Molecular characterization of virulence factors and antimicrobial susceptibility of thermophilic Campylobacter species associated with human clinical infections in Rwanda

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In Rwanda, limited data exist on their molecular characteristics and antimicrobial resistance (AMR) profiles. This study investigated the virulence gene profiles and antimicrobial susceptibility of thermophilic Campylobacter species isolated from human clinical infections in northern Rwanda. Methods A cross-sectional study was conducted between March and July 2025 among 200 symptomatic patients at Ruhengeri Hospital. Stool samples were cultured under microaerophilic conditions, and presumptive Campylobacter isolates were confirmed by PCR targeting the 16S rRNA and mapA genes. Virulence genes ( flaA , cadF , cdtA , cdtB , cdtC ) were characterized by PCR. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disc diffusion method against ciprofloxacin, erythromycin, tetracycline, gentamicin, and chloramphenicol following EUCAST guidelines. Results Sixteen (8%) thermophilic Campylobacter isolates were confirmed, predominantly C. jejuni (93.8%). All isolates harbored flaA and cadF genes, while cdtA and cdtB were present in 93.8% and cdtC in 81.3%. All isolates (100%) were resistant to erythromycin, 81.3% to ciprofloxacin (resistant/intermediate), and 75.0% to tetracycline, while complete sensitivity to chloramphenicol and 87.5% sensitivity to gentamicin were observed. Over half (56.3%) of isolates exhibited multidrug resistance (MDR), primarily to erythromycin, ciprofloxacin, and tetracycline. Conclusion The study reveals high virulence gene prevalence and alarming resistance rates among Campylobacter isolates from clinical infections in Rwanda. The universal resistance to erythromycin and high rates of ciprofloxacin and tetracycline resistance highlight the urgent need for enhanced AMR surveillance, rational antibiotic use, and One Health-based control strategies. The retained susceptibility to chloramphenicol and gentamicin offers limited but valuable therapeutic alternatives. Campylobacter jejuni antimicrobial resistance virulence genes Rwanda multidrug resistance Introduction Campylobacter is one of the leading causes of bacterial gastroenteritis, being associated with an estimated 96 million cases of foodborne illnesses annually [ 1 ]. Within this genus, C. jejuni and C. coli are the most frequently implicated in human infections [ 1 ]. The main reservoir of thermophilic Campylobacter is poultry, but other livestock including pigs and cattle have been implicated in transmission [ 2 ]. Human infection typically occurs via the consumption of contaminated foods, most notably undercooked poultry products as well as contaminated water or unpasteurized dairy products [ 2 ], [ 3 ]. Campylobacter infections are often self-limiting, but treatment is required in severe or prolonged infections and drugs of choice include macrolides and fluoroquinolones [ 4 ], although, tetracycline (TET), chloramphenicol (CHL) or gentamicin (GEN) can be used as alternatives [ 4 ], [ 5 ]. However, the rise of antimicrobial resistance (AMR) in Campylobacter poses a significant global public health concern [ 2 ], [ 5 ]. For instance, resistance rates to fluoroquinolones exceeding 60% have been reported in some parts of Africa and South America [ 5 ]. Resistance to macrolides, though lower, has reached 30% in some studies [ 6 ], [ 7 ]. This trend portends serious treatment challenges in the not-so-distant future with possible adverse outcomes and increased treatment costs particularly where there is multi-drug-resistance. Human campylobacteriosis is often complicated by the presence of various virulence genes that contribute to pathogenicity. Key genes include the cytolethal distending toxin (CDT), whose three distinct subunits cdt A, cdt B, & cdt C are associated with DNA damage, inflammation, and cellular dysfunction [ 8 ] The fla A gene, encoding the flagella protein, is crucial for motility and intestinal colonization [ 9 ] while cad F gene is essential for bacterial adhesion to epithelial cells, promoting invasion and infection establishment [ 10 ]. In Africa, due to inadequate surveillance systems, and limited laboratory capacity for isolation and characterization, there is a general underreporting on the burden of Campylobacter infections [ 11 ]. In Rwanda in particular, only two studies have ever described the burden of Campylobacter in humans; one among under-five children, that looked at the causes of diarrhea, reporting a prevalence of 15% [ 12 ], and another conducted recently in capital city (Kigali), described the burden of Campylobacter in the general urban population, reporting a prevalence of 7% [ 13 ]. This picture highlights the paucity of data on Campylobacter in our setting. Such gaps hinder our understanding of local disease epidemiology and how to optimally design and implement effective surveillance systems. Furthermore, inadequate data complicates efforts to control the spread of AMR through evidence-based antimicrobial stewardship initiatives. The current study, therefore, explored the virulence gene profiles of thermophilic Campylobacter species as well as the antimicrobial susceptibility patterns from human clinical isolates from another region of the country. Material and methods Study design and catchment area This was a cross-sectional study conducted between March and July 2025. Stool samples were obtained from symptomatic patients presenting at Ruhengeri hospital, a level-2 teaching, and referral health facility located in Musanze district. This hospital is the largest healthcare facility within the North and Western Provinces of Rwanda, serving a catchment area in excess of two million people (National Institute of Statistics of Rwanda, 2022). The hospital receives patients from all the neighboring districts such as Burera, Gakenke, Nyabihu, Rubavu and as far as parts of Eastern Democratic Republic of Congo. Study population and enrollment The study targeted patients of all ages presenting with gastrointestinal symptoms at the hospital. Research assistants introduced the study to prospective participants after which they requested informed consent (or parental assent in case of children). Individuals who declined to give consent were excluded from further involvement. Sample collection and handling Human stool samples were collected from patients presenting with gastrointestinal (± systemic) symptoms such as diarrhea, abdominal pain/cramping, nausea/vomiting, fever, malaise, or fatigue. Clinical, laboratory and socio-demographic data were recorded using a structured questionnaire specifically developed for the study (see supplementary file 1). Participants received clear instructions on how to collect between 2–5 gram of stool in sterile leak-proof containers. Samples were immediately delivered at the hospital laboratory where they were labeled with a unique study code and transported to the Microbiology Laboratory of the Institut d’Enseignement Supérieur de Ruhengeri (INES-Ruhengeri) for further processing. Isolation and phenotypic identification of Campylobacter spp. For each sample, approximately 1 gram of stool was enriched in Preston Broth supplemented with Campylobacter Growth Supplement SR0232E added to modified Preston Campylobacter Selective Supplement SR0204E and 5% defibrinated sheep blood SR0051B. Inoculated tubes were placed in an anaerobic jar and incubated at 37°C for 24 hours under microaerophilic conditions (5% O 2 , 10% CO 2 , 85% N 2 ) generated by CampyGen sachets. After incubation for 24 hours, a loopful of the enrichment broth was streaked onto modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) supplemented with SR155E. All plates were then incubated at 37°C for 48 hours under the same conditions as previously described [ 13 ]. All the above reagents were sourced from Oxoid Ltd, Basingstoke, England, United Kingdom. Presumptive Campylobacter colonies were sub-cultured onto 5% sheep blood agar plates and incubated under identical microaerophilic conditions at 37°C for an additional 48 hours. Colonies from the blood agar were then examined microscopically using Gram staining to confirm characteristic morphology (gram-negative, curved, or spiral rods). Subsequently, these isolates were subjected to standard catalase and oxidase biochemical tests. Only isolates displaying typical morphology and positive reactions for both tests were retained for further molecular characterization. These isolates were preserved in Muller Hinton broth supplemented with 25% glycerol and stored at -20°C as previously described [ 14 ], with the longest storage sample lasting approximately 1 month. Molecular methods: DNA extraction, Campylobacter confirmation, and characterization of virulence genes Prior to DNA extraction, isolates in storage were brought back into life through sub-culturing on 5% blood agar plates. Genomic DNA was extracted out of revived colonies by utilizing the Qiagen Dneasy Blood and