Development and application of real-time PCR assays for quantification of genes encoding tetracycline resistance

We report here the development, validation, and use of three real-time PCR assays to quantify the abundance of the following three groups of tetracycline resistance genes: tet(A) and tet(C); tet(G); and tet genes encoding ribosomal protection proteins, including tet(M), tet(O), tetB(P), tet(Q), tet(S), tet(T), and tet(W). The assays were validated using known numbers of sample-derived tet gene templates added to microbiome DNA. These assays are both precise and accurate over at least 6 log tet gene copies. New tet gene variants were also identified from cloned tet amplicons as part of this study. The utility of these real-time PCR assays was demonstrated by quantifying the three tet gene groups present in bovine and swine manures, composts of swine manure, lagoons of hog house effluent, and samples from an Ekokan upflow biofilter system treating hog house effluent. The bovine manures were found to contain fewer copies of all three groups of tet genes than the swine manures. The composts of swine manures had substantially reduced tet gene abundance (up to 6 log), while lagoon storage or the upflow biofilter had little effect on tet gene abundance. These results suggest that the method of manure storage and treatment may have a substantial impact on the persistence and dissemination of tet genes in agricultural environments. These real-time PCR assays provide rapid, quantitative, cultivation-independent measurements of 10 major classes of tet genes, which should be useful for ecological studies of antibiotic resistance.     

Antibiotic resistance genes in multidrug-resistant Enterococcus spp. and Streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation

AIMS: In this study, multidrug-resistant bacteria previously recovered from the indoor air of a large-scale swine-feeding operation were tested for the presence of five macrolide, lincosamide and streptogramin (MLS) resistance genes and five tetracycline (tet) resistance genes. METHODS AND RESULTS: Enterococcus spp. (n = 16) and Streptococcus spp. (n =16) were analysed using DNA-DNA hybridization, polymerase chain reaction (PCR) and oligoprobing of PCR products. All isolates carried multiple MLS resistance genes, while 50% of the Enterococcus spp. and 44% of the Streptococcus spp. also carried multiple tet resistance genes. All Enterococcus spp. carried erm(A) and erm(B), 69% carried erm(F), 44% carried mef(A), 75% carried tet(M), 69% carried tet(L) and 19% carried tet(K). All Streptococcus spp. carried erm(B), 94% carried erm(F), 75% carried erm(A), 38% carried mef(A), 50% carried tet(M), 81% carried tet(L) and 13% carried tet(K). CONCLUSIONS: Multidrug resistance among airborne bacteria recovered from a swine operation is encoded by multiple MLS and tet resistance genes. These are the first data regarding resistance gene carriage among airborne bacteria from swine-feeding operations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of multiple resistance genes reported here suggests that airborne Gram-positive bacteria from swine operations may be important contributors to environmental reservoirs of resistance genes.     

Characterization of a mobile and multiple resistance plasmid isolated from swine manure and its detection in soil after manure application

Aims: To isolate and characterize multiple antibiotic resistance plasmids found in swine manure and test for plasmid‐associated genetic markers in soil following manure application to an agricultural field. Methods and Results: Plasmids were isolated from an erythromycin enrichment culture that used liquid swine manure as an inoculant. Plasmids were transformed into Escherichia coli DH10β for subsequent characterization. We isolated and DNA sequenced a 22 102‐bp plasmid (pMC2) that confers macrolide, and tetracycline resistances, and carries genes predicted to code for mercury and chromium resistance. Conjugation experiments using an pRP4 derivative as a helper plasmid confirm that pMC2 has a functional mobilization unit. PCR was used to detect genetic elements found on pMC2 in DNA extracted from manure amended soil. Conclusions: The pMC2 plasmid has a tetracycline‐resistant core and has acquired additional resistance genes by insertion of an accessory region (12 762 bp) containing macrolide, mercury and chromium resistance genes, which was inserted between the truncated DDE motifs within the Tn903/IS102 mobile element. Significance and Impact of the Study: Liquid swine manure used for manure spreading contains multiple antibiotic resistance plasmids that can be detected in soil following manure application.     

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc(r)) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc(r) genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc(r) genes was quantified by real-time quantitative PCR. To confirm the Tc(r) gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc(r) genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc(r) genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.     

