Analysis of two sampling treatments of beef for microbiome studies based on metataxonomic.

Microbial enumeration by serial dilution is one of the best resources to estimate cellular density for microbiological analysis. However, for metataxonomic analysis, it is not clear if serially diluted samples may accurately be used for metataxonomic analysis to represent species composition in beef samples. In this study, the effect of sampling preparation of beef samples on the bacterial composition was evaluated by the comparison of dilution and exudate. Based on the obtained results, data obtained from the exudate of the samples were more robust in terms of number of generated reads, but no significant differences in terms of biological diversity were observed (P < .05, Wicoxon Test). Besides, both sample preparation procedures evidenced equivalent results of bacterial composition as well as its relative abundances. In conclusion, the use of exudate allows bacterial enumeration and metataxonomic analysis, which is interesting for the point of view of food microbiologists as cellular loads and microbial composition of culturable and unculturable bacteria could be compared.

Bioinformatics tools to assess metagenomic data for applied microbiology.

The reduction of the price of DNA sequencing has resulted in the emergence of large data sets to handle and analyze, especially in microbial ecosystems, which are characterized by high taxonomic and functional diversities. To assess the properties of these complex ecosystems, a conceptual background of the application of NGS technology and bioinformatics analysis to metagenomics is required. Accordingly, this article presents an overview of the evolution of knowledge of microbial ecology from traditional culture-dependent methods to culture-independent methods and the last frontier in knowledge, metagenomics. Topics that will be covered include sample preparation for NGS, starting with total DNA extraction and library preparation, followed by a brief discussion of the chemistry of NGS to help provide an understanding of which bioinformatics pipeline approach may be helpful for achieving a researcher's goals. The importance of selecting appropriate sequencing coverage and depth parameters to obtain a suitable measure of microbial diversity is discussed. As all DNA sequencing processes produce base-calling errors that compromise data analysis, including genome assembly and microbial functional analysis, dedicated software is presented and conceptually discussed with regard to potential applications in the general microbial ecology field.

Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (ß-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Identification and evaluation of the probiotic potential of Lactobacillus paraplantarum FT259, a bacteriocinogenic strain isolated from Brazilian semi-hard artisanal cheese.

This study aimed to identify a bacteriocinogenic Lactobacillus isolate (FT259) obtained from Brazilian semi-hard Minas type cheese and to evaluate its probiotic and antimicrobial potentials. The strain was identified by biochemical tests (at genus level), and by 16S rDNA sequencing combined with recA gene amplification (for species). To determine the inhibitory spectrum towards food borne pathogens and lactic acid bacteria, the spot-on-the-lawn assay was carried out. Moreover, the proteinaceous nature of the antimicrobial compound produced was evaluated by susceptibility to degradation by proteolytic enzymes. The isolated strain was tested for survival in acidified culture media (pH 2.0, 2.5 and 3.5), in vitro tolerance to bile salts and viability under gastric conditions. Adhesion of Lactobacillus paraplantarum FT259 to Caco-2 cells was evaluated by surface plate count on De Man, Rogosa, and Sharpe (MRS) agar and also by FISH method (fluorescent in situ hybridization) with the aid of Eub338 probe for fluorescence microscopy analysis. The isolate was identified as L. paraplantarum FT259 and it produced bacteriocins that inhibited the growth of Listeria monocytogenes, Listeria innocua and several lactic acid bacteria. It was also observed that L. paraplantarum FT259 tolerated exposure to pH 3.5, and bile salts 0.3% for up to 180 min. In experiments with simulated gastric juice, viable cells of L. paraplantarum FT259 decreased from 8.6 log CFU/mL to 3.5 log CFU/mL after 180 min. For the same strain, in studies with Caco-2 cells, 74% of adhesion was observed through plate count and FISH assays. It was also demonstrated isolated FT259 was susceptible to the majority the antibiotics tested. Overall, the results indicated L. paraplantarum FT259 is a potential probiotic and the production of bacteriocin may be an interesting feature for food applications.

The role of microorganisms in the biotransformation of psychoactive substances and its forensic relevance: a critical interdisciplinary review.

Microorganisms are widespread on the planet being able to adapt, persist, and grow in diverse environments, either rich in nutrient sources or under harsh conditions. The comprehension of the interaction between microorganisms and drugs is relevant for forensic toxicology and forensic chemistry, elucidating potential pathways of microbial metabolism and their implications. Considering the described scenario, this paper aims to provide a comprehensive and critical review of the state of the art of interactions amongst microorganisms and common drugs of abuse. Additionally, other drugs of forensic interest are briefly discussed. This paper outlines the importance of this area of investigation, covering the intersections between forensic microbiology, forensic chemistry, and forensic toxicology applied to drugs of abuse, and it also highlights research potentialities. Key points: Microorganisms are widespread on the planet and grow in a myriad of environments.Microorganisms can often be found in matrices of forensic interest.Drugs can be metabolized or produced (e.g. ethanol) by microorganisms.

