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Objectives In this study, 77 Enterobacter spp. isolates from a collection of 175 Gram-negative bacilli isolated from Tlemcen University Hospital Center (North-West of Algeria) were tested for antibiotic resistance, biocide tolerance and genetic determinants of antimicrobial resistance. Methods The isolates were identified by 16S rDNA gene sequencing. Biocide tolerance was determined by broth microdilution, and antibiotic resistance was determined by disk diffusion. Genetic determinants of resistance were studied by PCR amplification using suitable primers. Results The most common Enterobacter species was Enterobacter cloacae (58.4%), followed by Enterobacter hormaechei (24.7%). The most common antibiotic resistance was to ticarcillin either alone or in combination with clavulanic acid (70.1%), followed by cefepime (68.8%), cefotaxime (63.6%), ceftazidime (54.5%) and gentamicin (54.5%). Tobramycin was active against 87.0% of the isolates. Levels of biocide tolerance were high for hexachlorophene and to a lesser extent for benzalkonium chloride. The extended-spectrum β-lactamase genes blaTEM and blaCTX-M were detected in 44.2% and 36.4% of isolates, respectively. Other antimicrobial resistance genes (ARGs) frequently detected were aac(6ʹ)-Ib (57.1%) and sul2 (50.6%). Multidrug-resistant isolates carrying several ARGs were common. Significant positive correlations were detected for efflux pump genes with ARGs and also between ARGs. Conclusion The results of this study reveal thatEnterobacter spp. isolates from hospital settings are both resistant to clinically-used antibiotics and tolerant to biocides. Biocide tolerance could be an advantage for antibiotic-resistant strains in hospitals.

The aim of the present study was to determine the effect of high hydrostatic pressure (HP) and films activated with a combination of enterocin AS-48 and thymol (AF) on the microbial load and bacterial diversity of fruit puree (banana, apple, pear). HP and AF were applied singly, or in combination (AFHP). Samples were chill-stored. HP and AFHP treatments reduced viable counts significantly (P ≤ 0.05). The predominant bacterial groups in the puree were fam. Comamonadaceae (23.17%) and genera Methylobacterium (21.46%), Acidovorax (8.70%) and Sphingomonas (6.63%). Pseudomonas became relevant by the end of storage (10.50%). Most of the AF samples had higher relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas and lower relative abundance of Pseudomonas. Application of HP treatment markedly reduced the relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas. Lactobacillales increased in relative abundance upon application of HP treatment but not during storage, while Pseudomonas increased towards the end of storage. The combined treatment (AFHP) achieved greatest reduction in the relative abundances of Comamonadaceae and Methylobacterium. Results indicate that AFHP treatment had greatest effects on the bacterial diversity of the fruit puree and was also the most effective in keeping total aerobic mesophiles and Enterobacteriaceae below detectable levels.

European plum (Prunus domestica L.) is a tree cultivated in many countries for its edible fruits, and necessarily certain amounts of useless woody residues are generated every year during pruning works. The objectives of this research were to recover the phenolics present on that wood wastes and study their antimicrobial and antibiofilm activities against a selection of foodborne microorganisms, including multi-resistant strains. The isolation of components was carried out by combining fast centrifugal partition chromatography (FCPC) and semi-preparative high-performance liquid chromatography (HPLC), and the identification was achieved by conventional techniques. Nine compounds were identified: 2 flavan-3-ols [catechin (1) and epicatechin (2)]; 1 phenolic glucoside [annphenone (3)]; and 6 dimeric A-type proanthocyanidins (4–9). All of them have been reported in Prunus domestica by the first time in this work, with the exception of 1 and 2. Extracts, fractions and purified phenolics were submitted to antimicrobial and antibiofilm activity evaluations. The most significative antimicrobial effect was detected on the strain Enterobacter sp. UJA37p, previously isolated from organic tomato and with high tolerance to different biocides. All pure compounds showed MIC values of 100 μg/mL against this resistant bacterium. On the other hand, when bacteria were previously allowed to attach and form biofilm before treatments, we found interesting results in disruption of preformed biofilms with compound 7 [(+)-epiafzelechin-(2β→O→7,4β→8)-epicatechin], followed by compound 6 [(−)-ent-epiafzelechin-(2α→O→7,4α→8)-catechin] and compound 3 at a concentration of 0.1 μg/mL.

Acidity often prevents the undesirable microbial colonization both in fermented foods and under gastric conditions. Thus, the acid resistance of Lactobacillus pentosus strains used as starter cultures and/or probiotics requires further understanding. This was investigated by means of comparative proteomic approach using three strains representing the phenotypes: resistant (AP2-15), intermediate (AP2-18) and sensitive (LP-1) to acidic conditions. Proteomic analysis of constitutive phenotypes revealed that the intrinsic resistance of L. pentosus is associated with the over-production of three principal proteins: 2,3-bisphosphoglycerate- dependent phosphoglycerate mutase 2 (PGAM-d), elongation factor G and 50S ribosomal protein L10, and additionally on ATP synthase subunit beta and chaperone protein DnaK; they are associated with metabolic pathways of proteins and carbohydrates, energy production and stress responses. Suggested protein biomarkers for acid resistance in L. pentosus include elongation factor G and PGAM-d, both being abundantly found in the constitutive proteome of the resistant phenotype under standard and acidic conditions. Furthermore, L. pentosus strains pre-exposed to acids displayed enhanced probiotic function such as auto-aggregation ability via surface proteins. We conclude that pre-exposure of probiotic L. pentosus strains to acid may strategically enhance their performance as starter cultures and probiotics. (c) 2017 Elsevier Ltd. All rights reserved.

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.

A collection of 38 biocide-adapted strains with significant increases in their tolerance to biocides after step-wise exposure to these compounds were screened for sensitivity to essential oils and chemical preservatives. Several strains adapted to quaternary ammonium compounds (benzalkonium chloride, hexadecylpyridinium chloride or cetrimide) showed a generalized increase in the sensitivity to preservatives. Similar results were found among hexachlorophene- or chlorhexidine-adapted strains. Moreover, tolerance to hexadecylpyridinium chloride showed a very strong positive correlation with 4-hydroxybenzoic acid, thyme oil and sodium nitrite increased sensitivity, as well as a strong correlation with clove oil, potassium sorbate and potassium nitrate increased sensitivity. On the contrary, an increase in tolerance to preservatives was detected among triclosan-adapted strains. Results from this study suggest that exposure of bacteria from foods to biocides is not always associated with co-selection for other antimicrobial resistances, especially against essential oils or chemical preservatives used in the food industry.

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.

The purpose of the present study was to determine the effect of high hydrostatic pressure treatment (HHP) on the microbiota of mango pulp. Mango pulp was artificially contaminated with its own epiphytic microbiota to simulate a worst-case scenario of contamination during pulp preparation. Controls and samples treated by HHP (600 MPa, 8 min) were chill stored for 30 days. HHP treatment significantly (P Actinobacteria (45.63%), Firmicutes (42.55%), Proteobacteria (10.68%) and Bacteroidetes (1.0%) changed during storage, with a strong increase of Proteobacteria . HHP treatment also induced a strong increase in Proteobacteria , followed by a late recovery of Firmicutes and to a less extent Actinobacteria . Lactobacillus was the main operational taxonomic unit (OTU) detected both in controls during early storage and in HHP-treated samples during late storage. Results from the present study indicate how bacterial populations of both controls and HHP-treated mango pulp samples undergo complex changes during refrigeration storage.