Occurrence and Persistence of Erythromycin Resistance Genes (erm) and Tetracycline Resistance Genes (tet) in Waste Treatment Systems on Swine Farms

Animal manure from modern animal agriculture constitutes the single largest source of antibiotic resistance (AR) owing to the use of large quantities of antibiotics. After animal manure enters the environment, the AR disseminates into the environment and can pose a potentially serious threat to the health and well-being of both humans and animals. In this study, we evaluated the efficiency of three different on-farm waste treatment systems in reducing AR. Three classes of erythromycin resistance genes (erm) genes (B, F, and X) conferring resistances to macrolide–lincosamides–streptogramin B (MLS_B) and one class of tetracycline resistance genes (tet) gene (G) conferring resistance to tetracyclines were used as models. Real-time polymerase chain reaction assays were used to determine the reservoir sizes of these AR genes present in the entire microbiome. These classes of AR genes varied considerably in abundance, with erm(B) being more predominant than erm(F), erm(X), and tet(G). These AR genes also varied in persistence in different waste treatment systems. Aerobic biofiltration reduced erm(X) more effectively than other AR genes, while mesophilic anaerobic digestion and lagoon storage did not appreciably reduce any of these AR genes. Unlike chemical pollutants, some AR genes could increase after reduction in a preceding stage of the treatment processes. Season might also affect the persistence of AR. These results indicate that AR arising from swine-feeding operations can survive typical swine waste treatment processes and thus treatments that are more effective in destructing AR on farms are required.     

Acquired tetracycline and/or macrolide-lincosamides-streptogramin resistance in anaerobes

In general bacterial antibiotic resistance is acquired on mobile elements such as plasmids, transposons and/or conjugative transposons. This is also true for many antibiotic resistant anaerobic species described in the literature. Of the 23 different tetracycline resistant efflux genes identified, tet(B), tet(K), tet(L), and tetA(P) have been found in anaerobic species and six of the ten tetracycline resistant genes coding for ribosomal protection proteins, tet(M), tet(O), tetB(P), tet(Q), tet(W), and tet(32), have been identified in anaerobes. There are now three enzymes which inactivate tetracycline, of which the tet(X) has been identified in Bacteroides though is not functional under anaerobic growth conditions. A similar situation exists with the genes conferring macrolide-lincosamide-streptogramin (MLS) resistance. Of the 26 rRNA methylase MLS resistant genes characterized, five genes; erm(B), erm(C), erm(F), erm(G), and erm(Q), have been identified in anaerobes. In contrast, no genes coding for MLS resistant efflux proteins or inactivating enzymes have been described in anaerobic species. This mini-review will summarize what is known about tetracycline and MLS resistance in genera with anaerobic species and the mobile elements associated with acquired tetracycline and/or MLS resistance genes.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Development and comparison of SYBR Green quantitative real‐time PCR assays for detection and enumeration of sulfate‐reducing bacteria in stored swine manure

Aims: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real‐time PCR detection of sulfate‐reducing bacteria (SRB) in stored swine manure. Methods and Results: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR‐R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus‐like Group 1 SRB in manure. Conclusions: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.     

Tetracycline Residues and Tetracycline Resistance Genes in Groundwater Impacted by Swine Production Facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.

To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators—including inorganic ions, antibiotics, and antibiotic resistance genes—were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 μg/L.

Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes—tet(M), tet(O), tet(Q), and tet(W)—were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Persistence of Resistance to Erythromycin and Tetracycline in Swine Manure During Simulated Composting and Lagoon Treatments

