Abstract

Background: Rising global concern about antimicrobial resistance (AMR) has drawn attention to the use of antibiotics in livestock. This research was aimed at isolation and determination of the total coliform bacteria, characterization of the bacterial isolates, screening for multidrug resistant bacteria and the resistant genes from faecal samples of pig farms in Anambra State. Methods: A total of 400 pig faecal samples collected from 40 farms in three senatorial zones of Anambra State were subjected to microbiological analysis and the total coliform bacteria determined. Enteric bacteria from the faecal samples were isolated and identified based on their morphological and biochemical characteristics. Susceptibility of the isolates to different antibiotics were carried out using the standard Kirby‐Bauer disc diffusion method and isolates resistant to cephalosporins were further subjected to Double Disc Synergy Test (DDST) for the phenotypical detection of extended spectrum beta-lactamase (ESBL) production. The ESBL producers were subjected to molecular studies for the detection of ESBL genes using PCR protocols. Results: Mean Total coliform counts of the bacteria from pig faecal samples varied in the 3 senatorial zones. Gram-negative bacteria from the pig faecal samples include the genera Escherichia, Klebsiella, Citrobacter, Salmonella, Enterobacter and Proteus. There is a significant difference in resistance of the isolates to cefotaxime (p = 0.025) and streptomycin (p = 0.012) but no significant difference (p > 0.05) was observed on the rest of the antibiotics tested. Streptomycin (60%) was the most highly resisted while Imipenem (4%) was the least antibiotics. 54.6% of all the organisms were multidrug resistant while 43.7% were ESBL producers. Bla_CTX was present in ESBL-producing isolates. Conclusion: There is presence of pathogenic enteric organisms in pig faecal samples harbouring antimicrobial resistant genes. Prudent use of antibiotics in pig farms in Anambra State is therefore recommended to reduce the spread of antibiotic resistance.

1. Introduction

The pig industry in Nigeria is an important arm of the livestock sub-sector, and pig production has been ticked as a panacea to protein inadequacy. Pigs have high fecundity, high feed conversion efficiency, early maturity, short generation interval and relatively small space requirement ¹. The indigenous pigs have been recommended as a good alternative source of cheap, high quality animal protein that suits escalating human population ². The modes of productions of pigs are intensive, due to the rapidly increasing demand for livestock products necessitating the uncontrolled use of antimicrobials in pig farming ³. Pig farmers use antibiotics for treatment, metaphylaxis, prophylaxis and growth promotion in their farm animals ^{4, 5}. Global antimicrobial consumption of livestock is predicted to increase by two-thirds over the next 10 years. Within this sector, antimicrobial consumption is estimated to be highest in pigs, compared with chicken and cattle ⁶. In pigs, antibiotics can be applied to whole groups either by mixing antibiotics into feed (medicated feed) or adding antibiotic powder or solution into drinking water (medicated water) ⁷.

The antibiotics used in livestock fall into all the major classes of antibiotics used in clinical practice. Many of the antibiotics commonly used in animals are categorised as Critically Important Antimicrobials (CIA) for treating humans according to the WHO list of Antimicrobials for Human Medicine ⁸. Of particular concern has been the use of CIA, the last-resort antibiotics normally reserved for the most severe infections in humans, in livestock production. There is emerging evidence of the CIA resistance including a recent report describing the discovery of a plasmid-mediated colistin-resistant gene in commensal Escherichia coli from tests on pigs, pork products and humans in China ⁹.

Antibiotics administered to animals provide selective advantages for antibiotic resistant bacteria to develop in animal intestines, which end up in the manures and eventually in the environment ^{10, 11}. Antimicrobial resistance (AMR), one among the most common priority areas identified by both National and International agencies, is mushrooming as a silent pandemic ^{12, 13}. Global monitoring efforts on antibiotic consumption and antibiotic-resistant bacteria presently take place in clinical and public health laboratories, while they rarely focus on animal husbandry in most countries ¹⁴. Antimicrobial resistance among Escherichia coli and Enterobacteriaceae in general, isolated from both healthy and infected farm animals is common, and such pathogens constitute important public health hazard because they can transfer drug resistant genes through horizontal gene transfer to other pathogens, both of animal and human origin ¹⁵.

Extended Spectrum Beta- Lactamases (ESBL) are plasmid-borne genes which confer resistance to penicillins, cephalosporins and monobactams ¹⁶. The common ESBLs are of TEM, SHV, and CTX-M types, and are widely distributed among Enterobacteriaceae. The presence of ESBL-producing E. coli in food animals is a public health concern and high antimicrobial use (AMU) and inappropriate use of cephalosporins in animal production are considered to be linked with the emergence and high prevalence of ESBL-producing Escherichia coli (ESBL-E. coli) in animals ¹⁷. Transmission of ESBL genes from animals to humans can occur through food or direct contact ¹⁸.