In the present study, a collection of 76 biocide-sensitive bacterial strains isolated from organically produced food were adapted by repeated exposure to increasing concentrations of the quaternary ammonium compounds (QACs) benzalkonium chloride (BC) and hexadecylpyridinium chloride (HDP). The sensitivity of both wildtype strains and their corresponding QAC-adapted strains to other biocides and to antibiotics was studied. QAC tolerance increased in 88.2% of strains for BC and in 30.3% of strains for HDP, with increases in minimum inhibitory concentrations between 2 and over 100 fold. Adaptive resistance was stable after 20 subcultures in biocide-free medium for 7 and 5 of the BC- and HDP-adapted strains, respectively. Adaptation to BC and HDP also reduced the susceptibility to other biocides, mainly hexachlorophene (CF), didecyldimethylammonium bromide (AB), triclosan (TC) and chlorhexidine (CH). BC-adapted strains showed increased antibiotic resistance to ampicillin (AM) followed by sulfamethoxazol (SXT) and cefotaxime (CTX), and some showed increased sensitivity to ceftazidime (CAZ), CTX, AM and STX. Changes in antibiotic resistance in HDP-adapted strains were more heterogeneous and strain-dependent. Main efflux pump genes detected in QAC-adapted strains were acrB, sugE, norC, qacE and qacH, as well as antibiotic resistance genes aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ic, ant(4_)-Ia, lsa, mrsA/B, ereA, ermB and cat. Membrane anisotropy experiments revealed that QAC adaptation induced an increase in membrane rigidity in the case of BC, while response to HDP was more heterogeneous and strain-dependent. Growth capacity was significantly higher in some QAC-adapted strains and strain-dependent changes in heat tolerance were also detected in QAC-adapted strains. Gastric acid or bile resistances do not seem to be influenced by QAC adaptation.

A collection of 120 bacterial isolates from small medium enterprises involved in the production of cow milk and the manufacture of goat cheese were screened for sensitivity to biocides benzalkonium chloride (BC), cetrimide (CT), hexadecylpyridinium chloride (HDP), triclosan (TC), hexachlorophene (CF) and poly-(hexamethylen guanidinium) hydrochloride (PHMG). Nineteen isolates were selected according to biocide tolerance and identified by 16S rDNA sequencing as Lactococcus sp. (6) Enterococcus sp. (1), Lactobacillus sp. (4), Bacillus sp. (1) Escherichia sp. (5), Enterobacter sp. (1) and Helicobacter sp. (1). These were further characterised regarding antimicrobial resistance phenotype and genotype. Several isolates were multiply (3 or more) tolerant to biocides or resistant to antibiotics, but only two Escherichia sp. isolates and Enterobacter sp. were multiply resistant to biocides and antibiotics. Statistical analysis of biocide tolerance and antibiotic resistance revealed significant positive correlations between different biocides and between biocides and antibiotics. The biocide tolerance genes most frequently found were qacEΔ1 and qacA/B. The sulfonamide resistance gene sul1 was found in two Escherichia sp. isolates and in Enterobacter sp., all of which also carried qacEΔ1. Beta-lactam (blaCTX-M, blaPSE) and tetracycline resistance genes [tet(A), tet(C) and tet(D)] were detected. Efflux pump genes acrB and mdfA were found in most Gram-negative isolates. Results from the study suggest that exposure to biocides can indirectly select for antibiotic resistance.

Microbiota recovered from fresh produce (Romaine lettuce, endives and cucumbers) was allowed to form biofilms on stainless steel coupons. The formed biofilms were treated with benzalkonium chloride (BC) at three different concentrations (0.01, 0.1, and 1.0 g/l), enterocin AS-48 (50 μg/ml) and combinations of BC and enterocin AS-48. The single treatment with bacteriocin had no effect on viability of sessile bacteria. A high concentration of BC (1.0 g/l) was required to achieve 4.1 logs reduction of viable cell counts. The combination of BC (1.0 g/l) and enterocin AS-48 reduced viable cell counts below detectable levels. High-throughput sequencing analysis revealed that the formed biofilms were composed mainly by Proteobacteria of the genera Pseudomonas and Stenotrophomonas . Treatments with enterocin AS-48 and BC at sub-inhibitory concentrations only induced minor changes in the relative abundance of the different bacterial groups associated with biofilms.

This study aimed at determining the effect of high hydrostatic pressure (HHP) treatments on the bacterial biodiversity of processed peaches. Peach cubes in peach juice inoculated with epiphytic microbiota recovered from peach surfaces were pressurized at 600 MPa for 8 min at temperatures of 22 °C and of 45 °C. Non-pressurized samples were used as controls. Samples were chill stored for 15 days. Application of HHP treatments reduced viable counts of total aerobic mesophiles and yeasts and molds, and delayed recovery of survivors. Pyrosequencing analysis revealed that 35.7% of operational taxonomic units (OTUs) from peaches samples belonged to Actinobacteria (mainly Couchioplanes, Bifidobacterium and Propionibacterium), 31.1% to Firmicutes (mainly Streptococcus, Lactobacillus and Enterococcus) and 28.8% to Proteobacteria (mainly Reyranella, Methylobacterium, Acetobacter, Dyella, Sphingomonas, Tatumella, Enterobacter, Prevotella and Gluconobacter). During storage of control samples, the relative abundances of Gluconobacter and Lactobacillus increased. Application of HHP treatments reduced the relative abundance of Actinobacteria and Firmicutes while Proteobacteria increased. The opposite was observed during storage of the treated samples. Increasing the treatment temperature to 45 °C decreased the relative abundance of Actinobacteria. Results from the present study indicate that peach microbiota is strongly influenced by refrigeration storage and by application of HHP treatments.

The application of high hydrostatic pressure (HHP, 600 MPa, 8 min) on brined green asparagus and the changes in bacterial diversity after treatments and during storage at 4 °C (30 days) or 22 °C (10 days) were studied. HHP treatments reduced viable cell counts by 3.6 log cycles. The residual surviving population did not increase during storage at 4 °C. However, bacterial counts significantly increased at 22 °C by day 3, leading to rapid spoilage. The microbiota of green asparagus was composed mainly by Proteobacteria (mainly Pantoea and Pseudomonas), followed by Firmicutes (mainly Lactococcus and Enterococcus) and to a less extent Bacteroidetes and Actinobacteria. During chill storage of untreated asparagus, the relative abundance of Proteobacteria as well as Enterococcus and Lactococcus decreased while Lactobacillus increased. During storage of untreated asparagus at 22 °C, the abundance of Bacteroidetes decreased while Proteobacteria increased during late storage. The HHP treatment determined a reduction of the Proteobacteria both early after treatment and during chill storage. In the HHP treated samples stored at 22 °C, the relative abundance of Pseudomonas rapidly decreased at day 1, with an increase of Bacteroidetes. This was followed by a marked increase in Enterobacteriaceae (Escherichia) simultaneously with increase in viable counts and spoilage. Results from the study indicate that the effect of HHP treatments on the viability of microbial populations in foods also has an impact on the dynamics of microbial populations during the storage of the treated foods.

Food Research International Volume 89, 1 November 2016, Pages 790-796 Microbial diversity in pitted sweet cherries (Prunus avium L.) as affected by High-Hydrostatic Pressure treatment (Article) Toledo del Árbol, J.ab, Pérez Pulido, R.a, La Storia, A.b, Grande Burgos, M.J.a, Lucas, R.a, Ercolini, D.b, Gálvez, A.a a Área de Microbiología, Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Universidad de Jaén, Jaén, Spain b Department of Agricultural Sciences, Division of Microbiology, University of Naples Federico II, Via Università 100, Portici, Italy View references (42) Abstract Sweet cherries are a highly appreciated seasonal fruit rich in anthocyanins. The purpose of the present study was to determine the effect of High-Hydrostatic Pressure (HHP) processing on the microbiological quality and bacterial biodiversity of sweet cherries. Pitted cherries inoculated with their own epiphyte microbiota to simulate a worst-case scenario of contamination during preparation and processing were treated or not by HHP (600 MPa, 8 min) and stored at 4 °C for 60 days. HHP treatment reduced total viable counts by 4.65 log cycles. The surviving bacterial fraction did not increase significantly (p < 0.05) for the first 15 days of storage. Concentrations of yeasts and molds were reduced below detectable levels. Upon prolonged storage (60 days), microbial growth was observed. Bacterial biodiversity studied by high-throughput sequencing of the 16S rRNA gene revealed that Proteobacteria had highest relative abundance (88.70%) in the spiked cherries followed by Firmicutes (11.04%). Gluconobacter and Enterobacteriaceae together with Leuconostoc were the most abundant Operational Taxonomic Units (OTUs). Upon application of HHP treatment, 97.62% of OTUs from the surviving fraction belonged to Proteobacteria. The relative abundance of Enterobacteriaceae also decreased markedly while Acetobacteraceae (represented mainly by Gluconobacter) increased to 89.18%. Gluconobacter dominated during storage. Results from the present study provide insights on the microbiota of sweet cherries and the dynamics of the bacterial populations surviving HHP treatments that may be useful to improve the non-thermal preservation of cherries.

