Effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on the proliferation of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> and <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

While it is generally accepted that Propionibacterium acnes is involved in the development of acne, other bacteria including Staphylococcus epidermidis have also been isolated from the acne lesion. The interaction between Lactobacillus reuteri, a probiotic bacterium, and acnegenic bacteria is unclear. This study examined the effects of L. reuteri on the proliferation of P. acnes and S. epidermidis. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of P. acnes and S. epidermidis. The proliferation of P. acnes was decreased by 2-log scales after incubation with L. reuteri for 24 h. In addition, the proliferation of S. epidermidis was decreased by 3-log scales after incubation with L. reuteri for 24 h, whereas the growth of L. reuteri was unaffected by P. acnes or S. epidermidis. Among the L. reuteri strains examined, L. reuteri KCTC 3679 had the strongest inhibitory effect on the growth of P. acnes and S. epidermidis, followed by L. reuteri KCTC 3594 and L. reuteri KCTC 3678. Interestingly, reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. The most pronounced the antibacterial activities of L. reuteri were attributed to the production of organic acids. Overall, these results suggest that L. reuteri may be a useful probiotic agent to control the growth of bacteria involved in acne inflammation and prevent acne.     

Inhibitory effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on periodontopathic and cariogenic bacteria

The interaction between Lactobacillus reuteri, a probiotic bacterium, and oral pathogenic bacteria have not been studied adequately. This study examined the effects of L. reuteri on the proliferation of periodontopathic bacteria including Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, and Tannerella forsythia, and on the formation of Streptococcus mutans biofilms. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of periodontopathic bacteria and the formation of S. mutans biofilms. These antibacterial activities of L. reuteri were attributed to the production of organic acids, hydrogen peroxide, and a bacteriocin-like compound. Reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. In addition, L. reuteri inhibited the production of methyl mercaptan by F. nucleatum and P. gingivalis. Overall, these results suggest that L. reuteri may be useful as a probiotic agent for improving oral health.     

Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.     

Evaluation of Reuterin Production in Urogenital Probiotic Lactobacillus reuteri RC-14

Classified as a distinct species in 1980, Lactobacillus reuteri strains have been used in probiotic formulations for intestinal and urogenital applications. In the former, the primary mechanism of action of L. reuteri SD2112 (ATCC 55730) has been purported to be its ability to produce the antibiotic 3-hydroxypropionaldehyde (3-HPA), also known as reuterin. In the vagina, it has been postulated that probiotic Lactobacillus reuteri RC-14 does not require reuterin production but mediates a restoration of the normal microbiota via hydrogen peroxide, biosurfactant, lactic acid production, and immune modulation. The aim of the present study was to determine whether strain RC-14 produced reuterin. Using PCR and DNA dot blot analyses, numerous Lactobacillus species, including RC-14, were screened for the presence of the gene encoding the large subunit of glycerol dehydratase (gldC), the enzyme responsible for reuterin production. In addition, lactobacilli were grown in glycerol-based media and both high-performance liquid chromatography and a colorimetric assay were used to test for the presence of reuterin. L. reuteri RC-14 was determined to be negative for gldC sequences, as well as for the production of reuterin when cultured in the presence of glycerol. These findings support that the probiotic effects of L. reuteri RC-14, repeatedly demonstrated during numerous studies of the intestine and vagina, are independent of reuterin production.     

In Vitro Antimicrobial Activity and Downregulation of Virulence Gene Expression on <Emphasis Type="Italic">Helicobacter pylori</Emphasis> by Reuterin

Helicobacter pylori is an infectious agent commonly associated with gastrointestinal diseases. The use of probiotics to treat this infection has been documented, however, their potential antimicrobial metabolites have not yet been investigated. In the present study, the effect of reuterin produced by Lactobacillus reuteri on H. pylori growth and virulence gene expression was evaluated. It was observed that reuterin caused significant (P < 0.05) H. pylori growth inhibition at concentrations from 0.08 to 20.48 mM, with minimal inhibitory concentrations (MICs) of 20.48 mM for H. pylori ATCC700824 and 10.24 mM for H. pylori ATCC43504. In a reuterin bacterial killing assay, it was observed that half of the MIC value for H. pylori (ATCC700824) significantly (P < 0.01) reduced colony numbers from 5.65 ± 0.35 to 3.78 ± 0.35 Log₁₀ CFU/mL after 12 h of treatment and then increased them to 5.25 ± 0.23 Log₁₀ CFU/mL at 24 h; at its MIC value (20.48 mM), reuterin abrogated (P < 0.01) H. pylori (ATCC700824) growth after 20 h of culture. In addition, reuterin significantly (P < 0.01) reduced H. pylori (ATCC 43504) colony numbers from 5.65 ± 0.35 to 4.1 ± 0.12 Log10 CFU/mL from 12 to 24 h of treatment and abrogated its growth at its MIC value (10.24 mM), after 20 h of treatment. Reuterin did not alter normal human gastric Hs738.St/Int cell viability at the concentrations tested for H. pylori strains. Furthermore, 10 μM reuterin was shown to significantly (P < 0.01) reduce mRNA relative expression levels of H. pylori virulence genes vacA and flaA at 3 h post-treatment, whose effect was higher at 6 h post-treatment, as measured by RT-qPCR. The observed direct antimicrobial effect and the downregulation of expression of virulence genes on H. pylori by reuterin may contribute to the understanding of the mechanisms of action of probiotics against H. pylori.     

