A high number of multidrug-resistant and predominant genetically related cluster of Shigella flexneri strains isolated over 34 years in Brazil.

Shigella flexneri has been a major public health problem in developing countries. This work analyzed the frequency of 16 virulence genes, the genotypic diversity, and the antimicrobial resistance profiles of 130 S. flexneri strains isolated in Brazil. The ipaH gene was found in all the 130 strains. The frequencies of the other genes were variable ial (88.5%), sigA (82.3%), iuc (74.6%), virA (73%), pic (72.3%), virF (57.7%), sat (48.5%), ipaBCD (37%), sen (36%), set1A (35.4%), sepA (30%), set1B (30%), virB (14%), icsA (10%), and ipgD (5.4%). A total of 57 (43.8%) strains were multidrug-resistant. ERIC-PCR grouped 96 of the strains into a single cluster with ≥ 70.4% of similarity, 75 of these strains presented a similarity ≥ 80.9%. PFGE grouped 120 of the strains into a single cluster with 57.4% of similarity and 82 of these strains presented a similarity ≥ 70.6%. In conclusion, the high frequency of some virulence genes reinforces the pathogenic potential of the strains studied. The high rates of MDR strains are alarming once it may lead to failure when antimicrobial treatment is necessary. Genotype techniques reveled a major cluster with high genetic similarity including S. flexneri strains from the different Brazilian states and distinct years of isolation, showing that they probably emerged from a common ancestor.

Molecular Epidemiology and Resistance Profile of Campylobacter jejuni and Campylobacter coli Strains Isolated from Different Sources in Brazil.

Aims: The objectives of this study were to genotype a total of 48 Campylobacter jejuni and 39 Campylobacter coli strains isolated in Brazil from 1995 to 2016 by multilocus sequence typing (MLST) and to determine their resistance profile. The presence or points of mutation in the related resistance genes was verified. Results: By MLST, C. jejuni strains were typed into 36 STs and C. coli strains were typed into 27 STs. A total of 70.8% of C. jejuni and 35.9% of C. coli were resistant to at least one antimicrobial tested. The tet(O) gene was detected in 43.7% C. jejuni and in 12.8% C. coli. The ermB gene was not detected and one C. jejuni presented the mutation in the 23S rRNA gene. Besides, 58.3% C. jejuni presented the substitution T86I in the quinolone resistance-determining region of gyrA and 15.4% C. coli presented the substitution T38I. The cmeB gene was detected in 97.9% C. jejuni and in 97.4% C. coli. Conclusion: The presence of C. jejuni and C. coli resistant to some antimicrobial agents of clinical use is of public health concern. The presence of STs shared between Brazilian strains and isolates of different countries is of concern since it might suggest a possible spread of these shared types.

Molecular characterization of multidrug-resistant Shiga toxin-producing Escherichia coli harboring antimicrobial resistance genes obtained from a farmhouse.

Shiga toxin-producing Escherichia coli (STEC) colonize the gastrointestinal tract of animals; however, STEC may also cause severe diarrheal diseases. Food-producing animals have been acting as reservoirs and disseminators of multidrug-resistant (MDR) bacteria and antimicrobial resistance genes (ARGs); however, there are few studies characterizing molecularly bacterial isolates from sheep. Therefore, this study aimed to characterize E. coli isolates obtained from feces of sheep in a Brazilian farmhouse. A total of 14 MDR E. coli isolates were obtained from 100 feces samples, six of which were classified as non-O157 STEC (stx1, stx2 and ehxA). MDR E. coli isolates presented different ARGs [blaCTX-M-Gp9, blaCMY, blaSHV, qnrS, oqxB, aac(6')-Ib, tet(A), tet(B), tet(C), sul1, sul2, and cmlA] and plasmids (IncI1, IncFrepB, IncFIB, IncFIA, IncHI1, IncK, and ColE-like). In addition, mutations in the quinolone-resistance determining region of GyrA (Ser83Leu; Asp87Asn) and ParC (Glu84Asp) were detected. PFGE showed a high genetic diversity (30.9 to 83.9%) and thirteen STs were detected (ST25, ST48, ST155, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST2522, ST3270, ST5036, and ST7100). Subtyping of the fimH gene showed seven fimH-type (25, 32, 38, 41, 54, 61, and 366). The results found in the present study showed high genetic diversity among MDR ARGs-producing E. coli obtained from a farmhouse. This study reports for the first time, the presence of MDR STEC and non-STEC belonging to ST25, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST3270, ST5036, and ST7100 in sheep, and contributes to the surveillance studies associated with One Health concept.