Tissue kit (Qiagen, Hilden, Germany) as per manufacturer’s directions. A concentration and purity of extracted DNA was measured by a Qubit fluorometer (Thermo Fisher Scientific, USA), and DNA samples were stored at -20 o C until use. The presence of Campylobacter species was identified by use of the 16S rRNA gene and the genus identified by map A gene which confirms C.jejuni. Other thermophilic Campylobacter spp. were seen as isolates positive for the 16S rRNA gene but negative in map A gene. For virulence characterization, the genes, fla A, cad F, and the CDT genes were targeted. Each PCR mixture had a total volume 25µL. This master mix consisted of 3µL of 10x PCR buffer, 0.3µL of Taq thermostable DNA polymerase, 1.2µL of MgCl 2 , 2.5µL of dNTPs, 2 µL of template DNA, 0.5µL of forward primer (10 pmol/L) and 0.5µL of reverse primer (10 pmol/L). The volume of the last one was brought up to 25 mL with 15 µL of RNAse-free water as previously described [ 15 ]. Gene amplification was done using a thermal cycler with one cycle of denaturation stage at 95 o C for 10 min and 35 cycles which contained denaturation (30 sec) at 95 o C, anneal (90 sec) at 59 o C and extension (60 sec) at 72 o C. Samples were subjected to the final extension process (72 o C, 5 minutes) and it established that the production was complete of all PCR products. The reactions were subsequently stored in 4 o C until further determination [ 16 ]. Electrophoresis was performed by running the PCR products in a 1.5% (w/v) agarose gel prepared in 1x Tris-borate-EDTA (TBE) buffer and stained with ethidium bromide (0.5 g/mL). The amplicon bands were also compared to 100 bp DNA ladder (Thermoscientific) and observed under a UV transilluminator. Bands for the 16S rRNA (for Campylobacter genus) and map A gene (for C. jejuni) were characterized by 857 bp and 589 bp respectively. For virulence genes fla A, cad F, cdt A, cdt B and cdt C, bands were observed at 1200 bp, 500 bp, 370 bp, 620 and 182 bp respectively, as previously described [ 16 ]. Antimicrobial Susceptibility Testing (AST) The Kirby-Bauer disc diffusion method was used to establish antimicrobial resistance profiles as per recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [ 17 ]. Briefly, pure Campylobacter colonies were revived on blood agar. An inoculum was prepared through the suspension of colonies in normal saline to give a turbidity of 0.5 McFarland standard. The bacterial suspension was streaked on Muller Hinton Agar (MHA) with 5% sheep blood using sterile glass spreaders and plates were left to dry for 15 min. Antibiotic disks; (CIP, 5 µg), (ERY, 15 µg), (TET, 30 µg), (GEN, 10 µg), and (CHL, 30 µg) were then placed on the surface of the agar plates using sterile forceps, making sure to maintain a minimum distance of 24 mm between discs. The plates were subsequently incubated under microaerophilic conditions at 37 o C for 48 hours and inhibition zone diameters measured (in millimeters) using a ruler. EUCAST epidemiologic cutoffs (ECOFFs) were used to make interpretations as sensitive (S), intermediate (I ), or Resistant (R) [ 17 ]. Multidrug resistance (MDR) was defined by resistance to at least three antibiotics from different classes [ 17 ]. Data analysis Data entry and analysis were done using Statistical Package for Social Sciences (SPSS) (version 21.0, IBM Corp., Armonk, NY, USA). Descriptive statistics (including prevalence, proportions, and frequencies) were computed. Ethical considerations Ethics approval was obtained from the Research Ethics Committee of INES-Ruhengeri Institute of Applied Sciences as well as Ruhengeri district hospital (Ref 329/RL2TH/DG/2025). All participants provided written informed consent (or parental assent, in the case of minors). All patient information was coded and securely stored on a password-protected computer to ensure confidentiality. Results Socio-demographic characteristics of patients Over the 5-month period (March-July 2025), stool samples from 200 symptomatic participants were collected and analyzed. Majority of participants were female 145 (72.5%) with children < 5 year comprising 26.5% of the sample (n = 53). Approximately one quarter (24.5%) of participants had no formal education while 39.5% had completed primary education and 36% had completed secondary school education or higher. Prevalence of thermophilic Campylobacter species Culture and biochemical tests presumptively identified 18 (9%) Campylobacter isolates of which 16 (8%) were positively confirmed by molecular methods. Of these, 15/16 (93.8%) were confirmed to be C. jejuni while one isolate was not conclusively identified. Virulence gene characterization of thermophilic Campylobacter isolates Molecular screening of virulence genes from the 16 isolates revealed the presence of fla A and cad F genes in all isolates (100%). In contrast, cdt A and cdt C genes were present in 93.6% of isolates while presence of cdt B was demonstrated in only 81.3% of isolates (Table 1 ) Table 1 Distribution of virulence genes in thermophilic Campylobacter spp. from human stool samples from Rwanda (n = 16) Virulence gene No. positive Prevalence (%) fla A 16 100 cad F 16 100 cdt A 15 93.8% cdt B 15 93.8% cdt C 13 81.3% Antimicrobial susceptibility profiles of thermophilic Campylobacter isolates. All isolates (100%) were resistant to erythromycin. Taking both R & I categories, resistance to ciprofloxacin was also high at 81.3% while resistance to tetracycline was 75.0%. In contrast, all isolates were sensitive to chloramphenicol while sensitivity to gentamycin was 87.5%. Details are shown in Table 2 . Table 2 Antimicrobial susceptibility profiles of thermophilic Campylobacter isolates from Rwanda (n = 16) Number of isolates Antibiotic tested Sensitive (%) Intermediate (%) Resistant (%) Ciprofloxacin 3 (18.7) 8 (50) 5 (31.25) Erythromycin 0 0 16 (100) Chloramphenicol 16 (100) 0 0 Gentamicin 14 (87.5) 2 (12.5) 0 Tetracycline 4 (25) 2 (12.5) 10 (62.5) Of the 16 Campylobacter isolates tested against 5 antibiotics above, 2 isolates demonstrated resistance to one antibiotic each, while 5 isolates showed resistance to two antibiotics each. However, 9 isolates (56.3%) showed resistance to three or more antibiotics and were classified as MDR. The predominant MDR pattern consisted of resistance to ciprofloxacin, erythromycin, and tetracycline. Discussion This study provides critical insights into the molecular characteristics and antimicrobial resistance patterns of thermophilic Campylobacter species isolated from human clinical infections in Rwanda. The prevalence of 8% observed in this study aligns with the 7% reported in the recent study from Kigali [ 13 ], and remains lower than the 15% prevalence documented among under-five children reported ten years back [ 12 ]. This variation may reflect differences in study populations, geographic locations, or detection methods. The predominance of C. jejuni (93.8%) among confirmed isolates in our sample is consistent with global epidemiological patterns, where this species accounts for the majority of human campylobacteriosis cases [ 1 , 2 ]. Distribution of virulence genes and pathogenicity implications The universal presence (100%) of both fla A and cad F genes among all isolates is particularly noteworthy and consistent with findings from other studies from Asia [ 14 ] and South America [ 18 ]. The fla A gene, encoding the flagellin protein, is essential for bacterial motility and represents a critical determinant for intestinal colonization [ 9 ]. Without functional flagella, Campylobacter would be unable to navigate through the mucus layer to reach epithelial cells, making this gene indispensable for establishing infection. Similarly, the ubiquitous presence of cad F underscores its fundamental role in pathogenesis. This gene encodes an outer membrane protein that mediates bacterial adhesion to fibronectin on host epithelial cells, representing the initial step in colonization and subsequent invasion [ 10 ]. The universal presence of these two genes suggests that all isolates in this study possessed the basic molecular machinery necessary for successful host colonization and infection establishment. The CDT gene complex showed variable distribution, with cdt A and cdt B present in 93.8% of isolates, while cdt C was detected in only 81.3%. This differential distribution is intriguing and has important pathogenic implications. The cytolethal distending toxin represents a well-known toxin produced by C. jejuni and functions as a critical virulence factor. CdtB, the active subunit, possesses DNase activity that causes DNA damage, cell cycle arrest at the G2/M phase, and ultimately induces apoptosis in host cells [ 10 ]. Studies have demonstrated that CDT contributes to both the invasion of host cells and the prolongation of clinical symptoms [ 8 ]. The toxin operates as what has been termed a "tri-perditious toxin," impairing host defenses through three mechanisms: disrupting epithelial barriers, suppressing acquired immunity, and promoting pro-inflammatory responses [ 8 ]. The lower