The quorum sensing molecule C12-HSL promotes biofilm formation and increases adrA expression in Salmonella Enteritidis under anaerobic conditions.

Acyl-homoserine lactones (AHLs) are quorum-sensing signaling molecules in Gram-negative bacteria and positively regulate biofilm formation in Salmonella under specific conditions. In this study, biofilm formation in Salmonella enterica was evaluated at 28 and 37 °C, under aerobic and anaerobic conditions. Additionally, the influence of the N-dodecanoyl-DL-homoserine lactone (C12-HSL) on biofilm formation and the expression of genes related to the synthesis of structural components, regulation, and quorum sensing was assessed under anaerobiosis at 28 and 37 °C. Biofilm formation was found not to be influenced by the atmospheric conditions at 28 °C. However, it was reduced at 37 °C under anaerobiosis. C12-HSL enhanced biofilm formation at 37 °C under anaerobiosis and increased the expression of the adrA and luxS genes, suggesting an increase in c-di-GMP, a second messenger that controls essential physiological functions in bacteria. These results provide new insights into the regulation of biofilm formation in Salmonella under anaerobic conditions.

Biodegradation of polyurethanes by Serratia liquefaciens L135 and its polyurethanase: In silico and in vitro analyses.

Polyurethanes (PUs) are found in many everyday products and their disposal leads to environmental accumulation. Therefore, there is an urgent need to develop ecologically sustainable techniques to biodegrade and recycle this recalcitrant polymer and replace traditional methods that form harmful by-products. Serratia liquefaciens L135 secretes a polyurethanase with lipase activity, and this study explores the biodegradation of PUs by this bacterium and its enzyme through in silico and in vitro analyses. PUs monomers and tetramers were constructed in silico and tested with modeled and validated structure of the polyurethanase from S. liquefaciens. The molecular docking showed that all PUs monomers presented favorable interactions with polyurethanase (values of binding energy between -84.75 and -121.71 kcal mol-1), including PU poly[4,4'-methylenebis (phenyl isocyanate)-alt-1,4-butanediol/di (propylene glycol)/polycaprolactone] (PCLMDI). Due to repulsive steric interactions, tetramers showed less favorable interactions (values between 24.26 and -45.50 kcal mol-1). In vitro analyses evaluated the biodegradation of PUs: Impranil® and PCLMDI; this latter showed high binding energy with this polyurethanase in silico. The biodegradation of Impranil® by S. liquefaciens and its partially purified polyurethanase was confirmed in agar by forming a transparent halo. Impranil® disks inoculated with S. liquefaciens and incubated at 30 °C for six days showed rupture of the PU structure, possibly due to the formation of cracks visualized by scanning electron microscopy (SEM). PCLMDI films were also biodegraded by S. liquefaciens after 60 days of incubation, with the formation of pores and cracks visualized by SEM. The biodegradation may have occurred due to the action of polyurethanase produced by this bacterium. This work provides essential information on the potential of S. liquefaciens to biodegrade PUs through in silico analyses combined with in vitro analyses.

Exposure to cellulose acetate films incorporated with garlic essential oil does not lead to homologous resistance in Listeria innocua ATCC 33090.

Essential oils (EOs) have been considered potential green additives for active food packaging. However, sub-lethal concentrations of EOs may lead to bacterial resistance, which is a concern. In this sense, the effects of 1% (GEO1) and 10% (GEO10) of garlic EO in cellulose acetate-based films regarding homologous resistance in Listeria innocua were investigated after incubation at 37 °C/24 h and 7 °C/10 d. The films were also characterized and tested on sliced mozzarella cheese as interfold packaging for 8-days storage at 7 °C. The EO did not alter the mechanical properties of the films nor their thermal degradation profile. However, GEO10 was less permeable to water vapor than GEO1. When tested against L. innocua, the incubation at 7 °C enhanced the films' antimicrobial effect: log reductions of 4.3 and 5.7 were obtained for GEO1 and GEO10, respectively. Moreover, 86.3% of L. innocua cells were injured at sub-lethal level when exposed to GEO10. Despite this, no occurrence of homologous resistance was found. When the active films were tested on cheese against the natural microbiota, they resulted in slices of mozzarella with fewer contaminants, however the reduction was not significant. Nevertheless, we considered this an important finding to the food industry since this work suggested that GEO is a safe active compound from the point of view of homologous resistance to be used against Listeria.

