Bojana Bogovič Matijašić

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.

Fate of Listeria monocytogenes on Fully Ripened Greek Graviera Cheese Stored at 4, 12, or 25°C in Air or Vacuum Packages : In Situ PCR Detection of a Cocktail of Bacteriocins Potentially Contributing to Pathogen Inhibition

The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a five-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25 degrees C, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P < 0.05) at 4 degrees C under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogenes was. Although enterocin A-specific PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenes by the enterocin-producing strain was not enhanced compared with batches A and C, which also contained enterocin A, but in lower amounts. Additionally enterocins B, P, L50A, and L50B; lactococcin G; and plantaricin A genes were detected in all batches, suggesting that indigenous bacteriocin-producing lactic acid bacteria might contribute to Listeria inhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2 or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.

Microbial stability and safety of traditional Greek Graviera cheese: characterization of the lactic acid bacterial flora and culture-independent detection of bacteriocin genes in the ripened cheeses and their microbial consortia.

The microflora of four batches of traditional Greek Graviera cheese was studied at 5 weeks of ripening, and 200 lactic acid bacteria (LAB) isolates were phenotypically characterized and screened for antilisterial bacteriocins. The cheeses were also analyzed for organic acids by high-performance liquid chromatography and for the potential presence of 25 known LAB bacteriocin genes directly in cheese and their microbial consortia by PCR. All batches were safe according to the European Union regulatory criteria for Listeria monocytogenes, Salmonella, enterobacteria, and coagulase-positive staphylococci. The cheese flora was dominated by nonstarter Lactobacillus casei/paracasei (67.5%) and Lactobacillus plantarum (16.3%) strains, whereas few Streptococcus thermophilus (3.8%), Lactococcus lactis subsp. lactis (0.6%), and Leuconostoc (1.9%) organisms were present. Enterococcus faecium (9.4%) and Enterococcus durans (0.6%) were isolated among the dominant LAB from two batches; however, enterococci were present in all batches at 10- to 100-fold lower populations than mesophilic lactobacilli. Sixteen E. faecium isolates produced antilisterial enterocins. In accordance, enterocin B gene was detectable in all cheeses and enterocin P gene was present in one cheese, whereas the consortia of all cheeses contained at least two of the enterocin A, B, P, 31, L50A, and L50B genes. Plantaricin A gene was also amplified from all cheeses. Mean concentrations of lactic, acetic, citric, and propionic acids in the ripened cheeses exceeded 1.5% in total, of which approximately 0.9% was lactate. Thus, organic acid contents constitute an important hurdle factor for inhibiting growth of pathogens in traditional Graviera cheese products, with LAB bacteriocins, mainly enterocins, potentially contributing to increased cheese safety.

Lactobacillus Isolates from Weaned Piglets' Mucosa with Inhibitory Activity against Common Porcine Pathogens

Twelve lactobacilli isolates from mucosa of 3–5-week-old weaned pigs were found to exert good antimicrobial activity against common porcine pathogens (S. aureus, B. cereus, E. coli, C. perfringens). Two of them produced in addition to lactic acid also considerable amounts of acetic acid, and 6 of them produced hydrogen peroxide and metabolites other than organic acids. Isolates 4/26 and 2/25 (identified as L. crispatus or L. amylovorus) were inhibitory against most strains of S. aureus, B. cereus and E. coli, and especially the strain 4/26 survived well in simulated gastric and intestinal juice. Diarrhea-causing E. coli O8K88H9 Ent+ was successfully inhibited by the growing culture as well as by the catalase-treated and neutralized supernatant of L. reuteri 12/26. Mucin degradation and multiple resistance to antibiotics were not observed.

Lactobacillus gasseri LF221 and K7 — from isolation to application

The article presents research findings on two human strains with probiotic activity. On the basis of API 50 CHL fermentation pattern, PCR by species-specific primers and sequencing of the V2–V3 region of 16S rRNA both strains designated as LF221 and K7 were identified as members of the Lactobacillus gasseri species. Two LF221 bacteriocins, acidocin LF221 A and B were purified and sequenced. They were classified as members of the two-component class II bacteriocins. Among basic probiotic properties, the survival under conditions in gastro-intestinal tract, ability to adhere to cultured intestinal enterocytes and pig’s mucosa and stimulation of the immune response were demonstrated. In in vivo study of 24 weaned piglets, the survival rate of K7 Rifr and LF221 Rifr was quantified by selective enumeration on MRS agar with rifampicin. The survival of both strains was good (2.9 × 105 cfu of K7 Rifr /g faeces; 4.8 × 105 cfu of LF221 Rifr /g) and the LF221 Rifr /K7 Rifr viable cells were found either in the mucosa of duodenum, jejunum or in the ileum. The possible effect of K7 to inhibit adhesion of E. coli O8:K88 to enterocytes was studied on Caco-2 cultured cells, on tissue obtained from small intestines of pigs and in vivo on gnotobiotic piglets. Lactobacilli were found to be effective in reducing E. coli adhesion to enterocytes in Caco-2 model, but not on mucosa of pig’s jejunum under ex vivo conditions. Competitive exclusion, production of organic acids and stimulation of immune response, were involved in inhibition of E. coli by K7 strain in gnotobiotic piglets. Any inflammatory change in intestines of piglets treated with K7 was observed, which confirmed its safe use. Among the technological parameters the survival and activity of the strains during cheese-making are presented.

Complete nisin A gene cluster from Lactococcus lactis M78 (HM219853) — obtaining the nucleic acid sequence and comparing it to other published nisin sequences

Nisin is an antimicrobial peptide produced by Lactococcus lactis. It has a long history of safe use, mainly in food production. This bacteriocin has been studied from many aspects of genetics, biosynthesis, immunity, regulation and mode of action. The strain Lac. lactis M78 has already been described in previous studies as a good nisin A producer with equally good potential to be used in food production. The main objective of the present study was to determine the complete nucleic acid sequence of the nisin A gene cluster from this strain. This is the first time that all 11 genes that form the nisin A gene cluster were sequenced. The obtained sequence (GenBank: HM219853) was compared to other known nucleic acid sequences of bacteriocin nisin. The results of the comparison showed certain differences in sequences that might influence the structure and function of proteins involved in nisin biosynthesis, immunity and regulation.

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.

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.

Detection and quantification of probiotic strain Lactobacillus gasseri K7 in faecal samples by targeting bacteriocin genes

Lactobacillus gasseri K7 is a probiotic strain that produces bacteriocins gassericin K7 A and K7 B. In order to develop a real-time quantitative PCR assay for the detection of L. gasseri K7, 18 reference strains of the Lactobacillus acidophilus group and 45 faecal samples of adults who have never consumed strain K7 were tested with PCR using 14 pairs of primers specific for gassericin K7 A and K7 B gene determinants. Incomplete gassericin K7 A or K7 B gene clusters were found to be dispersed in different lactobacilli strains as well as in faecal microbiota. One pair of primers was found to be specific for the total gene cluster of gassericin K7A and one for gassericin K7B. The real-time PCR analysis of faecal samples spiked with K7 strain revealed that primers specific for the gene cluster of the gassericin K7 A were more suitable for quantitative determination than those for gassericin K7 B, due to the lower detection level. Targeting of the gassericin K7 A or K7 B gene cluster with specific primers could be used for detection and quantification of L. gasseri K7 in human faecal samples without prior cultivation. The results of this study also present new insights into the prevalence of bacteriocin-encoding genes in gastrointestinal tract.

Evaluation of Lactobacillus strains for selected probiotic properties

Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P < 0.05) in the number of non-toxigenic E. coli O157:H7 adhering to Caco-2 cells was observed with all lactobacilli. Three strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.