Fate of Chlortetracycline- and Tylosin-Resistant Bacteria in an Aerobic Thermophilic Sequencing Batch Reactor Treating Swine Waste

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25°C, 37°C, and 60°C decreased after treatment, but they were still abundant (102 to 108 most probable number ml−1) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25°C, 37°C, and 60°C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60°C and for tylosin-resistant populations grown at 37°C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.

[1]  P. Juteau Review of the use of aerobic thermophilic bioprocesses for the treatment of swine waste , 2006 .

[2]  P. Juteau,et al.  Swine waste treatment by self-heating aerobic thermophilic bioreactors. , 2004, Water research.

[3]  A. Magurran Ecological Diversity and Its Measurement , 1988, Springer Netherlands.

[4]  Awwa,et al.  Standard Methods for the examination of water and wastewater , 1999 .

[5]  J. Sofos,et al.  Antimicrobial resistance: Implications for the food system: An expert report, funded by the IFT Foundation , 2006 .

[6]  M. Sobsey,et al.  Microbial indicator reductions in alternative treatment systems for swine wastewater , 1998 .

[7]  H. Blöcker,et al.  Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. , 1989, Nucleic acids research.

[8]  Marilyn Roberts,et al.  Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance , 2001, Microbiology and Molecular Biology Reviews.

[9]  J. Swings,et al.  Molecular Diversity and Characterization of Tetracycline-Resistant Staphylococcus aureus Isolates from a Poultry Processing Plant , 2005, Applied and Environmental Microbiology.

[10]  S. Ladely,et al.  Effects of Tylosin Use on Erythromycin Resistance in Enterococci Isolated from Swine , 2004, Applied and Environmental Microbiology.

[11]  F. Lowy,et al.  Antimicrobial-resistant bacteria in the community setting , 2006, Nature Reviews Microbiology.

[12]  F. Aarestrup,et al.  Relationship between Copper, Glycopeptide, and Macrolide Resistance among Enterococcus faecium Strains Isolated from Pigs in Denmark between 1997 and 2003 , 2005, Antimicrobial Agents and Chemotherapy.

[13]  K. Carlson,et al.  Response of antibiotic resistance genes (ARG) to biological treatment in dairy lagoon water. , 2007, Environmental science & technology.

[14]  S. Dridi,et al.  Prevalence and antimicrobial resistance of Campylobacter coli isolated from fattening pigs in France. , 2004, Veterinary microbiology.

[15]  S. Costanzo,et al.  Ecosystem response to antibiotics entering the aquatic environment. , 2005, Marine pollution bulletin.

[16]  L. Whyte,et al.  Characterization of the Prokaryotic Diversity in Cold Saline Perennial Springs of the Canadian High Arctic , 2007, Applied and Environmental Microbiology.

[17]  F. Aarestrup,et al.  An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype typhimurium DT104. , 1999, The New England journal of medicine.

[18]  F. Aarestrup,et al.  Antimicrobial Growth Promoter Ban and Resistance to Macrolides and Vancomycin in Enterococci from Pigs , 2001, Journal of Clinical Microbiology.

[19]  J. Casadesús,et al.  Use of mixed infections to study cell invasion and intracellular proliferation of Salmonella enterica in eukaryotic cell cultures. , 2004, Journal of microbiological methods.

[20]  Yi-Wei Tang,et al.  Enterococcus faecium-Related Outbreak with Molecular Evidence of Transmission from Pigs to Humans , 2002, Journal of Clinical Microbiology.

[21]  I. Good THE POPULATION FREQUENCIES OF SPECIES AND THE ESTIMATION OF POPULATION PARAMETERS , 1953 .

[22]  F. Haesebrouck,et al.  Molecular Analysis of Human, Porcine, and Poultry Enterococcus faecium Isolates and Their erm(B) Genes , 2005, Applied and Environmental Microbiology.

[23]  J. Sutcliffe,et al.  Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system , 1996, Antimicrobial agents and chemotherapy.

[24]  R. Mackie,et al.  Molecular Ecology of Tetracycline Resistance: Development and Validation of Primers for Detection of Tetracycline Resistance Genes Encoding Ribosomal Protection Proteins , 2001, Applied and Environmental Microbiology.

[25]  B. White,et al.  Development, Validation, and Application of PCR Primers for Detection of Tetracycline Efflux Genes of Gram-Negative Bacteria , 2002, Applied and Environmental Microbiology.

[26]  T. Grebe,et al.  Detection of erythromycin-resistant determinants by PCR , 1996, Antimicrobial agents and chemotherapy.

[27]  H. N. Chinivasagam,et al.  Microbiological status of piggery effluent from 13 piggeries in the south east Queensland region of Australia , 2004, Journal of applied microbiology.

[28]  E. Bruck,et al.  National Committee for Clinical Laboratory Standards. , 1980, Pediatrics.

[29]  J. E. Olsen,et al.  Transmission routes of Salmonella Typhimurium DT 104 between 14 cattle and pig herds in Denmark demonstrated by molecular fingerprinting , 2006, Journal of applied microbiology.

[30]  R. Surampalli,et al.  Disinfection of swine wastewater using chlorine, ultraviolet light and ozone. , 2006, Water research.

[31]  J. Delgenès,et al.  Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis. , 2005, FEMS microbiology ecology.

[32]  E. Topp,et al.  Bacterial community dynamics in liquid swine manure during storage: molecular analysis using DGGE/PCR of 16S rDNA , 2001 .

[33]  W. Witte,et al.  Medical Consequences of Antibiotic Use in Agriculture , 1998, Science.

[34]  A. E. Greenberg,et al.  Standard methods for the examination of water and wastewater : supplement to the sixteenth edition , 1988 .

[35]  E. Topp,et al.  Temporal Dynamics and Impact of Manure Storage on Antibiotic Resistance Patterns and Population Structure of Escherichia coli Isolates from a Commercial Swine Farm , 2007, Applied and Environmental Microbiology.

[36]  J. Rood,et al.  Nomenclature for Macrolide and Macrolide-Lincosamide-Streptogramin B Resistance Determinants , 1999, Antimicrobial Agents and Chemotherapy.

[37]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[38]  Bent Halling-Sørensen,et al.  Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry. , 2003, Environment international.

[39]  M. Roberts Update on acquired tetracycline resistance genes. , 2005, FEMS microbiology letters.

[40]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[41]  F. Haesebrouck,et al.  Differentiation and identification of Enterococcus durans, E. hirae and E. villorum , 2002, Journal of applied microbiology.

[42]  A. Uitterlinden,et al.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA , 1993, Applied and environmental microbiology.