Characterization of isolated thermophilic campylobacters and associated risk factors in poultry farms of Uttarakhand, India

Campylobacters are the common commensals of poultry responsible for several cases of gastroenteritis in humans. The illness, if severe can result into complications causing a nervous disorder named Guillian Barre syndrome. Owing to its serious health implications, the study aimed to screen eight organized poultry farms and their environment (water, litter, manure, and feed) of Uttarakhand state, India for the presence of thermophilic Campylobacter species and their virulence and antibiotic resistance prole. It also undertook identication of risk factors associated with the occurrence of campylobacters in each farm using a questionnaire survey comprising eleven potential risk factors (other animals on farm, reuse of litters, use of foot bath, in house or branded feed, chlorination of water, distance of manure heap, housing system, ock size, oor type, shoe use by farm personnels, moist or dry litter and number of broiler oor).


Background
The incidences of food-borne illnesses are observed in developing as well as developed nations. These illnesses are mainly caused by pathogenic bacteria present in food (1,2). As per Center for Disease Control and Prevention (CDC), campylobacters stand as 4th major cause of food-borne illness (9%), 3rd major cause of hospitalization (15%) and 5th main cause of human deaths (6%) due to food-borne infections annually in the United States (3). India, a developing country, lacks a decent data on foodborne diseases as many cases go unreported. The Integrated Disease Surveillance Programme (IDSP) network, launched in India in 2004, highlights that food-borne outbreaks together with acute diarrhoeal diseases constitute nearly half of all reported outbreaks based on data collected from 2011-15 (4). Among the well known food-borne pathogens, thermophilic campylobacters namely Campylobacter jejuni (C.jejuni) and Campylobacter coli (C.coli) contribute approximately 95% of human infection (3). Two others, C. lari and C. upsaliensis also account for many diarrhoeal cases in humans (5,6). These microorganisms constitute the normal gastrointestinal micro ora in many animals,.especially birds (7). Poultry birds can be infected with the bacteria at a very high level without showing any visible clinical symptoms. Campylobacters, C. jejuni and C. coli are well adapted to birds because of their ability to grow at 41-42 (the approximate body temperature of a bird). These organisms have been frequently isolated from the caecal micro ora (8).
Intestinal content is thus one of the primary suspected source of meat contamination during slaughter.
Hence, managing Campylobacter spp. in the poultry reservoir is a crucial step in prevention and control of food-borne campylobacteriosis in humans. Other possible sources like contaminated drinking water, consumption of unpasteurized milk and ready to eat food products, faecal run-off of birds and domestic animals contaminating surface water and direct contact with animals are signi cant in transmitting illness to humans (9).
Campylobacter illness in humans occur worldwide with estimated 500 million infections annually (10). Although it is a self limiting disease, the emergence of antimicrobial resistance in campylobacters has become a concern for food safety. Development of antimicrobial resistant (AMR) campylobacters has been linked to the indiscriminate use of antimicrobials in food animal production system (poultry and swine) for disease prevention and growth promotion (11,12). Sub-therapeutic use of antimicrobials in food production systems is believed to create selection pressure and force microorganisms to develop resistance in order to survive (13). A rapid increase in the proportion of Campylobacter strains resistant to antimicrobial agents, particularly uoroquinolones and macrolides, has been reported in many countries (14,15,16,17). Nevertheless, there still exists paucity of data on the presence of antimicrobial resistant campylobacters and various risk factors responsible for the prevalence of these organisms in poultry production systems in India. Very few researchers have reported campylobacters in poultry (18,19,20,21) thus, more future research awaits in this direction.
Uttarakhand, an Indian state with high tourist footfall of around 34.36 million with foreign tourist visits over 0.13 million in 2017 (22) nds limited data on campylobacter presence in farms. To ll this knowledge gap, the present study was designed to estimate the occurrence of thermophilic campylobacters,virulence,antibiotic resistance and risk factor associated with campylobacters in eight commercial poultry farms located in Kumaon region of Uttarakhand.

