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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code OD2014

2. Project title

Effect of tetracycline use in animals on appearance and dissemination of resistance to third generation tetracyclines

3. Contractororganisation(s)

University of AberdeenRowett Institute of Nutrition and HealthGreenburn RoadBucksburnAberdeenAB21 9SB

54. Total Defra project costs £ 286,061(agreed fixed price)

5. Project: start date................ 01 April 2005

end date................. 31 December 2008 (amended)

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.BackgroundAntibiotics are probably one of the most successful forms of chemotherapy in the history of medicine and they have saved many millions of lives and placed under control the majority of infectious diseases that plagued the human history for many centuries. Initially, antibiotics were extremely efficient in clearing pathogens thus leading many to think that the infectious diseases would become a problem of the past and they will be eventually wiped out from the human populations. The emergence and rapid dissemination of antibiotic resistant pathogens and especially multi-drug resistant bacteria, however, exposed our lack of knowledge about the evolutionary processes taking place in microbial ecosystems. It is evident now that the microbial populations possess enormous metabolic diversity, from which they may mobilize the protective mechanisms allowing them to withstand the selective pressures imposed by natural forces as well as human interventions such as antibiotics. Another underestimated aspect was the extent of horizontal gene exchange in the microbial world. It is clear now that bacterial populations, including pathogens, are not isolated entities confined to a particular ecosystem. Gene flow between bacteria in different ecosystems is substantial and probably the entry of antibiotic resistance genes into pathogens is one of the final events culminating in difficult-to-treat bacterial infections. In this regard, the use of antibiotics in animal production may lead to the elevated pool of antibiotic resistant genes, from which they may enter the populations of human commensals and pathogens. ObjectivesThus our main idea to be tested in this project was that in animal production systems with considerable use of antibiotics the emergence, maintenance and dissemination of antibiotic resistance genes is higher compared to organic production systems with a limited antibiotic use. The focus of this investigation was 1. Cloning and sequence analysis of tigecycline resistance (TGR) genes: construction of the swine gut

microbial metagenome, screening the metagenome for resistance to tigecycline, subcloning and sequence analysis of TGR genes

2. Genetics of TGR: sequence analysis of regions surrounding TGR genes and conjugation experiments.

3. Ecology of TGR: primer design and real-time PCR detection of TGR genes in conventional and organically grown pigs, food products, and the environment.

MethodsThe main methodology proposed was based on a metagenomic approach, when the large DNA fragments isolated directly from pig faecal DNA are cloned into BAC vectors and screened in E. coli host. It allows the study of genes that reside in hard-to-grow bacteria and therefore reveals the uncultivated diversity of functional genes, including those conferring resistance to antibiotics. Then tigecycline-resistant clones were to be analysed in various ways including transposon mutagenesis, sequencing, genetic transfer, and

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study the mechanisms of resistance. Based on sequence data obtained, we planned to design the probes and primers for environmental studies of the incidence of TGR genes. The organic farm animals that would not have received GPA were sampled on May 15, 2005 (before the ban on GPA imposed from January 2006). The conventional farm was sampled on May 10, 2006, that is, five months after the ban of growth-promoting antibiotics. In this farm, the sows are prophylactically given trimediazine during the lactation period. The comparison was made between these two groups regarding the frequency of tetracycline resistance genes circulating.

ResultsIn the course of the pig gut metagenomic analyses we sampled the equivalent of 472 bacterial genomes from the pigs in organic and conventional farms but no tigecycline resistant clones were encountered. This suggests that the background level of tigecycline resistance is very low and we performed enrichment and selection for low-frequency tigecycline-resistant fecal bacteria. Using this approach we isolated a number of TGR bacteria. The aerobes were represented by Proteus mirabilis (IC50 – 30 µg/ml), Providencia sp. (IC50 – 17 µg/ml), Pseudomonas aeruginosa (IC50 >30 µg/ml), Staphylococcus lentus (IC50 – 10 µg/ml). Facultative anaerobes were represented by lactobacilli: L. delbruckii (IC50 >30 µg/ml), L. mucosae (IC50 -15 µg/ml), L. johnsonii (IC50 -17 µg/ml), and L. nantesii (IC50 - 30 µg/ml). The anaerobic isolate was identified as Prevotella sp. (IC50 - 15 µg/ml). Thus, although the initial background resistance to tigecycline was below the detection limit of metagenomic libraries, the resistant isolates, even to high concentrations of Tg, were easily selected. To access the genetics of TGR in these isolates, we performed analyses that included functional screening of clone libraries from the isolates for TGR genes, transposon mutagenesis to knock-out the TGR genes, and potential for genetic transfer of TGR genes to the corresponding related recipients of laboratory strains. The putative mechanisms of TGR were studied in kinetic studies with the use of efflux pump inhibitors and LC-MS/MS analyses of the antibiotic intracellularly and in the supernatant, as well as monitoring for the possible products of chemical modification of Tg. Despite the numerous attempts, no TGR genes were cloned and the contingency plan that was described in the proposal as In the worst case scenario, when no TGR genes are detected at all, the efforts will be mostly focused on evaluation of the pool of “classical” tetracycline resistance genes in organic and conventional swine farms was implemented. In the comparative study of organic and conventional pig metagenomic libraries, 132 clones resistant to the first and second generation tetracyclines were found among 10,400 clones analysed while the similarly sized library of 9,000 clones from the organic pigs resulted in just 10 resistant clones. This suggests that the average load of tetracycline resistance genes per average-sized gut bacterial genome of conventional pigs is about 2.54, e.g., every gut bacterium carries between 2 and 3 tetracycline resistance genes. In organic pigs, the load of tetracycline resistance genes is much lower, about 0.22 genes per genome, thus the majority of bacteria, ca. 78%, are essentially free from tetracycline resistance genes. This brought us to the conclusion that the consequences of antibiotic usage (animals which have only received antibiotics periodically over a prolonged period of time vs. those that until recently had received growth promoting antibiotics and are still receiving prophylactic antibiotics) has tremendous consequences in terms of tetracycline resistance gene pool in gut bacteria. It results in more than a 100-fold difference in tetracycline resistance gene concentration and, probably, not only the genes conferring resistance to this particular antibiotic. At the same time, the organic metagenomic library, which was an equivalent of about 45 gut bacterial genomes sampled, still harboured a sizable number of antibiotic resistance clones (equivalent of a quarter of gut bacteria carrying a single resistance determinant) and we performed further analyses of these clones by sequencing and transposon mutagenesis to find out what genes and genetic mechanisms may contribute to their maintenance in antibiotic-free animals. Sequencing of the organic tetracycline resistome is already finished and sequencing of the corresponding resistome from conventional pigs is in progress with results pending. Below are the results of analysis of the tetracycline resistome of organic pig gut. Among the ten clones, we encountered tet(C) four times, tet(40) three times, tet(W) two times, and also observed single incidences of two novel resistant determinants, galE-1 and galE-2.

The most frequently encountered tet(C) gene was found within a sequence that displayed more than 99% nucleotide sequence identity to the well-characterized mobilisable IncQ plasmid pSC101. This may explain the high frequency of the occurrence of the tet(C) gene in the metagenomic library. Moreover, we were able to reconstruct the insert as an autonomously replicating plasmid and perform experiments with the aim of estimating the fitness cost of maintaining such a plasmid in the antibiotic-free environment. For this, we transformed E. coli ATTC® 23734 strain with the reconstituted construct from the metagenomic library and tested the freshly transformed strain against the empty isogenic strain in an antibiotic-free mixed culture for 20 generations. The ratio of the plasmid-free and plasmid-harboring cells was 86% and 14%, respectively, suggesting that the metabolic cost of carrying this plasmid is substantial. The plasmid-carrying strain was then subjected to selection by tetracycline (5 μg/ml) for 100 generations and the competition experiment was repeated. This time the ratio of plasmid-free and plasmid-carrying isogenic strains was 66% and 44%, respectively, suggesting that even this fairly short selection is sufficient for cells to 'adapt' to carry this plasmid without excessive metabolic burden.

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The genetic context of the tet(40) gene in one case was associated within a putative transposon and in another - within a putative plasmid. In the former case it was present as a single resistance determinant and in the latter was linked to tet(W). The downstream region of tet(40) possessed a short 160 bp sequence which is perfectly conserved among all currently known tet(40) genes and therefore may serve as a hot spot for excision and integration of the gene thus contributing to its mobility.

Similarly to tet(40), the flanking regions of tet(W) showed sequence conservation among the genes analyzed in this study and of others in databases. The presence of such conserved core sequences may be indicative of their role in horizontal gene transfer. The gene was also associated with putative mobile genetic elements.

Conclusions1. At present the level of resistance to tigecycline among the pig commensal microbiota is low.

2. Tigecycline-resistant bacteria can easily emerge once the corresponding selective pressure is applied.

3. The mechanisms of tigecycline resistance in our isolates are different from that described in literature.

4. No genetic transfer of tigecycline resistance was detected under laboratory conditions.

5. Animals that have only received antibiotics periodically over a prolonged period of time harbor substantially lower numbers of tetracycline resistant gut bacteria than animals that until recently had received growth promoting antibiotics and are still receiving prophylactic antibiotics.

