Effect of High-Pressure Treatments on the Properties of Food Packaging Materials with or without Antimicrobials.

The aim of this research work was the comparative study of the different properties of interest in the case of plastic materials for food use before and after being subjected to treatment by high hydrostatic pressure (HHP) as well as the impact of additivation with antimicrobials. This method of food preservation is currently on the rise and is of great interest because it is possible to extend the shelf life of many foods without the need for the use of additives or thermal processing, as is the case with other preservation methods currently used. The effects of HHP treatment (680 MPa for 8 min) on plastic materials commonly used in the food industry were studied. These materials, in sheet or film form, were polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), multilayer polyethylene terephthalate-ethylene-vinyl alcohol copolymer-polyethylene (PET-EVOH-PE), multilayer polyethylene-polyethylene terephthalate (PE-PET), polyvinyl chloride aluminum (PVC-AL), and polylactic acid (PLA), which were provided by manufacturing companies in the sector. PE, PP, and PLA activated with tyrosol, zinc oxide, or zinc acetate were also tested. The phenomena and properties, such as overall migration, thermal behavior, oxygen barrier, and physical properties were analyzed before and after the process. The results show that the HHP process only slightly affected the properties of the materials. After pressurization, oxygen permeability increased greatly in PVC-AL (from 7.69 to 51.90) and decreased in PLA (from 8.77 to 3.60). The additivation of the materials caused a change in color and an increase in oxygen permeability. The additivated PE and PP showed migration values above the legal limit for certain simulants. The HHP treatment did not greatly affect the mechanical properties of the additivated materials. The main increases in the migration after HHP treatment were observed for PE activated with tyrosol or zinc oxide and for PS activated with zinc oxide. Activated PLA performed the best in the migration studies, irrespective of the HHP treatment. The results suggest that activated PLA could be used in HHP food processing as an inner antimicrobial layer in contact with the food packed in a container with the desired oxygen permeability barrier.

Correlations among Resistances to Different Antimicrobial Compounds in Salmonella Strains from Hen Eggshells.

Persistence of antibiotic-resistant Salmonella in the food chain may depend on strain tolerance to other antimicrobials and also on biofilm formation capacity. Yet, there is limited information on sensitivity of antibiotic-resistant Salmonella to other antimicrobials, such as phenolic compounds, chemical preservatives, or antimicrobial peptides. This study aimed at correlating antimicrobial resistance and biofilm formation capacity in antibiotic-resistant, biocide-tolerant Salmonella strains from hen eggshells. A collection of 21 strains previously selected according to their antibiotic resistance and biocide tolerance phenotypes were used for the present study. Strains were inspected for their biofilm formation capacity and for their sensitivity to (i) phenolic compounds (carvacrol, thymol), (ii) chemical preservatives (sodium lactate, trisodium phosphate), and (iii) cationic antimicrobials (polymyxin B, lysozyme-EDTA). Biofilm formation capacity was not correlated with antimicrobial resistances of the planktonic Salmonella. Polymyxin B and the lysozyme-EDTA combinations showed significant ( P < 0.05) positive correlations to each other and to sodium lactate. Significant ( P < 0.05) positive correlations were also observed for benzalkonium chloride and cetrimide with carvacrol, thymol, and trisodium phosphate, or between hexadecylpyridinium chloride and carvacrol. Antibiotic resistance also correlated positively with a higher tolerance to other antimicrobials (cefotaxime, ceftazidime, and ciprofloxacin with carvacrol, thymol, and trisodium phosphate; netilmicin with thymol and trisodium phosphate; tetracycline with carvacrol and thymol). These results must be taken into consideration to ensure a proper use of antimicrobials in the poultry industry, at concentrations that do not allow coselection of biocide-tolerant, antibiotic-resistant Salmonella.

Copper and Zinc Tolerance in Bacteria Isolated from Fresh Produce.

The continued agricultural exposure of bacteria to metals such as copper and zinc may result in an increased copper tolerance through the food chain. The aim of this study was to determine the Cu and Zn tolerance of bacteria from fresh produce (cucumber, zucchini, green pepper, tomato, lettuce, vegetable salad, broccoli, cabbage, carrot, green onion, onion, and mango). Isolates (506 aerobic mesophiles) from 12 different food produce products were tested for growth in a range of Cu and Zn concentrations. Selected isolates were identified using 16S rDNA sequencing, and the presence of metal resistance genes was studied using PCR amplification. More than 50% of the isolates had MICs for copper sulfate greater than 16 mM, and more than 40% had MICs greater than 4 mM for zinc chloride. Isolates with high levels of tolerance to Cu and Zn were detected in all the produce products investigated. A selection of 51 isolates with high MICs for both Cu and Zn were identified as belonging to the genera Pseudomonas (28), Enterobacter (7), Serratia (4), Leclercia (1), Bacillus (10), and Paenibacillus (1). A study of the genetic determinants of resistance in the selected gram-negative isolates revealed a high incidence of genes from the pco multicopper oxidase cluster, from the sil cluster involved in Cu and silver resistance, and from the chromate resistance gene chrB. A high percentage carried both pco and sil. The results suggest that Cu and Zn tolerance, as well as metal resistance genes, is widespread in bacteria from fresh produce.

