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

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

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

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

Biocide tolerance in Salmonella from meats in Southern Spain.

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

Biocide tolerance in Salmonella from meats in Southern Spain

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

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

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

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

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

Biocide and copper tolerance in enterococci from different sources.

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.

Antibacterial activity of carvacrol and 2-nitro-1-propanol against single and mixed populations of foodborne pathogenic bacteria in corn flour dough.

Cereal doughs are an important part of human diet, but at the same time can act as vehicles for the transmission of human pathogenic bacteria. In the present study, four pathogenic or toxinogenic bacteria (Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, Bacillus cereus and Staphylococcus aureus) were inoculated in a dough made from corn flour in combination with the single antimicrobial compounds carvacrol and 2-Nitro-1-propanol (2NPOH). Survival of single and mixed populations in the treated doughs incubated at 37 degrees C was followed by culture-dependent and independent methods (TTGE). All strains were completely inactivated within 24 h by the tested compounds at 5% final concentration, but showed variable inhibition at lower concentrations of 0.5% and 2%. Sensitivity to antimicrobial compounds was in general modified when strains were tested in cocultures compared with single cultures. B. cereus was more sensitive to carvacrol (minimum bactericidal concentration, MBC, 0.5%) in coculture with S. aureus. It was also more sensitive to 2NPOH in cocultures with S. aureus and with S. enterica (MBC, 2% in both cases). S. aureus was more resistant to carvacrol (MBC, 5%) in cocultures with B. cereus, E. coli, as well as S. enterica. However, sensitivity to 2NPOH was not modified in any of the coculture experiments (MBC, 2%). E. coli was more resistant to carvacrol (MBC, 5%) in cocultures with S. aureus and with S. enterica. Resistance of E. coli to 2NPOH also increased in cocultures with B. cereus (MBC, 5%) and with S. aureus (MBC, 2%), but not with S. enterica (MBC, 0.5%). S. enterica was more resistant to carvacrol (MBC, 5%) in cocultures with E. coli, but it was more sensitive to 2NPOH in cocultures with B. cereus as well as with S. aureus (MBC, 2% in both cases). TTGE analysis of survivors from cocultures treated with 2NPOH or carvacrol allowed a good estimation of the identity of survivors according to their DNA band patterns. Results from this study indicate that the efficacy of antimicrobials such as carvacrol and 2NPOH is greatly influenced by the complexity of the microbial populations under target and the relationships between individual populations.

Antistaphylococcal effect of enterocin AS-48 in bakery ingredients of vegetable origin, alone and in combination with selected antimicrobials.

The inhibitory effect of enterocin AS-48 against Staphylococcus aureus was investigated in various types of bakery ingredients. Antibacterial activity greatly depended on the food substrate, ranging from complete inactivation of S. aureus in liquid caramel (in which the bacterium survived poorly) to no significant inhibition (as in vanilla or chocolate creams). Significant reductions of viable counts in the range of 1.8 to 2.7 log units (P < 0.05) were achieved in substrates like pumpkin confiture or diluted almond cream stored at temperatures of 10 or 22 degrees C. Given the very low activity detected in chocolate substrates, enterocin AS-48 was tested in combination with other antimicrobials. Bactericidal activity increased markedly for the combinations of AS-48 and 0.1% eugenol (v/v), 0.5% 2-nitropropanol (v/v), or 3% Nisaplin (w/v). Enterocin AS-48 could be applied in combination with other antimicrobials for preservation of bakery ingredients against S. aureus.

Microbial diversity changes in soybean sprouts treated with enterocin AS-48.

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.

Enhanced bactericidal activity of enterocin AS-48 in combination with essential oils, natural bioactive compounds and chemical preservatives against Listeria monocytogenes in ready-to-eat salad.

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.

Antibacterial protection by enterocin AS-48 in sport and energy drinks with less acidic pH values.

The low pH and acid content found in sports and energy drinks are a matter of concern in dental health. Raising the pH may solve this problem, but at the same time increase the risks of spoilage or presence of pathogenic bacteria. In the present study, commercial energy drinks were adjusted to pH 5.0 and challenged with Listeria monocytogenes (drinks A to F), Staphylococcus aureus, Bacillus cereus, and Bacillus licheniformis (drink A) during storage at 37 degrees C. L. monocytogenes was able to grow in drink A and survived in drinks D and F for at least 2 days. Addition of enterocin AS-48 (1 microg/ml final concentration) rapidly inactivated L. monocytogenes in all drinks tested. S. aureus and B. cereus also survived quite well in drink A, and were completely inactivated by 12.5 microg/ml enterocin AS-48 after 2 days of storage or by 25 microg/ml bacteriocin after 1 day. B. licheniformis was able to multiply in drink A, but it was completely inactivated by 5 microg/ml enterocin AS-48 after 2 days of storage or by 12.5 microg/ml bacteriocin after 1 day. Results from the present study suggest that enterocin AS-48 could be used as a natural preservative against these target bacteria in less acidic sport and energy drinks.

