Advances in Microbiology, 2015, 5, 797-806
Published Online November 2015 in SciRes.
How to cite this paper: Vaishnavi, C., Singh, M. and Kapoor, P. (2015) Isolation of Campylobacters from Intestinal Tract of
Poultry in Northern Region of India. Advances in Microbiology, 5, 797-806.
Isolation of Campylobacters from Intestinal
Tract of Poultry in Northern Region of India
Chetana Vaishnavi*, Meenakshi Singh, Prashant Kapoor
Department of Gastroenterology Postgraduate, Institute of Medical Education and Research, Chandigarh, India
Received 16 October 2015; accepted 13 November 2015; published 16 November 2015
Copyright © 2015 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution International License (CC BY).
Campylobacter is one of the most common food-borne bacterial enteropathogens. We planned to
investigate the prevalence and antibiotic resistogram of Campylobacter in poultry in and around
Chandigarh. Poultry samples (n = 127) were obtained from slaughter houses/retail outlets and
cultured microaerophilically on Campylobacter media. The isolates were identified phenotypically
and by molecular investigation. Identification of specific genes to look for resistance to nalidixic
acid, ciprofloxacin, tetracyclin and streptomycin was also done. Campylobacter was isolated from
57/127 (44.9%) of the samples. The most frequent serotypes identified were B: 2, S: 27, Z5: 52 and
Z7: 57. All culture isolates (100%) were reconfirmed as Campylobacter by 16S rRNA polymerase
chain reaction. Molecular identification of isolates revealed the presence of C. jejuni in 45 (79.0%),
C. coli in 1 (1.8%) and co-infection of C. coli and C. jejuni in 11 (19.3%). No C. lari and C. upsaliensis
were detected. Antibiogram typing showed nalidixic acid resistance in 36.8%, ciprofloxacin resis-
tance in 35.0% and 31.5% resistance for both streptomycin and tetracyclin. A high level of Cam-
pylobacter prevalence was found among the poultry with C. jejuni being the most commonly iso-
lated species. Resistance to major antibiotics among Campylobacter isolates from poultry was also
very high. The study of prevalence of Campylobacter in poultry and its resistance to major antibio-
tics will help to plan risk burden strategies throughout the food chain.
Antibiotic Resistance, Campylobacter, Molecular Investigation, Phenotypic Identification, Poultry,
1. Introduction
Campylobacter has long been known as a part of the normal flora in the intestine of most animals including
poultry due to their high body temperature which provides an optimum growth milieu for this thermotolerant
Corresponding author.
C. Vaishnavi et al.
genus [1]. The organism has been found to be responsible for causing abortion in cattle and sheep and diarrhea
in cattle and pigs [2]. Human beings can acquire Campylobacter through consumption of raw or undercooked
meat and poultry [3], contaminated water and vegetables [4], unpasteurized milk [5], or by contact with fecal
matter from infected domestic pets or people [6]. As poultry meat is a good source of animal protein, it is easily
appealing to consumers and the consumption of which leads to infections. Poultry is therefore considered to be
the main reservoir for campylobacteriosis in humans. Poultry meat is of prime importance in food associated
disease in developed countries [7]. The most common Campylobacters causing human diseases are C. jejuni and
C. coli with rarely other spec ies like C. fetus, C. upsaliensis and C. lari [8] [9]. C. jejun i has been reported to be
the most commonly isolated species in chickens [1]. Thus, Campylobacter is one of the most common agents of
bacterial gastroenteritis and a major health burden for both developed and developing countries [10].
Campylobacter is an important bacterial cause of food-borne disease outbreaks in the Un ited States [11]. Stu-
dies on antibiotic resistan t pattern of Campylobacters from developing countries are sparse, due to lack of fund-
ing and facilities for culture. Ther e is a paucity of information fr om India regarding Campylobacter and their an-
tibiotic resistance pattern in poultry, probably due to the absence of surveillance in our region. Campylobacte r
infections remain a high research priority for improvement of strategies related to prevention and management
of the disease [9]. As poultry is th e main reservoir of Campylobacters for human trans mission, there is clearly a
need for local surveillance and control measures based on quantitative data of Campylobacter colonization in
poultry. The present work was carried out to study the prevalent antibiotic profile of Campylobacters isolated
from poultry in Chandigarh, a northern region of India.
