Fábio Campioni

Genetic diversity, virulence genes and antimicrobial resistance of Salmonella Enteritidis isolated from food and humans over a 24-year period in Brazil

Salmonellosis is a major health problem worldwide. Serovar Enteritidis has been a primary cause of Salmonella outbreaks in many countries. In Brazil, few molecular typing studies have been performed. The aims of this study were to molecularly type Salmonella Enteritidis strains isolated in Brazil in order to determine the genetic relationship between strains of food and human origin, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 128 S. Enteritidis strains isolated from human feces (67) and food (61) between 1986 and 2010 were studied. The genotypic diversity was assessed by ERIC-PCR and PFGE using XbaI, the antimicrobial resistance by the disc-diffusion assay and the presence of the SPI-1, SPI-2 and pSTV virulence genes assessed by PCR. The ERIC-PCR results revealed that 112 strains exhibited a similarity of >85.4% and the PFGE that 96 strains exhibited a similarity of >80.0%. Almost all strains (97.6%) harbored all 13 virulence genes investigated. Thirty-six strains (28.12%) were resistant to nalidixic acid. In conclusion, the nalidixic acid resistance observed after 1996 is indicative of an increase in the use of this drug. It may be suggested that these 128 strains might have descended from a common ancestor that differed little over 24 years and has been both contaminating food and humans and causing disease for more than two decades in Brazil.

Pulsed-Field Gel Electrophoresis characterization of Listeria monocytogenes isolates from cheese manufacturing plants in São Paulo, Brazil.

This study aimed to evaluate the occurrence of Listeria monocytogenes in cheese and in the environment of three small-scale dairy plants (A, B, C) located in the Northern region state of São Paulo, Brazil, and to characterize the isolates using conventional serotyping and PFGE. A total of 393 samples were collected and analyzed from October 2008 to September 2009. From these, 136 came from dairy plant A, where only L. seeligeri was isolated. In dairy plant B, 136 samples were analyzed, and L. innocua, L. seeligeri and L. welshimeri were isolated together with L. monocytogenes. In dairy plant C, 121 samples were analyzed, and L. monocytogenes and L. innocua were isolated. Cheese from dairy plants B and C were contaminated with Listeria spp, with L. innocua being found in Minas frescal cheese from both dairy plants, and L. innocua and L. monocytogenes in Prato cheese from dairy plant C. A total of 85 L. monocytogenes isolates were classified in 3 serotypes: 1/2b, 1/2c, and 4b, with predominance of serotype 4b in both dairy plants. The 85 isolates found in the dairy plants were characterized by genomic macrorestriction using ApaI and AscI with Pulsed Field Gel Electrophoresis (PFGE). Macrorestriction yielded 30 different pulsotypes. The presence of indistinguishable profiles repeatedly isolated during a 12-month period indicated the persistence of L. monocytogenes in dairy plants B and C, which were more than 100 km away from each other. Brine used in dairy plant C contained more than one L. monocytogenes lineage. The routes of contamination were identified in plants B and C, and highlighted the importance of using molecular techniques and serotyping to track L. monocytogenes sources of contamination, distribution, and routes of contamination in dairy plants, and to develop improved control strategies for L. monocytogenes in dairy plants and dairy products.

Genotypic diversity and virulence markers of Yersinia enterocolitica biotype 1A strains isolated from clinical and non-clinical origins.

Yersinia enterocolitica biotype 1A (B1A) strains are considered as non‐pathogenic; however, some reports have identified some strains as the causal agents of infection. In South America, few studies molecularly characterized the strains of this biotype. This work typed 51 B1A strains isolated from clinical and non‐clinical sources from Brazil and Chile by Enterobacterial Repetitive Intergenic Consensus‐PCR (ERIC‐PCR) to elucidate their genotypic diversity, and verify the distribution of 11 virulence markers by PCR. The strains were divided into two groups, ERIC‐A and ERIC‐B, clustered independently of their clinical or non‐clinical origin. No differences were observed in the frequencies of the virulence markers between clinical and non‐clinical strains. However, the genes ystB, hreP and myfA occurred exclusively in the strains of the group ERIC‐A. Some clinical and non‐clinical strains were clustered in the same genetic group and presented the same number of virulence markers, which might suggest the role of the environment and food as a potential source of infection for humans and animals. The results corroborate with the hypothesis that B1A strains are divided into two main clusters that differ in the frequency of some virulence markers, a fact observed for the first time in South American strains.

