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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 diagram of the CMY regions of Typhimurium strain YU39 and pX1::CMY transconjugants. Panel A) shows a schematic diagram of the CMY region in the pA/C plasmid of strain YU39 [5]. Panel B) depicts a large CMY region inserted into the intergenic region between 046 and 047 genes for IC2 transconjugant. Panel C) shows a short CMY region inserted into stbE gene for IIIC10 transconjugant. The PCR amplifications designed to assess the extension of the CMY regions are indicated by double arrowheads under the diagrams. The PCRs used to determine the pX1 CMY junctions are indicated by bars with circles.
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Figure 1: Schematic diagram of the CMY regions of Typhimurium strain YU39 and pX1::CMY transconjugants. Panel A) shows a schematic diagram of the CMY region in the pA/C plasmid of strain YU39 [5]. Panel B) depicts a large CMY region inserted into the intergenic region between 046 and 047 genes for IC2 transconjugant. Panel C) shows a short CMY region inserted into stbE gene for IIIC10 transconjugant. The PCR amplifications designed to assess the extension of the CMY regions are indicated by double arrowheads under the diagrams. The PCRs used to determine the pX1 CMY junctions are indicated by bars with circles.

Mentions: To address the extent of the CMY region transferred from pA/C to pX1 we used the PCR typing scheme developed in our previous studies (Figure 1A). Four of the pX1 transconjugants were positive for six of the seven genes present in the complete CMY region of pA/C (c. a. 12 kb), spanning from ISEcp1 to hypothetical protein 0093 (according to pSN254 annotation; GenBank:NC_009140); while the other four displayed a short version of the CMY region (c. a. 3 kb) including only ISEcp1, blaCMY-2, blc and sugE (Figure 1B and Figure 1C).


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 diagram of the CMY regions of Typhimurium strain YU39 and pX1::CMY transconjugants. Panel A) shows a schematic diagram of the CMY region in the pA/C plasmid of strain YU39 [5]. Panel B) depicts a large CMY region inserted into the intergenic region between 046 and 047 genes for IC2 transconjugant. Panel C) shows a short CMY region inserted into stbE gene for IIIC10 transconjugant. The PCR amplifications designed to assess the extension of the CMY regions are indicated by double arrowheads under the diagrams. The PCRs used to determine the pX1 CMY junctions are indicated by bars with circles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic diagram of the CMY regions of Typhimurium strain YU39 and pX1::CMY transconjugants. Panel A) shows a schematic diagram of the CMY region in the pA/C plasmid of strain YU39 [5]. Panel B) depicts a large CMY region inserted into the intergenic region between 046 and 047 genes for IC2 transconjugant. Panel C) shows a short CMY region inserted into stbE gene for IIIC10 transconjugant. The PCR amplifications designed to assess the extension of the CMY regions are indicated by double arrowheads under the diagrams. The PCRs used to determine the pX1 CMY junctions are indicated by bars with circles.
Mentions: To address the extent of the CMY region transferred from pA/C to pX1 we used the PCR typing scheme developed in our previous studies (Figure 1A). Four of the pX1 transconjugants were positive for six of the seven genes present in the complete CMY region of pA/C (c. a. 12 kb), spanning from ISEcp1 to hypothetical protein 0093 (according to pSN254 annotation; GenBank:NC_009140); while the other four displayed a short version of the CMY region (c. a. 3 kb) including only ISEcp1, blaCMY-2, blc and sugE (Figure 1B and Figure 1C).

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