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We analyzed the adhesion capacity to mucus of 31  Lactobacillus pentosus strains isolated from naturally fermented Aloreña green table olives using an immobilized mucin model. On the basis of their adhesive capacity to mucin, three phenotypes were selected for cell-wall protein proteomic analysis to pinpoint proteins involved in the adhesion process: the highly adhesive L. pentosus CF1-43 N (73.49% of adhesion ability), the moderately adhesive L. pentosus CF1-37 N (49.56% of adhesion ability) and the poorly adhesive L. pentosus CF2-20P (32.79% of adhesion ability). The results revealed four moonlighting proteins over-produced in the highly adhesive L. pentosus CF1-43 N, which were under/not produced in the other two L. pentosus strains (CF1-37 N and CF2-20P). These proteins were involved in glycolytic pathway (phosphoglycerate mutase and glucosamine-6-phosphate deaminase), stress response (small heat shock protein) and transcription (transcription elongation factor GreA). Furthermore, the relative fold change in gene expression analysis showed significant up-regulation of the genes coding for these four moonlighting proteins in the highly adhesive L. pentosus CF1-43 N versus the poorly adhesive L. pentosus CF2-20P and also in response to mucin for 20 h which clearly indicate the significant role of these genes in the adhesion capacity of L. pentosus . Thus, these proteins could be used as biomarkers for mucus adhesion in L. pentosus . On the other hand, mucin exposure induced other probiotic effects in L. pentosus strains, enhancing their co-aggregation ability with pathogens and possible inactivation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Despite increasing interest to investigate horizontal gene transfer as a leading cause of antibiotic resistance spread, the resistome is not only influenced by the influx and efflux of genes in different environments. Rather, the expression of existing genes under different stress conditions requires special attention. This study determined whether pre-adapting Lactiplantibacillus pentosus strains, isolated from Aloreña green table olives, to vegetable-based edible oils influence their phenotypic and genotypic responses to antibiotics. This has significant diet, food matrix, gut health, and food safety concerns. Pre-adapting L. pentosus strains to oils significantly changed their susceptibility profile to antibiotics. However, results generally differed among the three strains; although changes in the Minimum Inhibitory Concentration (MIC) of antibiotics occurred, it depended on the L. pentosus strain and the oil used for adaptation. The pre-adaptation of L. pentosus strains with olive, sunflower, argan and linseed oils induced gene expressions (e.g., rpsL, recA and uvrB) in several stress responses. Thus, to analyze this fact in-depth, transcriptional changes were reported in the selected potential probiotic L. pentosus CF2-10 adapted with olive or sunflower, rerouting its metabolic pathways to export toxic molecules through efflux pumps and ABC transporters. Pre-adaptation of some lactobacilli with olive or sunflower oils may represent a novel approach for manufacturing probiotic products with improved stability, functionality and robustness. • Oil adaptation induced different phenotypic and genotypic response to antibiotics. • Susceptibility to antibiotics was dependent on L. pentosus strain and oil used. • Oil adaptation induced differential gene expressions (e.g., rpsL, recA and uvrB). • Transcriptome data revealed enhanced export of toxic molecules after oil adaptation. • Olive or sunflower adaptation of L. pentosus as strategy to enhance robustness. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) isolated throughout the fermentation process of Aloreña table olives were found to be resistant at least to three antibiotics (Casado Muñoz et al., 2014); however, most were sensitive to the biocides tested in this study (with minimum inhibitory concentrations [MIC] below the epidemiological cut-off values). 2–15% of the isolates were found to be biocide resistant: Leuconostoc Pseudomesenteroides , which were resistant to hexachlorophene, and Lactobacillus pentosus to cetrimide and hexadecylpiridinium. We analyzed the effect of different physico-chemical stresses, including antimicrobials, on the phenotypic and genotypic responses of LAB, providing new insights on how they become resistant in a changing environment. Results indicated that similar phenotypic responses were obtained under three stress conditions: antimicrobials, chemicals and UV light. Susceptibility patterns to antibiotics changed: increasing MICs for ampicillin, chloramphenicol, ciprofloxacin, teicoplanin and tetracycline, and decreasing the MICs for clindamycin, erythromycin, streptomycin and trimethoprim in most strains. Statistically, cross resistance between different antibiotics was detected in all stress conditions. However, expression profiles of selected genes involved in stress/resistance response ( rpsL , recA , uvrB and srtA ) differed depending on the stress parameter, LAB species and strain, and the target gene. We conclude that, despite the uniform phenotypic response to stresses, the repertoire of induced and repressed genes differs. So, a search for a target to improve stress tolerance of LAB, especially those of importance as starter/protective cultures or probiotics, may depend on the individual screening of each strain, even though we could predict the antibiotic phenotypic response to all stresses. [ABSTRACT FROM AUTHOR]

