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Item Potential Control of Listeria monocytogenes by Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC Strains Isolated From Artisanal Cheese(Probiotics and Antimicrobial Proteins, 2019-03) Cavicchioli, Valéria Quintana; Camargo, Anderson Carlos; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoBacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC strains, previously isolated from artisanal cheese, were evaluated for their safety with the aim to determine whether they could be used as beneficial strains, especially in the control of Listeria monocytogenes. Both isolates survived simulated gastrointestinal conditions and showed high levels of auto- and co-aggregation with L. monocytogenes, although the hydrophobicity of cells varied. Using the agar-spot test with 33 commercial drugs from different groups, only anti-inflammatory drugs and drugs containing loratadine and propranolol hydrochloride were able to affect the growth of the tested strains. Both strains were resistant to 3 out of 11 antibiotics tested by the disc diffusion method, and low frequencies of antibiotic resistance-encoding genes were observed by PCR analysis. Tested strains neither presented biogenic amine-related genes nor produced these substances. Aside from some antibiotic resistance characteristics, the tested strains were considered safe as they lack other virulence-related genes. E. hirae ST57ACC and P. pentosaceus ST65ACC both presented beneficial properties, particularly their ability to survive gastrointestinal conditions and to aggregate with L. monocytogenes, which can facilitate the elimination of this pathogen. Further studies should be conducted to better understand these interactions.Item Lactobacillus curvatus UFV-NPAC1 and other lactic acid bacteria isolated from calabresa, a fermented meat product, present high bacteriocinogenic activity against Listeria monocytogenes(BMC Microbiology, 2019) Castilho, Nathália Parma Augusto; Colombo, Monique; Oliveira, Leandro Licursi de; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoBacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry.Three LAB strains (firstly named as Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles (rep-PCR, RAPD, bacteriocin-related genes) and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins.LAB isolates obtained from calabresa presented high inhibitory activity. Among these isolates, bacteriocins produced by L. curvatus 12, now named as L. curvatus UFV-NPAC1, presented the highest inhibitory performance and the purification procedures revealed four peptides with sequences not described for bacteriocins to date.Item Antibiotic resistance of Listeria monocytogenes isolated from meat-processing environments, beef products, and clinical cases in Brazil(Microbial Drug Resistance, 2015) Camargo, Anderson Carlos; Castilho, Natalia Parma Augusto de; Silva, Danilo Augusto Lopes da; Vallim, Deyse Christina; Hofer, Ernesto; Nero, Luís AugustoThe present study aimed to assess the antimicrobial resistance and the presence of virulence markers in 137 Listeria monocytogenes isolates obtained from meat-processing environments, beef products, and clinical cases. All isolates were subject to molecular serogrouping and their antibiotic resistance profiles were assessed against 12 antimicrobials. In addition, isolates were subjected to detection of virulence marker genes (inlA, inlC, inlJ). The isolates were classified into serogroups 4b, 4d, 4a, or 4c (46%), 1/2c or 3c (27%), 1/2a or 3a (13.9%), and 1/2b or 3b (13.1%). All tested isolates presented sensitivity to the majority of the tested antimicrobials, but most of them presented resistance or intermediate resistance to clindamycin (88.3%) and oxacillin (73.7%). Virulence markers were detected in all isolates, demanding further analysis to better characterize their pathogenic potential.Item Expression of genes associated with stress conditions by Listeria monocytogenes in interaction with nisin producer Lactococcus lactis(Food Research International, 2017-12-13) Miranda, Rodrigo Otávio; Campos-Galvão, Maria Emilene Martino; Nero, Luís AugustoThe use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2 log in BHI and 1 log in milk after 24 h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24 h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods.Item Genetic diversity and some aspects of antimicrobial activity of lactic acid bacteria isolated from goat milk(Applied Biochemistry and Biotechnology, 2015-01-31) Cavicchioli, Valéria Quintana; Dornellas, Wesley dos Santos; Perin, Luana Martins; Pieri, Fábio Alessandro; Franco, Bernadette Dora Gombossy de Melo; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoLactic acid bacteria (LAB, n = 57) were previously obtained from raw goat milk, identified as Lactococcus spp. (n = 24) and Enterococcus spp. (n = 33), and characterized as bacteriocinogenic. Fingerprinting by pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity, and 30 strains were selected and exhibited strong antimicrobial activity against 46 target strains (LAB, spoilage, and foodborne pathogens). Six strains (Lactococcus lactis: GLc03 and GLc05; and Enterococcus durans: GEn09, GEn12, GEn14, and GEn17) were selected to characterize their bacteriocinogenic features, using Listeria monocytogenes ATCC 7644 as the target. The six strains produced bacteriocins at higher titer when incubated in MRS at 37 °C up to 12 h, when compared to growth at 25 and 30 °C. The produced bacteriocins kept their antimicrobial activity after exposure to 100 °C for 2 h and 121 °C for 20 min; the antimicrobial activity was also observed after treatment at pH 2.0 to 10.0, except for GLc03. L. monocytogenes populations were reduced approximately two logs after treatment with cell-free supernatants from the selected strains. These data show that goat milk can contain a diverse microbiota able to inhibit