Piggery manure used for soil fertilization is a reservoir for
transferable antibiotic resistance plasmids
Chu Thi Thanh Binh, Holger Heuer, Martin Kaupenjohann & Kornelia Smalla
FEMS MICROBIOL. ECOL. 66:25-37
Overuse of antibiotics has been responsible for the spread of antibiotic resistance among bacteria all over the world. Continual use of antibiotics has maintained a strong selective pressure for the persistence of antibiotic resistance genes, while horizontal gene transfer has resulted in the spread of these genes across phylogenetically diverse bacteria (Witte, 1998; Rhodes et al., 2000; Schmidt et al., 2001; Tennstedt et al., 2003). Studies on plasmid content from manures have shown the presence of transferable plasmids carrying antibiotic resistance genes (Gotz et al., 1996; Smalla et al., 2000; Heuer et al., 2002; van Overbeek et al., 2002). This study looks at manures from 15 pig farms, where each farm represents a different size of herd or different quantity of meat production.
16 manure samples were taken from 15 different farms across Germany. Exogenous biparental matings were carried out in the laboratory by using E. coli CV601 as the recipient and manure as donor. Mixtures of recipient and donor were incubated overnight and then plated on agar supplemented with either amoxicillin, sulfadiazine or tetracycline.
A total of 228 transconjugants were picked. Eight antibiotics were tested on all transconjugants using the disc diffusion method (Barry et al.,). Based on different combinations of antibiotic resistance phenotypes, 37 unique patterns were found. 204 transconjugants showed sulfadiazine resistance. The frequent use of sulfadizine in animal husbandry may be the reason for this observation. 40 transconjugants showed resistance to six antibiotics and 4 were resistant to all 8 antibiotics used. This is a frightening scenario, since only 8 antibiotics were tested and many more resistance genes may be present on these plasmids. A previous study (Normark & Normark, 2002) had shown that selection for one antibiotic might co-select other antibiotics. The authors hypothesize that this may be the reason for the appearance of multiple antibiotic resistances on these plasmids. In order to make their study simpler, they decided to use a subset of the 228 transconjugants. Hence, one transconjugant was chosen per manure for each antibiotic resistance pattern. This gave them 81 plasmids which they decided to analyse further.
Plasmids extracted from transconjugants were dot-blotted and hybridized with probes specific for replicon sequences of the broad-host-range (BHR) plasmid classes IncN, IncW, IncP-1 and IncQ. 28 plasmids were found to be IncN, 1 was IncW, 13 were IncP-1, 19 were similar to the recently discovered pHHV216-like plasmids (Heuer et al., 2008) and 20 plasmids could not be assigned to any of the known Inc groups. Next the authors wanted to see which genes were conferring resistances to amoxicillin and sulfadiazine in these plasmids. Dot-blotted plasmid DNA was hybridized with labeled probes for bla-TEM, sul1, sul2 and sul3 genes. While bla-TEM genes are most often associated with resistance to amoxicillin, a combination of sul1, sul2 and sul3 genes may be responsible for sulfadiazine resistance. From this experiment they saw that all transconjugants with the amoxicillin resistance phenotype carried the bla-TEM gene, confirming the findings of Binh et al., who showed the frequent occurrence of bla-TEM genes in manure and amoxicillin resistance soils. An interesting observation was the repeated occurrence of these genes on similar plasmids, for example the occurrence of bla-TEM genes on all 28 IncN plasmids. The authors conclude that IncN plasmids that were captured from 10 different manures could be responsible for the dissemination of bla-TEM genes. Similarly, the sul2 gene was found on all 19 pHHV216-type plasmids captured from 6 manures and the sul1 gene was found on 12 of 13 IncP-1 plasmids. The authors state that their work shows that antibiotic resistance genes are associated preferably with BHR plasmids. Next the authors tested the transferability of the 81 plasmids by carrying out matings where they used their transconjugants as donors and E. coli J53 as the recipient. They found that 73 could be transferred to the recipient and only 8 could not. 6 of these 8 were the pHHV216-like plasmids.
In order to compare the method of direct PCR-based detection of plasmids in total DNA of manure to the method of plasmid capture, they used primers specific to repA for IncN, trfA2 for IncP-1, or oriV for IncQ and IncW for PCR of total DNA of manure.
No correlation was observed between the frequency of plasmid capture and plasmid abundance as noted from total DNA of manure. For example, although one third of the plasmids captured from 15 manures were characterized as IncN, this class of plasmid was detected in only 5 manures by PCR and Southern blot hybridization. The authors attribute this to the low abundance of IncN plasmids in manure, which could have resulted in making PCR based detection difficult. Using the exogenous plasmid isolation method, they were able to capture IncN plasmids from these soils. Thus, they suggest that the exogenous isolation method captures plasmids even when they are not abundant and PCR-based detection of plasmid types may not be as efficient.
This study is important because it shows how prevalent broad host range plasmids are. Moreover, association of antibiotic resistance genes with such plasmids ensures their rapid spread in an environment with antibiotics that maintain a strong selection. We get some idea of the prevalence of resistance to antibiotics in bacteria. All transconjugants were found to confer resistance to one or more antibiotics. Co-selection of antibiotics is also a phenomenon that we should be looking at closely.
Witte W. (1998): Medical consequences of antibiotic use in agriculture. Science 279: 996–997.
Rhodes G., Huys G., Swings J., McGann P., Hiney M., Smith P. & Pickup R.W.
(2000): Distribution of oxytetracycline resistance plasmids between Aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant Tet A. Appl Environ Microbiol 66: 3883–3890.
Schmidt A.S., Bruun M.S., Dalsgaard I. & Larsen J.L. (2001): Incidence, distribution, and spread of tetracycline resistance determinants and integron-associated antibiotic resistance genes among motile aeromonads from a fish farming environment. Appl Environ Microbiol 67: 5675–5682.
Tennstedt T., Szczepanowski R., Braun S., Puhler A. & Schlüter A. (2003): Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. FEMS Microbiol Ecol 45:239–252.
Gotz A., Pukall R., Smit E., Tietze E., Prager R., Tschape H., van Elsas J.D. & Smalla K. (1996) Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl Environ Microbiol 62: 2621–2628.
Smalla K., Heuer H., Gotz A., Niemeyer D., Krogerrecklenfort E. & Tietze E., 2000: Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66: 4854–4862.
Heuer H., Krogerrecklenfort E., Egan S. et al. (2002): Gentamicin resistance genes in environmental bacteria: prevalence and transfer. FEMS Microbiol Ecol 42: 28-302.
Van Overbeek L.S., Wellington E.M.H., Egan S., Smalla K., Heuer H., Collard J.M., Guillaume G., Karagouni A.D., Nikolakopoulou T.L. & van Elsas J.D. (2002): Prevalence of streptomycin-resistance genes in bacterial populations in European habitats. FEMS Microbiol Ecol 42: 277–288.
Normark B.H. & Normark S (2002): Evolution and spread of antibiotic resistance. J Intern Med 252: 91–106.
Heuer H., Kopmann C., Binh C.T. T., Top E.M., Smalla K.(2008): Spreading antibiotic resistance through spread manure: characteristics 1 of a novel 2 plasmid type with low %G+C content. In press.
Barry A. L., Garcia F., and Thrupp L.D. (1970): An improved single-disk method for testing the antibiotic susceptibility of rapidly-growing pathogens. Am. J. Clin. Pathol. 53:149-158.
Department of Biological Sciences,
University of Idaho