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Poult Sci 2007. 86:610-613
© 2007 Poultry Science Association
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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Influence of Temperature on Survival and Conjugative Transfer of Multiple Antibiotic-Resistant Plasmids in Chicken Manure and Compost Microcosms

J. Guan1, A. Wasty, C. Grenier and M. Chan

Ottawa Laboratory–Fallowfield, Canadian Food Inspection Agency, Nepean, Ontario, Canada K2H 8P9

1 Corresponding author: guanj{at}inspection.gc.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of this study was to determine if mobile plasmids carrying antibiotic-resistant genes could survive and be transferred in chicken manure maintained under conditions similar to those found in commercial cage layer operations and during composting. Escherichia coli J5 harboring a self-transmissible plasmid (RP4) and E. coli C600 harboring a mobile plasmid (pIE723) were used as plasmid donors; E. coli CV601 was used as a plasmid recipient. At 23°C both plasmids were transferred to E. coli CV601 in chicken manure and in compost microcosms that consisted of a mixture of chicken manure and peat. The transfer frequencies ranged from 8.1 x 10–5 to 2.4 x 10–3 per donor cell in manure and from 2.4 x 10–5 to 5.5 x 10–4 per donor cell in compost microcosms. After 45 d of incubation at 23°C, RP4, but not pIE723, was recovered by an exogenous isolation method although their E. coli hosts were not cultured from the microcosms. However, when the temperatures of the compost microcosms were elevated to 50°C or above, neither the plasmids nor their E. coli hosts could be detected. The results suggested that composting of chicken manure at high temperatures could help prevent the spread of antibiotic-resistant genes via plasmids in the environment.

Key Words: mobile plasmid • chicken manure • composting


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The intensive use of antibiotics in human therapy and agriculture has contributed to the emergence of antibiotic-resistant bacterial pathogens, and plasmid-mediated gene transfer plays an important role in the rapid dissemination of the resistance genes (Witte, 1998; Smalla et al., 2000). Previous studies demonstrated that antibiotic resistance genes transfer among commensal and pathogenic bacteria through mobile plasmids in poultry and ruminant digestion tracts (Poppe et al., 2005; McCuddin et al., 2006). These findings were corroborated by Smalla et al. (2000) who found high diversity and prevalence of mobile plasmids carrying antibiotic genes in pig manure. In addition, a study by Götz and Smalla (1997) demonstrated that pig manure enhances the transfer of mobile plasmids in soil. Exacerbated by the intensification of agriculture, the growth of farm animal populations, and the popularity of organic farming, the high prevalence of mobile plasmids and their efficient mobilization in animal manure presents the need for environmentally acceptable means to inactivate these plasmids before manure is applied to land as fertilizer or otherwise disposed of in the environment.

Bacterial pathogens such as Escherichia coli O157:H7 and Salmonella typhimurium survive in chicken manure at low temperatures (Himathongkham et al., 2000), but they die in compost managed to promote high temperatures (Jones and Martin, 2003). However, there is limited information on the transfer of mobile plasmids in manure under typical conditions in commercial egg layer operations or during composting. A self-transmissible plasmid (RP4) and a mobile plasmid (pIE723) were used as models for this study. Plasmid RP4 is an IncP-1 plasmid, which can replicate and be stably maintained in almost all gram-negative bacteria and may be transferred by conjugation to gram-positive bacteria, yeasts, and eukaryotic cell lines (Adamczyk and Jagura-Burdzy, 2003). Plasmid pIE723 is an IncQ plasmid, which is able to replicate in a wide range of gram-negative bacteria and can be efficiently mobilized by a number of self-transmissible plasmids, especially IncP plasmids (Rawlings and Tietze, 2001). Plasmid RP4 harbors genes for resistance to ampicillin, kanamycin, and tetracycline (Datta et al., 1971), and pIE723 harbors genes for resistance to gentamicin, kanamycin, and streptomycin (Recchia and Hall, 1995). This study was to determine the fate of the plasmids in chicken manure at 23°C and in manure compost at temperatures of 50°C and above.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids
Escherichia coli J5 harboring RP4 was used as both a donor and a helper; E. coli C600 harboring plasmid pIE723 was used as a donor; and E. coli CV601 (Götz and Smalla, 1997), which is resistant to 30 µg/mL of rifamycin, was used as a recipient for the study of conjugative transfer of the plasmids.