Probiotic bacterial cultures require resistance mechanisms to avoid stress-related responses under challenging environmental conditions; however, understanding these traits is required to discern their utility in fermentative food preparations, versus clinical and agricultural risk. Here, we compared the proteomic responses of Lactobacillus pentosu s MP-10, a potentially probiotic lactic acid bacteria isolated from brines of naturally fermented Aloreña green table olives, exposed to sub-lethal concentrations of antibiotics (amoxicillin, chloramphenicol and tetracycline) and biocides (benzalkonium chloride and triclosan). Several genes became differentially expressed depending on antimicrobial exposure, such as the up-regulation of protein synthesis, and the down-regulation of carbohydrate metabolism and energy production. The antimicrobials appeared to have altered Lb. pentosus MP-10 physiology to achieve a gain of cellular energy for survival. For example, biocide-adapted Lb. pentosu s MP-10 exhibited a down-regulated phosphocarrier protein HPr and an unexpressed oxidoreductase. However, protein synthesis was over-expressed in antibiotic- and biocide-adapted cells (ribosomal proteins and glutamyl-tRNA synthetase), possibly to compensate for damaged proteins targeted by antimicrobials. Furthermore, stress proteins, such as NADH peroxidase (Npx) and a small heat shock protein, were only over-expressed in antibiotic-adapted Lb. pentosu s MP-10. Results showed that adaptation to sub-lethal concentrations of antimicrobials could be a good way to achieve desirable robustness of the probiotic Lb. pentosu s MP-10 to various environmental and gastrointestinal conditions (e.g., acid and bile stresses). [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate biocide susceptibility in mesophilic and psychrotrophic pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which was representative of the region. To determine biocide resistance in pseudomonads, we determined for the first time the epidemiological cut-off values (ECOFFs) of benzalkonium, cetrimide, chlorhexidine, hexachlorophene, P3 oxonia, polyhexamethylene guanidine hydrochloride (PHMG), topax 66 and triclosan being generally very similar in different Pseudomonas spp. with some exceptions. Thus, resistance of pseudomonads was mainly shown to triclosan, and in lesser extent to cetrimide and benzalkonium chloride depending on the species, however they were highly susceptible to industrial formulations of biocides. By means of statistical analysis, positive correlations between antibiotics, biocides and both antimicrobials in pseudomonads were detected suggesting a co- or cross resistance between different antimicrobials in goat and lamb slaughterhouse environment. Cross-resistance between biocides and antibiotics in pseudomonads were especially detected between PHMG or triclosan and different antibiotics depending on the biocide and the population type. Thus, the use of those biocides as disinfectant in slaughterhouse zones must be carefully evaluated because of the selection pressure effect of antimicrobials on the emergence of resistant bacteria which could be spread to the consumer. It is noteworthy that specific industrial formulations such as topax 66 and oxonia P3 showed few correlations with antibiotics (none or 1–2 antibiotics) which should be taken into consideration for disinfection practices in goat and lamb slaughterhouse. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The distribution and quantification of tetracycline, sulfonamide and beta-lactam resistance genes were assessed in slaughterhouse zones throughout meat chain production and the meat products; this study represents the first to report quantitatively monitor antibiotic resistance genes (ARG) in goat and lamb slaughterhouse using a culture independent approach, since most studies focused on individual bacterial species and their specific resistance types. Quantitative PCR (qPCR) revealed a high prevalence of tetracycline resistance genes tetA and tetB in almost all slaughterhouse zones. Sulfonamide resistance genes were largely distributed, while beta-lactam resistance genes were less predominant. Statistical analysis revealed that resistant bacteria, in most cases, were spread by the same route in almost all slaughterhouse zones, except for tetB, blaCTX and blaTEM genes, which occurred in few zones as isolated ‘hot spots.’ The sum of all analyzed ARG indicated that slaughterhouse surfaces and end products act as reservoirs of ARG, mainly tet genes, which were more prevalent in slaughtering room (SR), cutting room (CR) and commercial meat products (MP). Resistance gene patterns suggest they were disseminated throughout slaughterhouse zones being also detected in commercial meat products, with significant correlations between different sampling zones/end products and total resistance in SR, CR and white room (WR) zones, and also refrigerator 4 (F4) and MP were observed. Strategically controlling key zones in slaughterhouse (SR, CR and WR) by adequate disinfection methods could strategically reduce the risks of ARG transmission and minimize the issues of food safety and environment contamination. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the phenotypic and genotypic antibiotic resistance profiles of pseudomonads isolated from surfaces of a goat and lamb slaughterhouse, which were representative of areas that are possible sources of meat contamination. Mesophilic (85 isolates) and psychrotrophic (37 isolates) pseudomonads identified at the species level generally were resistant to sulfamethoxazole, erythromycin, amoxicillin, ampicillin, chloramphenicol, trimethoprim, rifampin, and ceftazidime (especially mesophiles), as well as colistin and tetracycline (especially psychrotrophes). However, they generally were sensitive to ciprofloxacin, gentamicin, imipenem, and kanamycin regardless of species identity. Worryingly, in the present study, we found multidrug resistance (MDR) to up to 13 antibiotics, which was related to intrinsic and acquired resistance mechanisms. Furthermore, a link between various antimicrobial resistance genes was shown for beta-lactams and tetracycline, trimethoprim, and sulfonamides. The distribution and resistomebased analysis of MDR pseudomonads in different slaughterhouse zones indicated that the main sources of the identical or related pseudomonad strains were the animals (feet and wool) and the slaughterhouse environment, being disseminated from the beginning, or entrance environment, to the environment of the finished meat products. Those facts must be taken into consideration to avoid cross-contamination with the subsequent flow of mobile resistance determinants throughout all slaughterhouse zones and then to humans and the environment by the application of adequate practices of hygiene and disinfection measures, including those for animal wool and feet and also the entrance environment. [ABSTRACT FROM AUTHOR]

