Prevalence and Antimicrobial Resistance of Campylobacter Species in Diarrheal Patients in Mymensingh, Bangladesh

Campylobacter enteritis is the leading cause of gastroenteritis in humans worldwide including Bangladesh. The objectives of this study were to estimate the prevalence and antimicrobial-resistance status of Campylobacter spp. in human diarrheal samples collected from Surya Kanta Hospital, Mymensingh, Bangladesh. In this study, we evaluated a total of 330 clinical samples for the presence Campylobacter spp. via cultural and biochemical tests and molecular assays. Furthermore, antimicrobial susceptibility testing for Campylobacter species was accomplished by the standard agar disc diffusion technique against eight commercially available antimicrobial agents. A pretested semistructured questionnaire was used to capture the data on socioanthropological factors from the diarrheal patients. Pearson's chi-square test was performed, and a p value of <0.05 was considered for the level of significance. Nearly one in three diarrheal patients admitted in this hospital were infected with Campylobacter spp. Overall prevalence of Campylobacter spp. was estimated to be 31.5% (104/330) that comprised the prevalence of C. jejuni, 21.8% (n = 72), and C. coli, 9.6% (n = 32). Among the positive cases, the prevalence of Campylobacter was higher in the age group 0-5 years (52%) followed by 6-18 years (42.7%), 19-40 years (34.0%), 41-60 years (25.4%), and >60 years (10.5%). Age, family level's personal hygiene, and involvement with animal husbandry were captured as potential determinants to be associated with the Campylobacter positive status. Among the isolates, 27.3% (n = 20) of C. jejuni and 31.2% (n = 10) of C. coli demonstrated as multidrug-resistant (MDR) to three or more antimicrobial agents. The present study shows that Campylobacter spp. is most prevalent among the hospital-admitted diarrheal patients, and proper measures should be taken to reduce the burden focusing on the potential determinants.


Introduction
Campylobacter spp. are considered to be zoonotic pathogens that cause foodborne infection throughout the world [1,2]. These pathogens are the leading causative agents of sporadic bacterial diarrhea worldwide [3,4]. It was estimated that more than 16 million people got sick with nearly 37 thousand deaths in 2010 worldwide that were connected with Campylobacter spp. as a single infection [3]. Campylobacters are the most frequently isolated bacterial enteric pathogens both in developed and low-and middle-income countries (LMICs) [5]. In developed countries, Campylobacter jejuni and Campylobacter coli are the predominately isolated species associated with human bacterial diarrheal disease [6,7]. Primarily, most of the zoonotic Campylobacters are well adapted as a commensal in the gastrointestinal tract of poultry, cattle, sheep, and pigs [8] and can act as reservoirs [9]. Humans can be infected with Campylobacter via consumption of contaminated meat and poultry including dairy products [10,11]. Additionally, Campylobacter can contaminate the soil or water bodies to facilitate human infection or even direct human-to-human transmission [12].
Campylobacter infections exhibit variable clinical signs from sudden diarrhea to vomition, and abdominal cramps with onset of fever could continue at least for a week [13]. Campylobacter jejuni could cause severe chronic symptoms called Guillain-Barré syndrome (GBS) following the infection as a consequence of autoimmune disease [14].
C. jejuni is an important etiological agent of childhood diarrhea (25.5%) in Bangladesh [15]. However, the Guillain-Barré syndrome (GBS) which is connected to Campylobacter infection causes acute flaccid paralysis (AFP) established in Bangladesh with an estimated incidence rate of 3.25 cases/100,000 children under 15 years of age [16,17]. Many efforts have been made to minimize Campylobacter infection and its associated GBS risks without considering the source of introduction in the LMICs. Therefore, a significant burden of Campylobacter prevails in such counties [18], even in Bangladesh.
Earlier studies in Bangladesh reported a variable level of prevalence rate during the 1990s ranging from 17 to 26% with C. jejuni [19,20] and 9.45% with C. jejuni and 2.68% with C. coli spanning from 2005 to 2008 [21]. A few separate studies confirmed 8.5% prevalence for Campylobacter jejuni and Campylobacter coli in fecal specimens [22] and 15.3% and 11.3% prevalence of Campylobacter jejuni and Campylobacter coli, respectively [23]. However, a most recent study confirmed the prevalence of C. jejuni/coli as 28.3% in 2019 [24]. Nowadays, the widespread use of antimicrobial agents both in human and veterinary practices is a global concern [25]. This practice appears to be increased quickly in LMICs where rampant use of antibiotics is more common [26]. As a result, the resistance of Campylobacter species to antimicrobials has been documented throughout the world [27]. The emergence of antimicrobial-resistant Campylobacter species due to the overuse of antimicrobial agents in food animal production enables the spreading of antimicrobial-resistant bacteria [28]. This phenomenon highlights a serious impact on both veterinary and human health regarding food safety and public health issues.
In Bangladesh, several studies have been conducted with very few explored socioanthropological determinants of the occurrence of Campylobacter spp. along with antimicrobialresistant pattern of the isolates from human diarrheal patients. Therefore, the present study was designed to estimate the prevalence and antimicrobial resistance profile along with socioanthropological determinants of Campylobacter spp. infection in diarrheal patients from the Surya Kanta Hospital of Mymensingh district in Bangladesh.

