Role of Conjugative Plasmids in Antibiotic Resistance between Two Strains of E. coli

Two isolated bacterial strains ( E. coli 64, E. coli MM294), were purified, they are: gram-negative bacteria, circular, regular edged, thick somewhat glitter, viscous, and lactose fermenter. They belong to the family of Enterobacteriaceae E. coli 64 showed the resistant to all used antibiotics except Rifampicin (RA), Cephotaxime


Introduction
E. coli is a member of the genus Escherichia that includes in the family of Enterobacteriaceae (Ewing, W.H., 1986).It is a gram-negative, nonsporing, facultative rod that ferments lactose with gas formation within 48hr.at 35°C (Feng, P., et al., 2002).E. coli is the best studied bacterium and the experimental organism of choice for many microbiologists.It is a major inhabitant of the colon of human and other worm-blooded animals (Conway, P.L., 1995; Madigan, M.T., and Martinko, J.M., 2006).It is the major causative agent of urinary tract infection (UTI).UTI is inflammatory disease that involves areas ranging from the kidney to the urethra, with the urethra and bladder most commonly affected.The incidence of UTI is higher among women than men (Todar, K., 2007).Water contaminated with infected bacteria (E.coli) is a major source of UTI therefore E. coli is quite useful in the analysis of water for fecal contamination (Brenner, K. P., et al., 1996) (Salyers, A.A., et al., 2004).Plasmids are relatively small, circular DNA molecules that can exist independently of host chromosomes and are found in many bacteria including E. coli (they are also present in some yeasts and other fungi).Plasmids are responsible for resistance and transmitting the resistance to specific sensitive strain through a bridge formed by F-factor during a process called conjugation (Lederberg, J., 1952).Conjugation is happened between two mating types: donors or males, and recipients or females.The determinant of maleness is F-factor which is denoted by F and called the sex plasmid, genetically a male cell is designated F + , a female cell lacks the F plasmid and is designated F -. F-factor is responsible for cell attachment between specific bacterial strains.During conjugation male-female pairs are formed and in a way that is not completely understood, paring signals the replication of F and one copy of F is transferred to female (recipient) in about one minute.After conjugation the recipient cells called transconjugants (Lederberg, J., and Tatum, E., 1946; Holmes, R.K., and Jobling, M.G., 1996; Ryan, K.J., and Ray, C.G., 2004).5µg, Tobramycin (TM) 10µg, Amoxicillin (AMX) 10µg (Sigma) these disks were stored at 4°C) in disks diffusion method as the following: 0.1ml of each strain was cultured by spreading on the suface of nutrient agar plate, and then antibiotic disks were placed on the surface of agar using sterile forceps and incubated at 37°C for 24 hr.The results were indicated according to formation of inhibition zone around the disk for sensitive or not formation of inhibition zone around the disk for resist.(

Conjugation (in Liquid Media):
single colony of the donor and recipient strains was inoculated into nutrient broth (Biolife) separately and incubated at 37°C for 24 hr., the two strains were mixed in sterilized tube and incubated at 37°C for 2 hr.without shaking, sample was taken from conjugated tube and diluted properly and spread on a selective media (Nutrient agar containing 60 µg/ml tetracycline + 20 µg/ml refampicin) then incubated at 37°C for 24 hours.Controls were done by spreading each donor and recipient strains separately on Nutrient agar containing 60 µg/ml tetracycline + 20 µg/ml refampicin for identification of transconjugants.(William R. Will

Purification of Bacterial Strains:
The two strains (E. coli 64, and E. coli MM 294) were purified by cultivation on Macchonkey agar media, their morphological characteristics and properties were determined as: circular, regular edge, thick somewhat, glitter pink, viscous, and lactose fermenter.(Feng, P., et al., 2002).

Antibiotic
Sensitivity: The sensitivity to antibiotic was determined and the results indicate that E. coli 64 was sensitive to 5 antibiotics and resist to 14 antibiotics from 19 types of antibiotics used (Table 1).
For this reason this strain can be found in the urine sample of UTI patients.The results for E. coli MM294 indicate the sensitivity of this strain to 15 and resistance to 4 antibiotics (19 types of antibiotics were tested) (Table 1).For this reason this strain can't be isolated from urine samples of UTI patients but it can be found in lab as experimental strain, and here it can be considered as recipient strain in conjugation experiment while E. coli 64 can be considered as a donor strain.(Vidal, O., et al., 1998;Fux, C.A., et al., 2005).The tetracycline and refampicin were selected to be used as selectable markers in conjugation between E. coli 64 and E. coli MM294 because E. coli 64 is sensitive to reifampicin and resist to tetracycline while E. coli MM294 is opposite (Table 1).

