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Antimicrobial usage in pig production: Effects on Escherichia colivirulence profiles and antimicrobial resistance

Abstract
Antimicrobials (AM) are used for growth promotion and therapy in pig production. Its misuse has led to the development of resistant organisms. We evaluated Escherichia colivirulence genes, and compared phenotypic–genotypic antimicrobial resistance (AMR) patterns of faecal E. colifrom pigs receiving routine farm treatment without antimicrobial agents against pigs treated routinely with AM over 70 days. Recovered E. coliwere tested for AMR using disk diffusion and polymerase chain reaction. Virulence genes were detected in 24.8% of isolates from antimicrobial group and 43.5% from non-antimicrobial group (p= 0.002). The proportion of virulence genes heat-stable enterotoxins a & b (STa, STb), enteroaggregative heat stable enterotoxin 1 [EAST1] and Shiga toxin type 2e [Stx2e]) were 18.1%, 0.0%, 78.7% and 3.0% for antimicrobial group and 14.8%, 8.5%, 85.1% and 12.7% for non-antimicrobial groups, respectively. Resistance to oxytetracycline was most common (p= 0.03) in samples collected between days 10 and 21. Resistance shifted to amoxicillin on days 56–70, and trimethoprim resistance was observed throughout. Seventeen phenotypic AMR combinations were observed and eight were multidrug resistant. At least one tetracycline resistance gene was found in 63.9% of the isolates. tet (A) (23.3%) was most common in the antimicrobialgroup, whereas tet (B) (43.5%) was prevalent in the non-antimicrobial group. Usage or non-usage of antimicrobial agents in growing pigs does not preclude virulence genes development and other complex factors may be involved as previously described. Heavily used AM correspond to the degree of resistance and tetracycline resistance genes were detected during the growth phase.

Keywords: antimicrobial; Escherichia coli; microbial drug resistance; virulence.

Introduction
Escherichia coliis a major cause of diarrhoea in pigs (piglets and weaners) at different levels of intensity worldwide (Vu Khac et al. 2006). In piglets, E. colidiarrhoea may be followed by terminal septicaemia, which is an important cause of economic loss for pig producers globally (Toledo et al. 2012). The estimated pig population in South Africa as of 2010 was about 1.5 million (Meissner, Scholtz & Palmer 2013), while the population worldwide is about 1 billion. Pork serves as an important source of protein for human beings in developing countries or areas where pork consumption is not prohibited (Madzimure et al. 2012).
Diarrhoeagenic E. colipathovars involved in pig enteric infections include mainly enterotoxigenic E. coli(ETEC) encoding heat-stable enterotoxins a & b (STa, STb), enteroaggregative heat stable enterotoxin 1 [EAST1]) and/or heat-labile (LT) enterotoxins, causing secretory diarrhoea in newborn and weaned piglets (Gyles & Fairbrother 2010). In addition, Shiga toxin E. coli(STEC) strains encode the Shiga toxin type 2e (Stx2e) that causes oedema disease but not diarrhoea (MacLeod, Gyles & Wilcock 1991). Interestingly, some strains harbour both the Stx2e genes and enterotoxin genes capable of causing symptoms of both oedema disease and diarrhoea in the same animal (STEC/ETEC) (Barth, Schwanitz & Bauerfeind 2011). Many porcine ETEC andSTEC strains have fimbrial structures on their surface that like LT, STa and STb enterotoxins are usually plasmid mediated (Dubreuil, Isaacson & Schifferli 2016). These fimbriae are termed colonisation antigens and they enable the bacteria to colonise the epithelial surface of the pig’s small intestine, namely, F4 (K88), F5 (K99), F6 (P987), F18 and F41 usually found in pig ETEC (Blanco et al. 2006). Antimicrobial agents are frequently used in the treatment and control of these enteric infections in pigs.
A recent study has shown that administration of antimicrobial agents increases the riskof antimicrobial resistance (AMR) (Burow et al. 2014). Other factors like stress from temperature, crowding and management also seem to contribute to the occurrence of AMR in animals (Sørum & Sunde 2001). The commensal bacteria in animals may become a reservoir of resistance to genes for pathogenic bacteria. This may contaminate meat andmeat products meant for human consumption (Van den Bogaard & Stobberingh 2000). Recent reports have indicated that the prevalence of antimicrobial-resistant E. coliis on theincrease (Luppi et al. 2015; Toledo et al. 2012) and the infections caused by the resistant bacteria usually fail to respond to treatment by specific antimicrobial agents (Rice 2009). This may be associated with the increased proliferation of bacterial pathogens, re-infection rates, chronicity, opportunistic infections with resistant organisms and a reduced life span (Capita & Alonso-Calleja 2013).
Resistance to tetracycline determined phenotypically has been reported more frequentlyamong bacteria isolated from pigs than previously known (Tadesse 2012). The resistance is known to be inducible and occurs basically because of the acquisition of tetracycline (tet) or oxytetracycline (otr) genes (Roberts 2011) and many isolates from pigs have shown multidrug resistance genes located on plasmids (Lutz et al. 2011).
Escherichia coliinfections have been identified to be a challenge in the South African pig production industry (Fasina, Bwala & Madoroba 2015; Kanengoni et al. 2017). A recent study showed that the prevalence of ETEC, STEC and EAST1 and associated fimbrial genes in indigenous South African breeds was high (Mohlatlole et al. 2013), an outbreak of multidrug resistance coliceptisaemia in weanling pigs was reported (Ikwap et al. 2016) and an investigation on piglet mortality in a farm was characterised to be associated with STEC (Kanengoni et al. 2017). Treatment and control of disease outbreaks in the South African pig industry involves the use of antimicrobial agents (Henton et al. 2011). The purpose of this investigation was to determine the effect of antimicrobial treatment on the prevalence of virulence genes and AMR in intestinal E. coliin growing pigs.

