The human gastrointestinal tract and oral microbiota in inflammatory bowel disease: a state of the science review.

Inflammatory bowel disease (IBD) includes a spectrum of diseases from ulcerative colitis (UC) to Crohn's disease (CD). Many studies have addressed the changes in the microbiota of individuals affected by UC and CD. A decrease in biodiversity and depletion of the phyla Bacteroidetes and Firmicutes has been reported, among others. Changes in microbial composition also result in changes in the metabolites generated in the gut from microbial activity that may involve the amount of butyrate and other metabolites such as H2 S being produced. Other factors such as diet, age, or medication need to be taken into consideration when studying dysbiosis associated with IBD. Diverse bacterial species have been associated specifically or non-specifically to IBD, but none of them have been demonstrated to be its ethiological agent. Recent studies also suggest that micro-eukaryotic populations may also be altered in IBD patients. Last, but not least, viruses, and specially bacteriophages, can play a role in controlling microbial populations in the gastrointestinal tract. This may affect both bacterial diversity and metabolism, but possible implications for IBD still remain to be solved. Dysbiosis in the oral microbiome associated with IBD remains an emerging field for future research.

Virulence factors and antimicrobial resistance in Escherichia coli strains isolated from hen egg shells.

Eggs may contain extraintestinal pathogenic (ExPEC) and diarrheogenic (DEC) Escherichia coli which in addition may carry antibiotic resistance. The wide use of biocides and disinfectants in the food industry may induce biocide tolerance in bacteria. The aim of the present study was to evaluate biocide tolerance and antibiotic resistance in E. coli from hen egg shells. A total of 27 isolates obtained from a screening of 180 eggs were studied. Seven isolates carried both eae and bfpA genes of typical enteropathogenic E. coli (EPEC) strains, while 14 isolates only carried eae associated with atypical EPEC strains. Shiga toxin genes stx and stx2 were detected in four isolates. Heat-stable and heat-labile enterotoxin genes as well as aggR were also detected. Several isolates had minimum inhibitory concentrations (MICs) that were higher than the wild-type for the biocide hexadecylpyridinium chloride (HDP, 18.52%) or the commercial disinfectant P3 oxonia (OX, 14.81%). Antibiotic resistance was detected for ampicillin (37.03%), streptomycin (37.03%), tetracycline (37.03%), chloramphenicol (11.11%), nalidixic acid (18.51%) and trimethoprim-sulfamethoxazole (14.81%). Eight isolates (29.63%) were biocide tolerant and antibiotic resistant. Efflux pump genes detected included acrB (96.29%), mdfA (85.18%) and oxqA (37.03%), in addition to quaternary ammonium compound (QAC) resistance genes qacA/B (11.11%) and qacE (7.40%). Antibiotic resistance genes detected included blaCTX-M-2 (22.22%), blaTEM (3.70%), blaPSE (3.70%), tet(A) (29.63%), tet(B) (29.63%), tet(C) (7.40%), tet(E) (11.11%), aac(6')-Ib (3.70%), sul1 (14.81%), dfrA12 (3.70%) and dfrA15 (3.70%). Most isolates (96.30%) carried more than one genetic determinant of resistance. The most frequent combinations were efflux pump components acrB and mdfA with tetracycline resistance genes (33.33% of isolates). Isolates carrying QAC resistance genes also carried between 4 and 8 of the additional antimicrobial resistance genes investigated. Regardless of biocide tolerance and antibiotic resistance, all isolates were sensitive to carvacrol (0.25%), thymol (0.125%) and trisodium phosphate (1 to 1.5%), but they exhibited a heterogeneous response to sodium lactate and lysozyme-EDTA combinations that apparently were not related with antibiotic resistance. Results from the study reveal not only a low incidence of biocide tolerance but also the presence of multiple resistance strains carrying multiple genetic determinants of resistance.

Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria.

Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins.

Increased inactivation of exopolysaccharide-producing Pediococcus parvulus in apple juice by combined treatment with enterocin AS-48 and high-intensity pulsed electric field.

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

Assay of enterocin AS-48 for inhibition of foodborne pathogens in desserts.

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

Effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against the spoilage bacterium Lactobacillus diolivorans in apple juice.

