Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro

Aims:  To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro .
Methods and Results:  Strains CIDCA 133 ( Lactobacillus delbrueckii subsp. lactis ) and CIDCA 83114 ( Lactobacillus plantarum ) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells.
Conclusions:  Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen.
Significance and Impact of the Study:  Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.     

Improved treatment of vulvovaginal candidiasis with fluconazole plus probiotic Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14

Aims:  To determine the ability of probiotic lactobacilli to improve the treatment of vulvovaginal candidiasis (VVC) using a randomized, double-blind and placebo-controlled trial.
Methods and Results:  Fifty-five women diagnosed with VVC by vaginal discharge positive for Candida spp. (according to culture method) associated with at least one of the symptoms (itching and burning vaginal feeling, dyspareunia and dysuria), were treated with single dose of fluconazole (150 mg) supplemented every morning for the following 4 weeks with two placebo or two probiotic capsules (containing Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14). At 4 weeks, the probiotic treated group showed significantly less vaginal discharge associated with any of the above mentioned symptoms (10·3% vs 34·6%; P  =   0·03) and lower presence of yeast detected by culture (10·3% vs 38·5%; P  =   0·014).
Conclusion:  This study has shown that probiotic lactobacilli can increase the effectiveness of an anti-fungal pharmaceutical agent in curing disease.
Significance and Impact of the Study:  This novel finding of probiotic lactobacilli augmenting the cure rate of yeast vaginitis, not only offers an alternative approach to a highly prevalent condition that adversely affects the quality of life of women around the world, but also raises the question of how this combination works.     

Prebiotic effects of oligosaccharides on selected vaginal lactobacilli and pathogenic microorganisms

The purpose of this study was to select endogenous human vaginal lactobacilli strains on the basis of the main probiotic properties observed in the vaginal environment in order to use them for the evaluation of the potential prebiotic properties of oligosaccharides. From vaginal samples of 50 women with a normal flora, 17 lactobacilli strains were first isolated because of their high level of hydrogen peroxide production. Then six strains were selected mainly for their ability (i) to adhere to vaginal cells, (ii) to produce compounds in sufficient amount, such as lactic acid, having an inhibitory action on pathogens, and less importantly, (iii) to demonstrate arginine deiminase activity. These six strains were found to belong to three distinct species: Lactobacillus crispatus, L. jensenii and L. vaginalis. One strain of each species was chosen as a potential vaginal probiotic strain with regard to our criteria. These three strains were then used to evaluate the prebiotic properties of different oligosaccharide series: two fructooligosaccharide series (FOS Actilight and FOS Raftilose) and two glucooligosaccharide series varying by their osidic linkages (alpha-1,6/alpha-1,4 GOS and alpha-1,2/alpha-1,6/alpha-1,4 GOS). The test was based on the ability of the oligosaccharides to promote the growth of the three beneficial strains selected but not of pathogenic microorganisms often encountered in urogenital infections such as Candida albicans, Escherichia coli and Gardnerella vaginalis. Oligosaccharide hydrolysis was followed by HPLC analysis. This revealed that two oligosaccharide series (FOS Actilight DP3 and all alpha-1,6/alpha-1,4 GOS DP > or = 4) were used only by the lactobacilli strains, the pathogenic microorganisms being unable to metabolise them. The selected lactobacilli and oligosaccharides are good candidates for incorporation in a formula to prevent vaginal infections.     

In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation

Aim:  To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products.
Methods and Results:  An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5·8.
Conclusions:  L. agilis JCM 1048 and L. salivarius ssp . salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum.
Significance and Impact of the Study:  This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.     

Fermentation properties of gentio-oligosaccharides

AIMS: To investigate the fermentation properties of gentio-oligosaccharides (GOS), as compared to fructo-oligosaccharides (FOS) and maltodextrin in mixed faecal culture. METHODS AND RESULTS: The substrates were incubated in 24 h batch culture fermentations of human faecal bacteria. Fluorescent in situ hybridization was used to determine changes in populations of bifidobacteria, lactobacilli, clostridia, bacteroides, streptococci and Escherichia coli. Gas and short-chain fatty acid (SCFA) production was also measured. GOS gave the largest significant increases in bifidobacteria, lactobacilli and total bacterial numbers during the incubations. However, FOS appeared to be a more selective prebiotic as it did not significantly stimulate growth of bacterial groups which were not probiotic in nature. GOS and maltodextrin produced the highest levels of SCFA. Lowest gas production was seen with GOS and highest with FOS. CONCLUSIONS: GOS possessed bifidogenic activity in vitro. Although fermentation of GOS was not as selective as FOS, gas production was lower. Gas production is often seen as an undesirable side effect of prebiotic consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided the first data on fermentation of GOS in mixed faecal culture. The study has also used molecular microbiology methods (FISH) to quantify bacterial groups. The data extend our knowledge of the selectivity of fermentation of oligosaccharides by the gut microflora.     