prevalence of cdt C (81.3%) compared to cdt A and cdt B is particularly significant. While CdtA and CdtC function as binding subunits that facilitate the delivery of the toxic CdtB subunit into host cells, all three subunits are typically required for full toxin activity. The absence of cdt C in approximately 19% of isolates suggests these strains may have reduced cytotoxic capacity. However, some studies have shown that CDT variants with incomplete subunit profiles can still exhibit pathogenic activity, albeit at reduced levels. This genetic heterogeneity among clinical isolates may partially explain the spectrum of disease severity observed in campylobacteriosis, ranging from mild, self-limiting diarrhea to severe invasive disease. Interestingly, while CDT genes are considered important virulence factors, some studies have found no direct association between cdt B presence and invasive bloodstream infections versus gastroenteritis alone [ 16 ]. This suggests that disease severity and clinical outcomes depend on complex host-pathogen interactions beyond the mere presence of individual virulence genes. Host immune status, bacterial load, concurrent gut microbiota, and other environmental factors likely modulate the clinical manifestations of Campylobacter infections. Antimicrobial resistance: an alarming trend The antimicrobial susceptibility profile revealed in this study is deeply concerning and reflects the broader antimicrobial resistance capabilities acquired by Campylobacter in Africa as well as globally. The universal resistance to erythromycin (100%) is particularly alarming given that macrolides represent first-line treatment options for severe campylobacteriosis [ 4 ]. This finding diverges dramatically from the expected resistance rates and exceeds even the concerning 30–40% macrolide resistance previously reported in some African studies [ 6 , 20 ]. The complete resistance observed in our study suggests extensive selective pressure, potentially driven by their widespread use in human infections as well as animal agriculture in Rwanda. Likewise, the high resistance to ciprofloxacin, observed in 81.3% of isolates (combining resistant and intermediate categories), is equally troubling. Fluoroquinolones serve as alternative first-line agents for treating campylobacteriosis, particularly in adults where macrolides may be contraindicated [ 4 ]. In 2017, the WHO classified fluoroquinolone-resistant Campylobacter among priority pathogens against which new antibiotics were urgently needed. This was however reversed in the 2024 update following expert consensus [ 20 ]. Despite this change, the high rate of resistance documented in this study remains clinically significant as it mirrors trends observed across sub-Saharan Africa and South America, where resistance rates exceeding 60% have been reported [ 6 , 21 , 22 ]. Tetracycline resistance was observed in 75% of isolates (combining resistant and intermediate categories), and is consistent with resistance patterns recently reported elsewhere in Rwanda [ 13 ] and other African countries [ 21 ]. Tetracyclines are commonly used in veterinary medicine, and this high resistance likely reflects the agricultural use of these antimicrobials in livestock production, creating selective pressure that favors the spread of resistance. Encouragingly, all isolates remained sensitive to chloramphenicol, and 87.5% showed sensitivity to gentamicin. This preservation of susceptibility to these alternatives provides critical therapeutic options for managing severe infections. Chloramphenicol, despite concerns about potential side effects, remains effective and could serve as a valuable treatment option in cases where resistance to fluoroquinolones and macrolides precludes their use. The multidrug resistance (MDR) profile is particularly worrisome, with 56.3% of isolates showing resistance to three or more antimicrobials from different classes. The predominant MDR pattern involving ciprofloxacin, erythromycin, and tetracycline effectively eliminates the most commonly prescribed first-line treatment options. This pattern mirrors findings from the recent Kigali study [ 13 ] and highlights the rapid evolution of resistance in Rwandan Campylobacter populations. The high MDR prevalence suggests potential clonal spread of resistant strains or widespread horizontal gene transfer of resistance determinants within the bacterial population. Incidentally, the phenomenon of MDR Campylobacter has also been reported in other continents including Europe [ 22 ], South America [ 23 ]. Several factors may contribute to the elevated levels of AMR observed in this study. There is no doubt that indiscriminate use of antibiotics in human medicine, coupled with their prophylactic and growth-promoting application in animal agriculture, creates sustained selective pressure. Furthermore, in Rwanda, as in many African countries, weak regulatory frameworks for antibiotic stewardship, availability of over-the-counter antimicrobials without prescription, and limited diagnostic capacity leading to empirical treatments all contribute to the selection and spread of drug resistance. Additionally, we believe that the close proximity of humans to livestock in many rural Rwandan communities facilitates zoonotic transmission of resistant strains and vice versa. Study context and public health implications The location of this study in Musanze district, serving populations from North and Western Rwanda and parts of Eastern Congo, provides insights into Campylobacter epidemiology beyond urban centers like Kigali. The consistency of high resistance rates between this study and the Kigali study [ 13 ] suggests that AMR in Campylobacter is geographically widespread in Rwanda, rather than being confined to urban centers. This geographic distribution has important implications for national treatment guidelines and antimicrobial stewardship policies. Furthermore, the presence of highly virulent, multidrug-resistant Campylobacter strains in clinical settings represents a significant threat to public health. More worrying is that patients infected with MDR strains face limited therapeutic options, increased risk of prolonged illness & complications, higher healthcare costs or even death. The situation is particularly concerning for vulnerable populations including young children, elderly individuals, and immunocompromised patients who may easily develop severe or invasive disease. Study limitations Several limitations should be acknowledged. First, the relatively small sample size (n = 16 confirmed isolates) limits statistical power and generalizability of findings. Secondly, the study was conducted at a single hospital over five months, which may not capture seasonal variations in Campylobacter prevalence and may not be representative of the entire country. However, the high correlation between our findings and the Kigali study [ 13 ] (both in terms of prevalence and resistance profiles) appears to strengthen validity of our findings. In our study, molecular characterization was limited to PCR detection of selected virulence genes, although we believe that whole genome sequencing would have provided more comprehensive insights into resistance mechanisms, genetic diversity, and phylogenetic relationships. Unfortunately, our funding would not enable us to probe further than this. Finally, the study would have been more enriching if clinical outcome data were collected and correlated with virulence gene profiles and AMR patterns. Again, funding constraints precluded these efforts. Conclusion This study demonstrates a concerning prevalence of virulence genes and alarmingly high antimicrobial resistance rates among thermophilic Campylobacter species isolated from human clinical infections in Rwanda. The universal presence of fla A and cad F genes confirms that all isolates possessed essential colonization factors, while the high prevalence of CDT genes (> 80%) indicates significant cytotoxic potential. Most concerning is the universal resistance to erythromycin and high resistance to ciprofloxacin, which effectively eliminates them as first-line treatment options for severe campylobacteriosis. The high proportion of MDR isolates (56.3%) represents a critical threat to effective clinical management of these infections. These findings underscore the urgent need for enhanced surveillance systems, improved antimicrobial stewardship, and implementation of comprehensive One Health approaches to combat antimicrobial resistance. The preservation of chloramphenicol and gentamicin susceptibility offers alternative therapeutic options, but these must be judiciously protected through rational use policies. Without immediate action, the emergence and spread of multidrug-resistant Campylobacter strains threatens to undermine our ability to effectively treat these common but potentially serious infections. Abbreviations AMR: Antimicrobial Resistance AST: Antimicrobial Susceptibility Testing EUCAST: European Committee on Antimicrobial Susceptibility Testing MDR: Multidrug