Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan.

Background: The study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. Methodology: Frequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. Results: We found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. Conclusion: BAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Genomic and Phenotypic Insight into the Probiotic Potential of Lactic Acid Bacterial spp. Associated with the Human Gut Mucosa.

Commensal microbiome-based health support is gaining respect in the medical community and new human gut-associated Lactic Acid Bacteria (LAB) strains must be evaluated for their probiotic potential. Here we characterized the phenotype and genomes of human ileocecal mucosa-associated LAB strains using metagenomic sequencing and in vitro testing. The strains characterized belonged to the genus Enterococcus (Enterococcus lactis NPL1366, NPL1371, and Enterococcus mundtii NPL1379) and Lactobacillus (Lactobacillus paragasseri, NPL1369, NPL1370, and Lactiplantibacillus plantarum NPL1378). Genome annotation suggested bacterial adaptation to both human physiological and industrial manufacturing-related stressors. Genes for histidine kinases in enterococci and Na + /K + antiporters and F0F1 ATP synthases in Lactobacillus strains may support their tolerance to acid seen in vitro. The bile salt hydrolase (BSH) gene in Lp. plantarum and L. paragasseri may help explain their reported bile salt deconjugation and cholesterol-lowering behavior. Thioredoxin is the principal antioxidant system, and several oxidases and general stress-related proteins are found in lactobacilli, most notably in L. plantarum NPL1378. Multiple adhesion and biofilm-related genes were predicted in the LAB genomes. Adhesion and biofilm-related genes figured prominently in the genomes of enterococcal strains, especially E. lactis, corresponding to its biofilm formation capacity in vitro. Bacteriocin and secondary metabolite biosynthetic gene clusters in the sequenced genomes of E. lactis NPL1366 and Lp. plantarum NPL1378 may explain their in vitro pathogenic antagonism. Moreover, folate producing Lp. plantarum strain holds potential to be used in therapeutics or biofortification of food. All the strains were deemed safe through in vitro and in silico analysis. This basic genetic and phenotypic information supports their contention as probiotic adjuncts to conventional medical therapy.

Mechanism of the antidiabetic action of Nigella sativa and Thymoquinone: a review.

Introduction: Long used in traditional medicine, Nigella sativa (NS; Ranunculaceae) has shown significant efficacy as an adjuvant therapy for diabetes mellitus (DM) management by improving glucose tolerance, decreasing hepatic gluconeogenesis, normalizing blood sugar and lipid imbalance, and stimulating insulin secretion from pancreatic cells. In this review, the pharmacological and pharmacokinetic properties of NS as a herbal diabetes medication are examined in depth, demonstrating how it counteracts oxidative stress and the onset and progression of DM. Methods: This literature review drew on databases such as Google Scholar and PubMed and various gray literature sources using search terms like the etiology of diabetes, conventional versus herbal therapy, subclinical pharmacology, pharmacokinetics, physiology, behavior, and clinical outcomes. Results: The efficiency and safety of NS in diabetes, notably its thymoquinone (TQ) rich volatile oil, have drawn great attention from researchers in recent years; the specific therapeutic dose has eluded determination so far. TQ has anti-diabetic, anti-inflammatory, antioxidant, and immunomodulatory properties but has not proved druggable. DM's intimate link with oxidative stress, makes NS therapy relevant since it is a potent antioxidant that energizes the cell's endogenous arsenal of antioxidant enzymes. NS attenuates insulin resistance, enhances insulin signaling, suppresses cyclooxygenase-2, upregulates insulin-like growth factor-1, and prevents endothelial dysfunction in DM. Conclusion: The interaction of NS with mainstream drugs, gut microbiota, and probiotics opens new possibilities for innovative therapies. Despite its strong potential to treat DM, NS and TQ must be examined in more inclusive clinical studies targeting underrepresented patient populations.

Insights from metagenome-assembled genomes on the genetic stability and safety of over-the-counter probiotic products.

