Assessing the molecular epidemiology and antimicrobial susceptibility profiles of thermophilic campylobacter species from human and animal feces in South Korea and Tanzania

PhD Thesis

Campylobacter species cause human gastroenteritis and have developed resistance to existing antimicrobials. The epidemiology of Campylobacter infections is not fully understood due to a complex genome and the existence of various reservoirs. Poultry is the primary reservoir of Campylobacter but other domestic and wild animals have also been reported as contributing sources. Natural products are regarded as alternative treatments in the post-antibiotic era while the whole-genome sequencing (WGS) is seen as a promising technology towards deciphering the epidemiology and evolution of Campylobacter. The main objective of this research was to assess the molecular epidemiology and antimicrobial susceptibility profiles of thermophilic Campylobacter species from human and animal feces in South Korea and Tanzania. Specifically, this study aimed at 1) determining the antimicrobial resistance (AMR) profiles, virulence genes, and genetic diversity of thermophilic Campylobacter species isolated from a layer poultry farm in Korea; 2) determining the susceptibility of layer chicken-derived and reference Campylobacter strains to selected natural products and frontline antibiotics; and 3) carrying out genomic characterization of fluoroquinolone (FQ)-resistant thermophilic Campylobacter strains isolated from layer chicken faeces in Gangneung, Korea by whole-genome sequencing; and 4) carrying out molecular techniques for the detection of Campylobacter species from human stool and cattle faecal samples in Kilosa district, Tanzania. In the first study, 153 faecal samples were obtained from two layer chicken farms in Gangneung, South Korea. Isolation of Campylobacter was carried out by culture, followed by species confirmation with PCR and sequencing. Antimicrobial susceptibility tesing for six antimicrobials (sitafloxacin, ciprofloxacin, nalidixic acid, gentamicin, erythromycin, and tetracycline) was performed by broth microdilution. Three antimicrobial resistance (AMR) and nine virulence genes were screened by PCR. Genotyping was performed by flagellin A-restriction fragment length polymorphism (flaA-RFLP) and multilocus sequence typing (MLST). Of the 153 samples, Campylobacter spp. were detected in 55 (35.9%) with 49 (89.1%) and 6 (10.9%) being C. jejuni and C. coli, respectively. High-level resistance (MIC ≥ 32 μg/mL) was observed for ciprofloxacin (100%), nalidixic acid (100%), and tetracycline (C. jejuni: 93.9%; C. coli: 83.3%) but no resistance was observed for sitafloxacin. Sequencing confirmed mutations associated with quinolones (C257T in gyrA gene) and tetracycline [tet(O) gene] resistance. Multidrug resistance at a rate of 8.2% was exclusively recorded in C. jejuni. cstII, flaA, dnaJ, cadF, and cdtB were found in all Campylobacter isolates while the proportions for other genes (ciaB, pldA, and csrA) varied from 33.3 to 98 %. The flaA-RFLP typing resulted in 21 types for C. jejuni and five for C. coli: 5 while MLST showed 10 sequence types (STs) for C. jejuni and three STs for C. coli with CC-607 (ST 3611) and CC-460 (ST-460) being predominant. Among the 10 STs of C. jejuni, three were newly assigned. The second work determined the susceptibility of layer chicken-derived and reference Campylobacter strains to selected natural products and frontline antibiotics. The efficacy of selected natural products was assessed by broth microdilution and the optical density recorded by a microplate reader. Antibiotic resistance genes (tet(O) and gyrA) were characterized at the molecular level. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 25 to 1600 μg/mL. Cinnamon extract, cinnamon oil, (E)-Cinnamaldehyde, clove oil, eugenol, and baicalein had the lowest MIC and MBC values (25–200 μg/mL). Chicken-derived isolates were resistant to quinolones and tetracycline but sensitive to erythromycin and gentamicin. NPs were effective against both AMR and sensitive Campylobacter strains. The third study characterized FQ-resistant C. jejuni (200605) and C. coli (200605) strains of layer chicken origin by whole-genome sequencing using Illumina sequencing technology. A phylogenetic relationship to existing strains was also established. 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. No resistance to macrolides and aminoglycosides was found. Putative genes conferring resistance to doxycycline, minocycline; blaOXA-452, cephalosporin, and penam were also recorded in both strains. Virulence genes associated with motility, chemotaxis, and capsule formation were found in both strains. 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 of such rare STs. Lastly, 70 human stool and 30 cattle faecal samples were collected in Kilosa district, Tanzania. Species confirmation was conducted by polymerase chain reaction (PCR) and 16S rRNA sequencing while the phylogenetic analysis was done with 16S rRNA sequences. Campylobacter species detection rates were 65.7% (46/70) and 20.0% (6/30) in humans and cattle, respectively. In humans, C. concisus was the predominant species 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). Molecular detection methods need to be adopted in routine Campylobacter testing and surveillance studies because they provide results in short period of time. The findings of this study highlight (i) the usefulness of molecular techniques in emerging Campylobacter detection, (ii) the molecular epidemiology and antimicrobial resistance of Campylobacter from layers; (iii) the importance of some natural products as alternative to conventional antimicrobials in the control of Campylobacter infections; and (iv) WGS data of Campylobacter from layer chicken for better understanding the Campylobacter epidemiology and serve as a baseline for future studies. This study also identified new sequence types (STs) including ST-10645, ST-10647, ST-10648 that were isolated from layer chicken in Korea. I recommend further studies on (i) the synergism of natural products and existing antimicrobials; and (ii) chemical profiling of used plant extracts and their anti- adhesion effects to both biotic and abiotic surfaces.

PASET-RSIF