Lactic Acid Bacteria isolated from traditional and innovative alheiras as potential biocontrol agents.

From a selection of seven traditional and 14 innovative alheiras, 491 lactic acid bacteria (LAB) were isolated and tested for their antimicrobial activity against several food-borne pathogens. Among these, six strains revealed antimicrobial activity through potential bacteriocin production against 14 Listeria monocytogenes strains, Enterococcus faecalis ATCC 29212, Clostridium sporogenes ESB050, and Clostridium perfringens ESB054. Through whole genome sequencing (WGS), these strains were identified as Lactiplantibacillus plantarum (2), Leuconostoc mesenteroides (1), and Pediococcus acidilactici (3). Furthermore, several orthologues of class II bacteriocins genes were identified, including Plantaricin E, Plantaricin F, Pediocin PA, Enterocin X, Leucocin A, and Coagulin A. No virulence or antibiotic resistance genes' orthologues were detected by WGS analysis. However, the selected LAB strains showed variable phenotypic patterns related to virulence genes and antibiotic resistance when assessed through classical methodologies. None of these strains demonstrated the production of biogenic amines, gelatinase or DNase. Additionally, no hemolytic activity or lipase enzyme production was observed. However, only Lpb. plantarum 9A3 was sensitive to all tested antibiotics and was thus chosen for further examination. The bacteriocins produced by Lpb. plantarum (9A3) exhibited stability across a broad range of conditions, including temperatures from 4 to 100 °C, pH values ranging from 2 to 8, exposure to surfactants and detergents (Tween 20 and 80, SDS, EDTA 0.1, 2 and 5 mM, urea and sodium deoxycholate), and enzymes (papain and catalase). Their maximum activity (AU/mL = 12,800) against four L. monocytogenes strains was observed between 21 and 36 h of growth of Lbp. plantarum 9A3, indicating a bacteriostatic mode of action. Therefore, this strain appears to be a robust candidate for potential application as a protective strain to be used in the food industry. Not only is it safe, but it also produces stable bacteriocins (harbouring genes encoding for the production of three) effectively inhibiting significant pathogens such as L. monocytogenes and C. perfringens.

Antilisterial activity of bacteriocinogenic Pediococcus acidilactici HA6111-2 and Lactobacillus plantarum ESB 202 grown under pH and osmotic stress conditions.

Bacteriocin producing lactic acid bacteria (LAB) cultures can be used as biopreservatives in fermented food products; thus the food industry is interested in stable cultures that produce bacteriocins consistently. Inhibtion of Listeria spp. by bacteriocinogenic Pediococcus acidilactici and Lactobacillus plantarum (both isolated from fermented meats) was investigated under conditions of stress induced by low pH and high salt concentrations. Listeria monocytogenes serogroup IIb (from cheese), L. monocytogenes serogroup IVb (from cheese), L. monocytogenes serogroup IIb (from ground beef) and Listeria innocua NCTC 11288 were used as target strains. P. acidilactici and Lb. plantarum demonstrated antilisterial activity under the stress conditions investigated (pH 3.5; pH 8.5; 7.5% NaCl). However, activity was dependent on the stress conditions applied and on the target organism. L. monocytogenes serogroup IIb (from ground beef) and L. innocua C 11288 were, respectively the most sensitive and the most resistant to the cell-free supernatants produced by the LAB investigated.

Characterization of a Lactiplantibacillus plantarum R23 Isolated from Arugula by Whole-Genome Sequencing and Its Bacteriocin Production Ability.

Lactiplantibacillus plantarum is one of the lactic acid bacteria species most used as probiotics and starter cultures in food production. Bacteriocin-producers Lpb. plantarum are also promising natural food preservatives. This study aimed to characterize Lpb. plantarum R23 and its bacteriocins (R23 bacteriocins). The genome sequence of Lpb. plantarum R23 was obtained by whole-genome sequencing (WGS) in an Illumina NovaSeq platform. The activity of Lpb. plantarum R23-produced bacteriocin against two Listeria monocytogenes strains (L7946 and L7947) was evaluated, and its molecular size was determined by tricine-SDS-PAGE. No virulence or antibiotic resistance genes were detected. Four 100% identical proteins to the class II bacteriocins (Plantaricin E, Plantaricin F, Pediocin PA-1 (Pediocin AcH), and Coagulin A) were found by WGS analysis. The small (<6.5 kDa) R23 bacteriocins were stable at different pH values (ranging from 2 to 8), temperatures (between 4 and 100 °C), detergents (all, except Triton X-100 and Triton X-114 at 0.01 g/mL), and enzymes (catalase and α-amylase), did not adsorb to the producer cells, had a bacteriostatic mode of action and their maximum activity (AU/mL = 12,800) against two L. monocytogenes strains occurred between 15 and 21 h of Lpb. plantarum R23 growth. Lactiplantibacillus plantarum R23 showed to be a promising bio-preservative culture because, besides being safe, it produces a stable bacteriocin or bacteriocins (harbors genes encoding for the production of four) inhibiting pathogens as L. monocytogenes. Further studies in different food matrices are required to confirm this hypothesis and its suitability as a future starter culture.

Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics.

Probiotics are living microorganisms used as nutritional additives that confer health benefits on the host. Their use in food products is very attractive, especially if they could also inhibit important foodborne pathogens. In this study, antimicrobial activity against several foodborne pathogens was screened for 280 lactic acid bacteria (LAB) isolated from different food products and the probiotic characteristics of bacteriocinogenic isolates were evaluated. Seven out of 280 LAB isolates were selected due to their bacteriocinogenic properties and identified by 16S rRNA gene sequence analysis as Pediococcus pentosaceus (n = 6) and Lactobacillus plantarum (n = 1). Virulence factors and antibiotic resistances were not detected for any of the isolates. Except for L. plantarum R23, all the isolates were able to survive through the simulated gastrointestinal tract conditions. Only P. pentosaceus CFF4 was able to adhere to Caco-2 cells after the simulated gastrointestinal tract passage. In conclusion, even though in vivo studies should be performed, P. pentosaceus CFF4, which was also able to inhibit the growth of foodborne pathogens in vitro, seems to be a potential probiotic to be used in the food industry.

Characterization of anti-Listeria bacteriocins isolated from shellfish: potential antimicrobials to control non-fermented seafood.

This work had as main objectives to characterize two bacteriocins produced by lactic acid bacteria (LAB) previously isolated from non-fermented seafood, in order to evaluate their potential as new food protective agents. The two bacteriocinogenic isolates were identified by Polymerase Chain Reaction (PCR) using genus- and species-specific primers, and confirmed by 16S rDNA sequencing, as Enterococcus faecium and Pediococcus pentosaceus. The antimicrobial spectrum of each strain included several indicator microorganisms, some of them also isolated from seafood. Growth of Listeria innocua, L. monocytogenes, Staphylococcus aureus, Bacillus cereus and other LAB species were inhibited, although no inhibition of Gram-negative microorganisms was observed. Proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants confirming the proteinaceous nature of the inhibitors. The antimicrobial activity was maintained after treatment with NaCl, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 2 h or 5 h of exposure and both bacteriocins were stable over a wide range of pH and temperatures. Production of bacteriocin by E. faecium (bacALP7) was detected initially at exponential phase and reached a maximum activity of 25,600 AU/ml in the early stationary phase, whereas bacteriocin production by P. pentosaceus ALP57 (bacALP57) reached the maximum at exponential phase with 12,800 AU/ml. The bacteriocins did not kill L. monocytogenes ESB54 nor L. innocua 2030c however, cellular growth was reduced. The partially purified bacteriocins, bacALP7 and bacALP57, were below 6.5 kDa in size as determined by Tricine-SDS gel electrophoresis. E. faecium and P. pentosaceus contained DNA fragments corresponding in size to those recorded for enterocin B and pediocin PA-1, respectively. Sequencing of the fragments from both bacteriocins confirmed the homology. To our knowledge, for the first time two LAB producing bacteriocins similar to pediocin PA-1 and enterocin B, were isolated from non-fermented shellfish. The adaptation of the cultures to seafood matrices may be advantageous in terms of application as a biopreservation strategy for reduction of L. monocytogenes levels in seafood products.

Phenotypic and genetic heterogeneity of lactic acid bacteria isolated from "Alheira", a traditional fermented sausage produced in Portugal.

The aim of this study was to evaluate the phenotypic and genetic heterogeneity of lactic acid bacteria (LAB) isolated from "Alheira", a fermented sausage produced in Portugal. LAB were identified to genus and species level by phenotypic characteristics, using genus or species-specific primers and sequencing of the gene encoding 16S rRNA. Two-hundred and eighty-three isolates were grouped into 14 species. Lactobacillus plantarum was isolated from all sausages and Enterococcusfaecalis from most of the samples. Low numbers of Lactobacillus paraplantarum, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus sakei, Lactobacillus zeae, Lactobacillus paracasei, Leuconostoc mesenteroides, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella cibaria, Weissella viridescens and Enterococcus faecium were recorded. The genetic heterogeneity of L. plantarum and E. faecalis strains were determined by numerical analysis of DNA banding patterns obtained by RAPD-PCR. Strains of L. plantarum and E. faecalis were different from different producers. This study forms the basis from which starter cultures could be selected for production of "Alheira".

Influence of oregano essential oil on the inhibition of selected pathogens in "Alheira" during storage.

BACKGROUND: Plant-derived essential oils (EOs) have shown remarkable antimicrobial potential against spoil- age and pathogenic microorganisms in meat and meat products. The aim of this study was to investigate the influence of oregano EO on the inhibition of Salmonella Enteritidis, Listeria monocytogenes and Staphylo- coccus aureus in an internal mixture of “Alheira” during storage. METHODS: Different concentrations of oregano EO (4%, 1.5%, 0.5%, 0.195% and 0.0975%) were evaluated against the selected pathogens during 21 days of storage at 4°C. The pH and water activity values and lactic acid bacteria (LAB) counts were also evaluated. Finally, sensory assessment was performed. RESULTS: The antibacterial effect varied according to the oregano EO concentration used, and target pathogen. Oregano EO at 4% demonstrated the highest antimicrobial activity against all the pathogens tested. The low- est concentrations used (0.195% and 0.0975%) resulted in ~2–3 log reduction, but only for L. monocytogenes after 21 days of storage. Counts of LAB were ~109 CFU/ml for all samples and no differences in the pH and aw values were detected between samples. However, at a concentration of 0.195%, Oregano EO had a negative impact on consumer acceptance of “Alheira”. CONCLUSIONS: These results could be interesting for the meat industry, as a starting point for other studies that have now to concentrate on strategies to “mask” unpleasant sensorial alterations caused by EOs in “Alheira” and helping the industry to ensure the microbiological safety of its products.

Inhibitory Effect of Lactobacillus plantarum FL75 and Leuconostoc mesenteroides FL14 against Foodborne Pathogens in Artificially Contaminated Fermented Tomato Juices.

Tomatoes and tomato based-foods contain beneficial microorganisms and various organic acids that have important nutritional values for human. The objective of this study was to access the physiochemical properties of fermented tomatoes juices and to evaluate the competitiveness of lactic acid bacteria (LAB) against Listeria monocytogenes, Listeria innocua, and Salmonella spp., in artificially contaminated tomato juice. Microbial counting (LAB, fungi Salmonella spp., and Listeria spp.) was performed after fermentation and weekly during storage. Different organic acids (Lactic, succinic, and acetic) and ethanol were also monitored using HPLC method. Color parameters were also determined. The results showed an increase of lactic and acetic acid content, during fermentation and storage of juices inoculated with Lactobacillus plantarum and Leuconostoc mesenteroides at 25°C. Besides, citric acid and ethanol revealed higher content at the end of storage compared to that registered at 4°C. The pH from tomatoes juices decreased from an initial value of 4.5 to below 3.2. Alongside, foodborne pathogen population was significantly suppressed in tomatoes juices when the samples were coinoculated with LAB strains. Moreover, the inhibition of Salmonella species was faster compared to that of Listeria. After four weeks of storage at 4°C, Lb. plantarum and Lc. mesenteroides showed high survival rate, while pathogenic bacteria, yeasts, and molds cell numbers decreased drastically in all the contaminated vials. This work highlights the efficiency of Lb. plantarum and Lc. mesenteroides as potential starters for developing nutritious and safe fermented tomato juice products.

Hand hygiene management among nurses: collective health challenges.

OBJECTIVE: To describe the determining factors in hand hygiene management among nurses and identify associated collective health challenges. METHOD: Cross-sectional descriptive study. Data were collected using a questionnaire that was applied in four internal medicine units of a hospital of reference in Portugal. RESULTS: The sample was composed of 50 nurses aged 26 to 55 years (mean age of 34.88 years); 80% were women, 58% had a Bachelor's degree, and had 5-30 years of nursing practice (X̄ =11.94;±5.92). The vast majority of nurses (90%) reported complying with the existing recommendations on hand hygiene in pre-established moments. However, none of the nurses were able to identify all the moments for hand hygiene using water and soap or alcohol-based handrub. CONCLUSION: This study shows that continuous training, adequate materials/structures in the units, and redesigned administration/supervision practices are determining factors to achieve higher levels of adherence to hand hygiene among nurses, as well as increased quality and safety in care delivery, which is a current collective health challenge.

