Bacteriocins of Lactobacillus gasseri K7 – Monitoring of gassericin K7 A and B genes’ expression and isolation of an active component

The genome of Lactobacillus gasseri K7, isolated from baby's faeces, contains gene regions encoding two-component bacteriocins named gassericin K7 A (GenBank EF392861) and gassericin K7 B (GenBank AY307382). The strain has been known to exhibit bacteriocin activity in vitro, however, no data exist on the expression of particular genes of bacteriocins’ operons or on the activity of individual components of this bacteriocin complex, which has not been isolated so far. The objectives of this study were to examine bacteriocin genes’ expression during the growth of L. gasseri K7 and to isolate individual components in order to reveal the contribution of individual peptides to the overall bacteriocin activity. All eight target genes were expressed during exponential phase of growth in MRS broth. Mass spectrometry analysis revealed that the amino acid sequence of isolated peptide matched the deduced amino acid sequence of putative active peptide of gassericin K7 B (Gas K7 B_AcP) and GatX, a complementary peptide of gassericin T, previously supposed to have no antimicrobial activity. The isolated peptide showed a broad spectrum of antimicrobial activity. Furthermore, the isolation protocol developed in this study will enable to obtain a considerable amount of purified bacteriocins needed for further investigation of their functionality.     

Conjugative Plasmid from Lactobacillus gasseri LA39 That Carries Genes for Production of and Immunity to the Circular Bacteriocin Gassericin A

Gassericin A is a circular bacteriocin produced by Lactobacillus gasseri strain LA39. We found a 33,333-bp plasmid, designated pLgLA39, in this strain. pLgLA39 contained 44 open reading frames, including seven genes related to gassericin A production/immunity (gaa), as well as genes for replication, plasmid maintenance, and conjugative transfer. pLgLA39 was transferred from LA39 to the type strain of L. gasseri (JCM 1131) by filter mating. The transconjugant exhibited >30-fold-higher more resistance to gassericin A and produced antibacterial activity. Lactobacillus reuteri LA6, the producer of reutericin 6, was proved to harbor a plasmid indistinguishable from pLgLA39 and carrying seven genes 100% identical to gaa. This suggests that pLgLA39 might have been transferred naturally between L. gasseri LA39 and L. reuteri LA6. The seven gaa genes of pLgLA39 were cloned into a plasmid vector to construct pGAA. JCM 1131^T transformed with pGAA expressed antibacterial activity and resistance to gassericin A. pGAA was segregationally more stable than a pGAA derivative plasmid from which gaaA was deleted and even was more stable than the vector. This suggests the occurrence of postsegregational host killing by the gaa genes. pLgLA39 carried a pemIK homolog, and segregational stabilization of a plasmid by the pLgLA39-type pemIK genes was also confirmed. Thus, pLgLA39 was proved to carry the genes for at least two plasmid maintenance mechanisms, i.e., gaa and pemIK. Plasmids containing a repA gene similar to pLgLA39 repA were distributed in several L. gasseri strains.Lactobacillus species are normal inhabitants of the human gastrointestinal tract, and Lactobacillus gasseri is one of the most commonly detected of these species (37, 47). Health-promoting effects of this species, such as immunomodulation (35), suppression of Helicobacter pylori-induced interleukin-8 production (44), and improvement of intestinal conditions (34), have been reported, and some L. gasseri strains are used in commercial probiotic products.Bacteriocins are antimicrobial peptides, proteins, or protein complexes produced by bacteria and active mainly against related bacterial species (38). Several bacteriocins also inhibit the growth of food-borne pathogens, such as Listeria, Bacillus cereus, and Clostridium