Cloning and heterologous expression of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD): preliminary characterization

Purpose of work  

To clone, express and characterize uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD) from Lactobacillus reuteri.     

Characterization of a Reuterin-Producing Lactobacillus reuteri BPL-36 Strain Isolated from Human Infant Fecal Sample

A reuterin (3-hydroxypropinaldehyde, 3-HPA)-producing isolate from a human infant fecal sample was identified as Lactobacillus reuteri BPL-36 strain. The organism displayed a broad-spectrum antimicrobial activity. The gene (gdh) encoding a glycerol dehydratase subunit was detected by PCR, thus confirming its reuterin-producing ability. Reuterin concentration of 89.63?mM/mL was obtained in the MRS?Cglycerol medium after 16?h of incubation at 37?°C. The reuterin concentration required to inhibit the growth of Pseudomonas aeruginosa, Escherichia coli O157: H7, Salmonella typhi, Staphylococcus aureus, and Listeria monocytogenes was found to be 1.0, 2.0, 2.0, 4.0, and 10.0?AU/mL, respectively. Antimicrobial efficiency test using BPL-36 cell-free supernatant co-incubated along with different test pathogens was done. Viability of all the tested pathogens decreased with increasing contact time with the cell-free supernatant. S. typhi was observed to be the most susceptible among the tested organisms, and the number of viable cells hugely declined as the contact with cell-free supernatant continued, resulting in a reduction of 6 log cycles (100?% inhibition) of the cells after 4?h of treatment. Production of biogenic amines and degradation of mucin by the reuterin-producing BPL-36 strain were not detected.     

The plant Apolipoprotein D ortholog protects <Emphasis Type="Italic">Arabidopsis</Emphasis> against oxidative stress

Background  

Lipocalins are a large and diverse family of small, mostly extracellular proteins implicated in many important functions. This family has been studied in bacteria, invertebrate and vertebrate animals but little is known about these proteins in plants. We recently reported the identification and molecular characterization of the first true lipocalins from plants, including the Apolipoprotein D ortholog AtTIL identified in the plant model Arabidopsis thaliana. This study aimed to determine its physiological role in planta.     

Contribution of glutamate decarboxylase in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> to acid resistance and persistence in sourdough fermentation

Background

Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri.

Results

A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ?gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ?gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ?gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ?gadB after 5 cycles of fermentation in back-slopped sourdough fermentations.

Conclusions

The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.

     

Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria <Emphasis Type="Italic">Wolbachia</Emphasis>

Background  

Outer membrane proteins (OMPs) of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP) can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods.     

In vivo evaluation of reuterin and its combinations with suramin,melarsoprol, dl-α-difluoromethylornithine and bleomycin in mice infected with Trypanosoma brucei brucei

1. Trypanosoma brucei brucei -infected mouse models treated with a new antibiotic, reuterin, showed reduction of the levels of parasitemia and prolonged survival of the mice.2. Cures of the parasitemia were observed in groups of mice treated with combinations of reuterin and suramin or melarsoprol.3. Reuterin administered in combination with bleomycin or dl-α-difluoromethylornithine showed temporary remission of the parasitemia in groups of mice.     

The mycobacterial DNA-binding protein 1 (MDP1) from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> BCG influences various growth characteristics

Background  

Pathogenic mycobacteria such as M. tuberculosis, M. bovis or M. leprae are characterised by their extremely slow growth rate which plays an important role in mycobacterial virulence and eradication of the bacteria. Various limiting factors influence the generation time of mycobacteria, and the mycobacterial DNA-binding protein 1 (MDP1) has also been implicated in growth regulation. Our strategy to investigate the role of MDP1 in mycobacterial growth consisted in the generation and characterisation of a M. bovis BCG derivative expressing a MDP1-antisense gene.     