Resistant bacteria CDT: Cytolethal Distending Toxin ECOFFs: Epidemiological Cut-Offs values CIP: Ciprofloxacin ERY: Erythromycin TET: Tetracycline GEN: Gentamycin CHL: Chloramphenicol Declarations Ethical approval and consent to participate This study was conducted in accordance with the World Medical Association’s 1964 Declaration of Helsinki ( as amended 2013 ). Ethical clearance was obtained from the Research Ethics Committee of the Institut d’Enseignement Supérieur de Ruhengeri (INES-Ruhengeri) and permission to conduct the study was granted by the administrative management of Ruhengeri Level 2 Teaching Hospital (Ref.329/RL2TH/DG/2025). Written informed consent (or parental assent in case of minors) was obtained from all participants prior to enrollment. Clinical Trial Number: Not applicable Consent for publication: Not applicable Availability of data and materials The dataset generated and used for this study are available from the corresponding author upon reasonable request. Competing interest All authors declare that they do not have any conflict of interest (direct or indirect) regarding the publication of this work. Funding statement This work was partly funded by the PASET Regional Scholarship and Innovation Grant under its Junior Investigator Research Award (JIRA) under grant number RSIF/JIRA/002 Authors’ contribution EB, AS, COO, PO & NG conceived the study. EB, CM,TH, YI, AMH & AS performed the laboratory experiments. EB, AS, & NG wrote the draft manuscript. COO & EB performed critical revisions to produce the final manuscript. All authors read and approved the final version. Acknowledgments The authors acknowledge the University of Global Health Equity for providing the necessary time and academic facilities that enabled EB to complete his MSc studies in Clinical Microbiology. The authors also extend their gratitude to INES-Ruhengeri for fostering a conducive learning environment and providing the academic foundation that was crucial for this work. The authors also thank the administration and staff of Ruhengeri Level-2 Teaching Hospital for granting permission to conduct data collection. References Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global Epidemiology of Campylobacter Infection. Clin Microbiol Rev. July 2015;28(3):687–720. 10.1128/CMR.00006-15 . Gahamanyi N, Mboera LEG, Matee MI, Mutangana D, Komba EVG, Prevalence. Risk Factors, and Antimicrobial Resistance Profiles of Thermophilic Campylobacter Species in Humans and Animals in Sub-Saharan Africa: A Systematic Review; Int. J. 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Bacteriol., vol. 2014, p. 560617, 2014. 10.1155/2014/560617 WHO bacterial priority pathogens list. 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. Accessed: Oct. 5, 2025. Available: https://www.who.int/publications/i/item/9789240093461 Asuming-Bediako N, Parry-Hanson Kunadu A, Abraham S, Habib I. Campylobacter at the Human-Food Interface: The African Perspective. Pathog Basel Switz. June 2019;8(2):87. 10.3390/pathogens8020087 . García-Fernández A, Dionisi AM, Arena S, Iglesias-Torrens Y, Carattoli A, Luzzi I. Human Campylobacteriosis in Italy: Emergence of Multi-Drug Resistance to Ciprofloxacin, Tetracycline, and Erythromycin. Front. Microbiol., vol. 9, p. 1906, 2018. 10.3389/fmicb.2018.01906 Portes AB, Panzenhagen P, Pereira Dos Santos AM, Junior CA. C. Antibiotic Resistance in Campylobacter: A Systematic Review of South American Isolates, Antibiot. Basel Switz., vol. 12, no. 3, p. 548, Mar. 2023, 10.3390/antibiotics12030548 Additional Declarations No competing interests reported. Supplementary Files Supplementaryfile1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7843102","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":561680910,"identity":"ba3a6d95-5ad7-4e6e-886f-cfb10d0847bf","order_by":0,"name":"Ezechiel Bizimana","email":"","orcid":"","institution":"INES-Ruhengeri","correspondingAuthor":false,"prefix":"","firstName":"Ezechiel","middleName":"","lastName":"Bizimana","suffix":""},{"id":561680911,"identity":"df806f23-b784-47ca-809e-3fe15765fd41","order_by":1,"name":"Celestin Musabyumuremyi","email":"","orcid":"","institution":"INES-Ruhengeri","correspondingAuthor":false,"prefix":"","firstName":"Celestin","middleName":"","lastName":"Musabyumuremyi","suffix":""},{"id":561680913,"identity":"9c16d809-8a36-4938-aa5c-23afa58f4ce8","order_by":2,"name":"Thierry Habyarimana","email":"","orcid":"","institution":"INES-Ruhengeri","correspondingAuthor":false,"prefix":"","firstName":"Thierry","middleName":"","lastName":"Habyarimana","suffix":""},{"id":561680915,"identity":"bf467d73-d7b7-4a8d-9617-aed3dc9d80de","order_by":3,"name":"Patrick Orikiriza","email":"","orcid":"","institution":"University of Global Health Equity","correspondingAuthor":false,"prefix":"","firstName":"Patrick","middleName":"","lastName":"Orikiriza","suffix":""},{"id":561680916,"identity":"1ba5ef27-adf0-45e9-91d0-b93b5e939796","order_by":4,"name":"Yoranda 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Within this genus, \u003cem\u003eC. jejuni\u003c/em\u003e and \u003cem\u003eC. coli\u003c/em\u003e are the most frequently implicated in human infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The main reservoir of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e is poultry, but other livestock including pigs and cattle have been implicated in transmission [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Human infection typically occurs via the consumption of contaminated foods, most notably undercooked poultry products as well as contaminated water or unpasteurized dairy products [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eCampylobacter\u003c/em\u003e infections are often self-limiting, but treatment is required in severe or prolonged infections and drugs of choice include macrolides and fluoroquinolones [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], although, tetracycline (TET), chloramphenicol (CHL) or gentamicin (GEN) can be used as alternatives [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the rise of antimicrobial resistance (AMR) in \u003cem\u003eCampylobacter\u003c/em\u003e poses a significant global public health concern [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For instance, resistance rates to fluoroquinolones exceeding 60% have been reported in some parts of Africa and South America [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Resistance to macrolides, though lower, has reached 30% in some studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This trend portends serious treatment challenges in the not-so-distant future with possible adverse outcomes and increased treatment costs particularly where there is multi-drug-resistance.\u003c/p\u003e \u003cp\u003eHuman campylobacteriosis is often complicated by the presence of various virulence genes that contribute to pathogenicity. Key genes include the cytolethal distending toxin (CDT), whose three distinct subunits \u003cem\u003ecdt\u003c/em\u003eA, \u003cem\u003ecdt\u003c/em\u003eB, \u0026amp; \u003cem\u003ecdt\u003c/em\u003eC are associated with DNA damage, inflammation, and cellular dysfunction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] The \u003cem\u003efla\u003c/em\u003eA gene, encoding the flagella protein, is crucial for motility and intestinal colonization [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] while \u003cem\u003ecad\u003c/em\u003eF gene is essential for bacterial adhesion to epithelial cells, promoting invasion and infection establishment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Africa, due to inadequate surveillance systems, and limited laboratory capacity for isolation and characterization, there is a general underreporting on the burden of \u003cem\u003eCampylobacter\u003c/em\u003e infections [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In Rwanda in particular, only two studies have ever described the burden of \u003cem\u003eCampylobacter\u003c/em\u003e in humans; one among under-five children, that looked at the causes of diarrhea, reporting a prevalence of 15% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and another conducted recently in capital city (Kigali), described the burden of \u003cem\u003eCampylobacter\u003c/em\u003e in the general urban population, reporting a prevalence of 7% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This picture highlights the paucity of data on \u003cem\u003eCampylobacter\u003c/em\u003e in our setting. Such gaps hinder our understanding of local disease epidemiology and how to optimally design and implement effective surveillance systems. Furthermore, inadequate data complicates efforts to control the spread of AMR through evidence-based antimicrobial stewardship initiatives. The current study, therefore, explored the virulence gene profiles of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e species as well as the antimicrobial susceptibility patterns from human clinical isolates from another region of the country.