A concerted probiotic activity to inhibit periodontitis-associated bacteria.

Periodontitis can result in tooth loss and the associated chronic inflammation can provoke several severe systemic health risks. Adjunctive to mechanical treatment of periodontitis and as alternatives to antibiotics, the use of probiotic bacteria was suggested. In this study, the inhibitory effect of the probiotic Streptococcus salivarius subsp. salivarius strains M18 and K12, Streptococcus oralis subsp. dentisani 7746, and Lactobacillus reuteri ATCC PTA 5289 on anaerobic periodontal bacteria and Aggregatibacter actinomycetemcomitans was tested. Rarely included in other studies, we also quantified the inverse effect of pathogens on probiotic growth. Probiotics and periodontal pathogens were co-incubated anaerobically in a mixture of autoclaved saliva and brain heart infusion broth. The resulting genome numbers of the pathogens and of the probiotics were measured by quantitative real-time PCR. Mixtures of the streptococcal probiotics were also used to determine their synergistic, additive, or antagonistic effects. The overall best inhibitor of the periodontal pathogens was L. reuteri ATCC PTA 5289, but the effect is coenzyme B12-, anaerobiosis-, as well as glycerol-dependent, and further modulated by L. reuteri strain DSM 17938. Notably, in absence of glycerol, the pathogen-inhibitory effect could even turn into a growth spurt. Among the streptococci tested, S. salivarius M18 had the most constant inhibitory potential against all pathogens, followed by K12 and S. dentisani 7746, with the latter still having significant inhibitory effects on P. intermedia and A. actinomycetemcomitans. Overall, mixtures of the streptococcal probiotics did inhibit the growth of the pathogens equally or-in the case of A. actinomycetemcomitans- better than the individual strains. P. gingivalis and F. nucleatum were best inhibited by pure cultures of S. salivarius K12 or S. salivarius M18, respectively. Testing inverse effects, the growth of S. salivarius M18 was enhanced when incubated with the periodontal pathogens minus/plus other probiotics. In contrast, S. oralis subsp. dentisani 7746 was not much influenced by the pathogens. Instead, it was significantly inhibited by the presence of other streptococcal probiotics. In conclusion, despite some natural limits such as persistence, the full potential for probiotic treatment is by far not utilized yet. Especially, further exploring concerted activity by combining synergistic strains, together with the application of oral prebiotics and essential supplements and conditions, is mandatory.

Cell-free supernatant of Streptococcus salivarius M18 impairs the pathogenic properties of Pseudomonas aeruginosa and Klebsiella pneumonia.

M18 strain of Streptococcus salivarius is a bacterial replacement probiotic that has been suggested for use in the oral cavity. Here, we have shown that S. salivarius M18 cell-free supernatant reduced the growth of the two most common human pathogens Pseudomonas aeruginosa and Klebsiella pneumonia and sensitized the pathogenic bacteria to antibiotic. Besides, the supernatant inhibited biofilm formation of P. aeruginosa drastically. For pinpointing the biomolecular changes that occurred in P. aeruginosa incubated with the probiotic supernatant, attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used. Unsupervised learning algorithms, principal component analysis (PCA) and hierarchical cluster analysis (HCA), and intensity analyses of individual spectral bands exhibited comprehensive alterations in the polysaccharide and lipid contents and compositions of P. aeruginosa cultivated with S. salivarius M18 cell-free supernatant. These results indicate that S. salivarius M18 has the potential for the prevention or alleviation of different pathogen-induced infections along with the infections of oral pathogens.

Bacteria-targeting BSA-stabilized SiC nanoparticles as a fluorescent nanoprobe for forensic identification of saliva.

Forensic saliva identification represents an increasingly useful auxiliary means of crime investigations, particularly in sex crimes. Salivary bacteria detection techniques have been shown to be viable methods for identifying the presence of saliva. A one-pot method is described for the fabrication of bovine serum albumin-stabilized SiC nanoparticles (SiC@BSA NPs). The SiC@BSA NPs were conjugated to antibacterial peptide GH12 to allow for fluorometric detection and imaging of bacteria in saliva. More specifically, the nanoprobe, with fluorescence excitation/emission maxima at 320/410 nm, was used to detect the oral bacteria S. salivarius levels. The detection limit is 25 cfu·mL-1, and the assay can be performed within 40 min. The nanoprobe was also used to detect bacteria in forensic body fluids including blood, urine, and semen. In all cases, positive results were obtained with (mixed) samples containing saliva, while other saliva samples without saliva showed negative results. Fluorescent images of S. salivarius cells were obtained by implementing a high-content image analysis system. These results suggest that this new nanoprobe can be applied to screen for forensic saliva stains. Graphical abstractSchematic representation of the preparation of SiC@BSA-GH12 nanoprobe for fluorometric detection and imaging of S. salivarius in saliva.

Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2.

Lantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain, Streptococcus salivarius K12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCE The amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

Comparison of Tunable Diode Laser Absorption Spectroscopy and Isothermal Micro-calorimetry for Non-invasive Detection of Microbial Growth in Media Fills.

Two methods were investigated for non-invasive microbial growth-detection in intact glass vials as possible techniques for automated inspection of media-filled units. Tunable diode laser absorption spectroscopy (TDLAS) was used to determine microbially induced changes in O2 and CO2 concentrations within the vial headspaces. Isothermal microcalorimetry (IMC) allowed the detection of metabolic heat production. Bacillus subtilis and Streptococcus salivarius were chosen as test organisms. Parameters as robustness, sensitivity, comparability and time to detection (TtD) were evaluated to assess method adequacy. Both methods robustly detected growth of the tested microorganisms within less than 76 hours using an initial inoculum of <10CFU. TDLA turned out to be less sensitive than TDLA and IMC, as some false negative results were observed. Compared to the visual media-fill examination of spiked samples, the investigated techniques were slightly slower regarding TtD. Although IMC showed shorter TtD than TDLAS the latter is proposed for automating the media-fill inspection, as larger throughput can be achieved. For routine use either TDLA or a combination of TDLA and TDLA should be considered. IMC may be helpful for replacing the sterility assessment of commercial drug products before release.