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality.

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 °C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics' viability decreases conversely with the drying temperature. At temperatures close to 120 °C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 °C are required to ensure the microbial cells' viability and shelf-life in the powdered preparation and survival during food processing and storage.

Cocaine esterase occurrence in global wastewater microbiomes and potential for biotransformation of novel psychoactive substances.

The analysis of drugs in wastewater for forensic purposes has been constantly increasing and the investigation of the potential interaction between drugs or metabolites and sewage microbiota is important. The results demonstrated that cocaine esterase genes were widely distributed in 1142 global wastewater samples collected from 64 countries and linked to several bacterial species. In addition, in silico predictions indicated that carfentanil, 4F-MDMB-BINACA, 5F-MDMB-PICA, MDMB-4en-PINACA and mitragynine might also undergo microbial hydrolysis, in a similar fashion of cocaine degradation by cocaine esterase. In conclusion, it was demonstrated the microbial potential to hydrolyze drugs of abuse in wastewater environments, contributing to the critical evaluation of potential metabolites as biomarkers for microbial and human transformation of drugs in wastewater.

Beneficial effects of probiotics on the pig production cycle: An overview of clinical impacts and performance.

In pig nutrition, antibiotics are used to promote growth and/or to treat diseases in order to improve animal performance. However, due to the potential risk of cross selective pressure for antibiotic resistance among bacterial pathogens, the development of new nutritional additives is needed. Among them, probiotics are of great interest since they could improve the immune response, maintain animal intestinal health, and improve nutritional efficiency. Studies with probiotics have also demonstrated their antimicrobial effects on several pathogenic strains, emphasizing that the form of administration can enhance the beneficial effects. In view of the promising advances in probiotic research, it is opportune to highlight their capacity to modulate health and improve performance at all stages of pig production. Therefore, in this review, we will discuss the benefits of probiotics on physiological, immunological, and clinical aspects during different stages of the pig's life cycle. Specifically, probiotics improve performance during pregnancy, parturition and lactation in sows, they can improve immunohematological parameters and defenses in the growing phase, they can influence the quality of meat in the finishing phase and can also help in the reduction of environmental pollutants.

Vaginal lactobacilli as potential probiotics against Candida SPP.

Urogenital infections affect millions of people every year worldwide. The treatment of these diseases usually requires the use of antimicrobial agents, and more recently, the use of probiotic lactic acid bacteria (LAB) cultures for the management of vaginal infections has been extensively studied. In this work, 11 vaginal lactobacilli isolates, previously obtained from healthy patients, were studied to screen microorganisms with probiotic properties against Candida spp. The LAB were tested for their ability of auto-aggregation, co-aggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide (H2O2). All lactobacilli isolates tested were able to auto-aggregate (ranging from 25.3% to 75.4% assessed at 4 hours of incubation) and to co-aggregate with the four Candida species into different degrees; among them L. crispatus showed the highest scores of co-aggregation. The highest amount of lactic acid was produced by L. salivarius (13.9 g/l), followed by L. johnsonii (6.5 g/l), L. acidophilus (5.5 g/l), and L. jensenii (5.4 g/l). All isolates produced H2O2, but the highest levels (3 - 10 mg/l) were observed for L. acidophilus, L. crispatus, L. gasseri, L. johnsonii, and L. vaginalis. Only L. agilis, L. jensenii, L. johnsonii and L. ruminus were able to adhere to epithelial Caco-2 cells. Among the isolates evaluated, L agilis, L. jensenii, L. johnsonii, and L. ruminus exhibited simultaneously several desirable properties as potential probiotic strains justifying future studies to evaluate their technological properties in different pharmaceutical preparations for human use.

Analysis of vaginal lactobacilli from healthy and infected Brazilian women.

Culture-dependent PCR-amplified rRNA gene restriction analysis and culture-independent (PCR-denaturing gradient gel electrophoresis) methodologies were used to examine vaginal lactobacilli from Brazilian women who were healthy or had been diagnosed with vulvovaginal candidiasis (VVC) or bacterial vaginosis. Only Lactobacillus crispatus was detected accordingly by both methods, and H(2)O(2)-producing lactobacilli were not associated with protection against VVC.

Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation.

