Prevalence and antibiogram of Campylobacter spp. from cattle in Anand, Gujarat

Introduction

Foodborne infections remain a major global public health concern. Among foodborne pathogens, Campylobacter has emerged as one of the leading causes of bacterial gastroenteritis worldwide. In recent years, the global burden of Campylobacter infections has continued to increase due to factors such as globalization of food supply, intensive livestock production, and increased surveillance systems. According to recent global estimates, Campylobacter species are responsible for millions of cases of diarrhoeal disease each year and remain among the most frequently reported bacterial causes of foodborne illness in many countries (WHO, 2023; CDC, 2022) [2,5] . Campylobacter belongs to the family Campylobacteraceae, which also includes the genera Helicobacter, Arcobacter, and Wolinella. This family is classified within the epsilon subdivision of the class Proteobacteria. Advances in molecular taxonomy have expanded the genus, and currently more than 30 species of Campylobacter have been identified. Among them, Campylobacter jejuni and Campylobacter coli are the most significant species associated with human disease. These organisms are commonly present as commensals in livestock, poultry, and domestic animals. C. jejuni accounts for approximately 80-90% of human Campylobacteriosis cases, while C. coli contributes about 5-10% of infections worldwide (Man, de Steenhuijsen Piters & Bogaert, 2021) [1] . Campylobacter species are Gram-negative, microaerophilic, spiral-shaped or curved rods measuring approximately 0.5-8.0 μm in length and 0.2-0.5 μm in width.

Most strains possess a single polar flagellum, although some may have bipolar flagella. These bacteria exhibit a characteristic corkscrew-like motility with rapid darting movements and instantaneous directional changes. This motility allows the organism to penetrate the mucus layer and colonize the epithelial surfaces of the gastrointestinal tract (EFSA, 2022) [3] . Campylobacter organisms are widely distributed in the gastrointestinal tract of warm-blooded animals, including cattle, sheep, pigs, and poultry. In cattle, C. jejuni commonly colonizes the proximal small intestine and may be shed in feces, which contributes to environmental contamination. Human infections occur mainly through the foodborne route, particularly through consumption of contaminated poultry products, unpasteurized milk, or contaminated water. However, recent studies suggest that multiple food sources and environmental reservoirs may contribute significantly to human infections (EFSA & ECDC, 2023) [3] . The role of cattle in the epidemiology of Campylobacteriosis is not limited to contamination of milk or carcasses during slaughter. Cattle may also contribute to environmental contamination through manure disposal, slurry spreading on agricultural land, and abattoir effluents. These activities may contaminate soil, water bodies, and food products, thereby increasing the risk of transmission to humans.

Campylobacteriosis is an acute gastrointestinal infection characterized by symptoms such as diarrhoea, abdominal pain, fever, nausea, headache, and muscle pain. The incubation period generally ranges from 2 to 5 days, and the illness usually lasts about 5-7 days. Although most infections are self-limiting, some patients may develop severe post-infectious complications. One of the most serious sequelae is Guillain-Barré syndrome (GBS), an autoimmune neuropathy that can cause acute flaccid paralysis. Other complications include reactive arthritis and irritable bowel syndrome (CDC, 2022; WHO, 2023) [5,2] . In most cases, Campylobacter infections are self-limiting, and antimicrobial therapy is not required. However, antibiotic treatment may be recommended in severe or prolonged cases, especially among immunocompromised patients. Macrolides such as erythromycin or azithromycin are considered the drugs of choice. However, the increasing emergence of antimicrobial resistance, particularly to fluoroquinolones and tetracyclines, among Campylobacter isolates has become a significant global health concern (WHO, 2023; Tang et al., 2022) [2,4] . Considering the increasing public health importance of Campylobacter infections and the growing problem of antimicrobial resistance, continuous surveillance of this pathogen in foodproducing animals is essential. Therefore, the present study was undertaken to determine the prevalence and antibiotic resistance pattern of Campylobacter spp. isolated from cattle in Anand city, Gujarat, India, in order to understand their epidemiological significance and potential risk to human health.

Materials and Methods

Approximately, a total 200 faecal samples were collected aseptically and transported to the laboratory in an icebox for microbiological analysis. Samples were processed in laboratory immediately within two hour.