Effects of yeast probiotic formulation on viability, revival and protection against infection with Salmonella enterica ssp. enterica serovar Typhimurium in mice

Aims:  To compare the effects of five yeast probiotic formulations on viability, revival and washout kinetic in the digestive tract of mice, and the protection against an experimental infection with Salmonella enterica serovar Typhimurium.
Methods and Results:  The number of viable cells in five commercial probiotic products codified as A, B, C and D ( Saccharomyces boulardii – lyophilized) and E ( Saccharomyces cerevisiae – aqueous suspension) was determined, as well as revival and washout kinetic in mouse intestine. Protective capacity was evaluated by survival rate and histopathology of liver and intestine of mice treated with each product and then challenged with Salm . Typhimurium.
Conclusions:  Product A contained the highest number of viable cells and, fed to mice, gave the highest counts of viable yeasts and the longest persistence in faeces. Probably as a consequence, the highest survival and protection of intestinal and hepatic tissues were observed when product A was used for mouse treatment. Product E showed low counts in the formulation and was not recovered from mouse intestine.
Significance and Impact of the Study:  Formulation (lyophilization or aqueous suspension) is an important factor for revival and survival of a probiotic product in vivo and consequently for its protective properties.     

高效液相色谱法同时测定 保健食品中低聚果糖和低聚木糖

目的建立高效液相色谱法同时测定保健食品中低聚果糖(包括蔗果三糖、蔗果四糖及蔗果五糖)和低聚木糖(包括木二糖、木三糖、木四糖、木五糖、木六糖及木七糖)的含量。方法采用键合有酰胺官能团杂化填料的色谱柱,以乙腈和水为流动相,利用亲水保留色谱机制(HILIC)实现对低聚果糖和低聚木糖各单体的有效分离,结合示差检测以外标法定量。该方法同时利用木糖对低聚木糖各单体转换系数进行定量。结果低聚果糖和低聚木糖各单体线性关系良好,相关系数均大于0.998,平均加标回收率为95%以上,具有较高的准确度和良好的重现性。结论高效液相色谱法由于采用木糖对照品转换定量低聚木糖含量,减少低聚木糖各单体标准品在日常工作中的使用量,进而降低检测成本。该方法经济可靠,测定结果准确,适合于添加有低聚果糖和低聚木糖的功能性产品检测。     

In vitro fermentation of prebiotics by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> CFR 2194: selectivity,viability and effect of metabolites on β-glucuronidase activity

Prebiotic Fructooligosaccharides (FOS) escape metabolism in upper GI tract undergo microbial metabolism in colon and thereby influence the nature, type and number of intestinal microbiota to improve host’s health. The present study focuses on the ability of Lactobacillus plantarum CFR 2194 to utilize FOS as a selective carbon and energy source. The effect of fermentative metabolites of L. plantarum on the β-glucuronidase was also investigated. A total of 16 strains of lactobacilli were assessed for their ability to ferment oligosaccharides. L. plantarum CFR 2194, an isolate from kanjika was found to utilize FOS effectively. Lactic acid was the main metabolic end product, followed by acetic acid, butyric acid, formic acid and ethanol. The inhibitory effects of these metabolites have been confirmed through the reduction of β-glucuronidase activity. L. plantarum when co-cultured with β-glucuronidase producing E. coli, in a basal media containing FOS as an energy source, could inhibit the growth of the pathogen during the course of fermentation. The results showed that L. plantarum CFR 2194 has the ability to utilize the prebiotic FOS as a selective carbon and energy source. The organism could inhibit the growth of the pathogen which produces β-glucuronidase and lowered its activity by the metabolites of FOS which indicates the probable use of L. plantarum through dietary intervention in combating colon carcinogenesis.     

Lactobacillus rhamnosus GG inhibits invasion of cultured human respiratory cells by prtF1-positive macrolide-resistant group A streptococci

Aims:  This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1.
Methods and Results:  Eight prtF1 -positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced ( P  <   0·001) GAS invasion efficiency in both assays. No antibacterial activity of Lactobacillus GG against GAS was detected. Both L-LGG and HK-LGG and all prtF1 -positive GAS induced a strong agglutination reaction using Fn-coated particles.
Conclusions:  Lactobacillus GG exerts an antagonistic action against GAS by inhibiting cell invasion. Competitive binding of Lactobacillus GG and GAS to Fn might be involved in the inhibition process.
Significance and Impact of the Study:  The finding that Lactobacillus GG can prevent in vitro invasion of respiratory cells by GAS suggests new applications for this probiotic strain and warrants further studies of its capacity to prevent GAS throat infections.     