Furanone and phytol influence metabolic phenotypes regulated by acyl-homoserine lactone in Salmonella.

Salmonella is an important foodborne pathogen, and it is unable to produce the quorum sensing signaling molecules called acyl-homoserine lactones (AHLs). However, it synthesizes the SdiA protein, detecting AHL molecules, also known as autoinducer-1 (AI-1), in the external environment. Exogenous AHLs can regulate specific genes related to virulence and stress response in Salmonella. Thus, interfering with quorum sensing can be a strategy to reduce virulence and help elucidate the cell-to-cell communication role in the pathogens' response to extracellular signals. This study aimed to evaluate the influence of the quorum sensing inhibitors furanone and phytol on phenotypes regulated by N-dodecanoyl homoserine lactone (C12-HSL) in Salmonella enterica serovar Enteritidis. The furanone C30 at 50 nM and phytol at 2 mM canceled the alterations promoted by C12-HSL on glucose consumption and the levels of free cellular thiol in Salmonella Enteritidis PT4 578 under anaerobic conditions. In silico analysis suggests that these compounds can bind to the SdiA protein of Salmonella Enteritidis and accommodate in the AHL binding pocket. Thus, furanone C30 and phytol act as antagonists of AI-1 and are likely inhibitors of the quorum sensing mechanism mediated by AHL in Salmonella.

Enhancing cell resistance for production of mixed microbiological reference materials with Salmonella and coliforms by freeze-drying.

The reference material (RM) is a technical requirement for the quality assurance of analytical results and proficiency tests or interlaboratory comparisons. Microbiological RMs are most available in the dehydrated form, mainly by freeze-drying, and maintaining bacterial survival after preparation is a challenge. Thus, obtaining the most resistant cells is essential. Considering that bacteria present cross-response to dehydration after being submitted to an array of stress conditions, this study aimed to evaluate the influence of growth conditions on enterobacteria for the production of mixed microbiological RMs by freeze-drying in skim milk powder. Salmonella enterica serovar Enteritidis, Cronobacter sakazakii, Escherichia coli, and Citrobacter freundii were grown in a minimal medium with 0.5 M NaCl and 0 to 5.0 mM of manganese sulfate (MnSO4) until stationary phase. Salmonella Enteritidis presented an increased resistance to dehydration in the presence of Mn, while C. sakazakii was the most resistant to freeze-drying and further storage for 90 days. Mixed microbiological RMs were produced by freeze-drying and containing Salmonella Enteritidis and coliforms in skim milk powder with 100 mM of trehalose and the Salmonella survival rate was 91.2 to 93.6%. The mixed RM was stable after 30 days at -20 °C, and Salmonella and coliforms were detected by different methods being, the Rambach Agar the best for the bacterial differentiation. The results showed that the culture conditions applied in this study resulted in bacterial cells being more resistant to dehydration, freeze-drying, and stabilization for the production of mixed microbiological RMs more stable and homogeneous.

N-acyl-homoserine lactone produced by Rahnella inusitata isolated from the gut of Galleria mellonella influences Salmonella phenotypes.