Isolation and identification

Samples were processed to isolate the Campylobacter spp. as per the method described by Salihu et al. (2010) [31] The samples were enriched into 45 ml of Blood free Campylobacter broth base containing CCDA selective supplement (HiMedia Laboratories, Mumbai, India). Mixed properly and incubated at microaerophilic environment (85% N 2 , 5% O 2 and 10% CO 2 ) in water jacketed CO 2 incubator (NUAIRE, Polymouth, MN) at 42 °C for 48 hour. After enrichment culture, a loopful of enriched culture was streaked on blood free charcoal cefoperazone deoxycholate agar medium plates and incubated at microaerophilic environment at 42 °C for 48 hour. The typical dew drops like colonies were isolated on charcoal based media such as mCCDA.

Identification of Campylobacter species

The presumptive Campylobacter isolates were identified on the basis of culture characteristics, Gram staining morphology, Nitrate reduction test, Urease test, H 2 S production, Hippurate and Indoxyl acetate hydrolysis.

Template DNA preparation

A loopful of overnight growth culture of Campylobacter spp. was suspended in 100 µl of sterilized DNAse and RNAse-free milliQ water (Millipore, USA) in PCR tube. After proper mixing, the tube was then kept at 95 °C for 10 min in PCR tubes. Cell debris was sedimented by centrifugation at 10,000 rpm for 5 min and 3 µl of supernatant was used as a DNA template in PCR reaction mixture.

Confirmation and species identification of isolates using polymerase chain reaction (PCR)

The biochemically identified isolates were further employed for confirmation by PCR as genus Campylobacter, C. jejuni, C. coli and C. lari by amplifying specific target gene with species specific oligonucleotide primers. The details of primers are tabulated in Table 1. The DNA amplification for each primer pair was carried out in a Applied Biosystems 2720 Thermal Cycler in 25 μl reaction containing 3 μl of DNA template, 12.5 μl mastermix (Thermo Scientific, USA) (containing 0.05 unit/μl Taq DNA Polymerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP), 10 pmole of each forward and reverse primer (10 pmole/μl), and 7.5 ml nuclease free water with positive and negative control. The control strains of C. jejuni and C. coli isolates in our department were used for positive control whereas DNase free distilled water was used for negative control. The cycling protocol for the genus confirmation was standardized to set the PCR assay as initial denaturation at 95 °C for 5 min followed by 35 cycles with denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 30 s and final extension at 72 °C for 10 min. For hippuricase (hipO) gene of Campylobacter jejuni, cycling condition was optimized to initial denaturation at 95 °C for 5 min, followed by 35 cycles with denaturation at 95 °C for 45 s, annealing at 51 °C for 45 s and extension at 72 °C for 45 s and final extension at 72 °C for 7 min and for asperkinase A (askA) gene of C. coli cycling condition was optimized to initial denaturation at 95 °C for 5 min followed by 35 cycles with denaturation at 95 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 30 s and final extension at 72 °C for 7 min. On completion of the reaction, the amplified products were held briefly at 4 °C. Amplification of the PCR products were detected by electrophoresis in 1.5% agarose gel with ethidium bromide (10 μg/ml) in 0.5X TBE buffer (Sigma, USA) at 100 V for 40 min and documented in G: BOXF3 (SynGene, USA).

In vitro Antibiotic sensitivity pattern

The antimicrobial susceptibility testing of the Campylobacter isolates was performed to determine their resistance or sensitivity pattern using the disc diffusion method as originally described by William Bauer et al. (1966) [10] , with modifications according to recent Clinical and Laboratory Standards Institute (CLSI, 2023) guidelines. in vitro antibiotic susceptibility testing was carried out using commercially available antibiotic discs. The isolates were tested against commonly used antimicrobial agents including Ciprofloxacin (5 µg), Chloramphenicol (30 µg), Nalidixic acid (30 µg), Erythromycin (15 µg), Gentamicin (10 µg), Streptomycin (30 µg), and Tetracycline (30 µg). Pure isolates were inoculated into nutrient broth and incubated at 37 °C for 18-24 hours under microaerophilic conditions to obtain fresh bacterial growth. The broth cultures were adjusted to match 0.5 McFarland turbidity standard to ensure uniform inoculum density. A sterile cotton swab was then used to evenly spread the bacterial suspension on the surface of Mueller-Hinton agar plates supplemented with 5% sheep blood (HiMedia), which is recommended for susceptibility testing of Campylobacter species.After inoculation, the plates were allowed to dry for a few minutes, and antibiotic discs were carefully placed on the agar surface using sterile forceps within 15 minutes of inoculation. The plates were then incubated at 37 °C for 24 hours under microaerophilic conditions (5% O₂, 10% CO₂, and 85% N₂). Following incubation, the diameter of the zones of inhibition around each antibiotic disc was measured in millimeters using a ruler or caliper. The results were interpreted as Sensitive (S), Intermediate (I), or Resistant (R) according to the interpretative criteria recommended by Clinical and Laboratory Standards Institute (CLSI, 2023) or European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2024) guidelines.