6. When required, the enormous metabolic potential allows the microbial community to bring forth completely unexpected antibiotic resistance mechanisms such as the novel GalE mechanism found in this work.

Future workThe continuation of this work may include:

1. Clarification of TGR mechanisms in our TGR isolates. Presently, we cannot find any evidence that they are similar to the mechanisms described in literature.

2. Clarification of novel mechanisms of resistance to the second generation tetracyclines conferred by the galE genes.

3. Antibiotic selection: if the effect is limited to selection of resistance to a particular antibiotic used or the effect is broader due to co-selection and stimulation of gene transfer so the pool of other antibiotic resistance genes is also affected? The antibiotic that is used prophylactically in the conventional farm, trimediazine, consists of trimethoprim and sulfadiazine, both of which act on the same metabolic pathway and interfere with the synthesis of bacterial nucleic acids. Structurally, however, they are not related to tetracyclines and the mechanisms of co-selection may merit further investigation.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

Background

Beginning in the 1940s, the introduction of antibiotics into clinical practice led to a dramatic decrease in the occurrences of infectious diseases, and death associated with these diseases. The therapeutic action of

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antibiotics saved millions of lives by controlling and containing bacterial infections. During the last decades, however, the situation has changed dramatically because of the emergence of antibiotic resistant (ABR) infections. Currently this is one of the most challenging problems in public health care. The pharmaceutical industry responds to the challenge by introducing new antibiotics but the problem is that no new classes of antibiotics have been introduced into clinical practice during the last two decades; these are essentially derivatives of previously known antibiotics. At the stage, when some bacterial infections have become virtually untreatable by existing antibiotics, it is imperative to preserve the power of newly introduced antibiotics because the pipeline of novel antibiotics is running dry and only few alternatives exist to treat the human multiresistant infections.

At the time of their introduction in the early 50s, the first generation tetracyclines were very efficient and powerful antibiotics, active against broad range of Gram-positive and Gram-negative infections, and with few side effects. They were used broadly to treat human and animal infections as well as for non-therapeutic purposes such as growth promotion and prophylaxis in food animals. They were also widely used in aquaculture and horticulture. The consequence of such indiscriminate use of tetracyclines was that the genes conferring resistance to tetracyclines were disseminated to many bacterial genera, including pathogens, commensals, and bacteria in the environment (Chopra and Roberts 2001, Roberts, 2005). Unfortunately, the same problem resistance was encountered by the second generation semisynthetic tetracyclines such as doxycycline and minocycline, because mutations leading to the changed specificity can be easily derived from the pre-existing tetracycline resistance genes. The representative of the third generation of tetracyclines (also called glycylcyclines), tigecycline, was approved by FDA in 2005 and in 2006 in the EU. In a number of pre-approval studies, this antibiotic was shown to be effective against clinical isolates of Acinetobacter (Henwood et al., 2002), nontuberculous mycobacteria (Wallace et al., 2002), enterococci (Mercier et al., 2002; Lefort et al., 2003; Nannini et al., 2003), Staphylococcus aureus (Mercier et al., 2002; Petersen et al., 2002), Legionella pneumophila (Edelstein et al., 2003), Stenotrophomonas maltophilia (Betriu et al., 2002), and other clinical isolates (Betriu et al., 2002; Abbanat et al., 2003; Milatovic et al., 2003). These results suggest that tigecycline is a very valuable therapeutic option when dealing with multi-drug resistant infections such as MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae.

The first report on decreased susceptibility to glycylcyclines appeared in 2000 (Tuckman et al., 2000). Two veterinary Salmonella isolates carried the well-known tet(A) gene but with two mutations in the largest cytoplasmic loop of the efflux pump thus conferring novel specificity, this time against glycylcyclines. The authors also generated novel resistance in vitro, this time using another well-known tetracycline efflux pump encoded by the tet(B) gene. These results show that resistance to the third generation tetracyclines can be easily generated from the pre-existing genes conferring resistance to “old” tetracyclines. In this regard, the question “how many tet genes are around?” becomes crucial because the pool of these genes may serve as starting material for novel resistances to emerge. Obviously, the larger the pool of the “classical” tet genes, the higher is the probability of appearance of resistance to glycylcylines. Another part of the problem is the mobility of the tet genes. Both, tet(A) and tet(B), are located on transposons, Tn1721 and Tn10, respectively, and, in addition, tet(A) is located on a broad-host-range conjugative plasmid RP4. This could be potentially a problem in preventing the spread of glycylcycline resistance to other Gram-negative bacteria and other ecosystems. Tn1721, for example, was implicated in wide dissemination of tet(A) gene between the human and aquaculture environments in distinct geographical locations (Rhodes et al., 2000).

Other reports also point out that drug efflux pumps could be potential reservoirs of tigecycline resistance (Dean et al., 2003; Visalli et al., 2003). In Pseudomonas aeruginosa, the mechanism of decreased susceptibility to tigecycline is through so-called resistance nodulation division (RND) family of efflux pumps, in particular, the MexXY-OprM complex (Dean et al., 2003). However, the compensatory mechanisms of P. aeruginosa against tigecycline in the absence of MexXY-OprM included two other efflux pumps, MexAB-OprM and MexCD-OprJ, which, in norm, are “specialized” on the efflux of second generation semisynthetic tetracyclines, doxycycline and minocycline. All mutation/insertion events conferring the TGR phenotype were located either in a repressor protein (inactivation) or in regulatory regions (deletion/insertion), both leading to overexpression of the corresponding efflux pumps. In Proteus mirabilis, reduced susceptibility to tigecycline is also mediated by a drug efflux pump, encoded by the acrB homolog of E. coli (Visalli et al., 2003). AcrB is a transporter with the widest substrate specificity among multidrug pumps and can expel structurally unrelated antibiotics (including tetracyclines), disinfectants, dyes, detergents, and solvents (Nikaido, 1996; Zgurskaya and Nikaido, 2000). Since tetracyclines can co-select for the acrB gene as well, there could be a substantial reservoir of pre-selected TGR genes in places that use the first and second generation tetracyclines. Presently, no information on the lateral transfer potentials of genes encoding RND and AcrB is available. These fragmented observations lead us to think that the most likely mechanism of tigecycline resistance, at least in Gram-negative bacteria, can be the efflux of tigecycline from the cell.

Another aspect in the emerging tigecycline resistance is the possible co-selection of non-specific antibiotic resistance genes by other antibiotics (e.g., ‘older’ tetracyclines or structurally unrelated antibiotics) resulting in decreased susceptibility of microbiota to tigecycline. Since the microbial ecosystems are not isolated entities and there is always intensive horizontal gene exchange between the microbiota in different ecosystems,

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the antibiotic resistance genes that are pre-selected in one ecosystem may enter, even at low frequencies, but rapidly multiply in another ecosystem, if the corresponding selective pressure is applied. Within a microbial ecosystem, structurally different antibiotics may enhance the spread of resistance to other, structurally unrelated antibiotics. It was found, for example, that the sub-inhibitory concentrations of beta-lactams enhance the transfer of tetracycline resistance plasmids in Staphylococcus aureus by up to 1000-fold (Barr et al., 1986). Pre-growth of a donor Bacteroides strain on low concentration of tetracycline also seems to accelerate the mobilization of a resident non-conjugative plasmid by chromosomally encoded tetracycline conjugal elements (Valentine et al., 1988). The exposure of donor Bacteroides cells to low concentration of tetracycline appeared to be a pre-requisite for the excision of the CTnDOT family of conjugative transposons from the chromosome and conjugal transfer of the excised elements; virtually no transfer occurs without tetracycline induction of donor cells (Stevens et al., 1993; Whittle et al., 2002). Incorporation of tetracycline at sub-inhibitory concentrations in the mating medium also substantially enhances Tn916-mediated conjugal transfer (Showsh and Andrews, 1992). The similar stimulatory effect of tetracycline on conjugation transfer was also demonstrated for the conjugative transposon Tn925 (Torres et al., 1991). These in vitro results have been reproduced in in vivo models as well. In gnotobiotic mice, for example, the presence of a low concentration of tetracycline in drinking water increased the frequency of transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts about ten-fold (Doucet-Populaire et al., 1991). In gnotobiotic rats, selection for the resistant phenotype was the major factor causing higher numbers of Tn916 transconjugants in the presence of tetracycline (Bahl et al., 2004). Therefore, the enhancement of conjugal transfer of ABR-carrying transposons in the presence of sub-inhibitory concentration of antibiotics is not only an in vitro phenomenon but also takes place in gut ecosystems of animal models. For the first time these concerns of using antibiotics at low sub-inhibitory concentrations were addressed in Europe in 1986, when Sweden legislated totally against the use of antibiotics as growth promoters. In 1997 the EU banned avoparcin (related to the human antibiotic vancomycin) as a feed additive. In subsequent years, the EU banned virginiamycin, bacitracin, tylosin, and spiramycin, with the complete withdrawal of all low-dose growth-promoting antibiotics in 2006 (http://ec.europa.eu/food/food/animalnutrition/feedadditives/index_en.htm).

