Genomic features and heat resistance profiles of Escherichia coli isolated from Brazilian beef.

AIMS: Characterize Escherichia coli and E. coli -producing (STEC) isolates from Brazilian beef to determine heat resistance and the presence of the transmissible locus of stress tolerance (tLST). METHODS AND RESULTS: Twenty-two STEC previously isolated from beef and characterized as STEC by PCR were subjected to different heat survival challenges (60°C and 71°C). Furthermore, the three tLST-positive isolates and one tLST-negative isolate by PCR were selected for WGS analysis. Phenotypic results indicated that 3/22 (13.64%) were heat resistant, 12/22 (54.54%) were moderately resistant, and 7/22 (31.82%) were sensitive to heat treatments. WGS analyses showed that three isolates with heat resistance showed tLST with up to 80% and 42% of similarity by BLAST analysis, with the major tLST genes being responsible for the homeostasis module. However, WGS showed the absence of stx genes associated with tLST-positive isolates, albeit with virulence and resistance genes found in extraintestinal pathogenic E. coli (ExPEC). CONCLUSION: Our findings demonstrate the presence of heat-resistant E. coli as well as confirm some tLST genes in E. coli isolated from Brazilian beef.

Genomic analysis of Mycobacterium tuberculosis variant bovis strains isolated from bovine in the state of Mato Grosso, Brazil.

The species Mycobacterium tuberculosis variant bovis (M. tuberculosis var. bovis) is associated with tuberculosis, mainly in cattle and buffaloes. This pathogen has the potential to infect other mammals, including humans. Tuberculosis caused by M. tuberculosis var. bovis is a zoonosis clinically identical to tuberculosis caused by Mycobacterium tuberculosis, and the recommended treatment in humans results in the use of antibiotics. In this study, we used the whole genome sequencing (WGS) methodology Illumina NovaSeq 6000 System platform to characterize the genome of M. tuberculosis var. bovis in cattle circulating in Mato Grosso, identify mutations related to drug resistance genes, compare with other strains of M. tuberculosis var. bovis brazilian and assess potential drug resistance. Four isolates of M. tuberculosis var. bovis of cattle origin representing the main livestock circuits, which had been more prevalent in previous studies in the state of Mato Grosso, were selected for the genomic study. The genome sizes of the sequenced strains ranged from 4,306,423 to 4,332,964 bp, and the GC content was 65.6%. The four strains from Mato Grosso presented resistance genes to pncA (pyrazinamide), characterized as drug-resistant strains. In addition to verifying several point mutations in the pncA, rpsA, rpsL, gid, rpoB, katG, gyrB, gyrA, tlyA, embA, embB, embC, fgd, fbiB, and fbiC genes, these genes were similar to antibiotic resistance in more than 92% of the Brazilian strains. Therefore, our results indicated a high genetic diversity between our isolates and other M. tuberculosis var. bovis isolated in Brazil. Thus, multiple transmission routes of this pathogen may be present in the production chain. So, to achieve a bovine tuberculosis-free health status, the use of the WGS as a control and monitoring tool will be crucial to determine these transmission routes.

Salmonella Behavior in Meat during Cool Storage: A Systematic Review and Meta-Analysis.

The aim of the present study was to investigate Salmonella behavior in meat stored in cool conditions (between 0 °C and 7.5 °C), by employing a systematic review and meta-analysis. The data were obtained from research articles published in SciELO, PubMed, the Web of Science, and Scopus databases. The results of the retrieved studies were obtained from meat (beef, chicken, pork, poultry, and turkey), fish, shellfish, and broth media samples The data were extracted as sample size (n), initial concentration (Xi), final concentration (Xf), standard deviation (SD), standard error (SE), and microbial behavior effects (reduction or growth). A meta-analysis was carried out using the metaphor package from R software. A total of 654 articles were initially retrieved. After applying the exclusion criteria, 83 articles were selected for the systematic review, and 61 of these were used for the meta-analysis. Most studies were conducted at 0 °C to 4.4 °C storage temperatures under normal atmosphere package conditions. Salmonella Typhimurium, S. Enteritidis, and a cocktail (strain mixture) were inoculated at 5.0 and 6.0 log CFU mL−1. Articles both with and without the addition of antimicrobial compounds were found. Salmonella concentration decreases were observed in most studies, estimated for all study combinations as −0.8429 ± 0.0931 log CFU g−1 (95% CI; −1.0254, −0.6604) (p < 0.001), varying for each subgroup analysis. According to this survey, Salmonella concentration decreases are frequent during cool storage, although concentration increases and no bacterial inactivation were observed in some studies.

Whole-Genome Sequencing Analyses of Heat-Resistant Escherichia coli Isolated from Brazilian Beef.