Results and Discussion Prevalence of Campylobacter spp.

In the present study, an overall 4% prevalence of Campylobacter spp. was observed among the 200 samples examined. All the isolates were obtained from diarrhoeal faecal samples, while none of the samples collected from healthy cattle and calves were found positive for Campylobacter. According to the sample-wise analysis, the prevalence of Campylobacter was 8% in diarrhoeal faecal samples of cattle and 8% in diarrhoeal faecal samples of calves, whereas the prevalence in healthy cattle and calves was 0%. Species identification revealed that the prevalence of Campylobacter jejuni was 87.5%, while C. coli accounted for 12.5% of the isolates. This observation indicates that C. jejuni is the predominant species associated with bovine Campylobacteriosis, which is consistent with global reports that identify C. jejuni as the most frequently isolated species from animals and humans (Man et al., 2021; EFSA & ECDC, 2023) [1,3] .

The findings of the present study are comparable with the results reported by Chatur (2014) [13] , who observed 4% prevalence of Campylobacter in cattle samples. However, several other studies have reported higher prevalence rates. For example, Brown et al. (2004) [11] , Sato et al. (2004) [33] , Baserisalehi et al. (2005) [8] , Saito et al. (2005) [30] , Fernandez and Hitschfeld (2009) [18] , Ghane et al. (2010) [19] , Vilar et al. (2010) [38] and Eghbalia et al. (2011) [15] reported prevalence rates of 30%, 27.9%, 28.57%, 23.1%, 21.3%, 28.57%, 20.5% and 20%, respectively, which were considerably higher than those observed in the present study. Similarly, Tesfaye et al. (2005) [36] , Salihu et al. (2009) [32] and Uaboi-Egbenni et al. (2012) [37] reported 12.7%, 12.9% and 19.3% prevalence, respectively, which were also higher than the prevalence observed in the present study. The variation in prevalence rates among different studies may be attributed to differences in geographical location, sample size, diagnostic methods, animal management practices, environmental conditions, and seasonal variations (WHO, 2023) [2] . In contrast to the present findings, Rajendran et al. (2012) [28] did not detect Campylobacter in any of the cattle faecal samples examined. According to sample-wise analysis, Singh et al. (2011) [34] reported 6.25% prevalence, which was lower than that observed in the present study. However, Hannon et al. (2009) [20] and Ramonaite et al. (2013) [29] reported 76% and 78.6% prevalence, respectively, in cattle faecal samples, which were substantially higher than the results obtained in the present investigation. Recent surveillance reports also indicate that livestock, particularly cattle and poultry, serve as important reservoirs for Campylobacter species and play a significant role in the transmission of infection to humans through food and environmental contamination (EFSA & ECDC, 2023) [3] . In the present study, all Campylobacter isolates showed 100% resistance to Nalidixic acid. Resistance to Ciprofloxacin and Tetracycline was observed in 62.5% and 25% of isolates, respectively. On the other hand, all isolates were found to be sensitive to Chloramphenicol, Erythromycin, Gentamicin and Streptomycin sulphate. Six isolates (75%) were sensitive to Tetracycline, while three isolates (37.5%) were sensitive to Ciprofloxacin. Among the tested antibiotics, Chloramphenicol, Erythromycin, Gentamicin and Streptomycin sulphate showed the highest sensitivity, indicating their potential effectiveness against Campylobacter isolates in the present study. Similar findings have been reported by Afset and Maeland (2001) [7] , Sato et al. (2004) [33] and Eghbalia et al. (2011) [15] , who also observed high sensitivity of Campylobacter isolates to macrolides and aminoglycosides. With respect to Ciprofloxacin resistance, the findings of the present study are comparable with the observations of Elisabetta et al. (2014) [16] , who reported 62.76% resistance to Ciprofloxacin among Campylobacter isolates. However, other studies such as Parisi et al. (2007) [27] , Englen et al. (2007) [17] and Wonki et al. (2005) [39] reported 29%, 2.5% and 5.1% resistance, respectively, which were much lower than those observed in the present study. Recent global reports have highlighted an increasing trend of antimicrobial resistance in Campylobacter species, particularly against fluoroquinolones and tetracyclines. This rising resistance is mainly attributed to the extensive use of antibiotics in veterinary practice and livestock production, which may contribute to the emergence of resistant strains that can be transmitted to humans through the food chain (WHO, 2023; Tang et al., 2022) [2,4] . Therefore, continuous monitoring of antimicrobial resistance patterns in Campylobacter isolates from food-producing animals is essential to ensure effective treatment strategies and to minimize the risk of transmission of resistant strains to humans.

References

1. Man SM, de Steenhuijsen Piters WAA, Bogaert D. The microbiology of Campylobacter infection. Nat Rev Microbiol. 2021;19:720-736.
2. World Health Organization (WHO). Campylobacter (fact sheet). Geneva: WHO; 2023.
3. European Food Safety Authority, European Centre for Disease Prevention and Control. The European Union One Health 2022 zoonoses report. EFSA J. 2023;21(12).
4. Tang Y, Sahin O, Pavlovic N, LeJeune J, Carlson J. Antimicrobial resistance in Campylobacter: global trends and mechanisms. Front Microbiol. 2022;13.
5. Centers for Disease Control and Prevention (CDC). Campylobacter infection surveillance report. Atlanta: CDC; 2022.
6. Acik MN, Karahan M, Ongor H, Karagulle B, Cetinkaya B. Investigation of cytolethal distending toxin production and virulence genes in Campylobacter isolates from cattle. Rev Med Vet. 2013;164(5):272277.
7. Afset JE, Maeland JA. Erythromycin and ciprofloxacin resistant Campylobacter jejuni. J Norw Med Assoc. 2001;121:2152-2154.
8. Campylobacter spp. isolated from environmental samples. Indian J Med Microbiol. 2005;23(1):48-51.
9. Baserisalehi M, Bahador N, Kapadnis B. Isolation and characterization of Campylobacter spp. from domestic animal and poultry in south of Iran. Pak J Biol Sci. 2007;10(9):1519-1524.
10. Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol. 1966;45(4):493-496.
11. Brown PE, Christensen OF, Clough HE, Diggle PJ, Hart CA, Hazel S, et al. Frequency and spatial distribution of environmental Campylobacter spp. Appl Environ Microbiol. 2004;70:6501-6511.
12. Centers for Disease Control and Prevention (CDC). CDC estimates of foodborne illness in the United States. Atlanta: CDC; 2011. Available from: http://www.cdc.gov/foodborneburden/2011-foodborneestimates.html
13. Chatur YA. Molecular characterization, typing and antibiotic resistant pattern of Campylobacter spp. isolated from animal and human sources [M.V.Sc. thesis]. Anand (IN): Anand Agricultural University; 2014.
14. Dabiri H, Aghamohammad S, Goudarzi H, Noori M, Hedayati MA, Ghoreyshiamiri SM. Prevalence and antibiotic susceptibility of Campylobacter species isolated from chicken and beef meat. Int J Enteric Pathog. 2014;2(2):17087.
15. Eghbalia M, Baserisalehi M, Bahador N. Antimicrobial susceptibility of thermophilic Campylobacter isolated from environmental samples in Tonekabon. Int J Mol Clin Microbiol. 2011;1:21-24.
16. Elisabetta DG, Gabriella DS, Katiuscia Z, Alessandra A, Lorena S, Giuliano G, et al. Characterization of antimicrobial resistance patterns and detection of virulence genes in Campylobacter isolates in Italy. Sensors. 2014;14:3308-3322.
17. Englen MD, Hill AE, Dargatz DA, Ladely SR, Fedorka-Cray PJ. Prevalence and antimicrobial resistance of Campylobacter in US dairy cattle. J Appl Microbiol. 2007;102:1570-1577.
18. Fernandez H, Hitschfeld M. Occurrence of Campylobacter jejuni and Campylobacter coli and their biotypes in beef and dairy cattle from the south of Chile. Braz J Microbiol. 2009;40:450-454.
19. Ghane M, Bahador N, Baserisalehi M. Isolation, identification and characterization of Campylobacter spp. isolates from environmental samples in North Iran. Nat Environ Pollut Technol. 2010;9(4):823-828.
20. Hannon SJ, Allan B, Waldner C, Russell ML, Potter A, Babiuk LA, et al. Prevalence and risk factor investigation of Campylobacter species in beef cattle faeces from commercial feedlots in Alberta, Canada. Can J Vet Res. 2009;73:275-282.
21. Inglis GD, Kalischuk LD, Busz HW. Colonization of cattle intestines by Campylobacter jejuni and Campylobacter lanienae. Appl Environ Microbiol. 2005;71:5145-5153.
22. Khalifa NO, Afify JS, Nagwa SR. Zoonotic and molecular characterizations of Campylobacter jejuni and Campylobacter coli isolated from beef cattle and children. Glob Vet. 2013;11(5):585-591.
23. Linton D, Lawson AJ, Owen RJ, Stanley J. PCR detection, identification to species level and fingerprinting of Campylobacter jejuni and Campylobacter coli from diarrheic samples. J Clin Microbiol. 1997;35:2568-2572.
24. Linton D, Owen RJ, Stanley J. Rapid identification by PCR of the genus Campylobacter and five species enteropathogenic for man and animals. Res Microbiol. 1996;147:707-718.
25. Moore JE, Corcoran D, Dooley JS. Campylobacter. Vet Res. 2005;36:351-382.
26. On SLW. Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status and future prospects. Appl Microbiol. 2010;90:1-15.
27. Parisi A, Lanzilotta SG, Addante N, Normanno G, Di Modugno G, Dambrosio A, et al. Prevalence, molecular characterization and antimicrobial resistance of thermophilic Campylobacter isolates from cattle and poultry in Italy. Vet Res Commun. 2007;31:113-123.
28. Rajendran P, Babji S, George AT, Rajan DP, Kang G, Ajjampur SS. Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, South India. Indian J Med Microbiol. 2012;30(1):85-88.
29. Ramonaite S, Rokaityte A, Tamuleviciene E, Malakauskas A, Alter T, Malakauskas M. Prevalence, quantitative load and genetic diversity of Campylobacter spp. in dairy cattle herds in Lithuania. Acta Vet Scand. 2013;55:1-6.
30. Saito S, Yatsuyanagi J, Harata S, Ito Y, Shinagawa K, Suzuki N, et al. Campylobacter jejuni from poultry, cattle and human samples in Japan: comparison of serotypes and genotypes. FEMS Immunol Med Microbiol. 2005;45:311-319.
31. Salihu MD, Junaidu AU, Magaji AA, Rabiu ZM. Study of Campylobacter in raw cow milk in Sokoto State, Nigeria. Maxwell Sci Org. 2010.
32. Salihu MD, Abdulkadir JU, Oboegbulem SI, Egwu GO, Magaji AA, Lawal M, et al. Isolation and prevalence of Campylobacter species in cattle from Sokoto State, Nigeria. Vet Ital. 2009;45(4):501-505.
33. Sato K, Bartlett PC, Kaneene JB, Downes FP. Comparison of prevalence and antimicrobial susceptibilities of Campylobacter spp. from dairy herds in Wisconsin. Appl Environ Microbiol. 2004;70(3):1442-1447.
34. Singh H, Rathore RS, Singh S, Singh PC. Comparative analysis of cultural isolation and PCR assay for detection of Campylobacter jejuni. Braz J Microbiol. 2011;42:181-186.
35. Stanley K, Jones K. Campylobacter colonization in poultry: sources and transmission. Anim Health Res Rev. 2003;3:95-105.
36. Tesfaye K, Solomon G, Daniel A. Prevalence of thermotolerant Campylobacter species in food animals in Ethiopia. Ethiop J Health Dev. 2005;19(3):225-229.
37. Uaboi-Egbenni PO, Bessong PO, Samie A, Obi CL. Potentially pathogenic Campylobacter species among farm animals in South Africa. Afr J Microbiol Res. 2012;6(12):2835-2843.
38. Vilar MJ, Garcia-Pena FJ, Perez I, Dieguez FJ, Sanjuan ML, Rodriguez-Otero JL, et al. Presence of Listeria, Arcobacter and Campylobacter spp. in dairy farms in Spain. Berl Munch Tierarztl Wochenschr. 2010;123:5862.