A Study on Relationship of Plasmid with Antibiotic Resistance in Thermophilic Campylobacter spp. Isolates from Environmental Samples

MATERIALS AND METHODS

Organisms: Seventy isolates belonging to Camp. jejuni, Camp. coli and Camp. lari were isolated from environmental sources viz., animal feces (cow and horse) and sewage in Kazeroun, Iran, 2007. The method for isolation of the bacteria was prêt KB method (Baserisalehi et al., 2004). All these isolates were maintained in Burcella broth with 15% glycerol at -15°C.

Antibiotic susceptibility by disc diffusion method: The antimicrobial susceptibility pattern of the strains under study was studied by the disc diffusion method (Bauer et al., 1966). To perform the disc diffusion test, each culture was grown in 5 mL of Muller-Hinton broth until the turbidity corresponded to 0.5 MacFarland standard tube (1.5x10⁸ cells mL^-1). The suspension was spread inoculated using sterile cotton swab onto Muller-Hinton agar plate and various antibiotic discs were placed on it. After incubating the plates at 37°C under microaerophilic conditions for 48 h the inhibition zones were recorded.

The antibiotic discs included: chloramphenicol (30 μg), norfloxacin (10 μg), kanamycin (30 μg), co-trimoxazole (25 μg), cefotaxime (30 μg), ampicillin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), erythromycin (15 μg), gentamycin (10 μg), cephalexin (30 μg) (Hi Media Laboratories Limited, Mumbai).

Plasmid isolation: Plasmid was isolated using standard method recommended by Birnboim and Doly (1979). The quantitation and purity of DNA was done by determining 260/280 nm absorbance ratio spectra by spectrophotometer (UV-1601, Shimadzu).

Medium: Luria Bertani broth (LB) contained (g 100 mL^-1) Yeast Extract 0.5, NaCl 0.5 and Tryptone 1. The pH was adjusted to 7.2 and sterilized at 121°C for 20 min.

Reagents:

Single colony of each isolate was inoculated into 2 mL LB medium separately and incubated overnight at 37°C under microaerophilic conditions. The cells were harvested by centrifugation at 5000 rpm for 5 min at 4°C. The cell pellet was resuspended in 100 μL of solution I. Then 200 μL of solution II was added and the suspension was mixed gently and incubated on ice for 10 min. The solution III (150 μL) was added to the mixture and incubated on ice for 10 min. The mixture was centrifuged at 11500 rpm, 15 min at 4°C and the supernatant from each tube was transferred to fresh Eppendroff tubes. The mixture of phenol and chloroform in 1:1 proportion (500 μL) was added in the tubes and mixed properly. In each case the phases were separated by centrifugation at 11500 rpm, for 15 min at 4°C. Aqueous layer from each tube was transferred to fresh set of Eppendroff tubes and 1000 μL of 70% ethanol was added into each tube and incubated at room temperature for 10 min. The suspensions were centrifuged at 11500 rpm for 15 min at 4°C. The supernatant was removed by aspiration and discarded. The precipitated plasmid DNA was dried at room temperature for 30 min and dissolved in 50 μL TE buffer.

Confirmation of plasmid DNA by agarose gel electrophoresis: Electrophoresis of plasmid DNA was performed using horizontal gel electrophoresis. Agarose gel (0.8%) was prepared in TAE buffer (40 mM tris-HCl, 50 mM Sodium acetate, 1 mM EDTA; pH 8). Gel was run for two and half hours at 50 volts, stained for 30 min with ethidim bromide (0.5 μg mL^-1). The plasmids were visualized using UV light in Alpha imager gel documentation system (Alpha Innotech Corp., USA).