The growth-promoting use of antibiotics in the UK in 2005 was 14 tonnes of active ingredient, with no growth-promoters used in 2006 (VMD report, 2008, http://www.vmd.gov.uk/publications/annreps/annreps.htm). Still, the therapeutic use of antibiotics in animals remains substantial and the most frequenctly used antibiotic is tetracycline (Table 1). Table 1. Sales of total antimicrobial therapeutic products for animals by chemical grouping (tonnes active ingredient) during 2002 – 2007 in UK (VMD report, 2008).

Among the animal species, pig and poultry are the largest consumers of therapeutic antibiotics (Table 2).

Table 2. Total sales of therapeutic antimicrobials (tonnes active ingredient) for food-producing animals only during 2002 – 2007 in UK (VMD report, 2008).

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These considerations brought us to the idea to evaluate the pool of possible novel antibiotic resistance genes (e.g., encoding resistance to tigecycline) in a conventional pig farm that uses antibiotics for both therapeutic and prophylactic purposes. As a control, an organic pig farm with only periodic therapeutic use of antibiotics was proposed. Thus the main objective of this proposal was to evaluate the possible pool of novel antibiotic resistance genes in swineherds with different antibiotic use regimens, conventional and organic. The hypothesis to be tested was that in conventional herds the pool of antibiotic resistance genes is larger, thus increasing the probability of mutations/transpositions leading to the resistance to third generation tetracyclines. The pool of these mutant alleles was planned to be estimated through the metagenomic library analyses. Genetic transfer potential of these genes was to be evaluated by sequence analysis of the surrounding regions as well as in conjugation experiments. The possibility of dissemination of these genes was planned to be estimated through the quantitative real-time PCR detection in feed components, water, and environmental samples. Accordingly, the scientific objectives of this project were set up as follows:

1. Cloning and sequence analysis of tigecycline resistance (TGR) genes: construction of the swine gut microbial metagenome, screening the metagenome for resistance to tigecycline, subcloning and sequence analysis of TGR genes2. Genetics of TGR: sequence analysis of regions surrounding TGR genes and conjugation experiments.

3. Ecology of TGR: primer design and real-time PCR detection of TGR genes in conventional and organically grown pigs, food products, and the environment.

Methodology

Until recently, characterization of antibiotic resistance genes has been dependent on cultivation of bacteria under laboratory conditions. It is well known that only a small proportion (<1%) of bacteria from various ecosystems can be grown in culture (Amann et al., 1995) and therefore, the majority of antibiotic resistance genes circulating in ecosystems simply cannot be detected because of fastidious growth requirements. We proposed to hunt for novel tigecycline resistance genes through the analysis of metagenomes, when the large DNA fragments isolated directly from pig faecal DNA are cloned into BAC vectors and screened in E. coli host. This approach that has gained popularity in microbial ecology several years ago, does not rely on cultivation of bacteria but on cloning the environmental DNA for further structural and functional analyses (Handelsman, 2004). This approach was successfully used for characterization of some antibiotic resistance gene communities from the environment and humans (Song et al., 2005; Diaz-Torres et al., 2006; Walburton et al., 2008; Allen et al ., 2008). Another potential outcome of metagenome analysis was the possibility of discovery of novel mobile genetic elements. While a considerable knowledge exists on the occurrence and function of these elements among cultivated microbiota, the corresponding activities of uncultivated microbiota and the contribution of mobile genetic elements to in situ horizontal spread of antibiotic resistance and other genes are virtually unknown. This way we were able to access the pool of antibiotic resistance gene-tagged mobile genetic elements through the sequence analysis of surrounding regions as well as through functional analysis using standard conjugation procedure. Large-size inserts in BAC libraries allow cloning even of large conjugative transposons and plasmids

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in their entirety. Sequence analysis of the regions surrounding tigecycline resistance genes may also reveal physical linkage with other antibiotic resistance genes, if any. Other technical details of our experiments are described below.

Total DNA isolation. Thus total fecal DNA from five adult pigs in each group (conventional and organic) was used to generate metagenomic libraries based on a CopyControl™ BAC cloning kit (EPICENTRE®, USA). For this, faecal samples were collected from five organically reared and conventional adult pigs. The organic farm sampled was located north of Aberdeen (Cushnie). The fresh fecal samples were collected from five adults animals on May 15, 2005 (before the ban on GPA imposed from January 2006). We were informed that in this farm the animals are not given any antibiotic. We didn’t collect information on the use of antibiotics for disease treatment and so administration of antibiotics to these animals cannot be ruled out. The conventional farm sampled was located near Bristol and the fecal samples were collected from dry sows by our colleague from Bristol University, Dr. Marie Lewis, on May 10, 2006, that is, after the GPA ban. In this farm, the sows are prophylactically given trimediazine during the lactation period. In the case of disease the animals are prescribed antibiotics approved for the use in swine. The fresh fecal samples from these two farms were transported from the farm in the Cary Blair transport medium for anaerobic organisms (OXOID, UK). Faecal material, 5 g from each animal, was pooled, resuspended in 50 ml PBS buffer with 0.1% Tween 80, incubated for 1 h on ice with vigorous shaking and subjected to low-speed centrifugation to remove the debris (700g, 5 min). Supernatant was saved on ice and the same procedure was repeated with the pellet. Both supernatants were combined and centrifuged for 10 min at 10,000g. The pellet was washed twice with 100 ml PBS buffer and then washed twice with 100 ml TES (Tris/EDTA/Sucrose) buffer. The pellet was resuspended in 20 ml of lysis buffer (50 mM Tris pH 8, 1.25 mM EDTA pH 8, 0.5 M glucose, 20 mg/ml lysosyme, 25 μg/ml lysostaphine) and incubated for 1 h at 37ºC. Proteinase K was added to a final concentration of 100 μg/ml and the mix incubated for further 2 h at 55ºC prior to centrifugation for 5 min at 14,000g. The pellet was resuspended in 5 ml of TE buffer, n-lauryl sarcosine was added to the final concentration of 2.5% followed by incubation for 10 min 60ºC. The sample then was subjected to two phenol:chloroform:isoamyl alcohol (25:24:1) extractions. The liquid phase was transferred to a sterile 15 ml falcon tube and, after addition of RNase A at 50 mg/ml, incubated for 30 min at 37ºC. This was followed by a further phenol:chloroform:isoamyl alcohol extraction and DNA was finally precipitated in ethanol, washed with 70 % ethanol, air dried, and resuspended in 5 ml of TE buffer. High molecular weight (HMW) DNA was additionally purified using BD CHROMASPIN™-1000+TE (BD Biosciences, USA) columns according to the manufacturer’s instructions.Library construction. A CopyControl™ BAC cloning kit (EPICENTRE®, USA) was used to generate metagenomic libraries. Total gut metagenomic DNA was partially digested with HindIII (PROMEGA, UK) and the enzyme was heat-inactivated at 65ºC for 15 min. DNA was ethanol-precipitated, air-dried, and resuspended in 86 ml of deionised water. The insert DNA was ligated into the pCC1BAC™ vector, pre-cut with HindIII. Following overnight incubation at 16ºC, the ligation mix was heat-inactivated and desalted as recommended by the manufacturer. 2 ml of ligation was electroporated into 50 ml of electrocompetent E. coli Transformax™ EPI300™ cells (EPICENTRE®). Electroporation was done in a 1.0 mm cuvette using the geneZAPPER 450/2500 apparatus (IBI, USA) with the following settings: capacity - 21 μF, resistance - 200 Ohms, and voltage – 2,500 V. Clones were selected on LB agar plates containing 12.5 μg/ml of chloramphenicol (Cm). 9000 colonies were picked up using a BioRobotics BioPick colony picker (Genomic Solutions, USA) and arrayed into a 384-well microtiter plate containing 70 μl of the cryoprotective solution (2xLB medium supplemented with 10% glycerol). The cells were grown overnight at 37ºC and stored at -70ºC. Screening for antibiotic resistance was performed by printing on LB agar plates containing 5 μg/ml tetracycline (Tc), 3 μg/ml minocycline (Mn), 3 μg/ml doxycycline (Dx), and 0.75 μg/ml tigecycline (Tg). Antibiotics at these concentrations completely inhibited the growth of the negative control, host E. coli EPI300™ cells harbouring the ‘empty’ pCC1BAC™ vector.BAC DNA isolation. Five ml of LB containing 12.5 μg/ml Cm was inoculated with the biomass of a single colony and incubated overnight at 37ºC with shaking. Overnight culture was inoculated into five or ten ml of fresh medium with antibiotic and CopyControl™ Induction Solution (supplied with the cloning kit). Cultures were incubated at 37°C for 7 h with vigorous shaking (200 - 250 rpm), cells were harvested by centrifugation at 5000×g for 5 min and the bacterial pellets were stored at -20ºC prior to DNA isolation. BAC DNA was extracted using BACMAX™ DNA purification kit (EPICENTRE®) according to the manufacturer’s protocol.Transposon mutagenesis and sequencing. in vitro random insertion transposon mutagenesis was carried out using the EZ-Tn5™ <Kan-2> Insertion kit (EPICENTRE®) according to the manufacturer’s protocol. BAC DNA with transposon inserts was isolated using BACMAX™ DNA purification kit (EPICENTRE®) as recommended by supplier. Regions around the insertion sites were sequenced with KAN-2 FP1 Forward and KAN-2 RP1 Reverse primers supplied in the kit as described below.Standard molecular biology procedures. All chemicals, unless otherwise stated, were purchased from Sigma-Aldrich. Escherichia coli ATCC® 23724 (C600) and E. coli ATTC® 33694 (HB101) were purchased from the ATCC, E. coli HB101R (spontaneous rifampicin resistant mutant) was obtained during this work. E. coli strains were routinely cultured in LB medium at 37ºC. Enterococcus faecalis JH2-2 was grown on BHI medium at 37ºC. Standard PCR was performed with the BioTaqTM DNA (BIOLINE, UK) or GoTaqTM Flexi DNA (PROMEGA) polymerase kits. Annealing temperatures and extension times were optimised depending on the melting temperature of primers and the expected size of amplicons. Restriction digests were carried out using restriction