Enterocin AS-48 was tested in apple juice against the cider-spoilage, exopolysaccharide-producing strain Lactobacillus diolivorans 29 in combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode). A response surface methodology was applied to study the bactericidal effects of the combined treatment, with AS-48 concentration and HIPEF treatment time as process variables. At subinhibitory bacteriocin concentrations, microbial inactivation by the combined treatment increased as the bacteriocin concentration and the HIPEF treatment time increased (from 0.5 to 2.0 microg/ml and from 100 to 1000 micros, respectively). Highest inactivation (4.87 logs) was achieved by 1000 micros HIPEF treatment in combination with 2.0 microg/ml AS-48. While application of treatments separately did not protect juice from survivors during storage, survivors to the combined treatment were inactivated within the following 24 h of storage, and the treated samples remained free from detectable lactobacilli for at least 15 days at temperatures of 4 degrees C as well as 22 degrees C. The combined treatment could be useful for inactivation of exopolysaccharide-producing L. diolivorans in apple juice.

Inhibition of Salmonella enterica Cells in Deli-Type Salad by Enterocin AS-48 in Combination with Other Antimicrobials.

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.

Enhanced bactericidal effect of enterocin AS-48 in combination with high-intensity pulsed-electric field treatment against Salmonella enterica in apple juice.

The effect of the broad spectrum cyclic antimicrobial peptide enterocin AS-48 combination with high-intensity pulsed-electric field (HIPEF) treatment (35 kV/cm, 150 Hz, 4 micros and bipolar mode) was tested on Salmonella enterica CECT 915 in apple juice. A response surface methodology was applied to study the bactericidal effects of the combined treatment. The process variables were AS-48 concentration, temperature, and HIPEF treatment time. While treatment with enterocin AS-48 alone up to 60 microg/ml had no effect on the viability of S. enterica in apple juice, an increased bactericidal activity was observed in combination with HIPEF treatments. Survival fraction was affected by treatment time, enterocin AS48 concentration and treatment temperature. The combination of 100 micros of HIPEF treatment, 30 microg/ml of AS-48, and temperature of 20 degrees C resulted in the lowest inactivation, with only a 1.2-log reduction. The maximum inactivation of 4.5-log cycles was achieved with HIPEF treatment for 1000 micros in combination with 60 microg/ml of AS-48 and a treatment temperature of 40 degrees C. Synergism between enterocin AS-48 and HIPEF treatment depended on the sequence order application, since it was observed only when HIPEF was applied in the presence of previously-added bacteriocin. The combined treatment could improve the safety of freshly-made apple juice against S. enterica transmission.

Bacteriocin-producing Lactobacillus strains isolated from poto poto, a Congolese fermented maize product, and genetic fingerprinting of their plantaricin operons.

Thirty one bacteriocin-producing Lactobacillus isolates were identified among 135 lactobacilli isolated from the Congolese fermented maize product poto poto, during the preparation and from the finished product. Using species-specific PCR and 16S rRNA gene sequencing, 28 and 3 isolates were identified as L. plantarum and L. fermentum, respectively. Cluster analysis of RAPD-PCR fingerprints revealed two main groups (G1 and G2) plus the L. fermentum isolate C4-13. Group G1 contained 23 isolates with a similarity coefficient > 74.5%, and could be divided in two subgroups (G1-1, G1-2) each with several branches, plus the L. plantarum isolate C11. Group G2 contained 8 isolates with a similarity coefficient > 86%, with two main branches. Using PCR amplification with specific primers, several genes of the plantaricin cluster found in L. plantarum C11 were identified in the isolates. The number of genes that were detected varied between the strains. The L. fermentum isolate EC11 also contained the plnDEFG genes. PCR amplification of DNA from isolates with primers directed to the upstream and downstream region of the plantaricin cluster generated an amplicon identical to that obtained with DNA from the control strain L. plantarum WCFS1. Amplification products from the positive strains were used for restriction analysis with HindIII, EcoRI and KpnI in separate reactions. Cluster analysis of restriction profiles revealed high similarities for EcoRI and HindII digest profiles, and an identical profile for all KpnI digests. The L. fermentum EC11 isolate clustered with L. plantarum strains in a group with a high correlation coefficient. The results suggest a low degree of diversity in the plantarincin gene cluster. However, other strains that tested positive for individual plantaricin genes may present great heterogeneity in the plantaricin operons. Because of their broad spectra of inhibition (including Escherichia coli, Salmonella enterica, Enterobacter aerogenes, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis), isolates from the present study could be used to improve the safety and storage stability of poto poto.

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.

Efficacy of enterocin AS-48 against bacilli in ready-to-eat vegetable soups and purees.