The most studied mechanism of quorum sensing in Gram-negative bacteria is mediated by autoinducer 1 (AI-1), namely, acyl-homoserine lactone (AHL). This system allows communication among different bacterial species and regulates the expression of virulence genes in many pathogens. Although AHL-producing bacteria have been detected in the intestines of humans and other animals, no report was found about AHL-producing bacteria in the insect gut and the possible effects of these autoinducers on enteropathogenic bacteria. Therefore, this study aimed to identify AHL-producing bacteria in the gut of larvae of Galleria mellonella and to evaluate the influence of this quorum sensing signal on the regulation of adhesion and motility phenotypes in the intestinal pathogen Salmonella. Sequencing of the 16S rRNA gene, 16S rRNA gene-based phylogenetic analyses, and phenotypic characterization of gut isolates was performed. The profile of AHLs produced by the isolates was determined using thin-layer chromatography (TLC) and revealed with the biosensor strain Chromobacterium violaceum CV026. Sequencing, phylogenetic analyses and phenotypic characterization of gut isolates showed that the three AHL-producing strains belong to the species Rahnella inusitata, named GM34, GM56, and GM60. The TLC showed that R. inusitata produces a six-carbon AHL. In the presence of cell-free extract of R. inusitata containing AHL and under anaerobic conditions, Salmonella enterica increased the adhesion to stainless steel coupons and presented swarming motility. Extracts from the culture medium of R. inusitata isolates containing AHL increased the adhesion on stainless steel coupons and swarming motility of Salmonella enterica serovar Enteritidis PT4 under anaerobic conditions. The results suggest the possibility of communication between members of the G. mellonella intestinal microbiota with pathogens such as Salmonella.

Protein expression profiling of Staphylococcus aureus in response to the bacteriocin bovicin HC5.

Alternative strategies to antibiotic treatment are required to inhibit pathogens, including Staphylococcus aureus. Bacteriocins, such as the lantibiotic bovicin HC5, have shown potential to control pathogens. This study aims to evaluate the stress response of S. aureus to bovicin HC5 using a proteomic approach. Sublethal concentrations of the bacteriocin repressed the synthesis of 62 cytoplasmic proteins, whereas 42 proteins were induced in S. aureus COL. Specifically, synthesis of several proteins involved in amino acid biosynthesis, mainly products of ilv-leu operon, and DNA metabolism, such as DNA polymerase I, decreased following bovicin treatment while proteins involved in catabolism, mainly tricarboxylic acid cycle metabolism, and chaperones were over-expressed. The levels of CodY and CcpA, important regulators involved in the stationary phase adaptation and catabolite repression, respectively, also increased in the presence of the bacteriocin. These results indicate that stress caused by the sublethal concentration of bovicin HC5 in the cell membrane results in growth reduction, reduced protein synthesis, and, at the same time, enhanced the levels of chaperones and enzymes involved in energy-efficient catabolism in an attempt to restore energy and cell homeostasis. These results bring relevant information to amplify the knowledge concerning the bacterial physiological changes in response to the stress caused by the cell exposition to bovicin HC5. New potential targets for controlling this pathogen can also be determined from the new protein expression pattern presented. KEY POINTS: • Bovicin HC5 changed the synthesis of cytoplasmic proteins of S. aureus. • Bovicin HC5 interfered in the synthesis of proteins of amino acids biosynthesis. • Synthesis of chaperones enhanced in the presence of sublethal dosage of bovicin HC5.

Plant compounds and nonsteroidal anti-inflammatory drugs interfere with quorum sensing in Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative, saprophytic bacterium that can infect humans and its virulence may be regulated by quorum sensing via N-acyl homoserine lactones. A virtual screening study with plant compounds and nonsteroidal anti-inflammatory drugs for inhibition of C. violaceum quorum sensing system has been performed. In vitro evaluation was done to validate the in silico results. Molecular docking showed that phytol, margaric acid, palmitic acid, dipyrone, ketoprofen, and phenylbutazone bound to structures of CviR proteins of different C. violaceum strains. Phytol presented higher binding affinities than AHLs and furanones, recognized inducers, and inhibitors of quorum sensing, respectively. When tested in vitro, phytol at a non-inhibitory concentration was the most efficient tested compound to reduce phenotypes regulated by quorum sensing. The results indicate that in silico compound prospection to inhibit quorum sensing may be a good tool for finding alternative lead molecules.

Behavior of Salmonella Enteritidis and Shigella flexneri during induction and recovery of the viable but nonculturable state.

