Molecular detection of Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania

A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens but their contribution to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania using molecular techniques without culture. Seventy (70) faecal samples were collected from five diarrheic and 65 non-diarrheic human patients attending Kilosa District Hospital in Tanzania from July to October 2019. During the same period, 30 faecal samples were also collected from healthy cattle in the same district. Genus and species identification of Campylobacter was conducted on the samples using molecular techniques [the polymerase chain reaction (PCR) and 16S rRNA sequencing]. Phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively. There were five human diarrheic cases, four of which were positive for Campylobacter and of these, two were children ≤15 years of age. In humans, 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae, all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae. Phylogenetic analysis revealed that with the exception of C. lanienae, 16S rRNA sequences of Campylobacter species were closely related to the reference strains used (Percent identity: 90.51-96.56%). Based on our findings, we recommend that molecular techniques, mainly PCR be adopted for the direct detection of Campylobacter species during laboratory screening and surveillance studies.

Isolation and antibiotic sensitivity of Campylobacter species from fecal samples of broiler chickens in North West Province, South Africa

Background and Aim: Infections with Campylobacter species have gained recognition as the most frequent cause of foodborne gastroenteritis globally. Their significance in South Africa is still an area of study interest. This study was, therefore, carried out to determine the occurrence of Campylobacter species in chickens from North West Province of South Africa as well as their antibiotic sensitivity status. Materials and Methods: A total of 2400 chicken fecal samples were collected and pooled to a total of 480 samples from five registered active poultry abattoirs in the Ngaka Modiri Molema District of North West Province, South Africa. Polymerase chain reaction (PCR) was used for the detection of Campylobacter spp. targeting the 16S rRNA gene while antibiotic sensitivity was determined using disk diffusion inhibition test. Results: After isolation, a total of 26 samples were confirmed to be harboring Campylobacter jejuni by PCR and sequencing. C. jejuni was found to be the only isolate detected in all the fecal samples tested. The study further demonstrated that C. jejuni infections were highest in the summer season (3%) followed by autumn and winter at 1%, while there were none detected in the spring. The isolated C. jejuni-positive samples on disk diffusion inhibition test displayed resistance to nalidixic acid, tetracycline, erythromycin, and ciprofloxacin at 98%, 80%, 83%, and 21%, respectively. Conclusion: C. jejuni isolated in this study is known to cause disease in humans, and thus its occurrence requires application of "One Health" strategy to reduce the spread of this zoonotic pathogen in South Africa.

Molecular Detection of Fluoroquinolone Resistance among Multidrug-, Extensively Drug-, and Pan-Drug-Resistant Campylobacter Species in Egypt

In recent times, resistant foodborne pathogens, especially of the Campylobacter species, have created several global crises. These crises have been compounded due to the evolution of multidrug-resistant (MDR) bacterial pathogens and the emergence of extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Therefore, this study aimed to investigate the development of resistance and the existence of both XDR and PDR among Campylobacter isolates. Moreover, we explored the use of the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique for the detection of fluoroquinolone (FQ)-resistant Campylobacter isolates. A total of 120 Campylobacter isolates were identified depending on both phenotypic and genotypic methods. Of note, cefoxitin and imipenem were the most effective drugs against the investigated Campylobacter isolates. Interestingly, the majority of our isolates (75%) were MDR. Unfortunately, both XDR and PDR isolates were detected in our study with prevalence rates of 20.8% and 4.2%, respectively. All FQ-resistant isolates with ciprofloxacin minimum inhibitory concentrations ≥4 µg/mL were confirmed by the genetic detection of gyrA chromosomal mutation via substitution of threonine at position 86 to isoleucine (Thr-86-to-Ile) using the PCR-RFLP technique. Herein, PCR-RFLP was a more practical and less expensive method used for the detection of FQ resistant isolates. In conclusion, we introduced a fast genetic method for the identification of FQ-resistant isolates to avoid treatment failure through the proper description of antimicrobials.

Prevalence, Virulence, and Antimicrobial Resistance of Campylobacter Species Isolated From Carcasses of Camels Slaughtered in Slaughterhouses of Chaharmahal and Bakhtiari Province, 2018-2019

Background and aims: Gastritis is basically caused by Campylobacter coli and jejuni, and usually occures after the consumption of raw animal products. Methods: This study investigated the prevalence, virulence, and antimicrobial resistance of Campylobacter species isolated from slaughtered animals in Juneqan, Farrokhshahr, Saman, and Lordegan slaughterhouses in Chaharmahal and Bakhtiari Province of Iran. From 40 camels, 5 samples of liver, neck meat, kidney, heart, and rectal contents were taken from each carcass. The obtained samples were cultured and then the PCR was performed for them and, finally, the toxin genes of virulence and resistance against antibiotics were examined. Results: Out of 19 Campylobacter specimens isolated, 8 specimens were coli and 11 ones were jejuni. It was also found that the infection with Campylobacter in the carcasses was the highest in warmer seasons. Conclusion: The carcasses of slaughtered animals in slaughterhouses were likely a potential reservoir for coli and jejuni species, and their viscera and meat could have transmitted these bacteria to humans and animals.