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endonucleases purchased from PROMEGA, dephosphorylation reactions utilised shrimp alkaline phosphatase (ROCHE, UK), and ligations were performed with T4 DNA ligase (ROCHE). Sequencing reactions were read on an automated 8-capillary Beckman sequencer (Beckman, UK). Chromatograms were assembled using the Lasergene 6 package. Nucleotide and translated amino acid sequences were analyzed using on-line BLAST (http://www.ncbi.nlm.nih.gov/blast), PFAM (http://www.sanger.ac.uk/Software/Pfam) and PSORT (http://psort.nibb.ac.jp) programs. MIC determination. The MICs for Tc, Dx, Mn, and Tg were determined by inoculating 0.1 ml of overnight culture grown without antibiotics into 5 ml of corresponding media containing serial dilutions of antibiotic (0 – 20 μg/ml), in triplicate. Tubes were incubated at 37ºC for 16 h and the growth was monitored spectrophotometrically at 650nm (LKB Novaspec II, Pharmacia, Sweden). The lowest concentration of antibiotic inhibiting growth by 50% compared to control gave the corresponding MIC50 value. Efflux pump inhibitor studies. The effect of efflux pump inhibitors, phenyl-arginine-β-naphthylamide (PAβN), on the IC values was assessed as described above with the addition of PAβN at a final concentration of 25 μg/ml prior to overnight incubation. Conjugation experiments. Donor and recipient strains were grown overnight in LB with appropriate antibiotics. Overnight cultures were inoculated into fresh media without antibiotics, grown to the mid-exponential phase (OD650=0.4), and cells were collected by centrifugation at 1000xg for 15 min at 18ºC. The pellets were washed twice with 0.9% NaCl (1/4 of the original volume) and resuspended in 1/10 of the original volume of 0.9% NaCl. The donor and recipient cultures were then mixed in a 1:1 ratio according to the original OD values of cultures and centrifuged briefly. Most of the supernatant was decanted, the cells were resuspended in the remaining liquid and placed on the centre of a 0.2 mm-pore-size Millipore filter disc on a LB agar plate. After incubation for 16 h at 37ºC, cells were washed off with 0.9% NaCl and the serial dilutions were plated onto selective LB agar plates with appropriate antibiotics. Potential transconjugants were selected after 24 – 48 h incubation at 37ºC. Controls were treated similarly.Mobile element rescue. Recombinant BAC DNAs were isolated and the corresponding inserts were excised, self-ligated and transformed into chemically competent E. coli JM109 cells following manufacturers’ protocol (PROMEGA). Inserts were also transformed into the electrocompetent E. coli ATCC® 23724 and E. faecalis JH2-2 strains. Electrocompetent E. coli ATCC® 23724 were prepared using protocol as described in ( Sambrook et al., 1989) with the following modifications: bacteria were cultured in 2xYT medium and 10% glycerol was used as an electroporation solution. Electrocompetent E. faecalis JH2-2 cells were prepared as described in (Cruz-Rodz and Gilmore, 1990) with modifications. In brief, the biomass of a single colony was inoculated into 2 ml of M17 broth (OXOID) and incubated overnight at 37oC without aeration. This overnight culture was diluted 1:100 with 40 ml of fresh SGM17 broth (M17 broth with 0.5M sucrose and 8% glycine) and incubated for 21 h at 37oC without aeration. The cells were harvested by centrifugation at 1000g for 10 min at room temperature, washed three times with one volume of sterile ice-cold electroporation solution (0.5 M sucrose and 10 % glycerol), and resuspended in 1/100 of the original volume of electroporation solution. Cells were split into 40 ml aliquots. Plasmid DNA was mixed with the freshly prepared electrocompetent cells in a chilled tube and transferred to a chilled 2.0 mm electroporation cuvette. Electroporation was done with the same apparatus and settings as for E. coli. Immediately after the pulse the cell suspension was mixed with 0.98 ml of ice-cold SM17 MC (M17 broth with 0.5 M sucrose, 10 mM MgCl2 and 10 mM CaCl2) and kept on ice for 5 min. Subsequently cells were incubated at 37oC for 2 h without shaking. 100 ml of the mix was plated on SR agar medium (Cruz-Rodz and Gilmore, 1990) with appropriate antibiotic and incubated at 37oC for 48 h. Competition/fitness cost experiments. Rescued plasmids were electroporated into E. coli ATCC® 23724 cells and were tested against the isogenic background immediately or after selection with antibiotic. For the latter, plasmid-bearing cells were passaged for ~100 generations in LB in the presence of 5 μg/ml of tetracycline. For the competition experiment, the cultures were initially grown in 5 ml of LB overnight, without antibiotic in the case of E. coli ATCC® 23724 and with 5 μg/ml of tetracycline in the case of the isogenic strain carrying a plasmid. 50 μl of each overnight culture (~5x107 cells) were mixed together in 5 ml of antibiotic free LB medium and grown 27 h at 37oC with shaking. Serial dilutions were plated on LB agar plates, with (5 μg/ml) and without tetracycline to assess the ratio of plasmid-free and plasmid-carrying cells.LC-MS/MS. Triplicate cultures were set up by inoculating 5ml LB containing appropriate antibiotics with clones containing pBAC (negative control), galE-1, galE-2, tet(W), tet(X) or tet(C). After overnight growth, cultures were spun for 10 min at 5,400xg. The cleared supernatant was removed and 100 μl mixed with 900 μl of the acetonitrile in a 1.5 ml eppendorf tube. The remaining pellets were resuspended in 300 μl of water and sonicated for 10-15 sec (amplitude 9 microns). The resulting cell lysates were centrifuged at 4°C for 15 min at maximum speed to remove the cell debris. The cell lysate supernatant was removed and 100 μl mixed with 900 μl of the acetonitrile. Following addition of the acetonitrile the samples were mixed vigorously on whirlimixer for 15 sec, incubated for 10 min at room temperature and then spun down for 10 min at maximum speed. Final supernatants were transferred to fresh 1.5 ml eppendorf tubes. The acetonitrile contained the internal standard, demeclocycline (DMCTC), which was added at to a final concentration of 500 pg/μl prior to mixing with the experimental samples, to allow quantification of the tetracycline in each fraction.The concentration of antibiotics in each fraction was analysed by Liquid Chromatography – tandem Mass Spectrometry (LC-MS/MS) at room temperature, using an Agilent 1100 HPLC system (Agilent Technologies, Wokingham, UK) with a Jupiter 5 µm, C18 column (Phenomenex, Macclesfield, UK) and an organic mobile phase. Mobile phase solvents were a mixture of (A) and (B), where (A) was water containing 0.1% formic acid

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and (B) was acetonitrile containing 0.1% formic acid. The gradient programme started at 95% of (A) held for 5 minutes, followed by 3% of (A) held for 5 minutes, then at 95% of (A) and held for 4 min in preparation for the next injection. The flow rate was 300 µl/min and the injection volume was 5 µl. The LC eluent was directed into, without splitting, a Q-Trap triple quadrupole Mass Spectrometer (Applied Biosystems, Warrington, UK) with a Turbo Ion Spray source fitted in positive ion mode, for the detection and quantitation of the antibiotics. Antibiotics were detected using multiple reaction monitoring (MRM) transitions, which were calculated by infusing standards directly into the mass spectrometer, via a syringe pump, at a concentration of 5 ng/µl. Data was normalised according to the detection of antibiotics in the pGEM-T control, and tabulated as the mean of 6 replicates, from two independent growth experiments.Accession numbers. Sequence data generated in this work were deposited in GenBank under accession numbers: clone 7, FJ157999; clone 1, FJ158000; clone 2, FJ158001; clone 3, FJ158002; clone 9, FJ158003; clone 10, FJ158004; and clone 15, FJ158005. Other sequences will be deposited once the sequencing work is completed.

