Diverse Broad-Host-Range Plasmids from Freshwater Carry Few Accessory Genes

Broad-host-range self-transferable plasmids are known to facilitate bacterial adaptation by spreading genes between phylogenetically distinct hosts. These plasmids typically have a conserved backbone region and a variable accessory region that encodes host-beneficial traits. We do not know, however, how well plasmids that do not encode accessory functions can survive in nature. The goal of this study was to characterize the backbone and accessory gene content of plasmids that were captured from freshwater sources without selecting for a particular phenotype or cultivating their host. To do this, triparental matings were used such that the only required phenotype was the plasmid''s ability to mobilize a nonconjugative plasmid. Based on complete genome sequences of 10 plasmids, only 5 carried identifiable accessory gene regions, and none carried antibiotic resistance genes. The plasmids belong to four known incompatibility groups (IncN, IncP-1, IncU, and IncW) and two potentially new groups. Eight of the plasmids were shown to have a broad host range, being able to transfer into alpha-, beta-, and gammaproteobacteria. Because of the absence of antibiotic resistance genes, we resampled one of the sites and compared the proportion of captured plasmids that conferred antibiotic resistance to their hosts with the proportion of such plasmids captured from the effluent of a local wastewater treatment plant. Few of the captured plasmids from either site encoded antibiotic resistance. A high diversity of plasmids that encode no or unknown accessory functions is thus readily found in freshwater habitats. The question remains how the plasmids persist in these microbial communities.     

Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1β and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Short-term effects of amoxicillin on bacterial communities in manured soil

Antibiotic-resistant bacteria, nutrients and antibiotics that enter the soil by means of manure may enhance the proportion of bacteria displaying antibiotic resistance among soil bacteria and may affect bacterial community structure and function. To investigate the effect of manure and amoxicillin added to manure on soil bacterial communities, microcosm experiments were performed with two soil types and the following treatments: (1) nontreated, (2) manure-treated, (3) treated with manure supplemented with 10 mg amoxicillin kg(-1) soil and (4) treated with manure supplemented with 100 mg amoxicillin kg(-1) soil, with four replicates per treatment. Manure significantly increased the total CFU count and the amoxicillin-resistant CFU count of both soil types. However, only the soil with a history of manure treatment showed a significant increase in the relative number of amoxicillin-resistant bacteria as a result of amoxicillin amendment. The majority of plasmids exogenously isolated from soil originated from soil treated with amoxicillin-supplemented manure. All 16 characterized plasmids carried the bla-TEM gene, and 10 of them belonged to the IncN group. The bla-TEM gene was detected in DNA directly extracted from soil by dot-blot hybridization of PCR amplicons and showed an increased abundance in soil samples treated with manure. Molecular fingerprint analysis of 16S rRNA gene fragments amplified from soil DNA revealed significant effects of manure and amoxicillin on the bacterial community of both soils.     

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.     

Detection of conjugative plasmids and antibiotic resistance genes in anthropogenic soils from Germany and India

PCR typing methods were used to assess the presence of plasmids of the incompatibility (Inc) groups IncP, IncN, IncW, IncQ and rolling circle plasmids of the pMV158 type in total DNA extracts from anthropogenic soils from India and Germany. Ten different soils from two different locations in Germany, the urban park Berlin Tiergarten and the abandoned sewage field Berlin-Buch, and from four different locations in India were analysed. PCR amplification of the total DNA extracts revealed the prevalence of IncP-specific sequences in Berlin Buch and Indian soil samples. The detected IncP plasmids contained at least one transfer function, the origin of transfer, oriT. In contrast to IncP-specific sequences, IncQ, IncN, IncW and pMV158-specific sequences were never detected. The presence of ampC, tet (O), ermB, SHV-5, mecA, and vanA antibiotic resistance genes was also tested. Three Indian soil samples irrigated with wastewater contained the ampC gene, whereas the other resistance genes were not found in any of the samples. Detection of IncP trfA2 and oriT sequences by PCR amplification and hybridization is a clear indication that IncP plasmids are prevalent in these habitats. Exogenous plasmid isolation revealed conjugative plasmids belonging to the IncPbeta group encoding resistance to ampicillin.     

Spreading antibiotic resistance through spread manure: characteristics of a novel plasmid type with low %G+C content

Bioactive amounts of antibiotics as well as resistant bacteria reach the soil through manure fertilization. We investigated plasmids that may stimulate the environmental spread and interspecies transfer of antibiotic resistance. After treatment of two soils with manure, either with or without the sulfonamide antibiotic sulfadiazine, a significant increase in copies of the sulfonamide resistance gene sul2 was detected by qPCR. All sul2 carrying plasmids, captured in Escherichia coli from soil, belonged to a novel class of self-transferable replicons. Manuring and sulfadiazine significantly increased the abundance of this replicon type in a chemically fertilized but not in an annually manured soil, as determined by qPCR targeting a transfer gene. Restriction patterns and antibiograms showed a considerable diversity within this novel plasmid group. Analysis of three complete plasmid sequences revealed a conserved 30 kbp backbone with only 36% G+C content, comprised of transfer and maintenance genes with moderate homology to plasmid pIPO2 and a replication module ( rep and oriV ) of other descent. The plasmids differed in composition of the 27.0–28.3 kbp accessory region, each of which carried IS CR2 and several resistance genes. Acinetobacter spp. was identified as a potential host of such LowGC-type plasmids in manure and soil.     

Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5alpha by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant

The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281 bp fragment of trfA from all currently sequenced IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, α, β, γ and δ, were present in the wastewater treatment plant influent. Also sequences representing the recently described fifth subgroup, the ε subgroup, were detected in the wastewater. Thus, these results confirm the presence of at least five phylogenetically distinct subgroups of IncP-1 plasmids and represent the first time that sequences associated with plasmids of all of these five subgroups have been detected in a single setting. Additionally, the results confirm that wastewater constitutes a reservoir for the conjugative IncP-1 plasmids, which often harbor multiple antibiotic resistance genes.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract: Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.     

Comparative analysis of the structure of incompatibility plasmids N, P and W

Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.     

Manure and sulfadiazine synergistically increased bacterial antibiotic resistance in soil over at least two months

Manuring of arable soils may stimulate the spread of resistance genes by introduction of resistant populations and antibiotics. We investigated effects of pig manure and sulfadiazine (SDZ) on bacterial communities in soil microcosms. A silt loam and a loamy sand were mixed with manure containing SDZ (10 or 100 mg per kilogram of soil), and compared with untreated soil and manured soil without SDZ over a 2-month period. In both soils, manure and SDZ positively affected the quotients of total and SDZ-resistant culturable bacteria [most probable number (MPN)], and transfer frequencies of plasmids conferring SDZ resistance in filter matings of soil bacteria and an Escherichia coli recipient. Detection of sulfonamide resistance genes sul1, sul2 and sul3 in community DNA by polymerase chain reaction (PCR) and hybridization revealed a high prevalence of sul1 in manure and manured soils, while sul2 was mainly found in the loamy sand treated with manure and high SDZ amounts, and sul3 was not detected. By PCR quantification of sul1 and bacterial rrn genes, a transient effect of manure alone and a long-term effect of SDZ plus manure on absolute and relative sul1 abundance in soil was shown. The dynamics in soil of class 1 integrons, which are typically associated with sul1, was analysed by amplification of the gene cassette region. Integrons introduced by manure established in both soils. Soil type and SDZ affected the composition of integrons. The synergistic effects of manure and SDZ were still detectable after 2 months. The results suggest that manure from treated pigs enhances spread of antibiotic resistances in soil bacterial communities.     

Increased Abundance and Transferability of Resistance Genes after Field Application of Manure from Sulfadiazine-Treated Pigs

Spreading manure containing antibiotics in agriculture is assumed to stimulate the dissemination of antibiotic resistance in soil bacterial populations. Plant roots influencing the soil environment and its microflora by exudation of growth substrates might considerably increase this effect. In this study, the effects of manure from pigs treated with sulfadiazine (SDZ), here called SDZ manure, on the abundance and transferability of sulfonamide resistance genes sul1 and sul2 in the rhizosphere of maize and grass were compared to the effects in bulk soil in a field experiment. In plots that repeatedly received SDZ manure, a significantly higher abundance of both sul genes was detected compared to that in plots where manure from untreated pigs was applied. Significantly lower abundances of sul genes relative to bacterial ribosomal genes were encountered in the rhizosphere than in bulk soil. However, in contrast to results for bulk soil, the sul gene abundance in the SDZ manure-treated rhizosphere constantly deviated from control treatments over a period of 6 weeks after manuring, suggesting ongoing antibiotic selection over this period. Transferability of sulfonamide resistance was analyzed by capturing resistance plasmids from soil communities into Escherichia coli. Increased rates of plasmid capture were observed in samples from SDZ manure-treated bulk soil and the rhizosphere of maize and grass. More than 97% of the captured plasmids belonged to the LowGC type (having low G+C content), giving further evidence for their important contribution to the environmental spread of antibiotic resistance. In conclusion, differences between bulk soil and rhizosphere need to be considered when assessing the risks associated with the spreading of antibiotic resistance.     

Tanzawaic Acids,a Chemically Novel Set of Bacterial Conjugation Inhibitors

Bacterial conjugation is the main mechanism for the dissemination of multiple antibiotic resistance in human pathogens. This dissemination could be controlled by molecules that interfere with the conjugation process. A search for conjugation inhibitors among a collection of 1,632 natural compounds, identified tanzawaic acids A and B as best hits. They specially inhibited IncW and IncFII conjugative systems, including plasmids mobilized by them. Plasmids belonging to IncFI, IncI, IncL/M, IncX and IncH incompatibility groups were targeted to a lesser extent, whereas IncN and IncP plasmids were unaffected. Tanzawaic acids showed reduced toxicity in bacterial, fungal or human cells, when compared to synthetic conjugation inhibitors, opening the possibility of their deployment in complex environments, including natural settings relevant for antibiotic resistance dissemination.     

