., 2012; Berendonk et al., 2015).

The results of the present study agree with the reports by Klein (2018), that E. coli and K. oxytoca are commonly isolated bacteria in environmental polluted water. Bunkova et al., (2010) isolated Escherichia coli, Klebsiella oxytoca and Klebsiella pneumonia from poultry carcasses. Agapi et al., (2012) on spoilage microbiota associated with the storage of raw meat in different conditions, also isolated Salmonella spp and K. pneumoniae, which contribute to poultry foodborne outbreaks and poultry carcasses spoilage. These isolates are similar to the isolates obtained in the current study. K. pneumoniae has been proposed as a model organism for poultry diseases due to its presence in both the environment and in animal guts and in the development and spread of resistance (Tzouvelekis et al., 2012; Berendonk et al., 2015). The results of this study indicate poor microbiological quality of the drinking water that is supplied to the chickens. The isolates from the mouth and rectal swabs were different from the isolates from the water. The epidemiology and ecology of Klebsiella spp. and E. coli O157 suggest faecal contamination of feed or water may be a possible source of exposure of different microorganisms in poultry convectional houses and water resources. In the present study, rectal swabs showed higher contamination levels compared to other samples. This agrees with the report by (Adegunloye, 2006) that poultry faeces promote a significant growth of foodborne pathogens. The authors concluded that this prompt several foodborne diseases outbreak that result in devastating effects of mortalities in broilers. The environment of the poultry house can act as reservoir for pathogens (Gast, 2007). Magwedere et al., (2015) isolated several bacterial spp., including Escherichia spp and Klebsiella spp., in poultry feeds and water resources which are considered to be brought by faecal contamination in feeds and water. High levels of bacteria in drinking water have been shown to negatively impact productivity in poultry (Derouchey et al., 2004).

Amit et al., (2017) reported a high resistance of more than 70% of E. coli isolates to common antibiotics such as co-trimoxazole, ampicillin, penicillin and tetracycline. The study also reported a low resistance percentage of less than 30% to aminoglycosides such as ampicillin, amikacin and gentamicin. This antimicrobial resistance results affirms similarity to the results obtained in our study and support the reports from previous studies that there is high prevalence of bacterial resistant strains in poultry environments (Furtula et al., 2013 and Laube et al., 2014). Resistant bacteria proliferate and can also be transferred to humans through several routes such as direct contact of handlers, live animals and carcasses at poultry farms and slaughter houses. From the poultry water resources, the highest resistance was reported for Escherichia coli with 78% resistance to neomycin, penicillin, tetracycline, nalidixic acid, ciprofloxacin and ampicillin. Similar findings were reported in literature (Makhol et al., 2011).

Antibiotic resistance is of great public health concern, because the antibiotic resistant bacteria associated with the animals are mostly pathogenic to humans (Economou and Gousia, 2015; Friedman et al., 2016). According to Friedman et al., (2016), bacterial resistant strains are easily transmitted to humans via food chains and widely disseminated in the environment via animal wastes. The rising level of antibiotic-resistant bacterial pathogens hampers future treatment and prevention of infectious diseases in both animals and humans (Vincent et al., 2016). Marshall et al., (2011) also confirmed that the application of antimicrobials in animals, particularly in food animals, may leads to a development of resistant strains of bacteria, which propagates to infect both animals and man. Different resistance patterns of Escherichia coli to antibiotics reported in the current study are consistent with previous related studies on antimicrobial resistance (Kariuki and Dougan, 2014; Abbassi et al., 2017). Similar to the current study, studies have reported resistance to Neomycin, penicillin and co-trimoxazole (Nyabundi et al., 2017).

The current study revealed the presence of coliform bacteria in water, poultry rectal and mouth swabs confirming that chickens raised in the study area are susceptible to infection by this class of bacteria. This may partially be attributed to their rearing systems, especially the deep litter system which allows enteric microbial contamination within the poultry houses.