Four Escherichia coli isolates with moderate or high heat resistance were sequenced. Through sequencing, truncated transmissible locus of stress tolerance (tLST) variants tLST1 and tLSTa were identified in the three isolates. The most identified tLST genes (clpK and hsp) are responsible for the homeostasis module.

Salmonella in the processing line of farmed Tambatinga (Colossoma macropomum x Piaractus brachypomus) in Mato Grosso, Brazil: serotypes of occurrence and antimicrobial profile.

The objective of this study was to evaluate the dispersion dynamics and antimicrobial resistance profiles of Salmonella in the processing of Tambatinga (Colossoma macropomum x Piaractus brachypomus). Thirty fish were monitored during four processing stages (reception, first wash, evisceration, and prepackage area) in a fish slaughterhouse. One hundred and twenty fish surface samples were collected and tested through bacteriological analysis, PCR, serotyping, and antimicrobial resistance profile (disk-diffusion). Of these samples, 7.5% (9/120) were positive for Salmonella, with 0.83% being observed in the pre-packaging phase, indicating a low occurrence at this stage. All the analyzed stages were positive for Salmonella, with the prevalent serovars being Ndolo, Mbandaka, Typhimurium, Rough, and O:16. All strains were sensitive to various antimicrobials. Improvements in microbiological control during all processing stages should be implemented to ensure a Salmonella-free product.

Salmonella Schwarzengrund, Akuafo, and O:16 isolated from vacuum-packaged beef produced in the state of Mato Grosso, Brazil.

INTRODUCTION: Salmonella spp. is a pathogen associated with foodborne infections, mainly in foods of animal origin. In this context, the present study investigated the occurrence of Salmonella serotypes, genotypes and the antimicrobial resistance profiles of strains in fresh beef produced in Mato Grosso, Brazil. METHODOLOGY: A total of 107 samples from 13 different slaughterhouses in the Mato Grosso were analyzed. Suggestive Salmonella spp. colonies detected during the biochemical screening were submitted to DNA extraction, and hilA gene amplification was used for the PCR reaction. Antimicrobial resistance analyses were performed using 17 antimicrobial agents from eight different classes by the disk diffusion method. Strains exhibiting multiple drug resistances were submitted to PCR genotyping based on repetitive sequences (rep-PCR), using a commercial semiautomatic DiversiLab® system. RESULTS: A total of 5.6% (6/107) of the samples tested positive by the conventional method and were confirmed by PCR, namely two S. Akuafo, two non-typable Salmonella enterica strains, one Salmonella O:16 serovar, and one S. Schwarzengrund. The antimicrobial resistance profiles indicated resistance to gentamicin (30%), tetracycline, nitrofurantoin, and trimethoprim + sulfamethoxazole (16%). Genotyping indicated a 70% difference between S. Schwarzengrund and the non-typable Salmonella strains. No genetic similarities were observed between the six Salmonella isolates based on rep-PCR, including two S. Akuafo. CONCLUSIONS: The results obtained herein corroborate that Salmonella serovar Schwarzengrund is commonly isolated in animal products in the state of Mato Grosso, Brazil, also highlighting the presence of two unusual Salmonella serovars in beef (Akuafo and O:16).

Escherichia coli O26 and O113:H21 on Carcasses and Beef from a Slaughterhouse Located in Mato Grosso, Brazil.

Shiga toxin-producing Escherichia coli (STEC) is a group of emerging pathogens that can cause human diseases, including hemolytic uremic syndrome (HUS) and hemorrhagic colitis (HC). Monitoring slaughtering stages and checking contamination points are crucial for the production of safe food. In this context, the aim of this study was to verify contamination by STEC strains, to determine the contamination points and evaluate the resistance profile to 12 antimicrobials used in both veterinary and human medicine. A total of 80 samples were obtained from eight collection points (pen floor, rectum, hide, carcass swabs and esophagus, diaphragm, masseter, and retail beef tissue samples). The isolates were collected by dilution plating on MacConkey agar with sorbitol, cefixime, and tellurite and analyzed by multiplex polymerase chain reaction for virulence genes. Serotyping of non-O157 was performed, and testing for 12 antibiotics by disk diffusion was carried out. A total of 18 STEC strains were isolated, presenting different virulence profiles. Contamination by STEC was observed in the rectum (5/18), carcass surface (5/18), hide (3/18), diaphragm (2/18), retail beef (2/18), and masseter muscle (1/18). Pen floor swabs and esophagus tissues showed no STEC contamination. Moreover, three strains were identified as O26 and three as O113:H21 strains, which have been linked to HUS and HC outbreak cases in Brazil. All STEC isolates were susceptible to all evaluated antimicrobials, except streptomycin. The presence of STEC strains is a direct risk to the consumer, especially when isolated from retail beef, and contamination can occur during different slaughter stages. However, antimicrobial resistance profiles did not identify multidrug-resistant strains, limiting potential antimicrobial resistance transmission to other pathogens.