The broad-spectrum bacteriocin enterocin AS-48 was tested for biopreservation of ready-to-eat vegetable foods (soups and purees) against aerobic mesophilic endospore-forming bacteria. By adding AS-48 (10 microg/ml), Bacillus cereus LWL1 was completely inhibited in all six vegetable products tested (natural vegetable cream, asparagus cream, traditional soup, homemade-style traditional soup, vegetable soup, and vichyssoise) for up to 30 days at 6, 15, and 22 degrees C. A collection of strains isolated from spoiled purees showed slightly higher resistance to AS-48 in the order Paenibacillus sp. > Bacillus macroides > B. cereus, although they were also completely inhibited in natural vegetable cream by AS-48 at 10 microg/ml. However, cocktails of five or eight strains composed of B. cereus (three strains), B. macroides (two strains), and Paenibacillus sp., Paenibacillus polymyxa, and Paenibacillus amylolyticus showed higher bacteriocin resistance with AS-48 of up to 50 microg/ml required for complete inactivation in natural vegetable cream stored at 22 degrees C. Repetitive extragenic palindromic sequence-based PCR (REP-PCR) analysis showed that paenibacilli (along with some B. cereus) was the predominant survivor in the cocktails after bacteriocin treatment. To increase the effectiveness of enterocin AS-48, the bacteriocin was tested (at 20 microg/ml) against the eight-strain cocktail in natural vegetable cream in combination with other antimicrobials. The combination of AS-48 and nisin had a slight but significant additive effect. Bactericidal activity was greatly enhanced by phenolic compounds (carvacrol, eugenol, geraniol, and hydrocinnamic acid), achieving a rapid and complete inactivation of bacilli in the tested puree at 22 degrees C.

Differentiation and characterization by molecular techniques of Bacillus cereus group isolates from poto poto and dégué, two traditional cereal-based fermented foods of Burkina Faso and Republic of Congo.

Poto poto (a maize sourdough) and dégué (a pearl millet-based food) are two traditional African fermented foods. The molecular biology of toxigenic and pathogenic bacteria found in those foods is largely unknown. The purpose of this study was to study the phylogenetic relatedness and toxigenic potential of 26 Bacillus cereus group isolates from these traditional fermented foods. The relatedness of the isolates was evaluated with repetitive element sequence-based PCR (REP-PCR) and 16S rDNA sequencing analysis. A multiplex real-time PCR assay targeting the lef and capC genes of B. anthracis pXO1 and pXO2 plasmids and the sspE chromosomal gene of B. cereus and B. anthracis also was carried out. Melting curve analysis of the sspE amplification product was used to differentiate B. cereus from B. anthracis, and the presence of the B. cereus enterotoxin genes was determined with PCR amplification. Isolates had 15 different REP-PCR profiles, according to which they could be clustered into four groups. 16S rDNA sequencing analysis identified 23 isolates as B. cereus or B. anthracis and three isolates as B. cereus or Bacillus sp. Multiplex real-time PCR amplification indicated the absence of the lef and capC genes of B. anthracis pXO 1 and pXO2 plasmids, and melting curve analysis revealed amplification of the 71-bp sspE product typical of B. cereus in all isolates instead of the 188-bp amplicon of B. anthracis, confirming the identity of these isolates as B. cereus. Four isolates had amylolytic activity. All isolates had lecithinase activity and beta-hemolytic activity. Enterotoxin production was detected in two isolates. The emetic toxin gene was not detected in any isolate. The nheB toxin gene was detected in 19 isolates by PCR amplification; one of these isolates also contained the hblD (L1) gene. The cytotoxin K cytK-1 gene was not detected, but the cytK-2 gene was clearly detected in six isolates.

Plasmid profile patterns and properties of pediococci isolated from caper fermentations.

A collection comprising 14 isolates of Pediococcus pentosaceus and one Pediococcus acidilactici from the fermentation of caper fruits was studied. All isolates showed very similar fermentation profiles and produced a limited number of exoenzymes. All isolates carried large plasmids of diverse sizes between 20 and 55 kb, while some also contained smaller plasmids between 10 and 16 kb. Cluster analysis of plasmid profiles revealed four main groups with various degrees of similarities. All amino acid decarboxylation tests were negative, suggesting that pediococci are not involved in generation of biogenic amines. None of the isolates showed hemolytic activity. Antimicrobial resistance tests revealed that all isolates were sensitive to 11 different antimicrobials while being resistant to ciprofloxacin (MIC > or =2 mg/liter) and intrinsically resistant to vancomycin (MIC > or =16 mg/liter) and teicoplanin (MIC > or =16 mg/liter).

Microbiological study of lactic acid fermentation of Caper berries by molecular and culture-dependent methods.

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.