Prevalence, virulence gene profile and antibiogram of Campylobacter jejuni from fresh vegetables in Mymensingh, Bangladesh

: This study aimed to investigate Campylobacter jejuni, a major cause of food-borne bacterial infections worldwide, in fresh vegetables from five upzillas (Mymensingh, Trishal, Bhaluka, Muktagacha, and Fulbaria) in the Mymensingh district between July 2020 and April 2023. Using cultural, biochemical, and molecular techniques, 100 fresh vegetable samples (including tomato, carrot, cucumber, green chili, and coriander) were examined for C. jejuni . The isolates were further tested for virulence genes and antimicrobial susceptibility. Out of the 100 samples, 23% were confirmed as C. jejuni, by 16S rRNA gene-based polymerase chain reaction and all were found to be virulent with cytolethal distending toxins ( cdtA, cdtB and cdtC genes). Antibiotic susceptibility testing revealed resistance to amoxicillin (47.83%), tetracycline (43.48%), and streptomycin (39.13%) among the isolates. However, ceftriaxone and ciprofloxacin were effective against 47.83% and 43.48% of the isolates, respectively. Moreover, 52.17% of the isolates were sensitive to erythromycin. Alarmingly, 34.78% of the C. jejuni isolates exhibited multidrug resistance (MDR) with eight different antibiotic resistance patterns, including four MDR patterns. These findings highlight the presence of virulent and antibiotic-resistant C. jejuni in fresh vegetables, emphasizing the need for monitoring and control to ensure food safety and public health issues.


Introduction
Vegetables are widely recognized for their nutritional value, providing vitamins, fiber, micronutrients, and minerals essential for human health.Deficiencies in vitamins C and A can lead to various health issues, underscoring the importance of well-balanced diets with a high vegetable intake (Kalia and Gupta, 2006).Salad vegetables like tomatoes, carrots, cucumbers, green chilies, and coriander leaves are commonly consumed raw in traditional salad preparations worldwide.In today's society, food safety is a major concern, with microorganisms being a significant factor in food adulteration.Among the potential pathogen contaminants, Campylobacter spp. is particularly concerning.
Campylobacter is a well-known cause of bacterial infections, responsible for a majority of cases of acute gastrointestinal infection in humans globally (Allos, 1998).In developed countries, campylobacteriosis poses a serious public health risk and is a prevalent cause of gastroenteritis (Friedman et al., 2000).Among Campylobacter species, Campylobacter jejuni accounts for more than 95% of identified infections (Altekruse et al., 1999).The introduction of enteric pathogens, including Campylobacter, via fecal contamination from both urban and rural sources can happen at different stages, including field cultivation and food processing (Kumar et al., 2001).Campylobacter species are bacteria that are Gram-negative, non-spore-forming, and curved rod-shaped, measuring approximately 0.2 to 0.5 μm in width and about 0.5 to 5 μm in length (Doyle, 1990).They thrive in an environment with approximately 5% O 2 , 10% CO 2 , and 85% N 2 , which is considered ideal condition for their optimal growth (Forbes et al., 1998).Campylobacter is commonly found in the digestive tracts of poultry, cattle, and animal-derived food products and is often associated with cases of diarrhea (de Boer et al., 2000).According to Zia et al. (2003), Campylobacter spp. is highly pathogenic, causing severe diarrhea, reactive arthritis, and even Guillain-Barre syndrome.Campylobacteriosis, the infection caused by these bacteria, is a significant public health concern in many developed countries, and monitoring of infections and antibiotic resistance patterns is ongoing (Kabir et al., 2011).Thermotolerant Campylobacter is recognized as the most common bacterial cause of foodborne illnesses worldwide (Rossler et al., 2020).The increasing microbial resistance is a global concern due to their extensive use in both human and veterinary practices (Hassan et al., 2014).Reports of Campylobacter species' resistance to antimicrobial agents have been documented worldwide (Isenbarger et al., 2002;Rahman et al., 2021), with low and middle income countries (LMICs) experiencing a rapid escalation in resistance due to imprudent antibiotic usage (Englen et al., 2003).The excessive use of antimicrobial agents in food animal production has led to a rise in antimicrobial-resistant Campylobacter species, negatively impacting both human and animal health in terms of food safety and public health grounds (Engberg et al., 2004).Antibiotics are commonly employed in veterinary practices for livestock and poultry production as curative, preventative, and growth-promoting agents.Given the risks associated with Campylobacter contamination, strict sanitary measures, including personal hygiene and food safety, should prohibit the consumption of food products contaminated with this organism.Antibiotics may be used to treat human clinical cases of Campylobacter spp.infection following sensitivity testing (Karmaker et al., 2018).While a few studies have assessed the microbiological contamination of vegetables in Bangladesh (Nipa et al., 2011;Rahman and Noor, 2012;Ohiduzzaman et al., 2022), there are currently sparse studies on the occurrance of C. jejuni in fresh vegetables, its virulence gene profile, or its antibiograms.This study focuses on raw vegetables like cucumber, green chili, coriander, and tomato, commonly consumed in salads, which pose a significant risk for Campylobacter infection.The objective of this study is to isolate and identify C. jejuni from fresh vegetables obtained from various local markets, characterize its virulence, and assess trends in antibiotic resistance.