Plasmid curing
Plasmid curing by chemical agents: Three Camp. jejuni isolates (F44, P41 and W21) randomly were selected and subjected to plasmid curing by chemical agents and elevated temperature. Chemical agents used for plasmid curing were, Acridine orange, Acriflavine, Ethidium bromide (Intercalating dyes) and Rifampin. To perform plasmid curing, stock solutions of curing agents were prepared in distilled water. The curing agents were serially diluted in LB broth (1600 to 1.6 μg mL^-1). Overnight growth (0.1 mL) (adjusted to No. 0.5 McFarland tube 1.5x10⁸ cfu mL^-1) of each isolate was inoculated into each of the tubes containing 1 mL LB with increasing concentrations of curing agent and incubated at 37°C for 48 h. The Minimal Inhibitory Concentration (MIC) and the Minimal Bactericidal Concentration (MBC) were determined by observing, absence of growth in brain heart infusion broth and on nutrient agar at the lowest concentration of curing agent, respectively. The curing agents diluted to Subinhibitory Concentration (SIC) and were incorporated into the LB agar at SIC values. The overnight growth of resistant cultures was spread on LB agar with SIC of curing agent and incubated at 37°C for 2 days. The isolated colonies were picked up by sterile toothpicks and inoculated on another LB agar plate (50 colonies/plate). The plates were incubated at 37°C for 48 h and used as master plates. The colonies from each master plate were replicated on Luria agar with 30 μg mL^-1 chloramphenicol and on Luria agar with 15 μg mL^-1 Erythromycin and incubated at 37°C for 48 h. Those colonies, which failed to grow on selective medium, were regarded as cured colonies.

At the same time, a control for curing of each marker was maintained by inoculating non-cured culture on selective media to have a check on spontaneous loss of resistant markers.

Characterization of cured and non-cured Camp. jejuni isolates: Morphology of the cured and non-cured Camp. jejuni was evaluated using phase contrast microscope (Nikon, Japan). They were subjected to phenotypic identification tests recommended by Atabay and Corry (1997). These tests included, H₂S lead acetate strip, nitrate reduction, growth in 1% glycine and 3.5% NaCl, growth at different temperatures, viz., 25, 37 and 42°C and resistance to nalidixic acid (30 μg disc) and cephalothin (30 μg disc). Additional tests were hippurate hydrolysis, indoxyl acetate hydrolysis, urease production, alkaline phosphotase production and Glucose fermentation.

RESULTS

Antibiotic susceptibility of Campylobacter isolates: The results on antibiotic susceptibility of thermophilic Campylobacter isolates by disc diffusion method indicated that all the isolates of Campylobacter were sensitive to ciprofloxacin and resistant to cefotaxime, cephalexin and ampicillin. Besides, all the Camp. lari isolates were resistant to co-trimoxazole. All isolates of Camp. coli were sensitive to tetracycline. Amongst Camp. jejuni isolates, 74 and 70% of them were sensitive to gentamicin and kanamycin while 59 and 55% of them were sensitive to erythromycin and norfloxacin respectively. Less than 50% of the Camp. jejuni isolates were sensitive to chloramphenicol, tetracycline and co-trimoxazole and less than 50% isolates of Camp. coli were sensitive to rest of the antibiotics except co-trimoxazole and chloramphenicol. The number of Camp. coli isolates sensitive to antibiotics was relatively less than that of Camp. jejuni. Besides, less than 50% of Camp. lari isolates were sensitive to chloramphenicol, gentamicin, norfloxacin, kanamycin and erythromycin except tetracycline (Table 1).

Plasmid isolation: A set of forty isolates of Campylobacter was randomly selected and subjected to detection of plasmid by alkali lysis method. As shown in Table 2, 60% Camp. jejuni, 50% Camp. coli and 80% Camp. lari isolates harboured plasmids with ≥21 kb in size. The results indicated that the frequency of occurrence of the plasmid in Camp. lari isolates was relatively high while in Camp. coli was relatively low. Based on these observations, no correlation has been found between sources of isolates and presence of plasmids in Campylobacter isolates. Purity of plasmid DNA was found to be between 1.4-1.9 corresponding to 74.2-121 μg DNA mL^-1.

Plasmid curing: Curing is to confirm whether the genes for resistance are encoded by genomic DNA or plasmid DNA. Here, attempt was made to cure antibiotic resistant marker from Camp. jejuni isolates using chemical agents and physical agent (elevated temperature).

Camp. jejuni isolates F44, P41 and W21 were subjected to plasmid curing by chemical agents. The results obtained indicated that MIC, SIC and MBC values of rifampicin were relatively high while, that of Ethidium bromide were relatively low. The frequency of plasmid curing induced by rifampicin was 4%. Plasmid curing was observed only in Camp. jejuni F44. Acridine orange, Acriflavine and Ethidium bromide could not cure Camp. jejuni (Table 3).

However, erythromycin and chloramphenicol were considered as resistant markers for plasmid curing. But all cured Camp. jejuni showed loss of resistance to chloramphenicol (Table 4).

