In vitro model of human mammary gland microbial colonization (MAGIC) demonstrates distinctive cytokine response to imbalanced human milk microbiota.

Despite the established concept of the human mammary gland (MG) as a habitat with its own microbiota, the exact mechanism of MG colonization is still elusive and a well-characterized in vitro model would reinforce studies of the MG microbiota development. We aimed to establish and characterize an in vitro cell model for studying MAmmary Gland mIcrobial Colonization (MAGIC) model. We used the immortalized cell line MCF10A, which expresses the strong polarized phenotype similar to MG ductal epithelium when cultured on a permeable support (Transwell). We analyzed the surface properties of the MAGIC model by gene expression analysis of E-cadherin, tight junction proteins, and mucins and by scanning electron microscopy. To demonstrate the applicability of the model, we tested the adhesion capability of the whole human milk (HM) microbial community and the cellular response of the model when challenged directly with raw HM samples. MCF10A on permeable supports differentiated and formed a tight barrier, by upregulation of CLDN8, MUC1, MUC4, and MUC20 genes. The surface of the model was covered with mucins and morphologically diverse with at least two cell types and two types of microvilli. Cells in the MAGIC model withstood the challenge with heat-treated HM samples and responded differently to the imbalanced HM microbiota by distinctive cytokine response. The microbial profile of the bacteria adhered on the MAGIC model reflected the microbiological profile of the input HM samples. The well-studied MAGIC model could be useful for studies of bacterial attachment to the MG and for in vitro studies of biofilm formation and microbiota development.IMPORTANCEThe MAGIC model may be particularly useful for studies of bacterial attachment to the surface of the mammary ducts and for in vitro studies of biofilm formation and the development of the human mammary gland (MG) microbiota. The model is also useful for immunological studies of the interaction between bacteria and MG cells. We obtained pioneering information on which of the bacteria present in the raw human milk (HM) were able to attach to the epithelium treated directly with raw HM, as well as on the effects of bacteria on the MG epithelial cells. The MAGIC cell model also offers new opportunities for research in other areas of MG physiology, such as the effects of bioactive milk components on microbial colonization of the MG, mastitis prevention, and studies of probiotic development. Since resident MG bacteria may be an important factor in breast cancer development, the MAGIC in vitro tool also offers new opportunities for cancer research.

Genomic insights into antibiotic resistance and mobilome of lactic acid bacteria and bifidobacteria.

Lactic acid bacteria (LAB) and Bifidobacterium sp. (bifidobacteria) can carry antimicrobial resistance genes (ARGs), yet data on resistance mechanisms in these bacteria are limited. The aim of our study was to identify the underlying genetic mechanisms of phenotypic resistance in 103 LAB and bifidobacteria using whole-genome sequencing. Sequencing data not only confirmed the presence of 36 acquired ARGs in genomes of 18 strains, but also revealed wide dissemination of intrinsic ARGs. The presence of acquired ARGs on known and novel mobile genetic elements raises the possibility of their horizontal spread. In addition, our data suggest that mutations may be a common mechanism of resistance. Several novel candidate resistance mechanisms were uncovered, providing a basis for further in vitro studies. Overall, 1,314 minimum inhibitory concentrations matched with genotypes in 92.4% of the cases; however, prediction of phenotype based on genotypic data was only partially efficient, especially with respect to aminoglycosides and chloramphenicol. Our study sheds light on resistance mechanisms and their transferability potential in LAB and bifidobacteria, which will be useful for risk assessment analysis.

Safety evaluation of enterococci isolated from raw milk and artisanal cheeses made in Slovenia and Serbia.