Effects of Xylo-Oligosaccharides on Broiler Chicken Performance and Microbiota

In broiler chickens, feed additives, including prebiotics, are widely used to improve gut health and to stimulate performance. Xylo-oligosaccharides (XOS) are hydrolytic degradation products of arabinoxylans that can be fermented by the gut microbiota. In the current study, we aimed to analyze the prebiotic properties of XOS when added to the broiler diet. Administration of XOS to chickens, in addition to a wheat-rye-based diet, significantly improved the feed conversion ratio. XOS significantly increased villus length in the ileum. It also significantly increased numbers of lactobacilli in the colon and Clostridium cluster XIVa in the ceca. Moreover, the number of gene copies encoding the key bacterial enzyme for butyrate production, butyryl-coenzyme A (butyryl-CoA):acetate CoA transferase, was significantly increased in the ceca of chickens administered XOS. In this group of chickens, at the species level, Lactobacillus crispatus and Anaerostipes butyraticus were significantly increased in abundance in the colon and cecum, respectively. In vitro fermentation of XOS revealed cross-feeding between L. crispatus and A. butyraticus. Lactate, produced by L. crispatus during XOS fermentation, was utilized by the butyrate-producing Anaerostipes species. These data show the beneficial effects of XOS on broiler performance when added to the feed, which potentially can be explained by stimulation of butyrate-producing bacteria through cross-feeding of lactate and subsequent effects of butyrate on gastrointestinal function.     

Production of conjugated linoleic acid by probiotic Lactobacillus acidophilus La-5

Aims:  To study the ability of the probiotic culture Lactobacillus acidophilus La-5 to produce conjugated linoleic acid (CLA), which is a potent anti-carcinogenic agent.
Methods and Results:  The conversion of linoleic acid to CLA was studied both by fermentation in a synthetic medium and by incubation of washed cells. Accumulation of CLA was monitored by gas chromatography analysis of the biomass and supernatants. While the fermentation conditions applied may not be optimal to observe CLA production in growing La-5 cells, the total CLA surpassed 50% of the original content in the washed cells after 48 h under both aerobic and micro-aerobic conditions. The restriction of oxygen did not increase the yield, but favoured the formation of trans, trans isomers.
Conclusions:  The capability of L. acidophilus La-5 to produce CLA is not dependant on the presence of milk fat or anaerobic conditions. Regulation of CLA production in this strain needs to be further investigated to exploit the CLA potential in fermented foods.
Significance and Impact of the study:  Knowledge gained through the conditions on the accumulation of CLA would provide further insight into the fermentation of probiotic dairy products. The capacity of the nongrowing cells to produce CLA is also of great relevance for the emerging nonfermented probiotic foods.     

Synbiotics growth optimization of Bifidobacterium pseudocatenulatum G4 with prebiotics using a statistical methodology

Aims:  This study demonstrated the optimum growth of Bifidobacterium pseudocatenulatum G4 with prebiotics via statistical model.
Methods and Results:  Commercial prebiotics [inulin and fructooligosaccharide (FOS)], together with sorbitol, arabinan and inoculum rate, were tested by fractional factorial design to determine their impact on growth of Bif. pseudocatenulatum G4 in skim milk. At 48 h incubation, bacterial growth was mainly influenced by FOS and inoculum rate. Growth reduction was observed in all samples incubated for 72 h. Central composite design (CCD) was adopted using FOS and inoculum rate at 48 h incubation to develop the statistical model for optimization. The model predicted that 2·461 log CFU ml⁻¹ produced the optimum growth increase of Bif. pseudocatenulatum G4. The combination that produced the optimum point was 2·86% FOS (g/v) and 0·67% inoculum rate (v/v).
Conclusion:  At optimum combination of inoculum rate and FOS, validation experiments recorded 2·40 ± 10·02 log CFU ml⁻¹. The application in 1-l bioreactor for 24 h showed higher growth increase of 2·95 log CFU ml⁻¹.
Significant and Impact of the Study:  Response surface methodology approach is useful to develop optimum synbiotics combination for strain G4 with FOS.     