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eStudy design and catchment area\u003c/p\u003e \u003cp\u003eThis was a cross-sectional study conducted between March and July 2025. Stool samples were obtained from symptomatic patients presenting at Ruhengeri hospital, a level-2 teaching, and referral health facility located in Musanze district. This hospital is the largest healthcare facility within the North and Western Provinces of Rwanda, serving a catchment area in excess of two million people (National Institute of Statistics of Rwanda, 2022). The hospital receives patients from all the neighboring districts such as Burera, Gakenke, Nyabihu, Rubavu and as far as parts of Eastern Democratic Republic of Congo.\u003c/p\u003e \u003cp\u003eStudy population and enrollment\u003c/p\u003e \u003cp\u003eThe study targeted patients of all ages presenting with gastrointestinal symptoms at the hospital. Research assistants introduced the study to prospective participants after which they requested informed consent (or parental assent in case of children). Individuals who declined to give consent were excluded from further involvement.\u003c/p\u003e \u003cp\u003eSample collection and handling\u003c/p\u003e \u003cp\u003eHuman stool samples were collected from patients presenting with gastrointestinal (\u0026plusmn; systemic) symptoms such as diarrhea, abdominal pain/cramping, nausea/vomiting, fever, malaise, or fatigue. Clinical, laboratory and socio-demographic data were recorded using a structured questionnaire specifically developed for the study (see supplementary file 1). Participants received clear instructions on how to collect between 2\u0026ndash;5 gram of stool in sterile leak-proof containers. Samples were immediately delivered at the hospital laboratory where they were labeled with a unique study code and transported to the Microbiology Laboratory of the Institut d\u0026rsquo;Enseignement Sup\u0026eacute;rieur de Ruhengeri (INES-Ruhengeri) for further processing.\u003c/p\u003e \u003cp\u003eIsolation and phenotypic identification of Campylobacter spp.\u003c/p\u003e \u003cp\u003eFor each sample, approximately 1 gram of stool was enriched in Preston Broth supplemented with \u003cem\u003eCampylobacter\u003c/em\u003e Growth Supplement SR0232E added to modified Preston Campylobacter Selective Supplement SR0204E and 5% defibrinated sheep blood SR0051B. Inoculated tubes were placed in an anaerobic jar and incubated at 37\u0026deg;C for 24 hours under microaerophilic conditions (5% O\u003csub\u003e2\u003c/sub\u003e, 10% CO\u003csub\u003e2\u003c/sub\u003e, 85% N\u003csub\u003e2\u003c/sub\u003e) generated by \u003cem\u003eCampyGen\u003c/em\u003e sachets. After incubation for 24 hours, a loopful of the enrichment broth was streaked onto modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) supplemented with SR155E. All plates were then incubated at 37\u0026deg;C for 48 hours under the same conditions as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. All the above reagents were sourced from Oxoid Ltd, Basingstoke, England, United Kingdom. Presumptive \u003cem\u003eCampylobacter\u003c/em\u003e colonies were sub-cultured onto 5% sheep blood agar plates and incubated under identical microaerophilic conditions at 37\u0026deg;C for an additional 48 hours. Colonies from the blood agar were then examined microscopically using Gram staining to confirm characteristic morphology (gram-negative, curved, or spiral rods). Subsequently, these isolates were subjected to standard catalase and oxidase biochemical tests. Only isolates displaying typical morphology and positive reactions for both tests were retained for further molecular characterization. These isolates were preserved in Muller Hinton broth supplemented with 25% glycerol and stored at -20\u0026deg;C as previously described [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], with the longest storage sample lasting approximately 1 month.\u003c/p\u003e \u003cp\u003eMolecular methods:\u003c/p\u003e \u003cp\u003eDNA extraction, Campylobacter confirmation, and characterization of virulence genes\u003c/p\u003e \u003cp\u003ePrior to DNA extraction, isolates in storage were brought back into life through sub-culturing on 5% blood agar plates. Genomic DNA was extracted out of revived colonies by utilizing the Qiagen Dneasy Blood and Tissue kit (Qiagen, Hilden, Germany) as per manufacturer\u0026rsquo;s directions. A concentration and purity of extracted DNA was measured by a Qubit fluorometer (Thermo Fisher Scientific, USA), and DNA samples were stored at -20\u003csup\u003eo\u003c/sup\u003e C until use. The presence of \u003cem\u003eCampylobacter\u003c/em\u003e species was identified by use of the 16S rRNA gene and the genus identified by \u003cem\u003emap\u003c/em\u003eA gene which confirms \u003cem\u003eC.jejuni.\u003c/em\u003e Other thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e spp. were seen as isolates positive for the 16S rRNA gene but negative in \u003cem\u003emap\u003c/em\u003eA gene.\u003c/p\u003e \u003cp\u003eFor virulence characterization, the genes, \u003cem\u003efla\u003c/em\u003eA, \u003cem\u003ecad\u003c/em\u003eF, and the CDT genes were targeted. Each PCR mixture had a total volume 25\u0026micro;L. This master mix consisted of 3\u0026micro;L of 10x PCR buffer, 0.3\u0026micro;L of Taq thermostable DNA polymerase, 1.2\u0026micro;L of MgCl\u003csub\u003e2\u003c/sub\u003e, 2.5\u0026micro;L of dNTPs, 2 \u0026micro;L of template DNA, 0.5\u0026micro;L of forward primer (10 pmol/L) and 0.5\u0026micro;L of reverse primer (10 pmol/L). The volume of the last one was brought up to 25 mL with 15 \u0026micro;L of RNAse-free water as previously described [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGene amplification was done using a thermal cycler with one cycle of denaturation stage at 95\u003csup\u003eo\u003c/sup\u003eC for 10 min and 35 cycles which contained denaturation (30 sec) at 95\u003csup\u003eo\u003c/sup\u003eC, anneal (90 sec) at 59\u003csup\u003eo\u003c/sup\u003eC and extension (60 sec) at 72\u003csup\u003eo\u003c/sup\u003eC. Samples were subjected to the final extension process (72\u003csup\u003eo\u003c/sup\u003eC, 5 minutes) and it established that the production was complete of all PCR products. The reactions were subsequently stored in 4\u003csup\u003eo\u003c/sup\u003eC until further determination [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Electrophoresis was performed by running the PCR products in a 1.5% (w/v) agarose gel prepared in 1x Tris-borate-EDTA (TBE) buffer and stained with ethidium bromide (0.5 g/mL). The amplicon bands were also compared to 100 bp DNA ladder (Thermoscientific) and observed under a UV transilluminator. Bands for the 16S rRNA (for \u003cem\u003eCampylobacter\u003c/em\u003e genus) and \u003cem\u003emap\u003c/em\u003eA gene (for \u003cem\u003eC. jejuni)\u003c/em\u003e were characterized by 857 bp and 589 bp respectively. For virulence genes \u003cem\u003efla\u003c/em\u003eA, \u003cem\u003ecad\u003c/em\u003eF, \u003cem\u003ecdt\u003c/em\u003eA, \u003cem\u003ecdt\u003c/em\u003eB and \u003cem\u003ecdt\u003c/em\u003eC, bands were observed at 1200 bp, 500 bp, 370 bp, 620 and 182 bp respectively, as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimicrobial Susceptibility Testing (AST)\u003c/p\u003e \u003cp\u003eThe Kirby-Bauer disc diffusion method was used to establish antimicrobial resistance profiles as per recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Briefly, pure \u003cem\u003eCampylobacter\u003c/em\u003e colonies were revived on blood agar. An inoculum was prepared through the suspension of colonies in normal saline to give a turbidity of 0.5 McFarland standard. The bacterial suspension was streaked on Muller Hinton Agar (MHA) with 5% sheep blood using sterile glass spreaders and plates were left to dry for 15 min. Antibiotic disks; (CIP, 5 \u0026micro;g), (ERY, 15 \u0026micro;g), (TET, 30 \u0026micro;g), (GEN, 10 \u0026micro;g), and (CHL, 30 \u0026micro;g) were then placed on the surface of the agar plates using sterile forceps, making sure to maintain a minimum distance of 24 mm between discs. The plates were subsequently incubated under microaerophilic conditions at 37\u003csup\u003eo\u003c/sup\u003eC for 48 hours and inhibition zone diameters measured (in millimeters) using a ruler. EUCAST epidemiologic cutoffs (ECOFFs) were used to make interpretations as sensitive (S), intermediate (I ), or Resistant (R) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Multidrug resistance (MDR) was defined by resistance to at least three antibiotics from different classes [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eData entry and analysis were done using Statistical Package for Social Sciences (SPSS) (version 21.0, IBM Corp., Armonk, NY, USA). Descriptive statistics (including prevalence, proportions, and frequencies) were computed.