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Salmonella Dublin is a cattle-associated serovar sporadically causing disease in humans. S. Dublin strains isolated in Brazil and in other countries were analyzed to determine their phylogenetic relationships, the presence of genes, plasmids, genomic regions related to virulence and antimicrobial resistance genes repertoire, using WGS analyses. Illumina was used to sequence the genome of 112 S. Dublin strains isolated in Brazil from humans (n = 82) and animals (n = 30) between 1983 and 2016. Furthermore, 87 strains from other countries were analyzed. WGSNP analysis revealed three different clades, in which the strains from Brazil belonged to two clades, A and C. Most of the genes and genomic regions searched varied among the strains studied. The siderophore genes iroB and iroC were exclusively found in strains from Brazil and pegD gene, related to fimbrial adherence determinants, were positive in 124 strains from clades A and B but absent in all the strains from clade C (n = 71). Eleven plasmid replicons were found in the strains from Brazil, and nine were exclusively found in strains from other countries. The antimicrobial resistance genes mdsA and mdsB, that encode an efflux pump, were found in all the strains studied. The strains from Brazil carried other resistance genes, such as tet(A) (n = 11), tet(B) (n = 4) and tet(C) (n = 4), blaTEM-1 (n = 4), catA1 (n = 1), aadA1 (n = 1), and sul1 (n = 1). In conclusion, S. Dublin strains isolated in Brazil presented some few unique genes not found in strains from other countries and were allocated into two distinct clades with strains of human and animal origin epidemiologically related. This fact stresses the zoonotic potential of S. Dublin circulating in Brazil for more than 30 years.

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Salmonella Dublin is a strongly cattle-adapted serovar that has also been responsible for severe invasive infections in humans. Although invasive infections by non-typhoid Salmonella have increased in developed and in developing countries, in sub-Saharan Africa these infections have been frequently related to Salmonella Typhimurium strains from Sequence Type (ST) 313 that harbor a possible virulence marker, the bstA gene, broadly detected in S. Dublin strains. The aims of this study were to verify the frequency of bstA by PCR in 113 Salmonella Dublin strains isolated from humans (83) and animals (30) in Brazil and the expression by RT-PCR of bstA, sopE2 and fliC in six strains isolated from humans (4) and animals (2). Moreover, the invasion capacity in Caco-2 human epithelial cells and U937 human macrophages, plus in vivo virulence analysis in Galleria mellonella and the motility were verified for 20 S. Dublin strains isolated from humans (15) and animals (5). All studied strains presented the bstA gene. The relative expression rates ranged from 0.1 to 2.3 fold change for bstA and from no expression to 16.6 fold change for sopE2, while no expression was detected for fliC. The invasion in Caco-2 cells ranged from 54.0 to 88.9% and in U937 cells from 72.9 to 98.1% in the 20 strains studied. In addition, 17 strains presented a highly virulent profile in the G. mellonella model and 15 strains presented a non-motile profile. In conclusion, the presence and expression of bstA in the S. Dublin strains studied suggested that this gene may influence in the invasive characteristic of this serovar. The low expression of sopE2 in strains from human invasive cases suggested that its expression may not be a limiting factor to the invasion of S. Dublin strains. The absence of fliC expression and the low motility rates observed suggest that the flagella absence may favor the host immune system evasion by S. Dublin and the establishment of infection. Moreover, the high mortality rates observed in vivo in Galleria mellonella reinforce the pathogenic potential of S. Dublin strains.

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RESUMO

Salmonella Dublin is a strongly adapted serovar that causes enteritis and/or systemic disease with high rates of mortality in cattle and occasionally infects humans. Despite the importance of this serovar, there is a lack of studies in Brazil. The aim of this study was to characterize the genetic diversity of 112 S. Dublin strains isolated from humans and animals in Brazil by CRISPR and CRISPR-MVLST and the relatedness among strains by MLST. In addition, the frequency of some important virulence genes was verified. The strains studied belonged to nine different sequence types, being all of them single- or double-locus variants of the ST10. CRISPR discriminated the strains into 69 subtypes with a similarity ≥ 84.4% and CRISPR-MVLST into 72 subtypes with a similarity ≥ 84.7%. The virulence genes ratB, lpfA, mgtC, avrA, sopB, sopE2, sifA, sseA, ssrA, csgA, fliC, and sinH were found in all the strains studied, while spvB, spvC, sodCl, rpoS, sipA, sipD, invA, and hilA were detected in ≥ 93.7% of the strains. In conclusion, the high similarity among the strains reinforces the clonal nature of the strains of this serovar that may have descended from a common ancestor that little differed over 33 years in Brazil. CRISPR and CRISPR-MVLST showed to be good alternatives to type S. Dublin strains. MLST suggested that S. Dublin strains from Brazil were phylogenetically related to strains from other parts of the globe. Moreover, the high frequency of virulence genes among the strains studied reinforces the capacity of S. Dublin to cause invasive diseases.