Phenotypic Antimicrobial Susceptibility
On subjecting 42 revived isolates to disc diffusion test, forty one isolates (n=41, 97.6%) exhibited resistance to at least one antimicrobial on the disc diffusion assay and one isolate (ID.C4) was pansusceptible. Ten isolates (n=10, 23.80%) were multidrug resistant (MDR) exhibiting resistance to at least 3 or more antimicrobial classes. Three Campylobacter isolates were found resistant to four classes of antimicrobials while seven isolates showed phenotypic resistance to three classes of antimicrobials. However, twenty two isolates were found to be resistant to two classes of antimicrobials. β-lactam antimicrobials (cefoxitin, ceftriaxone and ampicillin) observed higher resistance than other classes studied . Highest frequency of resistance was found against cefoxitin (97.61%) followed by cipro oxacin (64.28 %), nalidixic acid (33.33 %), ampicillin (28.5%) and ceftriaxone (14.28%). Two isolates (4.76%) were resistant to tetracycline. However, only one isolate showed resistance to clindamycin, sulfafurazole and erythromycin. All isolates (n=42) were susceptible to levo oxacin and gentamicin.
A total of 16 different AMR combinations were detected of which, 8 resistance patterns were MDR represented by 10 isolates. The most common MDR patterns were AMP CX CIP TE (n=2) and AMP CX CIP (n=2). Distribution of antimicrobial resistance patterns across sample types and farm location is detailed in Table7.

Genotypic Characterization of AMR Determinants
Presence of four antibiotic resistance genes (ARGs) namely blaOXA-61, tet(O), cmeB and ermB conferring resistance to different classes of antibiotics were detected by speci c Antibiotic Resistance Genes-PCRs. Out of 41 isolates showing phenotypic resistance, 29 isolates showed presence of at least one resistance genes targeted (blaOXA-61, tet(O), cmeB and ermB. However, 12 resistant isolates did not harbour any of the four resistance genes. β-lactam resistance gene blaOXA-61 was detected in 18 (58.06%) out of 31 isolates showing phenotypic resistance. Resistance gene cmeB was detected in 19 uoroquinolone resistant isolates (79.16%) out of 24 tested. One lincosamide (Clindamycin) resistant isolate harboured cmeB gene. Tetracycline resistant tet(O) gene was detected in all isolates showing phenotypic tetracycline resistance (n=2). Macrolide resistance gene ermB was absent in a single erythromycin resistant isolate. Most prevalent resistance gene combination was blaOXA-61+ cmeB, which was detected in 11 isolates (Table 8).

Risk factor analysis
Of the 11 parameters studied as risk factors using a questionnaire distributed to farm owners, only one risk parameter i.e., feeding of branded feed was found to have signi cant association with Campylobacter presence in the examined broiler ocks (p-value 0.0047).

Discussion
The present study was designed to determine the prevalence of thermophilic campylobacters in poultry raised at farms and their living environment.