MLVA typing reveals higher genetic homogeneity among S. Enteritidis strains isolated from food, humans and chickens in Brazil in comparison to the North American Strains

Salmonella Enteritidis (S. Enteritidis) is a major causative agent of food-borne gastroenteritis associated with the consumption of contaminated poultry products. In this study we used multilocus variable number of tandem repeats (VNTRs) analysis (MLVA) to discriminate a total of 188 S. Enteritidis strains recovered from human (n=67), food (n=61) and chickens (n=60) during a 24 year period (1986 through 2010) in Brazil. MLVA profiles of the 188 strains from Brazil were compared to the MLVA profiles of 100 human clinical (n=52) and poultry-associated (n=48) strains isolated in North America between 1986 and 2008. MLVA typing led to classification of the 288 strains from Brazil and North America into two major clusters named A and B with 35% of similarity. Cluster A consisted of a vast majority of strains isolated from North America (n=71) and only three strains isolated from Brazil which included two pre-pandemic strains (SE5 and SE4). In contrast, cluster B consisted of all of the post-pandemic strains isolated from Brazil (n=185) and fewer strains isolated from North America (n=29). In general, MLVA typing showed that the North American strains were more genetically diverse whereas Brazilian strains were more genetically clonal. The clustering of pre-pandemic strains from Brazil with the North American strains suggests the possibility that the pre-pandemic strains were more likely genetically diverse; however after 1993 a new and prevalent subtype of S. Enteritidis was introduced in this country. This is the first study describing MLVA genotyping of the S. Enteritidis strains isolated from Brazil.

Comparison of four molecular methods to type Salmonella Enteritidis strains

This study compared the pulsed‐field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), multilocus variable‐number of tanden‐repeat analysis (MLVA), and multilocus sequence typing (MLST) methods for typing 188 Salmonella Enteritidis strains from different sources isolated over a 24‐year period in Brazil. PFGE and ERIC‐PCR were more efficient than MLVA for subtyping the strains. However, MLVA provided additional epidemiological information for those strains. In addition, MLST showed the Brazilian strains as belonging to the main clonal complex of S. Enteritidis, CC11, and provided the first report of two new STs in the S. enterica database but could not properly subtype the strains. Our results showed that the use of PFGE or ERIC‐PCR together with MLVA is suitable to efficiently subtype S. Enteritidis strains and provide important epidemiological information.

Genotyping of Yersinia enterocolitica biotype 1A strains from clinical and nonclinical origins by pulsed-field gel electrophoresis

Yersinia enterocolitica biotype 1A (B1A) strains are considered mainly nonpathogenic. However, some studies considered strains of this biotype to be the causal agents of infections in humans and animals. In South America, there are no studies that have compared clinical and nonclinical strains of B1A typed by pulsed-field gel electrophoresis (PFGE) and none that have compared the capability of different enzymes on typing these strains. This study typed 51 Y. enterocolitica B1A strains isolated in Brazil and Chile by PFGE, testing the enzymes XbaI, NotI, and XhoI. The resulting dendrograms discriminated the strains in 47, 40, and 49 pulsotypes generated by the cleavage with the enzymes XbaI, NotI, and XhoI, respectively. The majority of the strains were grouped independently of their clinical or nonclinical origins. The high discriminatory power of PFGE confirmed the heterogeneity of B1A strains but could not divide the strains studied into clusters that differed in the frequency of some virulence genes as observed in studies using other methodologies.

Prevalence of gyrA Mutations in Nalidixic Acid-Resistant Strains of Salmonella Enteritidis Isolated from Humans, Food, Chickens, and the Farm Environment in Brazil

Salmonella Enteritidis strains that are resistant to nalidixic acid and exhibit reduced susceptibility to fluoroquinolones have been increasing worldwide. In Brazil, few studies have been conducted to elucidate the quinolone resistance mechanisms of S. Enteritidis strains. This study analyzed the profile of gyrA, gyrB, parC, and parE mutations and plasmid-mediated quinolone resistance (PMQR) mechanisms in S. Enteritidis NalR strains isolated in Brazil. Moreover, the minimum inhibitory concentrations (MICs) of ciprofloxacin were evaluated in 84 NalR strains and compared with 20 NalS strains. The mutation profiles of the gyrA gene were accessed by high-resolution melting analysis and gyrB, parC, and parE by quinolone resistance-determining region sequencing. The MICs of ciprofloxacin were accessed with Etest®. The strains were divided into five gyrA melting profiles. The NalR strains exhibited the following amino acid substitutions: Ser97→Pro, Ser83→Phe, Asp87→Asn, or Asp87→Tyr. The average MICs of ciprofloxacin was 0.006 μg/ml in the NalS and 0.09 μg/ml in the NalR strains. No points of mutation were observed in the genes gyrB, parC, and parE. The qnrB gene was found in two strains. In conclusion, the reduced susceptibility to ciprofloxacin observed in NalR strains may cause treatment failures once this drug is commonly used to treat Salmonella infections. Moreover, this reduced susceptibility in these Brazilian strains was provided by target alteration of gene gyrA and not by mobile elements, such as resistance plasmids.

