No abstract

Cow raw milk cheese is widely eaten in Brazil. These products may be contaminated with pathogenic bacteria. In this work, we investigated the presence of Escherichia coli in raw milk cheese from different States in Brazil. From 147 "Minas" cheese samples, 28 cheeses were positive for E. coli. Among 39 E. coli isolates of the cheeses, one was positive for eae and negative for bpfA and efa1/lifA using PCR, and so was classified as atypical Enteropathogenic E. coli (aEPEC). Two other isolates were positive for extraintestinal pathogenic E. coli (ExPEC) genes. The aEPEC isolate belongs to serogroup O127 and was classified in A phylogenetic group, and ExPEC isolates were found in O73:H12 (EC-2 strain) and O64474:H8 (EC-9 strain) serotype. This ExPEC belongs to A and C phylogenetic group, respectively. Most of E. coli strains belonged to Clermont phylogenetic groups A (28.2%), C, and E (23.1%). Six strains (15.4%) of E. coli were positive for group B1 and two (5.1%) for B2. E. coli isolates presented an aggregative (46.0%) and diffuse (12.6%) adherence pattern to HeLa cells, and the other isolates did not show adhesion (41.4%). Four E. coli isolates (10.3%) were shown to produce moderate biofilm. The antimicrobial resistance rate was tetracycline (25.6%), followed by ampicillin (17.9%), cefoxitin (7.7%), nalidixic acid (5.1%), and amoxicillin-clavulanic acid (2.6%). One strain was resistant to three antimicrobials (tetracycline, ampicillin, and nalidixic acid). The presence of these microorganisms, the O127 strain, and a new serogroup in Brazil is a potential risk for public health.

Poultry litter is commonly used as fertilizer in agriculture. However, this poultry litter must be processed prior to use, since poultry have a large number of pathogenic microorganisms. The aims of this study were to isolate and genotypically and phenotypically characterize Escherichia coli from avian organic fertilizer. Sixty-four E. coli isolates were identified from avian organic fertilizer and characterized for ExPEC virulence factors, pathogenicity islands, phylogenetic groups, antimicrobial resistance, biofilm formation, and adhesion to HEp-2 cells. Sixty-three isolates (98.4%) showed at least one virulence gene (fimH, ecpA, sitA, traT, iutA, iroN, hlyF, ompT and iss). The predominant phylogenetic groups were groups A (59.3%) and B1 (34.3%). The pathogenicity island CFT073II (51.5%) was the most prevalent among the isolates tested. Thirty-two isolates (50%) were resistant to at least one antimicrobial agent. Approximately 90% of isolates adhered to HEp-2 cells, and the predominant pattern was aggregative adherence (74.1%). In the biofilm assay, it was observed that 75% of isolates did not produce biofilm. These results lead us to conclude that some E. coli isolates from avian organic fertilizer could be pathogenic for humans.

Infection with Shiga toxin-producing Escherichia coli (STEC) results in hemorrhagic colitis and can lead to life-threatening sequelae including hemolytic uremic syndrome (HUS). Conventional treatment is intravenous fluid volume expansion. Antibiotic treatment is contraindicated, due in part to the elevated risk of HUS related to increased Shiga toxin (Stx) release associated with some antibiotics. Given the lack of effective strategies and the increasing number of STEC outbreaks, new treatment approaches are critically needed. In this study, we used an antimicrobial peptide wrwycr, previously shown to enhance STEC killing without increasing Stx production, in combination with antibiotic treatments. Checkerboard and time-kill assays were used to assess peptide wrwycr-antibiotic combinations for synergistic STEC killing. Cytotoxicity and real-time PCR were used to evaluate Stx production and stx expression, respectively, associated with these combinations. The synergistic combinations that showed rapid killing, no growth recovery and minimal Stx production were peptide wrwycr-kanamycin/gentamicin. Transmission electron microscopy revealed striking differences in bacterial cell morphology associated with various treatments. This study provides proof of principle for the design of an antibiotic-peptide wrwycr combination effective in killing STEC without enhancing release of Shiga toxins. It also offers a strategy for the repurposing of antibiotics for treatment of STEC infection.

The Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family. The objective of this study was to detect the presence of diarrheagenic Escherichia coli (DEC) strains in avian organic fertilizer, and assess the potential damage they can cause in humans due to antimicrobial resistance. The presence of DEC pathotypes and phylogenetic groups were detected by multiplex-PCR. Phenotypic assays, such as tests for adhesion, cytotoxicity activity, biofilm formation and especially antimicrobial susceptibility, were performed. Fifteen DEC strains from 64 E. coli were isolated. Among these, four strains were classified as enteropathogenic (EPEC; 6.2%), three strains as Shiga toxin-producing (STEC; 4.7%), 10 strains as enteroaggregative (EAEC; 12.5%), but two of these harbored the eaeA gene too. The low number of isolated strains was most likely due to the composting process, which reduces the number of microorganisms. These strains were able to adhere to HEp-2 and HeLa cells and produce Shiga-toxins and biofilms; in addition, some of the strains showed antimicrobial resistance, which indicates a risk of the transfer of resistance genes to human E. coli. These results showed that DEC strains isolated from avian organic fertilizers can cause human infections.

The presence of pathogenic microorganisms in meat products may result in foodborne diseases and economic losses to their producers. Small industries in the region of Londrina, Paraná, produce sausages that are commercialized in free fairs, small markets, bars, and restaurants in the city. Although these industries are inspected by the Municipal Inspection Service of Londrina, there are no data about the pathogenic microorganisms present in these products. The objective of this study was to investigate the presence of Salmonella spp. in sausages produced and sold in the region of Londrina, Paraná, and identify eae, bfp, stx1, stx2, hlyA, ipaH, elt, est, aggR, aap, and AA probe genes in Escherichia coli strains isolated from these samples. Forty-six samples of three types of sausages (fresh pork, Tuscan, and Calabresa) produced by four different producers (brands A, B, C, and D) were analyzed. Salmonella spp. was isolated from 13 (28.3%) and E. coli from 33 (71.3%) of the analyzed samples. Seven (53.8%) of 13 samples contaminated with Salmonella spp. were from brand A. Salmonella spp. contamination was the highest in the Tuscan sausage samples (8/17, 41.7%) when compared with the fresh pork sausage samples of all brands analyzed. E. coli was isolated from 12 of 13 samples contaminated with Salmonella spp. One sample of Calabresa sausage was contaminated with atypical enteropathogenic E. coli serotype O108:H9 that has the eae and hlyA genes. The results suggest contamination of the processing plant and/or raw meat used in the manufacture of sausages. A better inspection of the industries is required to ensure that Good Manufacturing Practices are followed by which the contamination of products by pathogenic bacteria can be prevented. Key words: Pork meat. Enteropathogenic bacteria. Virulence genes. ResumoA presença de microrganismos patogênicos em produtos cárneos pode levar a doenças de origem alimentar e perdas econômicas aos seus produtores. Pequenas indústrias da região de Londrina, Paraná, produzem embutidos que são comercializados em feiras livres, pequenos mercados, bares e restaurantes da cidade. Embora essas indústrias sejam fiscalizadas pelo Serviço de Inspeção Municipal de Londrina, não existem dados sobre microrganismos patogênicos presentes nesses produtos. Os objetivos deste estudo foram pesquisar a presença de Salmonella spp. em amostras de linguiças produzidas e comercializadas na região de Londrina, Paraná, e identificar os genes de virulência eae, bfp, stx1, stx2, hlyA, ipaH, elt, est, aggR, aap e AA probe das cepas de E. coli isoladas dessas amostras. Quarenta e seis amostras de três tipos de linguiça (suína fresca, toscana e calabresa) produzidas por quatro diferentes produtores (marcas A, B, C e D) foram analisadas. Salmonella spp. foi isolada em 13 (28.3%) amostras e E. coli em 33 (71.3%) amostras. Das 13 amostras contaminadas com Salmonella spp., sete (53.8%) foram da marca A. A contaminação por Salmonella spp. foi maior nas amostras de linguiça toscana (8/17 -41.7%) quando comparad...

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Diarrheagenic (DEC) and avian pathogenic Escherichia coli (APEC) are associated with intestinal and extra-intestinal infections (ExPEC), respectively. We aimed to analyze the antimicrobial susceptibility, gene encoding virulence factors associated to DEC and APEC, and phylogenetic classification in E. coli isolated from 320 samples of feed and ingredients. Antimicrobial susceptibility was performed using the disk diffusion method and Multiple Antibiotic Resistance (MAR) Index and Multi-Drug Resistance (MDR) were calculated. Phylogenetic classification was performed on samples harboring DEC and/or APEC virulence-associated genes. A total of 110 E. coli strains were isolated in 15% (49/320) of the evaluated inputs (n=13 vegetable meal; n=33 animal meal, n=3 feed). In general, the isolates showed the highest rates of antimicrobial resistance to sulfonamide and cefazolin and 18% (20/110) were multi-drug resistant. MAR index of feed samples was the highest (0.467). Six and five strains had APEC and DEC virulence-associated genes, respectively, and belonging to phylogenetic groups A and B1. These findings point to the need for strict microbiological control during the production process of these foods.