Molecular characterization and toxigenic profiles of Bacillus cereus isolates from foodstuff and food poisoning outbreaks in Brazil.

Bacillus cereus sensu stricto (s.s.) is a well-known foodborne pathogen that produces a range of enterotoxins and is able to cause two different types of foodborne illnesses-the emetic and the diarrheal syndromes. In this study, 54 B. cereus s.s. strains isolated from foodstuff and foods involved in food poisoning outbreaks were characterized according to the presence of toxin-encoding genes, virulence-encoding genes, and panC typing. Most isolates were assigned to panC groups IV (61.1%) and III (25.9%), but members of groups II and V could also be found. Investigation of specific alleles revealed high numbers of isolates carrying toxin and other virulence genes including nheA (100%), nheB (100%), hblA (79.6%), hblC (79.6%), hblD (74.1%), cytK-2 (61.1%), clo (100%), pc-plc (75.9%), sph (68.5%), pi-plc (66.6%), hlyIII (62.9%), and hlyII (24.1%). All isolates were negative for ces and cytK-1. In summary, we detected various enterotoxin and other virulence factor genes associated with diarrheal syndrome in strains analyzed, implicated or not with food poisoning. Furthermore, the most isolates analyzed belong to high-risk phylogenetic groups' panC types III and IV. Our study provides a convenient molecular scheme for characterization of B. cereus s.s. strains responsible for food poisoning outbreaks in order to improve the monitoring and investigation and assess emerging clusters and diversity of strains.

Evaluation of MALDI-TOF MS as a tool for detection of Listeria spp. directly from selective enrichment broth from food and stool samples.

We evaluated the detection of Listeria spp. using MALDI-TOF MS directly in enrichment broths, without isolated colonies, with naturally contaminated food and stool samples. The success rate was 77%. Considering the reduced time for diagnosis and the success rate, this is a promising screening tool, but more tests are needed to determine its viability.

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR assay.

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC (the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88 Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method. In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide.

Next-generation sequencing virulome analysis of a Yersinia enterocolitica subsp. palearctica bioserotype 4/O: 3 ST18 isolated from human blood in Brazil

Abstract Yersinia enterocolitica is a widespread Gram-negative bacterium that causes gastrointestinal disease and other clinical manifestations in humans. Potentially pathogenic Y. enterocolitica has been isolated in Brazil, from human, environmental, food, and animal sources. Herein we report a genome sequence of Y. enterocolitica subsp. palearctica strain YE 19, serotype O:3, biotype 4, sequence type 18, with virulence determinants isolated from human blood in Rio de Janeiro in 2005. The results corroborate other findings that this strain harbors a set of virulence determinants that could play a role in host pathoadaptation and may also justify the successful dissemination of bioserotype 4/O:3 in Brazil. The presence of strains harboring all of these virulence genes in Brazil is a potential threat to young children and immunocompromised individuals, for whom yersiniosis are a significant source of morbidity and mortality. The results of a genomic data analysis will help understand the virulence of Brazilian strains and provide data for Y. enterocolitica studies worldwide.

Next-generation sequencing virulome analysis of a Yersinia enterocolitica subsp. palearctica bioserotype 4/O:3 ST18 isolated from human blood in Brazil.

Yersinia enterocolitica is a widespread Gram-negative bacterium that causes gastrointestinal disease and other clinical manifestations in humans. Potentially pathogenic Y. enterocolitica has been isolated in Brazil, from human, environmental, food, and animal sources. Herein we report a genome sequence of Y. enterocolitica subsp. palearctica strain YE 19, serotype O:3, biotype 4, sequence type 18, with virulence determinants isolated from human blood in Rio de Janeiro in 2005. The results corroborate other findings that this strain harbors a set of virulence determinants that could play a role in host pathoadaptation and may also justify the successful dissemination of bioserotype 4/O:3 in Brazil. The presence of strains harboring all of these virulence genes in Brazil is a potential threat to young children and immunocompromised individuals, for whom yersiniosis are a significant source of morbidity and mortality. The results of a genomic data analysis will help understand the virulence of Brazilian strains and provide data for Y. enterocolitica studies worldwide.

Characterization of epidemic clones of Listeria monocytogenes serotype 4b isolated from humans and meat products in Brazil.

INTRODUCTION: Listeria monocytogenes is an important foodborne pathogen and the 4b serotype is responsible for many cases of human listeriosis reported in Brazil. Several listeriosis outbreaks worldwide have involved a small number of well-defined clonal groups, designated as epidemic clones (ECs). METHODOLOGY: We studied 71 strains of serotype 4b, including 25 isolates from human cases of listeriosis and 46 from meat-based foods, collected in Brazil between 1977 and 2010. The presence of ECs (I and II) markers and virulence genes (inlA, inlB, ilnC, inlJ and actA) were evaluated by PCR assay. The genetic relationship of ECs-positive strains was assessed by pulsed field gel electrophoresis. RESULTS: ECI and ECII markers were found both in human and food strains, with 19.7% positive for the ECI marker and 40.8% for ECII. Most strains (97.2%) were positive for the virulence genes that were studied. Nevertheless, the actA gene amplicons showed two distinct sizes, with all ECI positive strains exhibiting a 105bp deletion. Pulsed field gel electrophoresis (PFGE) analysis allowed the recognition of highly related strains, particularly from two outbreaks of neonatal listeriosis in São Paulo State occurred in 1992 and 1997, both ECII-positive; and two ECI strains from a human case (1982) and from bovine meat (2009). CONCLUSIONS: The presence of ECs among clinical samples and beef isolates of serotype 4b from some regions of Brazil highlights the need for rigorous control of production procedures. Furthermore, the association of ECII with two nosocomial outbreaks suggests its ability to spread in these settings.

