Adhesion Forces and Coaggregation between Vaginal Staphylococci and Lactobacilli

Urogenital infections are the most common ailments afflicting women. They are treated with dated antimicrobials whose efficacy is diminishing. The process of infection involves pathogen adhesion and displacement of indigenous Lactobacillus crispatus and Lactobacillus jensenii. An alternative therapeutic approach to antimicrobial therapy is to reestablish lactobacilli in this microbiome through probiotic administration. We hypothesized that lactobacilli displaying strong adhesion forces with pathogens would facilitate coaggregation between the two strains, ultimately explaining the elimination of pathogens seen in vivo. Using atomic force microscopy, we found that adhesion forces between lactobacilli and three virulent toxic shock syndrome toxin 1-producing Staphylococcus aureus strains, were significantly stronger (2.2–6.4 nN) than between staphylococcal pairs (2.2–3.4 nN), especially for the probiotic Lactobacillus reuteri RC-14 (4.0–6.4 nN) after 120 s of bond-strengthening. Moreover, stronger adhesion forces resulted in significantly larger coaggregates. Adhesion between the bacteria occurred instantly upon contact and matured within one to two minutes, demonstrating the potential for rapid anti-pathogen effects using a probiotic. Coaggregation is one of the recognized mechanisms through which lactobacilli can exert their probiotic effects to create a hostile micro-environment around a pathogen. With antimicrobial options fading, it therewith becomes increasingly important to identify lactobacilli that bind strongly with pathogens

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro

Application of MLST and Pilus Gene Sequence Comparisons to Investigate the Population Structures of Actinomyces naeslundii and Actinomyces oris

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established

Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased

Putative Nanobacteria Represent Physiological Remnants and Culture By-Products of Normal Calcium Homeostasis

Putative living entities called nanobacteria (NB) are unusual for their small sizes (50–500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis

Characterization of Granulations of Calcium and Apatite in Serum as Pleomorphic Mineralo-Protein Complexes and as Precursors of Putative Nanobacteria

Calcium and apatite granulations are demonstrated here to form in both human and fetal bovine serum in response to the simple addition of either calcium or phosphate, or a combination of both. These granulations are shown to represent precipitating complexes of protein and hydroxyapatite (HAP) that display marked pleomorphism, appearing as round, laminated particles, spindles, and films. These same complexes can be found in normal untreated serum, albeit at much lower amounts, and appear to result from the progressive binding of serum proteins with apatite until reaching saturation, upon which the mineralo-protein complexes precipitate. Chemically and morphologically, these complexes are virtually identical to the so-called nanobacteria (NB) implicated in numerous diseases and considered unusual for their small size, pleomorphism, and the presence of HAP. Like NB, serum granulations can seed particles upon transfer to serum-free medium, and their main protein constituents include albumin, complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, as well as other calcium and apatite binding proteins found in the serum. However, these serum mineralo-protein complexes are formed from the direct chemical binding of inorganic and organic phases, bypassing the need for any biological processes, including the long cultivation in cell culture conditions deemed necessary for the demonstration of NB. Thus, these serum granulations may result from physiologically inherent processes that become amplified with calcium phosphate loading or when subjected to culturing in medium. They may be viewed as simple mineralo-protein complexes formed from the deployment of calcification-inhibitory pathways used by the body to cope with excess calcium phosphate so as to prevent unwarranted calcification. Rather than representing novel pathophysiological mechanisms or exotic lifeforms, these results indicate that the entities described earlier as NB most likely originate from calcium and apatite binding factors in the serum, presumably calcification inhibitors, that upon saturation, form seeds for HAP deposition and growth. These calcium granulations are similar to those found in organisms throughout nature and may represent the products of more general calcium regulation pathways involved in the control of calcium storage, retrieval, tissue deposition, and disposal