Virulence typing and antibiotic susceptibility profiling of thermophilic Campylobacters isolated from poultry, animal, and human species

Background and Aim: Campylobacteriosis finds its place among the four important global foodborne illnesses. The disease, though self-limiting, needs antibacterial therapy in extraintestinal complications. Therefore, the present study was designed to estimate the prevalence of thermophilic Campylobacters in poultry, animals, and humans of the Kumaon region of Uttarakhand. Materials and Methods: A total of 609 samples comprising of poultry ceca (n=116), poultry droppings (n=203), and feces of pigs (n=71), cattle (n=61), sheep (n=19), goat (n=17), human beings (n=88), and laboratory animals (n=34) (rats, rabbits, and guinea pigs) were collected. The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli were confirmed using multiplex polymerase chain reaction. The isolates were also screened for the presence of virulence genes, and their antibiotic susceptibility testing was done against eight antibiotics. Results: An overall prevalence of 6.24% was revealed with highest from poultry ceca (15.52%), followed by poultry droppings (5.91%), cattle feces (4.92%), human stools (3.40%), and pig feces (2.82%). The virulence genes, namely cadF, flaA, virB11, and pldA, were present in 38 (100%), 37 (97.37%), 7 (18.42%), and 14 (36.84%) isolates, respectively. All the isolates were resistant to nalidixic acid, while all were sensitive to erythromycin and co-trimoxazole. Conclusion: It was concluded that the animals and humans in the region harbored the thermophilic Campylobacters which may contribute to the human illness. Resistance shown among the isolates may complicate the antimicrobial therapy.


Introduction
Infections occurring due to the consumption of contaminated food are of a growing public health concern. These contaminated or unsafe foods pose a global threat affecting persons of all age groups. Food may get contaminated during any point of production and/or distribution by a number of microbes. Of all the food pathogens, Campylobacters have become a leading cause of enteric infections in both developing and developed countries. The Campylobacters have a broad animal reservoir. They are inhabitants of the intestinal tract of various domestic and wild animals, especially birds which are generally asymptomatic carriers. Therefore, inadequately cooked meat, particularly poultry, contaminated drinking water, unpasteurized milk, ready to eat food products, fecal runoff of birds and domestic animals contaminating surface water, and direct contact with animals act as the main source of the organism. Contaminated food is the primary mode of infection with poultry being the most common food source for humans [1]. Prevalence of Campylobacters in poultry has been reported by many authors [2][3][4]. In humans, the disease, campylobacteriosis is characterized by bloody diarrhea, fever, severe abdominal pain, and headache. Fever generally persists for up to 1 week in more than 90% of the patients; however, 50% of persons infected with Campylobacters remain asymptomatic [5]. It is a gastrointestinal disorder that generally affects infants, elderly people, and immunocompromised individuals. Most people suffering from campylobacteriosis recover within 2-5 days, but it may take up to 10 days in some cases. A very small dose of 500 cells may be infectious and sufficient to produce gastroenteritis. Common long-term complications of campylobacteriosis are Guillain-Barre syndrome, inflammatory bowel disease, bacteremia, rheumatoid arthritis, along with local complications such as cholecystitis, pancreatitis, peritonitis, massive gastrointestinal hemorrhage, thyroiditis, and prosthetic joint infection. Over 2.4 million persons annually or 0.8% of the total population [6] are affected making it a very important organism from a socioeconomic perspective. However, many of the cases go undiagnosed. The disease has a long back existence, and its recognition as a common infection is owed to improved laboratory methods. It still remains a high research priority to improve the strategies for management as well as prevention of the disease.
Molecular methods are useful in the identification of thermophilic Campylobacters as they enhance the sensitivity and specificity of the detection process. Polymerase chain reaction (PCR)-based species identification methods for Campylobacter spp. provide more reliable identification. Gorkiewicz et al. [7] recommended 16SrRNA sequence analysis as an effective, reliable, and rapid procedure for the specific identification of Campylobacters. Molecular techniques have come a long way in the characterization of microbes. Despite higher recovery rates of Campylobacters as foodborne pathogens, the specific virulence and pathogenic mechanisms of Campylobacter spp. infection is still poorly understood. The putative virulence factor for adhesion (cadF, dnaJ, jlpA, pldA, racR, and virB11) and invasion of epithelial cells (iamA, ciaB, and ceuE), toxin production (cdtA, cdtB, cdtC, and wlaN), and flagellar motility (flaA, flaB, flhA, flhB, flgB, flgE2, fliM, and fliY) are thought to be important virulence mechanisms. Different studies in this regard have indicated the role of different virulence markers for adherence, invasion, and colonization of the organism in humans and animals. These virulence-related factors contribute to survival and establishment of the disease in host, thus modulating the clinical presentation of the disease. Antimicrobial agents are used for the early recovery of extraintestinal infections in immunocompromised patients or whenever clinical therapy is needed. The emergence of antimicrobial-resistant (AMR) Campylobacters has alerted toward the chances of increased invasive illness. The resistance has been linked to the illicit use of antimicrobials in animal feeds, food animals, and flock treatment of animals. Few antibiotics such as macrolides (erythromycin [ERY]) and fluoroquinolones (ciprofloxacin [CIP]) used as drug of choice have shown resistance against Campylobacter spp. [8]. Newer antibiotics are being continuously added to the list every year.
The present study was, therefore, undertaken to characterize the isolated Campylobacters on the presence of virulence genes and the phenotypic antibiotic resistance.