ResultsOrganic swine faecal metagenomic library. Based on restriction digest of randomly chosen clones, the size of inserts ranged from 1.5 kb to 40 kb, with the average insert size in the library being ~15 kb. Thus the library with 9,000 clones analyzed represented approximately 45 bacterial genomes (assuming an average bacterial genome size of 3 Mb). Ten antibiotic resistant clones were isolated and purified on higher antibiotic concentrations, up to 10 μg/ml, to confirm the resistant phenotype. Most of the TcR clones (designated as 2, 3, 4, 5, 6, 7, and 10) were able to grown on 10 μg/ml of all three antibiotics tested. Clone 1 was able to grow on 10 μg/ml of Tc, but on only 5 μg/ml of Mn and Dx. Clones 9 and 15 were Tc sensitive but exhibited MnRDxR and DxR phenotypes, respectively. No clones resistant to tigecycline were found in this library.

Conventional swine faecal metagenomic libraries. The BAC library constructed from the fecal samples of five conventional adult pigs consisted of 10,400 clones with the average insert size 15kb, thus corresponding to the sampling of approximately 52 bacterial genomes. In this library, 132 resistant clones were distributed as follows: 98 triple resistances (Tc, Dx, and Mn resistant), 25 double resistances (13 – TcDx and 12 – DxMn resistant), 9 single resistances (7 clones resistant to Dx and 2 Mn-resistant clones). No Tg-resistant clones were encountered. Additional libraries were made in an attempt to clone tigecycline resistance gene(s) through the metagenomic approach. These libraries corresponded to screening of ca. 375 bacterial genomes for tigecycline resistance but no such clones were found. In total, we analyzed the equivalent of 427 bacterial genomes from the gut of conventional pigs and this suggest that the frequency of tigecycline resistance genes is below the detection limit of metagenomic libraries, e.g., <0.23% per genome. Another factor contributing to such low frequencies could be the heterologous expression problem in E. coli cells. Thus we decided to employ the back-up plan, which involved enrichment and selection for low-frequency tigecycline-resistant fecal bacteria.

Enrichment experiments. Fecal slurry from conventional pigs was inoculated in a 1:10 ratio into LB for aerobic or M2GSC for anaerobic growth and enrichment for Tg resistance was performed by growing the cultures with different concentrations of Tg. Strains isolated from these enrichment cultures were purified and characterized by sequencing the 16S rRNA genes and by IC determination. Aerobic Tg-resistant isolates were represented by Proteus mirabilis (IC – 30 µg/ml), Providencia sp. (IC – 17 µg/ml), Pseudomonas aeruginosa (IC >30 µg/ml), Staphylococcus lentus (IC – 10 µg/ml). Facultative anaerobes were represented by lactobacilli: L. delbruckii (IC >30 µg/ml), L. mucosae (IC -15 µg/ml), L. johnsonii (IC -17 µg/ml), and L. nantesii (IC - 30 µg/ml). The anaerobic isolate was identified as Prevotella sp. (IC - 15 µg/ml). Thus, although the initial background resistance to tigecycline was below the detection limit in metagenomic libraries, the resistant clones, even to high concentrations of Tg, were easily isolated. The representatives of each taxonomic group was chosen for cloning of tigecycline resistance genes as well as to assess the potentials of genetic transfer to other bacteria.

Screening of libraries from tigecycline-resistant isolates. Several types of libraries were constructed from seven of the TgR isolates selected from enrichment cultures (Proteus mirabilis, Providencia sp., Staphylococcus lentus, L. delbreuckii, L. mucosae, L. johnsonii, and Prevotella sp.). The small insert libraries (2-4kb) in E. coli using pUC19 vector (a high-copy number plasmid), as well as small insert libraries in pSMART-LC (low-copy number plasmid with transcription terminators) and pSMART-HC (high-copy number with transcription terminators) vectors. No TgR clones were detected, however, in any of the constructed libraries. To confirm that this was not due to the lack of expression of foreign genes in E. coli, the same libraries were tested for resistance to tetracycline. All the original TgR isolates were also resistant to tetracycline (Tc), as well as a selection of other antibiotics. Tetracycline resistant colonies were detected for 6 of the 7 isolates, illustrating that these genes were easily expressed in E. coli host. We concluded, therefore, that the resistance mechanisms in these TgR bacteria may require products of more than one gene. Mechanisms that have now been described in the literature often require the participation of up to three gene products, all of which contribute to the TgR phenotype. To address this technical challenge we considered several approaches; the first involved constructing metagenomic libraries from our TgR isolates using the pBAC vector system, which is capable of supporting large DNA inserts up to 100kb in size. However, recent information from the Klebsiella pneumonia sequencing project suggests that, at least in this bacterium, several genes conferring the TgR phenotype are located in different places within the genome. Even with the use of

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metagenomic libraries, cloning of such large DNA inserts including all genes involved in Tg R genes would be impossible. Hence our second and favoured research approach involved using the technique of transposon mutagenesis to firstly inactivate and then locate the TgR determinants. This strategy has been successfully used in other clinical bacteria and should identify the novel mechanisms of TgR.

Transposon mutagenesis. Because the attempt of cloning Tg-resistance genes from Tg-resistant bacterial isolates was not successful, transposon mutagenesis was used to locate the TgR determinants. The Proteus sp. isolate was taken as a first target for transposon mutagenesis using the EZ::TN <R6KγoriI/KAN-2> Tnp Transposome kit (EPICENTRE). The R6Kγori makes this transposon useful for “rescue cloning” of genomic DNA into which the transposon has been randomly inserted. In brief, Proteus sp. cells were electroporated with the transposome. The library of KanR Proteus containing the R6KγoriI/KAN-2 transposon was successfully created and almost 2000 clones were picked (5 x 384-well plates) and stored at -80˚C for further analysis. Subsequently the library was replica plated on tigecycline or kanamycine plates to find the KanRTgS phenotype, in such clones the transposon would be integrated into the TgR determinant thus rendering the clones sensitive to the antibiotic. Four clones with the increased sensitivity to Tg were identified. Total genomic DNA was isolated from these clones. The DNA was subsequently fragmented, end-repaired, self-ligated and electroporated to E. coli cells that express the pir gene product. When selected on Kan-containing plates only cells containing <R6KγoriI/KAN-2> transposon should grow. Unfortunately, we were unable to achieve the “rescue cloning” of the genomic DNA from TgS Proteus sp. and thus could not identify the locus into which the transposon integrated. We further focused on potential mobility of TgR genes.

Conjugation experiments. The TgR proteobacteria, Proteus mirabilis and Providencia sp., were tested in filter matings with several E. coli recipients with different restriction-modification and recombination genotypes. We did not observe the transfer of TgR to E. coli suggesting that the rates may be very low and most likely the deterimants do not reside on mobile elements.

Efflux pump inhibitor studies. Efflux pump inhibitors had no effect on the resistance phenotypes of P. mirabilis, S. lentus, or Providencia sp., thus the mechanism of resistance remains unidentified. The published works on the mechanisms of TgR in clinical isolates of P. mirabilis suggest the involvement of an efflux pump - AcrAB-TolC system (Visalli et al., 2003; Ruzin et al., 2005). Our TgR P. mirabilis and Providencia sp. isolates were tested for presence of the AcrB component of this system using specific PCR primers and the acrB gene was detected. Despite the fact that this part of the efflux pump system is known to be responsible for the Tg R phenotype and is detected in our isolates, the efflux pump inhibitor results indicate that it is not involved in TgR in these isolates. On the contrary, the efflux pump inhibitors substantially increased the succeptibility of TgR lactobacilli (L. delbreuckii, L. mucosae and L. johnsonii) to the antibiotic suggesting the involvement of efflux pump mechanisms in the TgR

phenotype. Some of our lactobacilli are the same species as described in databases (e.g., Lactobacillus delbrueckii) and we scanned the genomic data for the presence of the known tigecycline resistance genes such as encoding AcrAB, AdeIJK, AdeABC, MATE, and MexX. No homologous genes were found. We also designed a set of primers targeting acrB, adeA and mepA (MATE) and found no amplification signals either. Therefore, we concluded from the efflux pump inhibitor studies that: (i) TgR phenotype of our P. mirabilis, Providencia sp. and S. lentus isolates does not involve efflux pumps and most likely represents a novel mechanism of resistance; (ii) in lactobacilli, the efflux pumps are involved in resistance to tigecycline but they are different from the known AcrAB, AdeIJK, AdeABC, MATE, and MexX. The next step in clarification of mechanisms of tigecycline resistance was to develop LC-MS/MS technique to monitor the concentration of tigecycline in the medium and intracellularly as well as possible products of its modification if any.

LC-MS/MS experiments. We optimized the method for tigecycline concentration determination using LC-MS/MS technique. Data obtained in kinetic experiments, however, were not consistent, with broad variations in tigecycilne concentration between the replicates.

tet(X) detection. Since the tet(X) gene was implicated in tigecycline resistance (Moore et al., 2005), we designed PCR primers for its detection. We detected the presence of tet(X) in total fecal DNA from pigs from organic and conventional farms (same DNA preps were used in library construction).

