Genome analysis of Pseudomonas strain 4B with broad antagonistic activity against toxigenic fungi.

Pseudomonas sp. 4B isolated from the effluent pond of a bovine abattoir was investigated as antifungal against toxigenic fungi. The complete genome of Pseudomonas 4B was sequenced using the Illumina MiSeq platform. Phylogenetic analysis and genome comparisons indicated that the strain belongs to the Pseudomonas aeruginosa group. In silico investigation revealed gene clusters associated with the biosynthesis of several antifungals, including pyocyanin, rhizomide, thanamycin, and pyochelin. This bacterium was investigated through antifungal assays, showing an inhibitory effect against all toxigenic fungi tested. Bacterial cells reduced the diameter of fungal colonies, colony growth rate, and sporulation of each indicator fungi in 10-day simultaneous growing tests. The co-incubation of bacterial suspension and fungal spores in yeast extract-sucrose broth for 48 h resulted in reduced spore germination. During simultaneous growth, decreased production of aflatoxin B1 and ochratoxin A by Aspergillus flavus and Aspergillus carbonarius, respectively, was observed. Genome analysis and in vitro studies showed the ability of P. aeruginosa 4B to reduce fungal growth parameters and mycotoxin levels, indicating the potential of this bacterium to control toxigenic fungi. The broad antifungal activity of this strain may represent a sustainable alternative for the exploration and subsequent use of its possible metabolites in order to control mycotoxin-producing fungi.

Genomic analysis of Enterococcus durans LAB18S, a potential probiotic strain isolated from cheese.

Gut microbiota exerts a fundamental role in human health and increased evidence supports the beneficial role of probiotic microorganisms in the maintenance of intestinal health. Enterococcus durans LAB18S was previously isolated from soft cheese and showed some desirable in vitro probiotic properties, for that reason its genome was sequenced and evaluated for genes that can be relevant for probiotic activity and are involved in selenium metabolism. Genome sequencing was performed using the Illumina MiSeq System. A variety of genes potentially associated with probiotic properties, including adhesion capability, viability at low pH, bile salt resistance, antimicrobial activity, and utilization of prebiotic fructooligosaccharides (FOS) were identified. The strain showed tolerance to acid pH and bile salts, exhibited antimicrobial activity and thrived on prebiotic oligosaccharides. Six genes involved in selenium metabolism were predicted. Analysis of the SECIS element showed twelve known selenoprotein candidates. E. durans LAB18S was the only food isolate showing absence of plasmids, virulence and antimicrobial resistance genes, when compared with other 30 E. durans genomes. The results of this study provide evidence supporting the potential of E. durans LAB18S as alternative for probiotic formulations.

Proteomic study of Enterococcus durans LAB18S growing on prebiotic oligosaccharides.

This study evaluates the influence of prebiotic carbohydrates, namely fructooligosaccharides (FOS) and galactooligosaccharides (GOS), on the protein expression of Enterococcus durans LAB18S. The strain was cultivated in 10 g L-1 FOS, GOS or glucose (control) and cellular proteins were extracted for mass spectrometry analysis. A total of 771 proteins were identified and 135 E. durans proteins were validated by the Scaffold algorithm. The proteins were functionally categorized according to Gene Ontology terms. Both FOS and GOS were used as carbon source by E. durans LAB18S, upregulating the production of proteins that may be associated with intestinal mucosa adhesion, carbohydrate and nitrogen metabolism, and stress response. Cells grown with GOS showed an increased expression of the cell division protein divIVA, EF-Tu and glyceraldehyde 3-phosphate dehydrogenase that have been associated with epithelial cell adhesion. The use of FOS stimulated the production of proteins related to amino acid metabolism and energy conversion, and ClpX protein, which plays an important role in protein turnover. The results of this study indicate that FOS and GOS can be metabolized by E. durans and stimulate the microorganism to produce proteins related to some desirable characteristics for a probiotic strain.

Functional genome annotation depicts probiotic properties of Bacillus velezensis FTC01.

