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The objective of this work was to evaluate the microbiological quality of cheese produced by formal and informal micro-enterprises in Paipa, Colombia, to isolate potentially pathogenic bacteria and to determine their prevalence and resistance to antimicrobials such as antibiotics and biocides. Sixteen micro-enterprises of the seventy existing in the region were sampled during 3 years. Viable concentrations of aerobic mesophiles, total and fecal coliforms, Salmonella sp., Listeria monocytogenes, Staphylococcus sp., yeasts, and molds were determined. Seventy-three bacterial isolates were identified by 16S rRNA gene sequencing. The susceptibility of the isolates to antibiotics and biocides was determined. The results indicated that between 98 and 100% of the cheese samples (n = 48 samples) of formal and informal micro-enterprises presented populations of total and fecal coliforms and Staphylococcus sp. above the limits established by Colombian regulations and varied according to the micro-enterprise. The results also indicated that 56% of Staphylococcus isolates were S. aureus. L. monocytogenes was positive in 38% of the samples. Salmonella sp. was not detected. The coliforms that prevailed were Escherichia coli (25%), Citrobacter freundii (14%), and Proteus mirabilis (8%). All L. monocytogenes were sensitive to ampicillin but resistant to erythromycin and trimethoprim-sulfamethoxazole. S. aureus isolates were susceptible to most antibiotics, except tetracycline and erythromycin (7% resistance). Likewise, 30% of coliforms (n = 36) were multidrug-resistant to antibiotics but susceptible to biocides.

Fat type in diet is responsible for specific changes in gut microbiota (GM). Extra virgin olive oil (EVOO) has been shown to be beneficial for blood pressure and to produce effects on GM. To analyze the cause-effect relationship between intestinal microbial changes and blood pressure, we studied the effect of EVOO on fecal microbiota and systolic blood pressure (SBP) levels in spontaneously hypertensive rats (SHR). SHR were fed either an enriched EVOO diet or a standard diet for a period of 12 weeks. At the end of the experimental period, the microbial profiles in the feces were studied in both groups by using PCR-denaturing gradient gel electrophoresis. Real-time PCR was used to quantify the selected bacterial groups. The results demonstrated significant differences when using Lactobacillus (p

Quantitative real-time PCR may be a rapid and automated procedure for detection of bacterial pathogens from food samples. Nevertheless, when testing the effects of antimicrobials on the viability of bacterial pathogens in foods, we found that DNA from dead cells interfered greatly in the detection of viable Listeria monocytogenes after treatment with the broad-spectrum bacteriocin enterocin AS-48. To overcome this problem, a quantitative real-time PCR (qRT-PCR) assay based on bacterial mRNA was adapted to quantify viable L. monocytogenes in food after bacteriocin treatments. The procedure allowed a better and faster estimation of viable cells compared to PALCAM viable cell counts when the threshold level was 2 log units/g of food, while PALCAM viable count allowed detection of one log unit/g. This procedure may be useful to verify the efficacy of bacteriocins against L. monocytogenes in foods.

Geobacillus stearothermophilus is a thermophilic bacterium typically responsible for the flat-sour spoilage of low-acid canned food with high water activity. Control of vegetative cells and spores of G. stearothermophilus strains CECT 48 and CECT 49 by enterocin EJ97 produced by Enterococcus faecalis EJ97 is described. Both strains were highly sensitive to EJ97 in a culture medium. In samples from canned foods inoculated with a cocktail of vegetative cells or endospores of the two strains and stored at 45 °C for 30 days, viable cell counts were reduced below detection levels. The time course of microbial inactivation depended on the food sample and bacteriocin concentration. Dormant endospores were resistant to EJ97 short-time treatments (5 min), but endospores activated to germinate by heat became bacteriocin sensitive. The simultaneous application of enterocin EJ97 and heat treatments (90 and 95 °C) on dormant endospores had an increased antimicrobial effect that depended both on the bacteriocin concentration and the heat temperature. Results from this study strengthen the potential of enterocin EJ97 for biopreservation against G. stearothermophilus in canned vegetable foods and drinks.