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.

High survival rates of Campylobacter coli under different stress conditions suggest that more rigorous food control measures might be needed in Brazil.

Campylobacter spp. have been the most commonly reported gastrointestinal bacterial pathogen in many countries. Consumption of improperly prepared poultry meat has been the main transmission route of Campylobacter spp. Although Brazil is the largest exporter of poultry meat in the world, campylobacteriosis has been a neglected disease in the country. The aim of this study was to characterize 50 Campylobacter coli strains isolated from different sources in Brazil regarding the frequency of 16 virulence genes and their survival capability under five different stress conditions. All strains studied presented the cadF, flaA, and sodB genes that are considered essential for colonization. All strains grew at 4 °C and 37 °C after 24 h. High survival rates were observed when the strains were incubated in BHI with 7.5% NaCl and exposed to acid and oxidative stress. In conclusion, the pathogenic potential of the strains studied was reinforced by the presence of several important virulence genes and by the high growth and survival rates of the majority of those strains under different stress conditions. The results enabled a better understanding of strains circulating in Brazil and suggest that more rigorous control measures may be needed, given the importance of contaminated food as vehicles for Campylobacter coli.

Virulence-related genes, adhesion and invasion of some Yersinia enterocolitica-like strains suggests its pathogenic potential.

Yersina enterocolitica-like species have not been extensively studied regarding its pathogenic potential. This work aimed to assess the pathogenic potential of some Y. enterocolitica-like strains by evaluating the presence of virulence-related genes by PCR and their ability to adhere to and invade Caco-2 and HEp-2 cells. A total of 50 Y. frederiksenii, 55 Y. intermedia and 13 Y. kristensenii strains were studied. The strains contained the following genes: Y. frederiksenii, fepA(44%), fes(44%) and ystB(18%); Y. intermedia, ail(53%), fepA (35%), fepD(2%), fes(97%), hreP(2%), ystB(2%) and tccC(35%); Y. kristensenii, ail(62%), ystB(23%), fepA(77%), fepD(54%), fes(54%) and hreP(77%). Generally, the Y. enterocolitica-like strains had a reduced ability to adhere to and invade mammalian cells compared to the highly pathogenic Y. enterocolitica 8081. However, Y. kristensenii FCF410 and Y. frederiksenii FCF461 presented high invasion potentials in Caco-2 cells after five days of pre-incubation increased by 45- and 7.2-fold compared to Y. enterocolitica 8081, respectively; but, the ail gene was not detected in these strains. The presence of virulence-related genes in some of the Y. enterocolitica-like strains indicated their possible pathogenic potential. Moreover, the results suggest the existence of alternative virulence mechanisms and that the pathogenicity of Y. kristensenii and Y. frederiksenii may be strain-dependent.

Genotyping of Campylobacter coli strains isolated in Brazil suggests possible contamination amongst environmental, human, animal and food sources.

Campylobacter coli and Campylobacter jejuni are two of the most common causative agents of food-borne gastroenteritis in numerous countries worldwide. In Brazil, campylobacteriosis is under diagnosed and under-reported, and few studies have molecularly characterized Campylobacter spp. in this country. The current study genotyped 63 C. coli strains isolated from humans (n512), animals (n521), food (n510) and the environment (n520) between 1995 and 2011 in Brazil. The strains were genotyped using pulsed-field gel electrophoresis (PFGE), sequencing the short variable region (SVR) of the flaA gene ( flaA-SVR) and high-resolution melting analysis (HRMA) of the clustered regularly interspaced short palindromic repeat (CRISPR) locus to better understand C. coli genotypic diversity and compare the suitability of these three methods for genotyping this species. Additionally, the discrimination index (DI) of each of these methods was assessed. Some C. coli strains isolated from clinical and non-clinical origins presented ≥80 % genotypic similarity by PFGE and flaA-SVR sequencing. HRMA of the CRISPR locus revealed only four different melting profiles. In total, 22 different flaA-SVR alleles were detected. Of these, seven alleles, comprising gt1647­gt1653, were classified as novel. The most frequent genotypes were gt30 and gt1647. This distribution reveals the diversity of selected Brazilian isolates in comparison with the alleles described in the PubMLST database. The DIs for PFGE, flaA­SVR sequencing and CRISPR-HRMA were 0.986, 0.916 and 0.550, respectively. PFGE and flaA-SVR sequencing were suitable for subtyping C. coli strains, in contrast to CRISPR-HRMA. The high genomic similarity amongst some C. coli strains confirms the hypothesis that environmental and food sources potentially lead to human and animal contamination in Brazil.