Potential Horizontal Transfer Research Articles

Horizontal Transfer of the Tetracycline Resistance Gene tetM Mediated by pCF10 Among Enterococcus faecalis in the House Fly (Musca domestica L.) Alimentary Canal

The house fly (Musca domestica L.) alimentary canal was evaluated for the potential of horizontal transfer of tetM on plasmid pCF10 among Enterococcus faecalis. Two sets of experiments were conducted: (1) house flies without surface sterilization and (2) surface-sterilized flies. Both sets of flies were exposed to E. faecalis OG1RF:pCF10 as donor for 12 h and then E. faecalis OG1SSp as recipient for 1 h. Another group of flies received the recipient first for 12 h followed by exposure to the donor strain for 1 h. House flies were screened daily to determine the donor, recipient, and transconjugant bacterial load for up to 5 days. In addition, the sponge-like mouth parts used for food uptake (labellum) of surface-sterilized house flies were removed and analyzed for donors, recipients, and transconjugants, separately. In both groups of flies (n = 90 flies/group), transfer occurred within 24 h after exposure with a transconjugant/donor rate from 8.6 x 10(-5) to 4.5 x 10(1). Transconjugants were also isolated from the house fly labellum. Our data suggest that the house fly digestive tract provides a suitable environment for horizontal transfer of conjugative plasmids and antibiotic resistance genes among enterococci. Our results emphasize the importance of this insect as a potential vector of antibiotic-resistant bacterial strains.

Presence and potential for horizontal transfer of antibiotic resistance in oxidase-positive bacteria populating raw salad vegetables

To assess whether domestically grown fresh salad vegetables constitute a possible reservoir of antibiotic resistance for Canadian consumers, aerobic bacteria capable of forming colonies at 30 °C on nutrient-limited media were recovered from a single sampling of Romaine lettuce, Savoy spinach and alfalfa sprouts, then examined for their susceptibility to ten antibiotics and the carriage of potentially mobile R-plasmids and integrons. Of the 140 isolates resistant to one or more antibiotic, 93.5 and 90.0% were resistant to ampicillin and cephalothin; 35.7% to chloramphenicol, 10.0% to streptomycin, 4.2% to nalidixic acid, 4.2% to kanamycin, and 2.8% to gentamicin. Gram-positive isolates accounted for less than 4% of the antibiotic resistant strains. A small portion (23.1%) of the predominant oxidase-positive, gram-negative isolates was resistant to two or more antimicrobials. Members of the Pseudomonas fluorescens/putida complex were most prevalent among the 34 resistant strains identified. Sphingobacterium spp. and Acinetobacter baumanni also were detected. Ten of 52 resistant strains carried plasmids, 3 of which were self-transmissible and bore resistance to ampicillin and kanamycin. Eighteen of 48 gave PCR evidence for integron DNA. Class 2 type integrons were the most prevalent, followed by class 1. We conclude that the foods examined here carry antibiotic resistant bacteria at the retail level. Further, our determination that resistant strains contain integron-specific DNA sequences and self-transmissible R-plasmids indicates their potential to influence the pool of antibiotic resistance in humans via lateral gene transfer subsequent to ingestion.

Detection of feed-derived maize DNA in goat milk and evaluation of the potential of horizontal transfer to bacteria

The presence and the transforming capacity of feed-derived DNA in milk obtained from eight lactating goats fed on maize E176 silage were evaluated. The presence of single- and multi-copy maize genes was monitored by real-time PCR and conventional PCR. Chromosomal and plastid DNA extracted directly from maize flour and silage were readily amplifiable by conventional PCR, however, only chloroplast-specific gene fragments of 199 and 532 bp were detected in about 60 and 20%, respectively, of the milk samples analysed. Quantification by real time PCR yielded 9.5 (±6.7) × 102 plant gene copies/mL of milk sediment. In contrast, all milk samples were negative for the chromosomally located maize zein gene or the E176 specific cry1Ab transgene. The minimum concentration of plant DNA required for detection was 0.01 ng/mL raw milk for the chloroplast-specific fragment and 1 ng/mL for the cry1Ab transgene. The detection limit was determined by spiking milk samples with plant DNA prior to DNA extraction. The transformation capability of DNA in milk was evaluated after constructing a marker rescue system in Acinetobacter baylyi strain BD413 based on recombinational repair of the bla TEM gene. Two systems were developed that allowed the plant marker gene to recombine with the bacterial chromosome [A. baylyi BD413 (pUC-bla)] or plasmids [A. baylyi BD413 (pBBR1MCS-2Φ)]. The two systems showed the same efficiency of transformation, yielding 10−5 transformants per recipient cell (t/r) using plasmid pUC18 or a 1,873 bp fragment as donor DNA, and 3.5 × 10−11 t/r using DNA isolated from flour (E176). No transformants were detected when exposing the recipient bacterium to DNA extracted from maize (E176) silage or from milk obtained from goats feed maize (E176).

