Phenotypic analysis and whole genome sequencing of Campylobacter coli strains isolated from different sources over 16 year-period in Brazil

Author(s)

Carolina Nogueira Gomes

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2020-05-04

Examining board members

Juliana Pfrimer Falcão; Elaine Cristina Pereira de Martinis; Cristiano Gallina Moreira; José Freire da Silva Neto

• Advisor: Juliana Pfrimer Falcão

Abstract

Campylobacter spp., mainly the C. coli and C. jejuni species, are the most common cause of bacterial foodborne disease worldwide. In Brazil, there is a paucity of studies on C. coli, which makes it difficult to evaluate the involvement of this bacterium as a cause of diseases in humans and animals, as well as to determine the impact of its presence in food and the environment. The main aim of this study was to characterize and analyze comparatively by phenotypic tests and whole genome sequencing 63 C. coli strains isolated from clinical and non-clinical sources. The phenotypic tests were performed using 50 C. coli strains isolated from humans (12), animals (15), food (8) and the environment (15). In tests performed at both 37ºC and 4ºC, after 0.5 h and 24 hours of incubation, the majority of the C. coli studied grew more than or equal to the control strains Salmonella Typhimurium ATCC 14028 and C. jejuni ATCC 3329. After the first hour of incubation in BHI with a final concentration of 7.5% NaCl (wt/vol) and BHI pH=4.5 all the strains remained 100% viable. After two hours of incubation in BHI with a final concentration of 7.5% NaCl (wt/vol), a total of 13 strains including 12 (24%) C. coli strains plus the control strain S. Typhimurium ATCC 14028 showed 100% survival. Likewise, six C. coli strains plus the two controls strains studied showed 100% survival after 2 hours of incubation in BHI pH=4,5. A total of 23 strains exhibited growth after incubation at 42°C under aerophilic conditions for 24 hours and were select to oxidative stress tests for which 16 strains survived during 10 minutes of incubation. Seventeen C. coli strains showed invasion and survival percentage, in epithelial Caco-2 cells greater than or equal to the percentage (73.2%) showed by S. Typhimurium ATCC 14028 and 50% of the studied strains showed percentages of survival greater than or equal to the percentage presented by C. jejuni ATCC 33291 (64.6%). In the human macrophage U-937 survival assay, seven C. coli strains showed percentages of survival greater than or equal to the percentage showed by S. Typhimurium ATCC 14028 (73.2%) and all the strains studied showed percentages of survival greater than C. jejuni ATCC 33291 (30.4 %). In the chicken macrophage HD-11 survival assay, two strains showed percentages of survival greater than or equal to the percentage presented by S. Typhimurium ATCC 14028 (76.7 %) and 45 of 50 strains studied showed percentages of invasion and survival greater than or equal to the percentage presented by C. jejuni ATCC 33291(46%). The search of resistance genes in the 63 C. coli strains studied revealed the presence of blaOXA-605 e blaOXA-61 gene in 54% of them. The tet(O) gene was detected in 14 (22.2%) strains and QRDR gyrA mutations was detected in 8 (12.7%) strains. The cmeB gene was detected in 6 (9.5%) of the strains studied and the aadE-Cc, aph(3\')-IIIa, sat4, aad9 genes were detected in 4 (6.3%), 1 (1.6%), 1(1.6%) e 1(1.6%) strains, respectively. The MLST methodology identified 18 different STs being that 3 strains showed new STs not yet described in the database and 16 out of the 18 STs founded belong to the Clonal Complex (CC) 828. The phylogenetic analysis by cgMLST clustered the 63 C. coli strains into 5 main cluster which presented a high genomic diversity among them and a high genomic similarity among some strains isolated from different sources was observed. The phylogenetic analysis by cgMLST of the 63 C. coli strains isolated in Brazil in comparison to 3,401 isolates from different countries allows us to observe a wide distribution of the strains studied and a high genetic similarity among some strains isolated in Brazil. It can be concluded that the behavior of C. coli studied under different stress conditions is probably strain-dependent. The high invasion and survival capacity in Caco-2 epithelial cells and in U-937 and HD-11 macrophages reinforces the pathogenic potential of these strains. The high frequency of the blaoxa605 (blaoxa61) gene is of concern and may lead to therapeutic failure when treatment is done using β-lactams. The MLST and cgMLST analyzes showed a high similarity among some strains studied suggesting that the environment and food have been a possible source of contamination and / or transmission to humans and animals in Brazil. (AU)

• FAPESP's process: 15/23408-0 - Phenotypic analysis, genome and transcriptome sequencing of Campylobacter coli strains isolated from different sources during 16 years in Brazil
• Grantee: Carolina Nogueira Gomes
• Support Opportunities: Scholarships in Brazil - Doctorate