Biocide Tolerance and Antibiotic Resistance in Salmonella Isolates from Hen Eggshells.

The aim of the present study was to determine biocide tolerance and antibiotic resistance in Salmonella isolates from hen eggshells. A total of 39 isolates from hen eggshells, identified as either Salmonella spp. or Salmonella enterica according to 16S rDNA sequencing, were selected for biocide tolerance. Isolates with minimum inhibitory concentrations (MICs) above the wild-type MICs were considered to be biocide tolerant: benzalkonium chloride (BC, 7.7%), cetrimide (CT, 7.7%), hexadecylpyridinium chloride (HDP, 10.3%), triclosan (TC, 17.9%), hexachlorophene (CF, 30.8%), and P3-oxonia (OX, 25.6%). The resulting 21 biocide-tolerant isolates were further characterized. Most isolates (95.2%) were resistant to ampicillin, but only 9.5% were resistant to cefotaxime as well as to ceftazidime. Resistance to chloramphenicol (61.9%), tetracycline (47.6%), streptomycin (19.0%), nalidixic acid (28.6%), ciprofloxacin (9.5%), netilmicin (14.3%), and trimethoprim-sulfamethoxazole (38.1%) was also detected. Considering only antibiotics, 66.7% of isolates were multiresistant; furthermore, 90.5% were multiresistant considering antibiotics and biocides combined. Efflux pump and biocide tolerance genetic determinants detected included acrB (95.2%), oqxA (14.3%), mdfA (9.5%), qacA/B (4.8%), and qacE (9.5%). Antibiotic resistance genes detected included blaTEM (14.3%), blaCTXM-2 (4.8%), blaPSE (4.8%), floR (19.05%), tet(A) (9.5%), tet(C) (4.8%), dfrA12 (0.05%), and dfrA15 (0.05%). Significant positive correlations were detected between phenotypic tolerance/resistance to biocides, biocides and antibiotics, and also between antibiotics, suggesting that a generalized use of biocides could co-select antibiotic resistance.

Biocide tolerance in Salmonella from meats in Southern Spain.

Salmonella serovars sampled from meat products in Southern Spain (Andalucía) during the period 2002-2007 were analyzed in this study. The serovars most frequently detected (in order) were Typhimurium, Enteritidis, Derby, Anatum and Rissen. Isolates (n = 43) were tested for sensitivity to biocides, including the quaternary ammonium compounds benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and the bisphenols triclosan (TC) and hexachlorophene (CF). The minimum inhibitory concentration (MIC) for the quaternary ammonium compounds was in the range of 25 to 50 mg/L for most isolates, although a few isolates required much higher concentrations, up to 250 mg/L. Bisphenols showed higher inhibitory activity, with a MIC of 2.5 to 25 mg/L. A few isolates showed a "non-wildtype" MIC for TC of up to 250 mg/L. These results indicate a low incidence of tolerance towards quaternary ammonium compounds and triclosan among Salmonella from meats and meat products.

Biocide tolerance in Salmonella from meats in Southern Spain

Salmonella serovars sampled from meat products in Southern Spain (Andalucía) during the period 2002-2007 were analyzed in this study. The serovars most frequently detected (in order) were Typhimurium, Enteritidis, Derby, Anatum and Rissen. Isolates (n = 43) were tested for sensitivity to biocides, including the quaternary ammonium compounds benzalkonium chloride (BC), cetrimide (CT) and hexadecylpyridinium chloride (HDP), and the bisphenols triclosan (TC) and hexachlorophene (CF). The minimum inhibitory concentration (MIC) for the quaternary ammonium compounds was in the range of 25 to 50 mg/L for most isolates, although a few isolates required much higher concentrations, up to 250 mg/L. Bisphenols showed higher inhibitory activity, with a MIC of 2.5 to 25 mg/L. A few isolates showed a “non-wildtype” MIC for TC of up to 250 mg/L. These results indicate a low incidence of tolerance towards quaternary ammonium compounds and triclosan among Salmonella from meats and meat products.

Inactivation of Staphylococcus aureus in Oat and Soya Drinks by Enterocin AS-48 in Combination with Other Antimicrobials.