2. Materials and Methods
The study was approved by the Institute Ethics Committee of Post Graduate Institute of Medical Institute and
Research, Chandigarh, India and was conducted from May 2009 to January 2013.
2.1. Poultry Sample Collection
One hundred and twenty seven poultry samples from slaughter houses/retail outlets in and around Chandigarh
formed the basis of investigation. Lower intestinal portions of poultry were collected in campy-thioglycollate
media (HiMedia, India), transported in an ice bucket to the Microbiology Division of the Department of Gas-
troente rology and were i mmediatel y processed for t he isolation of Campylobacters. This was the same time period
(May 2009 and January 2013) and geographical area (Mauli Jagran a nd Indi ra colony ) from where the c ommunit y
fecal samples investigating for Campyloba cters were collected in an earlier study [9].
2.2. Culture for Campylobacters
Poultry intestine pieces were homogenized in physiological saline and vortex mixed for 1 min under sterile con-
ditions. The suspension was ino culated directly either on selective Campylobacter agar base media (Oxoid Ltd.,
Cheshire, England) containing antibiotics and 5% - 7% defibrinated sheep blood oron charcoal cefoperazone
deoxycholate agar (Oxoid Ltd., Cheshire, England) by quadrant streaking. The suspensions were als o passed
through filter membrane of pore size 0.45 µm and 0.65 µm (Millipore, USA) placed on media plates and kept as
such for one hour. The filters were then removed and discarded. All the plates were incubated under microaero-
philic conditions at 37˚C and 42˚C for 72 h.
2.3. Phenotypic Identification of Campylobacters
Predominant or pure growth of grey to ceramic colonies (Figure 1) was investigated by Gram stain and by bio-
chemical tests viz. catalase, oxidase and hippurate hydrolysis. Camp ylobacter species was suspected when the
isolate gave positive reaction with oxidase and/or hippurate (Figure 2) in addition to any other positive identifi-
cation tests.
2.4. Serotyping of C. jejuni
The pathogenic C. jejuni was serotyped on the basis of its heat stable “O” antigen extract and a passive hemag-
C. Vaishnavi et al.
Figure 1. Colonies of Campylobacter on Campylobacter agar
base media.
Figure 2. Formation of purple color indicative of positive hip-
purate hydrolysis.
glutination assay using 25 C. jejuni specific antisera (Denka-Seiken, Japan). For this procedure, antigen was
prepared by suspending a loopful of the organisms in physiological saline, heating at 100˚C for 1 h and centri-
fuging at 3287 g for 5 mins. The extracted antigen was suspended in 500 µl phosphate buffered saline. This
suspension was mixed with an equal volume of washed suspension of sheep erythrocytes (1% v/v). After sensi-
tization for 1 hour at 37˚C, erythrocytes were washed and resuspended to 500 µl. Twenty-five microliter each of
25 antisera were added in U-bottom microplates and an equal volume of sensitized sheep erythrocyte suspension
was added to each well. The plates were observed for agglutination after incubation at 37˚C for 30 m i ns .
2.5. Molecular Identification of Campylobacter Isolates
Campylobacter isolates were subjected to molecular identification for different species by polymerase chain
reaction (PCR). For isolation of DNA, heavy growth of Campylobacter isolate was suspended in 500 µl Tris-
EDTA buffer. The suspension was boiled at 100˚C for 10 mins and immediately transferred to an ice bath and
incubate d for 1 h. Next, t he suspensio n was centrifuge d at 6710 g for 2 mins and the su pernatant was se parated an d
C. Vaishnavi et al.
checked for DNA by 0.8% agarose gel electrophoresis. The primers (Table 1) used to identify Campylobacter
species and antibiotic r esistance b y amplifyin g specific g enes are the same as those mentioned in an earlier pub-
lication [9]. All the PCR products were analyzed by electrophoresis on 1.8% agarose gel containing 0.1 µg/ml
ethidium bromide.