Ethical approval
Samples were collected as per standard sample collection procedure.
In brief, the poultry ceca and poultry fecal samples were streaked directly onto the modified charcoal-cefoperazone-deoxycholate agar (mCCDA, HiMedia, India) plates and incubated at 42°C with 5% CO 2 in a CO 2 incubator for 48 h [10]. However, human and other animal fecal samples (1 g) 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 was incubated at the same time-temperature combination. The characteristic Campylobacter colonies (1-2 mm size, circular, flat to slightly raised, sticky, spreading, and shiny gray) were selected from each plate and tested biochemically.

Biochemical and molecular confirmation
All the presumptive Campylobacter isolates showing catalase and oxidase positive reaction while urease and TSI negative reaction were subjected to DNA isolation using Hi-PurA genomic DNA extraction kit (Hi-media).
A simplex PCR assay targeting the 16SrRNA [11] was used for the Campylobacter genus identification. The primer sequence and the cyclic conditions were used as per Linton et al. [11] for Campylobacter genus. All PCR confirmed Campylobacter isolates were stored as 20% glycerol stock at −80°C.

Multiplex PCR
Multiplex PCR was carried out for the identification of genus as well as species of the isolates. Multiplex PCR included amplification of cadF gene for genus identification [12], whereas hipO gene [11] and asp gene [11] amplification for the identification of Campylobacter jejuni and Campylobacter coli, respectively. The primer sequence and the cyclic conditions were used as per Linton et al. [11] and Nayak et al. [12] for Campylobacter genus and species, respectively.

Detection of virulence genes
All Campylobacter isolates were screened for the presence of various virulence genes by PCR. Virulence genes screened were flaA [13], virB11 [14], and pldA [15]. PCR reaction and cycling conditions were used as described earlier in respective references.

Antimicrobial susceptibility testing
The AMR profile of Campylobacter isolates was determined using standard Kirby-Bauer disc diffusion method as described by Taremi et al. [16]. A total of 38 revived isolates were tested against a panel of eight antibiotics that included ampicillin (AMP, 10 µg), gentamicin (GEN, 10 µg), ERY (15 µg), levofloxacin (LE, 5 µg), CIP (5 µg), nalidixic acid (NA, 30 µg), ceftriaxone (CTR, 30 µg), and co-trimoxazole (COT, 25 µg) (HiMedia). The isolates were revived on mCCDA plates supplemented with FD009 supplement. The growth suspension prepared in Tryptic soy broth and compared with 0.5 McFarland standard was spread on Mueller-Hinton agar plates supplemented with 7% sheep blood and incubated at 42°C in a CO 2 incubator at 5% CO 2 tension for 24 h. Zone diameter was measured and breakpoints were interpreted based on the recommendations of the Clinical and Laboratory Standards Institute standards for disc diffusion assay (CLSI 2016).  38 were confirmed as positive for Campylobacter yielding a prevalence of 6.24%.