\u003c/p\u003e \u003cp\u003eEthical considerations\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cp\u003e was obtained from the Research Ethics Committee of INES-Ruhengeri Institute of Applied Sciences as well as Ruhengeri district hospital (Ref 329/RL2TH/DG/2025). All participants provided written informed consent (or parental assent, in the case of minors). All patient information was coded and securely stored on a password-protected computer to ensure confidentiality.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSocio-demographic characteristics of patients\u003c/p\u003e \u003cp\u003eOver the 5-month period (March-July 2025), stool samples from 200 symptomatic participants were collected and analyzed. Majority of participants were female 145 (72.5%) with children\u0026thinsp;\u0026lt;\u0026thinsp;5 year comprising 26.5% of the sample (n\u0026thinsp;=\u0026thinsp;53). Approximately one quarter (24.5%) of participants had no formal education while 39.5% had completed primary education and 36% had completed secondary school education or higher.\u003c/p\u003e \u003cp\u003ePrevalence of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e species\u003c/p\u003e \u003cp\u003eCulture and biochemical tests presumptively identified 18 (9%) \u003cem\u003eCampylobacter\u003c/em\u003e isolates of which 16 (8%) were positively confirmed by molecular methods. Of these, 15/16 (93.8%) were confirmed to be \u003cem\u003eC. jejuni\u003c/em\u003e while one isolate was not conclusively identified.\u003c/p\u003e \u003cp\u003eVirulence gene characterization of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e isolates\u003c/p\u003e \u003cp\u003eMolecular screening of virulence genes from the 16 isolates revealed the presence of \u003cem\u003efla\u003c/em\u003eA and \u003cem\u003ecad\u003c/em\u003eF genes in all isolates (100%). In contrast, \u003cem\u003ecdt\u003c/em\u003eA and \u003cem\u003ecdt\u003c/em\u003eC genes were present in 93.6% of isolates while presence of \u003cem\u003ecdt\u003c/em\u003eB was demonstrated in only 81.3% of isolates (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of virulence genes in thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e spp. from human stool samples from Rwanda (n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVirulence gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. positive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrevalence (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003efla\u003c/em\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecad\u003c/em\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecdt\u003c/em\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecdt\u003c/em\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.8%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ecdt\u003c/em\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAntimicrobial susceptibility profiles of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eAll isolates (100%) were resistant to erythromycin. Taking both R \u0026amp; I categories, resistance to ciprofloxacin was also high at 81.3% while resistance to tetracycline was 75.0%. In contrast, all isolates were sensitive to chloramphenicol while sensitivity to gentamycin was 87.5%. Details are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial susceptibility profiles of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e isolates from Rwanda (n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eNumber of isolates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAntibiotic tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSensitive (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIntermediate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eResistant (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCiprofloxacin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (18.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 (50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5 (31.25)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eErythromycin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (100)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGentamicin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14 (87.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 (25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2 (12.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10 (62.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOf the 16 \u003cem\u003eCampylobacter\u003c/em\u003e isolates tested against 5 antibiotics above, 2 isolates demonstrated resistance to one antibiotic each, while 5 isolates showed resistance to two antibiotics each. However, 9 isolates (56.3%) showed resistance to three or more antibiotics and were classified as MDR. The predominant MDR pattern consisted of resistance to ciprofloxacin, erythromycin, and tetracycline.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides critical insights into the molecular characteristics and antimicrobial resistance patterns of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e species isolated from human clinical infections in Rwanda. The prevalence of 8% observed in this study aligns with the 7% reported in the recent study from Kigali [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and remains lower than the 15% prevalence documented among under-five children reported ten years back [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This variation may reflect differences in study populations, geographic locations, or detection methods. The predominance of \u003cem\u003eC. jejuni\u003c/em\u003e (93.8%) among confirmed isolates in our sample is consistent with global epidemiological patterns, where this species accounts for the majority of human campylobacteriosis cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDistribution of virulence genes and pathogenicity implications\u003c/p\u003e \u003cp\u003eThe universal presence (100%) of both \u003cem\u003efla\u003c/em\u003eA and \u003cem\u003ecad\u003c/em\u003eF genes among all isolates is particularly noteworthy and consistent with findings from other studies from Asia [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and South America [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The \u003cem\u003efla\u003c/em\u003eA gene, encoding the flagellin protein, is essential for bacterial motility and represents a critical determinant for intestinal colonization [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Without functional flagella, \u003cem\u003eCampylobacter\u003c/em\u003e would be unable to navigate through the mucus layer to reach epithelial cells, making this gene indispensable for establishing infection. Similarly, the ubiquitous presence of \u003cem\u003ecad\u003c/em\u003eF underscores its fundamental role in pathogenesis. This gene encodes an outer membrane protein that mediates bacterial adhesion to fibronectin on host epithelial cells, representing the initial step in colonization and subsequent invasion [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The universal presence of these two genes suggests that all isolates in this study possessed the basic molecular machinery necessary for successful host colonization and infection establishment.\u003c/p\u003e \u003cp\u003eThe CDT gene complex showed variable distribution, with \u003cem\u003ecdt\u003c/em\u003eA and \u003cem\u003ecdt\u003c/em\u003eB present in 93.8% of isolates, while \u003cem\u003ecdt\u003c/em\u003eC was detected in only 81.3%. This differential distribution is intriguing and has important pathogenic implications. The cytolethal distending toxin represents a well-known toxin produced by \u003cem\u003eC. jejuni\u003c/em\u003e and functions as a critical virulence factor. CdtB, the active subunit, possesses DNase activity that causes DNA damage, cell cycle arrest at the G2/M phase, and ultimately induces apoptosis in host cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Studies have demonstrated that CDT contributes to both the invasion of host cells and the prolongation of clinical symptoms [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The toxin operates as what has been termed a \"tri-perditious toxin,\" impairing host defenses through three mechanisms: disrupting epithelial barriers, suppressing acquired immunity, and promoting pro-inflammatory responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe lower prevalence of \u003cem\u003ecdt\u003c/em\u003eC (81.3%) compared to \u003cem\u003ecdt\u003c/em\u003eA and \u003cem\u003ecdt\u003c/em\u003eB is particularly significant. While CdtA and CdtC function as binding subunits that facilitate the delivery of the toxic CdtB subunit into host cells, all three subunits are typically required for full toxin activity. The absence of \u003cem\u003ecdt\u003c/em\u003eC in approximately 19% of isolates suggests these strains may have reduced cytotoxic capacity. However, some studies have shown that CDT variants with incomplete subunit profiles can still exhibit pathogenic activity, albeit at reduced levels. This genetic heterogeneity among clinical isolates may partially explain the spectrum of disease severity observed in campylobacteriosis, ranging from mild, self-limiting diarrhea to severe invasive disease.