Strategies for Pathogen Biocontrol Using Lactic Acid Bacteria and Their Metabolites: A Focus on Meat Ecosystems and Industrial Environments

The globalization of trade and lifestyle ensure that the factors responsible for the emergence of diseases are more present than ever. Despite biotechnology advancements, meat-based foods are still under scrutiny because of the presence of pathogens, which causes a loss of consumer confidence and consequently a fall in demand. In this context, Lactic Acid Bacteria (LAB) as GRAS organisms offer an alternative for developing pathogen-free foods, particularly avoiding Listeria monocytogenes, with minimal processing and fewer additives while maintaining the foods’ sensorial characteristics. The use of LAB strains, enabling us to produce antimicrobial peptides (bacteriocins) in addition to lactic acid, with an impact on quality and safety during fermentation, processing, and/or storage of meat and ready-to-eat (RTE) meat products, constitutes a promising tool. A number of bacteriocin-based strategies including the use of bioprotective cultures, purified and/or semi-purified bacteriocins as well as their inclusion in varied packaging materials under different storage conditions, have been investigated. The application of bacteriocins as part of hurdle technology using non-thermal technologies was explored for the preservation of RTE meat products. Likewise, considering that food contamination with L. monocytogenes is a consequence of the post-processing manipulation of RTE foods, the role of bacteriocinogenic LAB in the control of biofilms formed on industrial surfaces is also discussed.EEA FamailláFil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Blanco Massani, Mariana Raquel. Instituto Nacional de Tecnología Industrial; Argentina.Fil: Fontana, Cecilia Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Famaillá; ArgentinaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentin

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Antimicrobial potential of LEGUMES extracts against foodborne pathogens: A review

[EN] Background: Alternative protein sources are being investigated in response to increasing consumer demand for innovative and healthy food products of vegetable origin to replace non-sustainable animal exploitation. The Leguminosae family includes a wide variety of plants and nutritious seeds, very rich in protein with a high biological value, carbohydrates, vitamins and minerals. Not only the seeds but also the aerial parts, pods, hulls and roots have proved to be natural sources of antioxidants, and anti-inflammatory and antimicrobial compounds. Scope and approach: The present article overviews the antimicrobial potential of the most popular legumes worldwide against foodborne pathogens. Key findings and conclusions: According to the literature reviewed, soybean and chickpea are the two consumed legumes with the highest antimicrobial activity. Long-chain soy peptides (IKAFKEATKVDKVVVLWTA) have a high antimicrobial potential against both Gram-positive and Gram-negative bacteria at a concentration level of 37.2 ¿M. Also, a wide spectrum of proteins and peptides in raw chickpeas and processed extracts have exerted antimicrobial activity against foodborne pathogens when applied in the range 8¿64 ¿g/ml. These results open a new research line with good prospects regarding the development of a new generation of natural preservative ingredients and extracts to be included in novel formulated products. However, critical aspects, such as (i) the stability of antimicrobial activity during the shelf-life of newly formulated food products, and (ii) the microbial inactivation kinetics generated in novel matrices, should be covered prior to exploitation of legumes as sources of novel technological ingredients with antimicrobial potential.The present research work has been supported by funds provided by the Spanish Ministry of Economy and Competitiveness (MINECO) as the HELICOFOOD project, with reference AGL2014-53875-R. The post-doctoral contract of M.C. Pina-Pérez as Juan de la Cierva-Incorporación granted by the MINECO is also acknowledged.Pina Pérez, MC.; Ferrús Pérez, MA. (2018). Antimicrobial potential of LEGUMES extracts against foodborne pathogens: A review. Trends in Food Science & Technology. 72:114-124. doi:10.1016/j.tifs.2017.12.007S1141247

Evaluation of anti-Listeria meat borne Lactobacillus for biofilm formation on selected abiotic surfaces

The ability of meat borne anti-Listeria Lactobacillus to form biofilms under different in vitro conditions and on abiotic surfaces was investigated. Biofilm formation by the adhesion to polystyrene microtiter plates was determined, this being higher for Lactobacillus curvatus CRL1532 and CRL705 and Lactobacillus sakei CRL1862. The physicochemical properties of the cell surface were relatively hydrophilic and acidic in character; L. sakei CRL1862 exhibiting the strongest autoaggregation. The adhesion of lactobacilli to stainless steel (SS) and polytetrafluoroethylene (PTFE) supports at 10 °C was found to be maximal for L. sakei CRL1862 on SS after 6 days. When biofilm architecture was characterized by epifluorescence and SEM, L. sakei CRL1862 homogeneously covered the SS surface while cell clusters were observed on PTFE; the extracellular polymeric substance matrix adapted to the topography and hydrophilic/hydrophobic characteristics of each material. The feasibility of L. sakei CRL1862 to form biofilm on materials used in meat processing highlights its potential as a control strategy for Listeria monocytogenes biofilms.Fil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); ArgentinaFil: Castellano, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); ArgentinaFil: Vignolo, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); Argentin

Biofilms bacterianos: una amenaza para la seguridad en la industria cárnea. Evaluación de la capacidad formadora de biofilm de Lactobacillus bacteriocinogénicos sobre superficies industriales. Parte 1

