Prevention of the proliferation of oral pathogens due to prolonged mask use based on α-cyclodextrin and hydroxytyrosol mouthwash.

OBJECTIVE: Face masks help contain the aerosol-mediated transmission of infectious viral particles released from individuals via cough and sneezes. However, the prolonged use of face masks has raised concerns regarding oral hygiene. Here, we present a mouthwash formulation based on α-cyclodextrin and hydroxytyrosol that can maintain healthy oral microbiota. MATERIALS AND METHODS: We isolated and cultured Candida albicans, Staphylococcus aureus, and a mix of Streptococcus sp., Staphylococcus sp. and Neisseria sp. from oral and throat swabs. The microorganisms were cultured in a standard medium with or without the mouthwash. To evaluate the effect of the mouthwash on the oral microbiota, the DNA from the saliva of 3 volunteers that used the mouthwash was extracted. Then, the DNA was amplified using primer pairs specific for bacterial and fungal DNA. Twelve further volunteers were offered to use the mouthwash and a questionnaire was submitted to them to assess the possible beneficial effects of mouthwash on halitosis and other oral disturbances. RESULTS: The bacteria and fungi cultured in media containing the mouthwash showed a growth reduction ranging from 20 to 80%. The PCR amplification of fungal and bacterial DNA extracted from volunteers that used the mouthwash showed a reduction of both bacteria and fungi. Volunteers that used the mouthwash reported a tendency towards a reduction of halitosis, gingival and mouth inflammation, and dry mouth. CONCLUSIONS: The use of a mouthwash containing α-cyclodextrin and hydroxytyrosol is not aggressive against oral mucosa; it is safe and effective to reduce the bacterial and fungal load due to the continuous use of face masks.

Precise diagnosis of Neisseria macacae infective endocarditis assisted by nanopore sequencing.

Infective endocarditis caused by Neisseria macacae in humans is extremely rare. We presented here a case of N. macacae infective endocarditis in a 61-year-old man with a native aortic valve infection. N. macacae was isolated from blood culture and was detected by nanopore-based metagenomic sequencing in the vegetations. Finally, the patient recovered completely after surgery and antibiotic therapy.

Metabolic property of acetaldehyde production from ethanol and glucose by oral Streptococcus and Neisseria.

Acetaldehyde is known to be carcinogenic and produced by oral bacteria. Thus, bacterial acetaldehyde production might contribute to oral cancer. Therefore, we examined bacterial acetaldehyde production from ethanol and glucose under various conditions mimicking the oral cavity and clarified the metabolic pathways responsible for bacterial acetaldehyde production. Streptococcus mitis, S. salivarius, S. mutans, Neisseria mucosa and N. sicca were used. The bacterial metabolism was conducted at pH 5.0-8.0 under aerobic and anaerobic conditions. The production of acetaldehyde and organic acids was measured with gas chromatography and HPLC, respectively. Bacterial enzymes were also assessed. All of the bacteria except for S. mutans exhibited their greatest acetaldehyde production from ethanol at neutral to alkaline pH under aerobic conditions. S. mutans demonstrated the greatest acetaldehyde from glucose under anaerobic conditions, although the level was much lower than that from ethanol. Alcohol dehydrogenase and NADH oxidase were detected in all of the bacteria. This study revealed that oral indigenous bacteria, Streptococcus and Neisseria can produce acetaldehyde, and that such acetaldehyde production is affected by environmental conditions. It was suggested that alcohol dehydrogenase and NADH oxidase are involved in ethanol-derived acetaldehyde production and that the branched-pathway from pyruvate is involved in glucose-derived acetaldehyde production.

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance.

We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis.

Neisseria gonorrhoeae Crippled Its Peptidoglycan Fragment Permease To Facilitate Toxic Peptidoglycan Monomer Release.