Salmonella isolated from chicken carcasses from a slaughterhouse in the state of Mato Grosso, Brazil: antibiotic resistance profile, serotyping, and characterization by repetitive sequence-based PCR system.

Salmonella is one of the major causative agents of foodborne infections. Salmonellosis becomes more dangerous when strains resistant to several antibiotics are found in food, especially in chicken, one of the primary transmission vehicles of this pathogen for humans. The present study aimed to estimate the occurrence of Salmonella in chicken carcasses from the state of Mato Grosso, Brazil, as well as determine the antibiotic resistance profile and genotypic characteristic of multi-drug resistant (MDR) isolates. During a 15-month period, from 01/2014 to 05/2015, 850 samples of chilled fresh chicken carcasses were sampled from a slaughterhouse and submitted to Salmonella determinations according to the ISO-6579/2002 method, serotyping and multiplex PCR. The disc diffusion test was applied for 17 antibiotics, according to CLSI (2014). Five isolates were genotyped by repetitive sequence-based PCR using the semi-automated DiversiLab (bioMérieux®) system. The occurrence of Salmonella in chicken carcasses was of 3.7% (31/850), with only 4 strains (12.9%) presenting as MDR, and 6 strains (19.35%) displaying ESBL. The predominant serovars were Salmonella Infantis (35.4%, 11/31), and S. Abony (25.8%, 8/31), followed by serovars S. Agona (12.9%, 4/31), S. Schwarzengrund (9.7%, 3/31), S. Anatum and Salmonella enterica O:4,5 (6.5%, 2/31), and only one Salmonella enterica O:6,7 strain (3.2%, 1/31). All isolates were resistant to one to 5 classes of antibiotics in decreasing order: folate pathway inhibitors, ß-lactams (cephalosporins, penicillin, monobactams), tetracyclines, chloramphenicol, and gentamicin. However, strains sensitive to florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin were also found in this study. Genotyping revealed 98 to 99% homology between 3 Salmonella strains, which displayed high phenotypic resistance similarity to ß-lactams and folate pathway inhibitors. Detection of MDR non-typhoid Salmonella in chicken slaughterhouses with quality assurance systems such as Hazard Analysis and Critical Points and Implemented Good Manufacturing Practices is a concern, reinforcing the need for constant monitoring of these pathogens, with the purpose of safeguarding the safety of their products.

Hygienic sanitary conditions of vacuum packed beef produced by slaughterhouses qualified for export in the Mato Grosso state, Brazil / Condições higiênico sanitárias de carne embalada a vácuo produzida por abatedouros frigoríficos habilitados a exportação no estado de Mato Grosso, Brasil

ABSTRACT: Brazil is the largest exporter of beef of the world and Mato Grosso State is the highest beef producer in this country. To maintain product competitiveness and market expansion, sanitary hygienic control of the entire process is indispensable to ensure the attainment of harmless beef and quality. The objective of this study was to evaluate the hygienic sanitary conditions of vacuum-packed beef produced by establishments qualified for export in the state of Mato Grosso, Brazil. A total of 60 samples were submitted to coliforms counts at 35°C and at 45°C and E. coli. The mean contamination by at 35°C and coliforms at 45°C were 3,1 x 102MPN/g and 7.7MPN/g respectively. The presence of E. coli was verified in five samples, representing an occurrence of 8.3% (5/60), and Salmonella spp. in 5% (03/60) of the analyzed samples. The MPN (Most Probable Number) average of coliforms at 35°C and 45°C are in accordance to national and international legislation; however, the presence of Samonella spp., E. coli in some sample indicates a low risk of occurrence of salmonellosis and colibacillosis transmitted by the evaluated beef. However, transmission risk of these diseases cannot be ruled out, since the presence of E. coli does not depend on the amount of coliforms and national legal standards established for the group of thermotolerant coliforms.

RESUMO: O Brasil é o maior exportador de carne bovina do mundo e Mato Grosso é o maior produtor de carne bovina do país. Para manter a competitividade do produto e a expansão do mercado, o controle higiênico sanitário de todo o processo é indispensável para garantir a obtenção de carne inócua e de qualidade. O objetivo desse estudo foi avaliar as condições higiênico sanitárias da carne bovina resfriada embalada a vácuo, não maturada produzida por estabelecimentos detentores de Serviço de Inspeção Federal (SIF) no estado de Mato Grosso. Um total de 60 amostras foram submetidas a contagem de coliformes à 35°C, coliformes à 45°C, pesquisa de E. coli e Salmonella spp. A contaminação média por coliformes à 35°C e coliformes à 45°C da carne bovina mato-grossense inspecionada pelo SIF foi 3,1 x 102NMP/g e 7,7NMP/g, respectivamente. A presença de E. coli foi verificada em cinco amostras o que representou uma ocorrência de 8,3% (5/60) e Salmonella spp. em 5% (3/60). Foi avaliado que a média NMP de coliformes à 35°C e à 45°C atende a legislação nacional e internacional, no entanto, a verificação da presença de Salmonella spp., E. coli em algumas amostras, evidenciou um baixo risco de ocorrência de salmonelose ou colibacilose transmitidas pela carne. Ainda assim, não se pode descartar o risco de veiculação dessas doenças, uma vez que a presença de E. coli independe da quantidade de coliformes e dos padrões legais nacionais estabelecidos para o grupo dos coliformes termotolerantes.

