<Emphasis Type="Italic">Ureaplasma urealyticum</Emphasis> and <Emphasis Type="Italic">Mycoplasma hominis</Emphasis> Sensitivity to Bacteriocins Produced by Two Lactobacilli Strains

The purpose of the present study was to determine the inhibitory activities of two bacteriocins, produced by lactobacilli, against genital mycoplasmas. In this study, infections produced by genital mycoplasmas were studied; of these, 1.3% were caused by Mycoplasma hominis, 10.7% by Ureaplasma urealyticum and 5.6% by U. urealyticum + M. hominis. U. urealyticum was isolated from 75 out of 123 patients with genital mycoplasmas, while M. hominis was isolated from 9 patients (7.3%) and both U. urealyticum and M. hominis from 39 patients (31.7%). Bacteriocins, L23 and L60, produced by Lactobacillus fermentum and L. rhamnosus, respectively, appear to be two novel inhibitors of bacterial infection with potential antibacterial activity. Both bacteriocins proved to be active against 100% of strains tested; MICs of bacteriocin L23 ranged between 320 and 160 UA ml⁻¹ for 78% of the M. hominis strains and between 320 and 80 UA ml⁻¹ for 95% of the U. urealyticum strains. In addition, bacteriocin L60 was still active at 160 UA ml⁻¹ for a high percentage (56%) of M. hominis strains, and at 80 UA ml⁻¹ for 53% of the U. urealyticum strains. Interestingly, these antimicrobial substances produced by lactobacilli showed an inhibitory activity against genital mycoplasmas even when diluted. Altogether, our study indicates that the bacteriocins, L23 and L60, are good candidates for the treatment or prevention of genital infections in women.     

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.     

Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine

This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation.     

Enterococci from piglets — Probiotic properties and responsiveness to natural antibacterial substances

Fifty-five strains of enterococci isolated from the piglet intestine were characterized in vitro for probiotic activity. Identification of the isolates revealed Enterococcus faecium as the predominant species (84 %). Forty strains (73 %) were found to produce bacteriocin-like substances (only into solid media) with activity almost only toward Gram-positive genera. Thirty-eight % of strains were resistant to tetracycline, 27 % to chloramphenicol, 18 % to erythromycin and 16 % to vancomycin. In addition to control of strain safety, 6 % of isolates were β-hemolytic and 16 % produced gelatinase. Seven strains selected for further probiotic assays exhibited sufficient survival rate at pH 3.0 after 3 h, in the presence of 1 % ox-bile and lysozyme after 1 d (over 107 CFU/mL in all tests). The adhesion of tested strains to porcine and human intestinal mucus was found in a similar range (1.4–14.0 % and 1.4–17.6 %, respectively). In accordance with current research effort to use and/or to combine various health promoting substances, the sensitivity of all isolates toward plant extracts and toward bacteriocins produced by animal and environmental strains was determined. All enterococci were sensitive toward oregano and sage extracts and toward one (E. faecium EF55 — chicken isolate, activity of 25 600 AU/mL) of ten bacteriocin substances. It means that a similar anti-enterococcal potential of some bacteriocin substances may be observed as for certain plant extracts.     

The role of ‘soaking’ in spiteful toxin production in Pseudomonas aeruginosa

The ubiquitous production of antibacterial toxins, such as bacteriocins, is an ecologically significant class of interbacterial interactions that have primarily evolved through their indirect fitness benefits to the producer. Bacteria release bacteriocins into the environment at a cost to individual cell, but individual bacteriocin-producing cells are unlikely to gain any direct benefit from their own toxin; indeed, cell lysis is required in many species. There is a growing body of research describing the ecological conditions that can favour the evolution of bacteriocin production. However, an important aspect of many bacteriocins has yet to be investigated: the ability of bacteriocin-producing cells to neutralize toxin (‘soaking’) produced by other clonemates. By competing Pseudomonas aeruginosa bacteriocin-producing wild-type and ‘non-soaking’ strains against a bacteriocin-susceptible strain, we find that soaking markedly reduces the fitness of a bacteriocin-producing strain at both high and low frequencies.     

Lactobacillus rhamnosus L60, a potential probiotic isolated from the human vagina

The vagina has been increasingly viewed as an "ecosystem" whose normal microflora help protect it from invading pathogens, including those that cause urinary tract infections and sexually transmitted diseases. We tested new strains of lactobacilli as potential probiotics for maintenance of urogenital tract health, as well as prevention and therapy of urogenital infections. A strain of lactobacilli isolated from the vagina of nonpregnant, healthy, premenopausal women was identified as Lactobacillus rhamnosus L60 by 16S rDNA sequence homology. L60 was evaluated for antimicrobial activity, in vitro antibiotic resistance, autoaggregation, surface hydrophobicity, co-aggregation with other bacterial species, hydrogen peroxide (H(2)O(2)) production, and bacterial adherence. It displayed a wide spectrum of antimicrobial activity against urogenital pathogens, and resistance to antibiotics commonly prescribed for infections caused by these pathogens. L60 produced H(2)O(2), adhered to vaginal epithelial cells, co-aggregated with Escherichia coli and Candida albicans, and displayed self-aggregation. In view of these characteristics, L60 is considered a potential probiotic, and will be further evaluated for preventive and therapeutic application locally in the vaginal tract.     