As it was written in the original proposal, Potential delays could be expected only with Objective 1. These potential delays and ways to overcome them are discussed in Section 11 (see the metagenomic library construction section). No delays are expected in accomplishing Objectives 2 and 3.Objectives 2 and 3 are dependent on successful accomplishment of Objective 1. Once Objective 1 is accomplished, the two other objectives can be done independently. In this work we encountered exactly this problem with Objective 1: Cloning and sequence analysis of tigecycline resistance (TgR) genes: construction of the swine gut microbial metagenome, screening the metagenome for resistance to tigecycline, subcloning and sequence analysis of TgR

genes. Since no tigecycline resistance genes were cloned from the total fecal metagenomic DNA and tigecycline-resistant bacterial isolates, we implemented the approach, which in the original proposal was described as In the worst case scenario, when no TGR genes are detected at all, the efforts will be mostly focused on evaluation of the pool of “classical” tetracycline resistance genes in organic and conventional swine farms (see Approach 3).

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This methodology is well developed by the PI and preliminary data with swine farms in Illinois, USA suggest that no problems executing this part are expected. Thus the fecal metagenomic libraries from organic and conventional pigs were tested for the pool of genes encoding resistance against the first and second generation tetracyclines. Sequencing of the organic tetracycline resistome is already finished and sequencing of the corresponding resistome from conventional pigs has been commissioned and the results are pending. Below are the results of analysis of the organic pig tetracycline resistome.

Resistance to the first and second generation tetracyclines. Recombinant DNAs from resistant clones of the organic farm library were initially end-sequenced with vector-specific primers. Four of the 10 clones (2, 4, 5 and 6), with insert sizes of 9.3 kb, were identical and carried the tet(C) gene. Thus only one of them (clone 2) was sequenced to completion. Two out of the six remaining resistant clones (3 and 7), with the insert sizes of 9.7 kb and 3.7 kb, respectively, encoded identical tet(W) gene sequences on one end of the inserts. Thus the larger clone 3 was chosen for further sequence analyses by genome walking. End-sequencing of the remaining clones (1, 9, 10, and 15) did not reveal the presence of antibiotic resistance determinants and transposon mutagenesis was used to identify the genes responsible for the resistant phenotype. Antibiotic-sensitive mutants were sequenced from the ends of EZ-Tn5™ <Kan-2>. In the 4.8 kb insert of clone 1 the tet(40) gene, encoding the efflux of tetracycline from cells, was found, and the 9.2 kb insert of clone 10 harboured the tet(W) gene. The sequences surrounding Tn5 in clones 9 (4.9 kb) and 15 (9.5 kb) had no resemblance to any known tetracycline resistance genes and presumably encoded novel mechanisms of resistance.

tet(40) is a new TcR gene which was recently identified in the human faecal bacterium Clostridium saccharolyticum K10 and in clones from a human faecal metagenomic library (Kazimierczak et al., 2008). Comparison of the two translated tet(40) genes with database entries gave a 98% (clone 1) and 99% (clones 3 and 7) similarity with the Tet(40) protein (GenBank accession numbers AJ295238 and AM419751). The previously described tet(40) gene was located immediately downstream of the mosaic tet(O/32/O) gene (Kazimierczak et al., 2008) while in this study tet(40) was present as a single tetracycline resistance determinant in clone 1 and 1.2 kb downstream from tet(W) in clones 3 and 7(Fig. 1). In these clones, the tet(W) ORF had been interrupted during the cloning step and the tetracycline resistance phenotype was apparently achieved through the expression of tet(40) (Fig. 1). Analysis of the regions flanking tet(40) revealed no conservation in upstream sequences between clones 1 and 3, and these sequences were also different from the database sequence of tet(40) (GenBank accession numbers AJ295238 and AM419751), apart from a short 16 bp (clone 3) or 18 bp (clone 1) sequence containing the ribosome binding site immediately upstream of the start codon. The 160 bp region downstream of tet(40) was 100% identical not only between these clones but also with the corresponding region of the previously described tet(40) gene ((Kazimierczak et al., 2008).

The tet(W) gene was found in its entirety in clone 10, as an interrupted ORF in clone 3, and as a partial sequence in clone 7 (Fig. 1). Structurally, these genes were 96 - 99% identical to the previously described tet(W) gene (Scott et al., 2000). The 657 bp region upstream from the start codon in clone 10 was 99% identical to the corresponding regions in tet(W) described in Arcanobacterium pyogenes strains OX9 and OX4 (GenBank accession numbers DQ519394 and DQ517519; (Billington and Jost, 2006); Fig 1) while the similarity with the corresponding regions in six different genera of gut commensal bacteria was slightly lower at 93 - 96% (Kazimierczak et al., 2006). The 95 bp region following the tet(W) stop codon in clone 10 exhibited a 100% conservation with the corresponding regions in the A. pyogenes strains, OX4, OX9, and 5278-99 (GenBank accession number DQ519395, (Billington and Jost, 2006); Fig 1) and 97% similarity with the corresponding region of tet(32) in Streptococcus salivarus strain FStet12 (GenBank accession number DQ647324, (Patterson et al., 2007a). No similarity was detected to the tet(W) downstream regions conserved in human gut commensals (Kazimierczak et al., 2006). The G+C content of tet(W) was ~53%, which is not significantly different from the other OFRs in clones 3 and 7 but substantially higher than in clone 10 ORFs suggesting that a possible horizontal gene transfer event may have occurred in the latter case. Comparison of the regions flanking the tet(W) gene in clone 3 with database entries showed no conservation of the downstream region but the 351 bp upstream region was 94 – 100% similar to the corresponding regions in intestinal (Kazimierczak et al., 2006) and other bacteria carrying tet(W). As reported previously, the putative regulatory region, which contains a 14 aa leader peptide involved in transcription attenuation, is present in the 330 bp upstream region of tet(W) (Melville et al., 2004) (Fig. 1).

Novel genes conferring resistance to second generation tetracyclines. In clones 9 and 15 from the organic pig metagenomic library the transposon interrupted ORF2 (galE-1) and ORF4 (galE-2), respectively. The translated galE-1 encoded a 345 aa protein that shared 71% and 68% sequence identity with putative UDP-glucose 4-epimerases (UGE) from Bacteroides vulgatus ATCC8482 (YP_001301108) and Bacteroides thetaiotamicron VPI-5482 (AEM02206-08 Version 1_NP_809536), respectively. The protein product of galE-1 was therefore called UGE-1. The translated 342 aa sequence of galE-2 from clone 15 shared 72% and 65% sequence identity with proposed UDP-glucose 4-epimerases from Clostridium sp. strain SS2/1 (ZP_02440543) and Bacillus subtilis subsp. subtilis strain 168 (NP_391765). The protein product of galE-2 was therefore named UGE-2. galE-1 and galE-2 shared 56.2% sequence identity at the nucleotide level.

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The full ORFs of galE-1 and galE-2, together with the upstream regions, were amplified by PCR and cloned into the pGEMT-Easy vector. Since no Mn and Dx resistant phenotypes were expressed in this high-copy number vector, the inserts were re-cloned from pGEMT-Easy into the low-copy number BAC vector. This time the antibiotic resistance phenotype was expressed suggesting that the presence of a single ORF of galE-1 or galE-2

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Fig. 1. The structure of inserts conferring antibiotic resistance in the organic swine faecal metagenomic library. ORFs are represented by arrows. The shaded arrows within clone 3 indicate the area identical to the sequence of clone 7. The DNA% G+C content is shown above the ORFs and the percentage of similarity to database entries (where appropriate) is in brackets below the ORFs. Conserved regions are marked with: * - 158bp region downstream of tet(40); ** - 350bp within the core 657bp region upstream of tet(W); *** - 95bp region downstream of tet(W) as in A. pyogenes; **** - 657bp core region upstream of tet(W).

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was sufficient to confer antibiotic resistance. The IC50 for Mn and Dx in the case of galE-1 was 11 μg/ml and the IC50 for Dx in the case of galE-2 was 11 μg/ml. At present the mechanisms of resistance conferred by galE-1 and galE-2 are not clear since the experiments with efflux pump inhibitors and LC-MS/MS studies were not conclusive.

Genetic context of tetracycline resistant clones. In clones 1, 3, 7 and 10, apart from the known tetracycline resistance genes, the majority of other ORFs demonstrated weak or no similarity with database sequences (Fig. 1). The easily identifiable ORFs exhibited similarity with various mobile elements. ORF1 in clone 1 was similar to a transposase (YP_001707264), with a conserved transposase DDE domain at the C-terminus (Fig. 2). This D115(X77)D192(X47)E239 triad is also a conserved motif in transposases of IS982 and IS4 families of insertion sequences (12, 24) (Fig. 2). In clone 3, ORF7 was similar to the mobilization protein from Clostridium leptum DSM753 (ZP_02081735) and ORF8 - to the putative replication protein from Staphylococcus sciuri (NP_899174). Clone 7 (3.7 kb) was cloned as part of clone 3 (Fig. 1).

Fig. 2. Translated transposase sequences in clones 1 (A) and 10 (B). The residues in the DDE motif are indicated in boldface, characteristic residues upstream and/or downstream of the triad are in boldface italic, and regions N2, N3 and C1 are underlined.

