Introduction

Campylobacteriosis, a notable zoonosis, poses significant concerns for public health. The causative agent, Campylobacter, is a Gram-negative bacterium found in diverse environments, such as animal feces, water, and soil. This microorganism can inhabit a broad spectrum of animals, including farm animals, poultry, and wild species, which act as potential sources of human infection. Importantly, substandard hygiene practices in animal husbandry, poultry farming, and environmental management play crucial roles in the spread and proliferation of Campylobacter. On farms, insufficient biosecurity protocols, contaminated food and water supplies, and unhygienic animal living conditions can contribute to the dissemination of Campylobacter within and among animal groups. Furthermore, improper handling of waste and inadequate sanitation in livestock production facilities can result in environmental contamination, thereby increasing the risk of human exposure to the pathogen1,2,3,4. Campylobacter species can be shed in the feces of diseased, recovered, or asymptomatic animals, leading to contamination of the environment, which increases the transmission of infection between herds5,6.

Human campylobacteriosis primarily occurs through the consumption of contaminated food and beverages. Poultry and other meat products, particularly when undercooked or improperly managed, are major sources of infection. However, contaminated water, unpasteurized milk, and even fresh products can also transmit Campylobacter1,6,7. The consequences of Campylobacter infection can range from mild gastrointestinal illness to severe complications. While most cases resolve spontaneously, some individuals, such as young children, elderly individuals, and immunocompromised individuals, are at increased risk of developing severe infections, including Guillain–Barré syndrome and reactive arthritis8,9.

Polymerase chain reaction (PCR) is a powerful molecular technique for detecting and identifying Campylobacter species. 16S rRNA gene sequencing is a widely used method for species-level identification and differentiation10. Furthermore, PCR can be employed to detect the presence of virulence genes, which are crucial determinants of Campylobacter pathogenicity. These genes, such as the hipO, ceuE, and cadF genes, encode proteins involved in various virulence mechanisms, including iron acquisition, adhesion to host cells, and modulation of host immune responses. The presence or absence of these genes can provide valuable insights into the potential virulence and severity of Campylobacter infections10,11,12.

The emergence of antimicrobial resistance in Campylobacter poses a significant threat to public health. The indiscriminate use of antibiotics in both human and animal medicine has contributed to the development of drug-resistant strains, limiting treatment options and increasing the severity of infections13,14,15.

From a one health perspective, this study aimed to isolate Campylobacter from humans, animals and the environment. Then studying the sensitivity of isolates from different hosts to antibiotics. Finally, the genetic similarity of three representative isolates was studied to assess the potential of zoonotic transmission and support disease control strategies.

Methods

Ethical declaration

This study adhered to the ethical guidelines of the “Institutional Review Board” of New Valley University, Egypt (Approval Number: NVREC 0213-20,249). Informed consent was obtained from all farm owners prior to sample collection, ensuring their understanding of the study objectives and procedures.

Study area and design

The study was conducted in the New Valley Governorate, Egypt, from November 2023 to December 2024. This region encompasses a vast area with diverse agricultural practices, including livestock farming (Fig. 1).

Fig. 1
figure 1

Map of New Valley Governorate showing the locations of the study areas in the Governorate.

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Sampling

The total number of samples examined is 573 from animals, humans and the environment. Animal samples were firstly, rectal swabs from cows (n = 62), sheep (n = 75) and goats (n = 70). Secondly, the milk samples were from cows (n = 41), sheep (n = 36) and goats (n = 36). As for the human samples, they included: Firstly, 66 stool samples were collected from patients with intestinal disorders who frequent hospitals and laboratories in the study area. Secondly, 46 hand swabs from farm workers and people in contact with animals or their products. As for the environmental samples, they were as follows: 116 fecal samples, 10 water samples from animal courses and 15 wall swabs from animal residences. The samples were withdrawn according to the Campylobacter isolation protocol. All samples were collected under aseptic conditions, labelled and transferred in an ice box to the microbiology lab, Faculty of veterinary medicine New valley university with minimum delay as possible.