Characterization of two bacteriocins produced by Pediococcus acidilactici isolated from "Alheira", a fermented sausage traditionally produced in Portugal.

Lactic acid bacteria were isolated from "Alheira" sausages that have been sampled from different regions in Portugal. The sausages were produced according to different recipes and with traditional starter cultures. Two isolates (HA-6111-2 and HA-5692-3) from different sausages were identified as strains of Pediococcus acidilactici. Each strain produces a bacteriocin, designated as bacHA-6111-2 and bacHA-5692-3. Both bacteriocins are produced at low levels after 18 h of growth in MRS broth (3200 AU/ml against Enterococcus faecium HKLHS and 1600 AU/ml against Listeria innocua N27). BacHA-6111-2 and bacHA-5692-3 are between 3.5 kDa and 6.5 kDa in size, as determined by tricine-SDS-PAGE. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of cell-free supernatants with proteinase K, pronase and trypsin. No change in activity was recorded when treated with catalase. Both bacteriocins are sensitive to treatment with Triton X-114 and Triton X-100, but resistant to Tween 20, Tween 80, SDS, Oxbile, NaCl, urea and EDTA. The bacteriocins remained stable after 2 h at pH 6.0. A decrease in antibacterial activity was recorded after 60 min at 100 degrees C. After 60 min at 80 degrees C, 60 degrees C and 25 degrees C the antibacterial activity against L. innocua N27 decreased by 25%. Addition of bacHA-6111-2 and bacHA-5692-3 (1600 AU/ml) to a mid-log (5-h-old) culture of L. innocua N27 inhibited growth for 7 h. Addition of the bacteriocins (3200 AU/ml) to a mid-log (5-h-old) culture of E. faecium HKLHS repressed cell growth. The bacteriocins did not adhere to the surface of the producer cells. Both strains contain a 1044 bp DNA fragment corresponding in size to that recorded for pediocin PA-1. Sequencing of the fragments from both bacteriocins revealed homology to large sections of pedA (188 bp), pedB (338 bp) and pedC (524 bp) of pediocin PA-1 and the bacteriocins are considered similar to pediocin PA-1.

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays.

A total of 226 lactic acid bacteria (LAB) isolated from "Alheira", a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from "Alheiras" and to select strains that could be used in "Alheira" production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of "Alheira mass" (sterilized), at 4°C. The growth of L. innocua population was significantly suppressed in the paste of "Alheira" when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1×10(7)CFU/g after 28 days of incubation).

Role of flies as vectors of foodborne pathogens in rural areas.

This study aims to evaluate flies as a vector for foodborne pathogens. For this purpose, several flies were collected from different sites from rural areas. These flies were then analyzed for the presence of Enterobacteriaceae, Escherichia coli, Staphylococcus coagulase positive, and Listeria monocytogenes. Another aim of this study was to evaluate some virulence factors of the collected pathogens: susceptibility to some antibiotics and the presence of enterotoxigenic S. aureus. The results showed that flies in the presence of animals demonstrated a significantly higher prevalence of the studied pathogens than those collected in the kitchens, and kitchens situated in the closest proximity to the animal husbandry had a higher count than the kitchens in private houses. Enterobacteriaceae was the indicator organism with the highest microbial counts followed by E. coli and S. aureus. Listeria monocytogenes was not detected from any of the collected flies. The antimicrobial susceptibility test showed that the bacteria carried by the flies possessed multiantibiotic resistance profiles, and enterotoxin A was produced by 17.9% of the confirmed S. aureus isolates. These results demonstrate that flies can transmit foodborne pathogens and their associated toxin and resistance and the areas of higher risk are those in closer proximity to animal production sites.