Searching for bacteriocin-producing lactic acid bacteria in soil

A survey was conducted on the isolation and characterization of bacteriocin-producing lactic acid bacteria in soil. Forty-two acid-producing bacterial strains were isolated from 55 soil samples collected in Yamanashi prefecture, Japan. Investigation of antibacterial activities of isolates revealed that three isolates, Lactobacillus animalis C060203, Enterococcus durans C102901 and Leuconostoc mesenteroides subsp. mesenteroides C060204, showed antibacterial activities against the indicator strain of Lactobacillus sakei JCM 1157T. Bacteriocin from Enterococcus durans C102901 showed different characteristics from the known durancin L28-1A, produced by Enterococcus durans L28-1. Furthermore, this is the first report of a bacteriocin being produced by Lactobacillus animalis. Viewing from the species, bacteriocins from strains C102901 and C060203 showed high possibilities for the novel substances. These significant findings suggest that soil may be a common source for the isolation of novel bacteriocin-producing lactic acid bacteria.     

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

Synergistic Effect Between Two Bacteriocin-like Inhibitory Substances Produced by Lactobacilli Strains with Inhibitory Activity for <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis>

Group B streptococci (GBS) are bacterial species that colonize the vagina in pregnant women and as such may cause serious infections in neonates that passed through the birth channel. The objective of this work was to study the inhibitory activities produced by each bacteriocin-like inhibitory substance (BLIS) of Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23, and the effects of the combined BLIS-es of these lactobacilli on GBS. The interactions between the BLIS-es were assessed by qualitative and quantitative methods on agar plates. The minimum inhibitory concentrations (MICs) and fractional inhibitory concentrations (FICs) were determined by a modification of the broth microdilution and checkerboard methods, respectively. Antibiotic susceptibilities of all S. agalactiae strains were assayed and the results of these tests were evaluated for statistical significance. A 7.5% of GBS isolates were recovered from 760 pregnant women and 91% of those strains were susceptible to each BLIS produced by L. fermentum, L. rhamnosus, and also to a mixture of them. The comparisons among the BLIS-es showed statistically significant differences, with a combination of the BLIS-es from the two Lactobacillus species being better than the BLIS of each one alone (P < 0.05) as GBS growth inhibitors. Synergistic activities between the BLIS-es were found on 100% of susceptible GBS strains, MICs ranges of BLIS of L23 and L60 were 80–160 and 160–320 UA ml⁻¹, respectively. By the checkerboard method, the BLIS-es combination showed synergistic effect on all sensitive strains tested, with values of FICs ranging from 0.131 to 0.218. The BLIS-es produced by these lactobacilli of vaginal origin were able to inhibit S. agalactiae isolates. The results indicate that these strains may have probiotic potential for the control of GBS in women and may consequently prevent GBS infections in newborns.     

Genome sequence of the bacteriocin-producing oral probiotic Streptococcus salivarius strain M18

Streptococcus salivarius is a Gram-positive bacterial commensal and pioneer colonizer of the human oral cavity. Many strains produce ribosomally synthesized proteinaceous antibiotics (bacteriocins), and some strains have been developed for use as oral probiotics. Here, we present the draft genome sequence of the bacteriocin-producing oral probiotic S. salivarius strain M18.     

Isolation and characterization of bacteriocin‐producing bacteria from the gastrointestinal tract of broiler chickens for probiotic use

Aims: To isolate and characterize the bacteriocin‐producing bacteria (BPB) from the gastrointestinal tract of broiler chickens for probiotic use. Methods and Results: In total, 291 bacterial strains were isolated from broilers and screened for bacteriocin‐producing ability. The bacteriocins produced by Enterococcus faecium SH 528, Ent. faecium SH 632 and Pediococcus pentosaceus SH 740 displayed inhibitory activity against pathogens including Clostridium perfringens and Listeria monocytogenes. Activity of the bacteriocins remained unchanged after 30 min of heat treatment at 60°C or exposure to organic solvents, but diminished after treatment with proteolytic enzymes. PCR was used to detect the structural genes enterocin A and B in SH 528, enterocin L50 and P in SH 632, and pediocin PA‐1 in SH 740. Most of them were resistant to 0·5% bile salts and remained viable after 2 h at pH 3·0. Ent. faecium SH 528 exhibited the highest amylase activity among the strains tested. Conclusions: We selected Ent. faecium SH 528 and SH 632 and Ped. pentosaceus SH 740 by probiotic selection criteria including inhibition activity against pathogens. Significance and Impact of the Study: The isolated BPB could potentially be used in the poultry industry as probiotics to control pathogens.     