The use of antimicrobials in food animal production leads to the development of antimicrobial resistance (AMR), and animal manure constitutes the largest reservoir of such AMR. In previous studies, composted swine manure was found to contain substantially lower abundance of AMR genes that encode resistance to tetracyclines (tet genes) and macrolide–lincosamide–streptogramin B (MLS_B) superfamily (erm genes), than manures that were treated by lagoons or biofilters. In this study, temporal changes in AMR carried by both cultivated and uncultivated bacteria present in swine manure during simulated composting and lagoon storage were analyzed. Treatments were designed to simulate the environmental conditions of composting (55°C with modest aeration) and lagoon storage (ambient temperature with modest aeration). As determined by selective plate counting, over a 48-day period, cultivated aerobic heterotrophic erythromycin-resistant bacteria and tetracycline-resistant bacteria decreased by more than 4 and 7 logs, respectively, in the simulated composting treatment while only 1 to 2 logs for both resistant bacterial groups in the simulated lagoon treatment. Among six classes each of erm and tet genes quantified by class-specific real-time PCR assays, the abundance of erm(A), erm(C), erm(F), erm(T), erm(X), tet(G), tet(M), tet(O), tet(T), and tet(W) declined marginally during the first 17 days, but dramatically thereafter within 31 days of the composting treatment. No appreciable reduction of any of the erm or tet genes analyzed was observed during the simulated lagoon treatment. Correlation analysis showed that most of the AMR gene classes had similar persistence pattern over the course of the treatments, though not all AMR genes were destructed at the same rate during the treatments.     

Monitoring and Source Tracking of Tetracycline Resistance Genes in Lagoons and Groundwater Adjacent to Swine Production Facilities over a 3-Year Period

To monitor the dissemination of resistance genes into the environment, we determined the occurrence of tetracycline resistance (Tc^r) genes in groundwater underlying two swine confinement operations. Monitoring well networks (16 wells at site A and 6 wells at site C) were established around the lagoons at each facility. Groundwater (n = 124) and lagoon (n = 12) samples were collected from the two sites at six sampling times from 2000 through 2003. Total DNA was extracted, and PCR was used to detect seven Tc^r genes [tet(M), tet(O), tet(Q), tet(W), tet(C), tet(H), and tet(Z)]. The concentration of Tc^r genes was quantified by real-time quantitative PCR. To confirm the Tc^r gene source in groundwater, comparative analysis of tet(W) gene sequences was performed on groundwater and lagoon samples. All seven Tc^r genes were continually detected in groundwater during the 3-year monitoring period at both sites. At site A, elevated detection frequency and concentration of Tc^r genes were observed in the wells located down-gradient of the lagoon. Comparative analysis of tet(W) sequences revealed that the impacted groundwater contained gene sequences almost identical (99.8% identity) to those in the lagoon, but these genes were not found in background libraries. Novel sequence clusters and unique indigenous resistance gene pools were also found in the groundwater. Thus, antibiotic resistance genes in groundwater are affected by swine manure, but they are also part of the indigenous gene pool.     

Housefly Larva Vermicomposting Efficiently Attenuates Antibiotic Resistance Genes in Swine Manure,with Concomitant Bacterial Population Changes

Manure from swine treated with antimicrobials as feed additives is a major source for the expansion of the antibiotic resistance gene (ARG) reservoir in the environment. Vermicomposting via housefly larvae (Musca domestica) can be efficiently used to treat manure and regenerate biofertilizer, but few studies have investigated its effect on ARG attenuation. Here, we tracked the abundances of 9 ARGs and the composition and structure of the bacterial communities in manure samples across 6 days of full-scale manure vermicomposting. On day 6, the abundances of genes encoding tetracycline resistance [tet(M), tet(O), tet(Q), and tet(W)] were reduced (P < 0.05), while those of genes encoding sulfonamide resistance (sul1 and sul2) were increased (P < 0.05) when normalized to 16S rRNA. The abundances of tetracycline resistance genes were correlated (P < 0.05) with the changing concentrations of tetracyclines in the manure. The overall diversity and richness of the bacteria significantly decreased during vermicomposting, accompanied by a 100 times increase in the relative abundance of Flavobacteriaceae spp. Variations in the abundances of ARGs were correlated with the changing microbial community structure and the relative abundances of the family Ruminococcaceae, class Bacilli, or phylum Proteobacteria. Vermicomposting, as a waste management practice, can reduce the overall abundance of ARGs. More research is warranted to assess the use of this waste management practice as a measure to attenuate the dissemination of antimicrobial residues and ARGs from livestock production before vermicompost can be safely used as biofertilizer in agroecosystems.     