An in silico genome analysis of the probiotic Bacillus strain FTC01 was performed. The draft genome comprises 3.9 Mb, with a G + C content of 46.6% and a total of 3941 coding sequences. The species of strain FTC01 was defined as B. velezensis during GenBank genome annotation, following the current nomenclature. Eight gene clusters involved in the synthesis of non-ribosomal lipopeptides, polyketides and bacilysin were found, as well as part of the gene cluster involved in the synthesis of cyclic lipopeptide locillomycin. The production of lipopeptides surfactin and iturin by strain FTC01 was confirmed. In addition, a gene encoding a peptidylprolyl isomerase, involved in bacterial adhesion to the host tissue, beyond twelve genes responsible for acid tolerance and several hydrolase genes were found. These characteristics may help in host colonization and maintenance and may account for the probiotic properties observed for strain FTC01.

Genome analysis reveals insights into high-resistance and virulence of Salmonella Enteritidis involved in foodborne outbreaks.

Salmonella enterica serovar Enteritidis strain SE86 has been associated with several foodborne diseases occurring in Southern Brazil, becoming and important causative agent of human salmonellosis. In this work, the complete genome of the bacterium Salmonella Enteritidis SE86 was sequenced using the Illumina MiSeq platform. An in silico analysis of the SE86 genome was performed in order to compare it with different Salmonella strains as well as to investigate the presence of stress-resistance and virulence genes. This strain showed a variety of genes that can be involved in antimicrobial and biocide resistance, acid and thermal resistance as well as virulence and adhesion. These genetic features could explain its increased resistance and the prevalence of this strain in foodborne outbreaks in Southern Brazil.

Quantitative assessment of tolerance response to stress after exposure to oregano and rosemary essential oils, carvacrol and 1,8-cineole in Salmonella Enteritidis 86 and its isogenic deletion mutants ∆dps, ∆rpoS and ∆ompR.

This study assessed the influence of rpoS, dps and ompR genes on the tolerance response of Salmonella Enteritidis 86 (SE86) to homologous and heterologous stressing agents after exposure to essential oils (EOs) from Origanum vulgare L. (oregano; OVEO) and Rosmarinus officinalis L. (rosemary; ROEO) and their major constituents (ICs), carvacrol (CAR) and 1,8-cineole (CIN), respectively, by modelling the log reduction over time. Minimum inhibitory concentration values of OVEO (1.25 µL/mL), CAR (0.62 µL/mL), ROEO (20 µL/mL) and CIN (10 µL/mL) against SE86 were always one-fold higher than those against ∆dps, ∆rpoS and ∆ompR mutants. Exposure to the same concentration of OVEO, CAR, ROEO or CIN caused higher reductions (up to 2.5 log CFU/mL) in ∆dps, ∆rpoS and ∆ompR mutants than in SE86 in chicken broth. In assays with homologous stressing agents, ompR, dps and rpoS influenced the tolerance to OEs or ICs. After adaptation to OVEO, CAR, ROEO and CIN, osmotolerance and acid tolerance of SE86 were influenced by rpoS gene, while thermotolerance of SE86 was influenced by ompR. Tolerance of SE86 to sodium hypochlorite after adaptation to OEs or ICs was influenced by rpoS and dps. These findings quantitatively describe for the first time the influence of rpoS, dps and ompR genes on the tolerance of Salmonella Enteritidis to OVEO, CAR, ROEO and CIN.

Comparative proteomic analysis of foodborne Salmonella Enteritidis SE86 subjected to cold plasma treatment.

The increasing demand for high quality and safe food led to important technological innovations in food processing. Cold plasma appears as an emerging technology that has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food. In this study, the proteomic profile of Salmonella Enteritidis SE86 subjected to cold plasma treatment was investigated. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to cold plasma and approximately 100 µg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected for up to 20 min exposure to cold plasma, and 2 log reduction was achieved after 60 min. Overall, 1096 proteins were identified, with 249 detected only in plasma-treated samples, and 9 exclusive in non-treated control samples. The proteins uniquely detected in cold plasma-treated cells that showed higher abundance were glyoxalase I, ABC transporter substrate-binding protein and transcriptional activator OsmE, followed by some oxidoreductases. Proteins related with carbohydrate and nucleotide metabolism were mostly overexpressed in cold plasma treated cells, suggesting energy metabolism was increased.

Expression of essential genes for biosynthesis of antimicrobial peptides of Bacillus is modulated by inactivated cells of target microorganisms.

Certain Bacillus strains are important producers of antimicrobial peptides with great potential for biological control. Antimicrobial peptide production by Bacillus amyloliquefaciens P11 was investigated in the presence of heat-inactivated cells of bacteria and fungi. B. amyloliquefaciens P11 exhibited higher antimicrobial activity in the presence of inactivated cells of Staphylococcus aureus and Aspergillus parasiticus compared to other conditions tested. Expression of essential genes related to biosynthesis of the antimicrobial peptides surfactin (sfp), iturin A (lpa-14 and ituD), subtilosin A (sboA) and fengycin (fenA) was investigated by quantitative real-time PCR (qRT-PCR). The genes lpa-14 and ituD were highly expressed in the presence of S. aureus (inactivated cells), indicating induction of iturin A production by B. amyloliquefaciens P11. The other inducing condition (inactivated cells of A. parasiticus) suppressed expression of lpa-14, but increased expression of ituD. A twofold increase in fenA expression was observed for both conditions, while strong suppression of sboA expression was observed in the presence of inactivated cells of S. aureus. An increase in antimicrobial activity was observed, indicating that synthesis of antimicrobial peptides may be induced by target microorganisms.

Expression of ompR gene in the acid adaptation and thermal resistance of Salmonella Enteritidis SE86.

INTRODUCTION: The objective of this study was to evaluate the involvement of the ompR gene in the acid adaptation and thermal resistance of S. Enteritidis SE86, responsible agent of more than 95 % of investigated food-borne diseases, throughout the last decade in Southern Brazil. In this study, we constructed a mutant strain of S. Enteritidis SE86 (ΔompR) that was attenuated by a knockout technique. The OmpR protein expression was determined in a tagged (3XFLAG) strain of S. Enteritidis SE86. METHODOLOGY: The mutant strains were cultivated separately in nutrient broth and nutrient broth supplemented with 1% glucose (NBG) to induce acid adapted cells. The organisms were exposed to different temperature such as 37 ºC, 52 ºC, and 60ºC. The survival of the SE86 wild type (WT) and attenuated strain was determined by bacterial count, and the tagged protein (ompR::3XFLAG cat::FLAG) was detected by SDS-PAGE and immunoblotting with anti-FLAG antibodies. RESULTS: Results showed that when exposed at 52ºC, the acid-adapted SE86 WT cells were completely inactivated after 300 minutes; however, non-adapted cells (WT and ΔompR) and acid-adapted ΔompR demonstrated higher thermal sensitivity, since they were completely inactivated in 240 minutes. At 60ºC, the acid-adapted SE86 ΔompR also demonstrated higher sensitivity that SE86 WT, being totally inactivated after 15 minutes, while the WT cells were inactivated in 20 minutes. CONCLUSION: The acid adapted cells showed increased expression of OmpR when exposed to 52 ºC and 60ºC, this confirmed the requirement of acid adaptation  for S. Enteritidis SE86 to resist elevated temperatures.

Foodborne illnesses in Brazil: control measures for 2014 FIFA World Cup travellers.

Foodborne diseases are typically caused by the ingestion of food contaminated with micro-organisms or their toxins, resulting in gastrointestinal disorders and in some severe cases hospitalization and death. In Brazil, foodborne illnesses are caused mainly by Salmonella, Staphylococcus aureus and Escherichia coli. The most important contributing factors for outbreaks are exposure of foods to unsuitable temperatures, inadequate food preparation and contamination of raw material or water used to prepare food. Recently, aiming to prevent foodborne illnesses during the 2014 FIFA World Cup, Brazil has developed a risk-based evaluation tool able to assess and grade Brazilian food services in cities that will host football matches. This tool has been used by the Brazilian sanitary surveillance officers during the inspection of facilities where food services. This is considered an innovative preventative sanitary action because it was created based on scientific information, statistical calculation and on risks of foodborne diseases occurrence. In this mini-review we summarize general data, control measures and how travellers can prevent foodborne illness in Brazil during the 2014 FIFA World Cup.

Investigation of rpoS and dps genes in sodium hypochlorite resistance of Salmonella Enteritidis SE86 isolated from foodborne illness outbreaks in southern Brazil.

In Rio Grande do Sul, southern Brazil, Salmonella Enteritidis is one of the principal microorganisms responsible for foodborne disease. The present study was conducted to compare the sodium hypochlorite resistance of Salmonella Enteritidis SE86 with that of other strains of Salmonella Enteritidis isolated from different regions of the world and to investigate the involvement of the rpoS and dps genes in resistance to this disinfectant. We tested five Salmonella Enteritidis wild-type (WT) strains isolated from different countries, two mutant strains of Salmonella Enteritidis SE86, and two tagged (3XFLAG) strains of Salmonella Enteritidis SE86 for their resistance to sodium hypochlorite (200 ppm). The survival of the WT and attenuated strains was determined based on bacterial counts, and tagged proteins (Dps and RpoS) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with anti-FLAG antibodies. None of the WT strains of Salmonella Enteritidis were totally inactivated after 20 min. The SE86 strain lacking dps was more sensitive to sodium hypochlorite than was the WT SE86 strain, with a 2-log reduction in counts after 1 min. The RpoS and Dps proteins were actively expressed under the conditions tested, indicating that in Salmonella Enteritidis SE86 these genes, which are expressed when in contact with sodium hypochlorite, are related to oxidative stress.

Evaluation of bacterial multiplication in cleaning cloths containing different quantities of organic matter.

INTRODUCTION: To determine a proper length of time for cleaning cloth usage, the present work aimed to evaluate bacterial multiplication in artificially contaminated cleaning cloths containing different amounts of organic matter. METHODOLOGY: Cloths containing 1%, 5%, and 10% of bovine albumin were contaminated with Salmonella enteritidis 3091/05, Escherichia coli ATCC 25972, Staphylococcus aureus ATCC 25923, and Shigella sonnei CC07. They were incubated for different time periods at 30°C. Microbial multiplication was evaluated by bacterial counts and the ATP bioluminescence increase was monitored at sampling points. An ampicillin-resistant recombinant HSα E. coli strain was used as a pathogen surrogate to investigate the potential of microbial cloth dispersion. RESULTS: None of the strains showed expressive growth up to two hours of incubation. At three hours, the microorganisms demonstrated a slight increase, with E. coli ATCC 25972 showing a significant increase in cells (p < 0.05). The ATP bioluminescence did not increase during the incubation period and confirmed the microbial count results, demonstrating that the amounts of organic matter tested did not interfere with bacterial multiplication during the first three to four hours of incubation. The dispersion experiment indicated that a cleaning cloth contaminated with 104 CFU/cm² was able to spread 10² CFU/cm² of recombinant E. coli onto a stainless steel surface. CONCLUSION: Based on these results, we suggest that an appropriate period of time for using disinfected cleaning cloths is around two hours, not exceeding three hours of usage.

Diferentes pré-inóculos, temperaturas e tempos de incubação na produção aflatoxina B1 em arroz / Different previus inoculum, temperature and incubation time in the production of aflatoxin B1 in rice

Estudos indicam que um dos problemas mais sérios que afetam os manejos pré e pós-colheita do arroz é a presença de fungos das espécies Aspergillus ou Penicillium, potencialmente produtores de micotoxinas. Os objetivos deste trabalho foram avaliar a capacidade produtora de aflatoxina B1 de cepas isoladas do arroz e observar o efeito do pré-inóculo nas mesmas. Foram utilizados três isolados de Aspergillus flavus, conhecidamente já produtores, que foram testados nas temperaturas de 20 e 25°C, combinadas com tempos de incubação 11, 14 e 21 dias. Os pré-inóculos utilizados foram Yeast Extrat Sucrose (YES) e Czapeck Yeast Extrat (CYA). Todas as cepas retiradas do pré-inóculo em meio YES e inoculadas no arroz, em temperatura de 25°C/18 dias e 20°C/14 dias, produziram aflatoxina B1. O meio CYA apresentou menor desempenho, uma vez que as três cepas testadas não produziram aflatoxina B1 na combinação 20°C/14 dias. A 25°C/11 dias de incubação a aflatoxina B1 não foi detectada.

The production of aflatoxin in rice by three isolates of Aspergillus flavus was investigated for different culture conditions (temperature and incubation time) and previous inoculum (YES- Yeast Extrat Sucrose and CYA- Czapeck Yeast Extrat). All strains withdrawn from the previous inoculum medium YES and inoculated in the rice in temperature of 25°C/18 days and 20°C/14 days, produced aflatoxin B1. The medium CYA had lower performance since the three strains tested did not produce aflatoxin B1 in the combination 20°C/14 days. At 25°C/11 days of incubation time aflatoxin was not detectable.