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Construction of safe vectors for cloning of drug resistance genes in non pathogenic hosts

High Efficiency Cloning of Drug Resistance Genes Via Safe Vector Host Systems High Efficiency Cloning of Drug Resistance Genes Via Safe Vector Host
Shahrzad S. Connolly1,2 & Stephen N. Connolly2 1Department of Microbial Diseases, St John’s Institute of Dermatology, Guy’s, Kings College & St Thomas’s Hospitals, University of London, 2School of Science & Technology, University of Teesside, Borough Road, Middlesbrough, TS1 3BA, UK; ABSTRACT
Direct cloning of the genes originating from clinical isolates of coagulase negative staphylococci (CNS) conferring high level resistance to mupirocin (MupR, >2000 mgL-1) is described using a vector system based on the safe non-pathogen Staphylococcus carnosus. This approach eliminates the need for initial laborious sub-cloning steps via S.aureus and E.coli shuttle vectors. Thus, an 11.5 kb fragment of a S.epidermidis plasmid conferring MupR was cloned into a variant of the pCE10 vector and introduced into S.carnosus strains by a modified protoplast transformation procedure, with high efficiency (8x109) observed. In this way, straightforward cloning into a host able to express the gene without requiring genetic manipulation in a pathogenic strain curtails the wider dissemination of drug resistance elements. The opportunity for CNS gene expression in isogenic host species is afforded by this system. Evidence for the co-existence of elements conferring high level MupR both as a plasmid and an integrative form in the S.carnosus chromosome is also presented. Conjugative transfer of the hybrid plasmid to S.epidermidis also indicates successful cloning of the mobility (tras) genes as part of the MupR plasmid. 1. INTRODUCTION
Resistance to high (>512 mgL-1) levels of mupirocin (Mup), conferred by a variety of plasmids, has been described in pathogenic and coagulase negative staphylococcal (CNS) species of human origin associated with clinical isolates [1] [2] [3] while both high and low level (<256 mgL-1) resistance has been attributed to chromosomal resistant determinants [4] [5]. Large plasmids in the size range 23-68.4 kb have been reported in both S.aureus and CNS from various health care settings since the early 1990’s where the resistance predated the application of Mup [6]. Characterisation of these plasmids has demonstrated their different restriction profiles and ability to transfer to pathogenic strains by conjugation, particularly on the surface of skin. The mupirocin resistance (MupR) gene has previously been cloned as a 13.4 kb HindIII fragment of a S.aureus plasmid via a S.aureus-E.coli shuttle vector [7]. The present study involves an alternative strategy of direct cloning of the MupR gene from S.epidermidis via a 2.15 kb truncated S.carnosus vector based on pCE10 through modification of a protoplast transformation procedure with high efficiency. Thus although S.aureus and E.coli have been routinely employed as hosts, S.carnosus serves as a more amenable, non-hazardous and isogenic cloning and gene expression system due to its ease of transformation and non-pathogenic nature. The cloned MupR gene is stably maintained as part of a conjugative plasmid and also as an integral part of the chromosomal DNA in S.carnosus conferring high and low level resistance to Mup, respectively.  Corresponding author: Tel.: (0044) 1642-342509; Fax: (0044) 1642-34201; E-mail: shahrzad.connolly@tees.ac.uk High Efficiency Cloning of Drug Resistance Genes Via Safe Vector Host Systems
2. MATERIALS AND METHODS
The MupR plasmid was isolated from a clinical S.epidermidis strain, SCTD196, which also carried a penicillin resistance element, PnR. PnR was cured by sub-culturing SCTD196 and incubating at 40 oC. The characterisation of the MupR plasmids from SCTD196 involved conjugative transfer of a 34 kb plasmid to a plasmid free strain, NCTC8325, which was chromosomally resistant to tetracycline. Serial conjugative transfer of the 34 kb replicon rendered various recipients resistant to Mup at a high level (>2000 mgL-1). Curing experiments involved culturing of SCTD196 and the Mup resistant recipient NCTC8325 at 40 oC in the absence of the respective antibiotics. MupR plasmids were digested with EcoRI and HindIII (Sigma) according to the manufacturer’s instructions. Further characterisation of the S.carnosus MupR clones involved hybridisation of the EcoRI digested MupR plasmids with a 4 kb as well as a 13.4 kb labeled MupR DNA probe [1]. Bacterial strains and plasmids are shown in Table 1. Table 1: Characteristics of the strains employed Characteristics
