蝇蛆肠道微生物菌群对猪粪残留抗生素的降解及抗性基因的影响

【目的】过度使用抗生素作为动物饲料添加剂,导致畜禽粪便已成为抗生素抗性基因的主要蓄积库,为了研究蝇蛆(Musca domestica)对猪粪中残留抗生素及抗性基因的影响,本文动态采集了实际农场条件下蝇蛆转化过程中猪粪堆体及虫体样本。【方法】利用q PCR、液相色谱-电喷雾质谱、同位素内标法、Illumina高通量测序以及局部相似性研究蝇蛆生物转化过程中残留抗生素降解效能及相关抗性基因组变化的微生物生态机制。【结果】6 d周期内,猪粪中四环素、土霉素、金霉素、强力霉素、磺胺嘧啶、诺氟沙星、氧氟沙星、环丙沙星以及恩诺沙星等9种残留抗生素含量显著下降,累积减量为34.3%–58.1%,每日减量百分比介于7.8%–57.4%之间。猪粪中共检测到的158种抗性基因,其中有118种大幅衰减,衰减量平均达79.3%;23种抗性基因存在富集现象,富集倍数平均为3.48。在蝇蛆肠道的作用下,粪源微生物群落中Bacteroidetes相对丰度下降,Proteobacteria相对丰度增加,尤其是Ignatzschineria增幅最大。网络分析发现,抗性基因的增减与微生物群落的变化显著相关,与抗性基因衰减相关的微生物主要属于Clostridiales和Bacteroidales,而与抗性基因富集相关的微生物主要为Alcaligenaceae、[Weeksellaceae]及Bacillales。【结论】蝇蛆可有效削减猪粪中的残留抗生素及防控抗性基因向环境扩散。     

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

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

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.     

Piggery manure used for soil fertilization is a reservoir for transferable antibiotic resistance plasmids

In this study, the prevalence and types of transferable antibiotic resistance plasmids in piggery manure were investigated. Samples from manure storage tanks of 15 farms in Germany were analysed, representing diverse sizes of herds, meat or piglet production. Antibiotic resistance plasmids from manure bacteria were captured in gfp-tagged rifampicin-resistant Escherichia coli and characterized. The occurrence of plasmid types was also detected in total community DNA by PCR and hybridization. A total of 228 transconjugants were captured from 15 manures using selective media supplemented with amoxicillin, sulfadiazine or tetracycline. The restriction patterns of 81 plasmids representing different antibiotic resistance patterns or different samples clustered into seven groups. Replicon probing revealed that 28 of the plasmids belonged to IncN, one to IncW, 13 to IncP-1 and 19 to the recently discovered pHHV216-like plasmids. The amoxicillin resistance gene bla-TEM was detected on 44 plasmids, and sulphonamide resistance genes sul1, sul2 and/or sul3 on 68 plasmids. Hybridization of replicon-specific sequences amplified from community DNA revealed that IncP-1 and pHHV216-like plasmids were detected in all manures, while IncN and IncW ones were less frequent. This study showed that 'field-scale' piggery manure is a reservoir of broad-host range plasmids conferring multiple antibiotic resistance genes.     

Environmentally co‐occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context‐dependent fitness effects

Plasmids are important mobile elements that can facilitate genetic exchange and local adaptation within microbial communities. We compared the sequences of four co‐occurring pQBR family environmental mercury resistance plasmids and measured their effects on competitive fitness of a Pseudomonas fluorescens SBW25 host, which was isolated at the same field site. Fitness effects of carriage differed between plasmids and were strongly context dependent, varying with medium, plasmid status of competitor and levels of environmental mercury. The plasmids also varied widely in their rates of conjugation and segregational loss. We found that few of the plasmid‐borne accessory genes could be ascribed functions, although we identified a putative chemotaxis operon, a type IV pilus‐encoding cluster and a region encoding putative arylsulfatase enzymes, which were conserved across geographically distant isolates. One plasmid, pQBR55, conferred the ability to catabolize sucrose. Transposons, including the mercury resistance Tn5042, appeared to have been acquired by different pQBR plasmids by recombination, indicating an important role for horizontal gene transfer in the recent evolution of pQBR plasmids. Our findings demonstrate extensive genetic and phenotypic diversity among co‐occurring members of a plasmid community and suggest a role for environmental heterogeneity in the maintenance of plasmid diversity.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Phenotypic and genotypic bacterial antimicrobial resistance in liquid pig manure is variously associated with contents of tetracyclines and sulfonamides

Aims: Antibiotic residues as well as antibiotic‐resistant bacteria in environmental samples might pose a risk to human health. This study aimed to investigate the association between antibiotic residues and bacterial antimicrobial resistance in liquid pig manure used as fertilizer. Methods and Results: Concentrations of tetracyclines (TETs) and sulfonamides (SULs) were determined by liquid chromatography‐mass spectrometry in 305 pig manure samples; antibiotic contents were correlated to the phenotypic resistance of Escherichia coli (n = 613) and enterococci (n = 564) towards up to 24 antibiotics. In 121 samples, the concentration of the TET resistance genes tet(M), tet(O) and tet(B) was quantified by real‐time‐PCR. TETs were found in 54% of the samples. The median sum concentration of all investigated TETs in the positive samples was 0·73 mg kg^?1. SULs were found with a similar frequency (51%) and a median sum concentration of 0·15 mg kg^?1 in the positive samples. Associated with the detection of TETs and/or SULs, resistance rates were significantly elevated for several substances – some of them not used in farm animals, e.g. chloramphenicol and synercid. In addition, multiresistant isolates were found more often in samples containing antibiotics. Analysis of the resistance genes tet(M) and tet(O) already showed a significant increase in their concentrations – but not in tet(B) – in the lowest range of total TET concentration. Mean tet(M) concentrations increased by the factor of 4·5 in the TET concentration range of 0·1–1 mg kg^?1, compared to negative manure samples. Conclusions: Antibiotic contamination of manure seems to be associated with a variety of changes in bacterial resistance, calling for a prudent use of antibiotics in farm animals. Significance and Impact of the Study: This study provides an interdisciplinary approach to assess antimicrobial resistance by combining the microbiological analysis of bacterial resistance with high quality chemical analysis of antibiotic residues in a representative number of environmental samples.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

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.     

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying ≥ 2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.

     

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.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

Characterization of a Multiresistant Mosaic Plasmid from a Fish Farm Sediment Exiguobacterium sp. Isolate Reveals Aggregation of Functional Clinic-Associated Antibiotic Resistance Genes

The genus Exiguobacterium can adapt readily to, and survive in, diverse environments. Our study demonstrated that Exiguobacterium sp. strain S3-2, isolated from marine sediment, is resistant to five antibiotics. The plasmid pMC1 in this strain carries seven putative resistance genes. We functionally characterized these resistance genes in Escherichia coli, and genes encoding dihydrofolate reductase and macrolide phosphotransferase were considered novel resistance genes based on their low similarities to known resistance genes. The plasmid G+C content distribution was highly heterogeneous. Only the G+C content of one block, which shared significant similarity with a plasmid from Exiguobacterium arabatum, fit well with the mean G+C content of the host. The remainder of the plasmid was composed of mobile elements with a markedly lower G+C ratio than the host. Interestingly, five mobile elements located on pMC1 showed significant similarities to sequences found in pathogens. Our data provided an example of the link between resistance genes in strains from the environment and the clinic and revealed the aggregation of antibiotic resistance genes in bacteria isolated from fish farms.     

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

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

堆肥化处理对畜禽粪便中四环素类抗生素及抗性基因控制的研究进展

随着四环素类抗生素在畜禽养殖中的广泛应用,畜禽粪便已成为四环素类抗生素和抗性基因的重要富集位点,其未经处理直接施用具有潜在的生态环境和人类健康风险。堆肥化处理可有效消减畜禽粪便中的四环素类抗生素,并且对抗性基因的扩散和传播具有一定的控制效果。本综述比较了不同的堆肥化工艺对粪肥中四环素类抗生素消减的效果,并重点讨论了其微生物降解机理,总结了堆肥化处理对粪肥中四环素抗性基因消减的研究进展,进一步讨论了堆肥化处理过程中抗性基因变化的微生态机理与控制策略,最后提出了采用热水解等预处理工艺去除抗生素压力和采用厌氧堆肥化工艺增强抗性基因控制的技术建议,以及从动态的角度采用高通量的检测技术来解析抗性基因消减机制的研究策略建议。     

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.     

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene

Aims: Isolation and full sequence analysis of ColE‐type plasmid, which carries the qnrS2 gene. Methods and Results: Quinolone resistance (qnrS2) gene‐carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer‐walking approach. The total sizes of these plasmids (pAQ2‐1 and pAQ2‐2) were 6900 bp and 6903 bp, respectively, and they were 99·1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2‐type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1‐type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Conclusions: Two plasmids were assumed to be the same plasmid, and this identification of a plasmid‐mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. Significance and Impact of the Study: This is the first finding of the ColE‐type plasmid carrying the qnrS2 gene.     

Distribution of ten antibiotic resistance genes in <Emphasis Type="Italic">E. coli</Emphasis> isolates from swine manure,lagoon effluent and soil collected from a lagoon waste application field

The prevalence of ten antibiotic resistance genes (ARGs) was evaluated in a total of 616 Escherichia coli isolates from swine manure, swine lagoon effluent, and from soils that received lagoon effluent on a commercial swine farm site in Sampson County, North Carolina (USA). Isolates with ARGs coding for streptomycin/spectinomycin (aadA/strA and strB), tetracycline (tetA and tetB), and sulfonamide (sul1) occurred most frequently (60.6–91.3%). The occurrence of E. coli isolates that carried aadA, tetA, tetB, and tetC genes was significantly more frequent in soil samples (34.0–97.2%) than in isolates from lagoon samples (20.9–90.6%). Furthermore, the frequency of isolates that contain genes coding for aadA and tetB was significantly greater in soil samples (82.6–97.2%) when compared to swine manure (16.8–86.1%). Isolates from the lagoon that carried tetA, tetC, and sul3 genes were significantly more prevalent during spring (63.3–96.7%) than during winter (13.1–67.8%). The prevalence of isolates from the lagoon that possessed the strA, strB, and sul1 resistance genes was significantly more frequent during the summer (90.0–100%) than during spring (66.6–80.0%). The data suggest that conditions in the lagoon, soil, and manure may have an impact on the occurrence of E. coli isolates with specific ARGs. Seasonal variables seem to impact the recovery isolates with ARGs; however, ARG distribution may be associated with mobile genetic elements or a reflection of the initial numbers of resistant isolates shed by the animals.     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.