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.     

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.     

Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.     

The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant

The microbiota in the human gastrointestinal tract (GIT) is highly exposed to antibiotics, and may be an important reservoir of resistant strains and transferable resistance genes. Maternal GIT strains can be transmitted to the offspring, and resistances could be acquired from birth. This is a case study using a metagenomic approach to determine the diversity of microorganisms conferring tetracycline resistance (Tc(r)) in the guts of a healthy mother-infant pair one month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of Tc(r) genes. Fecal fosmid libraries were functionally screened for Tc(r), and further PCR-screened for specific Tc(r) genes. Tc(r) fosmid inserts were sequenced at both ends to establish bacterial diversity. Mother and infant libraries contained Tc(r), although encoded by different genes and organisms. Tc(r) organisms in the mother consisted mainly of Firmicutes and Bacteroidetes, and the main gene detected was tet(O), although tet(W) and tet(X) were also found. Identical Tc(r) gene sequences were present in different bacterial families and even phyla, which may indicate horizontal transfer within the maternal GIT. In the infant library, Tc(r) was present exclusively in streptococci carrying tet(M), tet(L) and erm(T) within a novel composite transposon, Tn6079. This transposon belongs to a family of broad host range conjugative elements, implying a potential for the joint spread of tetracycline and erythromycin resistance within the infant's gut. In addition, although not found in the infant metagenomic library, tet(O) and tet(W) could be detected in the uncloned DNA purified from the infant fecal sample. This is the first study to reveal the diversity of Tc(r) bacteria in the human gut, to detect a likely transmission of antibiotic resistance from mother to infant GITs and to indicate the possible occurrence of gene transfers among distantly related bacteria coinhabiting the GIT of the same individual.     

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.     

Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies

The abundance of six tetracycline resistance genes tet(O), tet(Q), tet(W), tet(M), tet(B) and tet(L), were quantified over time in wastewater lagoons at concentrated animal feeding operations (CAFO) to assess how feedlot operation affects resistance genes in downstream surface waters. Eight lagoons at five cattle feedlots in the Midwestern United States were monitored for 6 months. Resistance and 16S-rRNA gene abundances were quantified using real-time PCR, and physicochemical lagoon conditions, tetracycline levels, and other factors (e.g. feedlot size and weather conditions) were monitored over time. Lagoons were sorted according to antibiotic use practice at each site, and designated as 'no-use', 'mixed-use' or 'high-use' for comparison. High-use lagoons had significantly higher detected resistance gene levels (tet(R); 2.8 x 10(6) copies ml(-1)) relative to no-use lagoons (5.1 x 10(3) copies ml(-1); P < 0.01) and mixed-use lagoons (7.3 x 10(5) copies ml(-1); P = 0.076). Bivariate correlation analysis on pooled data (n = 54) confirmed that tet(R) level strongly correlated with feedlot area (r = 0.67, P < 0.01) and 'total' bacterial 16S-rRNA gene level in each lagoon (r = 0.51, P < 0.01), which are both characteristic of large CAFOs. tet(M) was the most commonly detected gene, both in absolute number and normalized to 16S-rRNA gene level, although tet(O), tet(Q) and tet(W) levels were also high in the mixed and high-use lagoons. Finally, resistance gene levels were highly seasonal with abundances being 10-100 times greater in the autumn versus the summer. Results show that antibiotic use strategy strongly affects both the abundance and seasonal distribution of resistance genes in associated lagoons, which has implications on water quality and feedlot management practices.     

Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tcr) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tcr gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tcr genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log10 of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r2 = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 microg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tcr genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tcr gene numbers in environmental and other samples.     

Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact

Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline 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 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.     

Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil

The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.     

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.     

Diverse tetracycline resistance genotypes of Megasphaera elsdenii strains selectively cultured from swine feces

A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 microg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.     

Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria

Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H(+) antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.     

Update on acquired tetracycline resistance genes

This mini-review summarizes the changes in the field of bacterial acquired tetracycline resistance (tet) and oxytetracycline (otr) genes identified since the last major review in 2001. Thirty-eight acquired tetracycline resistant (Tc(r)) genes are known of which nine are new and include five genes coding for energy-dependent efflux proteins, two genes coding for ribosomal protection proteins, and two genes coding for tetracycline inactivating enzymes. The number of inactivating enzymes has increased from one to three, suggesting that work needs to be done to determine the role these enzymes play in bacterial resistance to tetracycline. In the same time period, 66 new genera have been identified which carry one or more of the previously described 29 Tc(r) genes. Included in the new genera is, for the first time, an obligate intracellular pathogen suggesting that this sheltered group of bacteria is capable of DNA exchange with non-obligate intracellular bacteria. The number of genera carrying ribosomal protection genes increased dramatically with the tet(M) gene now identified in 42 genera as compared with 24 and the tet(W) gene found in 17 new genera as compared to two genera in the last major review. New conjugative transposons, carrying different ribosomal protection tet genes, have been identified and an increase in the number of antibiotic resistance genes linked to tet genes has been found. Whether these new elements may help to spread the tet genes they carry to a wider bacterial host range is discussed.     

Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins

Phylogenetic analysis of tetracycline resistance genes encoding the ribosomal protection proteins (RPPs) revealed the monophyletic origin of these genes. The most deeply branching class, exemplified by tet and otrA, consisted of genes from the antibiotic-producing organisms Streptomyces rimosus and Streptomyces lividans. With a high degree of confidence, the corresponding genes of the other seven classes (Tet M, Tet S, Tet O, Tet W, Tet Q, Tet T, and TetB P) formed phylogenetically distinct separate clusters. Based on this phylogenetic analysis, a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources was developed and characterized. A pair of degenerate primers targeted all tetracycline resistance genes encoding RPPs except otrA and tet, and seven other primer pairs were designed to target the specific classes. The primers were used to detect the circulation of these genes in the rumina of cows, in swine feed and feces, and in swine fecal streptococci. Classes Tet O and Tet W were found in the intestinal contents of both animals, while Tet M was confined to pigs and Tet Q was confined to the rumen. The tet(O) and tet(W) genes circulating in the microbiota of the rumen and the gastrointestinal tract of pigs were identical despite the differences in animal hosts and antibiotic use regimens. Swine fecal streptococci uniformly possessed the tet(O) gene, and 22% of them also carried tet(M). This population could be considered one of the main reservoirs of these two resistance genes in the pig gastrointestinal tract. All classes of RPPs except Tet T and TetB P were found in the commercial components of swine feed. This is the first demonstration of the applicability of molecular ecology techniques to estimation of the gene pool and the flux of antibiotic resistance genes in production animals.     

Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages

In order to study the prevalence and diversity of tetracycline resistant lactic acid bacteria (Tc(r) LAB) along the process line of two different fermented dry sausage (FDS) types, samples from the raw meat, the meat batter and the fermented end product were analysed quantitatively and qualitatively by using a culture-dependent approach. Both the diversity of the tet genes and their bacterial hosts in the different stages of FDS production were determined. Quantitative analysis showed that all raw meat components of both FDS types (FDS-01 and FDS-08) contained a subpopulation of Tc(r) LAB, and that for FDS-01 no Tc(r) LAB could be recovered from the samples after fermentation. Qualitative analysis of the Tc(r) LAB subpopulation in FDS-08 included identification and typing of Tc(r) LAB isolates by (GTG)5-PCR fingerprinting, plasmid profiling, protein profiling and a characterization of the resistance by PCR detection of tet genes. Two remarks can be made when the results of this analysis for the different samples are compared. (i) The taxonomic diversity of Tc(r) LAB varies along the process line, with a higher diversity in the raw meat (lactococci, lactobacilli, streptococci, and enterococci), and a decrease after fermentation (only lactobacilli). (ii) Also the genetic diversity of the tet genes varies along the process line. Both tet(M) and tet(S) were found in the raw meat, whereas only tet(M) was found after fermentation. A possible relationship was found between the disappearing of species other than lactobacilli and tet(S), because tet(S) was only found in lacotocci, enterococci, and streptococci. These data suggest that fermented dry sausages are among those food products that can serve as vehicles for Tc(r) LAB and that the raw meat already contains a subpopulation of these bacteria. Whether these results reflect the transfer of resistant bacteria or of bacterial resistance genes from animals to man via the food chain is difficult to ascertain and may require a combination of cultivation-dependent and cultivation-independent approaches.     

Disappearance of oxytetracycline resistance genes in aquatic systems

The disappearance of selected tetracycline resistance genes was investigated in different simulated receiving waters to determine conditions that maximize resistance gene loss after release. Wastewater from an operating cattle feedlot lagoon was provided to four pairs of duplicate 3-L flasks, and tet(O), tet(W), tet(M), tet(Q), and 16S rRNA gene levels were monitored over 29 days using real-time PCR. Treatments included simulated sunlight with 0, 25, and 250 microg L(-1) nominal oxytetracycline (OTC) levels, respectively, and 'dark' conditions. Gene disappearance rates were always highest when light was present, regardless of OTC level. First-order loss coefficients (k(d)) for the sum of resistance genes were 0.84, 0.75, and 0.81 day(-1) for 0.0, 25, and 250 microg L(-1) OTC treatments over the first 7 days after release, respectively, whereas k(d) was 0.49 day(-1) under dark conditions, which is significantly lower (P<0.10). k(d) varied fourfold among the four individual genes, although disappearance patterns were similar among genes. Results suggest that light exposure should be maximized in receiving waters in order to maximize resistance gene loss rate after release.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

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.     

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.