Antonio Cobo Molinos

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.

Effect of Immersion Solutions Containing Enterocin AS-48 on Listeria monocytogenes in Vegetable Foods

ABSTRACT The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 μg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 μg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15°C, as well as green asparagus at 15°C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22°C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.

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.

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.

Inhibition of Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by application of washing solutions containing enterocin AS-48 alone and in combination with other antimicrobials

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

Inactivation of Listeria monocytogenes in raw fruits by enterocin AS-48.

The purpose of this study was to determine the effect of enterocin AS-48 on Listeria monocytogenes CECT 4032 in fruits and fruit juice. Fruits were contaminated with a L. monocytogenes cell suspension, washed with enterocin AS-48 (25 microg/ml) or with sterile distilled water as control, and stored at different temperatures (-20, 6, 15, 22 degrees C). Washing treatments significantly inhibited or completely inactivated L. monocytogenes in strawberries, raspberries, and blackberries stored at 15 and 22 degrees C for up to 2 days and in blackberries and strawberries at 6 degrees C for up to 7 days. Washing treatments with enterocin AS-48 also reduced viable counts in sliced melon, watermelon, pear, and kiwi but did not avoid proliferation of survivors during storage at 15 and 22 degrees C. Added enterocin (25 microg/ml) completely inactivated L. monocytogenes in watermelon juice within 24 h. To enhance the antilisterial activity of treatments, enterocin AS-48 was tested in combination with other antimicrobial substances on sliced melon stored at 22 degrees C. The combinations of enterocin AS-48 and trisodium trimetaphosphate, sodium lactate, lactic acid, polyphosphoric acid, carvacrol, hydrocinnamic acid, p-hydroxybenzoic acid, n-propyl p-hydroxybenzoate, or 2-nitropropanol showed increased antilisterial activities compared with each antimicrobial tested separately. Washing treatments with enterocin AS-48 in combination with 12 mM carvacrol, as well as with 100 mM n-propyl p-hydroxybenzoate, avoided regrowth of Listeria during storage at 22 degrees C. Results from this study indicate that enterocin AS-48 alone or in combination with other preservatives could serve as an additional hurdle against L. monocytogenes in fruits and fruit juices.

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.

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.

A quantitative real-time PCR assay for quantification of viable Listeria monocytogenes cells after bacteriocin injury in food-first insights.

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.

Analysis of potential risks from the bacterial communities associated with air-contact surfaces from tilapia (Oreochromis niloticus) fish farming

Abstract Tilapia farming is a promising growing sector in aquaculture. Yet, there are limited studies on microbiological risks associated to tilapia farms. The aim of the present study was to analyse the bacterial communities from solid surfaces in contact with air in a tilapia farm in order to evaluate the presence of bacteria potentially toxinogenic or pathogenic to humans or animals. Samples from a local tilapia farm (tank wall, aerator, water outlets, sink and floor) were analyzed by high throughput sequencing technology. Sequences were assigned to operational taxonomic units (OTUs). Proteobacteria was the main phylum represented in most samples (except for one). Cyanobacteria were a relevant phylum in the inner wall from the fattening tank and the wet floor by the pre‐fattening tank. Bacteroidetes were the second phylum in relative abundance for samples from the larval rearing tank and the pre‐fattening tank and one sample from the fattening tank. Fusobacteria showed highest relative abundances in samples from the larval rearing tank and pre‐fattening tank. Other phyla (Verrucomicrobia, Actinobacteria, Firmicutes, Planktomycetes, Acidobacteria, Chloroflexi, Chlorobi, Gemmatiomonadetes or Fibrobacters) had lower relative abundances. A large fraction of the reads (ranging from 43.67% to 72.25%) were assigned to uncultured bacteria. Genus Acinetobacter (mainly A. calcoaceticus/baumanni) was the predominant OTU in the aerator of the fattening tank and also in the nearby sink on the floor. The genera Cetobacterium and Bacteroides showed highest relative abundances in the samples from the larval rearing tank and the pre‐fattening tank. Genera including fish pathogens (Fusobacterium, Aeromonas) were only detected at low relative abundances. Potential human pathogens other than Acinetobacter were either not detected or had very low relative abundances (< 0.01%). The results of the study suggest that the main risk factors to be monitored in tilapia farm are putative human pathogenic Acinetobacter and potential cyanotoxin‐producing cyanobacteria. HighlightsBacterial communities from air‐contact surfaces in a tilapia farm were investigated.Main phyla were Proteobacteria, Cyanobacteria, Bacteroidetes and Fusobacteria.Uncultured bacteria were highly represented.Acinetobacter was the main potentially human pathogen detected.Fish pathogens had low relative abundances or were undetected.