Antimicrobial Resistance among Enterobacteriaceae Found in Chicken and Cow Droppings and Their Public Health Importance

Introduction: The recent surge in the number of antimicrobial resistant cases from hospitals and communities has created a need to study the points and sources of exposure to certain bacteria and determine their susceptibility to commonly used antibiotics. This study aimed at identifying and screening for drug-resistant Enterobacteriaceae isolated from chicken droppings and cow dungs in Onitsha, Anambra state, in the South-Eastern part of Nigeria. Methods: This is a cross-sectional descriptive study which included 50 chickens and 50 cow dung samples collected from five poultry houses and cow ranches respectively using sterile swab sticks. The samples were transported to the laboratory and processed following standard microbiological protocols. Isolates in the samples were recovered using MacConkey Agar, Eosin Methylene Blue Agar and Salmonella-Shigella Agar following standard microbiological pro-cedures and then identified/characterized biochemically using commercial API 20E identification kits following the standard manufacturer’s protocol. Isolates were subjected to antibiotic susceptibility testing on Muller Hinton Agar using Kirby Bauer double-disc diffusion technique. The multiple antibiotics resistance index was determined as well. Isolates with reduced susceptibility genes, P. penneri, P. mirabilis and C. braakii were recovered from chicken droppings, whereas 13/43 (30.2%) Enterobacteriaceae including K. pneumoniae, S. enteritica, S. odorifera, E. coli, K. intermediate, P. stuartii, E. aerogenes, P. penneri, P. mirabilis and C. braakii were recovered from cow dungs. Two (12.5%) different isolates demonstrated metallo-beta-lactamase and ce-phalosporinase (AmpC) production. The isolates were susceptible to six antibiotics tested except Augmentin and Nitrofurantoin where the resistance is 100% and 85% respectively while Ceftriaxone and Ofloxacin had the best antibacterial activity against the isolates from both sites. Conclusion: The bacteria of public health importance isolated from these sites and their antibiogram profile have shown the need for proper monitoring and management of animal wastes in order to mitigate the threat to human health in the spirit of One Health as well as contribute to the fight against antibiotic resistance.


Introduction
The Enterobacteriaceae, a family of aerobic and Gram-negative rods that naturally inhabit the intestinal tract of humans and animals, have been implicated in many human diseases. This family of bacteria has recently been experiencing a rise in incidence of resistance to antibiotics, as reported in many countries of the world, thus posing a bigger threat to healthcare delivery [1] [2]. They are particularly of clinical importance in the cause of nosocomial and community acquired bacterial infections. With the continued economic activities in poultry practice, cattle ranching and increased exposure of both crop fields and humans to antibiotic-resistant bacteria present in chicken and cow excreta, human contacts with enterobacterial infections are inevitable and have constituted a threat to public health [3].
More so, previous studies have attributed the irrational use of antibiotics in the practice of animal husbandry as the reason for the emergence and spread of resistant bacteria [3] [4]. Resistance of the Enterobacteriaceae to commonly used antibiotics in the last decade has been of an alarming proportion, causing increased public health concerns [5] [6]. The mechanisms of resistance to such antibiotics are usually through efflux pumps, enzyme modification of the antibiotic, selective pressure and antibiotic inactivation [4] [5] [6] [7]. Commonly used antibiotics are becoming less useful owing to resistance and most of the antibiotics considered as last resort are also becoming ineffective for the same reason [8].
Furthermore, carbapenems are β-lactam group of drugs that are currently used as antibiotics of last resort for treating infection because of the problem of multidrug-resistance especially among Gram-negative rods [2] [9] [10]. This re-sistance has largely been attributed to the production or acquisition of Carbapenemases among enterobacteriaceae family [11]. Originally, organisms belonging to the enterobacteriaceae family were susceptible to carbapenems, but this is no longer true due to the emergence of Carbapenem resistant enterobacteriaceae in the last couple of years and so posing a serious health concern [10]. The Center for Diseases Control and Prevention (CDC) in 2013 reported that Carbapenemresistant enterobacteriaceae (CRE), which emerged within the past two decades, among other multidrug-resistant organisms, have remained the major cause of untreatable and hard-to-treat infections among hospitalized patients, and are considered an urgent threat to human health [2] [10]. Detecting CRE early in human and animal hosts is highly recommended in controlling infections by them as well as their spread [2].
Consequently, contamination of food and food-producing animals with MDR bacteria harboring MBLs and AmpC enzymes could be a source of antibiotic resistance [12]. Over the last few decades, several extended-spectrum β-lactamases (ESBL) and AmpC-producing Enterobacteriaceae (EPE) have emerged in both human and animal health management globally, with the animals being touted as the transmission link of ESBLs/AmpCs for humans [10] [11] [13]. In addition to this, Ejikeugwu et al., reported AmpC producing Enterobacteriaceae, as well as MDR and production of MBL amongst Klebsiella spp isolated from cow anal swabs in studies carried out in Nigeria [12] [14]. Although some studies have been carried out in Nigeria at poultry and animal houses, yet paucity of data is available on antimicrobial resistance resulting from poultry droppings and cow dungs especially in South-Eastern Nigeria; thus, creating a research gap for this study.
In this study, we proposed the hypothesis that antibiotic resistance in Enterobacteriaceae recovered from both poultry droppings and cow dungs could be attributed to intrinsic genetic factors possessed by these organisms that enhance the production of MBL and AmpC enzymes. Hence, this study was aimed at identifying and screening for drug-resistant Enterobacteriaceae isolated from chicken droppings and cow dungs in Onitsha, Anambra state, in the South-Eastern part of Nigeria.