Bacteria competing with the adhesion and biofilm formation by <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The activity of antagonistic substances produced by Pseudomonas aeruginosa and Lactobacillus acidophilus against the planktonic and sessile populations of Staphylococcus aureus strains was demonstrated. The strongest effects were caused by probiotic L. acidophilus strain — bacteriocin-like inhibitory substances (BLIS) positive. However, the S. aureus A3 growth, adhesion and biofilm formation was also limited by cell-free supernatant of L. acidophilus H-1 (BLIS negative). Moreover, competitive direct interactions were observed between staphylococci and the above bacteria, which influenced the formation of dualspecies aggregates on the surface.     

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.     

Characteristic of Bacteriocins of Lactobacillus rhamnosus BTK 20-12 Potential Probiotic Strain

Multidrug resistance (MDR) is a serious health threat throughout the world resulting in reduced efficacy of antibacterial, antiparasitic, antiviral, and antifungal drugs. One of the most promising concepts that may represent a good alternative to antibiotics can be the use of bacteriocins obtained from lactic acid bacteria. The L. rhamnosus BTK 20-12 strain was isolated from traditional Armenian naturally fermented salted cheese. The probiotic potential of the strain was approved. It was shown that strain produced at less two bacteriocins (BCN 1 and BCN 2) with different molecular weight (1427 Da and 602.6 Da, respectively). Bacteriocins inhibited the growth of multidrug-resistant bacteria of different etiologies and belong to different taxonomic groups with diverse efficiency and it depends on properties of bacteriocins, as well as from isolation sources of pathogens. Thus, bacteriocins of L. rhamnosus BTK 20-12 have protein-like nature and a broad range of activity and are excellent candidates for the development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics.

     

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.     

In vitro study on bacteriocin production of Enterococci associated with chickens

In recent years, the approach of using innovative strategies such as probiotics or bacteriocins for the prevention or treatment of bacterial infections has come into focus. The present study was undertaken to check in vitro ability of Enterococci-isolated from the gastrointestinal tract of chickens-to produce a bacteriocin-like substance and to describe some further probiotic properties in five selected Enterococcus faecium strains. All strains (n=17) were found to produce bacteriocin-like substances against 14 out of 20 indicator bacteria of animal, food or environmental origin. Selected E. faecium strains expressed sufficient survival by pH 3.0 after 3h, in the presence of 1% bile after 24h and they were sensitive to most of antimicrobials tested. All tested strains adhere to the human, canine and porcine intestinal mucus (between 1.5% and 9.2%). However, better adhesion ability was observed for the canine mucus. PCR detection of enterocin structural genes determined presence of enterocins A and P genes in all selected strains. Characterization of bacteriocin substance in detail was performed in E. faecium EF55. The EF55 strain produced a bacteriocin-like substance (during the late logarithmic and early stationary growth phase) with inhibitory activity mostly against Gram-positive bacteria (100-51,200 AU/mL) including Listeria monocytogenes. Proteinaceous character of the bacteriocin substance was confirmed (its inhibitory activity was lost after its treatment with proteases), it was found to be stable after heating (100 degrees C 10 min) and during 12 months storage at -20 degrees C. The highest inhibitory activity of bacteriocin produced by EF55 strain (growing in MRS) broth was achieved between pH 7.0 and 9.0.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.     

Antifungal activity of two Lactobacillus strains with potential probiotic properties

Aflatoxin (highly toxic and carcinogenic secondary metabolites produced by fungi) contamination is a serious problem worldwide. Modern agriculture and animal production systems need to use high-quality and mycotoxin-free feedstuffs. The use of microorganisms to preserve food has gained importance in recent years due to the demand for reduced use of chemical preservatives by consumers. Lactic acid bacteria are known to produce various antimicrobial compounds that are considered to be important in the biopreservation of food and feed. Lactobacillus rhamnosus L60 and Lactobacillus fermentum L23 are producers of secondary metabolites, such as organic acids, bacteriocins and, in the case of L60, hydrogen peroxide. The antifungal activity of lactobacilli strains was determined by coculture with Aspergillus section Flavi strains by two qualitative and one quantitative methods. Both L23 and L60 completely inhibited the fungal growth of all aflatoxicogenic strains assayed. Aflatoxin B (1) production was reduced 95.7-99.8% with L60 and 27.5-100% with L23. Statistical analysis of the data revealed the influence of L60 and L23 on growth parameters and aflatoxin B (1) production. These results are important given that these aflatoxicogenic fungi are natural contaminants of feed used for animal production, and could be effectively controlled by Lactobacillus L60 and L23 strains with probiotic properties.     

Interference competition and parasite virulence

Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections.