Antimicrobial use in swine production and its effect on the swine gut microbiota and antimicrobial resistance.

Antimicrobials have been used in swine production at subtherapeutic levels since the early 1950s to increase feed efficiency and promote growth. In North America, a number of antimicrobials are available for use in swine. However, the continuous administration of subtherapeutic, low concentrations of antimicrobials to pigs also provides selective pressure for antimicrobial-resistant bacteria and resistance determinants. For this reason, subtherapeutic antimicrobial use in livestock remains a source of controversy and concern. The swine gut microbiota demonstrates a number of changes in response to antimicrobial administration depending on the dosage, duration of treatment, age of the pigs, and gut location that is sampled. Both culture-independent and -dependent studies have also shown that the swine gut microbiota contains a large number of antimicrobial resistance determinants even in the absence of antimicrobial exposure. Heavy metals, such as zinc and copper, which are often added at relatively high doses to swine feed, may also play a role in maintaining antimicrobial resistance and in the stability of the swine gut microbiota. This review focuses on the use of antimicrobials in swine production, with an emphasis on the North American regulatory context, and their effect on the swine gut microbiota and on antimicrobial resistance determinants in the gut microbiota.

Temporal analysis of the effect of extruded flaxseed on the swine gut microbiota.

Flaxseed is a rich source of α-linolenic acid, an essential ω-3 fatty acid reported to have beneficial health effects in humans. Feeding swine a diet supplemented with flaxseed has been found to enrich pork products with ω-3 fatty acids. However, the effect of flaxseed supplementation on the swine gut microbiota has not been assessed to date. The purpose of this study was to investigate if extruded flaxseed has any impact on the bacterial and archaeal microbiota in the feces of growing-finishing pigs over a 51-day period, using denaturing gradient gel electrophoresis (DGGE) and real-time PCR. Bacterial DGGE profile analysis revealed major temporal shifts in the bacterial microbiota with only minor ones related to diet. The archaeal microbiota was significantly less diverse than that of Bacteria. The majority of bacterial DGGE bands sequenced belonged to the Firmicutes phylum while the archaeal DGGE bands were found to consist of only 2 species, Methanobrevibacter smithii and Methanosphaera stadtmanae. The abundance of Bacteroidetes decreased significantly from day 0 to day 21 in all diet groups while the abundance of Firmicutes was relatively stable across all diet cohorts and sampling times. There was also no significant correlation between pig mass and the ratio of Firmicutes to Bacteroidetes. While the addition of extruded flaxseed to the feed of growing-finishing pigs was beneficial for improving ω-3 fatty acid content of pork, it had no detectable impact on the fecal bacterial and archaeal microbiota, suggesting that extruded flaxseed may be used to improve meat quality without adverse effect on the swine gut microbiota or animal performance.

Temporal changes and the effect of subtherapeutic concentrations of antibiotics in the gut microbiota of swine.

The use of antibiotics in swine production for the purpose of growth promotion dates back to the 1950s. Despite this long history of use, the exact mechanism(s) responsible for the growth-promoting effects of antibiotics in swine remain largely unknown. It is believed, however, that growth promotion is due to antibiotics having a direct impact on the gut microbiota. In this study, the effect of two antibiotics on the swine gut microbiota over a 19-week monitoring period was investigated using Illumina-based sequencing. A shift in the relative abundance of several taxa and in 26 operational taxonomic units (OTUs) was observed in pigs fed subtherapeutic concentrations of tylosin (44-11 mg kg(-1) feed). Only minor alterations were noted with the administration of chlortetracycline at 5.5 mg kg(-1) feed. The most notable changes in the relative abundance of taxa and OTUs were noted between suckling piglets and postweaned pigs. Diversity was also reduced in the gut microbiota of suckling piglets as measured using the Shannon, Chao1, and phylogenetic diversity indices. These results show that the effect of antibiotics on the swine gut microbiota is variable based on dosage and duration and that the swine gut microbiota exhibits considerable resilience to long-term changes due to antibiotic perturbations.

Impact of subtherapeutic administration of tylosin and chlortetracycline on antimicrobial resistance in farrow-to-finish swine.

The use of antimicrobial agents in swine production at subtherapeutic concentrations for the purpose of growth promotion remains controversial due to the potential impact on public health. Beginning at weaning (3 weeks), pigs received either nonmedicated feed or feed supplemented with subtherapeutic levels of either tylosin (11-44 ppm) or chlortetracycline (5.5 ppm). After only 3 weeks, pigs given feed supplemented with tylosin had significantly higher levels of tylosin-resistant anaerobes (P < 0.0001) compared with the control group, increasing from 11.8% to 89.6%, a level which was stable for the duration of the study, even after a 2-week withdrawal prior to slaughter. Tylosin-fed pigs had a higher incidence of detection for erm(A), erm(F), and erm(G), as well as significantly (P < 0.001) higher concentrations of erm(B) in their feces. The continuous administration of chlortetracycline-supplemented feed, however, had no significant effect on the population of chlortetracycline-resistant anaerobes in comparison with nontreated pigs (P > 0.05). The resistance genes tet(O), tet(Q), and erm(B) were detected in all pigs at each sampling time, while tet(G), tet(L), and tet(M) were also frequently detected. Neither chlortetracycline nor tylosin increased the growth rate of pigs.