Prevalence of thermophilic campylobacters
The overall Campylobacter comprising poultry faeces (n=346) and environmental samples(n=199) was recorded as 12.29% (67/545). Other ndings reported from the studies conducted in broiler ocks have also reported almost similar overall prevalence. Chokboonmongkol et al. (23) reported 11.2% Campylobacter spp. prevalence in broiler ocks from Thailand while another study from Ecuador reported 12.4% prevalence of Campylobacter in broiler ocks (24). In India, limited studies have been done on Campylobacter prevalence in poultry. These studies have revealed prevalence ranging from 13.54-21.8% (13.54%,21; 15.89%,19; 21.8%, 25 14.28%,26 and 20%, 27). However, a much higher prevalence of Campylobacter as high as 72.2% from cloacal swab samples has also been documented from poultry by Vaz et al. (28). Another study by Ingresa et al. (29)  Only 4 isolates could be recovered from 199 environmental samples with a prevalence of 2.01%, which included 3 isolates from water and 1 isolate from litter sample However, Vaz et al. (28) recorded much higher 63.8% Campylobacter prevalence in litter samples from Brazilian broiler ocks. Similarly, Lisa et al. (30) reported 64.3%, 64.3% and 45.7% Campylobacter prevalence in soil, compost, and processed waste water respectively. Presence of campylobacters in environment is signi cant as campylobacters are able to form bio lms as a survival mechanism outside the host (31). Detection of Campylobacter spp. from water samples is important because all the birds in a ock drink water from the same waterer which aid in furthur spread within a ock. Also, capability to form a bio lm poses the risk of its presence in cold water inspite of chemical treatment (32).
Feed and manure samples of our study did not reveal any presence of Campylobacter spp. However, these sources cannot be neglected as a source of infection. Zero prevalence of Campylobacter in manure and feed samples could be due to less number of samples processed.
Interestingly, in this study majority of the isolates were identi ed as C. coli (76.11%) and only 23.88% isolates were C. jejuni. (Table4). C. jejuni is considered to be the predominant species colonizing poultry (33,34). Many studies ( 35,36,37) report the dominance of C.jejuni over C.coli in poultry. In India, Chattopadhyay et al. (38) and Rajendran et al. (39) also showed that C. jejuni were more frequent than C. coli in poultry faecal samples. However, in accordance to our study, many other authors have reported C.coli dominance. Pergola et al. (40) reported 70.71% prevalence of C. coli and 17.14% C. jejuni from cloacal swab samples. Monika (21) and 19) also reported higher C.coli presence of 75% and 67.44%,respectively of the total isolates recovered from poultry faeces of Uttarakhand. Also, Wieczorek et al (41) in their retrospective study of ve-years on prevalence and antimicrobial resistance of Campylobacter from poultry carcasses in Poland also found C.coli as a dominant species over C.jejuni In our opinion, the initial dominance of a species and further spread due to improper control measures can decide the higher presence of a species. Better colonization ability of either of the two species in poultry intestine and persistence in outside environment may decide the dominance.
No C. lari and C. upsaliensis were detected in this study. However, C. lari isolation from poultry is reported by some authors. Very few studies support the presence of C.lari in poultry isolates. Pillai (42) and 25 isolated 2 and a single isolate of C.lari from chicken samples in Bangalore and Bareillly respectively. Oyarzabal and Hussain (43) are of the opinion that, with the development of DNA based methods for the identi cation of isolates; C. lari has not been reported for more than 10 years in the United States, which suggests that previous reports may have been misidenti cations from the traditional biochemical tests which were used for species con rmation. Further studies on poultry using molecular diagnostic techniques would answer the same. Acke et al (44) reported that dogs are the main reservoirs for C. upsaliensis which could probably be the reason for non-isolation of this organism in our study.
No previous data on Campylobacter prevalence in poultry farms is available for selected locations except for Pantnagar and Haldwani. Probably Isolation of Campylobacter from the locations except the two (Pantnagar and Haldwani) has not been reported so far. Poultry farms at Pantnagar screened before have reported the prevalence rates of 16 % (45), 11.66 % (46) and 13.54 % (21). However, a lower prevalence of 6.9 % (19) and 5.34 % (47) also has been reported from Pantnagar. Rawat et al (20) reported 4.17 % Campylobacter prevalence in faecal samples of broilers collected from an organized farm of Pantnagar.