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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.

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Salmonella enterica serovar Dublin is a strongly adapted serovar that causes enteritis and/or systemic disease in cattle and results in high rates of mortality. Here, we report the draft genome sequences of 112 S. Dublin strains isolated from humans and animals in Brazil. These draft genome sequences will help enhance our understanding of this serovar in Brazil.

RESUMO

The gastroenteritis incidence caused by Salmonella Hadar has increased over the last decades worldwide. The uncontrolled use of antimicrobials for treating human patients and veterinary field contributes to increase the multidrug resistance of this serovar. In the present investigation, a total of 179 S. Hadar isolates from different sources of foodchain in Brazil were phage typed and analyzed for their antimicrobial resistance profile. The main S. Hadar phage types isolated were PT 38, PT 39, PT 40, PT 11, PT 34, PT 1 and PT 22. Others phage types as PT 13, PT 19, PT 21, PT 23, PT 31, PT 33 and PT 37 were obtained in low percentages. A total of 35,7 percent S. Hadar strains were resistant to two or more antimicrobials drugs. Furthermore, no resistance to third generation cephalosporin or ciprofloxacin was identified in these strains. Those results appoint to S. Hadar phage types circulating among animals, food and humans, as well as the increasing of multidrug resistance. The surveillance and monitoring of S. Hadar strains based on phage typing and antimicrobial resistance profile are useful for detecting outbreaks, identifying sources of infection and implementing prevention and control measures of salmonellosis.

A incidência de gastrenterite causada por Salmonella Hadar tem aumentado ao longo dos anos em todo o mundo. O uso indiscriminado de antimicrobianos na clínica humana e veterinária tem contribuído para o aumento da multiresistência deste sorovar. No presente estudo, 179 cepas de S. Hadar isoladas de diferentes fontes da cadeia alimentar no Brasil foram fagotipadas e analisadas quanto ao perfil de resistência antimicrobiana. Os principais fagotipos de S. Hadar isolados foram PT 38, PT 39, PT 40, PT 11, PT 34, PT 1 e PT 22. Outros fagotipos como PT 13, PT 19, PT 21, PT 23, PT 31, PT 33 e PT 37 foram obtidos em menores percentagens. Um total de 35,7 por cento das cepas avaliadas foi resistente a dois ou mais antimicrobianos. Por outro lado, não foi observada resistência a cefalosporinas de terceira geração ou ciprofloxacina. Esses resultados apontam para a circulação de fagotipos de S. Hadar entre animais, alimentos e seres humanos, bem como o aumento da multiresistência antimicrobiana. O monitoramento de cepas de S. Hadar baseado na fagotipagem e no padrão de resistência aos antimicrobianos são ferramentas úteis na detecção de surtos, identificação das fontes de infecção, além de auxiliar na implantação de programas de controle e prevenção de salmoneloses.

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Salmonella Typhimurium has become a widespread cause of salmonellosis among humans and animals worldwide. In Brazil, Salmonella Typhimurium (STM) is one of the most prevalent serovars isolated from food for human consumption. The uncontrolled sale and use of antimicrobials in agriculture and for treating human patients contributes to increase multidrug resistance of this serovar. In the present study, a total of 278 STM isolates from different sources and regions of Brazil over the period 1999 to 2004 were phage typed and analyzed for their antimicrobial resistance profile at Laboratory of Enterobacteria, Oswaldo Cruz Institute, FIOCRUZ. The main STM phage types isolated were DT 193 (64.3 percent), DT 19 (17.4 percent) and DT 18 (4 percent). Others phage types as DT 10 (2 percent), DT 27 (3.24 percent), DT 13 (0.36 percent), DT 22 (0.36 percent), DT 28 (0.36 percent), DT 29 (0.36 percent) and DT 149 (0.36 percent) were obtained in low percentages. A total of 54 percent STM strains were resistant to three or more antimicrobial classes, while no resistance to third generation cephalosporin or ciprofloxacin was identified in these strains. Those results show the STM phage types circulating among animals, food for human consumption and humans in Brazil as well as the increasing of multidrug resistance. The surveillance of STM strains based on phage typing and antimicrobial resistance profile are useful for detecting outbreaks, identifying sources of infection and implementing prevention and control measures.

Salmonella Typhimurium é considerada uma das principais bactérias causadoras de salmonelose nos animais e no homem em todo o mundo. No Brasil, Salmonella Typhimurium é um dos mais prevalentes sorovares isolados de alimentos para consumo humano. O uso indiscriminado de antibióticos em produtos agrícolas e no tratamento de pacientes humanos tem contribuído para aumentar a multirresistência desse sorovar a diversos antimicrobianos. No presente estudo, 278 cepas de STM foram selecionadas de diferentes fontes e regiões do Brasil, no período de 1999 a 2004 e realizadas a fagotipagem e análise do perfil de resistência antimicrobiana no Laboratório de Enterobactérias, Instituto Oswaldo Cruz, FIOCRUZ. Os principais fagotipos isolados foram DT 193 (64,3 por cento), DT 19 (17,4 por cento) e DT 18 (4 por cento). Os fagotipos DT 10 (2 por cento), DT 27 (3,24 por cento), DT 13 (0,36 por cento), DT 22 (0,36 por cento), DT 28 (0,36 por cento), DT 29 (0,36 por cento) e DT 149 (0,36 por cento) foram isolados em menores percentuais. Um total de 54 por cento das cepas de STM foi resistente a três ou mais classes de antimicrobianos e não foi observada resistência a cefalosporinas de terceira geração ou ciprofloxacina. Esses resultados indicam os principais lisotipos de Salmonella Typhimurium circulantes entre os animais, alimentos de consumo humano e seres humanos no Brasil, bem como o aumento da multirresistência antimicrobiana. O monitoramento de cepas de Salmonella Typhimurium baseado na fagotipagem e no padrão de resistência antimicrobiana são ferramentas úteis para detectar surtos, identificar a fonte de infecção e implementar programas de prevenção e controle de salmonelose.

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The presence of Vibrio spp. and Salmonella spp. in crabs marketed at the Bezerra de Menezes Ave., Fortaleza, State of Ceará, Brazil, was assessed between February and May, 2003. The number of individuals sampled in each one of the fifteen weekly samplings ranged between four and eight. Seven strains of Salmonella, from four different samplings, were identified, being five of them identified as serotype S. Senftenberg and two as S. Poona. All strains of Salmonella were sensitive to the tested anti-microbial drugs, with the exception of tetracycline and nalidixic acid, for which an intermediary sensibility was found. The MPN's for Vibrio ranged between 110/g and 110,000/g. Of the forty five Vibrio strains isolated from the crab samples, only 10 were identified up to the species level: two V. alginolyticus and eight V. parahaemolyticus. Bacteria belonging to the Enterobacteriaceae and Pseudomonaceae families were also identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pantoea agglomerans and Pseudomonas aeruginosa. The proper cooking of the animals is recommended in order to avoid problems for the consumers of this crustacean.

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RESUMO

Foram pesquisadas a presença de Vibrio spp. e de Salmonella spp. em caranguejos comercializados na Av. Bezerra de Menezes, Fortaleza, Ceará, Brasil, no período entre fevereiro e maio de 2003. O número de indivíduos em cada, das quinze coletas realizadas, semanalmente, variava entre quatro e oito dependendo do tamanho dos animais, totalizando um número de 90 (noventa) animais examinados. Foram identificadas sete cepas de Salmonella spp. provenientes de quatro coletas: cinco foram identificadas como sorovar S. Senftenberg e duas como S. Poona. Todas as cepas de Salmonella, isoladas das amostras de caranguejos, apresentaram sensibilidade aos antimicrobianos testados, com exceção de tetraciclina e ácido nalidíxico para os quais elas apresentaram uma sensibilidade intermediária. Os NMPs para Vibrio spp. variaram entre 110 e 110.000/g. Das quarenta e cinco cepas de Vibrio spp. isoladas das amostras de caranguejo, foram identificadas, até espécie, somente 10: duas de V. alginolyticus e oito de V. parahaemolyticus. Foram também identificadas bactérias pertencentes às famílias Enterobacteriaceae e Pseudomonadaceae: Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Pantoea agglomerans e Pseudomonas aeruginosa. Recomenda-se que a cocção dos animais seja bem feita a fim de se evitar problemas para os consumidores dos crustáceos.

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