L. monocytogenes, a common pathogen found in dairy products, and can be potentially employed in biopreservation of food produced under different processing conditions.Item In Vitro evaluation of bacteriocins activity against Listeria monocytogenes biofilm formation(Applied Biochemistry and Biotechnology, 2015-12-10) Camargo, Anderson Carlos; Paula, Otávio Almeida Lino de; Todorov, Svetoslav Dimitrov; Nero, Luís AugustoThe present study aimed to assess the activity of cell-free supernatant (CFS) containing bacteriocins on the formation and maintenance of biofilms developed by Listeria monocytogenes, and the associated effect of bacteriocins and ethylene-diamine-tetra-acetic acid (EDTA) on the formed biofilm. CFS from 9 lactic acid bacteria (LAB) strains was tested for inhibitory activity against 85 L. monocytogenes isolates and 21 LAB strains. Then, 12 L. monocytogenes strains were selected based on genetic profiles and sensitivity to CFS and were subjected to an in vitro assay to assess biofilm formation in microtiter plates, considering different culture media and incubation conditions. Based on these results, 6 L. monocytogenes strains were subjected to the same in vitro procedure to assess biofilm formation, being co-inoculated with CFS. In addition, these strains were subjected to the same in vitro procedure, modified by adding the CFS after biofilm formation. Relevant decrease in biofilm formation was observed in the first experiment, but CFS added after biofilm formation did not eliminate them. CFS from Lactobacillus curvatus ET31 were selected due to its anti-biofilm activity, being associated to EDTA at different concentrations and tested for biofilm control of three strains of L. monocytogenes, using the same in vitro procedure described previously. Concentrated bacteriocin presented poor performance in eliminating formed biofilms, and EDTA concentration presented no evident interference on biofilm elimination. Twelve selected L. monocytogenes strains were positive for investigated virulence makers and negative for luxS gene, recognized as being involved in biofilm formation. Selected L. monocytogenes strains were able to produce biofilms under different conditions. CFSs have the potential to prevent biofilm formation, but they were not able to destroy already formed biofilms. Nevertheless, low concentrations of CFS combined with EDTA caused a relevant reduction in already formed biofilms, but this association was not able to eliminate them. The activity of selected CFS was demonstrated against L. monocytogenes-formed biofilms, being more effective when associated to EDTA at different concentrations.Item PFGE characterisation and adhesion ability of Listeria monocytogenes isolates obtained from bovine carcasses and beef processing facilities(Meat Science, 2012-12) Galvão, Newton Nascentes; Chiarini, Eb; Destro, Maria Teresa; Ferreira, Márcia de Aguiar; Nero, Luís AugustoListeria monocytogenes is a pathogen capable of adhering to many surfaces and forming biofilms, which may explain its persistence in food processing environments. This study aimed to genetically characterise L. monocytogenes isolates obtained from bovine carcasses and beef processing facilities and to evaluate their adhesion abilities. DNA from 29 L. monocytogenes isolates was subjected to enzymatic restriction digestion (AscI and ApaI), and two clusters were identified for serotypes 4b and 1/2a, with similarities of 48% and 68%, respectively. The adhesion ability of the isolates was tested considering: inoculum concentration, culture media, carbohydrate source, NaCl concentration, incubation temperature, and pH. Each isolate was tested at 10⁸ CFU mL⁻¹ and classified according to its adhesion ability as weak (8 isolates), moderate (17) or strong (4). The isolates showed higher adhesion capability in non-diluted culture media, media at pH 7.0, incubation at 25°C and 37 °C, and media with NaCl at 5% and 7%. No relevant differences were observed for adhesion ability with respect to the carbohydrate source. The results indicated a wide diversity of PFGE profiles of persistent L. monocytogenes isolates, without relation to their adhesion characteristics. Also, it was observed that stressing conditions did not enhance the adhesion profile of the isolates.Item PFGE characterisation and adhesion ability of Listeria monocytogenes isolates obtained from bovine carcasses and beef processing facilities Author links open overlay panel(Meat Science, 2012-06-09) Galvão, Newton Nascentes; Chiarini, Eb; Destro, Maria Teresa; Ferreira, Márcia de Aguiar; Nero, Luís AugustoListeria monocytogenes is a pathogen capable of adhering to many surfaces and forming biofilms, which may explain its persistence in food processing environments. This study aimed to genetically characterise L. monocytogenes isolates obtained from bovine carcasses and beef processing facilities and to evaluate their adhesion abilities. DNA from 29 L. monocytogenes isolates was subjected to enzymatic restriction digestion (AscI and ApaI), and two clusters were identified for serotypes 4b and 1/2a, with similarities of 48% and 68%, respectively. The adhesion ability of the isolates was tested considering: inoculum concentration, culture media, carbohydrate source, NaCl concentration, incubation temperature, and pH. Each isolate was tested at 108 CFU mL− 1 and classified according to its adhesion ability as weak (8 isolates), moderate (17) or strong (4). The isolates showed higher adhesion capability in non-diluted culture media, media at pH 7.0, incubation at 25 °C and 37 °C, and media with NaCl at 5% and 7%. No relevant differences were observed for adhesion ability with respect to the carbohydrate source. The results indicated a wide diversity of PFGE profiles of persistent L. monocytogenes isolates, without relation to their adhesion characteristics. Also, it was observed that stressing conditions did not enhance the adhesion profile of the isolates.