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BRIEF COMMUNICATION
Year : 2014  |  Volume : 32  |  Issue : 4  |  Page : 425-429
 

Prevalence and antibiotic susceptibility of thermophilic Campylobacters from sources implicated in horizontal transmission of flock colonisation


1 Department of Microbiology, University of Pune, Ganeshkhind, Pune-411 007, Maharashtra, India
2 Institute of Bioinformatics and Biotechnology, University of Pune, Ganeshkhind, Pune-411 007, Maharashtra, India

Date of Submission04-Sep-2013
Date of Acceptance27-Mar-2014
Date of Web Publication4-Oct-2014

Correspondence Address:
B Kapadnis
Department of Microbiology, University of Pune, Ganeshkhind, Pune-411 007, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.142259

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 ~ Abstract 

Thermophilic Campylobacter are commonly associated with poultry as commensals of the avian gut and are the causative agent responsible for human Campylobacteriosis. This study aimed to establish the prevalence of Campylobacter spp. from environmental sources that have previously been implicated as sources of horizontal transmission. The highest prevalence of thermophilic Campylobacter was found in water samples (87.5%) and lowest from flies (7.2%). Only C. jejuni was isolated from all sources. A secondary aim was to provide a baseline of resistance profiles of Campylobacter spp. isolates obtained. Alarmingly all the C. jejuni isolates from environmental sources as well as humans were multi-drug resistant.


Keywords: Antibiotic resistance, campylobacter, horizontal transmission


How to cite this article:
Parkar S, Sachdev D, Kapadnis B. Prevalence and antibiotic susceptibility of thermophilic Campylobacters from sources implicated in horizontal transmission of flock colonisation . Indian J Med Microbiol 2014;32:425-9

How to cite this URL:
Parkar S, Sachdev D, Kapadnis B. Prevalence and antibiotic susceptibility of thermophilic Campylobacters from sources implicated in horizontal transmission of flock colonisation . Indian J Med Microbiol [serial online] 2014 [cited 2019 Aug 18];32:425-9. Available from: http://www.ijmm.org/text.asp?2014/32/4/425/142259



 ~ Introduction Top


Campylobacter jejuni and C. coli are the most commonly reported bacterial causes of acute gastroenteritis in humans in both developing and developed countries. [1] Campylobacters may cause a spectrum of illness in humans. The signs manifested by the patients include abdominal cramping and diarrhoea. Other extra intestinal diseases may result from Campylobacter infections including bacteraemia, endocarditis, meningitis, urinary tract infection (UTI), and Guillain-Barré syndrome, which are the acute paralytic diseases of the peripheral nervous system.

The most important sources of Campylobacter infections for humans are associated with poultry. [1] Various studies have demonstrated high levels of Campylobacter on broiler chickens from poultry farms [2] and on retail chickens. [3] The presence of Campylobacter in the agricultural context has widely been documented in scientific literature; however, there are few reports for the same from India. [4]

The treatment and clinical management of Campylobacteriosis in man is adversely affected due to the escalating rates of antibiotic resistance. [5] This is because antimicrobial resistance prolongs the infection and causes further complications in patients with bacteraemia. Infections due to antimicrobial resistant enteric bacteria are highest in the developing world, where the use of antimicrobial drugs in humans and animals is relatively unrestricted. [6] The unregulated use of antimicrobial agents in food animal production has led to the emergence and spread of antibiotic resistance among Campylobacter spp. After the approval and thereafter the subsequent use of fluoroquinolones in poultry in Europe and USA, there was an increase in fluoroquinolone resistance in Campylobacter spp. from animals and human patients. [7]

The epidemiology of Campylobacter in broiler production is not wholly understood to date. There is a difference of opinion over, which are the main sources for flock colonisation. [8] The transmission of Campylobacter occurs in the flock, after original introduction of the organism into that group and as intra-intestinal colonisation of its members commences. [9] Two types of transmission have been pursued by researchers. The first is vertical transmission from parent chicken to progeny. The second being horizontal transmission from external sources such as feed, water, domestic animals, insects, rodents and wild birds to chicken. Carryover from previous flocks and horizontal transmission via contaminated water, domestic and wild animals, personnel working in the broiler house, and the external environment has been implicated. [10],[11] Vertical transmission from Campylobacter-positive breeder flocks via the egg to their progeny has not been found to be very likely. [12] Therefore, based on preceding substantiation, and because, investigations on bacteriological, pathological, clinical, and epidemiological aspects of Campylobacters in India are moderately recent, the present study was undertaken to determine frequency of occurrence of pathogenic Campylobacters in the farm environment and to determine antimicrobial susceptibility patterns of the isolates.


 ~ Materials and Methods Top


During the study, 233 samples were collected from poultry farms and from several retail establishments located in Pune, India [Table 1]. Samples were collected from different wild birds and animals, poultry chickens, rodents, humans, water, soil, and flies. These were examined for presence of Campylobacter spp. All the samples were transported to the laboratory under refrigerated conditions, and microbiological analyses were carried out immediately. The poultry birds were sampled non-invasively in that there was no entry into the body cavity of the birds. All scientific ethical practices were respected. In this study, fresh faecal, cloacal swabs and feather swabs each were collected from the poultry chickens. Faecal samples were collected from wild birds in and around the poultry farm using a reported protocol. [13] The rectal contents were collected from rodents and analysed by reported protocol. [14] Flies and treated drinking water in the farm area were analysed using the protocol by Khalil et al., (1994). [15] Litter samples were obtained from poultry broiler house.
Table 1: Campylobacter jejuni isolated from farm sources

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For analysis of samples from humans and rodents about 0.5 g of rectal contents/faeces were directly plated on modified charcoal cefoperazone deoxycholate agar (mCCDA) plates. [14] This was followed by incubation at 42 ± 1°C for 48 h under the microaerobic conditions.

The swabs were transported in sterile Preston enrichment broth (PEB). Campylobacter was isolated from collected flies by dissecting them vertically and horizontally into four pieces followed by addition of each fly to 5 ml sterile PEB. Water was filtered through 0.45 μm millipore filters. The filter was removed and added to 50 ml sterile PEB. Preston Campylobacter selective enrichment broth (PEB) (HiMedia, Mumbai India) was supplemented with selective supplement (HiMedia, Mumbai India) containing polymyxin B (5 IU/ml), rifampicin (10 μg/ml), trimethoprim 10 (μg/ml) and cycloheximide (100 μg/ml) along with 10% horse blood (Haffkine Biopharmaceutical Ltd, Pune, India) for all isolations. Hundred microlitres were immediately cultured onto mCCDA with antibiotics. This was followed by incubation at 42 ± 1°C for 48 h under the microaerobic conditions (5% O 2 , 10% CO 2, and 85% N 2 ) in an anaerobic jar (Anaerobic System Mark VI, HiMedia, Mumbai India).

Presumptive thermotolerant Campylobacter colonies were picked based on their morphological characteristics. Biochemical tests, which consisted of hippurate hydrolysis, catalase test, indoxyl acetate hydrolysis and H 2 S test, were performed on colonies isolated from the blood agar plates. The HiCampylobacter™ Latex Test Kit (Hi-Media, India), a rapid latex agglutination test was used for confirmation of the isolates as thermophilic Campylobacters.

Templates for polymerase chain reaction (PCR) were obtained from single bacterial colony, which was selected for every isolate and suspended in sterile saline. Cells were centrifuged to obtain pellet and washed thrice with sterile water to remove media components. Purified DNA was prepared using commercial DNA isolation kits (Chromous Biotech (P) Ltd., India) according to manufacturer's instructions.

The presumptive Campylobacter were authenticated by the presence of a 450-bp amplicon obtained using the primers designed to specifically amplify coding regions from the flagellin gene by previously reported method [16] using.

Forward primer, Pg50 5'- ATGGGATTTCGTATTAAC-3' and Reverse primer, Pg3 5'-GAACTTTGAACCGATTTG-3'. Tubes were subjected to 25 cycles of 94°C for 1 min, 37°C for 1 min, and 72°C for 1 min, followed by a 5 min extension at 72°C. Primers were synthesised by BioResource Biotech (P) Ltd.

The 16S rRNA sequencing was carried out and sequenced data were subjected to Basic Local Alignment Search Tool (BLAST) analysis.

The minimum inhibitory concentration of Campylobacter was determined using the agar dilution method towards ampicillin, azithromycin, chloramphenicol, ciprofloxacin, doxycycline, gentamycin, nalidixic acid, norfloxacin, and tetracycline. There is a dearth of internationally validated criteria for breakpoints of susceptible or resistant isolates for Campylobacter. Consequently, where breakpoints from the Clinical and Laboratory Standards Institute (CLSI) were not available, established breakpoints were used. [17]


 ~ Results Top


In this study, 64 samples were collected from different wild birds and animals. Faecal samples were collected from 45 poultry chickens from the farm environments and neighboring retail markets, 10 rodents, and 15 humans. Alongside, water, soil, and flies were also examined for presence of Campylobacter spp. In total, 46 Campylobacter isolates were obtained from poultry farms as well as retail poultry. The highest prevalence was found in water samples (87.5%) followed by human faecal samples (80%), soil (50%) and poultry chickens (44.44%), wild birds (15.62%) and flies (7.2%). All rodent samples were negative for Campylobacter spp.