Prevalence of virulence genes
Total 48 Campylobacter isolates including 39 C. coli and 9 C. jejuni were included for virulence gene detection using PCR. Nineteen isolates (n=19) could not be revived and thus were not included in the virulence gene analysis. The genes associated with bacterial motility ( aA) and adhesion to epithelial cells (ca dF),were present in all (100%)the isolates.These genes are known to be conserved in Campylobacter spp. (48,49) and play a key role in the development of Campylobacter infection. The cdtB (97.9%) was second most prevalent gene. This gene along with cdtA and cdtC cytoxin gene has the ability to interfere with the division and differentiation of the intestinal crypt cells, ,thus has an important role in diarrhoea. This combination has been recorded with a prevalence of 96.6-97.6% in positive strains (50) which is in accordance with our study. It also suggests that the three genes( cdtA, cdtB and cdtC) should be included together in future studies for assessing toxic property.
The cgtB gene was found in 22.9% of the positive Campylobacter spp. isolates. Not much data is available on the presence of this gene in the campylobacters though this gene, as wlaN, also codes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide responsible for Guillain-Barré syndrome (GBS) (51) Other gene ciaB exhibited in 12.5% isolates. This gene is important for Campylobacter survival in the intestinal tract. The product of the ciaB marker, which play a role both in the intestinal invasiveness and in colonization of the epithelial cells (52), was identi ed in campylobacters by other authors also in a lower percentag (53,54).The presence of this gene is signi cant as it helps the organisms to overcome the stress conditions presented by the intestine and cause disease. Additionally, expression of ciaB has been observed to reduce under nutritional stress (55).
None of the Campylobacter isolate harboured wlaN gene. Many studies conducted on C. jejuni and C.coli have reported total absence of this gene (48,56,57).However, Kim et al. (58) identi ed the wlaN gene among 100% of 63 human and in 78.6% of 42 animal C. jejuni isolated tested in Korea. The product of the wlaN gene is also thought to be involved in development of of Guillain-Barre' syndrome after C. jejuni infection (49,58,59).
In our study, C.jejuni (cadF(100%), aA(100%), ciaB(66.66%), cdtB(88.88%) and cgtB(33.33%) ) possessed more number of virulent genes than C.coli (cadF and aA(100%), cdtB(100%) and cgtB(20.51%)). Moreover, ciaB gene presence ( responsible for both epithelial and intestinal mucosal invasion) only in C.jejuni isolates may suggest this species dominance over C.coli in being more pathogenic (60) and a cause for regulars diarrhoeal cases in humans (7). The virulent pro le of C. jejuni (59,61) showed that the greatest potential of this species over the other in causing clinical cases in humans (81.1%) (62) is due to the properties of invasion, colonization and toxin production which are essential to elicit its pathogenesis.
In contrast, C. Coli shows its priority is to ensure the survival through mechanisms (63).
Either of the virulence genes except wlaN were found in both faecal and environmental (water(n=3) and litter(n=1)) samples. This indicates potential risk to consumers.. Virulence genes cadF and aA were detected in all isolates (100%) recovered from all four farms. Pant (45) recorded 100% prevalence of aA and cadF genes in the isolates recovered from diverse sources collected from Udham Singh Nagar district. The presence of virulence genes such as cdtA and cdtB have been reported (46,64) who screened the sources from Pantnagar and nearby areas. Campylobacter isolates of the same region were also shown to express wlaN, iam, ciaB and dnaJ virulent genes (47).
Ten isolates of 41 (n=10, 23.80%) were multidrug resistant (MDR) exhibiting resistance to at least 3 or more antimicrobial classes. Only one isolate (ID.C4) was pan-susceptible. Higher resistance to β-lactam antimicrobials was detected in our study such as cefoxitin, ceftriaxone and ampicillin. Resistance to ampicillin (28.5%), a "critically important antimicrobial", crucial in human medicine is alarming, since it limits our options to treat critical human infections. Resistance was also detected against tetracycline (n=2) and clindamycin (n=1); antibiotics classi ed as "highly important" in human medicine according to WHO. Clinical management of Campylobacter infection becomes more di cult because of increasing development of resistance against antibiotics.
Of 16 different AMR combinations, 8 resistance patterns were MDR represented by 10 isolates. The most common MDR patterns were AMP CX CIP TE (n=2) and AMP CX CIP (n=2). Resistance pattern AMP CX CIP TE (n=2) had faecal origin and was identi ed from two separate locations,viz Haldwani and Pantnagar farm 2. Another MDR pattern AMP CX CIP (n=2) also had faecal origin. However, this pattern was identi ed from Bazpur and Bindukhatta farms. Distribution of antimicrobial resistance patterns across sample types and farm location is detailed in (Table 17). Most number of AMR patterns were detected from Bazpur farm (n=7), followed by Pantnagar farm 2 (n=6) and Bindukhatta farm (n=5). Four AMR patterns per farm were detected from Haldwani, Pantnagar farm 1 and Jawaharnagar farm. Signi cant diversity in the AMR patterns was detected across different farms and sample types. This may conclude the presence of genotypic diversity among the isolates circulating across locations and within a single location.