Filter Mating
To determine transfer frequencies of RP4 from E. coli J5 and pIE723 from E. coli C600 to E. coli CV601 on filter mating, the above 3 E. coli strains were cultured in 10 mL of Luria-Bertani (LB) broth (Fisher Scientific, Ottawa, Ontario, Canada) at 37°C with agitation at 200 rpm. After a 24-h incubation, the bacterial cells were collected by centrifugation at 10,000 x g for 10 min, and the cell pellet was washed and resuspended in sterile water. For filter mating, 0.1 mL of 1.0 x 108 cells of each of the 3 strains were mixed and plated onto cellulose nitrate filters (25 mm diameter and 0.45 µm pore size, Gelman Sciences, Ann Arbor, MI) that were laid on LB agar. After a 24-h incubation of the LB agar plates at 37°C, bacterial cells were washed from the filters with 1.0 mL of sterile 0.85% saline. The bacterial suspension was serially diluted and plated onto LB agar supplemented with 30 µg/mL of rifamycin, 50 µg/mL of kanamycin, and 10 µg/mL of tetracycline for E. coli CV601 harboring RP4 (RP4 transconjugant) or 50 µg/mL of streptomycin for E. coli CV601 harboring pIE723 (pIE723 transconjugant). Duplicate plates for each dilution were used for enumeration of bacteria during the entire study, and all antibiotics used in the study were purchased from Sigma (St. Louis, MO). Transconjugant colonies were enumerated after the plates were incubated at 37°C for 24 h. This mating experiment was repeated twice.

Conjugative Transfer of RP4 and pIE723 in Chicken Manure
Chicken manure was collected from specific-pathogen-free chickens housed in cages at the Ottawa Laboratory (Fallowfield), Canadian Food Inspection Agency. Moisture content of the manure was determined using an IR-35 moisture analyzer (Denver Instrument, Denver, CO), and was then adjusted to 65%. Each of the above 3 bacteria was grown in 1 L of LB broth overnight at 37°C, and bacteria were pelleted, washed, and resuspended in 10 mL of water. Approximately 1.0 x 1011 cells of each strain was inoculated into 100 g of chicken manure, which was contained in a 500-mL polypropylene straight-sided jar (Nalgene Labware, Rochester, NY). Another jar containing 100 g of uninoculated manure was included as a control. Three jars of inoculated manure and 1 control were incubated at 23°C for 7 d. One gram of manure was collected every 24 h from each jar for enumeration of the donors and the transconjugants using selective media. MacConkey agar (Becton, Dickinson, and Co., Oakville, Ontario, Canada) was supplemented with 1) 50 µg/mL of kanamycin and 10 µg/mL of tetracycline to select E. coli J5 harboring RP4; 2) 50 µg/mL of kanamycin and 50 µg/mL of streptomycin to select E. coli C600 harboring plasmid pIE723; 3) 50 µg/mL of kanamycin, 10 µg/mL of tetracycline, and 30 µg/mL of rifamycin to select RP4 transconjugant; and 4) 50 µg/mL of kanamycin, 50 µg/mL of streptomycin, and 30 µg/mL of rifamycin to select pIE723 transconjugant. Colonies were counted after the plates were incubated at 37°C for 24 h.