Bacteria may enter into a viable but nonculturable (VBNC) state as a response to stresses, such as those found in food processing. Cells in the VBNC state lose the ability to grow in a conventional culture medium but man recover culturability. The viability, culturability and intracellular reactive oxygen species (ROS) of Salmonella Enteritidis and Shigella flexneri were evaluated under stress conditions to induce a VBNC state. Cells were maintained under nutritional, osmotic and cold stresses (long-term induction) in Butterfield's phosphate solution plus 1.2 M of NaCl at 4°C and under nutritional and oxidative stresses (short-term induction) in 10 mM of H2O2. Culture media, recovery agents, sterilization methods of media and incubation temperature, were combined and applied to recover the culturability of the VBNC cells. Salmonella entered in the VBNC state after 135 days under long-term induction, while Shigella maintained culturability after 240 days. Under short-term induction, Salmonella and Shigella lose culturability after 135 and 240 min, respectively. Flow cytometric analysis revealed viable cells and intracellular ROS in both species in VBNC. It was not possible to recover the culturability of VBNC cells using the 42 combinations of different factors.

Carvacrol-loaded liposome suspension: optimization, characterization and incorporation into poly(vinyl alcohol) films.

The purpose of this study was to encapsulate carvacrol into liposomes in order to promote its application in active food packaging. Response surface methodology was used to evaluate the effect of the concentration of the liposomal components on its characteristics. The optimum formulation for the preparation of liposomes with the highest encapsulation efficiency (59.0 ± 1.99%) was found to be 3000 µg mL-1 of cholesterol and 4000 µg mL-1 of carvacrol. Carvacrol reduced the polydispersity index and increased the zeta potential and the thermal stability of liposomes. Fourier-transform infrared spectroscopy indicated that the interaction of carvacrol with liposomes occurred probably through hydrogen-bonding. The incorporation into liposomes maintained the antibacterial effect of carvacrol, but when in the film, carvacrol liposomes were not effective against the microorganisms tested. Liposomes may offer a viable option for stabilizing carvacrol, however, more studies are necessary to enable its application in food packaging.

Effects of sub-lethal doses of nisin on the virulence of Salmonella enterica in Galleria mellonella larvae.

Salmonella enterica is a pathogen that induces self-limiting gastroenteritis and is of worldwide concern. Nisin, an antimicrobial peptide, has emerged as an alternative for the control of microbial growth but its effect on the virulence of pathogenic bacteria is not yet well-explored. This work aimed to evaluate the virulence of S. enterica in the presence of sub-inhibitory nisin using the experimental model Galleria mellonella. Sub-inhibitory concentrations of nisin of 11.72 and 46.88 µM did not affect the cellular viability of S. enterica but promoted changes in gene expression within 1 h of treatment, with increases of up to 3-fold of pagC, 1.8-fold of invA and 2.3-fold of invF. Larvae of G. mellonella inoculated with S. enterica combined with nisin at 46.88 µM presented mortality, and TL50 noticeably increased to 50% and 80% at 24 and 48 h post-infection, respectively. Defence responses, such as melanisation, nodulation, pseudopodia, immune response, and expression of defence proteins of the larvae G. mellonella were enhanced when the treatments with S. enterica were combined with 11.72 or 46.88 µM nisin. These results show an increase in virulence of S. enterica by sub-MIC concentration of nisin that needs to be explored.

Inactivation of Salmonella Enteritidis on cherry tomatoes by ultrasound, lactic acid, detergent, and silver nanoparticles.

Ultrasound (US) combined with chemical agents could represent an effective method for decontaminating fruits and vegetables. This study aimed to evaluate the use of US (40 kHz for 5 min) alone or with 1% lactic acid (LA), 1% commercial detergent (DET), or 6 mg/L silver nanoparticles (AgNP, average diameter 100 nm) as an alternative treatment to 200 mg/L sodium dichloroisocyanurate for inactivating Salmonella enterica serovar Enteritidis present on cherry tomatoes. The interfacial tension between sanitizing solutions and bacterial adhesion was investigated. Sanitizers in solutions with DET and AgNP had lower surface tension. All treatments, except that with DET, reduced Salmonella Enteritidis by more than one logarithmic cycle. There was no significant difference between the mean values of log colony-forming units (CFU)/g reduction in all treatments. Transmission electron microscopy revealed the loss of the Salmonella Enteritidis capsule following treatment with US and with US + LA. Salmonella Enteritidis counts (2.29 log CFU/g) in cherry tomatoes were markedly reduced to safe levels by treatment with the combination of AgNP and US + LA (2.37 log CFU/g).

