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

Wiesner M, Fernández-Mora M, Cevallos MA, Zavala-Alvarado C, Zaidi MB, Calva E, Silva C - BMC Microbiol. (2013)

Bottom Line: The presence of pSTV in the recipients had little effect on the conjugation frequency.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.

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Affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, México. csilvamex1@yahoo.com.

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 (DH5α 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.

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Schematic representation for the insertion sites for the CMY region into the pX1 backbone. Panel A) depicts the insertion of the large CMY region into the intergenic region between 046 and 047 hypothetical proteins. Panel B) shows the insertion of the short CMY region into stbE. The numbers under the solid black arrows correspond to nucleotide numbers in the annotation of the reference pX1 pOU1114 (GenBank: DQ115387). The surrounding nucleotide sequences at the insertion points are shown. Underlined letters mark the insertion site of the CMY region, and below the nucleotide number in the annotation of pOU1114. The CMY region sequence is indicated in italics, and the duplicated sequences generated during the transposition events are highlighted in boldface.
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Figure 2: Schematic representation for the insertion sites for the CMY region into the pX1 backbone. Panel A) depicts the insertion of the large CMY region into the intergenic region between 046 and 047 hypothetical proteins. Panel B) shows the insertion of the short CMY region into stbE. The numbers under the solid black arrows correspond to nucleotide numbers in the annotation of the reference pX1 pOU1114 (GenBank: DQ115387). The surrounding nucleotide sequences at the insertion points are shown. Underlined letters mark the insertion site of the CMY region, and below the nucleotide number in the annotation of pOU1114. The CMY region sequence is indicated in italics, and the duplicated sequences generated during the transposition events are highlighted in boldface.

Mentions: For the characterization of pX1 transconjugants IC2, ID1 and IIID2, that were negative for the 046-047 region, we used a combination of primers from the CMY region along with the primers for 046-047 to determine if this was the site of insertion (Figure 1B; PCRs H and I). We successfully established that the IC2, ID1 and IIID2 transconjugants were positive for the CMY-046-047 junction (Table 3). Sequencing of these PCR products showed the exact insertion site for these pX1 transconjugants harboring a large CMY region. The schematic representation of the insertion of the CMY region into 046-047 in IC2 is presented in Figure 2A. Mapping according to the pOU1114 annotation revealed that the insertion site was in nucleotide 33,768. A repeat sequence of six nucleotides (TGAATA) flanking the CMY region was detected, corresponding to nucleotides 33,763 to 33,768 of pOU1114. We discovered that the hypothetical protein 0093 was truncated at nucleotide 4,168 removing 1,318 nucleotides of the complete ORF.


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

Wiesner M, Fernández-Mora M, Cevallos MA, Zavala-Alvarado C, Zaidi MB, Calva E, Silva C - BMC Microbiol. (2013)

Schematic representation for the insertion sites for the CMY region into the pX1 backbone. Panel A) depicts the insertion of the large CMY region into the intergenic region between 046 and 047 hypothetical proteins. Panel B) shows the insertion of the short CMY region into stbE. The numbers under the solid black arrows correspond to nucleotide numbers in the annotation of the reference pX1 pOU1114 (GenBank: DQ115387). The surrounding nucleotide sequences at the insertion points are shown. Underlined letters mark the insertion site of the CMY region, and below the nucleotide number in the annotation of pOU1114. The CMY region sequence is indicated in italics, and the duplicated sequences generated during the transposition events are highlighted in boldface.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222815&req=5

Figure 2: Schematic representation for the insertion sites for the CMY region into the pX1 backbone. Panel A) depicts the insertion of the large CMY region into the intergenic region between 046 and 047 hypothetical proteins. Panel B) shows the insertion of the short CMY region into stbE. The numbers under the solid black arrows correspond to nucleotide numbers in the annotation of the reference pX1 pOU1114 (GenBank: DQ115387). The surrounding nucleotide sequences at the insertion points are shown. Underlined letters mark the insertion site of the CMY region, and below the nucleotide number in the annotation of pOU1114. The CMY region sequence is indicated in italics, and the duplicated sequences generated during the transposition events are highlighted in boldface.
Mentions: For the characterization of pX1 transconjugants IC2, ID1 and IIID2, that were negative for the 046-047 region, we used a combination of primers from the CMY region along with the primers for 046-047 to determine if this was the site of insertion (Figure 1B; PCRs H and I). We successfully established that the IC2, ID1 and IIID2 transconjugants were positive for the CMY-046-047 junction (Table 3). Sequencing of these PCR products showed the exact insertion site for these pX1 transconjugants harboring a large CMY region. The schematic representation of the insertion of the CMY region into 046-047 in IC2 is presented in Figure 2A. Mapping according to the pOU1114 annotation revealed that the insertion site was in nucleotide 33,768. A repeat sequence of six nucleotides (TGAATA) flanking the CMY region was detected, corresponding to nucleotides 33,763 to 33,768 of pOU1114. We discovered that the hypothetical protein 0093 was truncated at nucleotide 4,168 removing 1,318 nucleotides of the complete ORF.

Bottom Line: The presence of pSTV in the recipients had little effect on the conjugation frequency.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos, México. csilvamex1@yahoo.com.

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 (DH5α 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.

Show MeSH
Related in: MedlinePlus