|Year : 2019 | Volume
| Issue : 3 | Page : 345-350
Detection and molecular typing of campylobacter isolates from human and animal faeces in coastal belt of Odisha, India
Nirmal Kumar Mohakud1, Saumya Darshana Patra2, Subrat Kumar2, Priyadarshi Soumyaranjan Sahu3, Namrata Misra4, Arpit Kumar Shrivastava2
1 Department of Paediatrics, Kalinga Institute of Medical Sciences, KIIT Deemed to be University, Bhubaneswar, Odisha, India
2 Department of Biotechnology, Infection Biology Laboratory, KIIT Deemed to be University, Bhubaneswar, Odisha, India
3 Department of Biotechnology, Infection Biology Laboratory, KIIT Deemed to be University, Bhubaneswar, Odisha, India; Department of Microbiology and Immunology, Medical University of the Americas, Devens, MA, USA
4 Department of Biotechnology, KIIT-Technology Business Incubator, KIIT Deemed to be University, Bhubaneswar, Odisha, India
|Date of Submission||08-Nov-2019|
|Date of Decision||04-Nov-2019|
|Date of Acceptance||18-Nov-2019|
|Date of Web Publication||05-Dec-2019|
Dr. Arpit Kumar Shrivastava
Department of Biotechnology, Infection Biology Laboratory, KIIT Deemed to be University, Bhubaneswar - 751 024, Odisha
Source of Support: None, Conflict of Interest: None
Introduction: Campylobacter-mediated diarrhoea is one of the major causes of gastroenteritis globally. A majority of the Campylobacter spp. that cause disease in humans have been isolated from animals. Faecal contamination of food and water is the identified frequent cause of human campylobacteriosis. Methodology: In the present study, faecal samples from patients with symptoms of acute diarrhoea (n = 310) and domestic animals including cows (n = 60), sheep (n = 45) and goats (n = 45) were collected from the same localities in the peri-urban Bhubaneswar city. Genomic DNA isolation followed by polymerase chain reaction and sequencing was employed to analyse Campylobacter spp.-positive samples. Results: Of the 460 faecal samples, 16.77% of human samples and 25.33% of animal samples were found to be positive for Campylobacter spp. Among animals, the isolation rate was highest in sheep followed by cows and goats with 9.33%, 8.66% and 7.33%, respectively. The highest number of Campylobacter-positive cases was diagnosed in infants of 2–5 years age. Concurrent infection of other pathogens in addition to Campylobacter spp. was frequently detected in the samples. Conclusion: The present study showed the incidence of Campylobacter infections in human and different animal species in and around Bhubaneswar, Odisha. The analysis suggested that domestic animals can be the potential sources for human campylobacteriosis in the region.
Keywords: Campylobacter spp., diarrhoea, Odisha, phylogenetic analysis
|How to cite this article:|
Mohakud NK, Patra SD, Kumar S, Sahu PS, Misra N, Shrivastava AK. Detection and molecular typing of campylobacter isolates from human and animal faeces in coastal belt of Odisha, India. Indian J Med Microbiol 2019;37:345-50
|How to cite this URL:|
Mohakud NK, Patra SD, Kumar S, Sahu PS, Misra N, Shrivastava AK. Detection and molecular typing of campylobacter isolates from human and animal faeces in coastal belt of Odisha, India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Apr 7];37:345-50. Available from: http://www.ijmm.org/text.asp?2019/37/3/345/272350
| ~ Introduction|| |
Campylobacter is recognised as a major pathogen contributing to the global burden of gastroenteritis., Particularly, in developing countries including India, Campylobacter spp. is reported as the second most common cause of bacterial enteritis., The Gram-negative pathogen exists as normal flora in the intestinal tracts of many animal and bird species without causing any serious disease symptoms.,, However, humans with Campylobacter infection experience acute watery or bloody diarrhoea, fever, weight loss and cramps. Along with gastrointestinal infections, Campylobacter also causes an array of clinical manifestations such as bacteraemia, sepsis, meningitis, endocarditis and myocarditis; Miller Fisher syndrome; haemolytic-uraemic syndrome; Guillain–Barre syndrome; brain abscesses; reactive arthritis and complications of the reproductive system as well.,
Several studies have reported that cattle, poultry and domestic animals are the major carriers of Campylobacter species., Animals containing Campylobacter pose a threat for human infections through ingestion of undercooked meat, contaminated carcasses, food and water, consumption of raw and unpasteurised milk and ready to eat food products, etc.,,, Although over the past few years, several intervention biocontrol measures, namely bacteriophage-based strategies and endolysins, have been employed at reducing the incidence of a number of food-borne infections and the transmission of enteric pathogens. Nevertheless, Campylobacter prevalence has been rising exponentially across both developed and developing countries. Currently, there are inadequate epidemiological data to provide an accurate assessment of the health implications of the infectious disease in developing countries, particularly in the Indian subcontinent.,
As rational control programs for any infectious diseases require an in-depth understanding of their epidemiology, the present study determines the prevalence of Campylobacter in the faecal samples of both human and major domestic animals in Bhubaneswar, Odisha.