Frequency of antibiotic resistance in a swine facility 2.5 years after a ban on antibiotics.

The addition of antibiotics to livestock feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations and agricultural ecosystems. The objective of this study was to assess the occurrence of resistance to chlortetracycline and tylosin among bacterial populations at the Swine Complex of McGill University (Province of Quebec, Canada) in the absence of antibiotic administration to pigs for 2.5 years prior to the beginning of this study. Feces from ten pigs born from the same sow and provided feed without antibiotic were sampled during suckling (n = 6 for enumerations, n = 10 for PCR), weanling (n = 10 both for PCR and enumerations), growing (n = 10 both for PCR and enumerations), and finishing (n = 10 both for PCR and enumerations). The percentage of chlortetracycline-resistant anaerobic bacterial populations (Tet(R)) was higher than that of tylosin-resistant anaerobic bacterial populations (Tyl(R)) at weanling, growing, and finishing. Prior to the transportation of animals to the slaughterhouse, resistant populations varied between 6.5 and 9.4 Log colony-forming units g humid feces(-1). In all pigs, tet(L), tet(O), and erm(B) were detected at suckling and weanling, whereas only tet(O) was detected at growing and finishing. The abundance of tet(O) was similar between males and females at weanling and growing and reached 5.1 × 10(5) and 5.6 × 10(5) copies of tet(O)/ng of total DNA in males and females, respectively, at finishing. Results showed high abundances and proportions of Tet(R) and Tyl(R) anaerobic bacterial populations, as well as the occurrence of tet and erm resistance genes within these populations despite the absence of antibiotic administration to pigs at this swine production facility since January 2007, i.e., 2.5 years prior to the beginning of this study. This work showed that the occurrence of bacterial resistance to chlortetracycline and tylosin is high at the Swine Complex of McGill University.

Impact of an aerobic thermophilic sequencing batch reactor on antibiotic-resistant anaerobic bacteria in swine waste.

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25 degrees C and 37 degrees C, resistant populations remained significant (10(4) to 10(5) most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37 degrees C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.

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 degrees C, 37 degrees C, and 60 degrees C decreased after treatment, but they were still abundant (10(2) to 10(8) 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 degrees C, 37 degrees C, and 60 degrees 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 degrees C and for tylosin-resistant populations grown at 37 degrees 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.

Influence of nutrients, hexadecane, and temporal variations on nitrification and exopolysaccharide composition of river biofilms.

Biofilms were cultivated on polycarbonate strips in rotating annular reactors using South Saskatchewan River water during the fall of 1999 and the fall of 2001. The reactors were supplemented with carbon (glucose), nitrogen (NH(4)Cl), phosphorus (KH(2)PO(4)), or combined nutrients (CNP), with or without hexadecane. The impact of these treatments on nitrification and on the exopolysaccharide composition of river biofilms was determined. The results showed that the biofilms had higher NH4(+) oxidation, NO3(-) production, and N2O production activities in fall 1999 than fall 2001 when grown with CNP but had higher activities in fall 2001 than fall 1999 when grown with individual nutrients. The exopolysaccharide amounts and proportions were generally higher in fall 1999 than fall 2001, as a consequence of the higher nutrient levels in the river water in the first year of this study. The addition of P and especially CNP stimulated NH4(+) oxidation by the biofilms, showing a P limitation in this river ecosystem. The presence of hexadecane negatively affected these activities and lowered the amounts of exopolysaccharides in CNP and P biofilms in fall 1999 but increased the biofilm activities and exopolysaccharide amounts in CNP biofilm in fall 2001. Antagonistic, synergistic, and independent effects between nutrients and hexadecane were also observed. This study demonstrated that the biofilm autotrophic nitrification activity in the South Saskatchewan River was limited by P, that this activity and the exopolysaccharide amounts and proportions were dependent on the nutrient concentrations in the river water, and suggested that exopolysaccharides may play a protective role for biofilm microorganisms against toxic pollutants.

Impact of seasonal variations and nutrient inputs on nitrogen cycling and degradation of hexadecane by replicated river biofilms.

Biofilm communities cultivated in rotating annular bioreactors using water from the South Saskatchewan River were assessed for the effects of seasonal variations and nutrient (C, N, and P) additions. Confocal laser microscopy revealed that while control biofilms were consistently dominated by bacterial biomass, the addition of nutrients shifted biofilms of summer and fall water samples to phototrophic-dominated communities. In nutrient-amended biofilms, similar patterns of nitrification, denitrification, and hexadecane mineralization rates were observed for winter and spring biofilms; fall biofilms had the highest rates of nitrification and hexadecane mineralization, and summer biofilms had the highest rates of denitrification. Very low rates of all measured activities were detected in control biofilms (without nutrient addition) regardless of season. Nutrient addition caused large increases in hexadecane mineralization and denitrification rates but only modest increases, if any, in nitrification rates, depending upon the season. Generally, both alkB and nirK were more readily PCR amplified from nutrient-amended biofilms. Both genes were amplified from all samples except for nirK from the fall control biofilm. It appears that bacterial production in the South Saskatchewan River water is limited by the availability of nutrients and that biofilm activities and composition vary with nutrient availability and time of year.