Members of the Campylobacter genus are gram-negative, curved and
S-shaped microaerophilic bacteria. They are responsible for human gastroenteritis
throughout the world and enteritis caused by them in some places is more
important than that of Salmonella and Shigella (Scotter et al.,
1993). Antimicrobial chemotherapy of patients with acute Campylobacter
enteritis involves treatment with erythromycin, tetracyclines and
fluoroquinolones (Alfredson et al., 2003). However, frequency of
occurrence of antibiotics resistance Campylobacter increasing in
developed and developing countries (Taylor and Courvalin, 1988), but antibiotics
therapy still is final remedy in case of patient with acute Campylobacter
enteritis. Resistance of Campylobacter spp. to a number of antibiotics,
such as tetracycline, erythromycin, ciprofloxacin, kanamycin,
nalidixic acid and chloramphenicol has been reported by Piddock
et al. (2000). Furthermore, the rate of fluoroquinolone-resistant
campylobacters in Thailand, from 0-84% increased during 1990-1995 (Isenbarger
et al., 2002). Resistance of Emergence and dissemination of antibiotic
resistance among Campylobacter spp. have been linked
to the use of antibiotics in veterinary medicine and use as
prophylactics and growth promoters in animal husbandry (Piddock
et al., 2000). The increasing rate of human infections caused
by antimicrobial-resistant strains of Camp. jejuni makes clinical
management of cases of campylobacteriosis more difficult, prolonging
illness and compromising treatment of patients with bacteremia
Despite, deficiency of data in case of the molecular pathogenesis of
campylobacters, many reports opined that antibiotic resistance markers
in campylobacters could be chromosomally or plasmid-mediated (Taylor and
Courvalin, 1988). For instance, tetracycline resistance in strains of
Campylobacter jejuni and Campylobacter coli was mediated
by plasmids (Taylor and Chau, 1996). Objectives of this study were assessed
to determined susceptibility of thermophilic Campylobacter isolates
to antibiotics. Afterward, plasmid isolation from pathogenic campylobacters
was carried out to achieved information concerning to the presence of
plasmid DNA in Campylobacter isolates and finally plasmid curing
was done to seek correlation between resistance to antibiotics and plasmid
occurrence in Camp. jejuni.
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
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.
||:50 mM Glucose, 10 mM Na2EDTA, pH 8 and 25
mM Tris-Cl, pH 8
||0.2N NaOH and 1% SDS
||3M Sodium acetate, pH 4.8-5.3
||10 mM Tris-Cl, pH 8 and 1mM Na2EDTA, pH 8
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 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.
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
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.
||Susceptibility of environmental campylobacters by disc
|*Ch: Chloramphenicol, Ce: Cephalexin, No: Norfloxacin,
Ka: Kanamycin, Co: Co-trimoxazole, Cf: Cefotaxime, Am: Ampicillin,
Ci: Ciprofloxacin, Te: Tetracycline, Er: Erythromycin, Ge: Gentamicin
||Plasmid isolation from Campylobacter isolates
|Plasmids isolated by Birnboim and Doly (1979) method
||Susceptibility of Campylobacter jejuni isolates
to chemical curing agents
|*MIC: Minimal Inhibitory Concentration, *MBC: Minimal
Bactericidal Concentration, *SIC: Subinhibitory Concentration
||Curing of plasmid in Campylobacter jejuni
|*chr: Chloramphenicol resistant marker, -:
No strain was cured
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
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.