Enterococci represent a significant part of the non-starter LAB microbiota of artisanal cheeses produced mainly from raw milk. Common approaches to safety evaluation of enterococci isolates include assessment of antimicrobial resistance and virulence potential. Hence, a collection of 47 (n = 22, Serbia; n = 25, Slovenia) dairy enterococcal isolates, of which E. faecalis (n = 28), E. faecium (n = 11), E. durans (n = 5), E. casseliflavus (n = 2), and E. gallinarum (n = 1), was analyzed. The susceptibility to 12 antimicrobials was tested using a broth microdilution method, and the presence of the selected antimicrobial resistance and virulence genes was investigated using PCR. Isolates were resistant to tetracycline (TET) (25.5%), erythromycin (ERY) (17.0%), gentamycin and chloramphenicol (CHL) (∼6%). No resistance to ampicillin (AMP), ciprofloxacin (CIP), daptomycin (DAP), linezolid (LZD), teicoplanin (TEI), tigecycline (TGC) and vancomycin (VAN) was detected. Among all the resistance determinants analyzed, ermB gene was detected most frequently. All 10 virulence genes analyzed were detected with a distribution of cpd (72.3%), cob and ccf (70.2%), gelE (68.1%), hyl (59.6%), agg (53.2%) and esp (46.8%). The genes encoding cytolysin (cylA, cylM and cylB) were amplified to a lesser extent (21.3%, 21.3% and 12.8%, respectively). However, due to the limited number of enterococci isolates analyzed in the present study, further studies are still required in order to better document the safety status of dairy enterococci.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool.

Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota.Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences.

Polysaccharide Hydrogels for the Protection of Dairy-Related Microorganisms in Adverse Environmental Conditions.

Adverse environmental conditions are severely limiting the use of microorganisms in food systems, such as probiotic delivery, where low pH causes a rapid decrease in the survival of ingested bacteria, and mixed-culture fermentation, where stepwise changes and/or metabolites of individual microbial groups can hinder overall growth and production. In our study, model probiotic lactic acid bacteria (L. plantarum ATCC 8014, L. rhamnosus GG) and yeasts native to dairy mixed cultures (K. marxianus ZIM 1868) were entrapped in an optimized (cell, alginate and hardening solution concentration, electrostatic working parameters) Ca-alginate system. Encapsulated cultures were examined for short-term survival in the absence of nutrients (lactic acid bacteria) and long-term performance in acidified conditions (yeasts). In particular, the use of encapsulated yeasts in these conditions has not been previously examined. Electrostatic manufacturing allowed for the preparation of well-defined alginate microbeads (180-260 µm diameter), high cell-entrapment (95%) and viability (90%), and uniform distribution of the encapsulated cells throughout the hydrogel matrix. The entrapped L. plantarum maintained improved viabilities during 180 min at pH 2.0 (19% higher when compared to the free culture), whereas, L. rhamnosus appeared to be less robust. The encapsulated K. marxianus exhibited double product yields in lactose- and lactic acid-modified MRS growth media (compared to an unfavorable growth environment for freely suspended cells). Even within a conventional encapsulation system, the pH responsive features of alginate provided superior protection and production of encapsulated yeasts, allowing several applications in lacto-fermented or acidified growth environments, further options for process optimization, and novel carrier design strategies based on inhibitor charge expulsion.

Evaluation of Dietary Supplements Containing Viable Bacteria by Cultivation/MALDI-TOF Mass Spectrometry and PCR Identification.