High hydrostatic pressure (HHP) treatments (singly or in combination with natural antimicrobials) were tested for stabilization of Manzanilla Aloreña 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 < 0.05) reduced the concentrations of aerobic mesophilic bacteria. Low-salt brined olives purged with N2 or supplemented with ascorbic acid and then pressurized for 5 min at 450 or 550 MPa were preserved for up to 5 months without spoilage, suggesting that the NaCl content in brines of packed Manzanilla Aloreña table olives could be reduced considerably by application of HHP as a stabilization treatment. [ABSTRACT FROM AUTHOR]

Abstract: Antimicrobial resistance of Lactobacillus pentosus (n=59) and Leuconostoc pseudomesenteroides (n=13) isolated from Aloreña green table olives (which are naturally-fermented olives from Málaga, 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≤2μg/ml), and also to chloramphenicol (MIC≤4μg/ml), gentamicin and erythromycin (MIC≤16μg/ml). However, they were phenotypically resistant to streptomycin (83–100%, MIC>256μg/ml), vancomycin and teicoplanin (70–100%, MIC>128μg/ml), trimethoprim (76% of Lb. pentosus and 15% of Lc. pseudomesenteroides, MIC>128μg/ml), trimethoprim/sulfomethoxazol (71–100%, MIC>4–64μg/ml) and cefuroxime (44% of Lb. pentosus and 85% of Lc. pseudomesenteroides, MIC>32–128μg/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>4–64μg/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. [Copyright &y& Elsevier]