Ethical approval
The Ethical Committee of the Bangladesh Agricultural University, Mymensingh, Bangladesh approved the study under reference no.AWEEC/BAU/2020 (12).

Collection and transportation of samples
One hundred (100) vegetable samples, comprising a mix of tomato, carrot, cucumber, green chili, and coriander, were collected and immediately transported to the Bacteriology Laboratory at the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh, while maintaining a cool chain system.The samples were promptly processed upon arrival to identify and isolate C. jejuni.

Isolation of C. jejuni
Isolation C. jejuni was carried out using the filtering technique (0.45 μm filter) as described by Shiramaru et al. (2012).Each vegetable sample was washed with water containing 0.1% peptone, and the filtered samples were placed on top of Blood Agar Base No. 2 with Skirrow Supplement.After pouring a portion of the peptone water over the filters, they were left at room temperature for 30 minutes.Subsequently, the filters were removed, and the plates were incubated under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ) at 37°C for 48 hours.The incubated media were examined for bacterial growth, and grey, flat, irregularly spreading colonies were observed on Skirrow blood agar.Gram's staining and microscopic observation confirmed the presence of Gram-negative curved bacteria.Oxidase and catalase tests were conducted on selected colonies that exhibited a Gram-negative curve in the smears and were catalase and oxidase positive.These selected colonies were sub-cultured onto Blood Agar Base No. 2 with Skirrow supplement to obtain single and pure colonies, which appeared as grey, flat, and irregularly spreading colonies on the surface of Skirrow blood agar.The resulting pure isolates were used for further research.

Molecular identification and virulence characterization by PCR
The DNA extraction from cultured bacteria followed the standard boiling method described by Hoshino et al. (1998).For this, 3-5 pure single colonies from Blood base agar were mixed with 250 µl of deionized water in an Eppendorf tube.The tubes were then placed in boiling water and boiled for 10 minutes, followed by immediate transfer to ice for 10 minutes to induce cold shock.Afterward, the tubes were centrifuged at 12,000 rpm for 12 min, and approximately 100 µl of the supernatant was collected as the DNA template for the PCR assay aimed at amplifying the targeted genes.PCR reactions to amplify various target genes in C. jejuni isolates were performed using a Thermocycler (2720 Thermal Cycler, Applied Biosystems, USA) with a 25 μl PCR mixture.The oligonucleotide primer sequences and corresponding target genes used for identifying and characterizing the virulence of C. jejuni isolates are listed in Table 1, while the thermal profiles applied for PCR amplification of various genes are presented in Table 2.The PCR products, including the 16S rRNA gene and hipO, cdtA, cdtB, cdtC genes, were separated on 1.5% and 2% agarose gels (Invitrogen, USA), respectively, and stained with ethidium bromide (0.5 μg/ml) (Sigma-Aldrich, USA).The gels were then visualized using an ultraviolet transilluminator (BDA digital, Biometra GmbH, Germany).

Multiple antimicrobial resistance index (MARI)
The formula used by Msolo et al. (2020) to calculate the multiple antimicrobial resistance index (MARI) for C. jejuni isolates is as follows: ARI a b where "a" is the number of antibiotics to which a particular isolate is found resistant, and "b" is the total number of antibiotics to which each individual isolate was evaluated.

Data management and statistical analysis
The data were recorded in a Microsoft Excel 2016 spreadsheet (Microsoft Office 2016, Microsoft, Los Angeles, CA, USA) and analyzed using SPSS version 20.Descriptive statistics, including frequency and percentage, were computed for the analysis.