MethodologyApprovals,

animal care and welfare

All pigs involved in the study were placed under a 24-hour monitoring programme conducted by the pig farm team (attendant and manager) for the duration of the study using the assessment and control of the severity of scientific procedures on laboratoryanimals scoring system and the guide to defining and implementing protocols for the welfare assessment of laboratory animals (Hawkins et al. 2011; Wallace et al. 1990). All piglets were housed in the farrowing unit with crates, creep area, heating lamps andunlimited access to the dam’s teats, creep feed and water ad libitum(Figure 1). A total of 4 out of 10 piglets were removed in the last 2 weeks of the study because of laboratory-confirmed colisepticaemia (oedema disease). For each animal to be removed by euthanasia (carbon dioxide asphyxiation in piglets or humane slaughter in weaners orgrowers) or sudden death, the humane endpoint was set with a Severity Index (SI) score of > 20 on the Laboratory Animal Science Association (LASA) Working Party Scale (Wallace et al. 1990) and/or a score of ≥6 on the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group Scale (Hawkins et al. 2011). No other unexpected occurrence was recorded in the course of the experiment. The situation that triggered the scores for removal includes pain incompatible with animal welfare like prostration, nervous manifestationthat affected normal movement and loss of the ability to ingest food for 24–48 hours.

 

 

Study design

A small-scale commercial pig farm was identified in the Gauteng province of South Africa, and two pregnant sows were monitored clinically and physiologically until the day of farrowing. Piglets (n= 10) were randomly selected (five from each sow together with their unselected litter mates) and placed into two groups. All 10 selected piglets were tagged into groups A (non-antimicrobial group: with five tagged piglets and other non-tagged litter mates that were kept in one farrowing pen under routine farm management practices but without any form of antimicrobial usage) and B (antimicrobial group: with five piglets and other non-tagged litter mates that were kept under the routine management practices of the farm, which included administration of multivitamins, deworming, tail docking, vaccination, provision of warmth and antimicrobial administration to the sick animals). Effort was made to ensure the prevention of cross-contamination from the environment and between the groups by leaving three farrowing pens vacant (5.4 m width) between the two groups ensuring caretakers attend to the non-antimicrobial group before the antimicrobial group daily.