Bacteriocins are antimicrobial peptides produced by bacteria. Among them, the enterococcal bacteriocin (enterocin) AS-48 stands for its peculiar characteristics and broad-spectrum antimicrobial activity. AS-48 belongs to the class of circular bacteriocins and has been studied in depth in several aspects: peptide structure, genetic determinants, and mode of action. Recently, a wealth of knowledge has accumulated on the antibacterial activity of this bacteriocin against foodborne pathogenic and spoilage bacteria in food systems, especially in vegetable foods and drinks. This work provides a general overview on the results from tests carried out with AS-48 in different vegetable food categories (such as fruit juices, ciders, sport and energy drinks, fresh fruits and vegetables, pre-cooked ready to eat foods, canned vegetables, and bakery products). Depending on the food substrate, the bacteriocin has been tested alone or as part of hurdle technology, in combination with physico-chemical treatments (such as mild heat treatments or high-intensity pulsed electric fields) and other antimicrobial substances (such as essential oils, phenolic compounds, and chemical preservatives). Since the work carried out on bacteriocins in preservation of vegetable foods and drinks is much more limited compared to meat and dairy products, the results reported for AS-48 may open new possibilities in the field of bacteriocin applications.

A collection of 26 enterococci isolated from dairy and meat products were tested for antimicrobial and proteolytic activity. Enterococcus faecium and E. faecalis were the most frequent species among tested enterococci, and 11 isolates produced antimicrobial compounds. Results revealed that 10 out of 11 enterococci synthesized enterocins showing antimicrobial activity against food-born pathogen such as Listeria monocytogenes and Staphylococcus aureus. The broadest spectrum of antimicrobial activity was detected in E. faecalis BGPT1-10P and BGPT1-78. E. faecalis BG221 showed antimicrobial activity that was not related to production of enterocin, H2O2 or organic acid. Twenty-five enterococci showed strong or moderate proteolytic activity towards β-casein. Two isolates, BGPT1-10P and BGPT1-78, showed the most intense hydrolysis of αs1-, β-, κ-casein fractions, total casein as well as gelatin. Extracellular BGPT1-10P and BGPT1-78 proteinases have a molecular mass of about 29 kDa. Bacteriocin production and proteinase activity of natural isolates of enterococci may be of technological interest in dairy and meat-fermented products.

The cyclic antibacterial peptide enterocin AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (PHBME) and with 2-nitropropanol against Salmonella enterica serovar Enteritidis CECT 4300 in Russian salad. In challenge tests on a cocktail of Salmonella strains (S. enterica ssp. enterica serotype Typhi CECT 409, S. enterica ssp. enterica serovar Choleraesuis CECT 915, S. enterica ssp. enterica serovar Enteritidis CECT 4300, S. enterica ssp. arizonae serovar Arizonae CECT 4395, and S. enterica ssp. salamae CECT 4000) in salad at 10°C, the combinations of PHBME and AS-48 (80 μg/g) or 2-nitropropanol and AS-48 (40 μg/g) reduced the concentrations of viable Salmonella from 4.27 to 4.75 log CFU/g down to the limit of detection for 7 days. Salmonella populations did not increase in control samples (without any antimicrobials or in the presence of AS-48 alone) probably due to the low pH of this type of salad and low temperature of incubation, but retained over 85% viability after 1 week. This work opens new possibilities to expand the spectrum of inhibition of enterocin AS-48 against Salmonella enterica.

A bacteriocin-producing lactic acid bacterium (strain 2.5) isolated from cow’s milk used in cheese production from Northern Morocco was selected for its strong anti-listerial activity. The producer strain was identified as Enterococcus faecalis by molecular methods. Strain 2.5 carried the genetic determinants for the two-peptide enterococcal bacteriocin enterocin 1071, and the active bacteriocin was purified to homogeneity by reversed-phase chromatography from culture broths of the producer strain. Strain 2.5 carried two plasmids (of ∼7 and 40 kb). Characterization of strain 2.5 at biosafety level indicated that this strain is non-haemolytic, and lacks the genetic determinants for most of the virulence factors described in enterococci (cylB, cylM, gelE, ace and agg) although it carried the genetic determinants cylA, efaAfs as well as determinants for the sex pheromone peptides cpd, cob, and ccf. Strain 2.5 was resistant to tetracycline, rifampicin, and ciprofloxacin, but it was sensitive to penicillin, ampicillin, vancomycin, and teicoplanin. Results from the present study support the potential role of strain 2.5 as an anti-listerial agent to be tested in traditional fermented foods.

