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SUSANE - Sustainable, sanitary and efficient
management of animal manure for plant nutrition
Impact of medicated feed on the development of antimicrobial resistant bacteria in
integrated pig-fish farms in Vietnam.
1. Introduction
Use of animal manure as fertilizer of aquaculture ponds is practiced widely in Southeast Asia. Pigs are raised in houses on the edge of ponds and pig manure is discharged directly into the fish pond to support growth of algae and aquatic plants Aquaculture, including integrated systems, is of increasing economical importance in many provinces in Vietnam. Fish farms using untreated livestock wastes as fertilizers are widespread. Moreover, development and spread of antimicrobial resistance has become a health problem in Vietnam as well as globally; a problem that is impacted by human and non- human antimicrobial usage. Antimicrobials are commonly used as feed growth promoters to improve pig productivity. The pig growth promoters are also used in integrated pig-fish farms, and this use may also increase the antimicrobial resistant level of bacteria in the harvested fish products which are used for human consumption. Unfortunately, there are very few evaluations of the resistance level of bacteria in fish product in Vietnam. Therefore, the sanitary component of SUSANE has carried out studies to throw light on the risk for increasing resistance of bacteria to medicine due the use of antibiotics and other antimicrobials in the swine production. This Newsletter presents a study of the impact of integrated pig-fish farming on the development of antimicrobial resistant bacteria in the aquatic pond environment. In coming newsletters we will present results from an ongoing study of resistant bacteria in fish from these fish ponds. 2. Design of the experiment
Manure from pigs was added to fish ponds in a controlled experiment where pigs were fed antimicrobials as growth promoters added to the feed (Fig. 1, sampling periods 2 and 4), and the antimicrobial résistance of bacteria in the manure and mixed water sediment samples were examined. The results of this study was compared with the antimicrobial resistance of bacteria from manure and mixed water sediments from a trial where the manure used was collected from pigs feed commercial feed without antimicrobial (Fig. 1, sampling periods 1 and 3). Resistance of strains of E. coli and Enterococcus spp. isolated from the samples was examined. These bacteria were selected for the study because both are internationally used as indicators of fecal pollution and in surveillance programs of development of antimicrobial 3. Resistance of E. coli
Level of antim icrobials resistant E.coil strains isolated
Level of antimicrobials resistant E.coli strains
from pig feces sam ples
isolated from water sediment sample
Period using feed for pig w ith or w ithout antimicrobials Period using f eed f or pig w ith or w ithout antimicrobials Figure 1: Level of antimicrobials resistant E. coli strains isolated from manure samples (left) and
water sediment samples of fish pond (right) NAL: Nalidixix acid; ENR: Enrofloxacin; TET: Tetracyclin Period 1, 3: Using feed for pig without antimicrobials Period 2, 4: Adding tetracycline (5 µg/kg pig weight/day) and enrofloxacin (0.45 µg/kg pig weight/day) in to feed for pig During four month from February to April 2009, water sediment samples of fish pond and pig manure samples were obtained a total of 13 times. In total, 634 Enterococcus spp. and 520 E. coli were obtained from all above pig manure samples (MS) and mixed water sediment samples (WS) for antimicrobial resistance testing. A significant temporal increase in nalidixic acid (NAL) and enrofloxacin (ENR) resistance were found for E. coli isolates obtained from two sample types (WS: NAL from 12.9% to 73.3% and ENR from 65% to 93.8%; MS: NAL from 38.3% to 81.7% and ENR from 66.7% to 98.3%). Consequently, the using antimicrobials in fish farming may represent a route of transmission of antimicrobial resistant bacteria and antimicrobial resistance genes from livestock to humans, e.g from pig, and fish products. 4. Resistance of enterococci
E. faecium
E. faecalis
E. faecium
E. faecalis
Enterococcus spp.
Enterococcus spp.
Table 1: Prevalences of resistance to enrofloxacin of enterococci isolated from manure samples
and water sediment samples
The tetracycline resistance prevalence among Enterococci spp. was 100% in isolates recovered from both sample types. The resistance prevalence for other antimicrobials shown by Enterococci spp. varied and was associated with the use or non-use of ENR in the pig feed (ENR resistance varied between 0% to 19.4% in manure samples and was 0% to 16,7% in water-sediment samples (table 1)); the MIC value of NAL varied from 61 µg/ml to 256 Species typing of all Enterococci spp strains was done by PCR to determine species of strictly fecal (E. faecalis and E. faecium) i.e. from pigs as well as species originating from the “aquatic environment. Species from the pigs and from the aquatic environment may show and develop different patterns of antimicrobial resistance. A total of 43 E. faecalis, 62 E. faecium and 199 strains of other Enterococcus spp. were obtained from water sediment samples; In pig manure samples, a total of 78 E. faecalis, 48 E. faecium and 204 strains of other Enterococcus spp. were recovered. Further analyses will be carried out to determine the resistance development of E. faecalis and E. faecium strains and to compare their pato- and genotypes with similar species isolates recovered from human specimens. 5. Conclusion
The conclusion of the project is that the use of antimicrobials as feed growth promoters for pigs raised in integrated pig- fish farming systems increases the prevalence of antimicrobial resistant bacteria in the pig manure as well as in the water and sediment of fish pond. The impact of such resistance development on food safety of fish produced and public health in general is uncertain, but is currently being investigated. Authors: PhD student: Dang Thi Thanh Son and Prof. Anders Dalsgaard
Edited by: Sven G. Sommer


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