Identification and characterization of a polyurethanase with lipase activity from Serratia liquefaciens isolated from cold raw cow's milk.

Lipases are associated with food spoilage and are also used in various biotechnological applications. In this study, we sought to purify, identify, and characterize a lipase from S. liquefaciens isolated from cold raw cow's milk. The lipase partially purified by ultrafiltration and gel filtration showed a specific activity of 2793 U/mg. By zymography, the enzyme presented approximately 65 kDa, and LC-MS/MS allowed the identification of a polyurethanase with a conserved domain of family I.3 lipase. The modeled and validated structure of polyurethanase was able to bind to different fatty acids and urethane by molecular docking. The polyurethanase showed optimum activity at pH 8.0 and 30 °C. In the presence of ions, activity was decreased, except for Ca2+, Mg2+, and Ba2+. Reducing agents did not alter the activity, while amino acid modifiers reduced enzyme activity. It is concluded that polyurethanase with lipase activity represents a potential enzyme for the deterioration of milk and dairy products, as well as a candidate for industrial applications.

Phenolic extract of Eugenia uniflora L. and furanone reduce biofilm formation by Serratia liquefaciens and increase its susceptibility to antimicrobials.

Serratia liquefaciens is a spoilage microorganism of relevance in the dairy industry because it is psychrotrophic, able to form biofilm, and produces thermoresistant proteases and lipases. Phenolic compounds and furanones have been studied as inhibitors of biofilm formation. In this study, the potential of the pulp phenolic extract of Eugenia uniflora L. orange fruits, also called pitanga, and furanone C30 on the inhibition of biofilm formation by S. liquefaciens L53 and the susceptibility to different antimicrobials were evaluated. The pulp phenolic extract of pitanga had a high total phenolic content, being mainly composed of glycosylated quercetins and ellagitannins. Sub-inhibitory concentrations of this extract and furanone reduced biofilm formation by S. liquefaciens on polystyrene and the amount of polysaccharides, proteins and extracellular DNA in the biofilms. These biofilms were also more susceptible to kanamycin. The combinations of furanone with phenolic extract of pitanga or kanamycin showed a synergistic effect with total growth inhibition of S. liquefaciens.

Comparison of stress conditions to induce viable but non-cultivable state in Salmonella.

Salmonella can enter on the viable but non-culturable state (VBNC), characterized by the loss of ability to grow in routine culture media hindering detection by conventional methods and underestimation of the pathogen. Despite advances in research done so far, studies comparing conditions that lead Salmonella into the VBNC state are scarce. The main objective of this study was to evaluate different stresses to induce Salmonella to the VNBC state. Osmotic (1.2 M NaCl), acid (peracetic acid, 5.66 mg/mL) and oxidative (hydrogen peroxide, 1.20 mg/mL) stress were used at 4 °C to induce Salmonella enterica serovars Enteritidis and Typhimurium to the VBNC state. The culturability loss was monitored in the brain heart infusion (BHI) broth and agar, and the viability was determined by fluorescence microscopy, using the Live/Dead® kit, and by flow cytometry. Besides, the morphological characterization by atomic force microscopy (AFM) was performed. Storage in 1.2 M NaCl at 4 °C induced the VBNC state in Salmonella cells for periods longer than 121 days, and the percentage of viable cells has reached above 80.9%. More aggressive stress conditions promoted by peracetic acid and hydrogen peroxide induced the VBNC state in periods of, at most 0.14 day, and resulted in percentages of 8.5% to 45.5% viable cells, respectively. The counts of viable cells in the flow cytometer corroborate the results obtained by microscopic counts. The VBNC cells obtained in 1.2 M NaCl at 4 °C showed morphological changes, reducing the size and changing the morphology from bacillary to coccoid. No morphological change was observed on the cells stressed by acid or oxidant compounds.