The insufficient quality of products containing beneficial live bacteria in terms of content and viability of labelled microorganisms is an often-reported problem. The aim of this work was to evaluate the quality of dietary supplements containing viable bacteria available in Slovenian pharmacies using plate counting, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and species- or subspecies-specific PCR with DNA isolated from consortia of viable bacteria, from individual isolates, or directly from the products. Twelve percent of the products (3 of 26) contained insufficient numbers of viable bacteria. Eighty-three of the labelled species (111 in total) were confirmed by PCR with DNA from the product; 74% of these were confirmed by PCR with DNA from viable consortium, and 65% of these were confirmed by MALDI-TOF MS analysis of colonies. Certain species in multi-strain products were confirmed by PCR with DNA from viable consortia but not by MALDI-TOF MS, suggesting that the number of isolates examined (three per labelled strain) was too low. With the exception of Lacticaseibacillus casei and closely related species (Lacticaseibacillus rhamnosus and Lacticaseibacillus zeae), PCR and MALDI-TOF identification results agreed for 99% of the isolates examined, although several MALDI-TOF results had lower score values (1.700-1.999), indicating that the species identification was not reliable. The species L. zeae, which appeared in 20 matches of the Biotyper analysis, was identified as L. rhamnosus by PCR. The MALDI-TOF MS analysis was also unsuccessful in detecting Lactobacillus acidophilus La-5 and Bacillus coagulans due to missing peaks and unreliable identification, respectively. Mislabelling was detected by both methods for two putative L. casei strains that turned out to belong to the species Lacticaseibacillus paracasei. PCR remains more successful in subspecies-level identification as long as the database of MALDI-TOF MS spectra is not expanded by building in-house databases. The lack of positive PCR results with viable consortia or colonies, but positive PCR results with DNA isolated directly from the products observed in 10% (11/112) of the labelled strains, suggests the presence of non-culturable bacteria in the products. MALDI-TOF MS is a faster and simpler alternative to PCR identification, provided that a sufficient number of colonies are examined. Generation of in-house library may further improve the identification accuracy at the species and sub-species level.

Characterization of antimicrobial resistance in lactobacilli and bifidobacteria used as probiotics or starter cultures based on integration of phenotypic and in silico data.

Lactic acid bacteria and bifidobacteria deliberately introduced into the food chain may act as a reservoir of antimicrobial resistance genes (ARGs), which is considered a safety concern. In the present study, resistance to antimicrobials of commercial probiotic strains, probiotic candidate strains, and starter cultures (n = 20) was characterised based on integration of phenotypic and in silico data. Minimum inhibitory concentrations (MICs) of 16 antimicrobials were determined for lactobacilli and bifidobacteria that were isolated from pharmaceutical products or obtained from the manufacturers or culture collections. Using different databases and bioinformatic tools, we predicted ARGs, mutations, genomic islands, and mobile genetic elements (MGEs) in their whole genome sequences. In addition, a comprehensive in silico analysis of the prevalence of the tetW gene and its genetic environment across lactobacilli and bifidobacteria (n = 1423) was conducted. Several strains exhibited phenotypic resistance to kanamycin, tetracycline, chloramphenicol, quinupristin-dalfopristin, ciprofloxacin, or neomycin. These resistances, however, did not always correspond to the presence of ARGs and vice versa. We detected an acquired tetW gene in four commercial strains of Bifidobacterium animalis subsp. lactis, whereas homologs of antimicrobial resistance (AR) proteins were predicted in all 20 proteomes. The prevalence of the tetW gene, which was often flanked by MGEs, was higher in analysed bifidobacteria (31.9%) than lactobacilli (6.3%). In addition, sequences flanking tetW were associated with putative genomic islands and were conserved in several strains, including potential pathogens. Our findings provide an insight into AR of probiotics, probiotic candidates, and starter cultures with an emphasis on tetracycline and into the safety of these strains in the context of AR.

Tetracycline resistance in lactobacilli isolated from Serbian traditional raw milk cheeses.

The aim of this study was to investigate the presence of tetracycline resistance in lactobacilli isolated from traditional Serbian white brined raw milk cheeses (Homolje, Sjenica, Zlatar). Isolation of presumptive lactobacilli was initially performed using MRS-S agar without tetracycline, or supplemented with 16 and 64 µg/mL of tetracycline. Rep-PCR (GTG)5 genotyping showed a high diversity of the isolates obtained, as examination of 233 isolates resulted in 156 different Rep-PCR fingerprints. Ninety out of 156 (57.69%) of the strains, representatives with different (GTG)5 fingerprints, were identified by MALDI-TOF MS as lactobacilli, while 66 out of 156 (42.31%) strains were identified as members of other LAB genera. All except one out of 90 Lactobacillus isolates further tested by microdilution method, demonstrated unimodal distribution of tetracycline MIC values which were equal to or lower from the breakpoint MIC values (EFSA in EFSA J 10: 1-10, 2012. 10.2903/j.efsa.2012.2740). Only one Lb. paracasei isolate showed the presence of tet(M) gene, while the other analyzed tet genes [tet(A), tet(B), tet(C) tet(K), tet(L), tet(O) and tet(W)] were not detected in any of the isolates. The results of this study indicates that lactobacilli from traditional Serbian raw milk cheeses do not present considerable tetracycline resistance reservoirs. For final conclusions about the safety of these autochthonous cheeses regarding the possible tetracycline resistance transferability, the assessment of the entire cheese microbiota is needed.