Shiga-toxin Producing Escherichia coli: Pathogenicity, Supershedding, Diagnostic Methods, Occurrence, and Foodborne Outbreaks.

Historically, Escherichia coli is among the most studied organisms and serves as the basis for understanding many fundamental biochemical and genetic concepts. In addition, it displays 9 pathogenesis groups, with the Shiga toxin-producing (STEC) group being the main representative regarding foodborne pathogenesis. Its typical characteristic is the presence of 2 distinct toxins and variants: stx1 (stx1a, stx1c, and stx1d), and stx2 (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g). The main challenge regarding the study of E. coli is the standardization of a high sensitivity method including all pathotypes, that allows for enrichment of STEC cells and a decrease of background microbiota. The ability of some E. coli cells belonging to other pathogenic groups, such as O104:H4, to acquire genes unique to the STEC group, increases the pathogenic power and the risk of new outbreaks related to these bacteria. In addition, animals with a high concentration of pathogenic E. coli cells present in feces (above 104 CFU/g), designated as supershedding animals, may be the primary transmission factor among ruminants. Therefore, the purpose of this review is to address pathogenicity factors and the importance of supershedding animals in the transmission of this pathogen, discussing the main methods currently applied, to focus on the occurrence of STEC in beef.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis.

Use of PCR for detection of bovine tuberculosis bacillus in milk of positive skin test cows / Uso da PCR para detecção de bacilo da tuberculose bovina em leite de vacas positivas ao teste cutâneo

The causative agent of bovine tuberculosis (BTB) is Mycobacterium bovis, a bacterium belonging to the M. tuberculosiscomplex (MTC). The definitive diagnosis is achieved through isolation and identification of M. bovis from clinical samples, using a combination of traditional culture and biochemical methods, which is considered the “gold standard”. This procedure is cumbersome and time-consuming. We evaluated a PCR assay for the direct detection of MTC DNA in milk of positive skin test cows, using primers that were previously tested and proven reliable to target the IS6110element. Milk previously seeded with M. bovis was used as the starting material, for standardization of the technique. The procedure involved extracting the DNA by enzymatic lysis (proteinase K and lysozyme), phenol, chloroform, isoamyl alcohol, followed by ethanol precipitation and PCR. The PCR assay allowed us to detect BTB in artificially contaminated milk, with a detection limit of 100 CFU/mL, and was also able to detect the bacillus in 50% (75/150) of the milk samples tested. This procedure could be used to assist the in vivo diagnosis of BTB, complementing sorological or microbiological tests, becoming an alternative option for epidemiological studies of BTB transmission and preventing contaminated milk from entering the food supply.

O agente causador da tuberculose bovina (BTB) é o Mycobacterium bovis, uma bactéria pertencente ao complexo M. tuberculosis (CMT). O diagnóstico definitivo é realizado através do isolamento e identificação de M. bovis em amostras clínicas, utilizando uma combinação de cultura bacteriológica e métodos bioquímicos, que são considerados como “padrão de ouro”. Entretanto, esses procedimentos são trabalhosos e demorados. No presente estudo, foi avaliado um ensaio de PCR para a detecção direta de DNA de CMT em leite de vacas positivas ao teste cutâneo, utilizando primerspreviamente testados e comprovadamente confiáveis, para amplificação da região de interesse IS6110. Leite previamen- te contaminado com M. bovis foi utilizado como material de partida para a padronização da técnica. O procedimento envolveu a extração do DNA por lise enzimática (proteinase K e lisozima), fenol, clorofórmio, álcool isoamílico, segui- do de precipitação com etanol e PCR. O ensaio de PCR detectou BTB em leite artificialmente contaminado, com um limite de detecção de 100 UFC/mL, e também foi capaz de detectar o bacilo em 50% (75/150) das amostras de leite testadas. A técnica de PCR pode ser utilizada para auxiliar o diagnóstico in vivo da BTB, além de complementar os tes- tes sorológicos ou microbiológicos, tornando-se uma alternativa para estudos epidemiológicos da transmissão de BTB, prevenindo que o leite contaminado entre na cadeia de alimentos.