Several studies have reported ESBL in pig farms where antibiotics are used in the animal production. ¹⁷ isolated ESBL- producing E. coli and reported that the most detected ESBL genes were bla_CTX-M-1, bla_TEM-52 and bla_CTX-M-14. ESBL-E. coli were detected in pig faecal samples collected from stable floors of 8 out of 10 farms in Spain ¹⁹. In a Danish study, ESBLs were detected in pigs on 15 out of 19 pig farms with high consumption of cephalosporins ²⁰. Of the one hundred and fourteen E. coli isolates recovered by ²¹ from pig and cattle farms, 72 (63.2%) isolates were positive for ESBLs with multiple resistance to the antibiotics tested. Due to the profitability of pig farming, there is a recent proliferation of pig farms in Anambra State. There is therefore need to evaluate antimicrobial resistance in these farms as a means of maintaining good farm practices and antimicrobial stewardship in the farms.

2. Materials and Methods

The study area of the pig farms is in Anambra State, Nigeria covering approximately 4416 km², and lies at Latitude of 6˚20'N and Longitude 7˚00'E ²². The farms were located in the three senatorial zones of Anambra State namely Anambra South, Anambra North and Anambra Central.

Forty of the randomly selected pig farms visited were made of either local mud bricks or concrete dwarf walls. They were divided into pens to enable separation of different categories of the pigs. The floors were either concrete floor or bare sandy floor, and water sources were boreholes or tanks filled by commercial water vendors. Feeding, medication and waste disposal methods were farm specific.

A total of 400 pig faecal samples were collected randomly from 40 farms sites from February, 2018- January, 2019, using sterile sample bottles. The sample bottles were labeled and transferred to Nnamdi Azikiwe University Microbiology Laboratory for processing within 24 h.

Mashed faecal sample (1.0g) was added into a test tube containing 9ml of 0.9% (w/v) saline water, 20% (v/v) glycerol, allowed to stand for 5mins and then stored at 4⁰C until required ²³.This served as stock culture.

The stock sample was diluted tenfold in phosphate buffered saline (PBS), pH 7.2 and 0.1ml of 10^-5 dilution plated onto sterile MacConkey agar (TM Media India) using spread plate technique and incubated at 37⁰C for 24h under aerobic condition. Duplicate plates were prepared. Developed colonies were counted and expressed as colony forming unit per gramme (cfu/g) of faecal samples. Candidate colonies were subcultured on sterile Nutrient agar medium.

The bacterial isolates were characterized based on their morphological and biochemical features following the methods described by ²⁴ and ²⁵. Biochemical tests carried out include Gram reaction, motility test, indole test, methyl red, Voges-Proskaeur test, citrate utilization test, catalase test, oxidase test and sugar fermentation tests. Selective and differential media like Eosin Methylene Blue Agar, Salmonella-Shigella Agar (Lab M), Sorbitol MacConkey Agar (Hi-Media), and Brain-heart infusion agar (BHIA) (TM Media India), were employed for further identification and confirmation of the isolates.

The antimicrobial susceptibility tests of the bacterial isolates were carried out following the methods described by Clinical and Laboratory Standards Institute ²⁶. The antibiotics discs (Oxoid Hampshire, UK) used include amoxicillin/clavulanic acid (30μg), cefotaxime (30μg), ceftazidime (30μg), ceftriaxone (30μg), streptomycin (10μg), gentamicin (10μg), tetracycline (30μg), ciprofloxacin (5μg), trimethoprim/sulfamethoxazole, (1.25/23.75 μg) and imipenem (10 μg). Susceptibility of the enteric bacterial isolates to these antibiotics was tested using the standard Kirby‐Bauer disc diffusion method. Two colonies of pure bacterial isolates were inoculated into test tubes containing 10ml of Mueller-Hinton broth and incubated for 18h at 37°C. The turbidity of the resulting bacterial suspension was adjusted to a 0.5 McFarland standard and 100μl of the suspension swabbed onto Mueller‐ Hinton agar plates using a sterile cotton swab stick. Antibiotic discs were placed on the plates, 20 mm apart using a sterile forcep, and the plates incubated aerobically at 37°C for 18h. The diameters of inhibition zones surrounding the antimicrobial discs were interpreted as resistant or susceptible according to the Clinical and Laboratory Standards Institute guidelines ²⁶. All tests were carried out in duplicates. Multidrug resistant (MDR) strains were identified as those resistant to at least three different classes of antibiotics.