Bacteriological and biochemical analysis of Campylobacter spp.

Campylobacter spp. were isolated according to the ISO 10,272–1 method16. Briefly, samples were enriched in Bolton broth (M1592 Himedia, India) supplemented with selective antibiotics and lysed horse blood, then incubated under microaerophilic conditions at 41.5 ± 1 °C for 48 h. Enriched cultures were streaked onto modified charcoal cefoperazone deoxycholate agar (M887I Himedia, India), and incubated under microaerophilic conditions (e.g., gas pack system) at 41.5 °C for 48 h to isolate presumptive Campylobacter colonies. Biochemical identification was carried out to the suspected colonies.

Molecular identification and characterization of Campylobacter spp.

PCR was performed to the randomly selected samples to perform molecular confirmation through the detection of the 16S rRNA gene for species identification and virulence-associated genes (hipO, ceuE, and cadF) via specific primers (Table 1)17,18,19,20. Genomic DNA was extracted via a QIAamp DNA Mini (catalog no. 51304). Emerald Amp GT PCR master mix (Takara, Code No. RR310A Kit) was used. The PCR products were analyzed via agarose gel electrophoresis and visualized via UV transillumination. The marker for the PCR products was a 100 bp DNA ladder.

Table 1 Oligonucleotide primer sequences.
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Phylogenetic analysis

Phylogenetic analysis was performed on a representative subset of isolates (one animal, one environmental, and one human isolate) on the basis of 16S rRNA gene sequences (Table 1)21. Sequence alignment was performed via CLUSTAL22, and phylogenetic trees were constructed via MEGA623 via the maximum likelihood, neighbor-joining, and maximum parsimony methods.

Antibiotic resistance profile of isolated Campylobacter spp.

Antimicrobial susceptibility testing was performed via the disc diffusion method. The CLSI (2020) and EUCAST (2021) recommended guidelines breakpoints were used for macrolides, while CLSI breakpoints for Enterobacteriaceae were used for other antibiotics24,25. The antimicrobial agents evaluated are listed in Table 2. The multidrug resistance (MDR) index was calculated26.

Table 2 Antibiotics used and the related breakpoints for interpretation of inhibition zone diameters.
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Statistical analysis

Statistical analysis was performed via SPSS, version 25 (IBM Corp., 2013). The data were analyzed via chi-square tests, and the significance level was set at p < 0.05. The data were treated as a complete randomization design according to Steel et al27.

Results

Prevalence

A total of 181 Campylobacter spp. isolates were recovered from 573 examined samples. The prevalence of Campylobacter spp. was at Animal samples level 32.9% in rectal swabs and 25.7% in milk samples. Additionally, it was at Human samples level 74.2% in stool samples and 4.4% in hand swabs. Moreover, at Environmental samples level the prevalence was 25.9% in fecal samples from animal litter, 10% in water samples, and 13.3% in wall swabs, as shown in (Table 3).

Table 3 Prevalence of Campylobacter species in different collected samples.
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Molecular characterization

Ten randomly selected isolates were evaluated by PCR for the presence of 16S rRNA and virulence genes (hipO, ceuE, and cadF). The prevalence of 16S rRNA was 90% (9/10), whereas the prevalence of virulence genes was 77.8% (7/9) for hipO, 22.2% (2/9) for ceuE, and 33.3% (3/9) for cadF, as shown in Table 4 and Fig. 2.

Table 4 Occurrence of virulence genes in 10 randomly selected Campylobacter spp. isolates.
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Fig. 2
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Agarose gel electrophoresis of amplified genes: 16S rRNA gene with a PCR product of 816 bp) (A), hipO gene with a PCR product of 323 bp (B), ceuE gene with a PCR product of 462 bp (C) and the cadF gene with a PCR product of 400 bp (D).

