Plasmids of Gram Positive Cocci as Tools for Genetic Engineering

Abstract

Summary More than 250 grams positive (cocci) indigenous clinical isolates were identified on the basis of morphological, growth (culture / colonial), biochemical and metabolite production characteristics. Among them 205 (82%) belonged to genus staphylococcus; 30 (12) to genus streptococcus and 15 (6%) genus micrococcus. Further studies have not only enabled us to search for the prevalence of the antibiotic resistance behaviour (patterns) among indigenous clinical staphylococci, but successful intergeneric transfer of gentamicin resistant markers of five isolates even been reported by filter paper mattings from staphylococcus aurous to klebsiella pneumonia. Follow up the gentamicin resistance transferability has been well established. Surprisingly 100% of the isolates have offered resistance via one or the other mode of action (viz by inhibiting protein synthesis, nucleic acid replication or cell wall synthesis. Present6 finding indicate towards the increasing (absolute) trend of drug resistance (against on or other antibiotic). The antibiotic used for screening included ampicillin, cephradine, erythromycin, gentamicin, neomycin, polymixin B, streptomycin and tetracycline. Resistance to polymixin B has been found to be found to the least 9%. We are also successful in isolating a few bacteriocins producing (7%) strains of staphylococci. However, there bacteriocins were only effective against closely related strains. It is interesting that the most all the bacteriocinogenic strains lost this property after curing; thereby indicating the bacteriocion regulation by plasmid-borne genes in the gram positive cocci in general. The alcidine orange and elevated temperature mediated plasmid curing experiments showed that in 20 culture one or more antibiotic resistance marker was found to be present on plasmid. Thus, some resistance determinants are located on the chromosome as well. However, the bacteriocinogenic determinants were found to be present solely on plasmid as all the 17 bacteriocinogenic isolates were cured after similar plasmid eliminating procedures. Most plasmids (narrow –host range) exist in only a limited number of closely related bacteria. However, the le4ss frequent plasmids are referred as broad-host range because they can be transferred to and maintained in bacteria of distant species and genera. Such plasmids are useful for establishing the genetic systems and drawing of genetic maps for several desirable bacteria and can also be used as shuttle vector in Genetic Engineering. We have been able to isolates the stable broad-host range plasmids (from our isolates) that have been transferred successfully into distant recipients by vivo gene manipulations. Most of the plasmids referred here, have been isolated and characterized on isolation and identification of staphylococcal plasmids employed in our studies was developed from mini-prep protocol. This method has enabled to analyse several isoaltes isoaltes simultaneously. The plasmids were characterized ( for the approximate molecular weight estimation) by horizontal agarose gel electrophoresis using EcoR Hind III lambda digest as ladder. It is interesting that natural broad-host range staphylococcal plasmids have been reported in Pakistan. We may speculate that Gram + plasmids (with particular reference to the staph. Aurous) carry a still broader host range trend of transmissibility and expression potential and thus, may prove to be the better shuttle vectors for genetic engineering technologies.