Minimum Inhibitory Concentration (MIC):
Each of E. coli 64 and E. coli MM294 was cultivated in different concentrations of tetracycline and refampicin agars after 24 hr. of incubation at 37°C (Table 2).
The study found that E. coli 64 grew at all tetracycline concentrations because E. coli 64 is resistant to it (Table 1) while it didn't grow at any concentrations of refampicin even at lowest concentration (10µg/ml).In contrary E. coli MM294 grew at 10. 20, 30, 40 µg/ml (Table 2) so this concentration of tetracycline can inhibits the growth of E.coli MM294, while it grew at all rifampicin concentration (resists to refampicin, Table 1).According to these results 20µg/ml of refampicin and 60 µg/ml tetracycline can be considered as MICs that prevent the growth of E. coli 64 and E. coli MM294.(Andrews, J. M., 2001).Conjugation: results in Table 3 illustrate the growth of cells in a petridish containing 60 µg/m. of tetracycline and 20 µg/ml of rifampicin after mixing the two bacterial strains (E. coli 64 and E. coli MM294) while there are no growth when each of bacterial strains has been cultivated separately in medium contains both quantity of antibiotics mentioned previously.

Antibiotic
Sensitivity of Transconjugants: to check whether cells in the media containing (60 µg/ml tetracycline + 20 µg/ml rifampicin) grew as a result of spontaneous mutation (mutant cells) or as a result of transfer of drug resistant plasmid from donor cells to Eng.&Tech.Vol.26.No.11,2008Role of Conjugative Plasmids in Antibiotic Resistance between Two Strains of E. coli 1433 recipient cells during conjugation (transconjugants), this can be done by making antibiotic sensitivity of transconjugants by selecting randomly 9 colonies and making antibiotic sensitivity test for each one of nine colonies to 6 types of antibiotic: Tetracycline, Amoxicillin, Neomycin, Chloramphenicol, Ampiciline, and Trimethoprim + Sulfamethorazole.Where E. coli 64 (donor cell) was resistant to each one of these 6 types of antibiotics while E. coli MM294 (recipient cell) was sensitive to all these 6 types of antibiotics.As shown in the Table 4 there are 7 colonies can resist to all these 6 types of antibiotics while there are 2 colonies from 9 colonies can resists to all six types of antibiotics except neomycin that inhibit the growth of each of colony number 2 and colony number 6.The results in Table 4 reveal that all these nine colonies are transconjugants (they are result of conjugation) and not mutant cells (result to spontaneous mutation), and it seems that more than one plasmid can participate in transferring drug resistance as following: Plasmid contains neomycin gene, Plasmid contains TE, AMC, C, AM, and SXT genes, and/or Plasmid contains all six genes of tested antibiotics.These results are in agreement with the study of Carrasco, C. E., and his colleagues in 1997.They reported the acquisition of tetracycline resistnace by E. coli HB101, which is sensitive to this antibiotic, and also they suggested that tropical waters exposed to sewage contamination may present a great danger to public health due to the survival of E. coli in these environments because of antibiotic resistance that may be readily transferred among the genera of Enterobacteriaceae , this fact has been studied extensively in 1983 through the researches of Bell, J. B., and his colleagues, and Hanahan, D.

Conclusion
The experimental results indicate the role of conjugative plasmids in spreading drug resistance among urinary tract infection bacterial strains (E.coli), The study predicts the danger role of E. coli MM294 on public health in case of discarding such experimental strain directly without killing into sewage leading to poisoning of food (vegetable) after washing with water known to receive sewage contamination.Therefore the course of treatment with antibiotics will be difficult due to antibiotic resistance phenomenon that convert such strains from recipients to transconjugants and make them resistant to wide number of antibiotics.Eng.&Tech.Vol.26.No.11,2008Role of Conjugative Plasmids in Antibiotic Resistance between Two Strains of E. coli