ABSTRACT
Integrated livestock-fish aquaculture utilizes animal excreta, urine, and feed leftovers as pond fertilizers to enhance the growth of plankton and other microorganisms eaten by the fish. However, antimicrobial-resistant bacteria may be transferred and develop in the pond due to selective pressure from antimicrobials present in animal feed, urine, and feces. In an experimental pig-fish farm located in periurban Hanoi, Vietnam, nine piglets were provided feed containing 5 μg of tetracycline (TET)/kg pig weight/day and 0.45 μg of enrofloxacin (ENR)/kg pig weight/day during the second and fourth (last) months of the experiment. The aim of this study was to determine the association between the provision of pig feed with antimicrobials and the development of antimicrobial resistance, as measured in a total of 520 Escherichia coli and 634 Enterococcus strains isolated from pig manure and water-sediment pond samples. MIC values for nalidixic acid (NAL) and ENR showed that E. coli and Enterococcus spp. overall exhibited significant higher frequencies of resistance toward NAL and ENR during the 2 months when pigs were administered feed with antimicrobials, with frequencies reaching 60 to 80% in both water-sediment and manure samples. TET resistance for both indicators was high (>80%) throughout the study period, which indicates that TET-resistant E. coli and Enterococcus spp. were present in the piglets before the initiation of the experiment. PCR-based identification showed similar relative occurrences of Enterococcus faecium, Enterococcus faecalis, and other Enterococcus spp. in the water-sediment and manure samples, suggesting that Enterococcus spp. isolated in the ponds originated mainly from the pig manure. The development of antimicrobial resistance in integrated animal husbandry-fish farms and possible transfers and the impact of such resistance on food safety and human health should be further assessed.
INTRODUCTION
Antimicrobial resistance has been recognized as an important global health problem over the past few decades (WHO, 2001). The main contributors to resistance are overuse of antibiotics in humans and animals, poor hygiene and sanitation, and inefficient prevention and control of infection in healthcare settings. Antimicrobial resistance has enormous health and economic impacts. Agriculture affects human health through both the consumption and production of food for the human diet. Manure from pig and cattle farms is commonly used as a substitute for inorganic nitrogen and phosphorus fertilizers for agricultural crops worldwide, especially in organic farming practices (Zilles JL, 2010). With the increasing consumer demand for organically produced food, the use of animal manure, which conforms to organic conventions, will likely increase in the future. According to the National Organic Program, raw manure may be used up to 90–120 d before harvest, depending on the crop, and composted manure may be applied at any time. There are no restrictions on the source of manure ( Federal Register 246 (2000).
Animal manure is an important reservoir of antibiotic-resistant bacteria, antibiotic-resistance genes (collectively known as the “resistome”), and pathogens (Heuer H, et al,2007). Although antibiotic use increases antibiotic-resistance genes and resistant bacteria in manure (Durso LM , et al, 2011) antibiotic-resistant bacteria are also abundant in manure from animals with no history of antibiotic treatment, indicating the natural presence of bacteria intrinsically resistant to antibiotics in animal gastrointestinal tracts ( D’Costa VM, et al.2011)
There is increasing concern about the use of manure as an agricultural amendment because of its possible contribution to the pool of resistance genes to resident soil bacteria and pathogens (2, 19). Antibiotic-resistance genes from the soil resistome can enter the food chain via contaminated crops or groundwater (5, 20), and have potential consequences for human health if transferred to human pathogens. Studies assessing the impact of fertilization with pig manure on the soil resistome have shown that excessive application of manure from farms with intensive sulfonamide use can lead to an increase of antibiotic-resistance genes in soil (2, 3); however, most studies have found that such increases are transient when the manure is applied at recommended rates (2, 21, 22). Cow manure from dairy farms, which use β-lactam antibiotics predominantly to prevent and treat diseases (23), is commonly used in crop production, but its impact on the soil resistome has yet to be investigated.
Along with its impact on the soil resistome, the application of manure can affect the composition and functional properties of soil microbial communities, as has been demonstrated by community fingerprinting (21, 24). Recent advances in DNA-based analysis, such as metagenomics and quantitative PCR (qPCR), offer greater precision in such studies, enabling identification of affected community members (25) and their resistance genes (4).
In the present study, we assessed the impact of pig dung on the composition and resistance profiles of bacterial communities in soil. Our results show that pig dung, contain microorganisms that are resistant to the antimicrobial drug.
