Antimicrobial Resistance of Escherichia coli and Salmonella isolated from Raw Retail Broiler Chickens in Zambia

Background Antimicrobial resistance (AMR) of foodborne pathogens is of public health concern, especially in developing countries like Zambia. This study was undertaken to determine the resistance profiles of Escherichia coli ( E. coli ) and Salmonella isolated from dressed broiler chickens purchased from open markets and supermarkets in Zambia. Results A total of 189 E. coli and five Salmonella isolates were isolated. Identification and confirmation of the isolates was done using Analytical Profile Index (API 20E) (Biomerieux ® ) and 16S rRNA sequencing. Antimicrobial susceptibility tests (AST) were performed using the Kirby Bauer disk diffusion technique using a panel of 10 different antibiotics and multiplex PCR was used to determine the presence of three target genes encoding for resistance: tetA, Sul1 and CTXM. AST results were entered and analyzed in WHONET 2018 software. A total of 189 E. coli and five Salmonella isolates were identified. Among the E. coli isolates, Tetracycline recorded the highest resistance of 79.4%, followed by Ampicillin 51.9%, Trimethoprim/Sulfamethoxazole 49.7%, Nalidixic Acid 24.3%, Chloramphenicol 16.4%, Cefotaxime 16.4%, Ciprofloxacin 10.1%, Colistin 7.4%, Amoxicillin/Clavulanic acid 6.9%, and Imipenem 1.1%. Two of the five Salmonella isolates were resistant to at least one antibiotic. Forty- seven (45.2%) of the isolates possessed at least one of the targeted resistance genes. study demonstrated the presence of AMR E. coli and Salmonella on raw from both

. Antibiotics are also used for growth promotion. However, if they are misused, they can lead to the development of resistance in organisms found in the chickens. In the absence of a national surveillance system on the use of antibiotics, it is impossible to know whether they are being used appropriately (WHO Global Report, 2014).
In monitoring development of AMR in bacteria, Escherichia coli (E. coli) is commonly used because it is part of the gut microbiota that have been shown to be a reservoir for antimicrobial resistance genes (Van Schaik, 2015;Yassin et al., 2017). Despite E. coli being an innocuous resident of the digestive system, it can also be pathogenic and cause severe intestinal and extra-intestinal diseases (Diarrassouba et al., 2007). It is estimated that every year, about 48 million people get sick from foodborne illness, the majority of these cases are hospitalized and some of these die (Torgerson et al., 2015). Pathogenic forms of E. coli such as Enterotoxigenic E. coli (ETEC) also cause significant diarrheal illness. It is the leading cause of travelers' diarrhea and other diarrheal illnesses in developing countries, especially among children (Nataro and Kaper, 1998).
E. coli infections can be treated using relevant antibiotics, but there is also accumulating evidence of the consequences of drug resistance. These consequences have a lot to do with the reduction in the efficiency of treatment with first-line drugs and limited choices after microbiological diagnosis (Clarke et al., 2012;Mshana et al, 2013).
Non-typhoidal Salmonella species are responsible for causing gastroenteritis and bacteremia, eventually leading to secondary infection. These bacteria are a problem in immune-compromised individuals such as patients with malignancy, human immunodeficiency virus, diabetes, and those receiving medication for anti-inflammatory diseases (Gordon, 2008).
In Zambia, recent findings showed that E. coli was among the most detected pathogens causing bacterial diarrheal disease in children between the ages of 0-59 months at the University Teaching Hospital (Chiyangi et al., 2017). This suggests that foodborne pathogens, poor hygiene and sanitation and other food safety risks such as the emergence of antimicrobial resistance (AMR) in foodborne pathogens are having a negative impact on public health (Mainda et al., 2015). There is currently limited data on AMR occurrence on food borne pathogens, in developing countries, including. Study of AMR in developing countries is important because information from such studies could enhance correct and controlled use of antibiotics in food production (Mshana et al, 2013;Chishimba et al., 2016).
This study, therefore, aimed at characterizing the phenotypes and genotypes of antimicrobial resistant E. coli and Salmonella in retail broiler chickens in Zambia.

Study Design
A cross-sectional study involving seven districts in Zambia that included Lusaka, Chilanga, Chongwe, Kafue, Choma, Kabwe and Kitwe was undertaken to investigate AMR E. coli and Salmonella in marketready broiler meat. The study was conducted between August 2017 and May 2018. Choma, Lusaka, Kabwe and Kitwe districts were purposely selected because as provincial headquarters, they are retail destinations for many poultry products from other districts while Chilanga, Chongwe and Kafue districts were included due to their proximity to Lusaka. The primary sampling units were the markets (broadly classified as Open markets and Supermarkets) and the secondary sampling units were individual dressed broiler chickens. An open market was defined as an unrestricted competitive market not housed in a building, where foodstuffs are sold often exposed and in which any buyer and seller was free to participate, while a supermarket was defined as a market housed in a closed building with modernized facilities. Proportion stratified random sampling was employed where Open markets and Supermarkets were the strata. For Lusaka province, from the information collected from

Sample size calculation
The sample size for estimation of a single proportion was calculated using Epi tools software (www.epitools.ausvet.com).The sample size was estimated using the following assumptions: assumed prevalence of AMR E. coli on dressed poultry =25%; confidence level=95%; level of precision=5%.
Using the above assumptions, the total number of supermarkets and open markets to be included in the study was calculated to be 63 markets, proportionally distributed as follows: 58.8% = Supermarkets; 41.2% = Open markets. For Lusaka districts, the estimated study population was supermarkets, all markets that were trading in dressed broiler chickens were included in the study, bringing the total number of markets to 92 markets (Table 6).