A genomic islet mediates flagellar phase variation in Escherichia coli strains carrying the flagellin-specifying locus flk.

The occurrence of unilateral flagellar phase variation was previously demonstrated in Escherichia coli strains carrying the non-fliC flagellin-specifying locus flk. In this study, we investigated the mechanism involved in this process. By using sequencing and sequence analysis, the flk region between the chromosomal genes yhaC and rnpB was characterized in all described flk-positive E. coli strains, including the H35 strain identified in this study (the other strains used are H3, H36, H47, and H53 strains), and this region was found to contain a putative integrase gene and flanking direct repeats in addition to the flk flagellin-specifying gene flkA and a fliC repressor gene, flkB, indicating that there is a typical genomic islet (GI), which was designated the flk GI. The horizontal transfer potential of the flk GI was indicated by detection of the excised extrachromosomal circular form of the flk GI. By generating fliC-expressing variants of H3 and H47 strains, unilateral flagellar phase variation in flk-positive strains was shown to be mediated by excision of the flk GI. The function of the proposed integrase gene was confirmed by deletion and a complementation test. The potential integration sites of the flk GI were identified. A general model for flagellar phase variation in flk-positive E. coli strains can be expressed as fliC(off) + flkA(on) --> fliC(on) + flkA(none). This is the first time that a molecular mechanism for flagellar phase variation has been reported for E. coli.

Toxicity, Uptake, and Transfer Efficiency of Fipronil in Western Subterranean Termite (Isoptera: Rhinotermitidae)

The potential horizontal transfer of nonrepellent termiticides has become an important paradigm to control termites in recent years. In this study, 14C-radiolabeled fipronil was used in a series of laboratory experiments to demonstrate the extent and ability of termites to transfer lethal amounts of fipronil to unexposed nestmates. Fipronil is an active and nonrepellent termiticide against western subterranean termites, Reticulitermes hesperus Banks, on sand at relevant doses. It exhibited delayed toxicity with the lowest LD50 ≈ap; 0.2 ng/termite expressed between day 4 and 7. Both continuous and brief exposures to fipronil-treated sand seriously impaired the termite’s ability to move and respond to a dodecatrienol trail, limiting potential horizontal transfer. In tunneling studies, fipronil prevented termite tunneling at concentrations as low as 0.5 ppm and was nonrepellent even at 500 ppm. Greater than 90% mortality was recorded by day 7 with concentrations ranging from 0.5 to 500 ppm. There was a linear relationship between the time of exposure and uptake of [14C]fipronil when termites were continuously exposed to 0.5, 1.0, and 5.0 ppm for 24 h. However, uptake discontinued when the termites were immobilized. Maximum transfer of fipronil from donors to recipients occurred within the first 24 h. Fipronil was transferred by body contact and trophallaxis did not play a major role in horizontal transfer. In successive transfer studies, there was not enough fipronil on recipients for them to serve as secondary donors and kill other termites. In a linear arena study, there was an inverse relationship between the amount of fipronil on dead termites and their distance from the treated zone. Maximum mortality was observed within 1.5 m from the treated zone. Results in our laboratory studies suggest that horizontal transfer was not a major factor contributing to the efficacy of fipronil in the field.

Toxicity, Uptake, and Transfer Efficiency of Fipronil in Western Subterranean Termite (Isoptera: Rhinotermitidae)

The potential horizontal transfer ofnonrepellent termiticides has become an important paradigm to control termites in recent years. In this study, 14C-radiolabeled fipronil was used in a series of laboratory experiments to demonstrate the extent and ability of termites to transfer lethal amounts of fipronil to unexposed nestmates. Fipronil is an active and nonrepellent termiticide against western subterranean termites, Reticulitermes hesperus Banks, on sand at relevant doses. It exhibited delayed toxicity with the lowest LD50 approximately 0.2 ng/termite expressed between day 4 and 7. Both continuous and brief exposures to fipronil-treated sand seriously impaired the termite's ability to move and respond to a dodecatrienol trail, limiting potential horizontal transfer. In tunneling studies, fipronil prevented termite tunneling at concentrations as low as 0.5 ppm and was nonrepellent even at 500 ppm. Greater than 90% mortality was recorded by day 7 with concentrations ranging from 0.5 to 500 ppm. There was a linear relationship between the time of exposure and uptake of [14C]fipronil when termites were continuously exposed to 0.5, 1.0, and 5.0 ppm for 24 h. However, uptake discontinued when the termites were immobilized. Maximum transfer of fipronil from donors to recipients occurred within the first 24 h. Fipronil was transferred by body contact and trophallaxis did not play a major role in horizontal transfer. In successive transfer studies, there was not enough fipronil on recipients for them to serve as secondary donors and kill other termites. In a linear arena study, there was an inverse relationship between the amount offipronil on dead termites and their distance from the treated zone. Maximum mortality was observed within 1.5 m from the treated zone. Results in our laboratory studies suggest that horizontal transfer was not a major factor contributing to the efficacy of fipronil in the field.