Sample collection

Rectal swabs were taken from all 10 piglets (four swabs per animal at each collection) with a sterile swab stick and each swab labelled with the specific pig identification number and age (days), and transported to the Agricultural Research Council-Onderstepoort Veterinary Research Feed and Food Analysis Laboratory (Bacteriology section) on ice. All samples were processed in the laboratory within 2 h of sample collection. The samples were collected periodically on days 1, 5, 10, 21, 28, 35, 56 and 70 from all pigs.

Classical microbiological analysis

Escherichia coli isolation and antimicrobial resistance testing

The swabs were streaked directly on MacConkey agar (Oxoid, Basingstoke, United Kingdom [UK]) plates and incubated aerobically overnight at 37 °C. Lactose fermenting colonies (n= 4–6) were selected and sub-cultured on MacConkey agar. The pure colonies were then transferred to nutrient agar (Oxoid, Basingstoke, UK) plates. The isolates onnutrient agar plates were subjected to an indole test together with other biochemical reactions for E. coliidentification. For this purpose, 10 mL of tryptone water was inoculated with pure culture and incubated over night at 37 °C. Kovacs reagent (one to two drops) was added and the formation of a red ring was indicative of E. coli. In addition, an Indole test, Methyl red test, Voges-Proskauer test and a Citrate utilization test (IMViC) were also conducted. Subcultures were also cultured on 5% sheep blood agar to determine the haemolytic characteristics of the E. coli. Escherichia coliATCC 25922 and E. coliO157 were used as controls. Antimicrobial susceptibility testing was done using the Kirby–Bauer disk diffusion method. The zones were interpreted according to the Clinical Laboratory Standards Institute (CLSI)guidelines (Clinical Laboratory Standards Institute [CLSI] 2015).
The following antimicrobial agent discs were selected according to standard regulations (CLSI 2015; Food and Drug Administration 2012; World Health Organization 2016): amoxicillin (AML) 10 µg; cefotaxime (CTX) 30 µg; oxytetracycline (OT) 30 µg; kanamycin (K) 30 µg; florfenicol (FFC) 30 µg; enrofloxacin (ENR) 5 µg and trimethoprim (W) 5 µg (Table 1). The criteria that were used for selecting the antimicrobial agents are the use on the farm during pig production and the recommendations for testing of bacteria from animals (CLSI 2015). Escherichia coliisolates were considered to be multidrug resistant in cases of resistance to three or more classes of antimicrobial agents.

Molecular characterisation of Escherichia coli isolates

DNA extraction and amplification using polymerase chain reaction

DNA from E. coliisolates was obtained using the cell-lysis method by boiling at 95 °C for 20 minutes to lyse the bacteria.
Escherichia coliisolates were tested for virulence genes and tetracycline resistance genes, tet (A, B, C and E) using sets of forward and reverse primers (Table 2). For detection of enterotoxin genes, STa, STb and heat-labile toxin (LT), a multiplex polymerase chain reaction (PCR) assay (Cheng et al. 2005) was adapted using a total 25 µL of reaction volume including the PCR master mix (DreamTaqTM Green PCR Master Mix), 0.3µL of each primer, nuclease free PCR water (Fermentas) and 3 µL deoxyribonucleic acid (DNA). Deoxyribonucleic acid amplification was carried out using Eppendorf Thermocycler (Eppendorf, Hamburg, Germany) and the cycling conditions were initial

denaturation at 94 °C for 3 min, followed by 10 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C – 56 °C (1 °C decrease for every two cycles) for 30 s and extension at 72 °C for 1 min, followed by another 22 cycles of similar thermocycling conditions but annealing at 56 °C, and a final extension at 72 °C for 10 min.