perfringens. Production of bacteriocin is thought to be a desired feature for probiotic strains, since bacteriocin is believed to provide an advantage for survival in the ecological niche and to prevent the growth of pathogens. Several L. gasseri strains are known to produce bacteriocins (18). The classification of bacteriocins remains controversial. We use the definition proposed by Maqueda et al. (30), where bacteriocins are classified into class I (lantibiotics), class II (nonlantibiotics), class III (large heat-labile bacteriocins), and class IV (circular bacteriocins linked at the N- and C-terminal ends). Among these, the class IV circular bacteriocins have attracted increasing attention, since they are the simplest prokaryotic representatives of the ubiquitous circular peptides with various physiological activities (6). Enterocin AS-48 from Enterococcus faecalis strain S-48 is the first and most vigorously characterized member of the class IV bacteriocins (30). L. gasseri strain LA39 (JCM 11657) produces a 58-amino-acid (aa) circular bacteriocin, gassericin A (18). Gassericin A is a representative of the non-AS-48-like circular bacteriocin group including butyrivibriocin AR10 from Butyrivibrio fibrisolvens AR10 (15) and carnocyclin A from Carnobacterium maltaromaticum UAL307 (32), as well as reutericin 6 from Lactobacillus reuteri LA6 (17) and acidocin B from Lactobacillus acidophilus M46 (26). The last two bacteriocins have nearly identical amino acid sequences to that of gassericin A. Though the number of reported circular bacteriocins has been increasing, their primary sequences and the genes responsible for production of and immunity to them are diversified (for a review, see reference 31). Recently, we isolated and sequenced seven genes (gaaBCADITE) from LA39 deduced to be responsible for production of and immunity to gassericin A (20). The gaa genes add new information to the complex world of the class IV bacteriocin genes.The structural gene of gassericin A, gaaA, was reported to be located on the chromosome of LA39 (19). However, the high amino acid sequence identity of gassericin A to reutericin 6 (100%) and to acidocin B (98%) suggests recent horizontal gene transfers of the relevant bacteriocin genes, possibly via mobile elements. In fact, the acidocin B genes were reported to be located on a plasmid, namely, pCV461 (26). Many Lactobacillus strains are known to harbor one or more plasmids of various sizes, and several Lactobacillus plasmids have been reported to contain genes for production of bacteriocins (48). To our knowledge, however, only three have been sequenced entirely: these are pLA103 from Lactobacillus acidophilus TK8912 (16), pRC18 from Lactobacillus curvatus (previously known as Lactobacillus casei) CRL705 (7), and pMP118 from Lactobacillus salivarius subsp. salivarius UCC118 (5). Thus, genetic information about bacteriocin-producing Lactobacillus plasmids is still limited. Furthermore, little has been known about plasmids of L. gasseri, even though the existence of plasmids in a few strains has been reported, including a 26.5-kb anonymous plasmid in strain ADH (27) and pK7 in strain K7 (28).Here we describe a 33.3-kb plasmid, designated pLgLA39, from L. gasseri LA39. The gaa genes are located on this plasmid. pLgLA39 carries a set of genes for conjugative transfer and was shown to be transmitted to another L. gasseri strain. L. reuteri LA6 also harbors a plasmid almost identical to pLgLA39. We demonstrated that production of gassericin A increased the apparent segregational stability of a plasmid carrying the gaa genes. A pemIK homolog in pLgLA39 was also functional as a plasmid-stabilizing mechanism. This is the first report describing the entire nucleotide sequence and detailed genetic analysis of an L. gasseri plasmid, which contains functional genes for circular bacteriocin production, conjugation, and plasmid maintenance.     