Multilocus sequence typing of Enterococcus faecalis from vegetable foods reveals two new sequence types.

A collection of 16 isolates of Enterococcus faecalis from different vegetable foods were characterized by multilocus sequence typing (MLST). One isolate belonged to sequence type (ST) 9 of the previously described clonal complex 9, which is frequently associated with hospital environments. The rest of the isolates were grouped into two new STs named 168 and 169. ST168 represented a singleton clone that included 14 isolates and seemed to be the predominant type among E. faecalis from vegetable samples. ST168 was closely related to ST72, differing only by one allele type. Singleton ST169 was not related to any of the previously described STs.

Inactivation of Geobacillus stearothermophilus in canned food and coconut milk samples by addition of enterocin AS-48.

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.

Combined physico-chemical treatments based on enterocin AS-48 for inactivation of Gram-negative bacteria in soybean sprouts.

Enterocin AS-48 was tested for decontamination of soybean sprouts against Gram-negative bacteria. Although treatment with bacteriocin alone had no effect on Salmonella enterica, a synergistic antimicrobial effect was detected at pH 9.0 and in combination with moderate heat treatment. Greatest inactivation was achieved for sprouts heated for 5 min at 65 degrees C in an alkaline (pH 9.0) enterocin AS-48 solution of 25 microg/ml. Bactericidal activity against S. enterica increased greatly when enterocin AS-48 was used in washing solutions in combination with several chemical compounds: EDTA, lactic acid, peracetic acid, polyphosphoric acid, sodium hypochlorite, hexadecylpyridinium chloride, propyl-p-hydroxybenzoate, and hydrocinnamic acid. The combined treatment of enterocin AS-48 and polyphosphoric acid was tested against several other Gram-negative bacteria inoculated on sprouts. The bacteria tested showed great differences in sensitivity to polyphosphoric acid, but synergism with enterocin AS-48 was confirmed in all cases. Combinations of enterocin AS-48 (25 microg/ml) and polyphosphoric acid in a concentration range of 0.1 to 2.0% significantly reduced or inhibited growth of the populations of S. enterica, Escherichia coli O157:H7, Shigella spp., Enterobacter aerogenes, Yersinia enterocolitica, Aeromonas hydrophila and Pseudomonas fluorescens in sprout samples stored at 6 degrees C and 15 degrees C. The combined treatment could therefore be applied to reduce the risks of Gram-negative pathogenic as well as spoilage bacteria on sprouts.

Detection of ebp (endocarditis- and biofilm-associated pilus) genes in enterococcal isolates from clinical and non-clinical origin.

A collection of enterococci from clinical samples, fruits and vegetables, water and soil was investigated for the presence of endocarditis- and biofilm-associated pilus (ebp) genes by PCR amplification and also by colony hybridisation. A high percentage of Enterococcus faecalis clinical isolates (94.59%) as well as all isolates from fruits and vegetables and two of the three isolates from water and soil carried ebpA, ebpB and ebpC genes. For E. faecium, PCR amplification revealed that over 81.81% of isolates from water and soil carried the three genes, compared to the lower incidence detected among isolates from vegetables (68.42%) and clinical samples (33.33%). The remaining E. faecium isolates tested negative both in the PCR and hybridisation tests. This is the first report of ebp detection among non-clinical E. faecium. Comparative sequence analysis of ebp genes revealed a higher similarity of clinical isolate E. faecalis EH17 with E. faecalis V583, while the non-clinical isolates E. faecalis V30C2 and E. faecium A9C4 grouped in a separate cluster. Greatest differences among clusters were observed for ebpC gene. Results from the present study suggest that enterococcal populations adapted to specific environments may also differ in the sequences of pili-encoding genes. Such variations may be important to produce functional pili and may have direct implications on the adhesion and colonisation capacity of isolates.

Application of bacteriocins in the control of foodborne pathogenic and spoilage bacteria.

Bacteriocins are antimicrobial peptides or proteins produced by strains of diverse bacterial species. The antimicrobial activity of this group of natural substances against foodborne pathogenic, as well as spoilage bacteria, has raised considerable interest for their application in food preservation. Application of bacteriocins may help reduce the use of chemical preservatives and/or the intensity of heat and other physical treatments, satisfying the demands of consumers for foods that are fresh tasting, ready to eat, and lightly preserved. In recent years, considerable effort has been made to develop food applications for many different bacteriocins and bacteriocinogenic strains. Depending on the raw materials, processing conditions, distribution, and consumption, the different types of foods offer a great variety of scenarios where food poisoning, pathogenic, or spoilage bacteria may proliferate. Therefore, the effectiveness of bacteriocins requires careful testing in the food systems for which they are intended to be applied against the selected target bacteria. This and other issues on application of bacteriocins in foods of dairy, meat, seafood, and vegetable origins are addressed in this review.