Biofilm formation is a matter of concern in food industries because biofilms facilitate the survival of pathogenic bacteria such as Listeria monocytogenes, which may contaminate food-processing equipment and products. In this study, nisin and two Enterococcus faecium strains were evaluated for their effect on biofilm formation by L. monocytogenes cultured in brain heart infusion broth and on stainless steel coupons. Elimination of preformed L. monocytogenes biofilms by peroxyacetic acid also was tested. Adhesion control experiments were performed with pure cultures of L. monocytogenes after swab collection of adhered cells, which were then enumerated on PALCAM agar plates and visualized by scanning electron microscopy. Formation of a biofilm was recorded when the number of adhered cells was at least 10(3) CFU/cm2. When L. monocytogenes was cocultured with E. faecium bac-, the number of adhered L. monocytogenes cells was 2.5 log lower (P = 0.002) when initially compared with the control culture, but after 6 h of incubation a biofilm was again detected. However, in coculture on stainless steel coupons, E. faecium bac+ inhibited L. monocytogenes adherence and did not allow biofilm formation for up to 48 h (P < 0.001). In the presence of nisin or after treatment with peroxyacetic acid, bacterial growth was reduced (P < 0.001) up to 4.6 and 5.6 log CFU/cm2, respectively, when compared with L. monocytogenes cultures on untreated coupons. However, after these treatments, cells were still present, and after 24 h of incubation, a renewed biofilm was detected in L. monocytogenes cultures treated with nisin. Although all tested conditions reduced L. monocytogenes growth to some extent, only coculture with E. faecium bac+ efficiently reduced biofilm formation, suggesting a potential control strategy for this pathogen.

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods.

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.

Development of Yoghurt with Juçara Pulp (Euterpe edulis M.) and the Probiotic Lactobacillus acidophilus La5.

Yoghurts are dairy products consumed worldwide and can be supplemented with substances that provide extra health benefits as well as probiotic strains. In this context, the present study aimed to prepare a yoghurt added of juçara (Euterpe edulis M.) pulp and the commercial probiotic strain Lactobacillus acidophilus La5. Moreover, the probiotic survival during storage and after in vitro exposure to simulated gastric and enteric conditions was evaluated. Four formulations of yoghurt were prepared: (a) natural yoghurt, (b) yoghurt added of probiotic, (c) yoghurt added of juçara pulp, and (d) yoghurt added of probiotic culture and juçara pulp. The preparations were evaluated for survival of probiotic strain during storage and its tolerance to gastric and enteric conditions in vitro. The probiotic population in yoghurt remained unchanged during 28 days of storage. In addition, juçara pulp increased the probiotic resistance to simulated gastric and enteric conditions in the first day of storage. These data indicate that juçara pulp is a potential ingredient for the production of probiotic yoghurts.

Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [Pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim.

Data on the prevalence and growth of Listeria monocytogenes in lightly preserved fish products from subtropical and tropical regions are very scarce. Our research describes L. monocytogenes that was detected in 5% of the packages of cold-smoked surubim, a native Brazilian freshwater fish that we analyzed, and shows that the strains isolated were of the same random amplified polymorphic DNA subtype as the strains that were isolated from the same factory 4 years earlier. A bacteriocinogenic strain of Carnobacterium piscicola (strain C2), isolated from vacuum-packed cold-smoked surubim, and two C. piscicola strains, isolated from vacuum-packed, cold-smoked salmon, were capable of limiting or completely inhibiting the growth of an L. monocytogenes (strain V2) isolated from surubim in fish peptone model systems incubated at 10 degrees C. Monocultures of L. monocytogenes reached 108 CFU/ml (g), whereas the growth of L. monocytogenes was completely inhibited by C. piscicola C2. The bacteriocinogenic C. piscicola A9b+ and its nonbacteriocinogenic mutant A9b- reduced maximum Listeria levels by 2 to 3 log units. Both bacteriocinogenic C. piscicola strains prevented listerial growth in cold-smoked fish juices (surubim and salmon). Although the carnobacteria grew poorly on cold-smoked surubim at 10 degrees C, the strains were able to reduce maximum Listeria counts by 1 to 3 log units in an artificially inoculated product (surubim). We conclude that Brazilian smoked fish products harbor L. monocytogenes and should be stabilized against the growth of the organism. C. piscicola C2 has the potential for use as a bioprotective culture in surubim and other lightly preserved fish, but further studies are required to optimize its effect.

Different methods to quantify Listeria monocytogenes biofilms cells showed different profile in their viability.

Listeria monocytogenes is a foodborne pathogen able to adhere and to form biofilms in several materials commonly present in food processing plants. The aim of this study was to evaluate the resistance of Listeria monocytogenes attached to abiotic surface, after treatment with sanitizers, by culture method, microscopy and Quantitative Real Time Polymerase Chain Reaction (qPCR). Biofilms of L. monocytogenes were obtained in stainless steel coupons immersed in Brain Heart Infusion Broth, under agitation at 37 °C for 24 h. The methods selected for this study were based on plate count, microscopic count with the aid of viability dyes (CTC-DAPI), and qPCR. Results of culture method showed that peroxyacetic acid was efficient to kill sessile L. monocytogenes populations, while sodium hypochlorite was only partially effective to kill attached L. monocytogenes (p < 0.05). When, viability dyes (CTC/DAPI) combined with fluorescence microscopy and qPCR were used and lower counts were found after treatments (p < 0.05). Selective quantification of viable cells of L. monocytogenes by qPCR using EMA revelead that the pre-treatment with EMA was not appropriate since it also inhibited amplification of DNA from live cells by ca. 2 log. Thus, the use of CTC counts was the best method to count viable cells in biofilms.