DNA Sequencing and Homologous Expression of a Small Peptide Conferring Immunity to Gassericin A,a Circular Bacteriocin Produced by Lactobacillus gasseri LA39

Gassericin A, produced by Lactobacillus gasseri LA39, is a hydrophobic circular bacteriocin. The DNA region surrounding the gassericin A structural gene, gaaA, was sequenced, and seven open reading frames (ORFs) of 3.5 kbp (gaaBCADITE) were found with possible functions in gassericin A production, secretion, and immunity. The deduced products of the five consecutive ORFs gaaADITE have homology to those of genes involved in butyrivibriocin AR10 production, although the genetic arrangements are different in the two circular bacteriocin genes. GaaI is a small, positively charged hydrophobic peptide of 53 amino acids containing a putative transmembrane segment. Heterologous expression and homologous expression of GaaI in Lactococcus lactis subsp. cremoris MG1363 and L. gasseri JCM1131^T, respectively, were studied. GaaI-expressing strains exhibited at least sevenfold-higher resistance to gassericin A than corresponding control strains, indicating that gaaI encodes an immunity peptide for gassericin A. Comparison of GaaI to peptides with similar characteristics found in the circular bacteriocin gene loci is discussed.Bacteriocins are antimicrobial peptides that act primarily against related bacterial species. The classification of bacteriocins remains controversial. Here, we use the classification of Maqueda et al. (30): class I (lantibiotics); class II (nonlantibiotics) with subclasses IIa (antilisteral pediocin-like bacteriocins), IIb (two-peptide bacteriocins), and IIc (leaderless bacteriocins); class III (large heat-labile bacteriocins); and class IV (circular bacteriocins linked at the N- and C-terminal amino acids).Nine class IV circular bacteriocins have been reported to date. They can be further divided into two major groups by using their primary structures, biochemical characteristics, and genetic arrangements. One group is the family of enterocin AS-48 (32), the first circular bacteriocin described (in 1994), which includes circularin A (25) and uberolysin (40). The other group is the family of gassericin A (19, 21), the second bacteriocin found (in 1998), which includes acidocin B (28), reutericin 6 (with a primary structure 100% identical to that of gassericin A) (22, 23), butyrivibriocin AR10 (17), and carnocyclin A, from Carnobacterium maltaromaticum UAL307 (33). The lantibiotic-like subtilosin A produced by Bacillus subtilis subsp. subtilis strain 168 (24) is an orphan member of the class IV bacteriocins. The gassericin A family of bacteriocins have been isolated from various bacterial species in several countries, suggesting the bacteriocin genes may be associated with transferable genetic elements.The bacteriocins of lactic acid bacteria (LAB) and bacteriocin-producing LAB strains isolated from foods are promising food preservative candidates, and strains of human origin are expected to be probiotics that could help to prevent the growth of harmful bacteria in food and the human intestine. Lactobacillus gasseri belongs to the Lactobacillus acidophilus group of LAB, which are natural inhabitants of the human intestinal tract (35), and many L. gasseri strains have been shown to produce bacteriocins (16, 20). Gassericin A was produced by L. gasseri LA39 isolated from the feces of a human infant; it has bactericidal activity against the food-borne pathogens Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus (16). Recently, using proteose peptone, some strains of L. gasseri containing LA39 were successfully cultured in reconstituted skim milk and cheese whey, where L. gasseri LA39 produced gassericin A; these low-cost, safe media could be used to improve the safety of biopreservation (1). Gassericin A has been purified and characterized, and its structural gene (gaaA) has been cloned and sequenced (21, 22). Determination of the complete chemical structure of gassericin A showed that the bacteriocin belongs to class IV and consists of 58 amino acid residues linked at the N and C termini (19). Little is known about the mechanisms of secretion and circularization of gassericin A and immunity to the circular bacteriocin.Here, we sequenced six genes surrounding gaaA thought to be related to production of and immunity to gassericin A and examined the homologous and heterologous expression of a small hydrophobic peptide, GaaI; we found that gaaI is an immunity gene providing protection against gassericin A.     

Wide-Inhibitory Spectra Bacteriocins Produced by Lactobacillus gasseri K7

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.     

Isolation and partial characterization of bacteriocins produced by Lactobacillus gasseri JCM 2124

Four antibacterially active peptides (B1 to B4) were purified from the culture broth of L. gasseri JCM 2124. The B2 peptide (gassericin B2) was determined to be 4400 Da by mass spectrometry and partially sequenced. Gassericin B2 did not show any sequence similarities to other known bacteriocins. The B1 and B3 peptides shared identical sequences with two peptides of a two-component bacteriocin from Lactobacillus acidophilus. However, synergistic activity upon complementation of B1 and B3 was not observed. Based on amino acid sequencing and molecular mass, it is suggested that B1 and B4 peptides were derived from B3 (gassericin B3).     

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.     

Anti‐listerial activity of bacteriocin‐producing Lactobacillus curvatus CWBI‐B28 and Lactobacillus sakei CWBI‐B1365 on raw beef and poultry meat

Aim: The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat. Methods and Results: The sakacin P and sakacin G structural genes were identified in Lact. curvatus CWBI‐B28 and Lact. sakei CWBI‐B1365 using PCR amplification, respectively. The effect of the two bacteriocinogenic strains either alone or together, and that of the nonbacteriocin‐producing strain Lact. sakei LMG17302, on the growth of L. monocytogenes was evaluated in beef and poultry meat. In raw beef, the pathogenic bacteria were inhibited by the bacteriocinogenic strains. The bacteriocinogenic strains had no activity in raw chicken meat when inoculated separately, while they showed a clear anti‐Listeria effect when applied together. Conclusion: Sakacin G producing Lact. sakei and sakacin P producing Lact. curvatus may be applied in raw beef to inhibit L. monocytogenes. In poultry meat, the inhibition of L. monocytogenes could only be achieved by a combined application of these bacteriocin‐producing strains. Significance and Impact of the Study: In some meat products, the combined application of different class IIa bacteriocin producing lactic acid bacterium can enhance the anti‐listerial activity.     

