IncP-1ε Plasmids are Important Vectors of Antibiotic Resistance Genes in Agricultural Systems: Diversification Driven by Class 1 Integron Gene Cassettes

Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut Braunschweig, Germany.
Frontiers in Microbiology (Impact Factor: 3.99). 01/2012; 3:2. DOI: 10.3389/fmicb.2012.00002
Source: PubMed


The role of broad-host range IncP-1ε plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1ε plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1ε plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1ε plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1ε plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1ε plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS1326, and in three of the plasmids the tetracycline resistance transposon Tn1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1ε plasmids in soil microcosms. Our data suggest that IncP-1ε plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

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Available from: Holger Heuer
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    • "Exposure to antibiotics can up-regulate the intI1 expression by triggering the SOS response and ultimately increase gene cassette recombination rates (Cambray et al., 2011; Hocquet et al., 2012) and might also lead to co-selection of antibiotic resistance, e.g., by heavy metals (Rosewarne et al., 2010). Furthermore, class 1 integrons are often located on MGEs such as transposons and plasmids, e.g., of the IncP-1ε incompatibility group, facilitating their transfer and spread within bacterial communities (Stokes and Gillings, 2011; Heuer et al., 2012; Gillings, 2014). Hence, due to their ability to foster bacterial adaptation to environmental perturbation, class 1 integrons might be used as a universal marker for selective pressure in the environment. "
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    ABSTRACT: Long-term irrigation with untreated wastewater can lead to an accumulation of antibiotic substances and antibiotic resistance genes in soil. However, little is known so far about effects of wastewater, applied for decades, on the abundance of IncP-1 plasmids and class 1 integrons which may contribute to the accumulation and spread of resistance genes in the environment, and their correlation with heavy metal concentrations. Therefore, a chronosequence of soils that were irrigated with wastewater from 0 to 100 years was sampled in the Mezquital Valley in Mexico in the dry season. The total community DNA was extracted and the absolute and relative abundance (relative to 16S rRNA genes) of antibiotic resistance genes (tet(W), tet(Q), aadA), class 1 integrons (intI1), quaternary ammonium compound resistance genes (qacE+qacEΔ1) and IncP-1 plasmids (korB) were quantified by real-time PCR. Except for intI1 and qacE+qacEΔ1 the abundances of selected genes were below the detection limit in non-irrigated soil. Confirming the results of a previous study, the absolute abundance of 16S rRNA genes in the samples increased significantly over time (linear regression model, p < 0.05) suggesting an increase in bacterial biomass due to repeated irrigation with wastewater. Correspondingly, all tested antibiotic resistance genes as well as intI1 and korB significantly increased in abundance over the period of 100 years of irrigation. In parallel, concentrations of the heavy metals Zn, Cu, Pb, Ni, and Cr significantly increased. However, no significant positive correlations were observed between the relative abundance of selected genes and years of irrigation, indicating no enrichment in the soil bacterial community due to repeated wastewater irrigation or due to a potential co-selection by increasing concentrations of heavy metals.
    Full-text · Article · Mar 2015 · Frontiers in Microbiology
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    • "What are the factors supporting the dissemination of ARGs? Among the genetic mechanisms, Heuer et al. (2012) identified IncP-1ε plasmids as important vectors for horizontal transfer of antibiotic resistance in agricultural systems. These plasmids are transferable to a wide range of Beta-and Gammaproteobacteria, with the concurrent transfer of ARGs. "
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    ABSTRACT: It becomes increasingly clear that the basis of antibiotic resistance problem among bacterial pathogens is not confined to the borders of clinical microbiology but has broader ecological and evolu-tionary associations. This Research Topic "Role and prevalence of antibiosis and the related resistance genes in the environ-ment" in Frontiers in Microbiology: Antimicrobials, Resistance, and Chemotherapy presents the examples of occurrence and diver-sity of antibiotic resistance genes (ARGs) in the wide range of environments, from the grasslands of the Colombian Andes, to the dairy farms and small animal veterinary hospitals in the United Stated, and to the various environments of Continental Europe and Indochina. Besides, various genetic mechanisms and selection/co-selection factors