| ~ Methodology|| |
Ethical approval provided by the Research Ethical Committee of Kalinga Institute of Medical Sciences Hospital, Bhubaneswar, was obtained prior to the study.
The study was conducted in and around Bhubaneswar, Odisha, India. Faecal samples from 310 patients presenting diarrhoea and 90 control samples were obtained from two major hospitals in Bhubaneswar over a period of 1 year from March 2016 to April 2017. Screening and detection of Campylobacter in animals involved collection of faecal samples from 150 domestic animals including cows (n = 60), sheep (n = 45) and goats (n = 45). To minimize the risk of environmental contamination, freshly excreted faecal material was carefully collected from faecal surfaces that had not contacted the ground. The samples were collected carefully using a spoon-attached sterile HiMedia sample collection tube from the dispersed sites across the study region and were transported on ice within 4 h of collection and stored at 4°C up to 1 week until analysed at the School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT University).
Genomic DNA extraction and quantification
The total of faecal genomic DNA was extracted from stool samples using QIAamp Fast DNA stool Mini Kit (Qiagen, Germany) following the manufacturer's instructions. The extracted DNA samples were quantified using a Nanodrop (Titertek Berthold, Germany) and were stored at −20°C until further processing.
Polymerase chain reaction and 16S rRNA sequence analysis
Campylobacter-specific 16s primers, [Table 1] were used for polymerase chain reaction (PCR) amplification. Genomic DNA isolated from faeces was used as a template and as a positive amplification control pathogen-specific genomic DNA isolated from pure cultures was used in PCR screening. Cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 45–60 s, extension at 72°C for 1 min and final extension at 72°C for 10 min. All PCR products were subjected to 1%–1.5% agarose gel electrophoresis for the confirmation of positive samples. PCR-positive products were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). The DNA sequencing was performed in the forward direction using the 16s rRNA forward primer [Table 1]. The sequencing was carried out in ABI 3730xl cycle sequencer. Sequence similarity search using BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the sample nucleotide sequence to known sequences in the NCBI GenBank database (http://www.ncbi.nlm.nih.gov).
|Table 1: Campylobacter specific primer sequences used in the polymerase chain reaction screening of faecal samples|
Click here to view
Phylogenetic and statistical analysis
The identification of phylogenetic neighbours was initially conducted by subjecting the partial 16S rRNA gene sequence to NCBI blast program against the database of type strains with validly published prokaryotic names. To determine the phylogenetic relationships of strains with its closest relatives, the partial 16S rRNA gene sequence of the strains was aligned using the CLUSTALW software program (Des Higgins at UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland). The aligned sequences were then used for reconstruction of the phylogenetic tree in tree-making algorithm methods of MEGA version 7 software. Evolutionary distance matrix was calculated using Kimura's two-parameter model method, and the tree was reconstructed using the neighbour-joining method. Descriptive statistics was done to calculate odds ratios, 95% confidence intervals and P values for significance calculation. The MEDCALC statistical software (MedCalc Software, Ostend, Belgium) was used to analyse the data.
| ~ Results|| |
Of the 460 faecal samples that were analysed, 52 of 310 (16.77%) human samples and 38 of 150 (25.33%) animal samples were found to be positive for Campylobacter spp.