Materials and Methods
2.1. Sample Collection and Shipment. Three hundred thirty (N = 330) stool specimens were collected randomly from patients suffering from different magnitudes of acute gastroenteritis symptoms including diarrhea admitted to the Surya Kanta Hospital, Mymensingh Medical College, Mymensingh, during the period from June 2019 to June 2020. A team composed of a physician and a lab technician were engaged in sampling and subsequent data collection. Aseptic measures were taken during the collection of samples. A single rectal swab sample was obtained from each patient, stored, and transported in Cary-Blair transport media. A unique identification number was given for each sample and transferred to the laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, maintaining with a cool chain (4-6°C).

Questionnaire Survey.
A semistructured interview questionnaire was developed and used for data collection from diarrheal patients on socioanthropological factors: age, sex, educational status, family level personal hygiene, and animal (ruminant, pets, and poultry) husbandry status including food habits. The questionnaire was translated into the local Bengali dialect for the face-to-face interview so that the respondent could easily understand its content.

Isolation and Identification.
Isolation and identification of Campylobacter spp. were accomplished through the filtration method [29]. In brief, each of the stool specimens was suspended in 500 μl of sterile saline. A portion of 100 μl of sample was spread onto the surface filters of blood agar base no. 2 including Skirrow supplements and permitted to stand for 30 min at room temperature. After 30 min, the filter was removed from the Skirrow blood agar and then the plates were incubated at 37°C for 48 h in microaerophilic condition (5% O 2 , 10% CO 2 , and 85% N 2 ). The incubated media were inspected for bacteria growth after 48 h. In Skirrow blood agar media, grey, flat, and irregularly spreading colonies were observed. The colonies were evaluated via Gram's staining, and Gram-negative curves were observed under the light microscope. Gram's stain positive colonies were further utilized in biochemical tests, namely, catalase, hippurate hydrolysis, and oxidase tests. The selected colonies that were found to be positive in Gram's staining and biochemical tests were further subcultured through the same procedure to get a pure colony. Thus, pure isolates obtained were used for further evaluations. Discrimination of Campylobacter isolates was completed based on growth characteristics as well as biochemical tests as per the standard protocols [30][31][32].

Molecular Identification by PCR.
Gram's staining and biochemical test positive isolates of the Campylobacter growth colony were validated by PCR assay as Campylobacter spp. The DNA materials were extracted from the pure colonies as per the procedure by Hoshino et al. [33]. Employing oligonucleotide primers, the genus of Campylobacter was confirmed through the amplification of the targeted 16S rRNA gene, as per the method labeled by Samosornsuk et al. [34] (Table 1).
After confirmation of Campylobacter spp. via 16S rRNA gene-based PCR assay, a cdtA gene-based multiplex PCR was done for the detection of different species of Campylobacter (i.e., C. jejuni, C. coli, and C. fetus) as per the protocol described by Linton et al. [36]. In these multiplex PCR assays, C. jejuni ATCC 33560, C. coli ATCC 33559, and C. fetus ATCC 27374 strains in DNA templates were utilized as a positive control. However, Escherichia coli ATCC 25922 2 BioMed Research International was utilized as a negative control. In the multiplex PCR assay, for those isolates confirmed as C. jejuni, their identity was further substantiated by a marker-based (hipO gene) PCR assay [36]. The sequences of the primers and parallel amplicon sizes including annealing temperatures of PCR are shown in Table 1. The PCR products were pictured in gel electrophoresis (1.5% agarose, Invitrogen, Carlsbad, CA, USA), and after coloring with ethidium bromide (0.5 μg ml -1 ) and decoloring with distilled water for 10 min, gel pictures were captured via a UV transilluminator (Biometra, Göttingen, Germany).