Listeria spp. em queijos Minas Frescal e avaliação das condições higiênicossanitárias de produção e comercialização em Viçosa, MG, Brasil / Listeria spp. in Minas Frescal cheeses and assessement of hygienic-sanitary conditions of production and commercialization in Viçosa, MG, Brazil

A produção de queijo Minas Frescal em propriedades rurais é uma atividade econômica importante em Minas Gerais. Entretanto, a presença de micro-organismos patogênicos compromete a segurança desses produtos, coloca a saúde pública em risco e ameaça esta atividade. Neste estudo, objetivou-se avaliar a ocorrência de Listeria spp. em queijos Minas Frescal, as condições de produção e comercialização desses produtos e relacioná-las à presença desses micro-organismos. Foram coletadas 161 amostras, 81 em agroindústrias e 80 em seus pontos de venda. Foram utilizados métodos analíticos oficiais estabelecidos pelo Ministério da Agricultura, Pecuária e Abastecimento para detecção e diferenciação das espécies de Listeria. A adoção das boas práticas durante a fabricação e a comercialização foi mensurada mediante avaliação in loco direcionada por listas de verificação pré-elaboradas. Constatou-se Listeria spp. em 14 amostras (8,7%) provenientes de oito das doze agroindústrias estudadas, quatro inspecionadas e quatro não inspecionadas. L. monocytogenes foi isolada em duas amostras. O percentual de atendimento às boas práticas de fabricação variou entre 28,3% e 51,1% e diversas irregularidades foram observadas durante a comercialização dos queijos. Os resultados indicam risco potencial do consumo deste tipo de produto e a necessidade de adoção das boas práticas de fabricação e comercialização para prevenir contaminações. (AU)

The production of Minas Frescal cheese on rural properties is an important economic activity in Minas Gerais, Brazil. However, the presence of pathogenic microorganisms compromises the safety of these products, puts public health at risk and threatens this activity. The purpose of this paper was to evaluate the conditions of production and commercialization of the Minas Frescal cheese produced in family agroindustries of Viçosa, MG and to connect them to presence of Listeria spp. A total of 161 samples of Minas Frescal cheese were collected, 81 which were in agroindustries and 80 in their respective market. Were adopted official analytical methods established by the Agriculture Ministry for the detection and differentiation of Listeria spp. The conditions of manufacture and commercialization were evaluated through observations directed by pre-elaborated checklists. Listeria spp. was detected in 4 samples (8.7%) from eight different establishment, four inspected and four non-inspected. L. monocytogenes was isolated in two samples. The compliance with good manufacturing practices ranged from 28.3% to 51.1% and several irregularities were observed during the marketing of the cheeses. These results indicate the potential risk of consumption of this product and the need to prevent contamination of this product in manufacturing and marketing stages. (AU)

Listeria spp. em queijos Minas Frescal e avaliação das condições higiênicossanitárias de produção e comercialização em Viçosa, MG, Brasil / Listeria spp. in Minas Frescal cheeses and assessement of hygienic-sanitary conditions of production and commercialization in Viçosa, MG, Brazil