The production of antimicrobial substances was studied among 195 bacterial isolates from retail table olives. A total 86 isolates tested positive, and they clustered in 10 groups according to their inhibitory spectra. Many isolates (38.37%) produced strong inhibition against all bacteria tested (Listeria innocua, Lactococcus lactis, Bacillus cereus, B. megaterium, Staphylococcus aureus, Micrococcus luteus, Enterococcus faecalis, and Escherichia coli). The selected bacterial isolates were Gram-positive bacteria with rod morphology (62.67%), short rods (26.65%) or cocci (10.67%). Isolates producing antimicrobial substances may be useful as starters to enhance control of table olive fermentation and improve the safety of retail table olives.

The complete nucleotide sequence of the small (5149 bp) and cryptic plasmid pS86 from Enterococcus faecalis ssp. faecalis S-86 has been determined. Sequence analysis revealed six putative open reading frames (ORFs) encoding polypeptides of 28.3, 11.5, 8.4, 65.1, 7.3, and 11.96 kDa each. Based on sequence similarity, two cassettes have been identified in pS86: ORF1 codes for the replication initiation protein (Rep); ORF4 codes for a putative mobilization protein that shows similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. No function could be assigned to the other putative ORFs found. According to our results, pS86 plasmid could use a theta-mode of replication, similar to the recently described theta-type replicons from pUCL287 (Tetragenococcus halophila) and pLA1 or pLA105 (Lactobacillus acidophilus) plasmids.

The bacteriocinogenic strain of Enterococcus faecalis EJ97 has been isolated from municipal waste water. It produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,340 Da) that is very stable under mild heat conditions and is sensitive to proteolytic enzymes. The amino acid sequence of the first 18 N-terminal residues of enterocin EJ97 indicates that it is different from other known protein sequences. Enterocin EJ97 is active on several gram-positive bacteria including enterococci, several species of Bacillus, Listeria, and Staphylococcus aureus. The producer strain is immune to bacteriocin. Enterocin EJ97 has a concentration-dependent bactericidal and bacteriolytic effect on E. faecalis S-47.

The inhibitory capacity of strain S-48 ofEnterococcus faecalis ssp.liquefaciens was studied. The strain produces a broad-spectrum peptide antibiotic (AS-48) that has been characterized elsewhere. The isolation of mutants from S-48 after mutagenic treatment revealed another inhibitory substance which remained masked in the wild strain. The protein nature and restricted spectrum of this substance points to its being a bacteriocin (Bc-48).

The bacteriocin AS-48 is a cationic peptide (7149 Da) having a broad antimicrobial spectrum, encoded by the 68 kb conjugative plasmid pMB2 from Enterococcus faecalis S-48. It is a unique peptide since it has a cyclic structure, which is achieved by the formation of a tail–head peptide bond after ribosomal synthesis (Galvez et al., 1989; Martinez-Bueno et al., 1994; Samyn et al., 1994). Preliminary CD and calorimetric studies (data not shown) pointed towards a highly helical and very stable three dimensional structure. All the information gathered until now indicates that the target of AS-48 is the cytoplasmic membrane in which it opens channels or pores, leading to dissipation of the proton motive force and cell death, which in some cases is also followed by bacterial lysis (Galvez et al., 1991). This peptide is a suitable tool for studying protein–membrane interactions, and it also offers promising perspectives for biotechnological applications. Knowledge of the 3D structure of AS-48 is a first step in the conduct of further structure–function studies. Here we report the complete1 H NMR assignment of its proton resonances together with the resulting secondary structure pattern as prerequisites for the determination of a high-resolution 3D solution structure.