Genotypic Characterization of AMR Determinants
Out of 41 isolates showing phenotypic resistance, 29 isolates showed presence of at least one resistance genes targeted (blaOXA-61, tet(O), cmeB and ermB). Most prevalent resistance gene combination was blaOXA-61+ cmeB, which was detected in 11 isolates. A variety of antimicrobial resistance genes (ARGs) conferring resistance to various classes of antibiotics detected in this study is a matter of concern because these antibiotics are frequently used in human medicine and also these resistant determinants can be transferred to susceptible bacterial population by horizontal gene transfer (HGT). Nesme and Simonet (69) reported that soil is prone to genetic exchange by means of horizontal gene transfer between ecologically distinct lineages present in other ecosystems. Kashoma et al. (68) reported antimicrobial resistance genes blaOXA-61 (52.6%), cmeB (26.3%), tet(O) (26.3%) and aph-3-1 (5.3%) in Campylobacter isolates.

Risk factor analysis
Out of 11 risk parameters tested, only feeding of branded feed was found to be signi cantly associated with Campylobacter colonization of the examined broiler ocks (p-value 0.0047). In a similar study, Hald et al. (70) reported that 35% Campylobacter positive ocks used purchased wheat. Authors further reported that farmers who purchased wheat from a feedstuff dealer (p value 0.026) had a higher risk of Campylobacter infections in their broiler ocks compared to farmers who fed home-grown wheat. Various studies have been conducted to determine potential risk factors for Campylobacter infection in poultry farms (70,71,72,73,74). Cardinale (75) reported that an elevated risk of Campylobacter infection at poultry farms was associated with several factors namely presence of other animals (mainly laying hens, cattle and sheep) in the farm, farm staff not wearing proper work clothing while working in poultry houses, uncemented poultry-house oors and the use of cartons that transport chicks from the hatchery to the farm as feed plates (rather than speci cally designed feed plates). However, thorough cleaning and disinfection of poultry-house surroundings and manure disposal outside the farm were associated with decreased ock risk. In our study, the strength of association of risk factors with the prevalence of Campylobacter organism could be better identi ed with more number of samples screened at much larger number of farm locations.

Conclusion
The study highlights the occurrence of food pathogens, Campylobacter jejuni and C.coli in the poultry farms and their environment of the state. The organisms possessed signi cant virulence genes capable of developing critical human illness. Moreover, their resistance for frequently used antibiotics and attaining multi drug resistance is a point of concern. Eight different multi drug resistant patterns point towards reinforcing strict regulations against frequent misuse of antibiotics in farms for commercial gains.
Majority of isolates possessing blaOXA-61+ cmeB gene combination may increase the peril by further possible horizontal spread in the surrounding micro ora. Evaluation of potential risk factors in colonization of campylobacters suggests a thorough examination of feed before use, though this nding needs a more detailed study with more number of samples. To conclude, improved biosecurity in farms is of paramount importance. Also, pre-harvest and post harvest interventions are valuable in reducing the risks linked with consumption /contamination of poultry meat.

Study Design and Sample collection
The present study was conducted in the Uttarakhand state of India. Samples were collected from eight poultry farms (n=8) farms located at Haldwani, Pantnagar, Kiccha, Ramnagar, Bazpur, Jawaharnagar and Bindukhatta regions of the state, India from September 2016 to May 2017. A total of 545 samples collected comprised poultry faeces (n=346) and environment samples (n=199). The environmental samples represented water (n=50), poultry feed (52), litter (51) and manure (46). Sterile 100 ml whirlPak bags (Nasco, Fort Atkinson, WI) were used to collect poultry faeces, poultry feed, litter and manure. The water samples were collected in 100ml sterile sample container (Abdos India). The samples were collected aseptically and immediately brought to the laboratory for processing as per previously published protocols (76,77,78).