Exogenous Isolation of Antibiotic Resistance Plasmids from Piggery Manure Slurries Reveals a High Prevalence and Diversity of IncQ-Like Plasmids

Antibiotic resistance plasmids were exogenously isolated in biparental matings with piggery manure bacteria as plasmid donors in Escherichia coli CV601 and Pseudomonas putida UWC1 recipients. Surprisingly, IncQ-like plasmids were detected by dot blot hybridization with an IncQ oriV probe in several P. putida UWC1 transconjugants. The capture of IncQ-like plasmids in biparental matings indicates not only their high prevalence in manure slurries but also the presence of efficiently mobilizing plasmids. In order to elucidate unusual hybridization data (weak or no hybridization with IncQ repB or IncQ oriT probes) four IncQ-like plasmids (pIE1107, pIE1115, pIE1120, and pIE1130), each representing a different EcoRV restriction pattern, were selected for a more thorough plasmid characterization after transfer into E. coli K-12 strain DH5α by transformation. The characterization of the IncQ-like plasmids revealed an astonishingly high diversity with regard to phenotypic and genotypic properties. Four different multiple antibiotic resistance patterns were found to be conferred by the IncQ-like plasmids. The plasmids could be mobilized by the RP4 derivative pTH10 into Acinetobacter sp., Ralstonia eutropha, Agrobacterium tumefaciens, and P. putida, but they showed diverse patterns of stability under nonselective growth conditions in different host backgrounds. Incompatibility testing and PCR analysis clearly revealed at least two different types of IncQ-like plasmids. PCR amplification of total DNA extracted directly from different manure samples and other environments indicated the prevalence of both types of IncQ plasmids in manure, sewage, and farm soil. These findings suggest that IncQ plasmids play an important role in disseminating antibiotic resistance genes.     

Increased Abundance of IncP-1β Plasmids and Mercury Resistance Genes in Mercury-Polluted River Sediments: First Discovery of IncP-1β Plasmids with a Complex mer Transposon as the Sole Accessory Element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1β replicon-specific sequences detected in total community DNA. The isolation of IncP-1β plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1β plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1β plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTΔP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1β plasmids R751 and pB8. However, this is the first example of an IncP-1β plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Safely Coupling Livestock and Crop Production Systems: How Rapidly Do Antibiotic Resistance Genes Dissipate in Soil following a Commercial Application of Swine or Dairy Manure?

Animal manures recycled onto crop production land carry antibiotic-resistant bacteria. The present study evaluated the fate in soil of selected genes associated with antibiotic resistance or genetic mobility in field plots cropped to vegetables and managed according to normal farming practice. Referenced to unmanured soil, fertilization with swine or dairy manure increased the relative abundance of the gene targets sul1, erm(B), str(B), int1, and IncW repA. Following manure application in the spring of 2012, gene copy number decayed exponentially, reaching background levels by the fall of 2012. In contrast, gene copy number following manure application in the fall of 2012 or spring of 2013 increased significantly in the weeks following application and then declined. In both cases, the relative abundance of gene copy numbers had not returned to background levels by the fall of 2013. Overall, these results suggest that under conditions characteristic of agriculture in a humid continental climate, a 1-year period following a commercial application of raw manure is sufficient to ensure that an additional soil burden of antibiotic resistance genes approaches background. The relative abundance of several gene targets exceeded background during the growing season following a spring application or an application done the previous fall. Results from the present study reinforce the advisability of treating manure prior to use in crop production systems.     

Exogenous isolation of conjugative plasmids from pesticide contaminated soil

Exogenous plasmid isolation method was used to assess conjugative plasmids conferring pesticide tolerance/multiple metal and antibiotic resistance from contaminated soil using bacteria detached from soil samples as a donor and rifampicin resistant E. coli HMS as a recipient strain on mineral salt agar medium supplemented with γ-HCH, and antibiotics ampicillin, tetracycline, chloramphenicol and kanamycin. Transconjugants were obtained on ampicillin (10?μg/ml) and tetracycline (20?μg/ml) amended MSA plates and frequency of ampicillin and tetracycline resistance gene transfer was 7.2?×?10(-6) and 9.2?×?10(-4) transconjugants/recipient, respectively. PCR typing methods were used to assess the presence of plasmids of the incompatibility groups IncP, IncN, IncW, IncQ and rolling circle plasmids of pMV158 type in DNA derived from transconjugants. All transconjugants were PCR amplified for the detection of Inc group plasmids and rolling circle plasmids of pMV158 family in which TM2, 3, 4, 11 and 12 (tet) transconjugants gave PCR products with the IncP-specific primers for both replication and transfer functions (trfA2 (IncP) and oriT (IncP)), while TM 14 (amp) gave an IncP specific PCR product for the replication gene trfA2 (IncP) only. TM15, 16, 18 and 21 (amp) gave a PCR product for the IncW-specific oriT (IncW). Out of 24 transconjugants, only TM 5 (tet) gave a PCR product with the pMV158 specific primer pair for oriT (RC). Our findings indicate that Inc group plasmids and rolling circle plasmids of pMV158 type may be responsible for transferring multiple antibiotic resistance genes among the bacterial soil community.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.