Release and persistence of extracellular DNA in the environment

The introduction of genetically modified organisms (GMOs) has called for an improved understanding of the fate of DNA in various environments, because extracellular DNA may also be important for transferring genetic information between individuals and species. Accumulating nucleotide sequence data suggest that acquisition of foreign DNA by horizontal gene transfer (HGT) is of considerable importance in bacterial evolution. The uptake of extracellular DNA by natural transformation is one of several ways bacteria can acquire new genetic information given sufficient size, concentration and integrity of the DNA. We review studies on the release, breakdown and persistence of bacterial and plant DNA in soil, sediment and water, with a focus on the accessibility of the extracellular nucleic acids as substrate for competent bacteria. DNA fragments often persist over time in many environments, thereby facilitating their detection and characterization. Nevertheless, the long-term physical persistence of DNA fragments of limited size observed by PCR and Southern hybridization often contrasts with the short-term availability of extracellular DNA to competent bacteria studied in microcosms. The main factors leading to breakdown of extracellular DNA are presented. There is a need for improved methods for accurately determining the degradation routes and the persistence, integrity and potential for horizontal transfer of DNA released from various organisms throughout their lifecycles.

Lettuce for human consumption collected in Costa Rica contains complex communities of culturable oxytetracycline- and gentamicin-resistant bacteria.

The present widespread use of antimicrobials in crop farming is based upon their successful application in human medicine. However, recent evidence suggests that the massive anthropogenic release of antimicrobials into the biosphere has selected for resistant bacteria and facilitated the transfer of resistance genes among them. This work deals with the examination of iceberg lettuce collected at 10 farms from two regions in Costa Rica. Farmers from nine sampling sites regularly apply commercial formulations containing gentamicin, oxytetracycline, streptomycin, or a combination of them without being able to indicate how often and how much of these products have been sprayed onto the crops. One organic farm was also investigated for comparative purposes. Oxytetracycline- and gentamicin-resistant bacteria were abundantly detected using selective enrichment cultures. Furthermore, colony mixtures from selective plates were characterized by chemotaxonomical and molecular fingerprinting methods. Both types of resistant communities accounted for a significant fraction of all culturable bacteria and included several resistance genes as well as factors for their potential horizontal transfer. Given the fact that lettuce is eaten raw, it may contribute to the dissemination of antimicrobial-resistant bacteria and/or their resistance genes from the environment to the microbial biota of the human intestine.

Macrolide and lincosamide resistance genes of environmental streptococci from bovine milk

Environmental streptococcus isolates from bovine milk were identified to the species and strain level and screened for resistance to macrolide and lincosamide antibiotics by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin and pirlimycin by broth microdilution assays. Presence of ribosomal methylase genes (ermA, ermB, ermC) and efflux pump genes (mefA/E, msrA/C) was detected by polymerase chain reaction (PCR). Resistance to pirlimycin (minimum inhibitory concentration (MIC) = 8microg/ml) was detected in 6 of 13 Enterococcus isolates that were identified as E. faecium by API20Strep typing. msrC was detected in 10 enterococcal isolates but the detection of msrC was not associated with phenotypic resistance. msrC negative isolates were reclassified as Enterococcus mundtii based on sequencing of housekeeping genes. Resistance to erythromycin and pirlimycin (MIC > 16microg/ml) was detected in 4 of 4 Streptococcus dysgalactiae and 12 of 20 Streptococcus uberis isolates and was encoded by ermB. All Streptococcus isolates tested negative for ermA, ermC, mefA/E and msrA/C. Among ermB positive streptococci, three alleles were identified based on a 527 bp gene fragment. Each allele was detected in at least two herds. The same alleles have also been detected in other bacterial species from bovine and non-bovine hosts and farm soil, suggesting a theoretical potential for horizontal transfer of macrolide resistance genes on dairy farms.

The Salmonella genomic island 1 is an integrative mobilizable element

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).