Conjugative Transfer of RP4 and pIE723 in Compost Microcosms During Simulated Composting
A compost microcosm (a small representative compost) was a 100-g mixture of chicken manure, peat, and water, which was incubated for composting reaction. The mixture had a carbon to nitrogen ratio of 25:1 and a moisture content of 65% (Guan et al., 2004). Eight compost microcosms were prepared; 6 were inoculated with the 3 bacteria as described above, and 2 uninoculated microcosms were included as controls. A set of 3 inoculated microcosms plus 1 control was incubated at 23°C or at simulated composting temperatures as described by Guan et al. (2004). On each of 15 sampling days during a 45-d period, two 1-g samples were collected from each microcosm. One sample was suspended in 9 mL of sterile 0.85% saline followed by vortexing at maximum speed for 2 min. The suspension was serially diluted and plated on selective media as described above for enumeration of donor and transconjugant cells. The other sample was suspended in 9 mL of LB broth supplemented with 50 µg/mL of kanamycin and 10 µg/mL of Fungizone (Squibb, Montreal, Quebec, Canada), and the suspension was incubated at 37°C for 24 h. After the incubation, 10 µL of the suspension was streaked on selective media for detection of the donor cells. For exogenous isolation of RP4 and pIE723, filter mating was conducted with 100 µL of the incubated compost suspension and 100 µL of the recipient E. coli CV601 suspension as described above. After 24 h of filter mating, RP4 and pIE723 transconjugants were cultured using Mac-Conkey agar supplemented with antibiotics as described above.

Compost Microcosms in Compost Bins
Three compost bins containing chicken manure and wood shavings were prepared as described by Guan et al. (2004). The depth of the compost in the bins was 100 cm, and temperatures were monitored at 3 levels: 20, 50, and 80 cm from the bottom of the bins. Compost microcosms were prepared as described above and contained in mesh bags. One inoculated microcosm and 1 control microcosm were placed at each of the 3 levels in each of the 3 compost bins. The experiment was terminated on d 57 after the compost temperatures had reached peaks of at least 50°C and then declined to ambient temperatures. The bins were disassembled, and the microcosms were collected and subjected to detection of the donor and the transconjugant bacteria and to exogenous isolation of RP4 and pIE723 as described above.

Colony Hybridization
Transconjugant colonies recovered from chicken manure and microcosms were confirmed to harbor RP4 and pIE723 by colony hybridization using probe trfA1: AACC-CCCAGCCGGAACTG and probe oriT: GCCGTTAGGC-CAGTTTCTCG, respectively (Götz et al., 1996). Probe labeling was carried out using the digoxigenin oligonucleotide 3'-end labeling kit (Roche Diagnostics Canada, Laval, Quebec, Canada). Colonies were lifted using a nylon membrane (Roche) according to the manufacturer’s instructions. Hybridization and probe detection were performed in accordance with the instructions of the DIG DNA labeling and detection kit (Roche).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The transfer frequencies of RP4 and pIE723 between the pure cultures of the E. coli strains were 8.67 x 10–1 and 6.22 x 10–1 per donor cell after the 24-h filter mating, respectively. When the 3 bacterial strains were inoculated into chicken manure, the concentrations of E. coli J5 harboring RP4 and E. coli C600 harboring pIE723 remained above 2.0 x 106 cfu/g of manure during the 7 d of incubation at 23°C (Figure 1Go). The transconjugants (i.e., E. coli CV601 harboring RP4 or pIE723) were detected 24 h after inoculation of the manure, and their concentrations gradually decreased from 1.0 x 106 to 5.0 x 103 cfu/g during incubation (Figure 1Go). The transfer frequencies ranged from 3.6 x 10–4 to 2.4 x 10–3 per donor cell for RP4 and from 8.1 x 10–5 to 2.1 x 10–3 per donor cell for pIE723. The E. coli transconjugants harboring RP4 and pIE723 were confirmed by colony hybridization using probes trfA1 and oriT, respectively. No E. coli harboring either plasmid was detected from any control samples in this study including chicken manure and compost microcosms.


Figure 1
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  		Figure 1. Enumeration of Escherichia coli J5 harboring RP4 (•), E. coli C600 harboring pIE723 ({blacksquare}), E. coli CV601 harboring RP4 ({circ}), and E. coli CV601 harboring pIE723 ({square}) in chicken manure that was incubated at 23°C. Data are mean log cfu/g of sample from 3 jars; standard deviations are indicated by error bars.