Isolation and identification of C. jejuni
Campylobacter spp.displayed distinct characteristics on Skirrow blood agar plates, showing grey, round, convex, smooth, and shiny colonies with regular edges after being incubated at 37°C for 48 h under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂).In Gram's staining, the organisms appeared as small, curved, Gram-negative cells arranged singly or in pairs, exhibiting a pink color.The purity of the organisms was confirmed and validated using specialized Blood Agar Base No. 2 media.In the catalase and oxidase tests, Campylobacter spp.isolates were found to be positive.The hippurate hydrolysis test indicated positive results only for C. jejuni, while C. coli was determined to be negative (Table 3).

Molecular detection by PCR
To confirm the presence of the Campylobacter genus, a 16S rRNA gene-based PCR was conducted.This PCR test generated a distinct amplification of 1530 bp in 23 different Campylobacter isolates (Figure 1).Furthermore, to validate the identity of the isolates as C. jejuni, a targeted hipO gene-based PCR was accomplished.The hipO gene-based PCR yielded specific amplification of 735 bp in all 23 Campylobacter isolates, confirming their identity as C. jejuni (Figure 2).

Virulence characterization of C. jejuni by cdt gene-based multiplex PCR assays
To characterize the virulence of C. jejuni, multiplex PCR assays targeting the cdtA, cdtB, and cdtC genes were performed.All 23 C. jejuni isolates showed specific amplification at 631 bp, 714 bp, and 524 bp for the cdtA, cdtB, and cdtC genes, respectively, indicating the presence of these virulence genes in all isolates (Table 4).The PCR results are presented in Figures 3, 4, and 5.

Antimicrobial susceptibility of C. jejuni isolated from fresh vegetable
The results of the antimicrobial susceptibility test conducted on the 23 C. jejuni isolates using eight commercially available antibiotics from six classes are summarized in Table 5.The antibiogram study revealed that 47.83% (11/23) of the C. jejuni were resistant to amoxicillin, and 43.48% (10/23) were resistant to tetracycline.Additionally, 39.13% (9/23) of the C. jejuni isolates showed resistance to streptomycin.Surprisingly, ceftriaxone and ciprofloxacin were effective against 47.83% (11/23) and 43.48% (10/23) of the isolates, respectively.Moreover, 52.17% (12/23) of the isolates were found to be sensitive to erythromycin.

Discussion
In this study, C. jejuni was isolated and characterized using various techniques, including a cultural featuresbased study, staining characteristics, biochemical tests, and PCR.To identify C. jejuni, the organism was cultured on Blood Agar Base No. 2 with Skirrow supplement, a selective agar media, under microaerophilic conditions (5% O 2 , 10% CO 2 , and 85% N 2 ).The filtration method (0.45 μm filter paper) was employed to isolate and identify C. jejuni, similar to experiments conducted by Haseena (2017) and Forbes et al. (1998).The colony characteristics of C. jejuni exhibited a pink or light pink color, gram-negative, and slightly curved shape, which were consistent with previous investigations by Kabir et al. (2011) and Karmaker et al. (2018).Biochemical tests were conducted for the identification of C. jejuni, and the results were in agreement with studies by Shiramaru et al. (2012) and Karmaker et al. (2018).The oxidase test showed a purple color shift in 23% (23/100) of the isolates, while the hippurate hydrolysis test revealed a strong blue or purple color in all isolates, confirming their identity as C. jejuni.The prevalence of C. jejuni in this study was found to be 23% (23/100) of the fresh vegetable samples tested.These findings align with previous studies conducted by Rahman et al. (2021), who reported a prevalence of 21.Osundiya et al. (2013), who associated MAR index values greater than 0.2 with higher-risk sources that are commonly treated with antibiotics.
Additionally, the study compared the MAR indices reported by Beshiru et al. (2022) and Fallah et al. (2022) with the permissible limit.Both of these studies reported MAR indices exceeding the permissible limit by 0.60 and 0.75 points, respectively.These results further underscore the concern of antibiotic resistance dissemination in various environments, which could have adverse effects on public health and food safety.