The demand for and acceptance of probiotics is determined by their quality and safety. Illumina NGS sequencing and analytics were used to examine eight marketed probiotics. Up to the species level, sequenced DNA was taxonomically identified, and relative abundances were determined using Kaiju. The genomes were constructed using GTDB and validated through PATRICK and TYGS. A FastTree 2 phylogenetic tree was constructed using several type strain sequences from relevant species. Bacteriocin and ribosomally synthesized polypeptide (RiPP) genes were discovered, and a safety check was performed to test for toxins, antibiotic resistance, and genetic drift genes. Except for two products with unclaimed species, the labeling was taxonomically correct. In three product formulations, Lactobacillus acidophilus, Limosilactobacillus reuteri, Lacticaseibacillus paracasei, and Bifidobacterium animalis exhibited two to three genomic alterations, while Streptococcus equinus was found in one. TYGS and GDTB discovered E. faecium and L. paracasei in distinctly different ways. All the bacteria tested had the genetic repertoire to tolerate GIT transit, although some exhibited antibiotic resistance, and one strain had two virulence genes. Except for Bifidobacterium strains, the others revealed a variety of bacteriocins and ribosomally synthesized polypeptides (RiPP), 92% of which were unique and non-homologous to known ones. Plasmids and mobile genetic elements are present in strains of L. reuteri (NPLps01.et_L.r and NPLps02.uf_L.r), Lactobacillus delbrueckii (NPLps01.et_L.d), Streptococcus thermophilus (NPLps06.ab_S.t), and E. faecium (NPLps07.nf_E.f). Our findings support the use of metagenomics to build better and efficient production and post-production practices for probiotic quality and safety assessment.

Probiotic profiling of bifidobacteria indigenous to the human intestinal mucosa shows alleviation of dysbiosis-associated pathogen biofilms.

The present study was undertaken to isolate bifidobacterial probiotics and characterize the biodiversity of mucosal bacteria in the human distal gut through 16S rRNA amplicon sequencing. Bifidobacterial strains obtained by selective culturing were investigated for biofilms and probiotic characteristics. Both culture-dependent and culture-independent approaches revealed substantial microbial diversity. Bifidobacterium strains yielded robust biofilms with predominantly exopolysaccharides and eDNA matrix. Microscopy revealed species-dependent spatial arrangement of microcolonies. Following probiotic profiling and safety assessment, the inter- and intra-specific interactions in in dual strain bifidobacterial biofilms were studied. As a species, only strains of B. bifidum exhibited exclusively inductive type of interactions whereas in other species, the interactions were more varied. On the other hand, in dual species biofilms, a preponderance of inductive interactions was evident between B. adolescentis, B. thermophilum, B. bifidum, and B. longum. The strong biofilm-formers also diminished pathogenic biofilm viability, and some were proficient in cholesterol removal in vitro. None of the strains exhibited harmful enzymatic activities associated with disease pathology. Interaction between biofilm-forming bifidobacterial strains provides an understanding of their functionality and persistence in the human host, and food or medicine. Their anti-pathogenic activity represents a therapeutic strategy against drug-resistant pathogenic biofilms.

Potentially probiotic Limosilactobacillus reuteri from human milk strengthens the gut barrier in T84 cells and a murine enteroid model.