The isolates were positively identified using the PCR-based assays for identification of Campylobacter spp. The PCR yielded the expected amplicon of product size of 450 bp, with the primers specific for flagellin gene as shown in [Figure 1].
Figure 1: Gel image depicting 450-bp amplicons obtained after PCR assay of genomic DNA of Campylobacter isolates, using the flagellin gene primer Lane1, C. coli MTCC 131; Lane 2, JQ972908; Lane3, JQ972909; Lane4, JQ972910; Lane5, JQ972911; Lane6, JQ972912; Lane7, JQ972901; Lane8, Thermo Scientific GeneRuler 100-bp Plus DNA Ladder (100-3000 bp)

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Partial 16S rRNA sequences of the 46 different isolates were obtained and subjected to BLAST analysis using the NCBI Blast software. The isolates were identified as C. jejuni according to the BLAST results as well as phenotyping.

The nucleotide sequences of 30 isolates were deposited in NCBI GenBank (accession numbers JQ972883-912). [Figure 1] represents the 450-bp amplicon obtained after PCR of these isolates. These results positively confirm the presence of Campylobacter spp. in the Indian poultry environment.

The proportion of isolates resistant to each antimicrobial agent for C. jejuni isolates [Table 2] from poultry farm was as follows: 100% for ciprofloxacin, norfloxacin and nalidixic acid, 97.82% for erythromycin and tetracycline, and 43.4% for gentamycin. All the isolates in this set were sensitive to ampicillin, azithromycin, chloramphenicol, clindamycin, doxycycline and streptomycin. The majority of the isolates were grouped into similar categories. In [Table 3] the multidrug resistance phenotypes of the isolates are elucidated. The isolates could be divided into 4 multidrug resistance profiles. Almost 52.2% and 43.5% of the isolates belonged to one of the two multidrug resistance phenotypes.
Table 2: Grouping of Campylobacter jejuni isolatesa based on MICb values of antibiotics

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Table 3: Antibiotic resistance profile of Campylobacter jejuni isolates

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 ~ Discussion Top


Previous studies in India have shown that 39.3% of the poultry, tested positive for Campylobacter in Calcutta, 64% in Vellore, 57% in Maharashtra and 17.14% in the Meghalaya-Assam region. [17] Our results from Pune, Maharashtra are in agreement with these reported values. However, only C. jejuni was isolated from the poultry farm environments. We do not conclude on this, because of biased detection in favour of this species. It must be noted that the prevalence of Campylobacters in poultry, depends on not only the isolation method but also flock size and type, geography, season, animal age, and number of animals investigated. We have no explanation as to why no other species were isolated. Nevertheless, this occurrence was reported in a similar study from New Zealand as well. [13]

The presence of Campylobacter in the agricultural context has widely been documented in scientific literature; however, there are few similar reports from India .[4] The highest prevalence was found in water samples (87.5%) followed by human faecal samples (80%), soil (50%) and poultry chickens (44.44%), wild birds (15.62%) and flies (7.2%). Though the presence of Campylobacter in 11% of rodents was detected in a similar study, we could not detect any Campylobacters from rodents. [18] Wild animals, wild birds, humans and even flies could be potential reservoirs of C. jejuni in poultry farms. It is thus postulated that either or all these reservoirs may be responsible for horizontal transmission of Campylobacter in the Indian poultry farm environment. Moreover, a large number of Campylobacter isolates obtained in this study showed the same multidrug resistance phenotype despite being isolated from different environmental sources. However, further molecular studies using flaA- RFLP or PFGE will be able to elucidate the route of transmission.

An important observation was that 80% of humans associated with poultry were found to be positive for Campylobacter. Campylobacter enteritis are usually treated using fluoroquinolones and macrolides. [1] Recent studies have reported the appearance of fluoroquinolones resistant Campylobacter spp. among poultry flocks, [7] necessitating the survey of prevalence of Campylobacter spp. in poultry and their antimicrobial resistances. Earlier reports from India show 30.6% and 35.83% of strains from retail poultry were multidrug resistant. [17],[19] However, we noted that alarmingly all the C. jejuni isolates from environmental sources as well as humans were multidrug resistant.

We wish to highlight these results due to a scarcity of Campylobacter research in India. Further, the results paint a dismal picture because there are no national surveillance studies and hardly any projects taken up to study the problem of Campylobacter. With a population of 1.2 billion people, the increasing rates of Campylobacter carriage in poultry, the alarmingly low infective dose of Campylobacter (only 500 bacterial cells) and the battery of complications following enteric infections, stringent measures must be taken to improve our understanding of Campylobacter in India.