Conclusions
The escalating bacterial resistance to antibiotics is a worldwide pandemic, posing a significant concern for public health.The extensive and indiscriminate use of antibiotics in food items is a key contributing factor to the development of resistance.The findings of the current study reveal the presence of virulent and multidrugresistant strains of C. jejuni in fresh vegetables within the study area.Considering the potential health risks, isolated cases should be carefully monitored, and risk factors must be minimized by promptly implementing stringent regulations and legislation.To mitigate the risk of C. jejuni infections from fresh vegetables, proactive measures should be adopted.Practicing proper hygiene, such as thorough washing of vegetables before cooking and ensuring they are properly cooked, can prove effective in preventing infections.Additionally, the adherence to food safety standards during the processing and handling of fresh vegetables is crucial to reduce the likelihood of contamination.In conclusion, addressing the issue of antibiotic-resistant C. jejuni in fresh vegetables necessitates a comprehensive approach involving both regulatory interventions and individual practices.
8%, andKarmaker et al. (2018), who reported a prevalence of 15.33%.The similarity in prevalence rates among these studies indicates a consistent occurrence of C. jejuni in fresh vegetables, highlighting the importance of monitoring and ensuring food safety practices to mitigate potential health risks associated with this pathogen.In this study, the confirmation of the Campylobacter genus was achieved using a 16S rRNA gene-based PCR, while the identification of C. jejuni was conducted through a targeted hippuricase gene-based (hipO) PCR test.The 16S rRNA gene-based PCR resulted in a characteristic amplification of 1530 bp in 23 different Campylobacter isolates, consistent with findings from previous studies byKabir et al. (2011) andRahman et al. (2021).The hipO gene-based PCR confirmed the identity of all 23 Campylobacter isolates as C. jejuni, as they demonstrated specific amplification at 735 bp.Additionally, the virulence of C. jejuni was assessed using a multiplex PCR assay targeting the cdtA, cdtB, and cdtC genes.All 23 C. jejuni isolates displayed specific amplification at 631 bp, 714 bp, and 524 bp, respectively, indicating the presence of cdtA, cdtB, and cdtC

. MDR and MAR profiles of C. jejuni isolated from fresh vegetables
The investigation's findings revealed that out of the 23 C. jejuni isolates, 34.78% (8/23, 95% CI: 18.81-55.11%)exhibitedmultidrug-resistant(MDR)phenotypes,with eight different antibiotic resistance patterns, including four MDR patterns.These MDR isolates were identified in 13.4% (3/23) of AMX-S-TE cases, which had the highest MDR pattern.Moreover, two isolates (AMX-TET-ER-GEN) showed resistance to four of the eight antibiotics tested, belonging to six distinct classes.The multiple antibiotic resistance (MAR) indices of the C. jejuni isolates ranged from 0.1 to 0.6.The MDR and MAR profiles of the C. jejuni isolates from fresh vegetables are presented in Table6and Figure6.

Table 6 . Phenotypic MDR profiles of C. jejuni isolates from fresh vegetables in Mymensingh district.
Karmaker et al. (2018), andRahman et al. (2021)ch studies conducted byKabir et al. (2015)andRahman et al. (2021).The antibiogram study was performed to assess the sensitivity and resistance pattern of the bacterial isolates to various antibiotics commonly used in the market.Among the 23 C. jejuni isolates, 47.83% (11/23) were found to be resistant to amoxicillin, and 43.48% (10/23) were resistant to tetracycline.Additionally, 39.13% (9/23) of the isolates exhibited resistance to streptomycin.Surprisingly, ceftriaxone and ciprofloxacin were effective against 47.83% (11/23) and 43.48% (10/23) of the isolates, respectively.Moreover, 52.17% (12/23) of the isolates were sensitive to erythromycin.These findings are similar to those reported byHakanen et al. (2003)andRahman et al. (2021), suggesting a consistent pattern of antibiotic resistance in C. jejuni isolates.Overall, the study utilized a range of techniques to characterize C. jejuni isolates, including PCR-based methods for genus and species confirmation, as well as virulence characterization, and an antibiogram study to assess antibiotic resistance patterns.The results provide valuable insights into the prevalence, virulence, and antibiotic resistance of C. jejuni in the tested fresh vegetable samples.The present study revealed a concerning observation of multidrug resistance (MDR) in the majority of C. jejuni isolates.Among the 23 Campylobacter isolates tested, 34.78% (8/23) were identified as multidrug resistant, defined as being resistant to three or more different antibiotics.This finding aligns with several earlier studies conducted byHakanen et al. (2003),McGill et al. (2006),Moore et al. (2006),Luangtongkum et al. (2009),Karmaker et al. (2018), andRahman et al. (2021), which also reported significant proportions of MDR C. jejuni isolates.Furthermore, the study assessed the Multiple Antibiotic Resistance (MAR) index to gain insights into the level of contamination and antibiotic use in the environment from which the isolates were obtained.The MAR index values are an important indicator of the potential risk of antibiotic resistance emergence in the environment.In this study, 21.74% (5/23) of the isolates had MAR indices greater than 0.2, indicating a higher risk of contamination from sources frequently exposed to antibiotics.In contrast, only 4.35% (1/23) of the isolates had MAR indices of 0.2 or less, suggesting lower risk of contamination from antibiotic-exposed sources.This finding is consistent with the observations of