Antimicrobial resistance in enterococci is a matter of concern. A collection of 272 strains (including 107 Enterococcus faecalis and 165 Enterococcus faecium strains) isolated from meat and dairy products, seafood, vegetable foods, wildflowers, animal feces (ewe, goat, horse, mule), and hospitals were tested for sensitivity to biocides of different classes (quaternary ammonium compounds, a bisphenol, and a biguanide) and copper sulfate. Most isolates were inhibited at 25 mg of benzalkonium chloride or cetrimide per liter or at 2.5 mg of hexadecylpyridinium chloride per liter. Few isolates had MICs higher than 25 mg/ liter for benzalkonium chloride (2.2%), cetrimide (0.74%), or hexadecylpyridinium chloride (0.37%), although they were all inhibited at 250 mg/liter. The population response to triclosan was very homogeneous, and most isolates (98.16%) were inhibited at 250 mg of triclosan per liter. Chlorhexidine showed the greatest variability, with MICs in a range from 2.5 to 2,500 mg/liter. Remarkably, 74.57% of isolates from clinical samples required 2,500 mg of chlorhexidine per liter for inhibition, compared to much-lower concentrations required for most isolates from other sources. Enterococci were inhibited by copper sulfate in a concentration range from 4 to 16 mM, with no bimodal distribution. However, most isolates required 12 mM (41.91%) or 16 mM (47.43%) for inhibition. The highest percentages of isolates requiring 16 mM CuSO4 were from vegetable foods, seafood, and wildflowers. The results from the present study suggest intermediate levels of copper tolerance and a low incidence of biocide tolerance in the enterococci investigated, except for chlorhexidine in clinical isolates. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: A collection of isolates from uncooked seafoods (molluscs, fish, and fish fillets) were identified as Enterococcus faecium species and studied in further detail. Isolates were clustered in well-defined genomic groups according to food origin after ERIC-PCR analysis. Four isolates (FR 1-2, FB 1-3-B, FB 3-1, FTA 1-2) decarboxylated lysine, ornithine, and tyrosine. Isolate FR 1-2 also decarboxylated histidine. Most isolates were sensitive to antibiotics of clinical use, but resistance was detected more frequently towards nitrofurantoin (50%), erythromycin (33.33%) or rifampicin (33.33%) to quinupristin/dalfopristin (12.5%). Resistance to β-lactams or vancomycin was not detected. The enterococcal antigen A was the presumed virulence trait detected most frequently. None of isolates carried haemolysin/cytolysin genes. Twelve isolates produced anti-listerial activity. Among them, seven isolates also produced bacteriocin-like inhibitory substances against other enterococci, and one isolate was also able to inhibit Staphylococcus aureus. Three isolates only were active against Listeria monocytogenes, and two only were active against enterococci. One bacteriocinogenic isolate carried the enterocin A structural gene, but genes corresponding to other enterocins (EntB, EntP, EntQ, Ent1071, EntL50A/EntL50B, and Ent31) were not detected. Bacteriocin-producing enterococci lacking undesirable traits (such as antibiotic resistance or biogenic amine production) or their produced bacteriocins could be potential candidates to aid in preservation of seafoods and other food products as well. [ABSTRACT FROM AUTHOR]

In this study, the survival and growth of seven probiotic Lactobacillus pentosus strains isolated from Aloreña green table olives in the presence of vegetable-based edible oils (i.e., sunflower, olive, linseed, soy, corn, almond and argan) and mint essential oil were determined for the first time. Slight decreases in bacterial viability were observed depending on the strain and oil exposure, mainly mint essential oil. However, pre-adaptating the strains to the corresponding oils significantly increased their cell viabilities. As such, this study examined whether pre-adaptating probiotic L. pentosus strains with oils will constitute a new strategy to increase stress resistance, e.g., acids (pH 1.5) or bile (up to 3.6%) in food production and/or during digestion, and improve functional probiotic properties. Improvements in stress resistance (acidic and bile conditions) were noticed in some pre-adapted strains with oils; further, pre-adaptations with olive, argan, sunflower and linseed oils induced gene expression (e.g., fus , rpsL, pgm, groEL, enol and prep) for moonlighting proteins involved in several stress responses and other functions. As such, pre-adaptation with vegetable edible oils may represent a novel approach for manufacturing probiotic products by improving the stability of bacteria during industrial processes that would otherwise reduce their viability and functionality. • Vegetable edible oils caused a slight decrease in Lactobacillus pentosus viability. • Adaptation with vegetable oils increased cell viability and stress resistance. • Adaptation with vegetable oils induced gene expression for stress proteins. • Adaptation with vegetable oils as a novel approach for probiotic manufacturing. [ABSTRACT FROM AUTHOR]

Lactobacillus pentosus MP-10 was isolated from brines of naturally fermented Alorefia 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. [ABSTRACT FROM AUTHOR]