Isolation of Campylobacter Species in the Stool of Under Five Children With Acute Diarrhea in a Tertiary Care Hospital of Bangladesh

Campylobacter species is the main etiology of gastroenteritis due to bacteria. To determine prevalence of Campylobacter species in stool of children less than five years of age with acute diarrhoea, this observational study was conducted in the Department of Microbiology, Sylhet M A G Osmani Medical College, Sylhet from January to December, 2017. Stool samples were collected from 162 under-five children with acute diarrhoea admitted in the Department of Paediatrics. Isolation of Campylobacter species were done by stool culture. About two third of the children were male (65.4%) and more than one fourth of the affected children (26.65%) were in age group 6-12 months. Campylobacter species was isolated in 24 (15%) sample and among them, Campylobacter jejuni were 22 (91.7%) and Campylobacter coli were 2 (8.3%). Isolation rate of Campylobacter species did not differ significantly between age group of 6-12 months and above 12 months (p=0.211) of age; male and female children (p=0.288); among socioeconomic status (p=0.673) and between residential status (p=0.108). Isolation rate of Campylobacter species are frequent among under five children with acute diarrhea and most of the children came from low socioeconomic background and were male. However, a large multicenter study needs to be conducted to generate more evidence regarding the issue. Bangladesh Med J. 2020 Sept; 49(3) : 11-16

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

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

Genomic Characterization of Fluoroquinolone-Resistant Thermophilic Campylobacter Strains Isolated from Layer Chicken Feces in Gangneung, South Korea by Whole-Genome Sequencing

Thermophilic Campylobacter species of poultry origin have been associated with up to 80% of human campylobacteriosis cases. Layer chickens have received less attention as possible reservoirs of Campylobacter species. Initially, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of two archived Campylobacter isolates (Campylobacter jejuni strain 200605 and Campylobacter coli strain 200606) from layer chickens to five antimicrobials (ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and gentamicin) were determined using broth microdilution while the presence of selected antimicrobial resistance genes was performed by polymerase chain reaction (PCR) using specific primers. Whole-genome sequencing (WGS) was performed by the Illumina HiSeq X platform. The analysis involved antimicrobial resistance genes, virulome, multilocus sequence typing (MLST), and phylogeny. Both isolates were phenotypically resistant to ciprofloxacin (MIC: 32 vs. 32 µg/mL), nalidixic acid (MIC: 128 vs. 64 µg/mL), and tetracycline (MIC: 64 vs. 64 µg/mL), but sensitive to erythromycin (MIC: 1 vs. 2 µg/mL) and gentamicin (MIC: 0.25 vs. 1 µg/mL) for C. jejuni strain 200605 and C. coli strain 200606, respectively. WGS confirmed C257T mutation in the gyrA gene and the presence of cmeABC complex conferring resistance to FQs in both strains. Both strains also exhibited tet(O) genes associated with tetracycline resistance. Various virulence genes associated with motility, chemotaxis, and capsule formation were found in both isolates. However, the analysis of virulence genes showed that C. jejuni strain 200605 is more virulent than C. coli strain 200606. The MLST showed that C. jejuni strain 200605 belongs to sequence type ST-5229 while C. coli strain 200606 belongs to ST-5935, and both STs are less common. The phylogenetic analysis clustered C. jejuni strain 200605 along with other strains reported in Korea (CP028933 from chicken and CP014344 from human) while C. coli strain 200606 formed a separate cluster with C. coli (CP007181) from turkey. The WGS confirmed FQ-resistance in both strains and showed potential virulence of both strains. Further studies are recommended to understand the reasons behind the regional distribution (Korea, China, and Vietnam) of such rare STs.