In clone 10, ORF3 was similar to the transposase from Lactobacillus helveticus DPC4571 (YP_001576992) and also contained the conserved DDE domain (Haren et al., 1999; Rezsohazy et al., 1993) (Fig. 2). The transposase homologues encoded by ORF3 in clone 10 and ORF2 in clone 1 shared only 20% identity.

In clones 1, 3 and 7 the upstream regions of tet(40) were not conserved except the 160 bp downstream region that was 100% identical between the clones and database entries (AJ295238 and AM419751). Apart from this limited conservation, the genetic background of clones 1 and 3 is completely different: transposase-like ORF2 in the former and mobilization and replication protein-resembling ORFs in the latter suggest that tet(40) may reside on different mobile elements.

Similarly to tet(40) the two tet(W) genes also resided in clones with different genetic background: in the probable plasmid in clone 3 and probable transposon in clone 10 (Fig 1). Thus tet(40) and tet(W) possessed the multiple dissemination/maintenance capabilites due to location on various mobile genetic elements.

Clone 9 encodes four ORFs (Fig. 1) and one of them, ORF2, was identified as a resistance gene by transposon mutagenesis. ORF1 showed 63% conservation with fructose-1,6-biphosphatase (Fujita et al., 1998) from Bacteroides fragilis YCH46 (YP_099100) while ORF3 and ORF4 displayed no discernible similarity to database entries. The genetic background in this case had no recognisable horizontal transfer capabilities.

Eight ORFs were identified in clone 15 with ORF4 encoding resistance to Dx. The closest similarity for ORF 1, 2, 3 and 5 was observed for proteins from Clostridium phytofermentans ISDg: ORF1 was similar to the coproporphyrinogen III oxidase (YP_001557676; (Sofia et al., 2001); ORF2 - to the beta-lactamase domain protein (YP_001557675); ORF3 - to the (p)ppGpp synthetase I of SpoT/RelA family of proteins (YP_001557674); and ORF5 - to the MATE efflux family protein (YP_001559167). The order of the first three ORFs was also conserved similarly to C. phytofermentans ISDg. The closest matches for ORF6 to ORF8 were the hypothetical proteins from C. thermocellum ATCC27405: ORF6 - to dihydrofolate reductase (YP_001037651), ORF7 - to thymidylate synthase (YP_001037652), and ORF8 - to metallo-beta-lactamase (YP_001037039). Although no mobility potential was detected, the genetic background in this clone is quite interesting because it carries multiple antibiotic resistance genes.

Clones 2, 4, 5 and 6 were identical and 99.7% similar to the IncQ plasmid pSC101 from Salmonella typhimurium (NC_002056; Bernardi and Bernardi, 1984). The differences included 28 single nucleotide polymorphisms as well as the presence of a 54 bp insertion sequence between the coordinates 7952 and 8005 in

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clone 2 compared to pSC101. Genetic transfer potential of this plasmid is well known as well as the mechanisms that prevent its loss from the host cell and stable maintenance in the absence of antibiotic selection (Ingmer et al., 2001).

Clone 3 also resembles a linearised plasmid: both insert ends carry parts of tet(W), interrupted by HindIII digest during the cloning step (Fig. 1). Thus we attempted to rescue to restore the original structures from clones 2 and 3.

Mobile element rescue experiments. Clones 2 and 3 were partially digested by HindIII and the appropriate bands were purified from agarose gels, self-ligated, and transformed into competent cells. A completely functional plasmid was recovered from clone 2 by transforming E. coli JM109 and the structure was verified by restriction and PCR analyses (Fig. 3).

In the case of clone 3 neither E. coli JM109 nor E. coli ATCC® 23724 produced any TcR transformants. Since sequence analysis had suggested that the plasmid may originate from a Gram-positive host, we also tested a Gram-positive recipient, E. faecalis JH2-2, but without success. Thus this plasmid may have a different host range precluding its replication/expression in the hosts we tested.

Competition experiments. The antibiotic resistance genes in the intestinal bacteria of organic pigs are not subjected to any obvious selective pressure but, nevertheless, they are stably inherited. This may indicate that the fitness cost of carrying these genes has been ameliorated over time and does not cause an excessive metabolic burden in the apparently antibiotic-free environment. To model this event, we transformed E. coli ATTC® 23734 strain with the rescued plasmid pKK2.1 and tested the freshly transformed strain against the empty isogenic strain in an antibiotic-free mixed culture for 20 generations. The ratio of the plasmid-free and plasmid-harbouring cells was 86% and 14%, respectively, suggesting that the metabolic cost of carrying this plasmid is substantial. The plasmid-carrying strain was then subjected to selection by tetracycline (5 μg/ml) for 100 generations and the experiment was repeated. This time the ratio of plasmid-free and plasmid-carrying isogenic strains was 66% and 44%, respectively, suggesting that even this fairly short selection is sufficient for cells to 'adapt' to carry this plasmid without excessive metabolic burden.

Fig. 3. The map of reconstructed plasmid from clone 2. ORFs are represented as solid arrows, and dotted arrows show the location of the primers used in PCR. Restriction sites tested are indicated.

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DiscussionThe question of what factors contribute to the emergence, maintenance and dissemination of antibiotic resistance genes remains contradictory (Aminov and Mackie, 2007). It is generally accepted that the main driving force is the use and overuse of antibiotics, which imposes selective pressure specific for a given antibiotic (Levy and Nelson, 1998; Chee-Sanford et al., 2001; Swartz, 2002). At the same time, antibiotic resistance genes may persist in apparently antibiotic-free environments (Alonso et al., 2001; Pallecchi et al., 2007; Thakur et al., 2007). In this work we attempted to test the hypothesis that resistance to novel antibiotics may more likely appear in agricultural settings with more frequent use of antibiotics.

Two swine production systems, one with the conventional use of antibiotics (presently, for prophylactic and therapeutic purposes) and another, an organic farm with intermittent therapeutic use of antibiotics, were chosen for this study. The fecal metagenomic libraries were made from five animals in each group and screened for resistance to the novel antibiotic, tigecycline, which was approved for clinical use in the EU in 2006. Despite that in total we sampled the equivalent of 472 bacterial genomes, no tigecycline-resistant clones were observed suggesting that the probability of encountering a TgR gene is below 2x10-3 per randomly sampled average-sized bacterial genome. Similarly, The Tigecycline Evaluation and Surveillance Trial (TEST) concluded that the level of tigecycline resistance among clinical isolates remains low and it has excellent in vitro activity against a broad spectrum of bacteria, including resistant strains (Bouchillon et al., 2008).

While the background frequencies of resistance to tigecycline are fairly low, the resistant isolates can be easily obtained when the selection by tigecycilne is applied to the gut microbiota. This way we obtained aerobic Tg-resistant isolates such as P. mirabilis, Providencia sp., P. aeruginosa, S. lentus: lactobacilli (L. delbruckii, L. mucosae, L. johnsonii, and L. nantesii); as well as an anaerobic isolate, Prevotella sp. Interestingly, the most frequently isolated resistant bacteria were lactobacilli, represented by the species typically inhabiting the gut. The level of resistance to tigecycline was high and even IC50 values were much higher than the typical MIC90 values established for pathogenic bacteria (</=2mg/L against Acinetobacter spp., K. pneumoniae, E. coli, Enterobacter spp. and Serratia marcescens and </=0.25mg/L against S. aureus, E. faecalis and Enterococcus faecium) (Bouchillon et al., 2008). Unfortunately, multiple attempts to gain insights into the genetic background of tigecycline resistance through the cloning, transposon mutagenesis and conjugation transfer approaches were not fruitful. When all possibilities were exhausted, the contingency plan in the proposal, which had been described as In the worst case scenario, when no TGR genes are detected at all, the efforts will be mostly focused on evaluation of the pool of “classical” tetracycline resistance genes in organic and conventional swine farms, was implemented.

When analysing pig gut metagenomic libraries, 132 clones resistant to the first and second generation tetracyclines were found among the 10,400 clones from the conventional animals while the similarly sized library of 9,000 clones from the organic pigs resulted in just 10 resistant clones. This suggests that the average load of tetracycline resistance genes per average-sized gut bacterial genome of conventional pigs is about 2.54, e.g., every gut bacterium carries between 2 and 3 tetracycline resistance genes. In organic pigs, the load of tetracycline resistance genes is much lower, about 0.22 genes per genome, thus the majority of bacteria, ca. 78%, are essentially free from tetracycline resistance genes. This brings us to the conclusion that the outcomes of different agricultural practices may result in considerable differences regarding the pool of antibiotic resistance genes circulating in the gut ecosystem of food animals. In particular, the microbiota of conventionally raised pigs possess substantially higher numbers of tetracycline resistant bacteria than organic animals and this probably is not limited by resistance to this particular antibiotic. Interestingly, the antibiotic that is used prophylactically in the conventional farm is structurally unrelated to tetracyclines and one of the possible explanation for the higher levels of tetracycline resistance genes in the conventional animals may include the process of co-selection.At the same time, the organic metagenomic library, which was an equivalent of about 45 gut bacterial genomes sampled, still harboured a sizable number of antibiotic resistance clones (equivalent of a quarter of gut bacteria carrying a single resistance determinant) and we performed further analysis of these clones to find out what genes and genetic mechanisms may contribute to their maintenance.