Analysis of the estimated health burden of antimicrobial resistance in Thailand from 2009 to 2010 revealed that at least 90 000 patients per year were hospitalized with infections caused by antimicrobial-resistant bacteria and approximately 30 000 of these patients died.2
Furthermore, antimicrobial-resistant bacterial infections resulted in at least 3 million excess days of hospitalization.2 The cost of antibiotics to treat these infections was US$200 million, resulting in a total cost of US$1.3 billion.2 Antimicrobial resistance is more prevalent in low- and middle-income countries, especially those in South and Southeast Asia.3 It is well recognized that antimicrobial-resistant pathogens are a common cause of hospital-acquired infections; however, an increasing number of community-onset extended-spectrum beta-lactamase (ESBL)-producing bacterial infections, especially those caused by ESBL-Enterobacteriaceae, have been reported in many countries, including Thailand.4–9 Risk factors for acquiring community-onset ESBL-producing Escherichia coli infections in Thailand include prior colonization with the bacterium and previous exposure to third-generation cephalosporins and fluoroquinolones.9 Fecal carriage of ESBL-producing enterobacteria has increased significantly worldwide, with developing countries (especially in South and Southeast Asia) being the most affected. In these isolates, CTX-M enzymes are the dominant type of ESBL.10 The prevalence of CTX-M-type ESBL-producing Enterobacteriaceae in feces of healthy individuals in three provinces of Thailand ranged from 29.3 to 76.2%.11–13 Risk factors associated with fecal carriage of these bacteria included the use of antibiotics without prescription, history of hospitalization, and the use of antibiotics within the last 3 months.11,12 The prevalence of colonization with ESBL-producing Enterobacteriaceae in the gastrointestinal tract of travelers from Sweden, the Netherlands, and Australia was 2.4–8.6% prior to travel, but increased to 30–49% following travel to Southeast Asia, India, and/or China.13–16 Risk factors in these cases included traveling to the Indian subcontinent and Asia, and taking antibiotics while traveling.13–16 Traveler’s diarrhea associated with ESBL-producing pathogens was also common among patients returning from India, Egypt, and Thailand.17 The aforementioned information indicated that the travelers probably acquired ESBL-producing Enterobacteriaceae from foods, water, and/or the environment in these Asian countries.
Antibiotic-resistant bacteria, including ESBL-producing Enterobacteriaceae, have been detected in food animals, meats, water, and the environment in many Asian countries, including China, Hong Kong, India, Bangladesh, and Malaysia, and could be reservoirs for colonization and infection of human beings.18–28 There are several previous microbiological surveys of food animals on farms, fresh meat samples from slaughterhouses and retail stores, exported fresh meat samples and vegetables, and stool samples from both healthy individuals and hospital patients from Thailand and several other countries.29–41 Almost all of these reports emphasized the ability of foodborne bacteria in causing gastrointestinal infections, especially Salmonella, Campylobacter, and Arcobacter species, though only a few studies described antibiotic-resistant E. coli. Many Salmonella species were isolated from these samples, most of which were resistant to antibiotics. Associations of antibiotic-resistant foodborne bacteria with food animals, fresh meats sold at retail stores, healthy Thais, and Thai and international patients were observed. Contamination with E. coli in chicken meat obtained from supermarkets in Bangkok during 2010–2011 was 53%.39 Some isolates of E. coli were resistant to ampicillin, cephalothin, gentamicin, co-trimoxazole, tetracycline, and/or ciprofloxacin, but all isolates were susceptible to ceftriaxone and carbapenems.39 A study of 240 E. coli strains isolated from Thai patients and healthy adults revealed that some E. coli isolates produced ESBLs and shared the same antibiotic resistance genes.41 The principal objective of our study focused on surveying foods along the production chain from farms to consumers and healthy individuals, including those who had direct contact with food animals, to determine the prevalence of important antibiotic-resistant bacteria that usually cause systemic infections in humans, especially ESBL-producing E. coli.
Aim of study
The overall objective is to determine the different types of antibiotics and their handling practices in pig production in Anambra state and the prevalence of resistance microorganisms from the pig dung.
SPECIFIC OBJECTIVES
1.To identify main sources and types of antibiotics used for prophylaxis and
treatment of infections by pig farmers in Anambra region Region.
- To assess the antibiotic handling practices of the pig farmers.
- To detect the resistant organisms in the pig dung.
- To evaluate the susceptibilities of isolated enterobacteria (E. coli, Enterobacter, Salmonellae, and Proteus vulgaris) to different antibiotics.
- to evaluate resistant of isolated enterobacteria (E. coli, Enterobacter, Salmonellae, and Proteus vulgaris) to different antibiotics.