In human faecal samples, overall 23.33% samples were found positive in the age group of 2–5 years, followed by 15.81% in the age age group of 0–2 years [Table 2]. Campylobacter infections were higher in female (22.88%) in comparison to males (13.02%). Campylobacter infection in humans was significantly associated with vomiting (P = 0.006) and fever (P = 0.005).
|Table 2: Distribution of incidences of Campylobacter in human and animal faeces|
Click here to view
In animals, the highest prevalence of pathogen was observed in sheep (9.33%), followed by cows (8.66%) and goat (7.33%). In congruence to human analysis, a similar correlation was observed in the case of animals. Furthermore, female animals displayed a higher prevalence (22%) as compared to males (16%). In relation to the age of animals sampled, adult animals displayed a higher percentage (26.95%) of positive samples than young ones [Table 2].
In addition to Campylobacter, a myriad of other pathogens such as Cryptosporidium, Rotavirus, Shigella, Enteropathogenic E. coli (EPEC), Adenovirus, Enterohemorrhagic E. coli, O157 and Shiga toxin-producing E. coli were also detected in the considered samples. [Table 3] shows the frequencies of detecting dual and/or multiple infectious aetiologies in human as well as animal faecal samples. A total of 36 cases with concurrent infections were obtained in human faeces, of which 18 presented infectious aetiology with three or more agents. The animal species also contained 16 coinfection cases in total, with 11 representing multiple aetiologic agents. The combination of Campylobacter, Cryptosporidium and EPEC was commonly observed in both human and animal faecal samples.
|Table 3: Combinations of two or more infectious etiologies detected along with Campylobacter in human and animal faecal samples|
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The sequences for 16S rRNA genes of 19 isolates of Campylobacter spp. from Bhubaneswar were submitted to GenBank under the accession number as listed in Supplementary [Table 1]. The 16S rRNA nucleotide sequence for Campylobacter strains was searched within GenBank and EzTaxon to identify the closest type strains of genus Campylobacter and their sequence similarities. In this study, based on the sequence similarity, phylogenetic analysis of 16S rRNA gene with 812 bp PCR product by multiple sequence alignment (neighbour-joining tree) in MEGA 7.0 version showed the specific detection of Campylobacter spp. In the present study, the phylogenetic tree was constructed based on the 19 Campylobacter spp. isolates that included human (n = 12), sheep (n = 4), goat (n = 2) and cow (n = 1) [Figure 1]. Groups A, B, C and D are composed of sequences showing similarity with Campylobacter type strains isolated from either human or animal isolates. Groups B and D comprise only human isolates and their sequences are identical to the type species Campylobacter concisus and Campylobacter jejuni, respectively, while group C represents the strains solely isolated from animal samples with sequence similarity to Campylobacter lanienae. Group A has sequence similarity with Campylobacter hyointestinalis and in the tree, it represents an interesting association that MN203696 and MN203681 isolates of human were more closely related with MN203692 and MN203694 isolates of goat and sheep, respectively as well as with MN203693 isolate of cow.