Microbes in Infant Gut Development: Placing Abundance Within Environmental, Clinical and Growth Parameters.

Sound and timely microbial gut colonization completes newborn's healthy metabolic programming and manifests in infant appropriate growth and weight development. Feces, collected at 3, 30, and 90 days after birth from 60 breastfed Slovenian newborns, was submitted to microbial DNA extraction and qPCR quantification of selected gut associated taxa. Multivariate regression analysis was applied to evaluate microbial dynamics with respect to infant demographic, environmental, clinical characteristics and first year growth data. Early microbial variability was marked by the proportion of Bacilli, but diminished and converged in later samples, as bifidobacteria started to prevail. The first month proportions of enterococci were associated with maternity hospital locality and supplementation of breastfeeding with formulae, while Enterococcus faecalis proportion reflected the mode of delivery. Group Bacteroides-Prevotella proportion was associated with infant weight and ponderal index at first month. Infant mixed feeding pattern and health issues within the first month revealed the most profound and extended microbial perturbations. Our findings raise concerns over the ability of the early feeding supplementation to emulate and support the gut microbiota in a way similar to the exclusively breastfed infants. Additionally, practicing supplementation beyond the first month also manifested in higher first year weight and weight gain Z-score.

Generation of Lactobacillus plantarum strains with improved potential to target gastrointestinal disorders related to sugar malabsorption.

Malabsorption of dietary sugars is a common cause of gastrointestinal discomfort, affecting up to one in three people with debilitating symptoms, such as abdominal pain, osmotic diarrhoea, bloating and flatulence. Besides dietary interventions, it has been suggested that ingestion of lactobacilli may alleviate these symptoms. The objectives of this study were to generate strains with improved potential to ameliorate sugar malabsorption related gastrointestinal disorders. Initial selection was made from 183 natural isolates of lactic acid bacteria, on the basis of broad sugar fermentation ability, absence of gas production, gastrointestinal survival and susceptibility to important medical antimicrobials. Two strains of L. plantarum (KR6 and M5) exhibited favourable characteristics for all criteria, and were further optimised through random mutagenesis and selection approaches. Ultraviolet light (UV) exposure resulted in mutants characterized by better survival (for 1.9 log and 1.4 log) in gastrointestinal conditions. Subsequent exposure to ethyl methanesulfonate (EMS) provided mutants with greater tolerance to glucose induced catabolic repression. UV and UV-EMS mutants of L. plantarum M5 showed improved adhesion ability. As a result of this optimisation, L. plantarum MP2026 and L. plantarum MP2420 have been identified as promising candidates for probiotics, intended for alleviation of gastrointestinal discomfort originating from sugar malabsorption.

Effects of synbiotic fermented milk containing Lactobacillus acidophilus La-5 and Bifidobacterium animalis ssp. lactis BB-12 on the fecal microbiota of adults with irritable bowel syndrome: A randomized double-blind, placebo-controlled trial.