contributing to the dissemination and maintenance of ARGs are presented. The topic is finalized by the mathematical modeling approach to access the probabil-ity of rare horizontal gene transfer (HGT) events in bacterial populations. The opinion article by Martínez (2012) summarizes our present understanding of the cycle of ARGs acquisition by bac-terial pathogens. The environmental microbiota harbors a vast diversity of genes, which we usually classify as conferring resis-tance to antibiotics. In natural ecosystems, however, their role may be different and not necessarily associated with this function. Yet, if the certain metabolic genes are acquired by commen-sal/pathogenic microbiota and appeared to be conferring selective advantage under the pressure of antibiotics, their primary func-tion under these new ecological circumstances becomes resistance to antibiotics. Moreover, upon the amplification under the antibi-otic selective pressure, these ARGs are released into the environ-ment thus contributing to the rise of antibiotic resistance in other ecological compartments. Evidence for the environmental contamination by ARGs can be seen in several articles of this Research Topic. For example, despite the low antibiotic usage in the grassland farms located in the Colombian Andes, there is a significant diversity of tetra-cycline resistance genes in the microbiota of the animal gut and the environment (Santamaría et al., 2011). But the diversity of the tet genes in the former ecosystem is higher thus sug-gesting the gene flow from the animals into the environment. Another study involved the isolation and characterization of the CTX-M [a major type of extended-spectrum beta-lactamase (ESBL)] producing Escherichia coli strains from soils, cattle, and the farm environment in the Burgundy region of France (Hartmann et al., 2012). Environmental and animal strains appeared to be clonally related. The study also suggests a long-term survival of the CTX-M-producing E. coli strains in soil since the last manure application has been done 1 year before the actual sampling. Czekalski et al. (2012) demonstrated the increased levels of multidrug-resistant bacteria and ARGs in Lake Geneva, Switzerland due to the discharge from the local wastew-ater treatment plant. Counterintuitively, wastewater treatment resulted in selection of extremely multidrug-resistant bacteria and accumulation of ARGs although the total bacterial load was substantially decreased. A less favorable situation with the treat-ment of wastewater is in Indochina, which includes Vietnam, Thailand, Cambodia, Lao PDR, and Myanmar. Suzuki and Hoa (2012) summarized the current knowledge regarding the pres-ence of quinolones, sulfonamides, and tetracyclines as well as the corresponding ARGs in this region. They concluded that: (1) no correlation exists between the quinolone contamination and quinolone resistance; (2) occurrence of the sul sulfonamide resistance gene varies geographically; and (3) microbial diversity relates to the oxytetracycline resistance level. Thames et al. (2012) used qPCR to investigate the effect of feeding milk replacers with various antibiotic doses on the excretion of ARGs by dairy calves. Interestingly, no significant differences have been found in the absolute numbers of ARGs excreted. After the normalization to the 16S rRNA genes the rel-ative tet(O) concentration appeared to be higher in animals fed the highest therapeutic doses of antibiotic. Besides, antibiotic feeding provided no obvious health benefits. The authors con-cluded that the greater than conventional nutritional intake in the study outweighs the previously reported health benefits of antibiotics. Ghosh et al. (2012) reported an interesting observa-tion regarding the carriage of multi-drug resistant enterococci by resident cats in small animal veterinary hospitals. Genotypically identical strains were isolated from cats and surfaces of cage door, thermometer, and stethoscope suggesting that the ani-mals may be involved in cross-contamination of the hospital environment. October 2014 | Volume 5 | Article 520 | 1
    Full-text · Article · Oct 2014 · Frontiers in Microbiology
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    • "These plasmids confer resistance to a broad spectrum of antibiotics. This is due to the presence of a class 1 integron that carries the gene sul1 responsible for sulphonamide resistance at its 5 ′ end, plus gene cassettes carrying several determinants of resistance to other antibiotics (Heuer et al., 2012). Plasmids belonging to subgroup IncP-1ε exhibit great diversity (Sen et al., 2011). "
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    ABSTRACT: The plasmids of the incompatibility (Inc) group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals, and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance, and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad-host-range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.
    Full-text · Article · Mar 2013 · Frontiers in Microbiology
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