Antimicrobial Sensitivity Testing.
Isolates of Campylobacter spp. were tested via the disk diffusion method [37] using eight (8) Figure S1-a). Subsequently, a cdtA gene-based multiplex PCR was accomplished for the detection of different genus of Campylobacter, viz., C. jejuni, C. coli, and C. fetus. In this PCR assay, C. jejuni and C. coli produced an amplicon size of 631 bp and 329 bp, respectively, as a verifying test for species confirmation (Supplementary Figure S1-b). However, the isolates of C. jejuni were further confirmed via a robust marker hipO gene-based PCR assay that presented a 735 bp amplicon size (Supplementary Figure S1- (Figure 1 and Table 3). A lower level of Campylobacter prevalence (21.5%) was documented in good hygienic practice patients and found to be statistically significant. Similarly, involvement of livestock (cattle, sheep, goat, and poultry) rearing was found to 3 BioMed Research International be risky practices as higher prevalence (43.2%) was recognized. Similarly, regarding food habits, Campylobacter was found to be more prevalent in those patients who ate home-prepared food than the patients who ate other sources of food. However, no statistical significance was found ( Table 2).

Discussion
We evaluated the prevalence, risk factors, and antimicrobial resistant status of Campylobacter spp. in diarrheal specimens collected from the patients admitted in Surya Kanta Hospital, Mymensingh, Bangladesh. The study confirmed that nearly one-third of the patients (31.5%) was found to be positive with Campylobacter spp., of which Campylobacter jejuni (21.8%) was captured as the key contributor of human bacterial diarrhea. The finding of this study is in accordance to similar studies conducted in Bangladesh [15,[19][20][21][22][23].
In this study, the prevalence of Campylobacter spp. was found higher in females than the males but not statistically significant. In Bangladesh, females are more likely to be at an increased risk of Campylobacter exposure as they are involved with household livestock rearing (cattle, sheep, goat, and poultry) than males. This finding conforms to the result of an earlier study conducted in Bangladesh [23]. However, improved personal hygiene practices would contribute towards the lower prevalence in males.
The distribution of prevalence of Campylobacter spp. was found to be significantly varied among the different age groups (p ≤ 0:001). In this study, the higher prevalence was observed in the youngest age group (0-5 years). Earlier studies also reported variable prevalence of Campylobacter as 25.5% [15] and 12.9% [41] but consistent with the most susceptible young patients. Campylobacter can cause infection in all age groups; however, the clinical presentation may differ by different age groups of patients established in previous studies [23,[42][43][44]. Children at a young age are not aware on hygiene and sanitation practices. Commonly, in the rural settings of Bangladesh, farmers keep the livestock close to their house which offers regular contact of young children with the animals which is likely to further the risk of exposure of infection.
Family-level personal hygiene and involvement with livestock rearing were found to be associated with a higher level of Campylobacter exposure. The main routes of transmission of Campylobacter are the consumption of poultry and involvement with poultry rearing activities [45] or exposures resulting from environmental contamination by cattle manure [46,47] that facilitate potential introduction in humans through the food chain [48]. However, exposure to livestock was documented as a significant association with higher Campylobacter positivity status [49]. Therefore, family-level involvement with livestock rearing including poor personal hygiene was presented to be a higher level of Campylobacter occurrence in this study.
Our study demonstrated that 27.8% (n = 20) and 31.2% (n = 10) of C. jejuni and C. coli, respectively, showed as  Figure 1: Distribution of prevalence with 95% confidence interval (CI) at different age groups in Surya Kanta Hospital, Mymensingh, from June 2019 to June 2020. The prevalence among the different age groups was found to be statistically significant from each other ( * p ≤ 0:001).  [23,50,[53][54][55]. The MDR noticed in this survey is likely to be a potential reflection of imprudent use of antimicrobials due to their easy accessibility at local drug stores throughout the country [21].
Based on the study findings, risk reduction measures to be taken to address health education for the household livestock keepers, personal hygiene, microbiological evaluation of the isolates through culture and molecular assessment, and prudent use antimicrobials are needed.

Conclusions
The present study shows occurrence of Campylobacter infection in diarrheal patients, of which Campylobacter jejuni was captured as an abundant species. The higher frequency was observed in 0-5 years age, family-level poor hygienic practices, and involvement of animal husbandry. Therefore, activities on awareness creating behavioral change relating to personal hygiene like hand washing and sanitization after using the toilet or animal contact are needed. Children 0-5 years of age should avoid contact with livestock. The treatment of diarrhea patients should be based on an updated database on the susceptibility status of Campylobacter spp. instead of clinical signs. A physician should consider that diagnosing diarrhea infection with Campylobacter spp. is crucial among the other enteric pathogens. Cognizing the burden of Campylobacter diarrheal disease is significant for framing effective control programs targeting the overall decline of diarrheal disease in all ages of people. Further investigations are required to substantiate the role of domestic animals in the spreading of Campylobacter spp. including species diversities/serotyping.

Data Availability
The tables, figures and texts in this research article contain the data that support the study conclusions.

Conflicts of Interest
The authors declare that no conflict of interest exists.