We conducted a randomized double-blind, placebo-controlled multicentric study to investigate the influence of a synbiotic fermented milk on the fecal microbiota composition of 30 adults with irritable bowel syndrome (IBS). The synbiotic product contained Lactobacillus acidophilus La-5, Bifidobacterium animalis ssp. lactis BB-12, Streptococcus thermophilus, and dietary fiber (90% inulin, 10% oligofructose), and a heat-treated fermented milk without probiotic bacteria or dietary fiber served as placebo. Stool samples were collected after a run-in period, a 4-wk consumption period, and a 1-wk follow-up period, and were subjected to real-time PCR and 16S rDNA profiling by next-generation sequencing. After 4wk of synbiotic (11 subjects) or placebo (19 subjects) consumption, a greater increase in DNA specific for L. acidophilus La-5 and Bifidobacterium animalis ssp. lactis was detected in the feces of the synbiotic group compared with the placebo group by quantitative real-time PCR. After 1wk of follow-up, the content of L. acidophilus La-5 and B. animalis ssp. lactis decreased to levels close to initial levels. No significant changes with time or differences between the groups were observed for Lactobacillus, Enterobacteriaceae, Bifidobacterium, or all bacteria. The presence of viable BB-12- and La-5-like bacteria in the feces resulting from the intake of synbiotic product was confirmed by random amplification of polymorphic DNA (RAPD)-PCR. At the end of consumption period, the feces of all subjects assigned to the synbiotic group contained viable bacteria with a BB-12-like RAPD profile, and after 1wk of follow-up, BB-12-like bacteria remained in the feces of 87.5% of these subjects. The presence of La-5-like colonies was observed less frequently (37.5 and 25% of subjects, respectively). Next-generation sequencing of 16S rDNA amplicons revealed that only the percentage of sequences assigned to Strep. thermophilus was temporarily increased in both groups, whereas the global profile of the fecal microbiota of patients was not altered by consumption of the synbiotic or placebo. In conclusion, daily consumption of a synbiotic fermented milk had a short-term effect on the amount and proportion of La-5-like strains and B. animalis ssp. lactis in the fecal microbiome of IBS patients. Furthermore, both synbiotic and placebo products caused a temporary increase in fecal Strep. thermophilus.

Vitamin D Status and Its Determinants in Healthy Slovenian Pregnant Women.

BACKGROUND/AIMS: Vitamin D deficiency is a common underdiagnosed condition. The aim of this was to analyze the status of vitamin D and its determinants in healthy Slovenian pregnant women. METHODS: A total of 132 volunteer pregnant women completed a questionnaire including baseline demographics, food frequency, physical activities; anthropometrical measurements, body mass index and levels of 25-(OH)D in serum were performed during the third trimester, and dietary intakes were assessed during the 27-28th week of gestation. RESULTS: Vitamin D deficiency was present in 14% while insufficiency was present in 41% of women. The risk for inadequacy was higher in women older than 30 years (p = 0.01), in those with less frequent outdoor physical activity (p = 0.01) and in pregnancies during the low sun exposure season (p = 0.04). Insufficiency was not significantly more frequent in less educated women, unemployed and in those living in urban area. The median value of vitamin D from habitual dietary intake was 1.5 µg/day (range 0.1-13.4) and did not influence 25-hydroxyvitamin D level (p = 0.91). CONCLUSIONS: The prevalence of vitamin D inadequacy was 55% and was dependent on age, season and outdoor physical activities. The results suggest a discrepancy between vitamin D intake through habitual diet and the reference needs.

Behavior of Staphylococcus aureus in culture broth, in raw and thermized milk, and during processing and storage of traditional Greek Graviera cheese in the presence or absence of Lactococcus lactis subsp. cremoris M104, a wild, novel nisin A-producing raw milk isolate.