|Figure 1: Neighbour-joining tree based on the analysis of 16S rRNA gene sequence showing the position and phylogenetic relationship between the Campylobacter spp. isolated from India. Only bootstrap values (expressed as percentage of 1000 replicates) >50% are shown at branch points. Bar, 0.0050, substitutions per nucleotide position|
Click here to view
| ~ Discussion|| |
Global incidences of acute gastroenteritis have dramatically increased over the past few years, particularly in developing countries. Being one of the established causes of gastroenteritis, Campylobacter spp. infection is also reported to be one of the most widespread infectious diseases in Asia. However, there is limited information about the true prevalence in this study region which is considered to be a developing province in India. In this study, the prevalence of Campylobacter infections in both humans and animals inhabiting the same locality is being reported for the first time, while few earlier studies have reported the prevalence of the pathogen in other states of India such as a study from Kolkata conducted from January 2008 to December 2010 and observed that 7.0% of gastroenteritis patients were culture positive for Campylobacter species. Likewise, another culture-independent real-time PCR study also reported 16.2% of positive cases of Campylobacter infection in hospitalized diarrhoea stool samples in the same region. In addition, few other studies elucidated the presence of campylobacteriosis to be around 10% in Puducherry and 4.5% from Vellore, South India. It was observed that 16.77% of diarrhoeic faecal samples from patients in Bhubaneswar, Odisha, showed the presence of Campylobacter spp., which was relatively higher as compared to the aforementioned reports in other parts of India which could be because of multiple factors such as hygiene level, nutrition, weather and multicultural population in the city., These findings are in solidarity with several studies in other developing counties such as China, Iran, Bangladesh, Pakistan, Egypt, Thailand, Nigeria and Jordan, in which Campylobacter spp. has also been found to be a common enteropathogen.,,,
While infection with Campylobacter species can occur in patients of all age groups, in the current study, the peak incidence of Campylobacter infection was observed in children of 2–5 years. These findings are consistent with similar studies from India where Campylobacter infection is also prevailing in children under the age of 5 years., Moreover, children ≤5 years have also been indicated as a group of high risk incurring campylobacteriosis in several other countries., A recent investigation from Denmark also demonstrated that infection is predominant in children (1–4 years) and young adults (15–24 years) than in other age groups. This could be due to partially established immune response, as well as improper hand hygiene and contact with animals and the environment. It could be considered that specific environmental, urban and sub-urban factors play a pivotal role in campylobacteriosis in children. The risk factor of campylobacteriosis in humans in association with faecal material of livestock animals has been extensively studied.
One of the channels for spreading of Campylobacter is faeces of wild and domestic animals and contaminated food and water. Against this backdrop, in the existing study, animal faecal samples of cow, sheep and goat along with the human faeces were investigated for the presence of Campylobacter in Bhubaneswar region. Among the animals, the highest prevalence of pathogen was observed in sheep followed by cows and goats. Female animals displayed a higher prevalence as compared to males. Based on the age of the sampled animals, adult ones displayed a greater percentage of positive samples than the younger counterparts. This observation is in contrast to previous studies performed in pigs and cattle where younger pigs tend to have elevated levels of Campylobacter than the adults.,
The phylogenetic tree depicts close proximity between the Campylobacter spp. obtained from human and animal isolates based on their evolutionary dynamics. The nucleotide sequences of the 16S rRNA genes of Campylobacter isolates of human and animals were determined and compared with sequences of Campylobacter-type strains. The results showed that the sequence identity was 96%–99% for the Campylobacter spp. local isolates and highest sequence similarity with C. concisus followed by C. jejuni, C. hyointestinalis and C. lanienae. In contrast to this, Al-Nasrawi who also identified Campylobacter spp. in the local isolates of human and birds in Iraq represented 99% sequence similarity with Campylobacter subantarctic, followed by C. jejuni, Campylobacter volucis and Campylobacter coli by 16S rRNA sequence analysis. Unlike the Groups B, C and D, sequences from Group A represent similarity with the C. hyointestinalis strains in both human and animal isolates. This can be attributed to the close proximity between the humans and animals and a possible zoonotic transmission.
| ~ Conclusion|| |
The current study focuses on the incidence of Campylobacter associated infections in humans and different animal species in and around Bhubaneswar, Odisha. The results corroborated with earlier reports that animals serve as the chief source of human campylobacteriosis. This is the pilot study that highlights the possible association of Campylobacter-related infection in both humans and domestic animals in this particular region. Further large scale studies are required to understand exact route of the anthromonotic and zoonotic transmission of Campylobacter spp. in the region.
The authors are grateful to Kalinga Institute of Medical Sciences (KIMS) Hospital, KIIT University, Bhubaneswar, to carry out this study. The help in animal sample collection and phylogenetic analysis from Mr. Ananta Panda is greatly appreciated. The present study is a pure exploratory in-house research project and is not supported by any funding grant-in-aid.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]