Enterocin AS-48 is a circular bacteriocin with strong anti- Listeria activity. The purpose of the present study was to evaluate the effect of the bacteriocin incorporated into different coating solutions on a cocktail of five L. monocytogenes strains previously inoculated on apple cubes. Coating solutions were made with chitosan, caseinate, alginate, k-carrageenate, xanthan gum, pectin, starch, carboxymethyl cellulose or methyl cellulose. Coatings were applied singly or combined with enterocin AS-48 at 20 or 40 μg/ml. Samples were stored at 4 °C for 7 days. The single application of coatings had almost no effect (as in alginate and methyl cellulose) or had a low effect on Listeria viability ( Listeria activity (up to 3.7 log cycles at day 7). Coatings dosed with 20-μg/ml enterocin AS-48 reduced viable Listeria counts gradually during storage in most cases, achieving significant reductions (p Listeria counts below detection levels from day 1 for most of the coatings tested. The combination of EDTA and AS-48 was also the most effective at time 0, achieving reductions of viable counts between 2.0 and 2.7 log cycles depending on the coating immediately after treatment compared with single coatings. Industrial relevance Results from the present study suggest the potential of edible coatings containing enterocin AS-48 and EDTA for inactivation of L. monocytogenes on apple surfaces. Since edible coatings are widely used on fruit surfaces, coatings activated with enterocin AS-48 and EDTA could find application as a hurdle against L. monocytogenes in fresh-cut apple pieces.

A cocktail of Leuconostoc mesenteroides, Leuconostoc gasicomitatum and Leuconostoc gelidum inoculated on autoclaved cherimoya pulp were treated by high hydrostatic pressure (HHP; 400, 500 or 600 MPa for 8 min) and with enterocin AS-48 (35 μg/g), singly or in combination. After treatments, samples were stored at 4 °C for 30 days or at 22 °C for 10 days. HHP treatments at 400 or 500 MPa reduced viable cell concentrations by 4.3 and 4.9 log cycles, respectively. No survivors were detected at 600 MPa. Bacteriocin addition in combination with HHP achieved an additional reduction of 0.6–0.9 log cycles after treatments at 400 or 500 MPa, and also reduced viable counts for most treatments during storage. In samples treated by HHP, regrowth of leuconostocs was delayed to days 15 (400 or 500 MPa) or 30 (600 MPa) of refrigeration storage, or day 3 at 22 °C (all treatments). Results from the study suggest that treatments at 600 MPa (8 min) can be useful to inactivate leuconostocs in cherimoya pulp.

Fermentation of caper berries is a traditional process relying on the indigenous microbiota. In this study, the application of Lactobacillus plantarum Lb9 isolated from spontaneous caper berry fermentation as starter culture was evaluated by means of culture-dependent and independent methods. The results obtained here indicated that L. plantarum Lb9 was able to carry on an accelerated lactic acid fermentation of caper berries, dominating the background microbiota present in the raw material until the end of fermentation even when fermentation broth was added with high salt concentration. Persistence of L. plantarum Lb9 in the starter-added fermentation was confirmed by RAPD-PCR of isolates from viable cell counting during the fermentation. TTGE analysis of the microbial community throughout fermentation process also indicated that lactobacilli were the predominant microbiota, and thus the microbial composition during the controlled fermentation was more homogeneous compared with spontaneous fermentations. Furthermore, the sensorial quality of the starter-added fermented capers was similar to the spontaneous fermented products. In conclusion, L. plantarum Lb9 was an adequate starter culture strain for caper berry fermentation prevailing over other bacteria present in the raw material. This fermentation ensured both the quality and safety of the end product.

Enterococcus faecalis and Enterococcus faecium isolated from various traditional fermented foods of both animal and vegetable origins have shown multidrug resistance to several antibiotics and tolerance to biocides. Reduced susceptibility was intra and inter-species dependent and was due to specific and unspecific mechanisms such as efflux pumps. EfrAB, a heterodimeric ABC transporter efflux pump, was detected in 100% of multidrug resistant (MDR) E. faecalis strains and only in 12% of MDR E. faecium strains. EfrAB expression was induced by half of minimum inhibitory concentration (MIC) of gentamicin, streptomycin and chloramphenicol. However, expression of efrA and efrB genes was highly dependent on the strain tested and on the antimicrobial used. Our results indicated that 3 mM EDTA highly reduced the MICs of almost all drugs tested. Nevertheless, the higher reductions (>8 folds) were obtained with gentamicin, streptomycin, chlorhexidine and triclosan. Reductions of MICs were correlated with down-regulation of EfrAB expression (10-140 folds) in all three MDR enterococci strains. This is the first report describing the role of EfrAB in the efflux of antibiotics and biocides which reflect also the importance of EfrAB in multidrug resistance in enterococci. EDTA used at low concentration as food preservative could be one of the best choices to prevent spread of multidrug resistant enterococci throughout food chain by decreasing EfrAB expression. EfrAB could be an attractive target not only in enterococci present in food matrix but also those causing infections as well by using EDTA as therapeutic agent in combination with low doses of antibiotics.

Africa has an age old history of production of traditional fermented foods and is perhaps the continent with the richest variety of lactic acid fermented foods. These foods have a large impact on the nutrition, health and socio-economy of the people of the continent, often plagued by war, drought, famine and disease. Sub-Saharan Africa is the world's region with the highest percentage of chronically malnourished people and high child mortality. Further developing of traditional fermented foods with added probiotic health features would be an important contribution towards reaching the UN Millennium Development Goals of eradication of poverty and hunger, reduction in child mortality rates and improvement of maternal health. Specific probiotic strains with documented health benefits are sparsely available in Africa and not affordable to the majority of the population. Furthermore, they are not used in food fermentations. If such probiotic products could be developed especially for household food preparation, such as cereal or milk foods, it could make a profound impact on the health and well-being of adults and children. Suitable strains need to be chosen and efforts are needed to produce strains to make products which will be available for clinical studies. This can gauge the impact of probiotics on consumers' nutrition and health, and increase the number of people who can benefit. Keywords: African traditional foods; Fermentation; GI infection; Malnutrition; Probiotics.

High hydrostatic pressure (HHP) treatments (singly or in combination with natural antimicrobials) were tested for stabilization of Manzanilla Alorena seasoned olives stored at 25 °C. HHP (5 min) was highly effective on yeast populations at 300 MPa or higher. No viable yeasts were detected in samples treated at 400 MPa for up to three months. Low levels of endospore-forming bacteria were always detected after HHP treatments. Addition of nisin to the brines reduced bacterial counts by 1.4 log cycles but it had no effect on yeasts when tested singly or in combination with HHP treatment (400 MPa, 5 min). Thyme oil had almost no effect on yeast concentrations, but rosemary oil reduced yeast viable counts progressively during storage. Essential oils in combination with HHP (400 MPa, 5 min) significantly (p

In the present study, a collection of 29 Gram-negative bacteria selected from organic foods according to antibiotics and biocide resistance were screened for multidrug efflux pumps and specific antibiotic resistance genes. The acrB pump gene of the AcrAB–TolC system was detected in all isolates. EfrAB determinants efrA and efrB were frequently detected (Enterobacter, Pantoea, Salmonella), while other efflux pump genes (norE, mepA mdeA, norC) were detected only in some Enterobacter and Pantoea isolates. Genes encoding resistance to quaternary ammonium compounds qacE and qacJ were detected only in Enterobacter isolates, while sugE was detected in Enterobacter and in one Pantoea isolate. The aminoglycoside-resistance ant(4_)-Ia gene was detected in Enterobacter, Pantoea agglomerans and Klebsiella oxytoca. The antibiotic resistance determinant most frequently found was lsa gene (Enterobacter, Pantoea and Salmonella isolates). Erythromycin resistance genes (ereA, ereB) were detected among Enterobacter, Klebsiella or Salmonella. The macrolide efflux pump msrA and the phosphotransferase type I mphA genes were detected in Salmonella. Other resistance determinants analyzed (mdfA, yhiUV, evgA, 455emeA, yvcC, qacA/B, qacC, qacG, qacH, norA, norB, norC, sdrM, sepA, mecA, emrE, smr, bla, cat, aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ib, aph(2_)-Ic, aph(2_)-Id, aph(3_)-IIIa, ermA, ermB, ermC, and mefA genes) were not detected in any isolate. Results from the present study suggest that the main mechanisms of antibiotic and biocide resistance in Gram-negative bacteria from organic foods rely on different kinds of efflux pumps of broad substrate specificity. The indiscriminate use of biocides in the food chain should be revised in order to minimize persistence of reservoirs of antimicrobial resistance in the food chain.

Antimicrobial resistance of Lactobacillus pentosus (n=59) and Leuconostoc pseudomesenteroides (n=13) isolated from Alorena green table olives (which are naturally-fermented olives from Malaga, Spain) to 15 antibiotics was evaluated. Most Lb. pentosus (95%) and all Lc. pseudomesenteroides were resistant to at least three antibiotics. Principal component analysis determined that the prevalence of antibiotic resistance in LAB throughout the fermentation process was highly dependent on the fermenter where the fermentation took place. All Lb. pentosus and Lc. pseudomesenteroides strains were highly sensitive to amoxicillin and ampicillin (MIC≤2 μg/ml), and also to chloramphenicol (MIC≤4 μg/ml), gentamicin and erythromycin (MIC≤16 μg/ml). However, they were phenotypically resistant to streptomycin (83-100%, MIC>256 μg/ml), vancomycin and teicoplanin (70-100%, MIC>128 μg/ml), trimethoprim (76% of Lb. pentosus and 15% of Lc. pseudomesenteroides, MIC>128 μg/ml), trimethoprim/sulfomethoxazol (71-100%, MIC>4-64 μg/ml) and cefuroxime (44% of Lb. pentosus and 85% of Lc. pseudomesenteroides, MIC>32-128 μg/ml). Lb. pentosus was susceptible to tetracycline and clindamycin, while 46% of Lc. pseudomesenteroides strains were resistant to these antibiotics. Only Lb. pentosus strains were resistant to ciprofloxacin (70%, MIC>4-64 μg/ml), although no mutations in the quinolone resistance determining regions of the genes encoding GyrA and ParC were found, thus indicating an intrinsic resistance. Similarly, no genes encoding possible transferable resistance determinants for the observed phenotypic resistance were detected by PCR. In some cases, a bimodal distribution of MICs was observed for some antibiotics to which both LAB species exhibited resistance. Nevertheless, such resistances resulted from an intrinsic mechanism, non-transferable or non-acquired resistance determinants which may in part be due to chromosomally encoded efflux pumps (NorA, MepA and MdeA). Results of the present study demonstrate that all Lb. pentosus and Lc. pseudomesenteroides strains lack transferable resistance-related genes (cat, bla, blaZ, ermA, ermB, ermC, msrA/B, ereA, ereB, mphA, mefA, tet(M), tet(O), tet(S), tet(W), tet(L), tet(K), aad(E), aac(6')-Ie-aph(2')-Ia, aph(2')-Ib, aph(2')-Ic, aph(2')-Id, aph(3')-IIIa, ant(4')-Ia, dfrA, dfrD, vanA, vanB, vanC and vanE) and should therefore, according to Qualified Presumption of Safety criteria, be considered safe for future application as starter cultures or as probiotics.

Enterocin AS-48 was tested singly or in combination with biocides on a cocktail of six Bacillus cereus strains in planktonic state and in biofilms formed on polystyrene microtiter plates. The biocides tested were benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, triclosan, chlorhexidine, polyhexamethylen guanidinium chloride and commercial sanitizers P3 oxonia and P3 topax 66. The numbers of survivors were determined after 60 min incubation with biocides or the biocide-bacteriocin combinations. Addition of enterocin AS-48 (25 mg/l) increased the inactivation of planktonic cells by the quaternary ammonium compounds, bisphenols and biguanines tested in a range of biocide concentrations from 0.25 to 2.5 g/l, and by 4 g/l polyguanine. Increased inactivation of the bacilli was also observed for the combination of enterocin AS-48 with 2.5% P3 oxonia, but not by P3 topax 66. In the sessile state, the bacilli were more resistant to biocides and also to the bacteriocin-biocide combinations. Hexadecylpyridium chloride was the most active biocide on biofilms in the single treatments. In the combined treatments with 50 mg/l bacteriocin, hexadecylpyridinium (2.5 g/l), polyhexamethylen guanidinium chloride (4 g/l) and P3 oxonia (2.5%) achieved complete inactivation of bacilli populations. P3 topax 66 showed the lowest performance among all treatments tested, either singly or in combination with bacteriocin. A cocktail of endospores was challenged with biocides and enterocin AS-48 for 60 min at temperatures of 22 °C, 40 °C, and 60 °C. Enterocin AS-48 did not significantly ( p > 0.05) reduce viable counts or increase the lethal effect of biocides. However, treatments with 5 g/l benzalkonium chloride at 60 °C, 2.5 g/l hexadecypyridinium at 60 °C or P3 oxonia at 0.025% and 60 °C or at 0.25% at 22–60 °C achieved complete inactivation of bacterial endospores, both singly and in combination with bacteriocin. Significant reductions of viable counts (1–2 log cycles) were also obtained for some treatments with cetrimide, triclosan or polyhexamethylen guanidinium chloride, but not for chlorhexidine (up to 5 g/l) or P3 topax 66 (up to 1%). Polystyrene surfaces dosed with enterocin AS-48 (25 or 50 mg/l) remained free of detectable bacilli from 2 to 24 h after being inoculated with a cocktail of endospores, but stainless steel surfaces dosed with 50 mg/l bacteriocin did not prevent bacterial growth from endospores. Results from this study suggest that enterocin AS-48 could be applied as enhancer of biocide activity against planktonic and sessile B. cereus cells.

Biocides have been employed for centuries, so today a wide range of compounds showing different levels of antimicrobial activity have become available. At the present time, understanding the mechanisms of action of biocides has also become an important issue with the emergence of bacterial tolerance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. While most of the mechanisms providing antibiotic resistance are agent specific, providing resistance to a single antimicrobial or class of antimicrobial, there are currently numerous examples of efflux systems that accommodate and, thus, provide tolerance to a broad range of structurally unrelated antimicrobials, both antibiotics and biocides. If biocide tolerance becomes increasingly common and it is linked to antibiotic resistance, not only resistant (even multi-resistant) bacteria could be passed along the food chain, but also there are resistance determinants that can spread and lead to the emergence of new resistant microorganisms, which can only be detected and monitored when the building blocks of resistance traits are understood on the molecular level. This review summarizes the main advances reached in understanding the mechanism of action of biocides, the mechanisms of bacterial resistance to both biocides and antibiotics, and the incidence of biocide tolerance in bacteria of concern to human health and the food industry.

Enterocin AS-48 was tested against planktonic Salmonella enterica UJ3197 in combination with polymyxin B and/or biocides. Enterocin AS-48 added at 50 mg/l or 100 mg/l in combination with 2 mg/l polymyxin B caused partial or complete growth inhibition of S. enterica UJ3197 at 24 h incubation. At 2 mg/l, polymyxin B did not enhance significantly the inhibitory effect of biocides against strain UJ3197, except for 25 mg/l cetrimide. The combination of polymyxin B (2 mg/l) and enterocin AS-48 (25 mg/l) significantly inhibited growth of strain UJ3197 in combination with 2.5 or 25 mg/l cetrimide, 25 mg/l hexadecylpyridinium chloride, 500 mg/l chlorhexidine, and also with 0.16 or 1.6 mg/l poly-(hexamethylen guanidinium) hydrochloride. Biofilms formed by four Salmonella strains (S62, S64, UJ3197, UJ3198) on polystyrene microtiter plates were treated with biocides singly or in combinations with polymyxin B (2 mg/l), enterocin AS-48 (25 or 50 mg/l) or both. Sessile salmonellae showed an increased tolerance to all biocides tested compared to planktonic cells. At the concentration tested, polymyxin B did not enhance the bactericidal activity for any of the biocides on sessile salmonellae. Enterocin AS-48 improved the activity of most biocides, but only for some of the strains and only for the highest bacteriocin concentration tested in most cases. The combinations of enterocin AS-48 and polymyxin B improved inactivation of sessile salmonellae for all biocides tested, although the degree of inactivation was highly dependent on strain and biocide. The combination of polymyxin B (2 mg/l) and enterocin AS-48 (50 mg/l) improved significantly (p

In order to investigate the prevalence of resistant bacteria to biocides and/or antibiotics throughout meat chain production from sacrifice till end of production line, samples from various surfaces of a goat and lamb slaughterhouse representative of the region were analyzed by the culture dependent approach. Resistant Psychrotrophs (n=255 strains), Pseudomonas sp. (n=166 strains), E. coli (n=23 strains), Staphylococcus sp. (n=17 strains) and LAB (n=82 represented mainly by Lactobacillus sp.) were isolated. Resistant psychrotrophs and pseudomonads (47 and 29%, respectively) to different antimicrobials were frequently detected in almost all areas of meat processing plant regardless the antimicrobial used, although there was a clear shift in the spectrum of other bacterial groups and for this aim such resistance was determined according to several parameters: antimicrobial tested, sampling zone and the bacterial group. Correlation of different parameters was done using a statistical tool "Principal component analysis" (PCA) which determined that quaternary ammonium compounds and hexadecylpyridinium were the most relevant biocides for resistance in Pseudomonas sp., while ciprofloxacin and hexachlorophene were more relevant for psychrotrophs, LAB, and in lesser extent Staphylococcus sp. and Escherichia coli. On the other hand, PCA of sampling zones determined that sacrifice room (SR) and cutting room (CR) considered as main source of antibiotic and/or biocide resistant bacteria showed an opposite behaviour concerning relevance of antimicrobials to determine resistance being hexadecylpyridinium, cetrimide and chlorhexidine the most relevant in CR, while hexachlorophene, oxonia 6P and PHMG the most relevant in SR. In conclusion, rotational use of the relevant biocides as disinfectants in CR and SR is recommended in an environment which is frequently disinfected.

Manzanilla Aloreña (or Aloreña) table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The aim of this study was to search for lactic acid bacteria (LAB) with technological properties of interest for possible inclusion in a starter or protective culture preparation or also as probiotics. A collection of 144 LAB obtained from Aloreña green table olives naturally-fermented by four small-medium enterprises (SMEs) from Málaga (Spain), including lactobacilli (81.94%), leuconostocs (10.42%) and pediococci (7.64%) were studied. REP-PCR clustering and further identification of strains by sequencing of phes and rpo genes revealed that all lactobacilli from the different SMEs were Lactobacillus pentosus. Pediococci were identified as Pediococcus parvulus (SME1) and leuconostocs as Leuconostoc pseudomesenteroides (SME1 and SME4). Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity specially for lactobacilli and also for leuconostocs. Some strains exhibit useful technological properties such as production of antimicrobial substances active against pathogenic bacteria such as Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus, Streptococcus mutans and Salmonella enterica, utilization of raffinose and stachyose, production of bile salt hydrolase, phytase and haeme-dependent catalase activities, growth at 10 °C and in the presence of 6.5% NaCl, good acidifying capacity and also resistance to freezing. However, none of the isolates showed protease or amylase activity, and also did not exhibit biogenic amine production from histidine, ornithine, cysteine or tyrosine. On the basis of data obtained, selected strains with potential traits were tested for their survival at low pH and their tolerance to bile salts, and the survival capacity demonstrated by some of the analysed strains are encouraging to further study their potential as probiotics.

The aim of this study was to select autochthonous microorganisms that are able to biosorb heavy metals in aqueous systems. Forty-eight strains, included fungi, yeasts and bacteria, were isolated from water and sludge samples from urban and industrial wastewater treatment plants in the province of Jaen, southern Spain. Purified isolates were screened on the basis of their resistance to Pb(II), Zn(II) and Ag(I). Ten strains were selected and identified by molecular techniques. Tolerance index and minimum inhibitory concentration (MIC) for the three metals were also determined. It can be noted that tolerance of the strains appears to be associated with the sites of their isolation. The best results were obtained for the yeast Trichosporon sp. 4S3 which showed the following MICs: 32–35 mM for lead, 16–18 mM for zinc and 3–5 mM for silver. The use of native strains can be an interesting alternative for bioremediation processes of heavy metals. This study increases the number of microbial strains that have the ability to resist relatively higher concentrations of metals, which may make them recommendable as potentially low-cost biosorbents. The nucleotide sequences of all the selected isolates were deposited in GenBank, which could be of great interest for other investigators as reference strains in bioremediation processes of heavy metals. 2012 Elsevier B.V. All rights reserved.

Inactivation of Staphylococcus aureus strains by high hydrostatic pressure (HHP) treatments applied singly or in combination with natural antimicrobials (nisin, enterocin AS-48, cinnamon oil and clove oil) was investigated in rice pudding. Treatments at 600 MPa for 10 min reduced initial populations of staphylococci (7.9 log CFU/g) below detectable levels of 1 log CFU/g in the puddings. Treatments at 500 MPa for 5 min (achieving a 2.9-log reduction of viable counts) were investigated singly or in combination with nisin (200 and 500 IU/g), enterocin AS-48 (25 and 50 μg/g), cinnamon oil (0.2%, vol/wt) or clove oil (0.25% vol/wt). The combined treatment of enterocin AS-48 (50 μg/g) and HHP caused a non-significant reduction of 0.4–0.6 log cycles compared to HHP alone. Additional reductions of 0.87, 1.3 and 1.8 log cycles were recorded for the combined HHP treatments with nisin (500 IU/g), cinnamon oil (0.2%) and clove oil (0.25%), respectively. During refrigeration storage for one week, viable counts in puddings from combined treatments were significantly lower compared to the single HHP treatments, e.g. 1.5–2.7 log cycles for HHP-nisin (500 IU/g), 1.1–1.3 log cycles for HHP-AS-48 (50 μg/g) or approx. 1.5 log cycles for HHP-cinnamon oil (0.2%). These results suggest that the time and intensity of HHP treatments required for inactivation of S. aureus in puddings can be reduced when HHP is applied in combination with selected natural antimicrobials.

Several studies have associated cannabis use with the development of schizophrenia. However, it has been difficult to disentangle the effects of cannabis from that of other illicit drugs, as previous studies have not evaluated pure cannabis users. To test whether the onset of cannabis use had an effect on the initiation of psychosis, we examined the time relationship between onset of use and onset of psychosis, restricting our analysis to a cohort of individuals who only used cannabis and no other street drugs.Fifty-seven subjects with non-affective psychoses who used cannabis prior to developing a psychosis were interviewed using the Diagnostic Interview for Genetic Studies (DIGS). The Family Interview for Genetic Studies (FIGS) was also used to interview a family informant about psychiatric illness in the patient and the entire family. Multiple linear regression techniques were used to estimate the association between variables.After adjusting for potential confounding factors such as sex, age, lifetime diagnosis of alcohol abuse or dependence, and family history of schizophrenia, the age at onset of cannabis was significantly associated with age at onset of psychosis (β=0.4, 95% CI=0.1-0.7, p=0.004) and age at first hospitalization (β=0.4, 95% CI=0.1-0.8, p=0.008). The mean time between beginning to use cannabis and onset of psychosis was 7.0±4.3. Age at onset of alcohol use was not associated with age at onset of psychosis or age at first hospitalization.Age at onset of cannabis is directly associated with age at onset of psychosis and age at first hospitalization. These associations remain significant after adjusting for potential confounding factors and are consistent with the hypothesis that cannabis could cause or precipitate the onset of psychosis after a prolonged period of time.