Samples from open markets
Forty-two open markets from Chilanga, Choma, Chongwe, Lusaka, Kafue, and Kitwe districts that had shops and/or tables trading in dressed broiler chickens were selected and included in the study. From each open market within Lusaka district that was sampled (n=24), three shops trading in dressed broiler chicken carcasses were selected and from each shop two-dressed broiler carcasses purchased (22×2×2=88). Also at the markets, three stands that traded in dressed broiler chickens, where available, were included in the study (one dressed broiler carcass from each stand/table). For other districts, all markets that traded in dressed broiler chickens were included in the study, bringing the total number of samples from open markets to 178.

Samples from the Supermarket
Fifty (50) supermarkets that traded in dressed broiler chickens from Chilanga, Choma, Chongwe, Lusaka, Kafue and Kitwe districts were selected. From each supermarket in all districts, a maximum of three different brands of dressed broiler chickens were sampled (one of each brand) were available. Therefore, the number of broiler carcasses from supermarkets in all supermarket in this study were 154.
Upon purchase, all samples were transported in a cooler box containing ice packs to the laboratory and processed within 8 hours. Laboratory isolation included a whole carcass rinse in buffered peptone water (Oxoid), pre-enrichment and subsequent incubation at 37 O C overnight that was done within eight hours in the laboratories within the area of sampling. Processing of pre-enriched broths was undertaken in the Public Health laboratory, School of Veterinary Medicine.

Laboratory Analysis
The methods proposed by The Food and Drug Administration's Bacteriological Analytical Manual (U.S. Food and Drug Administration, 2001) were used with a few modifications for the isolation of Salmonella and E. coli. Laboratory isolation included a whole carcass rinse in buffered peptone water (Oxoid) (Figure 1a-c), pre-enrichment and subsequent incubation at 37 O C overnight. During the carcass rinse technique, 450mL of sterile buffered peptone water was also poured into an empty bag that did not contain a carcass to act as a control. The rinsate was incubated overnight and later streaked onto MacConkey agar plate (Oxoid, UK) to ensure that the batch of bags was sterile and that the organisms isolated were indeed from the chicken carcasses and not the bags used for rinsing.
10µL of the incubated broth was then transferred to MacConkey agar (Oxoid UK) and resulting colonies were gram stained for detection of Gram-negative short rods, which were subsequently subcultured onto Eosin Methylene Blue (EMB) agar (Oxoid UK). Colonies that showed a metallic green sheen ( Figure 2) were subjected to biochemical tests for identification for E. coli isolates while 1ml of the incubated pre-enrichment broth was also transferred to Rappaport Vassiliadis (Oxoid UK) and later subcultured on Xylose-Lysine Deoxycholate agar (Oxoid UK). Pink and black colonies on XLD agar ( Figure 3) were then Gram stained and subjected to biochemical tests for identification of Salmonella using Analytical Profile Index (API 20E) (Biomerieux ® ). Further confirmation of the isolates was done using 16S rRNA sequencing (Weisburg et al., 1991). The Kirby-Bauer disk diffusion technique for AST was used on all confirmed Salmonella and E. coli isolates (Figure 4) using a panel of 10 different antibiotics (Kirby-bauer, 1961). The isolates were prepared by sub-culturing onto Blood agar (Oxoid UK) overnight at 37 o C. A Gram's stain was then done to identify the organisms and to check for purity. One or two colonies were then suspended in 4mL of 0.9% sodium chloride solution and their turbidity compared to that of a 0.5 McFarland's turbidity standard. An inoculum of the suspension was then spread on two Muellar Hinton agar (4 ml thickness) plates (Oxoid UK) until the entire surfaces of the plates were covered and 5 antibiotic wafers (Oxoid) placed on the surface of each of 2 plates using the applicator (Oxoid). Two plates were used for each isolate to accommodate the 10 antibiotics (Table 7) The list of antibiotics was prioritized based on the most frequently used in the poultry industry in Zambia and also based on the priority list by the WHO and OIE list of critical antibiotics (WHO, 2017). The plates were then incubated at 37 o C for 24hrs and the diameters of the zones of inhibition entered and analyzed in WHONET 2018 software.
Isolates that showed resistance to tetracyclines, sulphonamides and beta lactam antibiotics were then forwarded for molecular analysis that involved extraction of Deoxyribonucleic Acid (DNA) and checking for presence of target resistance genes. The process of DNA extraction involved the suspension of a few bacterial colonies in 100µL of nuclease free water and heating of the vials at 80 O C for 10 minutes. The suspension was then centrifuged at 60000G with temperature of 4°C for 3 minutes. Multiplex polymerase chain reaction (PCR) was performed to check presence of resistant genes of interest according to the method described by (Adesiji et al, (2014). The mastermix volumes and PCR reaction were as outlined in Table 8 and Table 9 below.
The target genes were selected based on the antimicrobial susceptibility results and the genes that were outlined as genes of importance for antimicrobial susceptibility. The 3 target genes were TetA (for tetracycline resistance), Sul1 (for sulfonamide resistance) and CtxM (for beta lactam resistance) ( Antimicrobial susceptibility A total of 189 E. coli and five Salmonella isolates were isolated and identified (Table 2). For E. coli isolates, Tetracycline, Trimethoprim-Sulfamethoxazole and Ampicillin recorded the highest resistance of 79.4% (n=150 isolates), 51.9% (n=98 isolates) and 49.7% (n=94 isolates), respectively (Table 3) while only Ampicillin and Tetracycline recorded resistance among the Salmonella isolates (Table 4).