Characterization of cured and original Camp. jejuni isolates: The results obtained from characterization of cured and original Camp. jejuni indicated that cured and original Camp. jejuni exhibited similar behavior regarding all tests as well as morphology.

Table 1:	Susceptibility of environmental campylobacters by disc diffusion method
*Ch: Chloramphenicol, Ce: Cephalexin, No: Norfloxacin, Ka: Kanamycin, Co: Co-trimoxazole, Cf: Cefotaxime, Am: Ampicillin, Ci: Ciprofloxacin, Te: Tetracycline, Er: Erythromycin, Ge: Gentamicin

Table 2:	Plasmid isolation from Campylobacter isolates
Plasmids isolated by Birnboim and Doly (1979) method

Table 3:	Susceptibility of Campylobacter jejuni isolates to chemical curing agents
*MIC: Minimal Inhibitory Concentration, *MBC: Minimal Bactericidal Concentration, *SIC: Subinhibitory Concentration

Table 4:	Curing of plasmid in Campylobacter jejuni
*chr: Chloramphenicol resistant marker, -: No strain was cured

DISCUSSION

Antimicrobial resistance property can come up through acquisition of genetic material encoding enzymes that inactivate a particular antibiotic (Hoffman, 1999). Gene resistance markers in campylobacters can be presented in plasmid, chromosome or both. For instance, erythromycin resistance has been reported previously as by chromosomal genes, whereas chloramphenicol resistance is plasmid encoded. It has been reported that resistance to chloramphenicol, kanamycin and tetracycline in Camp. jejuni is plasmid-mediated, while resistance to rest of the antibiotics is chromosomally mediated (Taylor and Courvalin, 1988; Taylor and Chau, 1996). On the other hand, several reports illustrated that some virulence factors of Campylobacter jejuni are associated with existence of the plasmid in the bacterium (Bacon et al., 2000; Tracz et al., 2005).

Present findings from this study indicated that out of all Campylobacter tested; plasmids were detected in 60% Camp. jejuni, 50% Camp. coli and 80% Camp. lari isolates with ≥21 kb in size. It means frequency of plasmid occurrence in Camp. lari is relatively high while, in Camp. coli was relatively low. Hence, it could be interpreted that occurrence of the plasmid in the Camp. lari isolates with high frequency might induce antibiotic resistant property to the bacterium.

Although Campylobacter jejuni is most important causes of bacterial diarrhea worldwide (Taylor, 1992), the details of its molecular pathogenesis are not well understood (Bacon et al., 2000). Thus, the present study was undertaken to carry out plasmid curing in order to achieve maximum information concerning to location of resistant markers in this bacterium.

Plasmid curing defined as a loss of plasmid from cell, which leads to loss of specific phenotypes such as drug resistance (Bouanchaud et al., 1969). To perform plasmid curing three strains of Camp. jejuni (F44, P41 and W21) isolates were subjected to plasmid curing to find out location of resistant markers of chloramphenicol and erythromycin. The result obtained indicated that the frequency of plasmid curing induced by elevated temperature and rifampicin was 8 and 4%, respectively while, Acridine orange, Acriflavine and Ethidium bromide could not cure Camp. jejuni. According to the data, only Camp. jejuni F44 was cured and the cured strain were sensitive to chloramphenicol and resistant to erythromycin. Therefore, it can be concluded that probably erythromycin resistant marker in Camp. jejuni is chromosomally mediated, while chloramphenicol resistant marker is plasmid mediated. This finding is supported by Wang and Taylor (1990). Although, according to present data chloramphenicol and erythromycin resistant markers in Camp. jejuni is plasmid and chromosomally mediated respectively, Dasti et al. (2007) reported, tetracycline resistance in Camp. jejuni, could be plasmid or chromosomally mediated.

The results obtained from characterization of cured and original strain of Camp. jejuni F44 using phenotypic identification tests indicated that no significant difference has been found between cured and original Camp. jejuni F44. Therefore, it can be concluded that the most of phenotypic characters of Camp. jejuni are associated with primary metabolism and its metabolites is chromosomally mediated.

Furthermore, present finding indicated that plasmid mediated of some antibiotic resistance markers in campylobacters cause transmission of the resistance markers among these bacteria and finally reach the human population by direct contact and via food products of animal origin.