Hand hygiene management among nurses: collective health challenges / Gestión del procedimiento de la higiene de las manos por enfermeros: desafíos para la salud colectiva / Gestão do procedimento higiene das mãos por enfermeiros: desafios para saúde coletiva

ABSTRACT Objective: To describe the determining factors in hand hygiene management among nurses and identify associated collective health challenges. Method: Cross-sectional descriptive study. Data were collected using a questionnaire that was applied in four internal medicine units of a hospital of reference in Portugal. Results: The sample was composed of 50 nurses aged 26 to 55 years (mean age of 34.88 years); 80% were women, 58% had a Bachelor's degree, and had 5-30 years of nursing practice (X̄ =11.94;±5.92). The vast majority of nurses (90%) reported complying with the existing recommendations on hand hygiene in pre-established moments. However, none of the nurses were able to identify all the moments for hand hygiene using water and soap or alcohol-based handrub. Conclusion: This study shows that continuous training, adequate materials/structures in the units, and redesigned administration/supervision practices are determining factors to achieve higher levels of adherence to hand hygiene among nurses, as well as increased quality and safety in care delivery, which is a current collective health challenge.

RESUMEN Objetivo: Describir los factores determinantes en la gestión de la higiene de las manos por enfermeros e identificar los desafíos inherentes para la salud colectiva. Método: Estudio descriptivo-transversal. Los datos se recogieron mediante un cuestionario en cuatro unidades de medicina interna de un hospital de referencia en Portugal. Resultados: se incluyó a 50 enfermeros con una edad media de 34,88 años (entre los 26 y 55 años), el 80 % del sexo femenino, el 58 % son licenciados y desempeñan sus funciones de 5 a 30 años (X̄ = 11.94; ± 5.92). De ellos, el 90 % considera que respeta las recomendaciones existentes sobre higiene de las manos en los momentos estipulados para tal efecto. Sin embargo, los enfermeros no identificaron ninguno de los momentos para realizar este procedimiento, con agua y jabón o solución antiséptica de base alcohólica. Conclusión: Las contribuciones como formación continua, adecuación de los materiales / estructuras de las unidades y reconstrucción de prácticas por gestores / supervisores emergen en este trabajo como factores determinantes para alcanzar niveles superiores de adhesión a la higiene de las manos por enfermeros, de calidad y seguridad, un desafío actual para la salud pública.

RESUMO Objectivo: Descrever fatores determinantes na gestão da higiene das mãos por Enfermeiros e identificar desafios inerentes para a saúde coletiva. Método: Estudo descritivo-transversal. Dados coletados por questionário em quatro unidades de medicina interna de um hospital de referência em Portugal. Resultados: incluídos 50 enfermeiros, idade média 34,88 anos (entre os 26 e 55 anos), 80% sexo feminino, 58% são licenciados e exercem funções entre 5-30 anos (X̄ =11.94;±5.92). Destes, 90% considera respeitar as recomendações existentes sobre higiene das mãos nos momentos estipulados para o efeito. Porém, nenhum dos momentos para a realização deste procedimento, com água e sabão ou solução antissética de base alcoólica, foi identificado por todos os enfermeiros. Conclusão: Contribuições como formação contínua, adequação dos materiais/estruturas das unidades e reconstrução de práticas por gestores/supervisores emergem neste trabalho como fatores determinantes para atingir níveis superiores de adesão à higiene das mãos por enfermeiros, de qualidade e segurança, desafio atual para a saúde pública.

The gellan gum biosynthetic genes gelC and gelE encode two separate polypeptides homologous to the activator and the kinase domains of tyrosine autokinases.

The high-molecular-weight exopolysaccharide gellan is an important commercial gelling agent produced in high yield by the Gram-negative bacterium Sphingomonas elodea ATCC 31461. The cluster of genes required for gellan biosynthesis contains the genes gelC and gelE. These encode for two polypeptides homologous to the activator domain and the kinase domain, respectively, of bacterial autophosphorylating tyrosine kinases involved in polysaccharide chain length determination. The GelC/GelE pair is an exception to the biochemically characterized Gram-negative tyrosine autokinases since it consists of two polypeptides instead of a single one. The deletion of gelC or gelE resulted in the abolishment of gellan in the culture medium confirming their role in gellan biosynthesis. In addition, ATP-binding assays confirmed the predicted ATP-binding ability of GelE. Interestingly, GelE contains an unusual Walker A sequence (ASTGVGCS), where the invariant lysine is replaced by a cysteine. This residue was replaced by alanine or lysine and although both mutant proteins were able to restore gellan production by complementation of the gelE deletion mutant to the production level observed with native GelE, only the mutated GelE where the cysteine was replaced by alanine was demonstrated to bind ATP in vitro. The importance of specific tyrosine residues present in the C-terminal domain of GelE in gellan assembly was also determined. The tyrosine residue at position 198 appears to be essential for the synthesis of high-molecular-weight gellan, although other tyrosine residues may additionally contribute to GelE biological function.