This study focused on biocide and antibiotic sensitivity in a collection of 378 isolates derived from 36 organic foods. Most isolates were sensitive to low concentrations of biocides, with MIC’s lower than 10 mg/l or 100 mg/l. Low percentages of isolates (0.3–5%) required higher biocide concentrations for inhibition, with MIC’s in the range of 1–10 g/l (as in the case of cetrimide, hexadecylpyridinium chloride, chlorhexidine or triclosan). Highest percentages of antibiotic resistance were detected for cefuroxime, followed by amoxicillin and erythromycin, while most isolates were sensitive to nitrofurantoin and ciprofloxacin. A selection of 64 isolates showing high MIC’s to biocides were subsequently identified by PCR amplification or by 16S rDNA sequencing. Most of the selected isolates belonged to Pantoea sp. and Enterobacter sp., followed by Bacillus cereus, Enterococcus faecium, Enterococcus sp., Bacillus sp., Klebsiella oxytoca and Salmonella. A total of 36 isolates (9.5%) with high MIC’s to at least one biocide were resistant to at least one of the antibiotics tested. Isolates with high MIC’s to multiple biocides were more frequently resistant to antibiotics as well. Results from this study indicate that organic foods may act as reservoirs for antibiotic-resistant bacteria and suggest that high levels of biocide tolerance could facilitate the prevalence of antibiotic-resistant strains.

Staphylococcus aureus was challenged with the cyclic bacteriocin AS-48 in four commercial sauces singly and in combination with sublethal heat treatments (50–60 °C, for 5 min) and with 2-nitro-1-propanol (2NPOH). Heating at 60 °C in combination with AS-48 (25 μg/ml) reduced initial counts (4.1–4.5 log CFU/ml) below detection limit (1.0 log CFU/ml), while a combined treatment at 55 °C inhibited proliferation of staphylococci in sauces for 24 h at 22 °C. In BHI broth, enterocin AS-48 (0–15 μg/ml) acted synergistically with 2NPOH (0–220 mM), reducing the concentrations of both antimicrobials required for inactivation of S. aureus. In sauces, enterocin AS-48 (25 μg/ml) plus 2NPOH (25 mM) reduced initial cell concentrations (4.1–5.0 log CFU/ml) below detectable levels within 8–24 h, depending on the sauce. The combinations of enterocin AS-48 and 2NPOH or sublethal heat are interesting for testing as part of hurdle technology aimed at reducing the risks for proliferation of S. aureus in sauces.

The effect of high hydrostatic pressure treatments on a cocktail of four Salmonella Enteritidis strains inoculated in commercial Spanish potato omelette (tortilla de patatas) was studied. Inactivation of Salmonella was influenced by treatment time and pressure. Treatments at 600 MPa for 5 or 8 min reduced the counts of Salmonella on selective medium (XLD agar) by 5.9 and 6.5 log cycles, respectively. Investigation of sublethally injured cells by growth on triple agar layer (TAL) plates or in non-selective medium (TSA) revealed that the population of sublethally injured cells able to grow on TSA could be 2.5 to 3.0 log cycles higher (depending on treatment) compared to non-injured cells. The TAL method only allowed recovery of a fraction of sublethally injured cells, suggesting different degrees of cell damage. In addition, tailings were observed in survival curves for treatments above 500 MPa both for selective and non-selective determinations. Omelette samples inoculated with Salmonella and treated at 500 MPa (5 min) or 680 MPa (8 min) were stored at 6 °C for 15 days. Counts of survivors on selective medium increased at day 3 of storage for the less intense HHP treatment (suggesting repair of sublethally injured cells), but not until day 10 for the 680 MPa treatment. Temperature abuse of the treated omelettes (6 h at 22 °C) applied at day 3 of storage increased the concentrations of cells able to grow on non-selective as well as on selective media, an effect that was more pronounced in the samples treated at 500 MPa for 5 min. The results suggest that temperature abuse may compromise the efficacy of HHP treatments against Salmonella in omelettes. Industrial relevance The efficacy of HHP treatments depends greatly on the food substrate and microorganisms. Therefore, the degree of protection afforded by HHP treatments for each foodborne pathogen and food system need to be determined experimentally. So far, there are no previous studies addressing the efficacy of HHP treatments on inactivation of Salmonella in Spanish omelettes. This type of RTE food is widely consumed not only in Spain, but also in many other countries, especially as an appetizer. Industrial application of HHP treatments on omelettes could improve their safety while at the same time enhancing the perception of this food product as a safer food.

The effect of enterocin AS-48 in controlling Lactobacillus sakei, Brochothrix thermosphacta , and Staphylococcus carnosus in a cooked ham system has been studied. AS-48 alone showed activity against Lactobacillus , with 60 μg g −1 reducing lactobacilli below detection levels from the beginning to end of storage at 5 °C. Combinations of 40 μg g −1 AS-48 with nitrate/nitrite, pentasodium tripolyphosphate, sodium pyrophosphate, sodium acetate, and sodium lactate reduced L. sakei below detection levels from the beginning to end of storage. Even 20 μg g −1 of enterocin combined with tripolyphosphate permanently eliminated L. sakei. Enterocin AS-48 (40 μg g −1 ) was also active against B. thermosphacta and S. carnosus , reducing both bacteria by more than 3 log in the cooked ham. Modified atmosphere (40% CO 2 /60% N 2 ) packaging affected neither bacterial growth nor AS-48 activity against any of the three bacteria. In contrast, storage under vacuum remarkably increased the growth and the inhibitory activity of the enterocin against B. thermosphacta and especially against L. sakei .

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.

Enterocin AS-48 was tested against rope-forming Bacillus subtilis CECT 4002 and Bacillus licheniformis CECT 20, as well as on Bacillus cereus and Bacillus pumilus strains in broth and in experimental dough from wheat flour. Vegetative B. subtilis cells in liquid broth were rapidly inactivated by AS-48 (7 AU/ml). In wheat dough, higher bacteriocin concentrations of 14 and 23 AU/g were required for inactivation of B. subtilis vegetative cells and endospores activated to germinate, respectively. B. cereus LWL1 and B. licheniformis CECT 20 were inactivated by AS-48 (14 AU/g) in doughs stored at 22 °C much faster compared to doughs stored at 10 °C. Strains of Bacillus pumilus were partially inactivated in dough by bacteriocin addition (14 AU/g). Results from this study indicate that enterocin AS-48 can reduce the populations of spoilage and potentially-toxigenic bacilli in wheat dough, decreasing the risks for spoilage and food intoxication.

Aloreña table olives are naturally fermented traditional green olives with a denomination of protection (DOP). The present study focused on Aloreña table olives manufactured by small and medium enterprises (SMEs) from Valle del Guadalhorce (Southern Spain) under three different conditions (cold storage, and ambient temperature fermentations in small vats and in large fermentation tanks). The microbial load of brines during fermentation was studied by plate counting, and the microbial diversity was determined by a culture-independent approach based on PCR-DGGE analysis. The viable microbial populations (total mesophilic counts, yeasts and molds, and lactic acid bacteria - LAB) changed in cell numbers during the course of fermentation. Great differences were also observed between cold, vat and tank fermentations and also from one SME to another. Yeasts seemed to be the predominant populations in cold-fermented olives, while LAB counts increased towards the end of vat and tank fermentations at ambient temperature. According to PCR-DGGE analysis, microbial populations in cold-fermented olives were composed mostly by Gordonia sp./Pseudomonas sp. and Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. together with halophilic archaea (mainly by haloarchaeon/Halosarcina pallida and uncultured archaeon/uncultured haloarchaeon/Halorubrum orientalis) and yeasts (Saccharomyces cerevisiae and Candida cf. apicola). Vat-fermented olives stored at ambient temperature included a more diverse bacterial population: Gordonia sp./Pseudomonas sp., Sphingomonas sp./Sphingobium sp./Sphingopyxis sp. and Thalassomonas agarivorans together with halophilic archaea and yeasts (mainly S. cerevisiae and C. cf. apicola, but also Pichia sp., and Pichia manshurica/Pichia galeiformis). Some LAB were detected towards the end of vat fermentations, including Lactobacillus pentosus/Lactobacillus plantarum and Lactobacillus vaccinostercus/Lactobacillus suebicus. Only the tank fermentation showed a clear predominance of LAB populations (Lactobacillus sp., Lactobacillus paracollinoides, and Pediococcus sp.) together with some halophilic archaea and a more selected yeast population (P. manshurica/P. galeiformis). The present study illustrates the complexity of the microbial populations in naturally-fermented Aloreña table olives.