Resistance Determinant
Mup, mupirocin; Pn, penicillin; Cm, chloramphenicol; Asa, arsenate; Asi, arsenite; Sb, antimony; Em, erythromycin; Tc, tetracycline An 11.5 kb HindIII fragment from the plasmid SCTD196 was inserted into a S. carnosus host strain via a modified 2.15 kb pCE10V plasmid. The same insert fragment was also cloned onto a 5.9 kb S.carnosus vector pCA43. Hybrid plasmids were introduced into S.carnosus, TM300, by a modification of the previously reported method of protoplast transformation using plasmid DNA concentrations in the range 250-2500 mgL-1 [7]. The protoplast suspension was incubated at 26-28 oC for 2 min with the DNA solution preheated to the same temperature prior to its addition. The mixture was cooled to room temperature and centrifuged at 7000 rpm for 25 min. Protoplast dilutions were inoculated on DM3 regeneration medium and incubated at 37 oC for 5 hours before the addition of 3 mL of softagar containing appropriate antibiotics. Plates were incubated for 3-4 days. Transformants were selected on Mup and erythromycin, Em, solid media, and confirmed according to restriction analysis and hybridisation to a MupR DNA probe. Plasmid DNA was isolated according to alkaline lysis with the addition of 5.2 mgL-1 lysostaphin (Sigma); purification was effected according to a standard phenol-chloroform method [8]. Electrophoresis was conducted on 0.8-1.2% agarose gels using Tris-borate-EDTA (Sigma). T4 ligase, DNA labeling and detection kits, as well as other reagents were obtained from Sigma. Other strains were routinely cultivated on Oxoid blood agar base containing Cm, 15 mgL-1, Em, 15 mgL-1, and Mup, 100-200 mgL-1. 3. RESULTS
Acquisition of the 34 kb plasmid by the recipient strains through conjugation and protoplast transformation conferred high level MupR. The resistance was stably maintained and serially transferable to new S.aureus strains by conjugation at the rate of 2x10-8 to 3x10-7. Loss of the plasmid under curing conditions rendered the strains sensitive to Mup. The efficiency of transformation of S.carnosus in molecular cloning of the MupR gene and protoplast transformation was 4.8x102 to 8x102 for concentrations of hybrid DNA up to 500 mgL-1. Significantly higher efficiencies of transformation in the order of 2x108 to 8x109 were obtained for DNA concentrations of between 1000-2500 mgL-1. Characterisation of the plasmids using HindIII and EcoRI demonstrated the presence of the same fragments from the MupR plasmid in both the clinical strain and the transconjugants; see Table 2 for the restriction profiles of the plasmids used in cloning. High Efficiency Cloning of Drug Resistance Genes Via Safe Vector Host Systems Table 2: Product of the restriction enzyme cleavage of plasmids Strain source of plasmid
Restriction enzyme
Mean restriction fragment size (kb)
Size (kb)
a pSSC40 recombinant generated through cloning into S.carnosus The hybrid plasmid pSSC40 was isolated from clones of S.carnosus and cleaved with HindIII resulting in fragments of 11.5 and 2.15 kb. These were separated by gel electrophoresis and transferred to a nylon membrane. The 11.5 kb HindIII fragment of the MupR plasmid from S.carnosus clones was hybridised to labeled 13.4 kb and 4 kb MupR gene probes, thus ascertaining the presence of the MupR gene in S.carnosus clones. These probes were also used to detect homologous sequences in the plasmids of transconjugants and the clinical strain. The 11.5 kb fragment from the transconjugants and the clinical strain hybridised to the Mup probes confirming the presence of the MupR gene in all resistant strains. Curing of the Mup resistant S.carnosus clones led to the complete loss of the extrachromosomal 34 kb plasmid, while the strains remained resistant to 500 mgL-1 of the antibiotic. A chromosomal DNA fragment of 10 kb in Mup resistant S.carnosus clones hybridised to the MupR probe indicating the presence of a copy of the MupR gene within the chromosome. The chromosomal MupR determinant was stably maintained under curing conditions and was not transferable by transconjugation.