Risk factors in enterococci isolated from foods in Morocco: determination of antimicrobial resistance and incidence of virulence traits.

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.

Inactivation of exopolysaccharide and 3-hydroxypropionaldehyde-producing lactic acid bacteria in apple juice and apple cider by enterocin AS-48.

The bacteriocin enterocin AS-48 was tested against exopolysaccharide producing lactic acid bacteria (LAB) strains of Lactobacillus collinoides, Lactobacillus dioliovorans and Pediococcus parvulus as well as two 3-hydroxypropionaldehyde (3-HPA)-producing Lb. collinoides strains causing apple cider spoilage. In fresh-made apple juice, a bacteriocin concentration of 2.5 microg/ml reduced the LAB viable cell counts below detection levels during the course of incubation at 10 and 22 degrees C for most strains tested, except for Lb. collinoides 5 and Lb. dioliovorans 29. These two strains were significantly inhibited at 10 degrees C by 5 microg/ml AS-48 or completely inactivated at 22 degrees C. In a commercial Basque apple cider, the added bacteriocin (2.5 microg/ml for Lb. collinoides strains 9 and 10, and 5 microg/ml for the rest of strains) completely inactivated all LAB strains tested during storage at 10 as well as 22 degrees C. In the commercial Asturian apple cider tested the LAB strains showed a poor capacity for survival, but the added bacteriocin was equally effective in reducing the numbers of survivors. When a cocktail of the five LAB strains was tested in commercial Basque apple cider, viable cell counts were reduced below detection levels after 2 days for a bacteriocin concentration of 12.5 microg/ml regardless of storage temperature. Comparison of RAPD-PCR profiles revealed that strain Lb. dioliovorans 29 was always the predominant survivor detected in bacteriocin-treated samples.

Comparative analysis of genetic diversity and incidence of virulence factors and antibiotic resistance among enterococcal populations from raw fruit and vegetable foods, water and soil, and clinical samples.

A comparative study was carried out among enterococci isolated from fruits and vegetable foods, water and soil, and clinical samples. Results indicate strong differences in the numbers of enterococcal species found in different environments as well as their abundance. While Enterococcus faecalis was clearly the predominant species in clinical samples, Enterococcus faecium predominated in vegetables, and it slightly outnumbered E. faecalis in water samples. Other species (Enterococcus hirae, Enterococcus mundtii, Enterococcus durans, Enterococcus gallinarum and Enterococcus casseliflavus) were found more frequently in vegetables, water, and specially in soil. Isolates from vegetable foods showed a lower incidence of antibiotic resistance compared to clinical isolates for most antimicrobials tested, especially erythromycin, tetracycline, chloramphenicol, ciprofloxacin, levofloxacin, gentamicin and streptomycin for E. faecalis, and quinupristin/dalfopristin, ampicillin, penicillin, ciprofloxacin, levofloxacin, rifampicin, choramphenicol, gentamicin and nitrofurantoin for E. faecium. E. faecium isolates from vegetable foods and water showed an average lower number of antibiotic resistance traits (2.95 and 3.09 traits for vegetable and water isolates, respectively) compared to clinical samples (7.5 traits). Multi-resistant strains were also frequent among clinical E. faecalis isolates (5.46 traits on average). None of E. faecalis or E. faecium isolates from vegetable foods, water and soil showed beta-haemolytic activity, while 25.64% of clinical E. faecalis did. A 51.28% of E. faecalis clinical isolates tested positive for the cylA, cylB, cylM set of genes, while some or all of these genes were missing in the rest of isolates. In clinical E. faecalis and E. faecium isolates, the genetic determinants for the enterococcal surface protein gene (esp), the collagen adhesin gene (ace) and the sex pheromone gene ccf (as well as cob in E. faecalis) showed a clearly higher incidence compared to isolates from other sources. E. faecalis isolates from vegetable foods and water had much lower average numbers of virulence genetic determinants per strain (4.23 and 4.0, respectively) compared to clinical isolates (8.71). Similarly, among E. faecium the lowest average number of traits per strain occurred in vegetable food isolates (1.72) followed by water (3.9) and clinical isolates (4.73). Length heterogeneity (LH)-PCR typing with espF-aceF-ccfF and espF-ccfF primers revealed genomic groups that clearly differentiated clinical isolates from those of vegetable foods, water and soil (except for two clinical isolates). The large differences found in the incidence of antibiotic resistance and virulence factors and in the genetic fingerprints determined by LH-PCR suggest a clear separation of hospital-adapted populations of enterococci from those found in open environments.