In an ecological study, a collection of Gram-positive bacteria were isolated from Gueddid, an artisanal Tunisian fermented meat. From these, 24 strains showed antimicrobial activity and identified as Enterococcus faecium using molecular methods. The enterococci strains were further investigated regarding their safety aspects and functional properties. All the isolates produce bacteriocins with inhibitory activity against several food spoilage bacteria and food borne pathogens, including Listeria spp. Enterococcus spp. and Staphylococcus aureus. One isolate was active against Escherichia coli CECT 877. The majority of the isolates tested positive upon PCR amplification of structural genes for enterocins A, B and P. Investigation of virulence factors by PCR amplification revealed the presence of genes encoding for gelatinase (gelE), enterococcal antigen (efaAfm), sex pheromone (cpd and ccf) and expression of cytolysin (the haemolytic component cylB) whereas, other presumed virulence genes encoding for (agg, esp, cylM, cylA, and cob) were not detected. The isolates were mostly resistant to erythromycin, rifampicin, ciprofloxacin, lavofloxacin, and nitrofurantoin. All of them showed sensitivity to several antibiotics (ampicillin, penicillin, vancomycin, chloramphenicol, teicoplanin, gentamicin, streptomycin, and quinupristin/dalfopristin). Tyrosine, lysine, ornithine and histidine were not decarboxylated by any enterococcal isolate. Nine of the antagonistic enterococci tested did not show any virulence traits or produced biogenic amines, and still had important technological properties. The safety aspects of these selected strains should be studied in deeper details in order to evaluate their potential for biotechnological applications.

Enterocin AS-48 was tested alone or in combination with chemical preservatives and/or heat against Listeria monocytogenes and Staphylococcus aureus in a cooked ham model system. AS-48 (20, 40 and 60 μg g −1 ) alone was active against L. monocytogenes at 5 and 15 °C, but it was not sufficient to avoid regrowth of Listeria during the 60 days storage. Combination of AS-48 (40 μg g −1 ) with nitrite/nitrate, pentasodium tripolyphosphate, sodium benzoate or potassium sorbate improved the anti-listeria effect during storage at 5 °C. The most effective combination was AS-48-nitrite/nitrate (0.007%) that reduced listeria below detection level from the beginning to end of storage. Although much more resistant, S. aureus was also inhibited by AS-48 alone at 5 °C, and especially in combinations with nitrite/nitrate, pentasodium tripolyphosphate, sodium lactate and sodium acetate. Best results against both pathogens were obtained when sodium pyrophosphate was applied in combination with 60 μg g −1 AS-48. Sub-lethal heat (60 °C, 2 min) clearly increased AS-48 activity against both Listeria and Staphylococcus .

Application of natural antimicrobial substances (such as bacteriocins) combined with novel technologies provides new opportunities for the control of pathogenic bacteria, improving food safety and quality. Bacteriocin-activated films and/or in combination with food processing technologies (high-hydrostatic pressure, high-pressure homogenization, in-package pasteurization, food irradiation, pulsed electric fields, or pulsed light) may increase microbial inactivation and avoid food cross-contamination. Bacteriocin variants developed by genetic engineering and novel bacteriocins with broader inhibitory spectra offer new biotechnological opportunities. In-farm application of bacteriocins, bacterial protective cultures, or bacteriophages, can decrease the incidence of food-borne pathogens in livestock, animal products and fresh produce items, reducing the risks for transmission through the food chain. Biocontrol of fungi, parasitic protozoa and viruses is still a pending issue.

The cyclic peptide bacteriocin enterocin AS-48 was tested (at final concentrations of 0.175, 0.613, and 1.05 AU/ml) against the exopolysaccharide-producing cider spoilage strain Pediococcus parvulus 48 in apple juice in combination with high-intensity pulsed electric field (HIPEF) treatment (35 kV/cm and 150 Hz for 4 mus and bipolar mode). The effect of the combined treatments was studied by surface response methodology, with AS-48 concentration and HIPEF treatment time as process variables. A bacteriocin concentration of 0.613 AU/ml in combination with HIPEF treatment time of 1,000 micros reduced the population of pediococci by 6.6 log cycles in apple juice and yielded an apple juice that was free from pediococci during a 30-day storage period at 4 and 22 degrees Celsius. In contrast, application of HIPEF treatment alone had no effect on the surviving pediococci during storage of juice at 22 degrees Celsius. The combined treatment significantly improved the stability of the juice against spoilage by exopolysaccharide-producing P. parvulus.

Seed sprouts may act as vehicles for foodborne pathogenic bacteria. In the present study, the effect of washing treatment with the enterococcal bacteriocin enterocin AS-48 on the microbiota of two batches of soybean sprouts was studied by culture-dependent and independent methods throughout storage at 10 degrees C. Viable cell counts of bacteriocin-treated samples revealed some modifications only for lactic acid bacteria and enterococci during storage. In the control samples from batch 1, the culture-independent DGGE analysis revealed species from genera Rahnella and Serratia as the predominant bacteria at early stages. Several bands corresponding to other genera (two Pantoea bands, one Escherichia band, and five Enterobacter bands) were also detected during storage of control samples, especially at days 3 and 5, while one Rahnella band disappeared. By contrast, some of the enterobacteria (Pantoea Escherichia and Enterobacter) were not detected or showed very faint bands in batch 1 bacteriocin-treated samples, in which two new and intense bands corresponding to genera Enterococcus and Leuconostoc were detected. Batch 2 showed a more homogeneous bacterial population, composed mainly by species of genus Enterobacter together with Pantoea. The major modifications detected in the bacteriocin-treated samples from batch 2 included the loss of one genus Enterobacter band at days 3, 5 and 7, and the detection of a new band corresponding to genus Leuconostoc at days 5 and 7. These results suggest that bacteriocin treatment disturbs the microbial balance in sprouts, producing changes in the microbial profile that cannot be detected by culture-dependent methods. The results also encourage the use of culture-independent methods to gain more insights into the global effects of bacteriocins in food systems.

Enterocin AS-48 (30-60 microg/g) significantly reduced viable counts of Listeria monocytogenes in Russian-type salad during one week storage at 10 degrees C. Antilisterial activity of AS-48 (30 microg/g) in salad was strongly enhanced by essential oils (thyme verbena, thyme red, Spanish oregano, ajowan, tea tree, clove, and sage oils tested at 1%, as well as with 2% rosemary oil). Antilisterial activity also increased in combination with bioactive components from essential oils and plant extracts, with other related antimicrobials of natural origin or derived from chemical synthesis (carvacrol, eugenol, thymol, terpineol, tyrosol, hydroxytyrosol, caffeic, ferulic and vanillic acid, luteolin, geranyl butyrate, geranyl phenylacetate, pyrocatechol, hydrocinnamic acid, tert butylhydroquinone, phenylphosphate, isopropyl methyl phenol, coumaric acid, and 2-nitropropanol), and with food preservatives (citric and lactic acid, sucrose palmitate, sucrose stearate, p-hydroxybenzoic methylester acid -- PHBME, and Nisaplin). AS-48 acted synergistically with citric, lactic acid, and PHBME. A mixed population of two L. monocytogenes strains was markedly reduced for one week in salads treated with AS-48 (30 microg/g) in combination with lactic acid, PHBME or Nisaplin. The increased bactericidal activity of these combinations is interesting to improve protection against L. monocytogenes during salad storage.

