Plasmids of Indigenous Pseudomonads: Molecular Characterization and Gene Manipulation

Abstract

The present research findings are concerned with the isolation of Pseudomonads from different pathological/diagnostic laboratories/hospitals of Karachi, and from environment (soil and water). More than 200 isolates were charecterized as Pseudomonas aeruginosa. All of the isolates were screened for drug resistance against different concentrations of eight antibodies. The result of the present studies are alarming because of the increasing trend of multiple drug resistance. It appears that Pseudomonas aeruginosa has developed very powerful resistance mechanisms against commonly used antibiotics due to their haphazard use and transmissible nature of resistance plasmids. Indeed, it is one of the most inherent antibiotic-resistant organism encountered in the clinical laboratory. Present study shows that resistance to ampicillin and cephalexin is the most common.All the isolates (100%) offered resitance against cephalexin (upto 500 ug/ mL) and 99% (upto 500 ug/mL) against ampicillin. However, gentamicin (an aminoglycoside), polymyxin B and ceftadime (3rd generation cephalosporin) resistance has been the least, yet 6% and 9% of the isolates were found to be resistant upto 500 ug/mL of gentamicin and ceftazidime respectively, indicating that resistance profile against these antibodies has gradually increased in Pseudomonas. Resistance to the drugs by common bacteria is worldwide, both in developed and developing countries. It has been argued that there is a direct relation between the antibiotic used and the frequency and kinds of antibiotic resistant strains in human beings. Bacteriocin (the protein antibiotics) are produced by a number of bacteria and in classical sense are active against closely related bacteria. Recent years have witnessed a tremendous interest in the bacteriocins of bacteria with refrence to their use as food bioprespective, in health care products (oral wash preprations, toothpastes, soaps etc) and cosmetics. All the isolates (211) were screened for bacteriogenesis potential. We are successful in isolating 67 bacterocin producing Pseudomonas (31.7%). They showed both intrageneric and intergeneric bacteriogenesis, the extent of antagonism was more aggressive among intergeneric bacteriogenesis than intrageneric. Fourteen, 19 and 20 isolates exhibited bioactivity against E.coli BU40, Ent.cloaceae and Klebsiella pneumoniae respectively. Sixty four were found to be active against S. aureus, 15 against S.pyogens and 63 against B.subtilis. These results illustrate the broad spectrum antagonism by Pseudomonas. The bacteriocins (after isolation/extraction) could be used against the indicator/sensitive strains belonging to different species and genera. The isolates were screened for hydrocarbon degradation using solid and liquid hydrocarbons. About 3% of the isolates were found to degrade phenanthrene while 100% of the isolates degraded octane (liquid hydrocarbon). Most of the novel pollution phenomena are related with the man-made organic chemicals like pesticides, plastics and other synthetics that persist, slowly degraded or are converted to less desirable residues. All the isolats have been monitored for metal resitance against six metals,. Most of the isolates were found to be resistant against chloride, nickle sulphate and lead acetate. Extrachromosomal location of different determinants was checked by different curing techniques. Hydrocarbon degradation, drug and bacteriocinogenic potential was lost after curing. Presence of plasmids was further confirmed by conjugation experiments. Unsuccesful results were obtained in case of transfer of degradative plasmids while successful intergeneric transfer (from Pseudomonas to E.coli BU40) of drug resistance and bacteriocinogenic plasmids was observed. Plasmids were isolated from the representative strains and transconjugants and subjected to agarose gel electrophoresis which revealed the plasmids of different molecular size. Molecular weight of the plasmids ranges from 12-21KB (in approxiamation). Cured isolates were also processed for plasmid isolation in order to confirm the elimination of plasmids. Nome of the cured isolate showed any plasmid band. All the transconjugants showed plasmid DNA bands while the recipient did not show the presence of any plasmid (after agarose gel electrophoresis), thereby confirming the transfer of plasmids from the donor to the recipient. Isolated and identified broad host-range bacteriocinogenic genetic factors (plasmids) could be used for genetic manipulations. Degradative plasmids could be used for biopesticide and hydrocarbon degradation to help in saving our polluted ecosystem from pollutants and toxic elements. Plasmid transfer among bacteria provides a mean for dissemination of antimicrobial resistance among clinically significant pathogens. We observed the transfer of gantamicin resistance plasmid ( from 2 Pseusdomonas strains) to E.coli FPL5014 cells. The presence of gene for gentamicin/ampicillin resistance in different Pseudomonas was confirmed by DNA hybridization with gentamicin probe (pGO500) using ECL kit. Infact, presence of cojugative and mobilizable drug resistance factors have been instrumental in posing problems related to the management of clinnico-medical therapy.