This study was conducted to evaluate the behavior of Staphylococcus aureus during processing, ripening, and storage of traditional Greek Graviera cheese in accordance with European Union Regulation 1441/2007 for coagulase-positive staphylococci in thermized milk cheeses. Lactococcus lactis subsp. cremoris M104, a wild, novel nisin A-producing (NisA+) strain, also was evaluated as an antistaphylococcal adjunct. A three-strain cocktail of enterotoxigenic (Ent+) S. aureus increased by approximately 2 log CFU/ml when co-inoculated (at approximately 3 log CFU/ml) in thermized Graviera cheese milk (TGCM; 63°C for 30 s) with commercial starter culture (CSC) and/or strain M104 at approximately 6 log CFU/ml and then incubated at 37°C for 3 h. However, after 6 h at 37°C, significant retarding effects on S. aureus growth were noted in the order TGCM + M104 > TGCM + CSC = TGCM + CSC + M104 > TGCM. Additional incubation of TGCM cultures at 18°C for 66 h resulted in a 1.2-log reduction (P < 0.05) of S. aureus populations in TGCM + M104. The Ent + S. aureus cocktail did not grow but survived during ripening and storage when inoculated (at approximately 3 log CFU/g) postcooking into Graviera mini cheeses prepared from TGCM + CSC or TGCM + CSC + M104, ripened at 18°C and 90% relative humidity for 20 days, and stored at 4°C in vacuum packages for 2 months. A rapid 10-fold decrease (P < 0.05) in S. aureus populations occurred within the first 24 h of cheese fermentation. Reductions of S. aureus were greater by approximately 0.4 log CFU/g in CSC + M104 than in CSC only cheeses, concomitantly with the presence of NisA + M104 colonies and nisin-encoding genes in the CSC plus M104 cheeses and their corresponding microbial consortia only. A high level of selective survival of a naturally nisin-resistant EntC z S. aureus strain from the cocktail was noted in CSC + M104 cheeses and in coculture with the NisA + M104 strain in M-17 broth. In conclusion, although S. aureus growth inhibition is assured during Graviera cheese ripening, early growth of the pathogen during milk curdling and curd cooking operations may occur. Nisin-resistant S. aureus strains that may contaminate Graviera cheese milks postthermally may be difficult to control even by the application of the NisA + L. lactis subsp. cremoris strain M104 as a bioprotective adjunct culture.

Improved Draft Genome Sequence of Probiotic Strain Lactobacillus gasseri K7.

Lactobacillus gasseri K7 is an isolate from infant feces and has in vitro and in vivo established probiotic properties. Here, we report the improved version of the draft genome sequence, which comprises 8 scaffolds (13 contigs), a total length of 1.99 Mb, and 1,841 predicted protein-coding sequences.

Engineering BmpA as a carrier for surface display of IgG-binding domain on Lactococcus lactis.

Basic membrane protein A (BmpA) is a potential carrier protein for surface display of the IgG-binding domain on Lactococcus lactis. We have shown that it can increase the adhesion of bacteria to the intestinal cell model by 1.3-fold and have improved BmpA-based surface display by engineering the BmpA molecule. The bulk of the BmpA molecule was shown to be important in surface display; however, limited shortening (variant Bmp1) resulted in a large increase in the surface display ability. The closeness of the N- and the C-terminals in the Bmp1 model and the inefficiency of the spacer suggest that the distance of the passenger from the membrane is not of prime importance in surface display.

Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry.

The basic requirement for probiotic bacteria to be able to exert expected positive effects is to be alive; therefore, appropriate quantification methods are crucial. Due to disadvantages of conventional microbiological methods, the bacterial quantification based on the nucleic acid detection is increasingly used. The objective of this study was to evaluate the possibility to use propidium monoazide (PMA) in combination with real-time polymerase chain reaction (PCR) method or LIVE/DEAD BacLight viability kit in combination with flow cytometry (FCM) for determination of probiotic bacteria in a lyophilised product containing Lactobacillus acidophilus LA-5 and Bifidobacterium animalis ssp. lactis BB-12. In addition, the viability of probiotic bacteria in lyophilised product during 3 months storage was investigated. In the product, the results of real-time PCR quantification of PMA-treated cells did not differ significantly from those of non-treated cells, which indicate that most of the bacterial cells retained the membrane integrity although they have lost the culturability. The results obtained by FCM analysis were comparable with those by PMA real-time PCR. In conclusion, the PMA real-time PCR and FCM determination of the viability of probiotic bacteria could complement the plate count method which considers only the culturable part of the population.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

Application of potential probiotic Lactobacillus fermentum AD1 strain in healthy dogs.