Study Setting
This is a cross-sectional descriptive study conducted from August, 2020 to April, 2021 at Nkwelle suburb, Onitsha North Local Government Area, Onitsha. Onitsha is a metropolitan city located near the River Niger, in Anambra state, South-Eastern of Nigeria. It lies within 6˚10'N 6˚47'E coordinates in a 36.12 km 2 landmass, and has a population of 561,066 [15].
The study included five different poultry and cow ranches with a capacity of 500 birds and 200 cows respectively. Simple random sampling was used to collect swabs of 50 chicken droppings and 50 cows dungs respectively. Information obtained from the farm attendants showed that, although, proper hygiene is Collection and transportation of fecal Samples Four heaped dessert-spoonful of fresh, warm dung pat on the ground samples were collected from adult cattle from 5 different areas where adult cows congregate. They were mixed and then a sterile swab stick was used to rob through the mixture.
Fifty (50) chicken and cow dung swab samples each, were collected from the poultry house and cow ranch in Nkwelle, Onitsha using sterile swab sticks and early in the morning before the attendants came for their routine cleaning and tending of the chicken and cows. Samples were collected by robbing a wet sterile swab stick on chicken and cow excreta dropped at different locations of the same poultry and ranch, for five different places. Each day, the collected samples were transported aseptically and processed within 2 hours of collection. The samples were processed at laboratory in Department of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Agulu of Nnamdi Azikiwe University, Awka.
Culture and purification of fecal Samples The chicken dropping and cow dung swab samples were each cultured in 5 ml double strength of nutrient broth (CM0003, Oxoid, UK) and incubated overnight at 30˚C. A loopful of the specimen was transferred aseptically onto Mac-Conkey agar (MAC) plates, Eosin Methylene Blue (EMB) agar, Cetrimide selective agar plates, and Salmonella-Shigella agar for the selective isolation of Klebsiella species, Escherichia coli and P. aeruginosa and Salmonella-Shigella species respectively and incubated at 30˚C for 18 -24 hours for phenotypic characterization. Phenotypically, Escherichia coli produces colonies with metallic green sheen on EMB agar and lactose-fermenting colonies on MAC; Klebsiella species produce small, circular, elevated and mucoid colony on MAC and non-metallic sheen mucoid colonies on EMB agar while P. aeruginosa isolates produce greenish pigmentation on Cetrimide selective agar [16].
Identification and confirmation of bacterial isolates using BioMerieux API 20E Kit After Gram staining and microscopic examination, the various isolates obtained were characterized biochemically using BioMerieux API-20E kit to further identify the organisms to species level. The pure cultures were further characterized using Analytical Profile Index (API) 20E test strip comprising of 20 micro-tubes seeded with dehydrated substrates for enzymes that are produced by Enterobacteriaceae family. To allow for some moisture during incubation, distilled water (5 ml) was spread onto the honey comb wells of the tray to ensure a humid environment in the incubation box before the strips were placed in the tray. Discrete colonies collected from 20 hours overnight culture plates were inoculated into 5 ml API 20E suspension medium, and emulsified to ensure a homogenous bacterial inoculum. Following the manufacturer's specifications, the preparations were carefully distributed into the tubes of the strips, ensuring that no air bubbles were left. The tube and cupule for GEL, VP and CIT tests were filled with the suspension while the inoculum was filled up to the tube in the rest of the test segments. To create an anaerobic environment for ADH, LDC, ODC, H2S and URE tests, the tubes were overlaid with mineral oil and the set up incubated for 24 hours at 37˚C. One drop each of TDA and James reagents was added in TDA and IND tubes respectively, one drop each of VP1 and VP2 was added in VP tube after the incubation period. The results were read and recorded after 10 minutes.
Antimicrobial Susceptibility Testing Antimicrobial Susceptibility testing was carried out on all the identified bac- Screening for Extended spectrum-β-lactamase production using ROSCO kits For the phenotypic detection of the various β-lactamases present in the strains, the double tablet synergy testing using subjective observations of synergy and the combination tablet method was used [13]. Attention was given to the differences in the zones of inhibition. The susceptibility tests were performed following the method M2A6 disc diffusion method on Mueller Hinton agar plate as recommended by the National Committee for Clinical Laboratory Standards [17]. A standardized (0.5 MacFrland) inoculum was swabbed onto the Mueller Hinton agar plate using sterile swabs and the discs were aseptically placed on the inoculated plates and pressed firmly onto the agar plate for complete contact while ensuring sufficient space between individual disc to allow for proper measurement of inhibition zones. The test isolates were tested against the following Screening for Amp-C β-lactamase production To test for Amp-C β-lactamase production, the test organisms were screened for presumptive AmpC production by testing their susceptibility to cefoxitin (30 μg) using Kirby Bauer disk diffusion. Following CLSI standard, isolates with an IZD of ≤ 18 mm were suspected to produce AmpC enzyme. Screening for Metallo-β-lactamase (MBL) production This was done by phenotypically screened for the production of MBL in the test isolates. Their susceptibility to imipenem (IPM), meropenem (MEM), and ertapenem (ETP) was also done according to the CLSI criteria. Test isolates with an inhibition zone diameter (IZD) of ≤23 mm were suspected to harbor MBL enzyme.
Determination of Multiple Antibiotic Resistance Index (MARI) The MARI was calculated using the formula: Number of antibiotics to which the isolates were resistant MARI Total number of antibiotics to which the isolates were subjected = The incidence of multidrug resistant isolates was calculated from the formula [15] Incidence of multidrug resistant isolates Number of isolates with MARI 0.3 100 Total number of Isolates ≥ = ×