The presence of toxicogenic Staphylococcus aureus in foods and the dissemination of methicillin-resistant S. aureus (MRSA) in the food chain are matters of concern. In the present study, the circular bacteriocin enterocin AS-48, applied singly or in combination with phenolic compounds (carvacrol, eugenol, geraniol, and citral) or with 2-nitro-1-propanol (2NPOH), was investigated in the control of a cocktail made from 1 methicillin-sensitive and 1 MRSA strains inoculated on commercial oat and soya drinks. Enterocin AS-48 exhibited low bactericidal activity against staphylococci in the drinks investigated when applied singly. The combinations of sub-inhibitory concentrations of enterocin AS-48 (25 µg/mL) and phenolic compounds or 2NPOH caused complete inactivation of staphylococci in the drinks within 24 h of incubation at 22 °C. When tested in oat and soya drinks stored for 7 d at 10 °C, enterocin AS-48 (25 µg/mL) in combination with 2NPOH (5.5 mM) reduced viable counts rapidly in the case of oat drink (4.2 log cycles after 12 h) or slowly in soya drink (3.8 log cycles after 3 d). The same combined treatment applied on drinks stored at 22 °C achieved a fast inactivation of staphylococci within 12 to 24 h in both drinks, and no viable staphylococci were detected for up to 7 d of storage. Results from the study highlight the potential of enterocin AS-48 in combination with 2NPOH for inactivation of staphylococci.

The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications.

Enterocin AS-48 is a circular bacteriocin produced by Enterococcus. It contains a 70 amino acid-residue chain circularized by a head-to-tail peptide bond. The conformation of enterocin AS-48 is arranged into five alpha-helices with a compact globular structure. Enterocin AS-48 has a wide inhibitory spectrum on Gram-positive bacteria. Sensitivity of Gram-negative bacteria increases in combination with outer-membrane permeabilizing treatments. Eukaryotic cells are bacteriocin-resistant. This cationic peptide inserts into bacterial membranes and causes membrane permeabilization, leading ultimately to cell death. Microarray analysis revealed sets of up-regulated and down-regulated genes in Bacillus cereus cells treated with sublethal bacteriocin concentration. Enterocin AS-48 can be purified in two steps or prepared as lyophilized powder from cultures in whey-based substrates. The potential applications of enterocin AS-48 as a food biopreservative have been corroborated against foodborne pathogens and/or toxigenic bacteria (Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella enterica) and spoilage bacteria (Alicyclobacillus acidoterrestris, Bacillus spp., Paenibacillus spp., Geobacillus stearothermophilus, Brochothrix thermosphacta, Staphylococcus carnosus, Lactobacillus sakei and other spoilage lactic acid bacteria). The efficacy of enterocin AS-48 in food systems increases greatly in combination with chemical preservatives, essential oils, phenolic compounds, and physico-chemical treatments such as sublethal heat, high-intensity pulsed-electric fields or high hydrostatic pressure.

Multilocus sequence typing and antimicrobial resistance in Enterococcus faecium isolates from fresh produce.

The purpose of the present study was to determine the relatedness of Enterococcus faecium isolates from fresh produce to E. faecium strains from other sources by using multi-locus sequence typing (MLST) and to determine the antimicrobial resistance of the isolates. MLST analysis of 22 E. faecium isolates from fresh produce revealed 7 different sequence types (ST 22, ST 26, ST 43, ST 46, ST 55, ST 94 and ST 296). Most isolates belonged to ST 296 (40.9 %), followed by ST 94 (27.3 %). All isolates were sensitive to vancomycin and to imipenem, and only one was resistant to ampicillin (MIC 32 mg/l). However, all were resistant to cefotaxime and ceftazidine. E. faecium isolates from fresh produce were inhibited by quaternary compounds (benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide), biguanides (chlorhexidine), polyguanides [poly-(hexamethylene guanidinium) hydrochloride], bisphenols (triclosan, hexachlorophene) and biocidal solutions of P3 oxonia and P3 topax 66. Didecyldimethylammonium bromide and triclosan were the least effective biocides in growth inhibition, while hexadecylpyridinium chloride was the most effective. Results from MLST typing and antibiotic resistance suggest that the studied E. faecium isolates from fresh produce are not related to the clinically-relevant clonal complex CC17.

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes.

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 µg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 µg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 µg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 µg/ml.

Genome sequence of Weissella thailandensis fsh4-2.

Weissella thailandensis fsh4-2 is a heterofermentative lactic acid bacterium isolated from the Korean fermented seafood condiment jeotkal. Here we report the draft genome sequence of W. thailandensis fsh4-2 (1,651 genes, 1,436 encoding known proteins, 183 encoding unknown proteins, 32 RNA genes), which consists of 50 large contigs of >100 bp.

Annotated genome sequence of Lactobacillus pentosus MP-10, which has probiotic potential, from naturally fermented Aloreña green table olives.

Lactobacillus pentosus MP-10 was isolated from brines of naturally fermented Aloreña green table olives. MP-10 has potential probiotic traits, including inhibition of human pathogenic bacteria, survival at low pH (1.5), and bile salt tolerance (3%). Here, we report for the first time the annotated genome sequence of L. pentosus.