Functional properties of free and encapsulated Lactobacillus reuteri DPC16 during and after passage through a simulated gastrointestinal tract

Lactobacillus reuteri DPC16 is a probiotic bacterium that has strong antimicrobial activities on pathogens, mainly due to its ability to produce reuterin, an antimicrobial compound. The objective of this study was to examine the ability of an encapsulation technique to protect the functional properties of cells of L. reuteri DPC16 during passage through a simulated GI tract. The functional properties of the cells were studied before and after passage through the tract. An alginate-skim milk encapsulation system was used to deliver the probiotic bacterium through the simulated GI tract, allowing for the release of the cells into the simulated colonic fluid. The cells were then isolated and cultured. The recovered cells showed no diminution in functional properties, including their growth kinetics, ability to adhere to epithelial cells and ability to inhibit the adhesion of E. coli to epithelial cells. The bacteriostatic and bactericidal properties of the recovered cells against some pathogens were significantly greater (P < 0.05) than those of the original cells. Production of reuterin by the recovered cells was significantly greater than that of the original cells when cultured in MRS medium in the absence of its metabolic precursor, glycerol. The results demonstrate significant consequences for the application of the encapsulation technique to protect and/or enhance the functional properties of the probiotic cells.     

Identification of genes involved in the mutualistic colonization of the nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> by the bacterium <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis>

Background  

Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.     

<Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>responds to heat shock,penicillin induced persistence,and IFN-gamma persistence by altering levels of the extracytoplasmic stress response protease HtrA

Background  

Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA.     

Sequence variability of <Emphasis Type="Italic">Campylobacter</Emphasis>temperate bacteriophages

Background  

Prophages integrated within the chromosomes of Campylobacter jejuni isolates have been demonstrated very recently. Prior work with Campylobacter temperate bacteriophages, as well as evidence from prophages in other enteric bacteria, suggests these prophages might have a role in the biology and virulence of the organism. However, very little is known about the genetic variability of Campylobacter prophages which, if present, could lead to differential phenotypes in isolates carrying the phages versus those that do not. As a first step in the characterization of C. jejuni prophages, we investigated the distribution of prophage DNA within a C. jejuni population assessed the DNA and protein sequence variability within a subset of the putative prophages found.     

Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits

AIMS: To determine the production of reuterin by lactobacilli isolates from pig faeces and to evaluate their potential as probiotic bacteria. METHODS AND RESULTS: Twenty-eight of 165 lactobacilli isolates produced reuterin in the presence of glycerol. Six isolates yielding high levels of reuterin with respect to type strain Lactobacillus reuteri CECT 925T were identified as Lact. reuteri. They were able to survive at pH 3 and subsequent exposure to cholic acid or oxgall, and presented bile salt hydrolase and bacteriocin-like activities. CONCLUSIONS: Reuterin production is a frequently found trait among lactobacilli isolated from pig faeces. Selected Lact. reuteri isolates were able to survive at conditions likely to be encountered throughout the gastrointestinal tract. SIGNIFICANCE AND IMPACT OF THE STUDY: High yields of reuterin may be obtained from selected isolates of Lact. reuteri. Probiotic characteristics of isolates studied in the present work suggest their application in food and feed.     

The role of iron uptake in pathogenicity and symbiosis in <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> TT01

Background  

Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺ and Fe²⁺ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.     

Biological characteristics and whole-genome analysis of the potential probiotic,Lactobacillus reuteri S5

Lactic acid bacteria are micro-organisms used for probiotic purposes and form major parts of human and mammalian intestinal microbiota, exerting important health-promoting effects on the host. Here, we evaluated Lactobacillus reuteri strain S5 isolated from the intestines of healthy white feather broilers. Lactobacillus reuteri S5 grew best after 20 h of incubation in MRS medium. Lactic acid production was 1·42 mmol l⁻¹ at 24 h, which was well tolerated. Activities of T-AOC, GSH-Px and T-SOD in the cell-free fermentation supernatant of L. reuteri S5 were higher than those in the bacteria, and the strain showed good hydrophobicity in vitro. The dominant carbon and nitrogen sources of L. reuteri S5 were glucose and soybean meal. A high-quality complete genome map of L. reuteri S5 was obtained using a Pacbio nanopore third-generation sequencing platform. The results showed that L. reuteri S5 possesses a complete primary metabolic pathway, encoding the main functional enzymes of the glycolysis pathway and pentose phosphate pathway. The genome contains genes encoding antioxidants and conferring tolerance to inorganic salt ions, acids and bile salts. This study shows that L. reuteri S5 is a probiotic strain with excellent probiotic characteristics and has great potential for the development of feed additives to promote animal health.