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Antimicrobial resistance

A total of 181 Campylobacter spp. isolates were evaluated for antibiotic susceptibility to 12 commonly used antibiotics in the study area. All the isolates were resistant to clindamycin. In contrast, the highest sensitivity was observed for imipenem and amikacin (100% sensitivity each). The isolates exhibited 96.1% sensitivity to gentamicin, 94.5% sensitivity to doxycycline, and 71.8% sensitivity to ciprofloxacin (Table 5). The mean MAR indices of Campylobacter spp. isolated from animal, environment and human samples were 0.393, 0.481, and 0.416, respectively (Table 6). Notably, the highest MAR index was observed in environmental isolates.

Table 5 Antibiotic sensitivity of animal, environmental, and human Campylobacter spp. isolates.
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Table 6 Mean MAR indices of the tested Campylobacter spp. isolates.
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Phylogenetic analysis

The 16S rRNA genes of three randomly selected isolates have been deposited in GenBank under accession numbers: PQ780749 for fecal isolates, PQ780750 for milk isolates, and PQ780748 for human stool isolates (Figs. 3 and 4). Phylogenetic analysis of sequences revealed high genetic similarity between the three isolates ranging from 99.8 to 100%. Additionally, 99.7–100% similarity was found with many other isolates in GenBank.

Fig. 3
figure 3

Phylogenetic tree of fecal, milk, and human stool Campylobacter spp. isolates based on 16S rRNA gene.

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Fig. 4
figure 4

Phylogenetic analysis of animal, human, and environment. Campylobacter spp. isolates based on 16S rRNA gene sequencing.

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Discussion

Campylobacteriosis is a major global public health concern and the leading cause of bacterial gastroenteritis, affecting millions of people annually and contributing to severe complications such as Guillain–Barré syndrome and reactive arthritis. Its burden extends beyond human health, causing infections in livestock, contaminating food and water sources, and imposing significant economic losses on healthcare systems, agriculture, and the food industry5,28,29.

The present results revealed that the prevalence of Campylobacter spp. in animal rectal swabs was 32.9%. These results were similar to those obtained in Kenya (30.9%)30. However, it was higher than the results of previous studies in Assiut, Egypt and Kenya, where the prevalence rates were 20.1% and 15.7%, respectively31,32. In contrast, it was lower than the prevalence reported in the United States, Bahama, and Mexico (80.7%)33, and 59.2% in northern Spain34.

The prevalence of Campylobacter spp. in milk was 25.7%. These results were similar to those reported in Italy (24.9%)35. However, lower prevalences of 12.6% and 2% were reported in Iraq and Egypt, respectively36,37. On the other hand, a higher prevalence rate was reported in Iran (65.8%)38.

The prevalence of Campylobacter in stool samples was 74.2%, which is similar to findings previously -reported in Egypt (76.9%)39. However, it was higher than other results in Egypt and Iran, where the prevalence rates were 66.6% and 8.4%, respectively6,8. In contrast, it is lower than the prevalence reported in other Egyptian studies (90.9%)40.

The results revealed that the prevalence of Campylobacter in hand swabs was 4.4%, which was similar to findings reported among handlers of raw poultry or meat9. However, this value was higher than that reported in a previous study in Ethiopia (1.8%)41. In contrast, it was lower than the prevalence (11.9%) reported in China42, whereas another study could not isolate Campylobacter from examined hand swab samples in Egypt43. There was a highly significant difference between the human stool and hand swabs (p < 0.001).

The prevalence of Campylobacter spp. in environmental fecal samples was 25.9%. These results were very close to those of two studies in Canada and Bangladesh (27% and 26.7%, respectively)29,44. However, it is lower than the prevalence reported in other studies in Sweden (78%) 7.

The prevalence of Campylobacter spp. in the water samples was 10%. These findings are similar to those reported in Malaysia (11.1%)45; however, these findings are lower than the prevalence reported in another study in South Africa (21.7%)3. Notably, no Campylobacter was detected in water samples in Bangladesh5. Moreover, the prevalence of Campylobacter spp. in wall swabs was 13.3%. This finding was similar to the prevalence of 15% reported in Canada46, and a previous study reported a prevalence of 15% in poultry farm environments47. However, it was lower than the prevalence reported in Malaysia (33.3%)45.