Neisseria gonorrhoeae (gonococci) and Neisseria meningitidis (meningococci) are human pathogens that cause gonorrhea and meningococcal meningitis, respectively. Both N. gonorrhoeae and N. meningitidis release a number of small peptidoglycan (PG) fragments, including proinflammatory PG monomers, although N. meningitidis releases fewer PG monomers. The PG fragments released by N. gonorrhoeae and N. meningitidis are generated in the periplasm during cell wall remodeling, and a majority of these fragments are transported into the cytoplasm by an inner membrane permease, AmpG; however, a portion of the PG fragments are released into the extracellular environment through unknown mechanisms. We previously reported that the expression of meningococcal ampG in N. gonorrhoeae reduced PG monomer release by gonococci. This finding suggested that the efficiency of AmpG-mediated PG fragment recycling regulates the amount of PG fragments released into the extracellular milieu. We determined that three AmpG residues near the C-terminal end of the protein modulate AmpG's efficiency. We also investigated the association between PG fragment recycling and release in two species of human-associated nonpathogenic Neisseria: N. sicca and N. mucosa Both N. sicca and N. mucosa release lower levels of PG fragments and are more efficient at recycling PG fragments than N. gonorrhoeae Our results suggest that N. gonorrhoeae has evolved to increase the amounts of toxic PG fragments released by reducing its PG recycling efficiency. IMPORTANCE: Neisseria gonorrhoeae and Neisseria meningitidis are human pathogens that cause highly inflammatory diseases, although N. meningitidis is also frequently found as a normal member of the nasopharyngeal microbiota. Nonpathogenic Neisseria, such as N. sicca and N. mucosa, also colonize the nasopharynx without causing disease. Although all four species release peptidoglycan fragments, N. gonorrhoeae is the least efficient at recycling and releases the largest amount of proinflammatory peptidoglycan monomers, partly due to differences in the recycling permease AmpG. Studying the interplay between bacterial physiology (peptidoglycan metabolism) and pathogenesis (release of toxic monomers) leads to an increased understanding of how different bacterial species maintain asymptomatic colonization or cause disease and may contribute to efforts to mitigate disease.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria.

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.

How to get (a)round: mechanisms controlling growth and division of coccoid bacteria.

Bacteria come in a range of shapes, including round, rod-shaped, curved and spiral cells. This morphological diversity implies that different mechanisms exist to guide proper cell growth, division and chromosome segregation. Although the majority of studies on cell division have focused on rod-shaped cells, the development of new genetic and cell biology tools has provided mechanistic insight into the cell cycles of bacteria with different shapes, allowing us to appreciate the underlying molecular basis for their morphological diversity. In this Review, we discuss recent progress that has advanced our knowledge of the complex mechanisms for chromosome segregation and cell division in bacteria which have, deceptively, the simplest possible shape: the cocci.

Effects of high hydrostatic pressure on bacterial growth on human ossicles explanted from cholesteatoma patients.

BACKGROUND: High hydrostatic pressure (HHP) treatment can eliminate cholesteatoma cells from explanted human ossicles prior to re-insertion. We analyzed the effects of HHP treatment on the microbial flora on ossicles and on the planktonic and biofilm states of selected isolates. METHODOLOGY: Twenty-six ossicles were explanted from cholesteatoma patients. Five ossicles were directly analyzed for microbial growth without further treatment. Fifteen ossicles were cut into two pieces. One piece was exposed to HHP of 350 MPa for 10 minutes. Both the treated and untreated (control) pieces were then assessed semi-quantitatively. Three ossicles were cut into two pieces and exposed to identical pressure conditions with or without the addition of one of two different combinations of antibiotics to the medium. Differential effects of 10-minute in vitro exposure of planktonic and biofilm bacteria to pressures of 100 MPa, 250 MPa, 400 MPa and 540 MPa in isotonic and hypotonic media were analyzed using two patient isolates of Staphylococcus epidermidis and Neisseria subflava. Bacterial cell inactivation and biofilm destruction were assessed by colony counting and electron microscopy. PRINCIPAL FINDINGS: A variety of microorganisms were isolated from the ossicles. Irrespective of the medium, HHP treatment at 350 MPa for 10 minutes led to satisfying but incomplete inactivation especially of gram-negative bacteria. The addition of antibiotics increased the efficacy of elimination. A comparison of HHP treatment of planktonic and biofilm cells showed that the effects of HPP were reduced by about one decadic logarithmic unit when HPP was applied to biofilms. High hydrostatic pressure conditions that are suitable to inactivate cholesteatoma cells fail to completely sterilize ossicles even if antibiotics are added. As a result of the reduced microbial load and the viability loss of surviving bacteria, however, there is a lower risk of re-infection after re-insertion.

Nutrient agar with sodium chloride supplementation for presumptive detection of Moraxella catarrhalis in clinical specimens.