Enterocin AS-48 was tested against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes in different kinds of desserts. The highest activity against S. aureus was detected in baker cream. However, in yogurt-type soy-based desserts and in gelatin pudding, AS-48 (175 arbitrary units [AU]/g) reduced viable cell counts of S. aureus by only 1.5 to 1.8 log units at most. The efficacy of AS-48 in puddings greatly depended on inoculum size, and viable S. aureus counts decreased below detection levels within 24 h for inocula lower than 4 to 5.5 log CFU/g. For L. monocytogenes, bacteriocin concentrations of 52.5 to 87.5 AU/g reduced viable counts below detection levels and avoided regrowth of survivors. The lowest activity was detected in yogurt-type desserts. For B. cereus, viable cell counts were reduced below detection levels for bacteriocin concentrations of 52.5 AU/g in instant pudding without soy or by 175 AU/g in the soy pudding. In gelatin pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10 degrees C or after 48 h at 22 degrees C. Bacteriocin addition also inhibited gelatin liquefaction caused by the proteolytic activity of B. cereus.

The cyclic bacteriocin enterocin AS-48 was tested on a cocktail of two Geobacillus stearothermophilus strains in canned food samples (corn and peas), and in coconut milk. AS-48 (7 microg/g) reduced viable cell counts below detection levels in samples from canned corn and peas stored at 45 degrees C for 30 days. In coconut milk, bacterial inactivation by AS-48 (1.75 microg/ml) was even faster. In all canned food and drink samples inoculated with intact G. stearothermophilus endospores, bacteriocin addition (1.75 microg per g or ml of food sample) rapidly reduced viable cell counts below detection levels and avoided regrowth during storage. After a short-time bacteriocin treatment of endospores, trypsin addition markedly increased G. stearothermophilus survival, supporting the effect of residual bacteriocin on the observed loss of viability for endospores. Results from this study support the potential of enterocin AS-48 as a biopreservative against G. stearothermophilus.

A collection of 25 isolates from foods of animal origin (including mainly milk and cheese, together with meat and ham) was studied. Enterococci were identified at species levels as E. faecalis (9 isolates) and E. faecium (16 isolates). Investigation of virulence factors by PCR amplification revealed incomplete sets of cytolysin genes both in E. faecalis and E. faecium isolates. Among E. faecalis, PCR amplification revealed a high incidence of genes encoding for enterococcal surface protein esp (7/9 isolates), enterococcal antigen efaAfs (6/9), aggregation substance agg (2/9) and sex-pheromone encoding genes ccf, cob, cpd (which were detected in 9, 5 and 3 out of 9 isolates, respectively). By contrast, only esp (7/16 isolates) and efaAfm (10/16) were detected among E. faecium. Antibiotic resistance detected at higher frequencies included rifampicin, nitrofurantoin, ciprofloxacin and levofloxacin. Vancomycin resistance was also detected among E. faecalis and E. faecium. E. faecalis isolates showed decarboxylating activity mostly for tyrosine (5/9 isolates), while E. faecium isolates showed a broader decarboxylating capacity, involving tyrosine (11/16 isolates) ornithine (6/16), lysine (4/16) and histidine (3/16). Six isolates produced bacteriocins, and genes encoding for enterocins A, B, P, L50, and 1071 were detected. Many isolates tested positive for several of the traits investigated, which raises concerns about their possible role as reservoirs for dissemination of antibiotic resistance and virulence traits in foods.

This study demonstrates the optimization of AS-48 production by strain Enterococcus faecalis A-48-32 using a whey-derived substrate, Esprion-300 (E-300). To optimise AS-48 production, various factors such as E-300 concentration, glucose, pH and inoculum size were adjusted. The following optimal conditions were established: a growth medium comprising 5% E-300 and 1% glucose, inoculated with 8% producer strain and incubated at 28 °C with a controlled pH of 6.55. Under these conditions a maximum activity of 360 AU mL −1 was attained after 18 h and remained relatively constant for 24 h. The critical factors for optimal AS-48 production in this medium were the pH stabilization at 6.55, in addition to a 1% glucose concentration.

Enterocin AS-48 is a broad-spectrum cyclic antimicrobial peptide produced by Enterococcus faecalis. In the present study, the bacteriocin was tested alone and in combination with other antimicrobials for decontamination of Bacillus inoculated on alfalfa, soybean sprouts and green asparagus. Washing with enterocin AS-48 solutions reduced viable cell counts of Bacillus cereus and Bacillus weihenstephanensis by 1.0-1.5 and by 1.5-2.38 log units right after application of treatment, respectively. In both cases, the bacteriocin was effective in reducing the remaining viable population below detection levels during further storage of the samples at 6 degrees C, but failed to prevent regrowth in samples stored at 15 or 22 degrees C. Application of washing treatments containing enterocin AS-48 in combination with several other antimicrobials and sanitizers (cinnamic and hydrocinnamic acids, carvacrol, polyphosphoric acid, peracetic acid, hexadecylpyridinium chloride and sodium hypochlorite) greatly enhanced the bactericidal effects. The combinations of AS-48 and sodium hypochlorite, peracetic acid or hexadecylpyridinium chloride provided the best results. After application of the combined treatments on alfalfa sprouts contaminated with B. cereus or with B. weihenstephanensis, viable bacilli were not detected or remained at very low concentrations in the treated samples during a 1-week storage period at 15 degrees C. Inhibition of B. cereus by in situ produced bacteriocin was tested by cocultivation with the AS-48 producer strain E. faecalis A-48-32 inoculated on soybean sprouts. Strain A-48-32 was able to grow and produce bacteriocin on sprouts both at 15 and 22 degrees C. At 15 degrees C, growth of B. cereus was completely inhibited in the cocultures, while a much more limited effect was observed at 22 degrees C. The results obtained for washing treatments are very encouraging for the application of enterocin AS-48 in the decontamination of sprouts. Application of washing treatments containing AS-48 alone can serve to reduce viable cell counts of bacilli in samples stored under refrigeration, while application of combined treatments should be recommended to avoid proliferation of the surviving bacilli under temperature-abuse conditions.

The bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.

A collection of enterococci isolated from meat, dairy and vegetable foods from Morocco including 23 Enterococus faecalis and 15 Enterococcus faecium isolates was studied. All isolates were sensitive to ampicillin, penicillin, and gentamicin. Many E. faecalis isolates were resistant to tetracycline (86.95%), followed by rifampicin (78.26% ciprofloxacin (60.87%), quinupristin/dalfopristin (56.52%), nitrofurantoin (43.47%), levofloxacin (39.13%), erythromycin (21.73%), streptomycin (17.39%), chloramphenicol (8.69%), vancomycin (8.69%), and teicoplanin (4.34%). E. faecium isolates showed a different antibiotic resistance profile: a high percentage were resistant to nitrofurantoin (73.33%), followed by erythromycin (66.60%), ciprofloxacin (66.66%), levofloxacin (60.00%), and rifampicin (26.66%), and only a very low percentage were resistant to tetracycline (6.66%). One isolate was resistant to vancomycin and teicoplanin. The incidence of virulence factors was much higher among E. faecalis isolates, especially for genes encoding for sex pheromones, collagen adhesin, enterococcal endocarditis antigen, and enterococcal surface protein. Isolates with multiple factors (both antibiotic resistance and virulence traits) were also more frequent among E. faecalis isolates, in which one isolate cumulated up to 15 traits. By contrast, several isolates of E. faecium had only very few unwanted traits as compared to only two isolates in E. faecalis. The high abundance of isolates carrying virulence factors and antibiotic resistance traits suggests that the sanitary quality of foods should be improved in order to decrease the incidence of enterococci.