E. coli isolates from broiler carcasses obtained from open markets had a higher resistance of 91.7%
(n=88) while supermarkets recorded 83.9% (n=78). The overall resistance for both the open markets and supermarkets was 88% (n=166). Four out of five Salmonella isolates were from supermarkets (n=4).

Determination of Antimicrobial resistance genes
One hundred and four isolates were analyzed for the presence of resistant genes and 45.2% (n=47) showed the presence of at least one of the targeted genes. The Beta-lactam gene (CtxM) was the most detected in most of the isolates from supermarkets while the Tetracycline gene (TetA) was the most detected in isolates from open markets (Table 5).

Discussion
The resistance to tetracyclines, sulfonamides and beta-lactam antibiotics was generally high. This could be attributed to the use of antibiotics in both livestock and humans which is not well regulated and is subject to misuse and abuse, especially among small poultry producers. In Zambia, there is poor regulation of veterinary drugs and antibiotics, whereby farmers are able to purchase antibiotics over the counter without a prescription (Mainda, 2016;Manyi-Loh et al., 2018). Further, the poor hygienic processing methods that are employed by small and medium scale producers may facilitate the contamination of the carcasses with AMR organisms. The handling of the carcasses during slaughter, rinsing, transportation and sale may all also introduce resistant organisms from humans and the environment. Broiler carcasses that originate from commercial abattoirs, however, may get contaminated mostly from the abattoir bench surfaces and intestines of the broilers during processing (Voidarou et al., 2011). Some isolates from carcasses from supermarkets were highly resistant to Nalidixic acid, in addition to Tetracycline and Trimethoprim/sulfamethoxazole. This could be as a result of the use of fluoroquinolone antibiotics such as Enrofloxacin and Ciprofloxacin during poultry production at commercial level, which are currently on the Zambian market. Similar trends have been noticed in other parts of the world (Donado-Godoy et al., 2012). Open markets that were surrounded by over-populated areas recorded the highest number of resistant isolates from dressed chickens.
These were also the areas where some of the broiler carcasses were sold mostly on tables and thus subject to contamination and proliferation of intrinsic bacteria in the absence of the cold chain.
Moreover, most traders in the open markets sourced their birds from small producers who probably abused or misused antibiotics (Apata, 2009). It has also been documented that Tetracycline and Sulfadimidine are among the most commonly used antibiotics for therapy, especially at small-scale production (Mainda et al., 2015). Over-time, farmers have learnt about these drugs and tend to selfprescribe whenever they have a disease situation when raising the birds. This overuse and misuse of antibiotics in livestock production has been reported to cause antimicrobial resistance (Lowe, 1982;Ngoma et al., 1993;Koluman and Dikici, 2013;Kalonda et al., 2015;Ayukekbong, Ntemgwa and Atabe, 2017). Of the three resistant genes that were targeted, the most detected was that of the beta-lactams (CtxM gene). The phenotypic and genotypic results of resistance profiles were similar, confirming the efficiency of the Kirby-bauer disk diffusion method. This implies that the beta-lactam gene of interest that was targeted is similar to the one that was found in other countries were similar studies were undertaken (Adesiji, Deekshit and Karunasagar, 2014;Chishimba et al., 2016;Ramachandran, Bhanumathi and Singh, 2017). Though the other two genes for resistance to sulfonamides and tetracyclines (Sul1 and TetA) were also detected, the detection rates were not as high as that of the beta-lactam gene of interest. These discrepancies could be attributed to differences in target sequences of the resistant genes that were being targeted.

Conclusions
Tetracyclines, beta-lactams, sulfonamide and fluoroquinolone antibiotics recorded the highest resistance. This could be attributed to both the overuse of these drugs for therapeutic reasons at both commercial and small-scale levels of production. The presence of these resistant organisms both in open markets and supermarkets is a major public health concern because this could lead to the spread of resistance to humans in households where these carcasses end up. The spread of this resistance to pathogenic bacteria is a major public health concern. There is need to regulate the use of these antibiotics during production. There is also need to do more molecular work that can give a complete understanding of the actual genes that are conferring resistance in Zambia. An understanding of the genes will be beneficial in the event that there is need for new drug formulation or combinations.