Association Between Infection With Campylobacter Species and the Risk of Esophageal Cancer: A Hospital-Based Case–Control Study in Thailand

Background Previous studies have shown the association between Campylobacter species infection and environmental factors, poor oral hygiene in particular, is linked to an increased risk of esophageal cancer (EC). However, no study has reported on these factors in Thai. Thus, this study objective was to evaluate the impact of the relationship between Campylobacter infection and environmental factors on EC incidence in the population Thailand. Methods Data from a case–control study were collected from 105 newly diagnosed EC cases and 105 controls recruited from 2007 to 2017. Infection with Campylobacter spp. was detected in the Formalin-fixed paraffin-embedded (FFPE) tissue of EC taken from gastroesophageal biopsy specimens obtained from the participants and evaluated using a TaqMan® real-time PCR. Multivariable logistic regression was performed to calculate the odds ratios (ORs) and perform data analysis. Results Smoking, alcohol use, a family history of cancer, history of gastroesophageal reflux disease, poor oral hygiene and Campylobacter spp. infection were shown to be significant risk factors for EC (p < 0.05). The combination of poor oral hygiene and infection with Campylobacter spp. constituted significant risk for EC (p < 0.001). In addition, the risk of EC in subjects co-infected with C. rectus and C. concisus practiced poor oral hygiene was even higher and was significant (ORadj = 4.7, 95%CI: 2.41–9.98, p = 0.003). Conclusions In Thailand, poor oral hygiene and infection with Campylobacter spp. was associated with an increased risk of EC.

#9: Diagnosis of Campylobacter upsaliensis and Helicobacter canis by plasma-based Next-Generation Sequencing for Microbial Cell-free DNA in twin brothers with XLA

Background X-linked agammaglobulinemia is a primary immunodeficiency associated with mutations in the B cell tyrosine kinase (BTK) gene leading to failure in B cell maturation and defective antibody production. Campylobacter and closely related Helicobacter species can cause persistent bacteremia, enteritis and cellulitis in patients with XLA and are difficult to diagnose and eradicate. In this context, detection of circulating microbial cell-free DNA (mcfDNA) by next-generation sequencing (NGS) can be used to identify infectious agents in susceptible immunocompromised hosts. Methods We report 4-year-old fraternal twins with XLA presenting with disparate clinical manifestations due to C. upsaliensis and coinfection with C. upsaliensis and H. canis respectively. Results Patient #1 was diagnosed with XLA at 11 months-old and was started on weekly immunoglobulin replacement. At 2.5 yo he developed recurrent fever, abdominal pain and diarrhea. Endoscopy/colonoscopy revealed focal ileocolitis without ulceration or granulomas. Stool infectious workup, including bacterial stool cultures, was negative. At 3 yo he developed gram- rod bacteremia that was not further identified but successfully treated with ceftriaxone. He presented 2 months later with fever and diarrhea; blood cultures were negative, but stool culture grew C. jejuni. His symptoms recrudesced after two months and he was treated for presumed recurrent Campylobacter infection. Intermittent fevers and diarrhea recurred, and repeat stool culture grew C. upsaliensis identified by MALDI-TOF resulting in a 3-month course of azithromycin. Stool PCR remained positive for Campylobacter species after one month of therapy. Fever and diarrhea recurred after completion of therapy and stool culture again grew C. upsaliensis but sensitivities could not be obtained. McfDNA testing confirmed C. upsaliensis and therapy with ertapenem, ciprofloxacin, amoxicillin and doxycycline was initiated. He remained symptom free three months into therapy. Patient #2 was diagnosed with XLA at 1 yo and started weekly immunoglobulin replacement. At 3.5 yo he developed fever, erythema nodosum (EN) and arthritis. Given his twin’s diagnosis of Campylobacter enteritis with likely bacteremia, a presumptive diagnosis of Campylobacter related reactive arthritis and EN was made. Treatment with Naprosyn and 14 days of azithromycin failed to prevent the return of EN post therapy. He had multiple courses of azithromycin each followed by return of EN rash. Following completion of therapy, he presented with high fevers, worsening rash, leukocytosis and elevated ESR. Blood and stool cultures were obtained and returned negative. Despite completion of a 3 month course of azithromycin, stool PCR remained positive for Campylobacter species and his symptoms persisted. The recrudescence of fevers and worsening rash 2 months after completion of therapy prompted repeat blood and stool cultures that returned negative. McfDNA testing was obtained in that context and identified Helicobacter canis and Campylobacter upsaliensis. Treatment with ertapenem, ciprofloxacin, amoxicillin and doxycycline was initiated and he remained symptom free three months into therapy. Conclusions Physicians should be aware of the varied presentations of chronic Campylobacter and Helicobacter infection in XLA patients. Plasma NGS for circulating mcfDNA in immunocompromised patients offers a rapid, non-invasive means of detecting these fastidious organisms that can be difficult to diagnose using more conventional means.