Solution Structure of Acidocin B,a Circular Bacteriocin Produced by Lactobacillus acidophilus M46

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.     

DNA analysis of the genes encoding acidocin LF221 A and acidocin LF221 B,two bacteriocins produced by<Emphasis Type="Italic"> Lactobacillus gasseri</Emphasis> LF221

Lactobacillus gasseri LF221, an isolate from the feces of a child, produces two bacteriocins. Standard procedures for molecular techniques were used to locate, clone and sequence the fragments of LF221 chromosomal DNA carrying the acidocin LF221 A and B structural genes, respectively. Sequencing analysis revealed the gene of acidocin LF221 A to be an open reading frame encoding a protein composed of 69 amino acids, including a 16-amino-acid N-terminal extension. The acidocin LF221 B gene was found to encode a 65-amino-acid bacteriocin precursor with a 17-amino-acid N-terminal leader peptide. DNA homology searches showed similarities of acidocin LF221 A to brochocin B, lactococcin N and thermophilin B, whereas acidocin LF221 B exhibited some homology to lactacin F and was virtually identical to gassericin X. The peptides encoded by orfA1 and orfB3 showed characteristics of class II bacteriocins and are suspected to be the complementary peptides of acidocin A and B, respectively. orfA3 and orfB5 are proposed to encode putative immunity proteins for the acidocins. Acidocin LF221 A and acidocin LF221 B are predicted to be members of the two-component class II bacteriocins, where acidocin LF221 A appears to be a novel bacteriocin. L. gasseri LF221 is being developed as a potential probiotic strain and a food/feed preservative. Detailed characterization of its acidocins is an important piece of background information useful in applying the strain into human or animal consumption. The genetic information on both acidocins also enables tracking of the LF221 strain in mixed populations and complex environments.     

Identification and antimicrobial susceptibility of porcine bacteria that inhibit the growth of Brachyspira hyodysenteriae in vitro

Aims: To identify bacilli, lactic acid bacteria and bifidobacteria that inhibit the growth of Brachyspira hyodysenteriae. Methods and Results: A total of 80 isolates were obtained from various porcine intestinal compartments using selective conditions and grouped into 15 similarity clusters based on whole‐cell protein profiles. Random amplified polymorphic DNA PCR patterns identified 24 genotypes. 16S rDNA sequencing assigned all genotypes, except eight aerobes, to established species (Bacillus subtilis, Enterococcus faecium, Lactobacillus salivarius, Lactobacillus mucosae, Lactobacillus reuteri, Lactobacillus amylovorus, Bifidobacterium thermophilum). According to their minimum inhibitory concentrations, four strains (Ent. faecium, Lact. reuteri, Lact. amylovorus, Bif. thermophilum) were susceptible to all clinically relevant antibiotics. Two lactobacilli showing multiresistance harboured the erm(B) determinant. A cross‐section of eight representative strains was examined for growth suppression of two strains of Brach. hyodysenteriae, the aetiological agent of swine dysentery, and compared with intestinal strains derived from other animal sources. The Brachyspira strains were inhibited by strains of Lact. salivarius, Bif. thermophilum, Ent. faecium and B. subtilis. Conclusions: Three porcine strains of Ent. faecium, Bif. thermophilum and B. subtilis were found to be suitable as probiotic candidates because of their well‐established identity, antibiotic susceptibility and antagonistic activity. Significance and Impact of the Study: For the first time, antagonistic activity of well‐characterized porcine strains against Brach. hyodysenteriae is presented. These findings suggest that certain intestinal strains might have a potential as probiotic feed additives for prevention of swine dysentery.     