AIMS: At conception, the infant gut barrier is immature, gradually developing with regular intake of maternal milk. This study addressed whether the barrier-strengthening effect of breast feeding might be attributable, at least in part, to autochthonous beneficial human milk bacteria. METHODS AND RESULTS: Twelve bacterial strains from the breast milk of Pakistani mothers who underwent cesarean delivery (NPL-88, NPL-157, NPL-179, NPL-181, NPL-388 (Limosilactobacillus reuteri), NPL-76, NPL-495, NPL-504 (Limosilactobacillus fermentum), NPL-415 (Lactobacillus pentosus), NPL-412, NPL-416 (Lactiplantibacilllus plantarum) and NPL-374 (Bifidobacterium longum) were shortlisted based on their tolerance to acidic pH (2.8-4.2) and bile (0.1-0.3%). The effect of these bacteria on gut barrier function in the presence and absence of pathogens was assessed as changes in transepithelial electrical resistance (TEER) in the human T84 colonic epithelial cell line and in murine enteroid-derived monolayers (EDMs). The TEER of T84 cells monolayers rose in the presence of most of the human milk strains, being most pronounced in case of L. reuteri NPL-88 (34% within five h), exceeding the effect of the well-known probiotic L. acidophilus (20%). qRT-PCR, western blot and immunofluorescent staining associated the increase in TEER with enhanced expression of tight junction proteins. Pretreatment of murine EDMs with NPL-88 also largely prevented the ability of the pathogen, Salmonella, to decrease TEER (87 ± 1.50%; P < 0.0001, n = 4). CONCLUSIONS: Human milk lactic acid bacteria are potential probiotics that can strengthen gut barrier function and protect breastfed neonates against enteric infections.

The Weissella Genus: Clinically Treatable Bacteria with Antimicrobial/Probiotic Effects on Inflammation and Cancer.

Weissella is a genus earlier considered a member of the family Leuconostocaceae, which was reclassified into the family Lactobacillaceae in 1993. Recently, there have been studies emphasizing the probiotic and anti-inflammatory potential of various species of Weissella, of which W. confusa and W. cibaria are the most representative. Other species within this genus include: W. paramesenteroides, W. viridescens, W. halotolerans, W. minor, W. kandleri, W. soli, W. ghanensis, W. hellenica, W. thailandensis, W. fabalis, W. cryptocerci, W. koreensis, W. beninensis, W. fabaria, W. oryzae, W. ceti, W. uvarum, W. bombi, W. sagaensis, W. kimchi, W. muntiaci, W. jogaejeotgali, W. coleopterorum, W. hanii, W. salipiscis, and W. diestrammenae. Weissella confusa, W. paramesenteroides, W. koreensis, and W. cibaria are among the few species that have been isolated from human samples, although the identification of these and other species is possible using metagenomics, as we have shown for inflammatory bowel disease (IBD) and healthy controls. We were able to isolate Weissella in gut-associated bacteria (post 24 h food deprivation and laxatives). Other sources of isolation include fermented food, soil, and skin/gut/saliva of insects/animals. With the potential for hospital and industrial applications, there is a concern about possible infections. Herein, we present the current applications of Weissella on its antimicrobial and anti-inflammatory mechanistic effects, the predisposing factors (e.g., vancomycin) for pathogenicity in humans, and the antimicrobials used in patients. To address the medical concerns, we examined 28 case reports focused on W. confusa and found that 78.5% of infections were bacteremia (of which 7 were fatal; 1 for lack of treatment), 8 were associated with underlying malignancies, and 8 with gastrointestinal procedures/diseases of which 2 were Crohn's disease patients. In cases of a successful resolution, commonly administered antibiotics included: cephalosporin, ampicillin, piperacillin-tazobactam, and daptomycin. Despite reports of Weissella-related infections, the evolving mechanistic findings suggest that Weissella are clinically treatable bacteria with emerging antimicrobial and probiotic benefits ranging from oral health, skin care, obesity, and inflammatory diseases to cancer.

Compositional Quality and Possible Gastrointestinal Performance of Marketed Probiotic Supplements.