During the screening for resistance to tetracyclines in organic pigs we encountered tet(C) four times, tet(40) three times, tet(W) two times, and also observed single incidences of two novel resistant determinants, galE-1 and galE-2. Additionally, it is likely that not all tetracycline resistance genes were detected because of limitations imposed by heterologous gene expression in the E. coli host. Thus our results suggest that even in organic animals the pool of antibiotic resistance genes remains substantial.

The most frequently encountered tet(C) gene was found within a sequence that displayed more than 99% nucleotide sequence identity to the well characterised mobilisable IncQ plasmid pSC101 (Bernardi and Bernardi, 1984). This may explain the high frequency of the occurrence of the tet(C) gene in the metagenomic library. Moreover, we were able to reconstruct the insert as an autonomously replicating plasmid and perform experiments with the aim to estimate the fitness cost of maintaining such a plasmid in the antibiotic-free environment. As expected, the fresh E. coli transformant exhibited low competitiveness but following the limited antibiotic selection for plasmid carriage for about 100 generations the fitness cost was substantially reduced.

SID 5 (Rev. 3/06) Page 18 of 22

Although we cannot directly extrapolate the results of these in vitro competition experiments back to the original host and ecosystem, nevertheless this finding may be a crucial explanation for the persistence of this antibiotic resistance gene in the antibiotic-free environment.

One of the recently discovered tetracycline resistance genes, tet(40), was encountered on three occasions. In the previous work, it was identified in a human bacterial isolate and in the human gut metagenomic library, and in both cases it was linked to another tetracycline resistance gene, the mosaic tet(O/32/O) and present on the conjugative transposon TnK10 (Kazimierczak et al., 2008). The three separate incidences of its cloning in the organic pig gut metagenome suggest that the gene is common among pig intestinal bacteria as well. Moreover, we also encountered two different genetic contexts for this gene, one on a putative transposon and another on a putative plasmid. In the former case it was present as a single TcR gene and in the latter was linked to tet(W). Although we were unable to reconstruct clone 3 as an autonomously replicating plasmid, most probably due to the absence of a suitable host, its structure strongly suggests that originally it was a 9.7 kb plasmid, which was linearized during the cloning step. The location on mobile genetic elements may contribute to the dissemination and maintenance of tet(40) in different ecosystems. Also, the downstream region of tet(40) possessed a short 160 bp sequence that is perfectly conserved among all currently known tet(40) genes. The G+C content of the inserts with tet(40) was fairly high in this study (45 – 58%) compared to C. saccharolyticum, a low G+C bacterium. Thus it appears that tet(40) and its immediate flanking regions originated from a common source and disseminated to other bacteria in different environments. The short conserved sequence may serve as a hot spot for excision and integration of the gene thus contributing to its mobility and maintenance.

tet(W) is one of the most widely disseminated tetracycline resistance genes (Roberts, 2005; Patterson et al., 2007b) and the conservation of its flanking regions was recently described in multiple species of gut bacteria (Kazimierczak et al., 2006). In this study we found two incidences of tet(W) - the clone 3 tet(W) which had higher identity with the genes described in (Kazimierczak et al., 2006) and the clone 10 tet(W) which was closer to the genes described in (Billington and Jost, 2006). The flanking sequences of the tet(W) genes described here were different. The DNA% G+C content of tet(W) in clone 10 is substantially higher (53.1%) than the rest of the clone (<37%) indicating a possible horizontal gene transfer event. Here, similarly to tet(40), the tet(W) flanking regions showed sequence conservation among the genes analysed in this study and of others in databases. This especially concerns tet(W)-4 from A. pyogenes strains OX9, OX4 and 5278-99 (GenBank accession numbers DQ519394, DQ517519 and DQ519395, respectively. The presence of such conserved core sequences may be indicative of their role in horizontal gene transfer. Similarly to tet(40), the genetic context of tet(W) was associated with potential mobility, through two different mechanisms, plasmid and transposon-mediated.

Two novel genes, galE-1 and galE-2, that confer resistance to the second generation tetracyclines were encountered during our analysis of the organic pig faecal metagenomic library. Interestingly, the resistance phenotype was expressed only when a low copy number of the genes was present in the cell. The genes do not display any similarity to known tetracycline resistance genes but they share 65 – 72% aa identity with UGEs and correspondingly possess the conserved GalE domain characteristic for these proteins (COG1087, (Fry et al., 2000; Thoden et al., 1997). The metabolic function of UGE is the interconversion of UDP-glucose and UDP-galactose and the latter is used as a monomer in the biosynthesis of extracellular lipopolysaccharides and capsular polysaccharides. Interestingly, screening a sludge metagenomic library for resistance to menadione resulted in the cloning of the similar gene (Mori et al., 2008). Structurally, menadione is very different from minocycline and doxycycline and it is unlikely that the resistance mechanism to such structurally different compounds may involve a similar enzymatic modification mechanism. Besides, using LC-MS/MS, we were unable to demonstrate any modification of doxycycline upon incubation with recombinant E. coli cells expressing galE-1 or galE-2. We hypothesise therefore that, as described for menadione resistance , resistance to the second generation tetracyclines conferred by galE-1 or galE-2 may be due the enhanced permeability barrier. Further work is required for more detailed characterization of the resistance mechanisms encoded by these genes. Although no mobility potential was detected in both cases, the genetic background in the galE-2 clone is quite interesting because it carries multiple antibiotic resistance genes.

Conclusions1. At present the level of resistance to tigecycline among the pig commensal microbiota is low.

2. Tigecycline-resistant bacteria can easily isolated once the corresponding selective pressure is applied.

3. The mechanisms of tigecycline resistance in our isolates are different from that described in literature.

4. No genetic transfer of tigecycline resistance was detected.

5. Animals that have only received antibiotics periodically over a prolonged period of time harbor substantially lower numbers of tetracycline resistant gut bacteria than animals that until recently had received growth promoting antibiotics and continue to receive prophylactic antibiotics.

6. When required, the enormous metabolic potential allows the microbial community to bring forth completely unexpected antibiotic resistance mechanisms such as GalE thus protecting against the second generation tetracyclines.

SID 5 (Rev. 3/06) Page 19 of 22

Future work

The continuation of this work may include:1. Clarification of TGR mechanisms in our TGR isolates. Presently, we cannot find any evidence that they

are similar to the mechanisms described in literature.2. Clarification of novel mechanisms of resistance to the second generation tetracyclines conferred by the

galE genes.3. Antibiotic selection: if the effect is limited to selection of resistance to a particular antibiotic used or the

effect is broader due to co-selection and stimulation of gene transfer so the pool of other antibiotic resistance genes is also affected? In our experiments the animals that receive a structurally unrelated prophylactic antibiotic, trimediazine, demonstrated the higher frequency of tetracycline resistance genes.

Knowledge transfer

The results of this work were presented at:1. The Fifth RRI/INRA Gut Microbiology meeting held in Aberdeen in June 21-23, 2006.2. The OBC 2-2 meeting, which was held in Okazaki, Japan, in September 11-16, 2006.3. EHRLICH II, 2nd World Conference on Magic Bullets, Oct 3-5, 2008 Nurnberg, Germany 4. The multilateral workshop Challenges for the reduction of antimicrobial use in animal production, which was held in Tsukuba, Japan, in November 11, 2008.

Sequence data generated in this work were deposited in a public database (GenBank, accession numbers: clone 7, FJ157999; clone 1, FJ158000; clone 2, FJ158001; clone 3, FJ158002; clone 9, FJ158003; clone 10, FJ158004; and clone 15, FJ158005). Other sequences will be deposited once the sequencing work and analyses are completed.

Published articles:Aminov RI, Mackie RI. 2007. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol. Lett. 271:147-161.Kazimierczak, K.A. and Scott, K.P. (2007) Antibiotics and resistance genes: influencing the microbial ecosystem in the gut. Adv. Appl. Microbiol. 62:269-292.Kazimierczak, K.A., Rincon, M.T., Patterson, A.J., Martin, J.C., Young, P., Flint, H.J. and Scott, K.P. (2008) A new tetracycline efflux gene, tet(40), is located in tandem with tet(O/32/O), in a human gut Firmicute bacterium and in metagenomic library clones. Antimicrob. Agents Chemother. 52:4001-4009.Katarzyna A. Kazimierczak, Karen P. Scott, Denise Kelly and Rustam I. Aminov. 2009. Tetracycline resistome of the organic pig gut. Appl. Environ. Microbiol. 75:1717-22.Aminov, R.I. 2009. The role of antibiotics and antibiotic resistance in nature. Environ. Microbiol. Published Online: Jul 6 2009.

We also intend to submit a manuscript that describes the tetracycline resistome of the conventional pig gut, dependent on the novelty of the sequence data.

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