Draft Genome Sequences of 256 Salmonella enterica subsp. enterica Serovar Enteritidis Strains Isolated from Humans, Food, Chickens, and Farm Environments in Brazil

ABSTRACT Salmonella enterica subsp. enterica serovar Enteritidis emerged in the late 1980s as the most isolated Salmonella serovar worldwide. Here, we report the draft genomes of 256 S. Enteritidis strains isolated from humans, food, chickens, and farm environments in Brazil. These draft genomes will help enhance our understanding of this serovar in Brazil.

Changing of the Genomic Pattern of Salmonella Enteritidis Strains Isolated in Brazil Over a 48 year-period revealed by Whole Genome SNP Analyses

Salmonella Enteritidis became the main serovar isolated from gastroenteritis cases in Brazil after the 90’s. In this study we used whole genome sequence analysis to determine the phylogenetic relationships among a collection of strains isolated in Brazil to identify possible genomic differences between the strains isolated in the pre and post-epidemic period. Also, we compared our data from strains isolated in Brazil to strains available in the public domain from other South American countries. Illumina technology was used to sequence the genome of 256 Salmonella Enteritidis strains isolated over a 48 year-period in Brazil, comprising the pre- and post-epidemic period. Phylogenetic analyses revealed distinct lineages for strains isolated before and after 1994. Moreover, the phage region SE20 that may be related to the emergence of Salmonella Enteritidis worldwide was present only in strains of the post-epidemic cluster. In conclusion, our results showed that the genomic profile of Salmonella Enteritidis strains isolated in Brazil shifted after 1994, replaced by a global epidemic group of strains. It may be hypothesized that the presence of the prophage SE20 might have conferred to these strains a better ability to colonize chicken and consequently to infect and cause disease in humans, which might better explain the increase in the number of S. Enteritidis cases in Brazil and other South American countries. However, to verify this hypothesis further studies are needed.

Genotypic Resistance to Quinolone and Tetracycline in Salmonella Dublin Strains Isolated from Humans and Animals in Brazil

Resistance of Salmonella Dublin strains to quinolones and tetracycline has been increasing worldwide. Studies regarding the genotypic resistance traits of strains of this serovar isolated in Brazil are scarce. This study aims to examine the genetic characteristics of Salmonella Dublin strains isolated in Brazil, which are associated with resistance to quinolone and tetracycline. The minimum inhibitory concentrations (MICs) of nalidixic acid, ciprofloxacin, and tetracycline of the 10 strains sensitive and 21 strains resistant to quinolone and tetracycline were determined using Etest.® The mutation profiles of the gyrA, gyrB, parC, and parE genes were accessed by sequencing, while the presence of plasmid-mediated quinolone resistance and tet genes was analyzed by PCR. Quinolone-resistant strains presented the amino acid substitutions Ser96→Tyr, Ser96→Phe, Asp107→Asn, or Asp108→Gly on the gyrA gene, and the Ser224→Phe and Glu231→Asp mutations on the gyrB gene. The qnrA, tet(A), and tet(B) genes were detected in 5, 13, and 6 strains, respectively. Analysis of the MIC values revealed that 1 and 3 strains presented intermediate and resistant MIC profiles to nalidixic acid, respectively; 6 strains presented intermediate MIC profile to ciprofloxacin; and 13 strains presented resistant MIC profile to tetracycline. In the Salmonella Dublin strains studied, quinolone resistance was mainly related to mutation points that led to target alteration in the gyrA and gyrB genes, while tetracycline resistance was associated with the presence of tet(A) and/or tet(B) genes, with the highest resistance levels detected in strains bearing the tet(B) gene. The presence of the aforementioned genotypic resistance traits in Salmonella Dublin strains isolated over 33 years in Brazil indicates that ciprofloxacin or tetracycline therapy against such strains may fail.