Erythromycin resistance and virulence genes in Enterococcus faecalis from swine in China

This study aims to describe the erythromycin resistance phenotypes and genotypes, and the prevalence of virulence genes of Enterococcus faecalis isolated from swine in China. A total of 117 nonreplicate E. faecalis isolates, obtained from 502 clinical samples taken from different pig farms between 2007 and 2009 were included in the study. Minimum inhibitory concentrations were determined using the broth microdilution method. All of the isolates were screened for the presence of seven virulence genes (ace, asa1, cylA, efaA, esp, gelE, and hyl). In addition, the DNA from rythromycin-resistant isolates were amplified with primers specific for erythromycin resistance erm(A), erm(B), erm(C), mef(A/E), and msr(C) genes. Results show that erythromycin, tylosin, and ciprofloxacin resistance rates in E. faecalis were 66.67% (n=78), 66.67% (n=78), and 64.10% (n=75), respectively. About 69.23% of isolates (n=81) were positive for gelE, 48.72% (n=57) for ace, 15.38% (n=18) for efa, 7.69% (n=9) for asa1, and 6.84% (n=8) for esp. Among the erythromycin-resistant isolates, erm(B) (n=54) was the most prevalent resistance gene, followed by erm(A) (n=37). A significant correlation was found between the presence of the gelE virulence gene and erythromycin resistance (P<0.05). These findings suggest that enterococci from swine should be regarded with caution because they can be reservoirs for antimicrobial resistance and virulence genes.     

Application of group specific amplified rDNA restriction analysis to characterize swine fecal and manure storage pit samples

Group specific amplified ribosomal-DNA restriction analysis was evaluated as a method to rapidly assess microbial community structure in swine fecal and manure storage pit samples. PCR primer sequences were evaluated for their specificity to ribosomal DNA from selected bacterial groups by optimizing annealing temperatures and determining specificity using a set of primer target and non-target organisms. A number of primer sets were identified targeting the following groups: Bacteroides-Prevotella, clostridial clusters I and II, clostridial clusters IX and XI, clostridial clusters XIVa and XIVb, Lactobacillus, Desulfovibrionaceae and Streptococcus-Lactococcus, as well as an universal primer set to represent total populations. Each bacterial group was digested with at least three restriction enzymes. We applied the group specific amplified ribosomal-DNA restriction analysis to swine fecal and manure storage pit samples obtained on two separate occasions. Fecal and manure storage pit samples obtained on the same day were more similar to each other than to any other samples. Results were consistent with 16S ribosomal DNA sequencing data from bacterial isolates and clones obtained from swine feces and manure storage pit. The group specific amplified ribosomal-DNA restriction analysis technique was able to rapid detect gross bacterial community differences among swine fecal and manure storage pit samples and determine groups of interest for more detailed examination.     

Positive and negative selection towards tetracycline resistance genes in manure treatment lagoons

Aims: To evaluate the role of manure treatment lagoons of swine operations in the fate of faeces‐ and feed‐borne tetracycline‐resistant genes (TRG). Methods and Results: Samples of feed, faeces, lagoon liquid and lagoon sediment in farm’s vicinity were collected at three swine operations varied on their operational practices and analysed on the presence and frequencies of incidence of sixteen TRG in upstream sources (feed, faeces) and downstream receptacles (lagoon liquid and sediments). The highest frequency of TRG incidence was observed in a farm with extensive antibiotic usage and the lowest in the antibiotic‐free farm. The study revealed a decrease in TRG richness and diversity in the downstream habitats of each farm. The observed TRG diverged into two groups, the persistent genes that were detected both upstream and downstream, and the transient genes that were detected in the upstream habitats but became nondetectable in the lagoons. Quantitative PCR analysis revealed that upstream concentrations and abundances (determined as TRG/16S rRNA gene ratios) of transient and persistent TRG were similar; however, the former were attenuated in the lagoons to the levels below the detection limit, whereas the latter were ～ 100‐1000 fold amplified in their (mostly) liquid phases and were also detected in farms’ vicinities. Conclusion: Manure lagoons of swine operations imposed both positive and negative selection towards faeces‐ and feed‐borne TRG that, respectively, caused either their proliferation or attenuation in those environments. Significance and Impact of the Study: The study reveals that discharge of antibiotic resistance genes from swine farms to the environment is linked to their positive selection (defined as an impact leading to proliferation of those genes) in manure lagoons.     

Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection

A macroarray system was developed to screen environmental samples for the presence of specific tetracycline (Tc(R)) and erythromycin (erm(R)) resistance genes. The macroarray was loaded with polymerase chain reaction (PCR) amplicons of 23 Tc(R) genes and 10 erm(R) genes. Total bacterial genomic DNA was extracted from soil and animal faecal samples collected from different European countries. Macroarray hybridization was performed under stringent conditions and the results were analysed by fluorescence scanning. Pig herds in Norway, reared without antibiotic use, had a significantly lower incidence of antibiotic resistant bacteria than those reared in other European countries, and organic herds contained lower numbers of resistant bacteria than intensively farmed animals. The relative proportions of the different genes were constant across the different countries. Ribosome protection type Tc(R) genes were the most common resistance genes in animal faecal samples, with the tet(W) gene the most abundant, followed by tet(O) and tet(Q). Different resistance genes were present in soil samples, where erm(V) and erm(E) were the most prevalent followed by the efflux type Tc(R) genes. The macroarray proved a powerful tool to screen DNA extracted from environmental samples to identify the most abundant Tc(R) and erm(R) genes within those tested, avoiding the need for culturing and biased PCR amplification steps.     

Reduction of the resistome risk from cow slurry and manure microbiomes to soil and vegetable microbiomes

In cow farms, the interaction between animal and environmental microbiomes creates hotspots for antibiotic resistance dissemination. A shotgun metagenomic approach was used to survey the resistome risk in five dairy cow farms. To this purpose, 10 environmental compartments were sampled: 3 of them linked to productive cows (fresh slurry, stored slurry, slurry-amended pasture soil); 6 of them to non-productive heifers and dry cows (faeces, fresh manure, aged manure, aged manure-amended orchard soil, vegetables-lettuces and grazed soil); and, finally, unamended control soil. The resistome risk was assessed using MetaCompare, a computational pipeline which scores the resistome risk according to possible links between antibiotic resistance genes (ARGs), mobile genetic elements (MGEs) and human pathogens. The resistome risk decreased from slurry and manure microbiomes to soil and vegetable microbiomes. In total (sum of all the compartments), 18,157 ARGs were detected: 24% related to ansamycins, 21% to multidrugs, 14% to aminoglycosides, 12% to tetracyclines, 9% to β-lactams, and 9% to macrolide–lincosamide–streptogramin B. All but two of the MGE-associated ARGs were only found in the animal dejections (not in soil or vegetable samples). Several ARGs with potential as resistome risk markers (based on their presence in hubs of co-occurrence networks and high dissemination potential) were identified. As a precautionary principle, improved management of livestock dejections is necessary to minimize the risk of antibiotic resistance.     

Detection of a common and persistent tet(L)-carrying plasmid in chicken-waste-impacted farm soil

The connection between farm-generated animal waste and the dissemination of antibiotic resistance in soil microbial communities, via mobile genetic elements, remains obscure. In this study, electromagnetic induction (EMI) surveying of a broiler chicken farm assisted soil sampling from a chicken-waste-impacted site and a marginally affected site. Consistent with the EMI survey, a disparity existed between the two sites with regard to soil pH, tetracycline resistance (Tc(r)) levels among culturable soil bacteria, and the incidence and prevalence of several tet and erm genes in the soils. No significant difference was observed in these aspects between the marginally affected site and several sites in a relatively pristine regional forest. When the farm was in operation, tet(L), tet(M), tet(O), erm(A), erm(B), and erm(C) genes were detected in the waste-affected soil. Two years after all waste was removed from the farm, tet(L), tet(M), tet(O), and erm(C) genes were still detected. The abundances of tet(L), tet(O), and erm(B) were measured using quantitative PCR, and the copy numbers of each were normalized to eubacterial 16S rRNA gene copy numbers. tet(L) was the most prevalent gene, whereas tet(O) was the most persistent, although all declined over the 2-year period. A mobilizable plasmid carrying tet(L) was identified in seven of 14 Tc(r) soil isolates. The plasmid's hosts were identified as species of Bhargavaea, Sporosarcina, and Bacillus. The plasmid's mobilization (mob) gene was quantified to estimate its prevalence in the soil, and the ratio of tet(L) to mob was shown to have changed from 34:1 to 1:1 over the 2-year sampling period.     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.