Isolation and molecular confirmation
All the isolates produced a genus-specific amplicon of 816 bp in 16SrRNA Campylobacter genus-specific PCR (Figure-1).

Multiplex PCR for species identification
On performing the multiplex PCR, all the 38 isolates amplified cadF gene and produced 400 bp amplicon suggesting the isolates belonging to genus Campylobacter. C. coli and C. jejuni species targeting asp gene (500 bp) and hipO gene (735 bp), respectively, were amplified in 29 (n=38) and 9 (n=38) isolates revealing a prevalence of 76.32% and 23.68%, respectively (Figure-2).

AMR
Antibiotic resistance profile Campylobacter isolates were determined using eight antibiotics according to the CLSI 2015. Comparison of antibiotic susceptibilities of the isolates from different sources is shown in Tables-4 and 5. All isolates (100%, n=38) were resistant to NA, while all    were sensitive to ERY and COT. Only 2 (5.3%) C. coli isolates were resistant to AMP and 1 (2.6%) C. jejuni was resistant to GEN. Of all the isolates, 13.16%, 18.42%, 23.68%, 36.84%, and 23.68% were intermediately resistant to AMP, GEN, LE, CIP, and CTR, respectively, probably reflecting a shift toward resistance.

Discussion
Campylobacter is one of the leading causes of gastrointestinal illnesses worldwide. The present study was done to determine the prevalence of thermophilic Campylobacters (C. jejuni and C. coli), their presence of virulence genes, and the antimicrobial susceptibility of the obtained isolates. Of 609 samples screened, 38 were detected to be positive for Campylobacter spp. showing the overall prevalence rate to be 6.24%. The findings were in accordance with the work of Rajagunalan [17] and Pandey [18] who accounted 6.9% and 5.34% prevalence rate of Campylobacter spp. in the same area, respectively. However, there are reports of a bit higher prevalence in the same study region 16% [19], 11.66% [20], and 13.54% [21], and the reason probably could be the     This study also showed that C. coli (29/38, 76.32%) was more prevalent than C. jejuni (9/38, 23.68%) isolates which was incorcondance with Rajagunalan [17] and Kumar [20]. In contrast to these findings, many workers including Rajendran et al. [22] and Deckert et al. [23] have reported the higher presence of C. jejuni than C. coli. The variation in the prevalence of C. jejuni and C. coli could be due to difference in the samples, animal species screened, and the geographical location of the study. The use of growth promoters and increased AMR in C. coli has also been reasoned for the variation [24]. Among different sample sources, the highest isolation rate was observed from poultry ceca (15.52%), followed by poultry droppings (5.91%), cattle feces (4.92%), human stools (3.40%), and pig feces (2.82%).
The observations were in agreement with the findings of Humphrey et al. [25], who observed that, other than poultry digestive tract, digestive tracts of cattle, pigs, and human beings also act as the significant reservoir for Campylobacter species. The isolation rate in poultry ceca was higher (15.52%) than poultry droppings (5.91%). The difference could be because organisms undergo stress after excretion which may affect its survival and recovery rate. Moreover, keeping period of a bird in a flock enhances the colonization of Campylobacters. The collection of fecal and cecal samples from the same bird might give a clear picture of the prevalence of the organism.
None of the isolates were recovered from sheep and goat feces. Low prevalence of Campylobacters in sheep and goat has been reported by Salihu et al. [26], Zweifel et al. [27], and Cortes et al. [28] who reported a very low prevalence of Campylobacter spp. in sheep and goats as compared to other animals. As a contradiction to this finding, Lazou et al. [29], Mpalang et al. [30], and Karikari et al. [31] have recorded high prevalence in sheep and goat. The fecal samples of laboratory animals also did not yield any isolate which was dissimilar to the findings of Jensen et al. [32] and Nkogwe et al. [33] who reported C. jejuni infection in rats. Our findings in case of sheep, goat, and laboratory animals differed probably due to a lower number of samples screened. Moreover, better husbandry practices [26] followed could have resulted in the absence of Campylobacters. The laboratory animals were kept in a strict hygienic environment separated from other animals in the medical as well as veterinary colleges which could be the reason for the absence of Campylobacter organism from these samples.  Virulence typing was performed using four genes as targets. Of all the 38 isolates, all the isolates (100%) showed the presence of cadF genes. The other three flaA, virB11, and pldA genes were present in 37 (97.37%), 7 (18.42%), and 14 (36.84%) isolates, respectively.