 
The E. coli harboring RP4 or pIE723 survived for at least 27 d in compost microcosms that were incubated at 23°C. The counts of the 2 bacteria decreased from 5.1 x 109 to 1.0 x 107 cfu/g from d 0 to 7, and then remained above 1.0 x 106 cfu/g until d 18. Plasmid RP4 transconjugant was recovered between d 5 and 21 at levels that ranged from 2.0 x 102 to 1.6 x 104 cfu/g, and pIE723 transconjugant was recovered between d 7 to 18 at levels that ranged from 2.0 x 102 to 5.0 x 103 cfu/g (Figure 2AGo). The transfer frequencies ranged from 5.9 x 10–5 to 9.1 x 10–4 per donor cell for RP4 and from 2.4 x 10–5 to 5.5 x 10–4 per donor cell for pIE723. The 2 plasmids were recovered by exogenous isolation from d 0 to 27, but only RP4 was recovered on d 45 (data not shown). In comparison, the numbers of the 2 E. coli donors rapidly decreased during the first 3 d of incubation as the temperatures increased from 23 to 45°C, and were not detected when the temperature exceeded 50°C (Figure 2BGo). The RP4 or pIE723 transconjugants were not recovered during the entire simulated composting process, and neither plasmid was recovered by exogenous isolation from compost microcosms when temperatures were above 50°C. During the self-heated composting, temperatures throughout the compost bins reached 50°C within 11 d. No E. coli harboring RP4 or pIE723 were recovered from any of the microcosms collected from the 3 bins on the termination day. Furthermore, the 2 plasmids were not recovered from any of these microcosms by exogenous isolation.


Figure 2
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  		Figure 2. Enumeration of Escherichia coli J5 harboring RP4 (•), E. coli C600 harboring pIE723 ({blacksquare}), E. coli CV601 harboring RP4 ({circ}), and E. coli CV601 harboring pIE723 ({square}) in compost microcosms incubated at (A) 23°C and (B) simulated composting temperatures ({blacktriangleup}). Data are mean log cfu/g of sample from 3 microcosms; standard deviations are indicated by error bars.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The conjugative transfer of RP4 and pIE723 between E. coli cells occurred in chicken manure that contained 65% moisture content and was incubated at 23°C. These conditions were similar to those in cage layer manure in many poultry buildings. During the first 7 d of incubation, transfer of the 2 plasmids occurred earlier and at higher frequencies in manure compared with compost microcosms. The difference might result from better survival of bacterial hosts and better cell-to-cell contact in manure than in compost microcosms (Inoue et al., 2005) and suggested that the peat in compost microcosms might interfere with the plasmid transfer. Transfer frequencies of RP4 were higher than those of pIE723 both in manure and in compost microcosms, because pIE723 mobilization requires RP4 or other self-transmissible plasmids (Rawlings and Tietze, 2001). Transfer frequencies of RP4 in compost microcosms were similar to those found in activated sludge (Geisenberger et al., 1999). After 45 d of incubation at 23°C, RP4 was recovered by exogenous isolation, although its E. coli hosts could not be cultured from the microcosms. This host-independent survival of RP4 indicated the transfer of the plasmid into indigenous bacteria in the microcosms.

Although E. coli transconjugants harboring RP4 or pIE723 were never detected in compost microcosms at temperatures above 50°C, it was not known if the plasmids could be transferred into uncultivated bacteria during composting where bacterial communities are highly diverse and dynamic (Miyatake and Iwabuchi, 2005; Tiquia, 2005). However, neither of the 2 plasmids was recovered by the exogenous isolation method from the end products of compost that had reached temperatures of 50°C or above. In contrast, such exogenous isolation methods have proven to be efficient for recovery of mobile genetic elements from various environmental samples without cultivation of the indigenous bacteria (Smalla and Sobecky, 2002). Thus, composting temperatures above 50°C could be expected to destroy plasmids carrying antibiotic-resistant genes in chicken manure.


   ACKNOWLEDGMENTS
 
Sincere appreciation is expressed to the Fertilizer Section, Plant Health and Protection Division in the Canadian Food Inspection Agency for providing funds; to B. Watson from Eastern Cereal and Oilseed Research Center, Agriculture and Agri-Food Canada, and K. Smalla from Federal Biological Research Centre for Agriculture and Forestry, Germany, for providing the bacterial strains.