4. DISCUSSION
Molecular cloning and the expression of staphylococcal genes has been achieved via S.aureus and E.coli plasmids as
shuttle vectors for the transformation of S.carnosus protoplasts [7] [9]. Other approaches, including electroporation
for the insertion of exogenous DNA into cells and transcutaneous electrical nerve stimulation for horizontal gene
transfer from CNS, have also been used with various frequencies of transformation involving S.aureus as the
recipient [10] [11] [12] [13]. The limitations presented by handling hazardous pathogenic strains, including
S.aureus, and the non-isogenic character of both S.aureus and E.coli have led to the promotion of S.carnosus as the
preferred host system for expressing staphylococcal genes. The major advantage of S.carnosus is that it provides a
safe host for gene expression due to its non-pathogenic nature. Recognition of the inert properties of S.carnosus in
humans and animals has led to the development and optimisation of sophisticated techniques for its transformation
[14]; though these methods require expertise and technology that is not widely available to all laboratories. In
contrast, protoplast transformation is more amenable and provides an accessible procedure for cloning into
S.carnosus. The introduction of hybrid DNA into S.carnosus protoplasts has generally relied on small concentrations
of transforming open-circular DNA of low molecular weight resulting in low efficiency transformation [15] [7].
Significantly improved efficiency of transformation of the order of 8x109 has been achieved by application of DNA
at higher concentrations (1000-2500 mgL-1). Other modifications include preheating the DNA suspension to 26-28
oC before its addition to the protoplast suspension maintained at the same temperature; this is in contrast to previous
reports of protoplast transformation and electroporation [15] [16] [7] [14].
The cloned MupR gene is stably maintained in S.carnosus as a 13.65 kb plasmid as well as an integral element
within the chromosomal DNA. High level MupR (>2000 mgL-1) is associated with the gene located on the plasmid,
whereas resistance at lower levels (<500 mgL-1) is attributed to chromosomal determinants. Curing and conjugal
transfer experiments demonstrate that the 13.65 kb plasmid encodes MupR and carries the essential elements for its
own mobilisation. High level Mup resistance was lost during curing with concomitant loss of the 13.65 kb element,
while the S.carnosus strains remained resistant to lower levels of Mup. This suggests the presence of a stable
chromosomal location for the resistance genes which was further confirmed by hybridisation of the EcoRI digests of
chromosomal DNA with a MupR probe. Chromosomally resistant S.carnosus strains which were cured of their
plasmid (pSSC40) did not transfer Mup resistance through conjugation with S.epidermidis and S.aureus, in contrast
to S.carnosus strains which participated in serial transconjugation.
High Efficiency Cloning of Drug Resistance Genes Via Safe Vector Host Systems
The presence of chromosomal determinants encoding both low and high level Mup resistance in S.aureus has been
reported [17] [5], however, high level resistance has been mainly attributed to the presence of various conjugative
plasmids in S.aureus and CNS [1] [18] [4]. In contrast, the present study demonstrates that the MupR element is
maintained in S.carnosus as both a plasmid and a chromosomal determinant. The protoplast transformation
procedure described here serves as a versatile and efficient system for the cloning and expression of staphylococcal
genes within an isogenic, apathogenic host.