The identification of a low molecular mass bacteriocin,rhamnosin A,produced by Lactobacillus rhamnosus strain 68

Aims: This study focuses on the isolation and characterization of a peptide with bacteriocin‐like properties isolated from Lactobacillus rhamnosus strain 68, previously identified by 16S rRNA gene sequencing and originating from human gastrointestinal flora. Methods and Results: The peptide was isolated from a supernatant of bacteria maintained under restrictive conditions by a combination of ethanol precipitation and reversed‐phase chromatography. The molecular mass of the peptide as assessed by mass spectrometry was 6433·8 Da. An isoelectric point of 9·8 was determined by 2D‐PAGE. The peptide designated rhamnosin A inhibited Micrococcus lysodeikticus ATCC 4698 but did not inhibit Lactobacillus plantarum 8014 or Lact. plantarum 39268. Inhibitory activity against M. lysodeikticus at concentrations used in this study was shown to be bacteriostatic rather than bacteriolytic or bactericidal. Rhamnosin A retained biological activity after heat treatment (95°C, 30 min) but was sensitive to proteolytic activity of pepsin and trypsin. Conclusions: The N‐terminal sequence of rhamnosin A, as determined by Edman degradation and in more detail by blast analysis, did not show identity with any currently available Lact. rhamnosus HN001‐translated protein sequences, nor any significant similarity with other sequences in the nonredundant protein sequence database. Being a small, heat‐stable, nonlanthionine‐containing peptide, rhamnosin A should be categorized as a class II bacteriocin. Significance and Impact of the Study: This study describes a partial bacteriocin sequence isolated from Lact. rhamnosus 68 and broadens our understanding of bacteriocins.     

Growth,acid production and bacteriocin production by probiotic candidates under simulated colonic conditions

Aims

The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA‐1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine.

Methods and Results

The three bacteriocin‐producing strains were grown in Macfarlane broth and in De Man–Rogosa–Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co‐culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant.

Conclusions

The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon.

Significance and Impact of the Study

This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.     

Atopic dermatitis‐mitigating effects of new Lactobacillus strain,Lactobacillus sakei probio 65 isolated from Kimchi

Aims

Atopic dermatitis (AD) is an inflammatory skin disease. Probiotics have been reported to modulate immune responses and thus are now being suggested as potential treatments for allergies. In this study, we investigated the inhibitory effects of Lactobacillus sakei probio 65 isolated from Kimchi on artificially inducing AD in NC/Nga mice.

Methods and Results

Oral administration of viable or heat‐inactivated Lact. sakei probio 65 improved the condition of skin and reduced scratching frequency. Serum levels of IgE and cutaneous T‐cell‐attracting chemokine (CTACK) were significantly decreased by this therapy. Dead Lact. sakei probio 65 also decreased IL‐4 and IL‐6 serum concentrations. Moreover, both live and dead Lact. sakei probio 65 inhibited the expression of Thymus and activation‐regulated chemokine and CTACK in AD‐like skin lesions. The increased levels of Foxp3 expression in the lesional skin and ears were also suppressed by Lact. sakei probio 65. In addition, Lact. sakei probio 65 inhibited β‐hexosaminidase release and the secretion of IL‐4, TNF‐α and IL‐6 from RBL‐2H3 cells.

Conclusions

Oral treatment with both viable and heat‐inactivated Lact. sakei probio 65 inhibits skin inflammation and AD‐like skin lesions, as well as mast cell activation.

Significance and Impact of the Study

Lactobacillus sakei probio 65 has an inhibitory effect on atopic dermatitis‐like skin lesions and may represent an effective new anti‐inflammatory agent.     

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell‐free supernatants containing bacteriocins, added to 3‐h‐old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto‐aggregation was strain‐specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co‐aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9–64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain‐dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco‐2 cells was within the range reported for Lact. rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.     

Bacteriocin Production, Antibiotic Susceptibility and Prevalence of Haemolytic and Gelatinase Activity in Faecal Lactic Acid Bacteria Isolated from Healthy Ethiopian Infants

The objective of this study was to characterise lactic acid bacteria (LAB) isolated from faecal samples of healthy Ethiopian infants, with emphasis on bacteriocin production and antibiotic susceptibility. One hundred fifty LAB were obtained from 28 healthy Ethiopian infants. The isolates belonged to Lactobacillus (81/150), Enterococcus (54/150) and Streptococcus (15/150) genera. Lactobacillus species were more abundant in the breast-fed infants while Enterococcus dominated the mixed-fed population. Bacteriocin-producing LAB species were isolated from eight of the infants. Many different bacteriocins were identified, including one new bacteriocin from Streptococcus salivarius, avicin A (class IIa) from Enterococcus avium, one class IIa bacteriocin from Enterococcus faecalis strains, one unknown bacteriocin from E. faecalis and two unknown bacteriocins from Lactobacillus fermentum strains and the two-peptide gassericin T from Lactobacillus gasseri isolate. Susceptibility tests performed for nine antibiotics suggest that some lactobacilli might have acquired resistance to erythromycin (3 %) and tetracycline (4 %) only. The streptococci were generally antibiotic sensitive except for penicillin, to which they showed intermediate resistance. All enterococci were susceptible to ampicillin while 13 % showed penicillin resistance. Only one E. faecalis isolate was vancomycin-resistant. Tetracycline (51 %) and erythromycin (26 %) resistance was prevalent among the enterococci, but multidrug resistance was confined to E. faecalis (47 %) and Enterococcus faecium (33 %). Screening of enterococcal virulence traits revealed that 2 % were β-haemolytic. The structural genes of cytolysin were detected in 28 % of the isolates in five enterococcal species, the majority being E. faecalis and Enterococcus raffinosus. This study shows that bacteriocin production and antibiotic resistance is a common trait of faecal LAB of Ethiopian infants while virulence factors occur at low levels.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Isolation and Partial Amino Add Sequence of Bacteriocins Produced by Lactobacillus acidophilus

Bacteriocins produced by Lactobacillus acidophilus JCM 1023, JCM 1028, JCM 1021, JCM 1229, and JCM 5342 were active against closely related lactobacilli. These bacteriocins were purified and partial sequenced. Bacteriocin activities of L. acidophilus JCM 1023 and JCM 1028 were associated with two components. On the basis of N-terminal amino acid sequencing and the molecular masses, it is interpreted that these two-component bacteriocins are identical to acidocin J1132, a bacteriocin from L. acidophilus JCM 1132 [Tahara et al., Appl. Environ. Microbiol., 62, 892–897 (1996)]. Other bacteriocins were single-peptide bacteriocins.     

The PTS transporters of <Emphasis Type="Italic">Lactobacillus gasseri</Emphasis> ATCC 33323

Background  

Lactobacilli can utilize a variety of carbohydrates which reflects the nutrient availability in their respective environments. A common lactobacilli in the human gastrointestinal tract, Lactobacillus gasseri, was selected for further study. The currently available annotation of the L. gasseri ATCC 33323 genome describes numerous putative genes involved in carbohydrate utilization, yet the specific functions of many of these genes remain unknown.     

Development of a chemically defined medium for the production of enterolysin A from Enterococcus faecalis B9510

Aim

This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510.

Methods and Results

The nutritional requirements of E. faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca‐pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M‐17 showed that growth and bacteriocin production in SDM was similar to that in M‐17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS‐PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin.

Conclusions

A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium.

Significance and Impact of the Study

The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.     

Isolation and characterization of a proteinaceous antifungal compound from Lactobacillus plantarum YML007 and its application as a food preservative

Korean kimchi is known for its myriad of lactic acid bacteria (LAB) with diverse bioactive compounds. This study was undertaken to isolate an efficient antifungal LAB strain among the isolated kimchi LABs. One thousand and four hundred LABs isolated from different kimchi samples were initially screened against Aspergillus niger. The strain exhibiting the highest antifungal activity was identified as Lactobacillus plantarum YML007 by 16S rRNA sequencing and biochemical assays using API 50 CHL kit. Lact. plantarum YML007 was further screened against Aspergillus oryzae, Aspergillus flavus, Fusarium oxysporum and other pathogenic bacteria. The morphological changes during the inhibition were assessed by scanning electron microscopy. Preliminary studies on the antifungal compound demonstrated its proteinaceous nature with a molecular weight of 1256·617 Da, analysed by matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF). The biopreservative activity of Lact. plantarum YML007 was evaluated using dried soybeans. Spores of A. niger were observed in the negative control after 15 days of incubation. However, fungal growth was not observed in the soybeans treated with fivefold concentrated cell‐free supernatant of Lact. plantarum YML007. The broad activity of Lact. plantarum YML007 against various food spoilage moulds and bacteria suggests its scope as a food preservative.

Significance and Impact of the Study

After screening 1400 kimchi bacterial isolates, strain Lactobacillus plantarum YML007 was selected with strong antifungal activity against various foodborne pathogens. From the preliminary studies, it was found that the bioactive compound is a low molecular weight novel protein of 1256·617 Da. Biopreservative potential of Lact. plantarum YML007 was demonstrated on soybean grains, and the results point out YML007 as a potent biopreservative having broad antimicrobial activity against various foodborne pathogens.