Purification and characterization of antimicrobial peptides from fish isolate Carnobacterium maltaromaticum C2: Carnobacteriocin X and carnolysins A1 and A2.

Carnobacterium maltaromaticum C2, isolated from Brazilian smoked fish (Surubim, Pseudoplatystoma sp.), was found to exert antimicrobial activity against Listeria monocytogenes, an important foodborne pathogen. In this study, the bacteriocins produced by C. maltaromaticum C2 were purified via an extraction with XAD-16 resin, a C18 solid phase extraction, followed by reversed-phase fast protein liquid chromatography. The purified active fractions were characterized using tandem mass spectrometry, permitting the identification of multiple bacteriocins. Carnobacteriocins BM1, B1, and a variant of carnobacteriocin B2 were all found, providing much of the antilisterial activity. Additionally, we herein report the first isolation of the previously predicted antimicrobial peptide carnobacteriocin X. Moreover, C. maltaromaticum C2 produces a novel two-component lantibiotic, termed carnolysin, homologous to enterococcal cytolysin. This lantibiotic is antimicrobially inactive when tested against the non-bacteriocinogenic strain C. maltaromaticum A9b-, likely requiring an additional proteolytic cleavage to reach maturity.

Effect of Bacteriocins and Conditions that Mimic Food and Digestive Tract on Biofilm Formation, In Vitro Invasion of Eukaryotic Cells and Internalin Gene Expression by Listeria monocytogenes.

In this report, Listeria monocytogenes isolates were evaluated for their ability to form biofilms, for adhesion/invasion of eukaryotic cells and for differential expression of internalin A (inl A) gene, which is related to virulence potential. The presence of bacteriocins of lactic acid bacteria and incubation at 5 °C were the main factors that influenced biofilm formation by L. monocytogenes, in comparison with BHI (control). In general, adhesion and invasion of Caco-2 cells were significantly lower in low pH (4.5), in incubation at 5 °C and in the presence of Oxgall 0.3 %. On the other hand, two L. monocytogenes isolates (INCQS 353 and Reg 26c) showed higher invasion rates when cultivated in the presence of NaCl 5 % (P < 0.05). One L. monocytogenes isolate (H-2) showed the strongest ability to form biofilm and to invade Caco-2 cells, under selected conditions, suggesting there is a relationship between biofilm formation and virulence potential. For all isolates, expression of inl A gene was down-regulated by the presence of bacteriocins, Oxgall 0.3 %, pH 4.5 and incubation at 5 °C. Nonetheless, for one L. monocytogenes isolate (HU 471), expression of inl A gene was eight times higher in the presence of sucrose, indicating that food components can increase the infectiveness of L. monocytogenes.

Effect of galactooligosaccharides and Bifidobacterium animalis Bb-12 on growth of Lactobacillus amylovorus DSM 16698, microbial community structure, and metabolite production in an in vitro colonic model set up with human or pig microbiota.

A validated in vitro model of the large intestine (TIM-2), set up with human or pig faeces, was used to evaluate the impact of potentially probiotic Lactobacillus amylovorus DSM 16698, administered alone (i), in the presence of prebiotic galactooligosaccharides (GOS) (ii), and co-administered with probiotic Bifidobacterium animalis ssp. lactis Bb-12 (Bb-12) (iii) on GOS degradation, microbial growth (L. amylovorus, lactobacilli, bifidobacteria and total bacteria) and metabolite production. High performance anion exchange chromatography revealed that GOS degradation was more pronounced in TIM-2 inoculated with pig faeces than with human faeces. Denaturing gradient gel electrophoresis profiling of PCR-amplified 16S rRNA genes detected a more complex Lactobacillus spp. community in pig faecal material than in human faecal inoculum. According to 16S rRNA gene-targeted qPCR, GOS stimulated the growth of lactobacilli and bifidobacteria in faecal material from both materials. The cumulative production of short chain fatty acids and ammonia was higher (P < 0.05) for pig than for human faeces. However, lactate accumulation was higher (P < 0.05) in the human model and increased after co-administration with GOS and Bb-12. This study reinforced the notion that differences in microbiota composition between target host organisms need to be considered when animal data are extrapolated to human, as is often done with pre- and probiotic intervention studies.