Isolation and Molecular Con rmation
Poultry faecal samples were streaked directly onto the modi ed Charcoal-Cefoperazone-deoxycholate agar (mCCDA, Hi media) plates and incubated at 42°C with 5% CO 2 in a CO 2 incubator for 48 hrs (OIE terrestrial manual 2008). The poultry feed and the environmental samples however were initially enriched in 9 ml Bolton broth (Oxoid, UK) supplemented with 5% sheep blood. Thereafter, a loopful of the enriched broth suspension was streaked onto mCCDA plates and were incubated at same time-temperature combination. The characteristic campylobacter colonies (1-2 mm size, circular, at to slightly raised, sticky, spreading and shiny grey) were selected from each plate and tested biochemically. All the presumptive Campylobacter isolates that were catalase and oxidase positive while urease and TSI negative were subjected to DNA isolation using heat-shock method. A simplex PCR and a multiplex PCR assay targeting the 16SrRNA(79 ) and lipid gene lpxA (80) respectively were used for the Campylobacter genus and species identi cation. The primer sequence and the cyclic conditions used were as per references (79 , 80 for Campylobacter genus and species, respectively). All PCR con rmed Campylobacter isolates were stored as 20% glycerol stock at -80°C.

Antibiotic Susceptibility Testing (AST)
The antimicrobial resistance (AMR) pro le of Campylobacter isolates was determined using standard Kirby-Bauer disc diffusion method. A total of 42 isolates out of 67 isolates could be recovered for antimicrobial sensitivity testing. Zone diameter was measured and break points were interpreted based on the recommendations of Clinical and Laboratory Standards Institute standards for disk-diffusion assay (81). The isolates showing resistance to three or more classes of antimicrobials were classi ed as Multidrug Resistant (MDR) (82).
The isolates with intermediate level of resistance were categorized as susceptible to avoid overestimation of resistance.
Detection of antimicrobial resistance genes (ARGs) Campylobacter isolates were screened for the presence of ve antimicrobial resistance genes coding resistance to the antimicrobials used. PCR was performed to detect the presence of β-lactam resistance coding blaOXA-61 gene (83), gentamicin resistance coding aphA-3-1 gene (84), tetracycline resistance coding tet(O) gene (84), macrolide resistance coding ermBgene (85)) and a multidrug resistance gene cmeB coding for uroquinolone and lincosamide antibiotics (83). PCR reaction and cycling conditions were used as described in respective references.

Detection of virulence genes
All Campylobacter isolates were screened for the presence of various virulence genes by PCR. Virulence genes screened were CadF(86), aA and CiaB (56) , cdtB(87), wlaN (88) and cgtB (89) . PCR reaction and cycling conditions were used as described earlier in respective references.

Risk factor analysis
A questionnaire was prepared to study various risk factors associated with the Campylobacter prevalence in the poultry farms. All farm owners were requested to respond to the questionnaire (Table 1). However, no records were taken if an owner showed unwillingness to answer the questionnaire.

Statistical analysis
Univariate analysis was used to analyze differences in the proportion of Campylobacter in various poultry farms. The statistical signi cance level was de ned as a two-tailed p ≤ 0.05. All data analysis was carried out using Statistix7 software (Tallahassee, Florida, US).

Declarations Ethics approval
The study was performed under the project on Zoonotic diseases.The project involves routine commection of probable samples for isolation and identi cation of food pathogens. The samples were analyzed using referred analytical methods. Therefore no informed consent was obtained.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its additional information les. Strains are available from the corresponding author on request.

Competing interests
The authors declare that they have no competing interests.

Funding
The funds for the research work was provided by the Indian Council of Agricultural Research, New Delhi, India to carry out the research work under a project on "Outreach Programme on Zoonotic Diseases (F.No. 14(1)2009-ASR-IV).
Author's contribution GG collected the samples and analyzed the samples. M designed the study. DK performed analysis of the data and AKU provided help as and when required and edited the manuscript. All authors have read and approved the nal manuscript.

1.
No. of broiler flocks As informed