The cadF and flaA genes responsible for the expression of adherence and colonization were highly detectable in all the isolates. The presence of cadF gene highly conserved in Campylobacters and regarded as genus-specific has been reported by Konkel et al. [34] and Wieczorek et al. [35]. This protein is important for full binding capacity of the Campylobacters to the host intestinal epithelial cells. Rozynek et al. [36] and Wieczorek and Osek [37] have also identified this virulence gene to be present in the feces of poultry, animal, and human isolates. The presence of flaA gene which determines flagellar motility responsible for motility and colonization of enterocytes is one of the best-studied virulence markers and in this study was present in 37 (97.37%) isolates. Previous studies have also indicated that the detection rate of flaA gene is high (>95%).
The virB11 and the pldA (phospholipase A) genes are responsible for invasion and survival within the host cells. The virB11 gene was rarest among all the studied genes and was present in the isolates obtained from poultry droppings (16.67%), poultry ceca (16.67%), cattle feces (33.33%), and human stools (33.33%). Bang et al. [38] also found only 7.5% of the isolates from pigs and cattle to be positive for virB11. Studies conducted on poultry, animals, and humans by Biswas et al. [15], Talukder et al. [39], Koolman et al. [40], and Jribi et al. [41] also, in fact, did not find the presence of virB11 gene. The pldA gene was also detected in 36.84% isolates. Melo et al. [42] also detected the presence of pldA gene in 63.65% of the C. jejuni strains isolated from chicken meat. The presence of these genes poses a potential hazard to human health.
Antibiotic susceptibility profile of all the 38 isolates (29 C. coli and 9 C. jejuni) revealed 100% resistance to NA. Only 2 (5.3%) C. coli and 1 (2.6%) C. jejuni were resistant to AMP and GEN, respectively. Of all the isolates, 13.16%, 18.42%, 23.68%, 36.84%, and 23.68% were intermediately sensitive to AMP, GEN, LE, CIP, and CTR, respectively, probably reflecting a shift toward resistance. The variation in the antimicrobial sensitivity pattern of the Campylobacter isolates was noticed among various reports. A significant increase in the resistance for NA (46.7% of the isolates) and CIP (52.2%) was also observed in Spain [43]. However, in our study, all the isolates were found to be resistant to NA but sensitive to CIP and LE. Furthermore, 100% resistance to cephalothin and COT and 100% sensitivity to AMP, GEN, and ERY were reported [17]. Only 71.4% of isolates had sensitivity against NA. 80% and 77% of Campylobacter isolates obtained from Thailand and India, respectively, were found to be resistant against fluoroquinolones [44]. Higher resistance rates to CIP (95.8-99%, 85.4%, and 91%) have been reported in China [45], the United Arab Emirates [46], and South Africa [47], respectively. The development of resistance in the poultry and animals of the study area is a potential threat to human health. There is much fear that this resistance may spread to environment [48] which may further lead to difficult to treat cases.

Conclusion
The present study highlights the existence of thermophilic Campylobacters in poultry, animals, and even in human samples. The recovery of positive isolates from humans emphasizes human-animal proximity that must have led to the transmission. The presence of virulence genes in the isolates marks their role in the establishment of the disease and thus modulates the clinical presentation in the host. Increasing antibiotic resistance against quinolone reflects the misuse/ overuse of the antibiotics in the area.

Authors' Contributions
NR 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 final manuscript.