Se investigó la capacidad formadora de biofilms en cepas de bacterias lácticas (BL) con actividad anti-Listeria en medios de cultivos y sobre superficies industriales a diferentes temperaturas. Lactobacillus curvatus CRL705, CRL1532 y Lactobacillus sakei CRL1862, aislados de productos cárneos fermentados Argentinos, fueron inoculados sobre chips de acero inoxidable (AI) y TeflónTM (T) en medios MRS, MRS sin Tween (-tMRS) y sin Tween/glucosa/MnSO4 (-tgmMRS) incubados a 10 ºC y 30 ºC durante diferentes tiempos y analizados mediante epifluorescencia y microscopía electrónica de barrido (MEB). Las BL evaluadas revelaron una superficie relativamente hidrofílica y de carácter acídico, exhibiendo L. sakei CRL1862 la mayor capacidad autoagregante. La adhesión a las superficies abióticas evaluadas resultó máxima para L. sakei CRL1862 sobre AI a los 6 días (10 ºC) y a los 3 días (30 ºC). La arquitectura de los biofilms formados por L. sakei CRL1862 caracterizada microscópicamente (por epifluerescencia y MEB), evidenció la formación de una homogénea cubierta biofilmogénica sobre la superficie de AI, mientras que grupos celulares irregulares se observaron sobre la superficie de T. Se detectó una adaptación a la topografía y características hidrofílicas/hidrofóbicas del AI y T de la matriz polimérica extracelular producida por los lactobacilos evaluados. La capacidad biofilmogénica de L. sakei CRL1862 sobre superficies frecuentemente usadas en la industria frigorífica pone en evidencia su potencial como estrategia de control de biofilms de Listeria monocytogenes.Fil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentin

Control of Listeria monocytogenes biofilms on industrial surfaces by the bacteriocin-producing Lactobacillus sakei CRL1862

The effect of the bacteriocin-producing Lactobacillus sakei CRL1862 and its bacteriocin in the control of Listeria biofilm formation on industrial surfaces at 10?C was investigated. A screening among different Listeria species was performed allowing selecting L. monocytogenes FBUNT for its use as a biofilm producer on stainless steel (SS) and polytetrafluoroethylene (PTFE) surfaces. Three conditions were simulated to evaluate the ability of the bacteriocinogenic strain to displace, exclude and compete pathogen biofilm formation. Lactobacillus sakei CRL1862 effectively inhibited biofilm formation by L. monocytogenes FBUNT through the three assayed mechanisms, pathogen inhibition being more efficient on PTFE than on SS surface. Moreover, co-culture of L. monocytogenes FBUNT with the bacteriocin-producer displayed the highest efficacy reducing the pathogen by 5.54 ± 0.12 and 4.52 ± 0.01 on PTFE and SS, respectively. Industrially, the pre-treatment with L. sakei CRL1862 or its bacteriocin (exclusion) constitutes the most realistic way to prevent pathogen biofilm settlement. The use of bacteriocins and/or the bacteriocin-producer strain represents a safe and environmentally-friendly sanitation method to mitigate post-processing food contamination.Fil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Leclercq, Alexandre. Instituto Pasteur; FranciaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentin

Strategies for Pathogen Biocontrol Using Lactic Acid Bacteria and Their Metabolites: A Focus on Meat Ecosystems and Industrial Environments

The globalization of trade and lifestyle ensure that the factors responsible for the emergence of diseases are more present than ever. Despite biotechnology advancements, meat-based foods are still under scrutiny because of the presence of pathogens, which causes a loss of consumer confidence and consequently a fall in demand. In this context, Lactic Acid Bacteria (LAB) as GRAS organisms offer an alternative for developing pathogen-free foods, particularly avoiding Listeria monocytogenes, with minimal processing and fewer additives while maintaining the foods’ sensorial characteristics. The use of LAB strains, enabling us to produce antimicrobial peptides (bacteriocins) in addition to lactic acid, with an impact on quality and safety during fermentation, processing, and/or storage of meat and ready-to-eat (RTE) meat products, constitutes a promising tool. A number of bacteriocin-based strategies including the use of bioprotective cultures, purified and/or semi-purified bacteriocins as well as their inclusion in varied packaging materials under different storage conditions, have been investigated. The application of bacteriocins as part of hurdle technology using non-thermal technologies was explored for the preservation of RTE meat products. Likewise, considering that food contamination with L. monocytogenes is a consequence of the post-processing manipulation of RTE foods, the role of bacteriocinogenic LAB in the control of biofilms formed on industrial surfaces is also discussed

Ability of meat borne Lactobacillus sakei CRL1862 bacteriocins to inhibit Listeria monocytogenes biofilm on industrial abiotic surfaces