The local pharmacies and shops are brimming with various probiotic products that herald a range of health benefits. The poor quality of probiotic products in both dosage and species is symptomatic of this multi-billion-dollar market making it difficult for consumers to single out reliable ones. This study aims to fill the potential gap in the labeling accuracy of probiotic products intended for human consumption. We describe a combinatorial approach using classical culture-dependent technique to quantify and molecular techniques (16 s rRNA gene sequencing, multilocus sequence, and ribotyping) for strain recognition of the microbial contents. The full gamut of probiotic characteristics including acid, bile and lysozyme tolerances, adhesiveness, anti-pathogenicity, and degree of safeness were performed. Their capacity to endure gastro-intestinal (GIT) stresses and select drugs was assessed in vitro. Our results forced us to declare that the local probiotic market is essentially unregulated. Almost none of the probiotic products tested met the label claim. Some (11%) have no viable cells, and a quarter (27%) showing significant inter-batch variation. A lower microbial count was typical with undesirables constituting a quarter of the total (~ 27%). Half of the products contained antibiotic-resistant strains; the unregulated use of these probiotics carries the risk of spreading antibiotic resistance to gut pathobionts. Poor tolerance to gut conditions and mediocre functionalism make the case worse. The current regulatory systems do not take this discrepancy into account. We recommend an evidence-based regular market surveillance of marketed probiotics to ensure the authenticity of the claims and product effectiveness.

A potentially probiotic strain of Enterococcus faecalis from human milk that is avirulent, antibiotic sensitive, and nonbreaching of the gut barrier.

Human milk is a key source of promising probiotic lactic acid bacteria. The Enterococcus species, because of their dual commensal and pathogenic nature, demand critical safety analysis to establish them as probiotic candidates. In this study, eighteen E. faecalis strains from human milk of mothers living in Pakistan were typed at the strain level by riboprinting. The typed strains were then evaluated in vitro for physiological safety and the presence of transmissible antibiotic resistance genes, adhesion genes, biogenic amines, and virulence factors. Selected strains were then checked for tolerance to gastrointestinal acid and bile as criteria for probiotic efficacy. Molecular typing revealed that the strains fell into five distinct clusters or ribotypes. Testing revealed that they were non-hemolytic; however, all strains had gelatinase activity except NPL-493. The isolates were susceptible to most clinically important antibiotics except streptomycin. Molecular screening for antibiotic resistance genes, adhesion genes, biogenic amines, and virulence factors indicated that none of the strains possessed resistance genes for aminoglycosides, vancomycin, bacitracin, tetracycline, or clindamycin. Most virulence factors were absent except for the genes gelE and efaAs associated with gut adhesion and translocation, which were present in all except NPL-493. Strain NPL-493 was the most promising probiotic candidate demonstrating significant tolerance to the acid, bile, and digestive enzymes in the human GIT and antibacterial activity against multiple pathogens. The study concluded that E. faecalis NPL-493 from human milk was safe among all the strains and could be considered a potential probiotic.

Aggrandizement of fermented cucumber through the action of autochthonous probiotic cum starter strains of Lactiplantibacillus plantarum and Pediococcus pentosaceus.

PURPOSE: Cucumber fermentation is traditionally done using lactic acid bacteria. The involvement of probiotic cultures in food fermentation guarantees enhanced organoleptic properties and protects food from spoilage. METHODS: Autochthonous lactic acid bacteria were isolated from spontaneously fermented cucumber and identified to species level. Only strains adjudged as safe for human consumption were examined for their technological and functional characteristics. Strain efficiency was based on maintaining high numbers of viable cells during simulated GIT conditions and fermentation, significant antioxidant activity, EPS production, nitrite degradation, and antimicrobial ability against Gram-positive and Gram-negative foodborne pathogens. RESULT: Two strains, Lactiplantibacillus plantarum NPL 1258 and Pediococcus pentosaceus NPL 1264, showing a suite of promising functional and technological attributes, were selected as a mixed-species starter for carrying out a controlled lactic acid fermentations of a native cucumber variety. This consortium showed a faster lactic acid-based acidification with more viable cells, at 4% NaCl and 0.2% inulin (w/v) relative to its constituent strains when tested individually. Sensory evaluation rated the lactofermented cucumber acceptable based on texture, taste, aroma, and aftertaste. CONCLUSION: The results suggest that the autochthonous LAB starter cultures can shorten the fermentation cycle and reduce pathogenic organism' population, thus improving the shelf life and quality of fermented cucumber. The development of these new industrial starters would increase the competitiveness of production and open the country's frontiers in the fermented vegetable market.

Metataxonomic analysis of microbiota from Pakistani dromedary camelids milk and characterization of a newly isolated Lactobacillus fermentum strain with probiotic and bio-yogurt starter traits.

This study was undertaken to investigate the starter and probiotic potential of lactic acid bacteria isolated from dromedarian camel's milk using both culture-dependent and -independent approaches and metataxonomic analysis. Strains of lactic acid bacteria recovered were examined in vitro for tolerance to gastric acidity, bile, and lysozyme. Bile salt hydrolysis, serum cholesterol-lowering, oxalate degradation, proteolytic activity, exopolysaccharide production, and cell surface characteristics necessary for colonizing intestinal mucosa were also evaluated. A single strain of the species, Lactobacillus fermentum named NPL280, was selected through multivariate analysis as it harbored potential probiotic advantages and fulfilled safety criteria. The strain assimilated cholesterol, degraded oxalate, produced exopolysaccharides, and proved to be a proficient alternate yogurt starter with good viability in stored bio-yogurt. A sensorial analysis of the prepared bio-yogurt was also found to be exemplary. We conclude that the indigenous L. fermentum strain NPL280 has the desired traits of a starter and adjunct probiotic culture for dairy products.

Mining indigenous honeybee gut microbiota for Lactobacillus with probiotic potential.

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.

Evaluation of the probiotic and postbiotic potential of lactic acid bacteria from artisanal dairy products against pathogens.

INTRODUCTION: Probiotic and postbiotic potential of thirty-two strains of lactic acid bacteria (LAB), obtained earlier from artisanal dairy sources in Pakistan, have been investigated against major multi-drug resistant (MDR) and food borne pathogenic bacteria. METHODOLOGY: LAB strains were identified by 16S rRNA gene sequencing and their antibacterial activity was assessed by the microdilution method. Four LAB isolates, Weissella confusa PL6, Enterococcus faecium PL7, and Lactobacillus delbrueckii PL11 and PL13 were shortlisted. Their ability to degrade lactose and safety for human consumption in terms of hemolysis and antibiotic susceptibility were assessed in vitro. The antibacterial components in the cell-free supernatants (CFSs) of isolate cultures were characterized biochemically by HPLC. RESULTS: Acid neutralization but not protease treatment abolished the antibacterial activity of CFSs. Lactic, acetic and propionic acids were the main acids in the CFSs, and acid production peaked in the stationary phase of growth. The antibacterial activity of the LAB cultures resulted from secretion of organic acids that lowered the pH. The strains exhibited variable ability to degrade lactose and were non-hemolytic and susceptible to the most common antibiotics. CONCLUSIONS: These LAB strains are probiotic candidates for further investigation of their postbiotic role in naturally preserving processed foods and for attenuation of lactose intolerance.

Generation of Lactose- and Protease-Positive Probiotic Lacticaseibacillus rhamnosus GG by Conjugation with Lactococcus lactis NCDO 712.

Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L. rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L. lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L. rhamnosus LAB49 effectively degraded ß-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GMOs), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture.IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.

Lactobacillus commensals autochthonous to human milk have the hallmarks of potent probiotics.

Maternal milk is an important source of essential nutrients for the optimal growth of infants. Breastfeeding provides a continuous supply of beneficial bacteria to colonize the infant gastrointestinal tract (GIT) and offers health benefits for disease prevention and immunity. The purpose of this study was to isolate novel probiotic strains from the breast milk of native Pakistani mothers and to evaluate their probiotic potential. We isolated 21 strains of bacteria from the colostrum and mature milk of 20 healthy mothers, who had vaginal deliveries and were not taking antibiotics. After phenotypic and genotypic characterization, these isolates were tested for survival in the GIT using in vitro acid and bile tests. Nine strains showing good acid tolerance were assessed for their growth rate, bile resistance and ability to hydrolyze bile salts. Out of the four Lactobacillus isolates adjudged to be most promising as probiotics, three were Lactobacillus fermentum strains and one was a strain of Lactobacillus oris. This study demonstrates that human milk is a viable source of commensal bacteria beneficial to both adults and babies.

Lactobacillus fermentum strains of dairy-product origin adhere to mucin and survive digestive juices.

Introduction. There is an ever present need to isolate and characterize indigenous bacterial strains with potential probiotic health benefits for humans.Aim. Lactobacillus fermentum of dairy origin was focused because of its propensity to adhere to the intestinal glycoprotein, mucin.Methodology. The lactobacillus strains were screened for mucin adhesion, resistance to low pH and bile, autoaggregation, hydrophobicity, and survival in an in vitro digestion model. The cholesterol-lowering and oxalate-degrading effects of selected strains were also determined. Safety was assessed for haemolytic, mucinolytic and gelatinase activity, biogenic amine production, antibiotic resistance and phenol resistance. Expression of the 32-mmub adhesion-related gene was also measured following strain exposure to simulated gastrointestinal tract (GIT) digestion.Results. The selected mucin-adhesive strains were tolerant to acid (pH 3.0) and bile (0.25 %) and demonstrated >85 % survival following simulated human digestion in the presence of milk. The digestive treatment did not affect the adhesive potential of PL20, and PL27, regardless of the food matrix. The simulated digestion had less effect on their adhesion than on the type strain and it also did not correlate with the mmub gene expression level as determined by qPCR. The selected strains exhibited cholesterol removal (36-44 %) and degraded oxalate (66-55 %). Neither of these strains exhibited undesirable characteristics.Conclusion. These preliminary findings suggest a functionality in the two strains of L. fermentum with high colonization potential on GIT mucosal membranes and possible health-promoting effects. This prima facie evidence suggests the need for further studies to test these probiotic candidates as live biotherapeutic agents in vivo.

Biofilm development in L. fermentum under shear flow & sequential GIT digestion.

The objective of this study was to investigate biofilm formation by Lactobacillus fermentum under physiologically relevant shear conditions both in the presence and absence of a food matrix and under simulated conditions of digestion. This was done using batch and flow biofilms of L. fermentum strains under conditions simulating digestion in the human gastrointestinal tract and shear flow using a high throughput platform BioFlux 1000Z system. The putative probiotic strain, PL29, was found to be capable of adhesion and biofilm formation in mucin-coated microfluidic channels under liquid flow conditions mimicking those of the GIT. Based on these in vitro measurements, we conclude that L. fermentum strain PL29 could be an effective probiotic for human consumption.

Cholate-stimulated biofilm formation by Lactococcus lactis cells.

Bile acid resistance by Lactococcus lactis depends on the ABC-type multidrug transporter LmrCD. Upon deletion of the lmrCD genes, cells can reacquire bile acid resistance upon prolonged exposure to cholate, yielding the ΔlmrCD(r) strain. The resistance mechanism in this strain is non-transporter based. Instead, cells show a high tendency to flocculate, suggesting cell surface alterations. Contact angle measurements demonstrate that the ΔlmrCD(r) cells are equipped with an increased cell surface hydrophilicity compared to those of the parental and wild-type strains, while the surface hydrophilicity is reduced in the presence of cholate. ΔlmrCD(r) cells are poor in biofilm formation on a hydrophobic polystyrene surface, but in the presence of subinhibitory concentrations of cholate, biofilm formation is strongly stimulated. Biofilm cells show an enhanced extracellular polymeric substance production and are highly resistant to bile acids. These data suggest that non-transporter-based cholate resistance in L. lactis is due to alterations in the cell surface that stimulate cells to form resistant biofilms.