Nanotechnology presents an innovative strategy to combat the spread of antibiotic resistant bacteria and their resistance genes throughout different ecosystems. To address this challenge, nanoparticles (silver, gold, zinc and copper) alone or in combination with metallo-β-lactamase inhibitor/antimicrobial-based formulation (EDTA/HLE) showed antimicrobial activity against antibiotic resistant Enterococcus sp. and Pseudomonas sp. strains. Furthermore, the observed synergistic effect was detected notably for silver, zinc or copper nanoparticles with EDTA (ethylenediaminetetraacetic acid) and silver nanoparticles with HLE against planktonic Enterococcus sp. strains, or gold nanoparticles+EDTA or HLE against Pseudomonas sp. Regarding activity against bacterial biofilms, zinc nanoparticles combined with either of the reagents caused strong inhibition of developing biofilms of antibiotic resistant Enterococcus sp. Pseudomonas sp. strains, while preformed biofilms were mainly inhibited by silver nanoparticles+reagent. Microscopic analyses confirmed that the antimicrobial activity of nanoparticles was caused by adsorption to the bacterial cell surface, and further enhanced by chelating agents. Hence, we can conclude that nanoparticles+EDTA or HLE could represent a good alternative to limit the spread of antibiotic resistant bacteria in the food chain and the environment. • Antimicrobial effect of nanoparticles against multiresistant food chain bacteria. • Synergistic effect of nanoparticles and EDTA/HLE against multiresistant bacteria. • Nanoparticles combined with EDTA/HLE inhibited developing and preformed biofilms. • Silver and zinc oxide nanoparticles combined with EDTA/HLE were the most effective. • A novel strategy to limit the spread of multiresistant bacteria in the environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Yeasts are eukaryotic microorganisms which have a long history in the biotechnology of food production, as they have been used since centuries in bread-making or in the production of alcoholic beverages such as wines or beers. Relative to this importance, a lot of research has been devoted to the study of yeasts involved in making these important products. The role of yeasts in other fermentations in association with other microorganisms - mainly lactic acid bacteria - has been relatively less studied, and often it is not clear if yeasts occurring in such fermentations are contaminants with no role in the fermentation, spoilage microorganisms or whether they actually serve a technological or functional purpose. Some knowledge is available for yeasts used as starter cultures in fermented raw sausages or in the production of acid curd cheeses. This review aimed to summarize the current knowledge on the taxonomy, the presence and potential functional or technological roles of yeasts in traditional fermented plant, dairy, fish and meat fermentations. [ABSTRACT FROM AUTHOR]

We evaluated the efficacy of a new disinfectant product, HLE, to inhibit multiple species of planktonic and biofilm bacterial cultures. The HLE disinfectant comprised of EDTA, lactic acid and hydrogen peroxide, and our data indicated that the disinfectant had effective antimicrobial and anti-biofilm activity even at low concentrations (0.15% to 0.4% HLE, v/v). Furthermore, the HLE disinfectant destabilized biofilm structures eradicated them due to the synergistic effect of EDTA and both antimicrobials (lactic acid and hydrogen peroxide), as revealed by confocal laser scanning microscopy. Additionally, sub-inhibitory concentrations of HLE disinfectant, with EDTA as an efflux pump inhibitor, inhibited the expression of multidrug EfrAB, NorE and MexCD efflux pumps in both planktonic and biofilm cultures. This could provide an alternative way to disinfect surfaces to avoid spreading multi-drug resistant strains in the food chain and the environment by decreasing efflux pump expression and consequently reducing the antibiotic selective pressure caused by systemic antibiotics and disinfectant use. [ABSTRACT FROM AUTHOR]

Lactobacillus pentosus MP-10, isolated from brines of naturally fermented Aloreña green table olives, exhibited high probiotic potential. The genome sequence of L. pentosus MP-10 is currently considered the largest genome among lactobacilli, highlighting the microorganism’s ecological flexibility and adaptability. Here, we analyzed the complete genome sequence for the presence of acquired antibiotic resistance and virulence determinants to understand their defense mechanisms and explore its putative safety in food. The annotated genome sequence revealed evidence of diverse mobile genetic elements, such as prophages, transposases and transposons involved in their adaptation to brine-associated niches. In-silico analysis of L. pentosus MP-10 genome sequence identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) as an immune system against foreign genetic elements, which consisted of six arrays (4–12 repeats) and eleven predicted cas genes [CRISPR1 and CRISPR2 consisted of 3 (Type II-C) and 8 (Type I) genes] with high similarity to L. pentosus KCA1. Bioinformatic analyses revealed L. pentosus MP-10 to be absent of acquired antibiotic resistance genes, and most resistance genes were related to efflux mechanisms; no virulence determinants were found in the genome. This suggests that L. pentosus MP-10 could be considered safe and with high-adaptation potential, which could facilitate its application as a starter culture and probiotic in food preparations. [ABSTRACT FROM AUTHOR]