REFERENCES
[1] S. Connolly, W. C. Noble, and I. Phillips, “Mupirocin resistance in coagulase-negative staphylococci”, Journal of Medical Microbiology”, Vol. 39, pp. 450-453, 1993. [2] J. G. Hurdle, A. J. O’Neill, L. Mody, I. Chopra, and S. F. Bradley, “In vivo transfer of high-level resistance from Staphylococcus epidermidis to methicillin-resistant Staphylococcus aureus associated with failure of mupirocin prophylaxis”, Journal of Antimicrobial Chemotherapy, Vol. 56, pp. 1166-1168, 2005. [3] S. S. Connolly and S. N. Connolly, “Prophylactic nurturing of antibiotic resistance”, 13th International Congress on Infectious Diseases, p. 172, Kuala Lumpur, Malaysia, 2008. [4] S. Fujimura, A. Watanabe, and D. Beighton, “Characterization of the mupA gene in strains of methicillin-resistant Staphylococcus aureus with low level of resistance to mupirocin”, Antimicrobial Agents and Chemotherapy, Vol. 45, pp. 641-642, 2001. [5] E. E. Udo, N. Al-Sweih, and B. C. Noronha, “A chromosomal location of the mupA gene in Staphylococcus aureus expressing high-level mupirocin resistance”, Journal of Antimicrobial Chemotherapy, Vol. 51, pp. 1283-1286, 2003. [6] M. Rahman, S. Connolly, W. C. Noble, B. Cookson, and I. Phillips, “Diversity of staphylococci exhibiting high-level resistance to mupirocin”, Journal of Medical Microbiology, Vol. 33, pp. 97-100, 1990. [7] S.Connolly, I. Phillips, and W. C. Noble, “Cloning and expression of the mupirocin resistance gene in Staphylococcus carnosus”, Medical Microbiology Letters, Vol. 3, pp. 409-415, 1994. [8] P. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Vol. 1, CSHL Press, pp. 135-137, [9] R. J. Williams, S. P. Nair, B. Henderson, K. T. Holland, and J. M. Ward, “Expression of the S.aureus hysA gene S.carnosus from a modified E.coli-staphylococcal shuttle vector, Plasmid, Vol. 47, No. 3, pp. 241-245, 2002. [10] S. Schenk and R. A. Laddaga, “Improved method for electroporation of Staphylococcus aureus”, FEMS microbiology Letters, Vol. 73, No. 1-2, pp. 133-138, 1992. [11] O.Chesneau, R. Lailler, A. Derbise, and N. El Solh, “Transposon of IS1181 in the genomes of Staphylococcus and Listeria”, FEMS Microbiology Letters, Vol. 177, No. 1, pp. 93-100, 1999. [12] K. Wishart, A. Loughrey, R. B. McClurg, C. E. Goldsmith, B. C. Millar, J. Rao, B. Sengupta, J. S. Dooley, R. J. Rooney, and J. E. Moore, “Lack of horizontal gene transfer of methicillin resistance genetic determinants from PBP2a-positive, coagulase-negative staphylococci to methicillin-sensitive Staphylococcus aureus using transcutaneous electrical nerve stimulation (TENS) , British Journal of Bioscience, Vol. 64, No.1, pp 6-9, 2007. [13] T. Mahmood, T. Zar, and S. M. Saqlan Naqvi, “Multiple pulses improve electroporation efficiency in Agrobacterium tumefasciens”, Electronic Journal of Biotechnology, Vol. 11, No. 1, 2007. [14] J. Lofblom, N. Kronqvist, M. Uhlen, S. Stahl, and H. Wernerus, “Optimization of electroporation-mediated transformation”, Journal of Applied Microbiology, Vol. 102, No. 3, pp. 736-747, 2007. [15] F. Gotz and B. Schumacher, “Improvements in protoplast transformation in Staphylococcus carnosus”, FEMS Microbiology Letters, Vol. 40, pp. 285-288, 1987. [16] G. R. Kraemer and J. J. Iandolo, “High frequency transformation of Staphylococcus aureus by electroporation”, Current Microbiology, Vol. 21, pp. 373-376, 1990. [17] E. E. Udo, L. E. Jacob, and B. Mathew, “Genetic analysis of methicillin-resistant Staphylococcus aureus expressing high- and low-level mupirocin resistance”, Journal of Medical Microbiology, Vol. 50, pp. 909-915, 2001. [18] J. E. Hodgson, S. P. Curnock, K. G. H. Dyke, R. Morris, D. R. Sylvester, and M. S. Gross, “Molecular characterization of the gene encoding high-level mupirocin resistance in Staphylococcus aureus J2870”, Antimicrobial Agents and Chemotherapy, Vol. 38, No. 5, pp. 1205-1208, 1994.

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