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Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

ABSTRACT: BACKGROUND: Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5? and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. RESULTS: YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. CONCLUSIONS: We showed that the transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure.

SUBMITTER: Wiesner M

PROVIDER: S-EPMC4222815 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

Wiesner Magdalena M Fernández-Mora Marcos M Cevallos Miguel A MA Zavala-Alvarado Crispín C Zaidi Mussaret B MB Calva Edmundo E Silva Claudia C

BMC microbiology 20131121

<h4>Background</h4>Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) ...[more]

PMID: 24262067

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Project description:The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5M-NM-1 harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol or streptomycin exposure were compared to cells left untreated at logarithmic phase using Illumina sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, blaCMY-2, aadA, and aacA. Antibiotic treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the up-regulation of floR and numerous chromosomally encoded genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators. Bacterial strains and growth conditions. E. coli strain DH5M-NM-1 harboring pAR060302 was grown in 10 mL DifcoTM Luria-Bertani (LB) broth aliquots at 37M-BM-: C with shaking until an OD600 of 0.5. A total of 8 cultures were independently grown representing two biological replicates per condition tested. Six of the cultures were amended, 2 cultures per antibiotic, with ampicillin (50 M-BM-5g/mL final concentration), florfenicol (30 M-BM-5g/mL final concentration), or streptomycin (50 M-BM-5g/mL final concentration) and allowed to incubate at 37M-BM-: C with shaking for an additional 30 min. Two cultures were not amended with any antibiotic. Cells were pelleted and RNA was purified using a commercially available RNA extraction kit (Qiagen). RNA preparations were then subjected to a DNase treatment to eliminate DNA contamination from the sample (Qiagen). A treatment was also included to deplete ribosomal RNA using a commercially available kit (MicrobExpress, Ambion). The two biological replicates for each growth condition were pooled for sequencing. Ilumina sequencing for transcriptome mapping. cDNA libraries were generated with an insert size of 100 bp and sequenced with 76-base cycles of single-end reads using a Genome Analyzer II (Illumina) platform according to manufacturerM-bM-^@M-^Ys protocols at the Biomedical Genomics Center (University of Minnesota, Minneapolis, Minnesota, USA). Approximately 160,000 plasmid-mapped reads each were obtained for the ampicillin and streptomycin treated samples, and 260,000 plasmid-mapped reads each for the control and florfenicol treatment samples. Genome-mapped read counts were as follows: control, ~6.4 million reads, florfenicol treatment, ~5.7 million reads, ampicillin treatment, ~1.7 million reads, and streptomycin treatment, ~5.2 million reads. We only used those reads uniquely mapped on plasmid or chromosomal DNA for global normalization and further analysis. RNAseq data analysis. cDNA reads were trimmed so that the quality at each base position was above 30 (~15-20 bp) and then mapped either to the E. coli K-12 MG1655 published genome sequence (Genbank accession no. NC_000913) or to the pAR060302 published sequence (Genbank accession no. NC_092692) using BOWTIE. The E. coli strain DH5M-NM-1 has an incomplete annotation and for this reason the E. coli K-12 annotation was used, representing an estimation of differentially expressed genes due to exposure of antimicrobials. The reads mapped per kilobase of gene per million (RPKM) reads was calculated using either the E. coli chromosome or the pAR060302 annotation and was used for global normalization. The per kilobase cDNA length normalized the effect of different length of cDNAs such that the sequence reads have a equal chance to map on the long cDNA regions and the short cDNA regions. After RPKM normalization, each sample is comparable to each other. An R package, DEGseq, was used to identify differentially expressed genes between the control and each antibiotic treatment condition. A cutoff of q-value < 0.05 and a fold change of > 3 were used to measure statistical significance.

Project description:Multiwalled carbon nanotubes (MWCNTs) regularly enter aquatic environments due to their ubiquity in consumer products and engineering applications. However, the effects of MWCNT pollution on the environmental microbiome are poorly understood. Here, we evaluated whether these carbon nanoparticles can elevate the spread of antimicrobial resistance by promoting bacterial plasmid transfer, which has previously been observed for copper nanomaterials with antimicrobial properties as well as for microplastics. Through a combination of experimental liquid mating assays between Pseudomonas putida donor and recipient strains with plasmid pKJK5::gfpmut3b and mathematical modeling, we here demonstrate that the presence of MWCNTs leads to increased plasmid transfer rates in a concentration-dependent manner. The percentage of transconjugants per recipient significantly increased from 0.21 ± 0.04% in absence to 0.41 ± 0.09% at 10 mg L-1 MWCNTs. Similar trends were observed when using an Escherichia coli donor hosting plasmid pB10. The identified mechanism underlying the observed dynamics was the agglomeration of MWCNTs. A significantly increased number of particles with >6 μm diameter was detected in the presence of MWCNTs, which can in turn provide novel surfaces for bacterial interactions between donor and recipient cells after colonization. Fluorescence microscopy confirmed that MWCNT agglomerates were indeed covered in biofilms that contained donor bacteria as well as elevated numbers of green fluorescent transconjugant cells containing the plasmid. Consequently, MWCNTs provide bacteria with novel surfaces for intense cell-to-cell interactions in biofilms and can promote bacterial plasmid transfer, hence potentially elevating the spread of antimicrobial resistance. IMPORTANCE In recent decades, the use of carbon nanoparticles, especially multiwalled carbon nanotubes (MWCNTs), in a variety of products and engineering applications has been growing exponentially. As a result, MWCNT pollution into environmental compartments has been increasing. We here demonstrate that the exposure to MWCNTs can affect bacterial plasmid transfer rates in aquatic environments, an important process connected to the spread of antimicrobial resistance genes in microbial communities. This is mechanistically explained by the ability of MWCNTs to form bigger agglomerates, hence providing novel surfaces for bacterial interactions. Consequently, increasing pollution with MWCNTs has the potential to elevate the ongoing spread of antimicrobial resistance, a major threat to human health in the 21st century.

Dataset Information

Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

Publications

Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

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