A produção de queijo Minas Frescal em propriedades rurais é uma atividade econômica importante em Minas Gerais. Entretanto, a presença de micro-organismos patogênicos compromete a segurança desses produtos, coloca a saúde pública em risco e ameaça esta atividade. Neste estudo, objetivou-se avaliar a ocorrência de Listeria spp. em queijos Minas Frescal, as condições de produção e comercialização desses produtos e relacioná-las à presença desses micro-organismos. Foram coletadas 161 amostras, 81 em agroindústrias e 80 em seus pontos de venda. Foram utilizados métodos analíticos oficiais estabelecidos pelo Ministério da Agricultura, Pecuária e Abastecimento para detecção e diferenciação das espécies de Listeria. A adoção das boas práticas durante a fabricação e a comercialização foi mensurada mediante avaliação in loco direcionada por listas de verificação pré-elaboradas. Constatou-se Listeria spp. em 14 amostras (8,7%) provenientes de oito das doze agroindústrias estudadas, quatro inspecionadas e quatro não inspecionadas. L. monocytogenes foi isolada em duas amostras. O percentual de atendimento às boas práticas de fabricação variou entre 28,3% e 51,1% e diversas irregularidades foram observadas durante a comercialização dos queijos. Os resultados indicam risco potencial do consumo deste tipo de produto e a necessidade de adoção das boas práticas de fabricação e comercialização para prevenir contaminações.

The production of Minas Frescal cheese on rural properties is an important economic activity in Minas Gerais, Brazil. However, the presence of pathogenic microorganisms compromises the safety of these products, puts public health at risk and threatens this activity. The purpose of this paper was to evaluate the conditions of production and commercialization of the Minas Frescal cheese produced in family agroindustries of Viçosa, MG and to connect them to presence of Listeria spp. A total of 161 samples of Minas Frescal cheese were collected, 81 which were in agroindustries and 80 in their respective market. Were adopted official analytical methods established by the Agriculture Ministry for the detection and differentiation of Listeria spp. The conditions of manufacture and commercialization were evaluated through observations directed by pre-elaborated checklists. Listeria spp. was detected in 14 samples (8.7%) from eight different establishment, four inspected and four non-inspected. L. monocytogenes was isolated in two samples. The compliance with good manufacturing practices ranged from 28.3% to 51.1% and several irregularities were observed during the marketing of the cheeses. These results indicate the potential risk of consumption of this product and the need to prevent contamination of this product in manufacturing and marketing stages.

Changes in the composition and architecture of staphylococcal biofilm by nisin.

Bacterial biofilms are involved in various medical infections and food contamination episodes and, for this reason, it is of great importance to developing new strategies of its prevention and control. The subinhibitory concentration of nisin was determined, and its effect against Staphylococcus aureus and Staphylococcus epidermidis biofilms was evaluated. Results obtained by confocal laser microscopy demonstrated morphological changes in the architecture of the structure of biofilms. The main components (polysaccharides, proteins, and extracellular DNA (eDNA)) of the biofilm matrix were determined by spectrophotometry and showed that the formation of staphylococcal biofilms in the presence of nisin results in a less dense matrix structure with modification in its constituents. These results contribute to increase the knowledge of the composition and architecture of the extracellular matrix of biofilms of S. aureus, as well as evidence that the investigation of alternative products to assist in the control and combat of biofilms is a promising strategy.

Quorum Sensing and Spoilage Potential of Psychrotrophic Enterobacteriaceae Isolated from Milk.

The 16S rDNA of six psychrotrophic Enterobacteriaceae isolated from cold raw milk were sequenced and the isolate 039 was identified as Pantoea sp., isolates 059, 068, and 071 were identified as Hafnia alvei, 067 was identified as Enterobacter sp., and 099 was identified as Aeromonas hydrophila. They presented different spoilage potentials in milk with A. hydrophila 099 being the most deteriorative. Only Pantoea sp. 039 was not able to induce the quorum sensing monitor strains of acyl homoserine lactones (AHLs). The halI gene, which encodes the AHL synthase in H. alvei, was identified in the isolates 059, 067, 068, and 071. After initial sequencing characterization and cloning, this gene showed its function by the heterologous synthesis of N-hexanoyl-DL-homoserine lactone and N-3-oxohexanoyl-L-homoserine lactone in Escherichia coli. In addition to producing AHLs, A. hydrophila 099 produced AI-2 in higher level than the assay's positive control Vibrio harveyi BB120. Therefore, Enterobacteriaceae strains isolated from cooled raw milk produce a rich array of signaling molecules that may influence bacterial traits in the milk environment.