\u003c/p\u003e \u003cp\u003eInterestingly, while CDT genes are considered important virulence factors, some studies have found no direct association between \u003cem\u003ecdt\u003c/em\u003eB presence and invasive bloodstream infections versus gastroenteritis alone [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This suggests that disease severity and clinical outcomes depend on complex host-pathogen interactions beyond the mere presence of individual virulence genes. Host immune status, bacterial load, concurrent gut microbiota, and other environmental factors likely modulate the clinical manifestations of \u003cem\u003eCampylobacter\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003eAntimicrobial resistance: an alarming trend\u003c/p\u003e \u003cp\u003eThe antimicrobial susceptibility profile revealed in this study is deeply concerning and reflects the broader antimicrobial resistance capabilities acquired by \u003cem\u003eCampylobacter\u003c/em\u003e in Africa as well as globally. The universal resistance to erythromycin (100%) is particularly alarming given that macrolides represent first-line treatment options for severe campylobacteriosis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This finding diverges dramatically from the expected resistance rates and exceeds even the concerning 30\u0026ndash;40% macrolide resistance previously reported in some African studies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The complete resistance observed in our study suggests extensive selective pressure, potentially driven by their widespread use in human infections as well as animal agriculture in Rwanda. Likewise, the high resistance to ciprofloxacin, observed in 81.3% of isolates (combining resistant and intermediate categories), is equally troubling. Fluoroquinolones serve as alternative first-line agents for treating campylobacteriosis, particularly in adults where macrolides may be contraindicated [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In 2017, the WHO classified fluoroquinolone-resistant \u003cem\u003eCampylobacter\u003c/em\u003e among priority pathogens against which new antibiotics were urgently needed. This was however reversed in the 2024 update following expert consensus [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Despite this change, the high rate of resistance documented in this study remains clinically significant as it mirrors trends observed across sub-Saharan Africa and South America, where resistance rates exceeding 60% have been reported [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTetracycline resistance was observed in 75% of isolates (combining resistant and intermediate categories), and is consistent with resistance patterns recently reported elsewhere in Rwanda [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and other African countries [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Tetracyclines are commonly used in veterinary medicine, and this high resistance likely reflects the agricultural use of these antimicrobials in livestock production, creating selective pressure that favors the spread of resistance.\u003c/p\u003e \u003cp\u003eEncouragingly, all isolates remained sensitive to chloramphenicol, and 87.5% showed sensitivity to gentamicin. This preservation of susceptibility to these alternatives provides critical therapeutic options for managing severe infections. Chloramphenicol, despite concerns about potential side effects, remains effective and could serve as a valuable treatment option in cases where resistance to fluoroquinolones and macrolides precludes their use. The multidrug resistance (MDR) profile is particularly worrisome, with 56.3% of isolates showing resistance to three or more antimicrobials from different classes. The predominant MDR pattern involving ciprofloxacin, erythromycin, and tetracycline effectively eliminates the most commonly prescribed first-line treatment options. This pattern mirrors findings from the recent Kigali study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and highlights the rapid evolution of resistance in Rwandan \u003cem\u003eCampylobacter\u003c/em\u003e populations. The high MDR prevalence suggests potential clonal spread of resistant strains or widespread horizontal gene transfer of resistance determinants within the bacterial population. Incidentally, the phenomenon of MDR Campylobacter has also been reported in other continents including Europe [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], South America [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral factors may contribute to the elevated levels of AMR observed in this study. There is no doubt that indiscriminate use of antibiotics in human medicine, coupled with their prophylactic and growth-promoting application in animal agriculture, creates sustained selective pressure. Furthermore, in Rwanda, as in many African countries, weak regulatory frameworks for antibiotic stewardship, availability of over-the-counter antimicrobials without prescription, and limited diagnostic capacity leading to empirical treatments all contribute to the selection and spread of drug resistance. Additionally, we believe that the close proximity of humans to livestock in many rural Rwandan communities facilitates zoonotic transmission of resistant strains and vice versa.\u003c/p\u003e \u003cp\u003eStudy context and public health implications\u003c/p\u003e \u003cp\u003eThe location of this study in Musanze district, serving populations from North and Western Rwanda and parts of Eastern Congo, provides insights into \u003cem\u003eCampylobacter\u003c/em\u003e epidemiology beyond urban centers like Kigali. The consistency of high resistance rates between this study and the Kigali study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] suggests that AMR in \u003cem\u003eCampylobacter\u003c/em\u003e is geographically widespread in Rwanda, rather than being confined to urban centers. This geographic distribution has important implications for national treatment guidelines and antimicrobial stewardship policies. Furthermore, the presence of highly virulent, multidrug-resistant \u003cem\u003eCampylobacter\u003c/em\u003e strains in clinical settings represents a significant threat to public health. More worrying is that patients infected with MDR strains face limited therapeutic options, increased risk of prolonged illness \u0026amp; complications, higher healthcare costs or even death. The situation is particularly concerning for vulnerable populations including young children, elderly individuals, and immunocompromised patients who may easily develop severe or invasive disease.\u003c/p\u003e \u003cp\u003eStudy limitations\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, the relatively small sample size (n\u0026thinsp;=\u0026thinsp;16 confirmed isolates) limits statistical power and generalizability of findings. Secondly, the study was conducted at a single hospital over five months, which may not capture seasonal variations in \u003cem\u003eCampylobacter\u003c/em\u003e prevalence and may not be representative of the entire country. However, the high correlation between our findings and the Kigali study [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] (both in terms of prevalence and resistance profiles) appears to strengthen validity of our findings. In our study, molecular characterization was limited to PCR detection of selected virulence genes, although we believe that whole genome sequencing would have provided more comprehensive insights into resistance mechanisms, genetic diversity, and phylogenetic relationships. Unfortunately, our funding would not enable us to probe further than this. Finally, the study would have been more enriching if clinical outcome data were collected and correlated with virulence gene profiles and AMR patterns. Again, funding constraints precluded these efforts.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates a concerning prevalence of virulence genes and alarmingly high antimicrobial resistance rates among thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e species isolated from human clinical infections in Rwanda. The universal presence of \u003cem\u003efla\u003c/em\u003eA and \u003cem\u003ecad\u003c/em\u003eF genes confirms that all isolates possessed essential colonization factors, while the high prevalence of CDT genes (\u0026gt;\u0026thinsp;80%) indicates significant cytotoxic potential. Most concerning is the universal resistance to erythromycin and high resistance to ciprofloxacin, which effectively eliminates them as first-line treatment options for severe campylobacteriosis. The high proportion of MDR isolates (56.3%) represents a critical threat to effective clinical management of these infections. These findings underscore the urgent need for enhanced surveillance systems, improved antimicrobial stewardship, and implementation of comprehensive One Health approaches to combat antimicrobial resistance. The preservation of chloramphenicol and gentamicin susceptibility offers alternative therapeutic options, but these must be judiciously protected through rational use policies. Without immediate action, the emergence and spread of multidrug-resistant \u003cem\u003eCampylobacter\u003c/em\u003e strains threatens to undermine our ability to effectively treat these common but potentially serious infections.