Percentage susceptibility (PS) was calculated thus;

(1)

ESBL production by the isolates was detected phenotypically using the double disc synergy test (DDST) method described by ²⁷. Amoxicillin-clavulanic acid discs (30 µg) were aseptically placed at the centre of a Mueller-Hinton plate inoculated with the test bacterium. Ceftazidime (30 µg) and cefotaxime (30 µg) single antibiotic discs were each placed adjacent to the central disc (amoxicillin-clavulanic acid) at a distance of 15 mm from the central disc and 30mm from each other. The plates were incubated at 37^oC for 18 h. A ≥ 5 mm increase or difference in the inhibition zone diameter for either of the cephalosporins (ceftazidime and cefotaxime) tested in combination with amoxicillin-clavulanic acid compared to its zone when tested alone is confirmatory of ESBL production phenotypically. All tests were carried out in duplicates.

The presence of selected ESBL genes in five (5) of the resistant isolates namely: NGN4, NGN2(3p) NGN2(p) EFF4 and DFC4 was determined by multiplex PCR assays.

Genomic DNA of the isolates was extracted using Quick-DNA^TM Fungal/Bacterial Miniprep Kit (Zymo Research) according to protocols earlier described by ²⁸.

A 12.5μl of One Taq Quick-Load 2X Master Mix with Standard Buffer (New England Biolabs Inc.), 0.5μl each of forward and reverse primers, 8.5μl of Nuclease free water and 3μl of DNA template were used to prepare 25μl reaction volume of the PCR cocktail. The reaction mixture was gently transferred to an Eppendorf nexus gradient Master cycler (Germany) for the PCR process. The primers TEM, SHV, CTX, were used with amplification conditions as shown in Table 1. PCR products were separated on a 2% agarose gel and amplified bands were visualized alongside a 100bp DNA ladder with Ethidium bromide.

Data on bacterial count and antimicrobial resistance were analyzed for significant differences using one-way Analysis of Variance (ANOVA) by Student-Newman-Keul (SNK) test at 95% confidence level. IBM SPSS statistics version 20 was used for the ANOVA.

3. Results

The mean total coliform counts (TCC) obtained from the faecal samples of the pigs in the studied farms in the three senatorial zones are shown in Figure 1. The highest total coliform count was observed in Farm B in Anambra North while the least was obtained in Farm AH in Anambra South. Univariate analysis of variance of the total coliform counts showed a significant difference (p < 0.05) in the counts obtained in the senatorial zones.

Out of the 400 pig faecal samples from the forty farms in Anambra State, a total of 300 gram negative organisms were isolated and identified. They include Escherichia coli (140), Klebsiella pneumoniae (85), Citrobacter freundi (30), Salmonella enterica (17), Enterobacter cloacae (15) and Proteus vulgaris (13). Escherichia coli had the highest percentage occurrence (46%), while Proteus vulgaris had the least (4.3%).

Antimicrobial resistance patterns of the bacterial isolates from the pig faecal samples from the three senatorial zones are presented in Table 2. Imipenem (4%) was the least resisted antibiotics in all the senatorial zones studied at 4%, 5.5% and 2.73% for Anambra North, Anambra Central and Anambra South respectively. Amoxicillin (67%) was the most resisted antibiotics in Anambra North while Streptomycin was the most resisted antibiotics in Anambra Central (64.4%) and Anambra South (61.8%). A one way analysis of variance showed no significant difference (p = 0.354, 0.439, 0.192, 0.130, 0.501, 0.196, 0.808, 0.984) in resistance to imipenem, amoxicillin/clavulanic, ceftazidime, tetracycline, gentamicin, ceftriaxone, ciprofloxacin, trimethoprim/sulfamethoxazol respectively but significant difference (p = 0.012, 0.025) in resistance to streptomycin and cefotaxime respectively in the three senatorial zones studied.

Table 3 shows the percentage of bacterial isolates exhibiting multidrug resistance extended spectrum beta-lactamase. More than half of the Escherichia coli isolates were observed to be multidrug resistant. A total of 131 (43.7%) of the bacterial isolates were ESBL producers. E. coli was the highest ESBL producer while E. cloacae and P. vulgaris were the least. Plate 1 shows the ‘key hole’ effect characteristic of ESBL-producing isolates in the DDST. The appearance is due to the synergistic effect produced between amoxicillin-clavulanic acid (central disc) and third generation cephalosporins (ceftazidime and cefotaxime). PCR screening of the five phenotypically confirmed ESBL-producing isolates for bla_CTX-M, bla_TEM and bla_SHV genes showed that bla_CTX-M was present in 2 (DFC4 and EFF4) out of the 5 isolates screened (Plate 2B). Bla_TEM and Bla_SHV were not detected in the bacterial isolates (Plates 2C and 2D).