Received for publication August 23, 2006. Accepted for publication December 6, 2006.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adamczyk, M., and G. Jagura-Burdzy. 2003. Spread and survival of promiscuous IncP-1 plasmids. Acta Biochim. Pol. 50:425–453.[ISI][Medline]

Datta, N., R. W. Hedges, E. J. Shaw, R. B. Sykes, and M. H. Richmond. 1971. Properties of an R factor from Pseudomonas aeruginosa. J. Bacteriol. 108:1244–1249.[Abstract/Free Full Text]

Geisenberger, O., A. Ammendola, B. B. Christensen, S. Molin, K.-H. Schleifer, and L. Eberl. 1999. Monitoring the conjugal transfer of plasmid RP4 in activated sludge and in situ identification of the transconjugants. FEMS Microbiol. Lett. 174:9–17.[ISI][Medline]

Götz, A., R. Pukall, E. Smit, E. Tietze, R. Prager, H. Tschape, J. D. van Elsas, and K. Smalla. 1996. Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR. Appl. Environ. Microbiol. 62:2621–2628.[Abstract]

Götz, A., and K. Smalla. 1997. Manure enhances plasmid mobilization and survival of Pseudomonas putida introduced into field soil. Appl. Environ. Microbiol. 63:1980–1986.[Abstract]

Guan, J., J. L. Spencer, M. Sampath, and J. Devenish. 2004. The fate of a genetically modified Pseudomonas strain and its transgene during composting of poultry manure. Can. J. Microbiol. 50:415–421.[ISI][Medline]

Himathongkham, S., H. Riemann, S. Bahari, S. Nuanualsuwan, P. Kass, and D. O. Cliver. 2000. Survival of Salmonella typhimurium and Escherichia coli O157:H7 in poultry manure and manure slurry at sublethal temperatures. Avian Dis. 44:853–860.[ISI][Medline]

Inoue, D., K. Sei, S. Soda, M. Ike, and M. Fujita. 2005. Potential of predominant activated sludge bacteria as recipients in conjugative plasmid transfer. J. Biosci. Bioeng. 100:600–605.[ISI][Medline]

Jones, P., and M. Martin. 2003. A review of the literature on the occurrence and survival of pathogens of animals and humans in green compost. The waste and resource action programme. http://www.wrap.org.uk Accessed Nov. 2003

McCuddin, Z. P., S. A. Carlson, M. A. Rasmussen, and S. K. Franklin. 2006. Klebsiella to Salmonella gene transfer within rumen protozoa: Implications for antibiotic resistance and rumen defaunation. Vet. Microbiol. 114:275–284.[ISI][Medline]

Miyatake, F., and K. Iwabuchi. 2005. Effect of high compost temperature on enzymatic activity and species diversity of culturable bacteria in cattle manure compost. Bioresour. Technol. 96:1821–1825.[ISI][Medline]

Poppe, C., L. C. Martin, C. L. Gyles, R. Reid-Smith, P. Boerlin, S. A. McEwen, J. F. Prescott, and K. R. Forward. 2005. Acquisition of resistance to extended-spectrum cephalosporins by Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey poultry intestinal tract. Appl. Envrion. Microbiol. 71:1184–1192.[Abstract/Free Full Text]

Rawlings, D. E., and E. Tietze. 2001. Comparative biology of IncQ and IncQ-like plasmids. Microbiol. Mol. Biol. Rev. 65:481–496.[Abstract/Free Full Text]

Recchia, G. D., and R. M. Hall. 1995. Plasmid evolution by acquisition of mobile gene cassettes: Plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid pIE723. Mol. Microbiol. 15:179–187.[ISI][Medline]

Smalla, K., H. Heuer, A. Götz, D. Niemeyer, H. Krogerrechlenfort, and E. Tietze. 2000. Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl. Envrion. Microbiol. 66:4854–4862.[Abstract/Free Full Text]

Smalla, K., and P. A. Sobecky. 2002. The prevalence and diversity of mobile genetic elements in bacterial communities of different environmental habitats: Insights gained from different methodological approaches. FEMS Microbiol. Ecol. 42:165–175.

Tiquia, S. M. 2005. Microbial community dynamics in manure composts based on 16S and 18S rDNA T-RFLP profiles. Environ. Technol. 26:1101–1113.[Medline]

Witte, W. 1998. Medical consequences of antibiotic use in agriculture. Science 279:996–997.[Free Full Text]





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