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAMR: Antimicrobial Resistance\u003c/p\u003e\n\u003cp\u003eAST: Antimicrobial Susceptibility Testing\u003c/p\u003e\n\u003cp\u003eEUCAST: European Committee on Antimicrobial Susceptibility Testing\u003c/p\u003e\n\u003cp\u003eMDR: Multidrug Resistant bacteria\u003c/p\u003e\n\u003cp\u003eCDT: Cytolethal Distending Toxin\u003c/p\u003e\n\u003cp\u003eECOFFs: Epidemiological Cut-Offs values\u003c/p\u003e\n\u003cp\u003eCIP: Ciprofloxacin\u003c/p\u003e\n\u003cp\u003eERY: Erythromycin\u003c/p\u003e\n\u003cp\u003eTET: Tetracycline\u003c/p\u003e\n\u003cp\u003eGEN: Gentamycin\u003c/p\u003e\n\u003cp\u003eCHL: Chloramphenicol\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the World Medical Association\u0026rsquo;s 1964 Declaration of Helsinki (\u003cem\u003eas amended 2013\u003c/em\u003e). Ethical clearance was obtained from the Research Ethics Committee of the Institut d\u0026rsquo;Enseignement Sup\u0026eacute;rieur de Ruhengeri (INES-Ruhengeri) and permission to conduct the study was granted by the administrative management of Ruhengeri Level 2 Teaching Hospital (Ref.329/RL2TH/DG/2025). Written informed consent (or parental assent in case of minors) was obtained from all participants prior to enrollment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dataset generated and used for this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they do not have any conflict of interest (direct or indirect) regarding the publication of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partly funded by the PASET Regional Scholarship and Innovation Grant under its Junior Investigator Research Award (JIRA) under grant number RSIF/JIRA/002\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEB, AS, COO, PO \u0026amp; NG conceived the study. EB, CM,TH, YI, AMH \u0026amp; AS performed the laboratory experiments. EB, AS, \u0026amp; NG wrote the draft manuscript. COO \u0026amp; EB performed critical revisions to produce the final manuscript. All authors read and approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the University of Global Health Equity for providing the necessary time and academic facilities that enabled EB to complete his MSc studies in Clinical Microbiology. The authors also extend their gratitude to INES-Ruhengeri for fostering a conducive learning environment and providing the academic foundation that was crucial for this work. The authors also thank the administration and staff of Ruhengeri Level-2 Teaching Hospital for granting permission to conduct data collection.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKaakoush NO, Casta\u0026ntilde;o-Rodr\u0026iacute;guez N, Mitchell HM, Man SM. Global Epidemiology of Campylobacter Infection. Clin Microbiol Rev. July 2015;28(3):687\u0026ndash;720. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/CMR.00006-15\u003c/span\u003e\u003cspan address=\"10.1128/CMR.00006-15\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGahamanyi N, Mboera LEG, Matee MI, Mutangana D, Komba EVG, Prevalence. Risk Factors, and Antimicrobial Resistance Profiles of Thermophilic Campylobacter Species in Humans and Animals in Sub-Saharan Africa: A Systematic Review; Int. J. 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Bacteriol., vol. 2014, p. 560617, 2014. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2014/560617\u003c/span\u003e\u003cspan address=\"10.1155/2014/560617\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWHO bacterial priority pathogens list. 2024: Bacterial pathogens of public health importance to guide research, development and strategies to prevent and control antimicrobial resistance. Accessed: Oct. 5, 2025. Available: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/publications/i/item/9789240093461\u003c/span\u003e\u003cspan address=\"https://www.who.int/publications/i/item/9789240093461\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsuming-Bediako N, Parry-Hanson Kunadu A, Abraham S, Habib I. Campylobacter at the Human-Food Interface: The African Perspective. Pathog Basel Switz. June 2019;8(2):87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/pathogens8020087\u003c/span\u003e\u003cspan address=\"10.3390/pathogens8020087\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-Fern\u0026aacute;ndez A, Dionisi AM, Arena S, Iglesias-Torrens Y, Carattoli A, Luzzi I. Human Campylobacteriosis in Italy: Emergence of Multi-Drug Resistance to Ciprofloxacin, Tetracycline, and Erythromycin. Front. Microbiol., vol. 9, p. 1906, 2018. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2018.01906\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2018.01906\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePortes AB, Panzenhagen P, Pereira Dos Santos AM, Junior CA. C. Antibiotic Resistance in Campylobacter: A Systematic Review of South American Isolates, Antibiot. Basel Switz., vol. 12, no. 3, p. 548, Mar. 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/antibiotics12030548\u003c/span\u003e\u003cspan address=\"10.3390/antibiotics12030548\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Campylobacter jejuni, antimicrobial resistance, virulence genes, Rwanda, multidrug resistance","lastPublishedDoi":"10.21203/rs.3.rs-7843102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7843102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCampylobacter\u003c/em\u003e species are among the leading causes of bacterial gastroenteritis globally. In Rwanda, limited data exist on their molecular characteristics and antimicrobial resistance (AMR) profiles. This study investigated the virulence gene profiles and antimicrobial susceptibility of thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e species isolated from human clinical infections in northern Rwanda.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA cross-sectional study was conducted between March and July 2025 among 200 symptomatic patients at Ruhengeri Hospital. Stool samples were cultured under microaerophilic conditions, and presumptive \u003cem\u003eCampylobacter\u003c/em\u003e isolates were confirmed by PCR targeting the 16S rRNA and \u003cem\u003emapA\u003c/em\u003e genes. Virulence genes (\u003cem\u003eflaA\u003c/em\u003e, \u003cem\u003ecadF\u003c/em\u003e, \u003cem\u003ecdtA\u003c/em\u003e, \u003cem\u003ecdtB\u003c/em\u003e, \u003cem\u003ecdtC\u003c/em\u003e) were characterized by PCR. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer disc diffusion method against ciprofloxacin, erythromycin, tetracycline, gentamicin, and chloramphenicol following EUCAST guidelines.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSixteen (8%) thermophilic \u003cem\u003eCampylobacter\u003c/em\u003e isolates were confirmed, predominantly \u003cem\u003eC. jejuni\u003c/em\u003e (93.8%). All isolates harbored \u003cem\u003eflaA\u003c/em\u003e and \u003cem\u003ecadF\u003c/em\u003e genes, while \u003cem\u003ecdtA\u003c/em\u003e and \u003cem\u003ecdtB\u003c/em\u003e were present in 93.8% and \u003cem\u003ecdtC\u003c/em\u003e in 81.3%. All isolates (100%) were resistant to erythromycin, 81.3% to ciprofloxacin (resistant/intermediate), and 75.0% to tetracycline, while complete sensitivity to chloramphenicol and 87.5% sensitivity to gentamicin were observed. Over half (56.3%) of isolates exhibited multidrug resistance (MDR), primarily to erythromycin, ciprofloxacin, and tetracycline.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe study reveals high virulence gene prevalence and alarming resistance rates among \u003cem\u003eCampylobacter\u003c/em\u003e isolates from clinical infections in Rwanda. The universal resistance to erythromycin and high rates of ciprofloxacin and tetracycline resistance highlight the urgent need for enhanced AMR surveillance, rational antibiotic use, and One Health-based control strategies. The retained susceptibility to chloramphenicol and gentamicin offers limited but valuable therapeutic alternatives.\u003c/p\u003e","manuscriptTitle":"Molecular characterization of virulence factors and antimicrobial susceptibility of thermophilic Campylobacter species associated with human clinical infections in Rwanda","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-18 17:25:52","doi":"10.21203/rs.3.rs-7843102/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c81002a2-8cb8-47b7-92f8-ccc1a662730d","owner":[],"postedDate":"December 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T08:44:27+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-18 17:25:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7843102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7843102","identity":"rs-7843102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}