4. Discussion

Pig farming is presently gaining interest among livestock farmers and one of the challenges of pig farming is effective pig waste management so as not to constitute public health issues. The mean estimates of coliform counts varied in the three senatorial zones studied (Figure 1) and reason for the difference could not be adduced. Earlier studies also have reported mean total coliform counts of 3.84 x10⁴ cfu g-¹ from dairy cow manure ²⁹. and 4.2 x 10⁵ cfu g^-1 from piggery effluents ³⁰. Since animal manure has been reported to have a higher impact on the abundance and diversity of antibiotic resistance genes in soil than chemical fertilizers, it is likely that the coliform bacteria observed in this study may be harbouring antibiotic resistance genes ^{31, 32}.

The bacteriological analysis of the faecal samples revealed the presence of several Gram negative bacteria species. The bacterial organisms isolated is in line with the works of other researchers ^{33, 34, 35}, who reported the isolation of E. coli, K. pneumoniae, S. enterica and other enteric bacterial organisms from various pig farms. ³⁵, however, reported recovering Enterobacter asburiae and Providencia spp. which were not recovered in this study. Antimicrobial resistance is a growing public health threat worldwide, and members of the Enterobacteriaceae are among the clinically important bacteria that are rapidly developing resistance to available antibacterial agents ^{36, 37}. The organisms isolated in this study have been implicated in various disease conditions in human and livestock populations and in the hosting and exchange of resistance determinant ^{35, 38}.

The Gram negative bacterial isolates showed varying susceptibility and resistance to tested antibiotics [Table 2]. Although resistance to antibiotics is a natural phenomenon, their overuse and misuse in humans and animals have significantly escalated the antibiotic resistance levels ^{39, 40}. As suggested by ⁴¹, the resistance in the bacterial isolates observed in this study could be attributed to indiscriminate use of these antibiotics in piggery.

There was marked resistance to amoxicillin and streptomycin in the study zones. Similar studies in pig farms by ^{35, 42}, also observed resistance to amoxicillin, tetracycline, streptomycin ,trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin in Enterobacteria tested. The heavy use of these antibiotics in treatment of animal infection could have resulted in the development of resistance by organisms constantly exposed to the antibiotics. It has been reported that factors behind the emergence and spread of resistant bacteria are complex, and include misuse of antibiotics and co-selection, whereby using one antibiotic selects for resistance to other antibiotics ⁴³.

Imipenem (a carbapenem) was the least resisted antibiotics in this study similar to the report of ⁴², who studied E. coli from an intensive pig production system in South Africa. They observed high resistance to critically important antibiotics for human and animal use but low resistance to imipenem. Notwithstanding the low percentage resistance to carbapenem as recorded in this study, the emergence of carbapenem resistance is grave, as WHO classifies them as critically important antibiotics and to be used as the last-resort antibiotics for treating a wide range of infections caused by multidrug-resistant Gram-negative bacteria ⁴³.

Extensive use of antimicrobial agents, particularly β-lactams and tetracyclines, in pig production, have been reported ⁴⁴, to be responsible for the emergence of multidrug-resistant (MDR) enteric bacteria, and this may have contributed to the high multidrug resistance in bacterial isolates observed in this study. ^{42, 45} also reported the presence of multidrug resistant Escherichia coli from pigs farms and pock.

The most important mechanism of resistance to third-generation cephalosporins among members of the Enterobacteriaceae, particularly Escherichia coli and Klebsiella pneumoniae, is the production of ESBL ⁴⁶. In this study, 43.7% of all the isolates were ESBL producers (Table 3, Figure 1). Several authors have isolated ESBL- producing enteric bacteria not only from pigs but in other livestock ^{35, 43, 47}. Bla_CTX-M was present in two of the screened ESBL producers (DFC4 and EFF4) [Plate 2B] but bla_TEM and bla_SHV were not detected. This observation agrees with the work of ³⁵, who reported that bla_CTX-M was present in all but one of their ESBL-producers. ⁴⁸, also detected bla_CTX-M producing Klebsiella spp. and E. coli in their study and concluded that pig farm environment is a source of beta-lactamase producing bacterial organisms.

There is presence of Gram-negative bacteria in pig faecal samples from the various farms studied and the improper use of antibiotics in pig farming may have resulted in antimicrobial resistance observed in these isolates. The presence of plasmid-borne resistance genes in the bacterial isolates is of great concern, as that may facilitate the spread of antibiotic resistance in the environment when faecal samples containing these genes are used as manure in crop production. There is therefore need for an improvement in farm management in order check the spread of antibiotic resistance.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References