Data Analysis
All the data collected was summarized and tabulated using Microsoft excel software 2016. The results were calculated using percentages and presented in tables, while the Multiple Antibiotic Resistance index (MAR index) was calculated for each isolate and tabulated in the result section.

Results
A total of 43 (69.4%) Enterobacteriaceae recovered from this study out of 62 (100%) Gram-negative bacteria (GNB) isolates obtained from this study. Table 1 shows the frequency of Gram-negative bacteria recovered from the samples collected from 40 (80%) chicken droppings and 22 (44%) cow dungs. K. pneumoniae and E. coli had the highest frequency in chicken droppings (15%), while K. pneumoniae and E. cloacae had the highest frequency in cow dung (18.1%). The Enterobacteriaceae recovered all showed to be lactose fermenters with pink colonies on MAC, whereas non-lactose fermenters showed pale colonies on MAC and were all negative for oxidase test. Characterization of the isolates biochemically using the API ® 20E kit identified the isolates accordingly (Table 1). Table 2 and Table 3 show the antibiogram profiles of the isolates to the tested antibiotics together with their respective MARI. All the isolates from chicken dropping, except Citrobacter braaki, were resistant to Augmentin while the isolates Serratia odorifera and Enterobacter cloacae were resistant to all the antibiotics tested ( Table 2). Ceftriaxone and Ofloxacin had the best antibacterial activity against the isolates from both sites. All the isolates from cow dungs were resistant to Augmentin while Shewanella putrefaciens was resistant to all the antibiotics tested ( Table 2). Table 2 and Table 3 show the multidrug resistant      profile of the isolates with most of the isolates showing resistance to three or more antibiotics (above 0.2) hence are considered as multi-antibiotics resistant strains. Table 4 shows the results of the screening tests for AmpC production among the 16 MDR isolates. From the test, Escherichia coli, Proteus penneri, Pantoea spp, Shewanella putrefaciens were positive for AmpC production. The results of the screening tests for Metallo-β-lactamase (MBL) production (Table   5) among the 16 multidrug-resistant isolates revealed that Serratia odorifera and Enterobacter cloacae were positive for Metallo-β-lactamase (MBL) production.