Indigestible carbohydrates alter the intestinal microbiota but do not influence the performance of broiler chickens

Aims:  Prebiotics are a potential alternative to in-feed antimicrobials to improve performance of chickens. We investigated the effects of mannanoligosaccharide (MOS) and fructooligosaccharide (FOS) on growth, performance and the intestinal microbiota.
Methods and Results:  Cobb 500 birds were fed either: Control, starter diet without antimicrobials; ZnB, Control + 50 ppm zinc bacitracin; MOS, Control + 5 g kg⁻¹ MOS; or FOS, Control + 5 g kg⁻¹ FOS. An energy metabolism study was conducted and intestinal microbial communities assessed by T-RFLP and Lac PCR-DGGE. Diet did not influence performance. Ileal microbial communities were significantly different in ZnB-fed birds compared to all diets, and FOS-fed chickens compared to Control. MOS-fed chickens had a different caecal profile to ZnB and FOS-fed birds. Consensus Lac PCR-DGGE profiles indicated Lactobacillus communities clustered according to diet with Lactobacillus johnsonii characteristic of ZnB diet. Control and MOS-fed chickens displayed significantly different jejunal Lactobacillus profiles to each other whilst ileal profiles were different between MOS and FOS-fed birds.
Conclusion:  Prebiotics influenced the intestinal microbiota, but did not affect performance.
Significance and Impact of the Study:  In light of pressure for in-feed antimicrobial withdrawal, the impact of alternative compounds on the intestinal microbiota and bird performance is critical to the poultry industry.     

Quantification of anammox populations enriched in an immobilized microbial consortium with low levels of ammonium nitrogen and at low temperature

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).     

FISH analysis of Lactobacillus biofilms in the gastrointestinal tract of different hosts

Aims: To investigate the spatial organization of endogenous and exogenously applied Lactobacillus communities at specific locations in the adult gastrointestinal tract of different hosts. Methods and Results: Samples of the human, murine and avian gastrointestinal tract of subjects that received or not received a Lactobacillus probiotic were analysed by fluorescence in situ hybridization (FISH) with rRNA‐targeted probes. High levels of endogenous lactobacilli were observed on the nonsecretory, stratified squamous epithelia present in the forestomach of mice and crop of chickens, respectively. These epithelial associations showed characteristics of bacterial biofilms, i.e. bacteria attached to a surface and embedded in a matrix of extracellular polymeric substances. In other regions of the analysed intestines, lactobacilli seemed to occur mainly as dispersed bacterial cells or as microcolonies. Exogenous administration of a Lactobacillus probiotic did increase the levels of loosely adherent Lactobacillus cells detected. However, the probiotic strains were unable to establish themselves inside the gastrointestinal biofilms. Conclusions: Gastrointestinal biofilms of lactobacilli occur only in specific niches in certain hosts, such as the murine forestomach and avian crop. Significance and Impact of the Study: Biofilm formation by lactobacilli in specific parts of animal gastrointestinal tracts was documented for the first time by FISH.     

Diversity among Lactobacillus paracasei phages isolated from a probiotic dairy product plant

Aims:  To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei .
Methods and Results:  Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk.
Conclusions:  Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making.
Significance and Impact of the Study:  This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.     

Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Bifidobacterium and Lactobacillus DNA in the human placenta

Aims:  Bifidobacteria and lactobacilli are part of the human normal intestinal microbiota and may possibly be transferred to the placenta. It was hypothesized that intestinal bacteria or their components are present in the placenta and that the foetus may be exposed to them. We investigated the presence of bifidobacteria and lactobacilli and their DNA in the human placenta.
Methods and Results:  We studied 34 human placentae (25 vaginal and nine caesarean deliveries) for the presence Bifidobacterium spp. and Lactobacillus rhamnosus. Cultivation was used for the detection of viable cells and genus and species-specific PCR for the detection of DNA. No bifidobacteria or lactobacilli were found by cultivation. Bifidobacterial DNA was detected in 33 and L. rhamnosus DNA in 31 placenta samples.
Conclusions:  DNA from intestinal bacteria was found in most placenta samples. The results suggest that horizontal transfer of bacterial DNA from mother to foetus may occur via placenta.
Significance and Impact of the Study:  Bacterial DNA contains unmethylated CpG oligodeoxynucleotide motifs which induce immune effects. Specific CpG motifs activate Toll-like receptor 9 and subsequently trigger Th-1-type immune responses. Although the newborn infant is considered immunologically immature, exposure by bacterial DNA may programme the infant's immune development during foetal life earlier than previously considered.     

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims:  To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages.
Methods and Results:  We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei , Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet (M) and erm (B).
Conclusions:  This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy.
Significance and Impact of the Study:  Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.