Research on microbial biofilms has been documented in many areas, with particular emphasis in control strategies for either preventing or remediating pathogenic biofilm colonization. Listeria monocytogenes is well known for its ability to form biofilm and to establish harborages on food-processing equipment, making its eradication even more difficult, which may led to food products contamination. Many lactic acid bacteria (LAB) are known for their ability to inhibit growth of spoilage and pathogenic microorganisms by producing antimicrobial compounds in planktonic stage, but biofilm biocontrol is not well documented. In this work biofilm formation of Listeria monocytogenes FBUNT on stainless steel (SS) and polytetrafluoroethylene (PTFE) and the ability to be inhibited by Lactobacillus sakei CRL1862 bacteriocin at low temperature was investigated. Listeria biofilm formation was tested on LB broth and LB diluted (1/10) (dLB). Petri dish containing 19 mL of media (individually) and chips (2.5 cm2) of each inert materials were inoculated with 0.1 mL of L. monocytogenes and incubated at 10 °C during 6 days; fluorescence microscopy after staining with acridine orange was used to evaluate developed biofilm. Inhibitory effect of L. sakei CRL1862 bacteriocin (266.66 AU/mL) was examined at 0 and 6 h on L. monocytogenes in biofilm by counts on BHI agar. Results showed epifluorescence microcopy pictures for L. monocytogenes only few scattered cells were adhered on both, SS and PTFE surfaces after growth on LB medium. Chips incubated on dLB medium appeared homogeneously and almost entirely covering SS surface while cell clusters were observed on PTFE. When bacteriocin effect on L. monocytogenes FBUNT biofilm was evaluated, a 1-log and 3-log reduction at 0 and 6 h, respectively, was produced. From this study, the effectiveness of the bacteriocin produced by meat borne L. sakei CRL1862 to suppress Listeria biofilm developed on industrial surfaces was demonstrated. Due to pathogen biofilms resistance to antimicrobials and sanitizing agents frequently used in the industry, this study is relevant to food microbiology providing information for biocontrol strategies to destroy or avoid the building of pathogen biofilms on processing surfaces and equipment used during the manufacturing of meat and meat products.Fil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Agüero, Efraín Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Castellano, Patricia Haydee. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaIV International Symposium on Lactic Acid Bacteria: Food, Health and ApplicationsSan Miguel de TucumánArgentinaConsejo Nacional de Investigaciones Científicas y Técnicas. Centro de Referencia para Lactobacilo

Vancomycin and nisin A are effective against biofilms of multi-drug resistant Staphylococcus aureus isolates from human milk.

Human milk provides complete nutrition for infants and at the same time promotes the growth of specific bacteria in the infant gastrointestinal tract. Breastfeeding can often be discontinued due to mastitis which is an inflammation of the breast tissue. We isolated 18 Staphylococcus aureus strains from milk donated by healthy (n = 6), subclinical (n = 6), and mastitic (n = 6) mothers, two strains of which were VISA (Vancomycin Intermediate S. aureus). All tested strains (n = 12) were able to form biofilms. We then examined the impact of nisin A and vancomycin alone and in combination on biofilm formation and eradication of selected strains (n = 8). We observed strain-specific responses, with the combinatorial treatment at 1/4X MIC (for both singularly) significantly inhibiting biofilm formation for seven out of eight strains when compared with nisin A or vancomycin alone. None of the selected treatments were able to eradicate pre-formed biofilms. Finally, we selected two strains, namely a VISA (APC3814H) and a strong biofilm former (APC3912CM) and used confocal microscopy to evaluate the effects of the antimicrobial agents at 1X MIC on biofilm inhibition and eradication. All treatments inhibited biofilm formation of APC3814H but were ineffective in eradicating a pre-formed biofilm. Single treatments at 1X MIC against APC3912CM cells did not prevent biofilm formation whereas combination treatment caused increased death of APC3912CM cells. Finally, the combination treatment reduced the thickness of the pre-formed APC3912CM biofilm as compared with the single treatments

Antilisterial efficacy of Lactobacillus bacteriocins and organic acids on frankfurters. Impact on sensory characteristics

Dipping solutions containing bacteriocins produced by Lactobacillus curvatus CRL705 and Lactobacillus sakei CRL1862 (Bact705/1862), nisin and organic acids (lactic acid, LA; acetic acid, AA) were tested alone or in combination against Listeria monocytogenes inoculated by immersion on vacuum-packaged frankfurters stored at 10 °C during 36 days. LA/AA solution (2.5% v/v each) reduced pathogen population by 1.50 log10 CFU/ml during storage. Semi-purified Bact705/1862 prevented L. monocytogenes growth, while nisin was not able to avoid its regrowth after 20 days. The combined addition of Bact705/1862 + LA/AA was the most effective approach for pathogen reduction below detection level from day 6 to final storage. Frankfurters treated with Bact705/1862 + LA/AA compared to fresh-purchased samples did not show significant differences in flavor, juiciness, color intensity and overall preference at 22 days-storage at 5 °C. Meat processors should not only validate the antimicrobial efficacy of combined treatments but also their sensory impact on the product, which is directly related to consumer acceptability.Instituto de Tecnología de AlimentosFil: Castellano, Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Peña, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Pérez Ibarreche, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Carduza, Fernando Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; ArgentinaFil: Soteras, Trinidad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Tecnología de Alimentos; ArgentinaFil: Vignolo, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia para Lactobacilos; Argentin