PCR is a valuable tool for the molecular confirmation of Campylobacter spp., including species identification and the detection of virulence genes10,11. In the present study, 90% of the randomly selected isolates were positive for the 16S rRNA gene. Among the positive isolates, 77.8% (7/9) harbored the hipO gene, 22.2% (2/9) harbored the ceuE gene, and 33.3% (3/9) harbored the cadF gene. These findings are consistent with previous studies reporting the presence of the hipO gene in 75% of isolates48. However, other studies have reported higher detection rates for the ceuE, hipO, and cadF genes49,50. The distribution of genes reflects both species identity and isolation source and the differences between them are always statistically significant. In general, it can be said that The variation in the distribution of these genes may be attributed to factors such as sample sources (e.g., animal. Environment, food and human) Campylobacter serotype, geographical location and genetic mutations12,48,49.

The high genetic similarity between the three isolates implies that cattle are an important reservoir for human campylobacteriosis. Additionally, these results showed similarities ranging from 99.7 to 100% with many isolates of human and animal origin, such as human stool isolates from the United Kingdom, human stool isolates from a child in Brussels, Belgium, and broiler cloacal swabs from Sweden (Accession Nos. AL111168, HE978252 and AF550627). The phylogenetic relatedness of C. jejuni isolates from animals and humans further supports cross-species transmission of this pathogen. These findings are in agreement with those of previous studies that documented zoonotic transmission of Campylobacter51,52.

The present data revealed that Campylobacter isolates presented 100% resistance to clindamycin, followed by high resistance rates to erythromycin, amoxicillin-clavulanic acid (85.64%), and azithromycin (70.2%). These findings are consistent with those of several previous studies15. However, they contradict other studies that reported higher susceptibility to these antibiotics53. Conversely, the highest sensitivity levels were observed for imipenem, amikacin (100%), gentamicin (96.1%), doxycycline (94.5%), and ciprofloxacin (71.8%). These results align with other studies that reported high sensitivity to imipenem, amikacin, and gentamicin53. Our results revealed that 90.1% of the Campylobacter spp. isolates exhibited multidrug resistance (MDR), defined as resistance to three or more classes of antibiotics. This finding is consistent with other studies that reported high levels of MDR in Campylobacter32. In fact, the widespread prevalence of multidrug resistant Campylobacter in the food chain, human, animal or environment has a significant impact on both public health and veterinary in terms of the difficulty of treatment and the increased cost. Consequently, effective strategies to fight antibiotic resistance include responsible antibiotic use and the development of alternative treatments are vital13,54.

Our data revealed that the MAR indices of Campylobacter spp. isolated from animal, environmental and human samples were 0.393, 0.481, and 0.416, respectively. These findings are consistent with previous studies that reported mean MDR indices of 0.49 for animal isolates55, 0.4 for environmental isolates and 0.3–0.4 for human isolates56. Notably, the mean MDR index was highest for the environmental isolates, suggesting a relatively high level of antibiotic resistance among Campylobacter strains from the environment. Several factors might explain the high antibiotic resistance of environmental isolates, including exposure to antibiotics in the environment through pollution, horizontal gene transfer between bacteria, natural selection due to constant exposure to low levels of antibiotics, and the presence of diverse microbial communities that act as reservoirs for resistance genes14,57.

Conclusions

This study demonstrated a significantly high prevalence of MDR Campylobacter spp. in various samples. Phylogenetic analysis revealed a high degree of genetic similarity among isolates from different sources, including animal, environmental, and human samples. This finding underscores the potential for zoonotic transmission of Campylobacter spp. between animals, the environment, and humans. Future studies should focus on comparative genomics to identify zoonotic markers and assess the role of agricultural practices in the evolution of C. jejuni. Additionally, implementing measures to restrict the use of antibiotics as growth promoters and for prophylaxis on animal farms is crucial to combat antimicrobial resistance. Finally, mitigating the spread of diseases depends critically on a One Health strategy that combines environmental, human, and animal monitoring. Additionally, educating the public on safe food handling practices is essential to prevent human infection.