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| Author(s): |
Felipe Pinheiro Vilela
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | Ribeirão Preto. |
| Institution: | Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) |
| Defense date: | 2021-04-23 |
| Examining board members: |
Juliana Pfrimer Falcão;
Monique Ribeiro Tiba Casas;
Mariza Landgraf
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| Advisor: | Juliana Pfrimer Falcão |
| Abstract | |
Infections caused by non-typhoid Salmonella enterica serovars are among the main causes of foodborne diseases worldwide. Salmonella enterica subspecies enterica serovar Infantis (S. Infantis) is a non-typhoid and ubiquitous serovar, able to infect a broad range of hosts besides humans and is among the most isolated serovars in the world. In Brazil, although S. Infantis strains have demonstrated a high prevalence, few studies exclusively characterized an expressive number of strains of this serovar. In this way, the present study aimed to characterize the genotypic diversity, to verify the presence of virulence markers and the phenotypic and genotypic antimicrobial resistance profiles of S. Infantis strains isolated from diverse sources in Brazil. A total of 80 S. Infantis strains isolated from food (n=27), the environment (n=24), humans (n=19), animals (n=7) and animal ration (n=3) between 2013 and 2018 from nine Brazilian states were studied. Antimicrobial susceptibility testing was performed by disk-diffusion for 18 antimicrobials. The strains studied were molecularly typed by Pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed and its data were used for the search of 12 virulence genes, acquired resistance genes and chromosomal point mutations associated to antimicrobial resistance. In adittion, the strains were also characterized by Multi-locus sequence typing (MLST), core genome MLST (cgMLST) and the analyses of Single-nucleotide polymorphisms (SNPs) and Clustered regularly interspaced short palindromic repeats (CRISPR) using WGS data. A total of 72 strains (90%) showed resistance or intermediate resistance profiles to at least one of the drugs tested and 31 (38,8%) showed resistance to three or more antimicrobials of three different drug classes. The most frequent resistant rates among the strains studied were ampicillin (57,8%), piperacillin (51,3%), tetracycline (37,5%), chloranphenicol (35,0%), cefotaxime (30,0%), cefazoline (26,3%) and ceftriaxone (23,8%). It is also important to notice that 42,5% of the strains exhibited intermediate resistance profiles to ciprofloxacin. PFGE divided the 80 S. Infantis strains into 43 PFGE-types, three distinct clusters with a ≥80% similarity among the strains comprised in each group and an overall similarity among all strains of ≥78,2%, and also presented a discriminatory index (DI) of 0,966. Among the 12 virulence markers searched, invA, sopB, sopD, sopE2, sipA, sipD, flgK, flgL, fljB, sifA and ssaR were present in all strains, while spvB was absent in all strains analyzed. Strains also harbored aminoglycoside resistance genes aac(6\')-Iaa (100%) and aadA12 (2.5%), β-lactam resistance genes blaTEM-1 (40%), blaCTX-M-8 (11.3%), blaCMY-2 (10.0%) and blaCMY-61 (1.3%), and resistance genes dfrA8 (37.5%), tet(A) (36.3%) and floR (36.3%), which are capable to confer resistance to diaminopyrimidine compounds, tetracyclines and amphenicols, respectively. All strains presented the point mutations Gln624→Lys in gyrB gene and Thr57→Ser and Thr255→Ser in parC gene, while a single strain presented the point mutation Val702→Ala in parC gene, which are all capable to confer resistance to quinolones and fluoroquinolones. Also, a single strain showed mutation Asp28→Tyr in pmrA gene, which is capable to confer resistance to antimicrobial peptides. All strains studied belonged to ST32 as demonstrated by MLST. The SNP analysis and cgMLST clustered the strains into two and three distinct clusters, respectively, and in both methodologies above 70% of the strains were grouped into the same cluster. CRISPR analysis clustered all strains into the same cluster, with an overall similarity iv among all strains of ≥80.7% and a 0,696 DI. In conclusion, the high prevalence of virulence markers detected, related to motility, cell invasion and survival within phagocytic cells, reinforces the pathogenic potential of this serovar to cause infections in humans, as well as the risk of its presence in food, and in environmental and veterinary sources. The high rates of strains presenting phenotypic and genotypic antimicrobial resistance alert for the potential risk of therapeutic failure in S. Infantis infections in humans when therapy is necessary. The absolute presence of ST32 revealed among the strains studied by MLST demonstrates the dominance of this ST among strains of serovar Infantis and the capacity of this methodology to precisely identify strains of this serovar. PFGE showed an adequate discriminatory capacity to differentiate the strains and its results suggest the circulation of a prevalent subtype of S. Infantis in different sources and states of the country. Similarly, cgMLST and SNP and CRISPR analyses suggest that the majority of the S. Infantis strains studied descend from a prevalent subtype that has been contaminating humans, animals, the environment and food, corroborating with PFGE results. Together, the results obtained reinforce the potential hazard that the S. Infantis strains of clinical and non-clinical sources studied may represent for the public health and food safety fields in Brazil. (AU) | |
| FAPESP's process: | 19/06947-6 - Molecular characterization of the genotypic diversity, resistance profile and pathogenic potential of Salmonella Infantis strains isolated from multiple sources in Brazil |
| Grantee: | Felipe Pinheiro Vilela |
| Support Opportunities: | Scholarships in Brazil - Master |