 
 ~ References Top

1.Allos BM. Campylobacter jejuni Infections: Update on emerging issues and trends. Clin Infect Dis 2001;32:1201-6.  Back to cited text no. 1
[PUBMED]    
2.Stern NJ, Clavero MR, Bailey JS, Cox NA, Robach MC. Campylobacter spp. in broilers on the farm and after transport. Poult Sci 1995;74:937-41.  Back to cited text no. 2
    
3.Zhao C, Ge B, De Villena J, Sudler R, Yeh E, Zhao S, et al. Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in retail chicken, turkey, pork, and beef from the Greater Washington, D. C., Area. Appl Environ Microbiol 2001;67:5431-6.  Back to cited text no. 3
    
4.Baserisalehi M, Al-Mahdi AY, Kapadnis BP. Antimicrobial susceptibility of thermophilic Campylo-bacter spp. isolated from environmental samples. Indian J Med Microbiol 2005;23:48-51.  Back to cited text no. 4
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5.Murphy GS, Echeverria P, Jackson LR, Arness MK, LeBron C, Pitarangsi C. Ciprofloxacin- and azithromycin-resistant Campylobacter causing traveler's diarrhea in U. S. troops deployed to Thailand in 1994. Clin Infect Dis 1996;22:868-9.  Back to cited text no. 5
    
6.Altekruse SF, Stern NJ, Fields PI, Swerdlow DL. Campylobacter jejuni: An emerging foodborne pathogen. Emerg Infec Dis 1999;5:28-35.  Back to cited text no. 6
    
7.Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, Teixeira P. Campylobacter spp. as a foodborne pathogen: A review. Front Microbiol 2011;2:200.  Back to cited text no. 7
    
8.Humphrey T, O'Brien S, Madsen M. Campylobacters as zoonotic pathogens: A food production perspective. Int J Food Microbiol 2007;117:237-57.  Back to cited text no. 8
    
9.Lindblom GB, Sjögren E, Kaijser B. Natural Campylobacter colonization in chickens raised under different environmental conditions. J Hyg (Lond) 1986;96:385-91.  Back to cited text no. 9
    
10.Petersen L, Nielsen EM, On SL. Serotype and genotype diversity and hatchery transmission of Campylobacter jejuni in commercial poultry flocks. Vet Microbiol 2001;82:141-54.  Back to cited text no. 10
    
11.Callicott KA, Friethriksdóttir V, Reiersen J, Lowman R, Bisaillon JR, Gunnarsson E, et al. Lack of evidence for vertical transmission of Campylobacter spp. in chickens. Appl Environ Microbiol 2006;72:5794-8.  Back to cited text no. 11
    
12.Jacobs-Reitsma WF, Van de Giessen AW, Bolder NM, Mulder RW. Epidemiology of Campylobacter spp. at two Dutch broiler farms. Epidemiol Infect 1995;114:413-21.  Back to cited text no. 12
    
13.Adhikari B, Madie P, Connolly J, Davies P, Layland M, Rogers L. Wild birds, flies and rodents as reservoirs of campylobacter spp. on dairy farm; 2002. MAF Technical Paper 2002/18.  Back to cited text no. 13
    
14.Hansson I, Pudas N, Harbom B, Engvall EO. Within-flock variations of Campylobacter loads in caeca and on carcasses from broilers. Int J Food Microbiol 2010;141:51-5.  Back to cited text no. 14
    
15.Khalil K, Lindblom, GB, Mazhar K, Kaijser B. Flies and Water as reservoirs for bacterial enteropathogens in urban and rural areas in and around Lahore, Pakistan. Epidemiol Infect 1994;113:435-44.  Back to cited text no. 15
    
16.Oyofo BA, Thornton SA, Burr DH, Trust TJ, Pavlovskis OR, Guerryl P. Specific Detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction. J Clin Microbiol 1992;30:2613-9.  Back to cited text no. 16
    
17.Parkar SF, Sachdev D, DeSouza N, Kamble A, Suresh G, Munot H, et al. Prevalence, seasonality and antibiotic susceptibility of thermophilic Campylobacters in ceca and carcasses of poultry birds in the "live-bird market. Afr J Microbiol Res 2013;7:2442-53.  Back to cited text no. 17
    
18.Adhikari B, Connolly JH, Madie P, Davies PR. Prevalence and clonal diversity of Campylobacter jejuni from dairy farms and urban sources. N Z Vet J 2004;52:378-83.  Back to cited text no. 18
    
19.Jain D, Sinha S, Prasad KN, Pandey CM. Campylobacter species and drug resistance in a north Indian rural community. Trans R Soc Trop Med Hyg 2005;99:207-14.  Back to cited text no. 19
    


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