(1) Identificati on of Campylobacter species: PCR assay was done using specific primers to the unique regions
of Campylobacter genus and to the unique regions of different Campylobacter species. Species-specific identi-
fication of C. jejuni, C. coli, C. lari and C. upsaliensis was done by amplifying the hippuricase (hipO) gene, the
aspartokinase (aspK) gene, the serine hydroxymethyl transferase (glyA) gene and li pop oly sacc haride (lpxA) gene
(2) Detection of antibiotic resistance: Nalidixic acid resistance was identified by amplifying specific gyrA
gene using primers gyrA forward and gyrA reverse. Ciprofloxacin (gyrA) resistance gene in Campylobacter
isolates was investigated by Mismatch Amplification Mutation Assay (MAMA) using a conserved forward primer,
Campy MAM A gyrA1 and a mutation detection reverse primer, Campy MAMA gyrA5. An annealing temperature
of 50˚C was used to give 265 bp products which indicated the presence of the Thr-6 to Ile (A CA ATA) mu-
tation in the Campylobac ter gyrA gene. Tetracyclin resistance was detected by using specific primers to amplify
tetO gene. Streptomycin resistance was detected by amplifying the strA gene .
3. Results
3.1. Phenotypic Methods
Campylobacter was isolated from 57/127 (44.9%) of the poultry samples by culture. Eighteen (31%) of the C.
jejuni isolates were serotyped. The serotype of the tested organism was determined as positive based on agglu-
tination obtained over the bottom of well and as negative when button formation occurred (Figure 3). If the or-
ganism reacted to multiple antiser a, it was determined as multiple serotypes. The most f requent serotypes of C.
difficile from poultry isolates in descending order were B: 2, S: 27, Z5: 52, V: 32 and Z7: 57 (Table 2).
3.2. Molecular Methods
All the 57 (100%) culture positive isolates were reconfirmed as Campylobacter species by molecular investigation
for 16S rRNA, hipO, aspK, glyA and lpxA genes. PCR revealed the presence of C. jejuni i n 45 ( 79.0%) , C. coli in
1 (1.8%) and c o-infection of C. coli and C. jejuni in 11 (1 9.3%) of the c ultures but we re negat ive for C. lari and C.
upsaliensis. Antibiogram typing done by molecular methods showed nalidixic acid r esistance in 36.8% (21/57),
ciprofloxacin resistance in 35.0% (20/57) and 31.5% (18/57) resistance each for streptomycin and tetracycline
(Figure 4).
Table 1. Primers used for amplification of target genes of Campylobacter.
S. No. Target genes Primer Sequences References
R-5’ GTAACTAGTTTAGTATTCCGC 3’ Linton et al. [32]
2 hipO (C. jejuni) F-5’ GGAGAGGGTTTGGGTGGT-3’
R-5’-AGCTAGCCTCGCATAATAACTTG-3’ Linton et al. [32]
3 aspK (C. coli)
R-5’ TACACATAATAATCCCACCC 3’ Yamazaki-Matsune et al. [34]
5 lpxA (C. upsaliensis)
Yamazaki-Matsune et al. [34]
6 Tetracyclin (tetO)
Ng et al. [35]
7 Nalidixic acid (gyrA) F-5’ GCT CTT GTT TTA GCT TGATGCA 3’
R-5’ TTG TCG CCA TC CTA CAGCTA 3’ Jesse et al. [36]
8 Ciprofloxacin (MAMA gyrA) F-5’ TTT TTA GCA AAG ATT CTG AT 3’
Zirnstein et al. [37]
9 Streptomycin (strA) F-5’ CCAATCGCAGATAGAAGGCAAG 3’
R-5’ ATCAACTGGCAGGAGGAACAGG 3’ Maidhof et al. [38]
C. Vaishnavi et al.
Figure 3. Microplate showing agglutination and button formation for C. jejuni isolates.
Table 2. Number of C. jejuni isol ates showing multiple serotypes (serotypes are given in alphabetic order and the most fre-
quent ones are marked in bold).
Serogroup No. of C. jejuni isolates (multiple serotypes)
Group A: 1, 44 3
Group B: 2 19
Group C: 3 6
Group D: 4, 13, 16, 43, 50 6
Group E: 5 3
Group F: 6, 7 6
Group G: 8 4
Group I: 10 6
Group J: 11 7
Group K: 12 6
Group L: 15 7
Group N: 18 6
Group O: 19 6
Group P: 21 5
Group R: 23, 36, 53 6
Group S: 27 12
Group U: 31 6
Group V: 32 9
Group Y: 37 5
Group Z: 38 4
Group Z2: 41 7
Group Z4: 45 7
Group Z5: 52 12
Group Z6: 55 6
Group Z7: 57 9
C. Vaishnavi et al.
Figure 4. PCR detection of gyrA genes for resistance of nalidixic acid (620 bp) and ciprofloxacin (265 bp) re-
spectively. Lane 1—M ar k er ; L ane 2Positive control; Lanes 3 to 10—Campylobacter isolates.
4. Discussion
Campylobacter is a major health burden for both developed and developing countries. Most of the Camp y lobac-
ter isolates causing human gastro enteritis are thermo-tolerant variety and can grow even at incubation tempera-
tures of 42˚C. Farm animals and wild birds are the primary sources contributing to human infections of Campy-
lobacter due to consumption of contaminated water. Drinking water gets contaminated from septic seepage and
waste water intrusion into ground water sources. Handling and eating undercooked poultry have consistently
been shown to be important ris k factor s in food-borne illness due to Campylobacter [3]. A survey of raw poultry
demonstrated that 50% - 70% of raw chickens tested at the retail level were contaminated with Campylobacter
C. jejuni has been reported to be the most isolated species in chicken [1]. Yu et al. [13] reported the first rec-
ognized major C. jejuni outbreak in a middle School in Incheon, associated with contaminated chicken in Korea
where an attack rate of 11.6% occurred with 40.3% stool samples positive for C. jejuni. The authors state that
the raw chickens used in the chicken soup with ginseng were supplied frozen (6˚C) in the morning of July 1 by
a company which was free of food hygiene violations. The chickens had been slaughtered and processed on June
29 and were deemed acceptable for use until July 9 under refrigeration. Despite this, the chicken soup prepared
and consumed on July 1, 2009 was considered to be the source of human infection [13]. Yano et al. [14] moni-
tored C. jejuni in four chicken farms during the period 2003 to 2006 to elucidate the mechanisms of transmission.
A total of 20 6 C. jejuni isolates were obtained with C. jejuni coming from common sources external to the farms.
In the present study, prevalence of Campylobacters was 44.9% in the poultry in and around Chandigarh region
and C. jejuni was the most prevalent Campylobacter. This is in contrast to another study from the same region
carried out during the same time period among human beings where a low prevalence of Campylobacter was
seen [9].
Typing methods have a significant role in the identification, monitoring, and prevention of Campylobacter
infections. Combination of phenotypic and genotypic methods can improve species discrimination of pathogens
such as Campylobacter . A genotyping method like PCR has been found to be an efficient and reliable typing
method with greater discriminatory power providing superior results [15]. Lawson et al. [16] used genetic tar-
gets aspK and hipO and Bang et al. [17] used 16S rRNA to identify C. coli and C. jejuni. The same molecular
methods were also used in the present study.
Serotyping methods can be us ed to differentiate clonally related isolates fro m unrelated ones due to different
characteristics. Penner hemaglutination assay is a serotyping method widely used for characterizing Campylo-
bacter isolates. The C. jejuni capsular polysaccharides (CPS) are the primary serodeterminant of the Penner
scheme. On and off CPS expression by C. jejuni suggests that this tactic might have a role in Campylobacter vi-
rulence [18]. Based on their geographic locations, the serotype prevalence of Campylobacter differs across
countries. The most predominant serotypes in Japan are B, D, and L, while those in Denmark are serotypes B, A,
and D [19] [20]. Ishihara et al. [21] identified 18 serotypes among the C. jejuni isolates from humans with major
C. Vaishnavi et al.
ones being B, D and R. In another study from Thailand [22], C. jejuni isolates from poultry were classified into
10 Penner serotypes viz. A, C, I, K, B, E, S, D, L and R. The most frequent serotypes in the present study were
B: 2 , S: 27, Z5: 52 and Z7: 57 in descending order. Thus serotype B seems to be more common among the Cam-
pylobacter isolates as also seen in our earlier study in humans [9] and Campylobacter from poultry could be the
source of infection among the human population studied from the same region [9].