Bacteria belonging to the genus Lactobacillus are used as starter cultures or that develop naturally as fermenting microbiota in the production of various foods. On the detrimental side, lactobacilli may act as reservoir of antibiotic resistance genes, which can spread to commensal bacteria in humans or animals, or to food-associated pathogens. In the last decade, advances in molecular biology and in genome sequencing have provided more information on antibiotic resistances in foodborne bacteria. The aim of this review was to consider and provide an up-to-date status on phenotypic and genotypic antibiotic resistance profiles in Lactobacillus species from fermented foods and also to highlight new information on the distribution of glycopeptide and chloramphenicol resistance genes in Lactobacillus genomes. In silico screening of vanZ (glycopeptide resistance) and cat (chloramphenicol resistance)-like sequences in Lactobacillus species isolated from fermented foods revealed for the first time the occurrence of vanZ and cat genes in Lactobacillus species being highly conserved genes in the chromosome of each species, presumably non-transferable. Further studies involving genome sequences of Lactobacillus isolated from fermented foods, especially those relying on spontaneous fermentation, is crucial to increase knowledge on the potential presence and spread of antibiotic resistance genes via the food route. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• High microbial diversity within the porcine slaughterhouse and pork products. • Bacteria and stress genes were more prevalent in the first slaughterhouse zones. • SOS are the most prevalent genes, possibly associated with genomic changes. • Porcine-type-C oncovirus was mainly found at the end and pork products. • HLE disinfection strategy may avoid gene spread and microbial contaminants. The use of shotgun metagenomic sequencing to understand ecological-level spread of microbes and their genes has provided new insights for the prevention, surveillance and control of microbial contaminants in the slaughterhouse environment. Here, microbial samples were collected from products and surrounding areas though a porcine slaughter process; shotgun metagenomic DNA-sequencing of these samples revealed a high community diversity within the porcine slaughterhouse and pork products, in zones originating from animal arrival through to the sale zones. Bacteria were more prevalent in the first zones, such as arrival- and anesthesia-zones, and DNA viruses were prevalent in the scorching-and-whip zone, animal products and sale zone. Data revealed the dominance of Firmicutes and Proteobacteria phyla followed by Actinobacteria , with a clear shift in the relative abundance of lactic acid bacteria (mainly Lactobacillus sp.) from early slaughtering steps to Proteobacteria and then to viruses suggesting site-specific community compositions occur in the slaughterhouse. Porcine-type-C oncovirus was the main virus found in slaughterhouse, which causes malignant diseases in animals and humans. As such, to guarantee food safety in a slaughterhouse, a better decipher of ecology and adaptation strategies of microbes becomes crucial. Analysis of functional genes further revealed high abundance of diverse genes associated with stress, especially in early zones (animal and environmental surfaces of arrival zone with 57,710 and 40,806 genes, respectively); SOS responsive genes represented the most prevalent, possibly associated with genomic changes responsible of biofilm formation, stringent response, heat shock, antimicrobial production and antibiotic response. The presence of several antibiotic resistance genes suggests horizontal gene transfer, thus increasing the likelihood for resistance selection in human pathogens. These findings are of great concern, with the suggestion to focus control measures and establish good disinfection strategies to avoid gene spread and microbial contaminants (bacteria and viruses) from the animal surface into the food chain and environment, which was achieved by applying HLE disinfectant after washing with detergent. [ABSTRACT FROM AUTHOR]

In silico analysis of Lactobacillus pentosus MP-10 plasmids (pLPE-1 to pLPE-5) suggests that plasmid-borne genes mediate the persistence of lactobacilli during olive fermentation and enhance their probiotic properties and their competitiveness in several ecological niches. The role of plasmids in the probiotic activities of L. pentosus MP-10 was investigated by plasmid-curing process which showed that plasmids contribute in increased metal tolerance and the biosequestration of several metals such as iron, aluminium, cobalt, copper, zinc, cadmium and mercury. Statistically significant differences in mucin adhesion were detected between the uncured and the cured L. pentosus MP-10, which possibly relied on a serine-rich adhesin (sraP) gene detected on the pLPE-2 plasmid. However, plasmid curing did not affect their tolerance to gastro-intestinal conditions, neither their growth ability under pre-determined conditions, nor auto-aggregation and pathogen co-aggregation were changed among the cured and uncured L. pentosus MP-10. These findings suggest that L. pentosus MP-10 plasmids play an important role in gastro-intestinal protection due to their attachment to mucin and, thus, preventing several diseases. Furthermore, L. pentosus MP-10 could be used as a bioquencher of metals in the gut, reducing the amount of these potentially toxic elements in humans and animals, food matrices, and environmental bioremediation. [ABSTRACT FROM AUTHOR]