Discussion
The nonstop incidence of antimicrobial resistance caused by selective pressure, The evaluation of the antimicrobial resistance profiles of the recovered isolates under study is paramount as antibiotic-resistant bacteria in animal excreta are an emergent concern. The resistance profiles of all Enterobacteriaceae isolates were evaluated by exposure to eight antibiotics of four different classes for the phenotypic characterization of the isolates. These were chosen to represent the main antibacterial classes used in human medicine and livestock production in Nigeria. The resistance of all isolates from chicken dropping, except Citrobacter braaki, to Augmentin as well as the resistance of Serratia odorifera and Enterobacter cloacae to all the antibiotics tested can be attributed to the use of extended spectrum cephalosporins such as cefoxitin and cefotazim in livestock. The results of this research buttress the findings of [11] who reported that the presence of Carbapenemases producing Enterobacteriaceae in animals is becoming worrisome. These organisms, all of which are of public health importance, are in line with the report [2] who reported that Carbapenem-resistant Enterobacteriaceae are organisms of medical importance. As well as [18] in Southern China who recorded high level of Carbapenem-resistant Acinetobacter spp. from clinical infection and fecal survey samples in Portugal [3]. The antibiogram profiles of the bacterial isolates from both sites revealed that some of the bacterial isolates were highly resistant to commonly used agents although some of the isolates were sensitive to Ofloxacin and moderately sensitive to Ceftazidime. Most of the isolates were resistant to three or more antibiotic classes hence are considered as multi-antibiotics resistant strains. The results of isolates from chicken dropping show that only one isolate, Providencia stuartii, gave a MARI of <0.20 as it had a MARI of 0.13 with others having 0.20 and above while Ewingella americana and Providencia stuartii both had a MARI of 0.13 among organisms isolated from the cow dung. The multiple antibiotics resistance index (MARI) is a protocol used to explain the spread of bacteria resistance and resistant genes in any bacterial population [19] [20]. Generally, Multiple antibiotics resistance index above 0.20 means that bacteria isolates originating from such an environment has been exposed to indiscriminate use of several antibiotics in time past [19] [21]. The multi-drug resistance to the antibiotics of different classes observed in this study may be due to the increasing administration of quinolones to treat avian infections [3]. The unnecessary use of antibiotics for enhancement of growth and prevention of diseases in farm animals has impressed selective pressures that induce more resistance among bacteria in the community. From the sixteen isolates examined phenotypically for the production of metallo β-lactamase (MBL), two (12.5%) isolates were positive for the production of this enzyme. The production of MBL is this study is similar to a previous study conducted by Ejikeugwu et al. [12] in which MBL was detected in Klebsiella species isolated from cow anal swabs. Only two AmpC producing Enterobacteriaceae was detected when the isolates were phenotypically screened for the enzyme. This is similar to an earlier report by Ejikeugwu et al. [14] in which AmpC enzymes were significantly detected in the E. coli and Klebsiella species isolated from cow anal swabs from an abattoir in Abakaliki, Nigeria. These results illustrate that the AmpC and metallo β-lactamase (MBL) producing species isolated in this study are multidrug resistant. They also produce AmpC and metallo β-lactamase (MBL) en-zymes which allow them to be resistant to the 2 nd and 3 rd generation cephalosporins which are clinically used to manage and treat serious bacterial infections. Furthermore, all the isolates screened phenotypically for ESBL production showed negative, this is in contrast to the review done by Madec et al. [11] where a study conducted among 699 S. enterica isolates from 1152 retail chickens reported a 24.6% rate of ESBL producers in Shanghai, China. This current study is relevant and a springboard to the increasing prevalence of antibiotic resistance in the non-nosocomial environment such as abattoir. More so, it provides acceptance to the possible abuse and irrational use of antibiotics in animal husbandry and for other non-clinical purposes. Hitherto, fecal excrement of chicken and cows in constant contact with humans who are husbandry rearers, pose high risk of cross contamination and further affects the antimicrobial resistance status and ultimately, the public health standards of nations.
Study Limitations: The molecular characterization of the isolates to further confirm the identity of the isolates wasn't conducted at the time of this writing due to limited funding. Also, the genes responsible for drug resistance could not be identified for the same financial constraints.

Conclusions
The bacteria of public health importance isolated from these sites and their antibiogram profile have shown the need for proper monitoring and management of animal wastes in order to mitigate the threat to human health in the spirit of One Health as well as contribute to the fight against antibiotic resistance.
What is known about this topic? World over:  Resistance of the Enterobacteriaceae to commonly used antibiotics in the last decade has been of an alarming proportion, causing increased public health concerns.  The last few years have witnessed the proliferation of several extended-spectrum β-lactamases (ESBL) and AmpC-producing Enterobacteriaceae (EPE) in both human and animal health management globally.  Animals have been touted as the transmission link of ESBLs/AmpCs for humans and demanded urgent response. What this study adds This study has showed that:  There is the presence of resistant strains among enterobacteriaceae found in chicken and cow droppings.  The need for proper monitoring and management of animal wastes in order to mitigate the threat to human health in the spirit of One Health.  More evidence that animals could serve as the transmission link of ESBLs/ AmpCs in humans.
Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Agulu Anambra state Nigeria and the Physician Assistantship/Public Health Department of Central University, Accra, Ghana. We would like to acknowledge all members of our team and staff of the laboratory for their continuous commitment to our research efforts.

Conflicts of Interest
None to declare.

Funding
The study did not receive external funding; instead, it was self-funded.