Another problem of great concern at global level is the acquisition of resistance to multiple antibiotics by
Campylobacters due t o unre gula ted use of a ntim icrobi al ag ents i n foo d ani mal pr oducti on [23]. Several resistance
genes underlie the emergence of multidrug-resistant Campylobacter. Macrolides and fluroquinolones are com-
monly prescribed for campylobacteriosis. But, resistance to t hese and other a ntibiot ics also occurs . In Net herland s
almost 30% of Campylobacter isolates were resistant to fluroquinolones [24]. An increase in f luroquinolone re-
sistance in Campylobacter spp. from Europe and USA has a lso been re porte d [25] due t o their u se in foo d anim als
[26]. Since erythromycin is the drug of choice for the treatment of Campylobacter infections the prevalence of
resistance to this anti microbial, especially among str ains isolated from food, is a cause of concern. Previous stu-
dies on the susceptibility of Campylobacter to macrolides showed that the rate of resistant isolates was at a low
level and did not e xceed 1% [27]-[29]. However, a relatively high level of resistance to st reptomycin (22.8%) was
reported in Poland [30]. In the present study antibiotic resistance was 36.8% for nalidixic acid, 35.0% for ci-
profloxacin, and 31.5% for both streptomycin and tetracyclin . The findings of the present investigation are co n-
sistent with previous results as a relatively high level of resistance to streptomycin was seen. Isolates from chicken
broilers were 67% resistan t to tetracyclin [31]. The MAMA PCR has been considered as a valuable and reliable
alternative tech ni q ue to sequencing for det e ction of the T h r-86 Ile mutation for ciprofloxacin [31].
5. Conclusion
This study indicates that in northern region of India, there is an increasing emergence of antibiotic resistance
among Campylobacter strains in poultry. It is important to reduce the contamination rates by Campylobacter in
poultry by plannin g risk bur den strate gies thr oughout t he food cha in. One l imita tion of the pr esent st udy is that we
cannot investigate Campylobacte rs in domestic poultry re ar ing, which can also be a source of infection to human
beings. New strategies for containing Campylobacter infections will likely includ e limiting consumption of an-
tibiotics by animals, disinfection of their food and water, treatment of their manure, and isolation of the conta-
giously ill. Other strategies like irradiation of food s of animal origin may become a feasible method of control of
the bacterial contamination of foods. Integrated efforts must be done in order to encourage the appropriate use of
antimicrobials in food animals and for the implementation of a surveillance system of drug resistance in Cam-
The authors are grateful to Prof. Rama Chaudhry for providing DNA material of C. upsaliensis and C. lar i as
controls. Mr. Prashant Kapoor and Mr. Vikram Singh are acknowledged for their technical support.
This work wa s s upported by the Indian Council of Medi c a l Research, New Del hi.
Transparency Declaration
There is no conflict of interest.
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List of Abbreviations
1) %—Percentage
2) µg—Microgram
3) µl—Microliter
4) aspK—aspartokinase
5) C. coliCampylobacter coli
6) C. fetusCampylobacter fetus
7) C. jejuniCampylobacter jejuni
8) C. lariCampylobacter lari
9) C. upsaliensisCampylobacter upsaliensis
10) CPS—Capsular polysaccharides
11) DNADeoxyribonucleic acid
12) EDTA—Ethylenediaminetetraacetic acid
13) g—gravity
14) glyA—Glycine
15) gyrA—Gyrase A
16) h—hour
17) hipO—hippuricase
18) Ile—Isoleucine
19) lpxA—lipopolysaccharide
20) MAMA—Mismatch Amplifica tion Mutation Assay
21) mins—minu tes
22) ˚C—Degrees Celsius
23) PCR—Polymerase chain reaction
24) rRNA—Ribosomal ribonucleic acid
25) strA—streptomycin A gene
26) tetO—tetracyclin O gene
27) Thr—Threonine