Phenotypic and genotypic analysis of bio-serotypes of Yersinia enterocolitica from various sources in Brazil.

INTRODUCTION: Yersinia enterocolitica is a well-known foodborne pathogen widely distributed in nature with high public health relevance, especially in Europe. METHODOLOGY: This study aimed to analyze the pathogenic potential of Y. enterocolitica isolated strains from human, animal, food, and environmental sources and from different regions of Brazil by detecting virulence genes inv, ail, ystA, and virF through polymerase chain reaction (PCR), phenotypic tests, and antimicrobial susceptibility analysis. Pulsed-field gel electrophoresis (PFGE) was used for the assessment of phylogenetic diversity. RESULTS: All virulence genes were detected in 11/60 (18%) strains of serotype O:3, biotype 4 isolated from human and animal sources. Ten human strains (4/O:3) presented three chromosomal virulence genes, and nine strains of biotype 1A presented the inv gene. Six (10%) strains were resistant to sulfamethoxazole-trimethoprim, seven (12%) to tetracycline, and one (2%) to amikacin, all of which are used to treat yersiniosis. AMP-CEF-SXT was the predominant resistance profile. PFGE analysis revealed 36 unique pulsotypes, grouped into nine clusters (A to I) with similarity ≥ 85%, generating a diversity discriminatory index of 0.957. Cluster A comprised all bio-serotype 4/O:3 strains isolated from animal and humans sources. CONCLUSIONS: This study shows the existence of strains with the same genotypic profiles, bearing all virulence genes, from human and animal sources, circulating among several Brazilian states. This supports the hypothesis that swine is likely to serve as a main element in Y. enterocolitica transmission to humans in Brazil, and it could become a potential threat to public health as in Europe.

Caracterização fenotípica e genotípica de cepas de yersinia enterocolitica isoladas de diferentes origens no Brasil / Phenotypic and genotypic characterization of Yersinia enterocolitica strains isolated from different origins in Brazil

Desde a sua inclusão na família Enterobacteriaceae, em 1972, Yersinia enterocolitica vem sendo extensivamente estudada, por se tratar de um patógeno de natureza zoonótica com relevância em saúde pública, amplamente distribuído na natureza em reservatórios aquáticos e fontes animais. Tendo o suíno como principal reservatório, por ser portador de cepas dos mesmos biosorotipos encontrados em humanos, Y. enterocolitica possui diversos genes de patogenicidade, tanto cromossomiais (inv, ail e ystA) como plasmidiais (virF), que lhes confere a capacidade de invadir e sobreviver em um organismo hospedeiro. Esse estudo teve como objetivo analisar cepas de Y. enterocolitica oriundas de fontes animal, alimentar, ambiental e humana de diferentes regiões do Brasil, para a detecção dos genes de virulência supracitados através da técnica da PCR em conjunto com testes fenotípicos, além da análise da susceptibilidade aos antimicrobianos através do método de disco-difusão para que, junto com os resultados da PCR, pudesse ser avaliado seu potencial patogênico. Para a avaliação da diversidade filogenética das cepas foram empregadas as técnicas de ERIC-PCR e do PFGE. Em um total de 60 amostras, foram encontradas 11(18 por cento) amostras pertencentes ao sorotipo O:3, biotipo 4 de origens humana e animal possuidoras de todos os genes de virulência. Dez amostras humanas (O:3/B4) apresentaram somente os 3 genes cromossomiais, e 9 amostras pertencentes ao biotipo 1A apresentaram o gene inv. No que tange à resistência aos antimicrobianos, 60 (100 por cento) amostras foram resistentes à ampicilina, 58(97 por cento) à cefalotina, 6(10 por cento) ao sulfametoxazol-trimetoprim, 7(12 por cento) à tetraciclina e 1(2 por cento) à amicacina. O perfil de resistência predominante foi AMP-KF-RL. Cinco amostras apresentaram resistência a cinco antibióticos, sendo a amostra YE42 resistente a 3 classes de antibióticos diferentes. O ERIC-PCR revelou a existência de 32 ERIC genótipos (EGT), gerando um índice discriminatório de diversidade de 0,924. Quarenta amostras pertencentes ao sorovar O:3 biotipo 4 de origens humana, animal, alimentar e ambiental ficaram agrupadas dentro de 12 EGTs em um conjunto com uma similaridade de 85 por cento. Já as amostras do Biotipo 1A isoladas de alimentos e ambiente são extremamente heterogênicas sorologicamente, e apresentaram padrões de banda mais diversificados. A análise do PFGE nos revelou a existência de nove clusters (A até I) agrupando os isolados com similaridade ≥ 85 por cento. Nestes clusters, foram agrupados trinta e seis padrões únicos de banda (PPT), gerando um índice discriminatório de diversidade de 0,957. O cluster A englobou todas as amostras do sorovar O:3 biotipo 4 isoladas de animal e de casos humanos. Com base nos resultados, conclui-se que cepas com os mesmos perfis genotípicos, isolados de humanos e animais, do sorovar O:3 e biotipo 4, encontram-se circulantes por vários estados do Brasil. Além disso, com base no exposto, sugere-se que o suíno possa funcionar como principal elemento na cadeia de transmissão de Y. enterocolitica para o homem, por albergar cepas dos mesmos genótipos encontrados em infecções humanas.

Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil.

INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC). RESULTS: Among the strains tested, serovar L4b (60.3%) was the most prevalent, followed by serovar 1/2a (20.6%), 1/2b (13.2%) and the more uncommon serovars 1/2c, 3b and 4ab (5.9%). All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5%) showed resistance to rifampin, and two (3%) were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2 µg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

Antimicrobial susceptibilities of Listeria monocytogenes human strains isolated from 1970 to 2008 in Brazil / Susceptibilidade antimicrobiana de cepas humanas de Listeria monocytogenes isoladas no período de 1970 a 2008 no Brasil

INTRODUCTION: Listeria monocytogenes is the causative agent of listeriosis, a foodborne illness that affects mainly pregnant women, the elderly and immunocompromised patients. The primary treatment is a combination of ampicillin with an aminoglycoside, in addition to a second-choice drug represented by chloramphenicol, erythromycin, tetracycline and rifampicin. The aim of this study was to analyze the antimicrobial susceptibility profile of strains isolated from human sources in the last four decades. METHODS: Sixty-eight strains were selected from the culture collection of the Laboratory of Bacterial Zoonoses/LABZOO/FIOCRUZ isolated in different regions of Brazil from 1970 to 2008 and primarily isolated from cerebrospinal fluid and blood culture. Susceptibility tests to antimicrobials drugs were evaluated using the criteria established by Soussy using the Kirby-Bauer method and E-Test strips were used to determine the minimum inhibitory concentration (MIC). RESULTS: Among the strains tested, serovar L4b (60.3 percent) was the most prevalent, followed by serovar 1/2a (20.6 percent), 1/2b (13.2 percent) and the more uncommon serovars 1/2c, 3b and 4ab (5.9 percent). All strains were susceptible to ampicillin, cephalothin, erythromycin, gentamicin, teicoplanin and vancomycin. Only one strain (1.5 percent) showed resistance to rifampin, and two (3 percent) were resistant to trimethoprim-sulfamethoxazole. MICs with values up to 2μg/ml reinforce the need for microbiological surveillance. CONCLUSIONS: The study demonstrated low prevalence of strains resistant to the antimicrobial drugs indicated in the treatment of human listeriosis. Monitoring antimicrobial resistance profile is still very important to determine adequate treatment, especially in immunocompromised patients.

INTRODUÇÃO: Listeria monocytogenes é o agente etiológico da listeriose, doença de origem alimentar que acomete principalmente grávidas, pacientes imunodeprimidos e idosos. O tratamento primário é a associação de ampicilina a um aminoglicosídeo além de outros, em segunda escolha, representados por cloranfenicol, eritromicina, tetraciclina e rifampicina. O presente estudo teve como objetivo analisar o perfil de susceptibilidade aos antimicrobianos de amostras de origem humana isoladas nas últimas quatro décadas. MÉTODOS: Foram selecionadas 68 cepas provenientes de casos clínicos humanos ocorridos em diferentes regiões do país no período de 1970-2008. A susceptibilidade aos antimicrobianos testados foi determinada através dos critérios estabelecidos por Soussy pelo método de Kirby-Bauer e a concentração mínima inibitória realizada através do E-Test. RESULTADOS: A amostragem constituiu-se de 68 cepas, isoladas principalmente de líquido cefalorraquidiano, e hemocultura no período, pertencentes ao Laboratório de Zoonoses Bacterianas/LABZOO/Fiocruz. O sorovar L4b (60,3 por cento) foi o mais prevalente, seguido do sorovar 1/2a (20,6 por cento), 1/2b (13,2 por cento) e aqueles mais raros representados por 1/2c, 3b e 4ab (5,9 por cento). Todas as cepas foram sensíveis à ampicilina, cefalotina, eritromicina, gentamicina, teicoplanina e vancomicina. Apenas uma cepa (1,5 por cento) apresentou resistência à rifampicina, enquanto duas (3 por cento) foram resistentes à associação de sulfametoxazol-trimetoprim. CONCLUSÕES: Apesar de o estudo ter demonstrado uma baixa prevalência de amostras resistentes aos antimicrobianos indicados na terapêutica da listeriose humana, o sistema de monitoramento do perfil de resistência antimicrobiana é de extrema importância para a orientação do tratamento adequado, principalmente nas infecções em pacientes imunocomprometidos.