Probiotic utilization is becoming increasingly popular in veterinary medicine. However, only few probiotic products are available commercially for use in dogs in our market. Therefore, the aim of our study was to determine the properties of new potential probiotic Lactobacillus fermentum AD1 strain-own canine isolate and to investigate its effect on several microbiological and biochemical parameters in healthy dogs. The strain expressed in vitro survival by pH 3.0 after 3h (86.8%) and in the presence of 1% bile (75.4%). The AD1 strain adhered to the canine and human intestinal mucus. It was sensitive to commonly used antimicrobials. Fifteen healthy dogs were supplemented with 10(9)L. fermentum AD1 for 7 days. At the end of AD1 strain application, numbers of faecal lactobacilli and enterococci increased significantly in the canine faeces. Significant increase of total protein and total lipid and significant reduction of glucose in serum of dogs were noted. These data indicate that L. fermentum AD1 survive transit through the canine gastrointestinal tract, and populate the colon and probably increased absorption of some nutrients. Whether longer time of its application lead to the same results as well as its potential to improve immune function in dogs remains to be determined.

Ability of Lactobacillus gasseri K 7 to inhibit Escherichia coli adhesion in vitro on Caco-2 cells and ex vivo on pigs' jejunal tissue.

The ability of Lactobacillus (Lb.) gasseri K 7 to inhibit adhesion of Escherichia coli O8:K88 to intestinal mucosa was studied on cultured Caco-2 cells and ex vivo on pigs' small intestinal tissue. Lactobacilli were added simultaneously with E. coli, before E. coli and after E. coli for competition, exclusion and displacement assays. The concentration of lactobacilli on fully differentiated Caco-2 cells was 4.5+/-0.3 x 10(8) cfu/well, while the concentration of E. coli varied from 1.5 x 10(6) to 4.3 x 10(8) cfu/well. The number of E. coli adhered to Caco-2 monolayer (cfu/well) was lineary correlated (R(2)=0.97) to the concentration of added cells. In the assay simulating exclusion, E. coli adhesion was reduced by Lb. gasseri K 7 strain by 0.1 to 0.6 log cfu/well. The binding of E. coli was inhibited even more when incubated simultaneously with lactobacilli, particularly at the lowest concentration of E. coli (ratio E. coli/lactobacilli 1:248), where five-times reduction (or 0.7 log) was observed. When adhesion to tissue derived from pigs' jejunum was tested, concentration of E. coli was constant (6.9+/-0.14 x 10(7) cfu/ml), while the concentration of Lb. gasseri K 7 was 5.9 x 10(7) and 1.3 x 10(7) cfu/ml in two independent experiments, respectively. The adhesion of E. coli and Lb. gasseri K 7 cells to jejunal mucosa was similar (1.0+/-0.17 x 10(6) and 1.54+/-0.10 x 10(6) cfu/cm(2)) when the concentrations of single strains in suspensions were approximately the same. No significant competition, exclusion or displacement of E. coli by lactobacilli was observed on jejunal tissue. In conclusion, Lb. gasseri K 7 was found to be effective in reducing E. coli adhesion to Caco-2 enterocytes, but it was not able to do so in ex vivo conditions tested for pig jejunal tissue.

Chromosomal location of the genetic determinants for bacteriocins produced by Lactobacillus gasseri K7.

The production of similar or even identical bacteriocins by different lactic acid bacteria is not a rare event. To take advantage of this finding, genetic determinants of the Lactobacillus K7 bacteriocins were tested for putative homologies with previously described bacteriocins of the Lactobacillus acidophilus group through polymerase chain reaction (PCR). Among specific primer pairs of seven known bacteriocins, derived from their respective sequences, only acidocin LF221 A and B primers amplified fragments in chromosomal DNA of K7 strain that revealed strong similarity over small regions of LF221 bacteriocins. Treatment of Lactobacillus K7 with ethidium bromide and mitomycin C was ineffective in generating non-bacteriocinogenic derivatives and had no impact on plasmid loss either. Classification studies elucidated Lactobacillus K7 as a member of the Lactobacillus gasseri species.