We previously reported that Nissui nutrient agar (N medium) promoted the growth of Moraxella catarrhalis but not commensal Neisseria spp. In the present study, we examined which constituent of N medium was responsible for the selective growth of M. catarrhalis using 209 M. catarrhalis and 100 commensal Neisseria spp. clinical strains. We found that peptone, but not meat extract or agar of N medium, had growth-promoting or growth-inhibiting ability with respect to M. catarrhalis and commensal Neisseria spp. Thus, we investigated the amino acid content of N peptone and found it had higher concentrations of amino acids than other commercial peptone products. On varying the sodium chloride concentration of reconstituted N medium, we noted that the concentration was an important factor in bacterial growth differences. Varying the sodium chloride concentration of other commercial nutrient agars achieved similar results to those for N medium. This is, to our knowledge, the first study observing that sodium chloride concentration is responsible for difference in growth between the two organisms. We also successfully isolated colonies of M. catarrhalis from respiratory specimens on N medium, whereas the growth of commensal Neisseria spp. was inhibited, and by adding bovine hematin and ß-NAD we were able to isolate Haemophilus influenzae colonies as efficiently as with a chocolate agar. In conclusion, nutrient agar can be used as a medium for the preferential isolation of M. catarrhalis from upper respiratory tract specimens.

Biochemical and genomic analysis of the denitrification pathway within the genus Neisseria.

Since Neisseria gonorrhoeae and Neisseria meningitidis are obligate human pathogens, a comparison with commensal species of the same genus could reveal differences important in pathogenesis. The recent completion of commensal Neisseria genome draft assemblies allowed us to perform a comparison of the genes involved in the catalysis, assembly and regulation of the denitrification pathway, which has been implicated in the virulence of several bacteria. All species contained a highly conserved nitric oxide reductase (NorB) and a nitrite reductase (AniA or NirK) that was highly conserved in the catalytic but divergent in the N-terminal lipid modification and C-terminal glycosylation domains. Only Neisseria mucosa contained a nitrate reductase (Nar), and only Neisseria lactamica, Neisseria cinerea, Neisseria subflava, Neisseria flavescens and Neisseria sicca contained a nitrous oxide reductase (Nos) complex. The regulators of the denitrification genes, FNR, NarQP and NsrR, were highly conserved, except for the GAF domain of NarQ. Biochemical examination of laboratory strains revealed that all of the neisserial species tested except N. mucosa had a two- to fourfold lower nitrite reductase activity than N. gonorrhoeae, while N. meningitidis and most of the commensal Neisseria species had a two- to fourfold higher nitric oxide (NO) reductase activity. For N. meningitidis and most of the commensal Neisseria, there was a greater than fourfold reduction in the NO steady-state level in the presence of nitrite as compared with N. gonorrhoeae. All of the species tested generated an NO steady-state level in the presence of an NO donor that was similar to that of N. gonorrhoeae. The greatest difference between the Neisseria species was the lack of a functional Nos system in the pathogenic species N. gonorrhoeae and N. meningitidis.

Chloramphenicol is a substrate for a novel nitroreductase pathway in Haemophilus influenzae.

The p-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to Haemophilus influenzae and Neisseria meningitidis; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with H. influenzae cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several Neisseria species and most chloramphenicol-susceptible Haemophilus species, but not Escherichia coli or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than H. influenzae have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of H. influenzae and Neisseria species is unknown. Further understanding of the H. influenzae chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.

Clarithromycin treatment selects for persistent macrolide-resistant bacteria in throat commensal flora.

The aim of the study was to determine the effect of clarithromycin treatment on resistance development in the commensal throat flora. Alpha-haemolytic streptococci and Neisseria spp. were isolated from patients receiving clarithromycin for eradication of Helicobacter pylori. The treatment resulted in an immediate increase in the number of macrolide-resistant streptococci, which remained for one year after treatment, but declined to background level three years later. The most prevalent resistance gene was mef(A). Neisseria spp. were less affected by the treatment: the number of resistant isolates increased in only in one case during treatment. In conclusion, a one-week standard therapy with clarithromycin selects for an increased prevalence of macrolide-resistant streptococci that persisted for more than one year.

Biofilm dispersal of Neisseria subflava and other phylogenetically diverse oral bacteria.

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of Neisseria subflava and were incubated under conditions of low vibration. N. subflava colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10(2) to 10(4) small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of N. subflava single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Streptococcus mitis, and a prevalent but previously uncultured oral Streptococcus sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.

Mechanisms other than penicillin-binding protein-2 alterations may contribute to moderate penicillin resistance in Neisseria meningitidis.

Penicillin resistance in Neisseria spp is thought to be generated by the interspecies transfer of genetic material from naturally penicillin-resistant, commensal species. We examined a series of successive transformants with increasing levels of penicillin resistance, obtained by co-cultivation of Neisseria meningitidis derivatives with Neisseria polysaccharea. Our results suggest that, in addition to the well-known decrease in penicillin affinity of penicillin-binding protein-2 (PBP-2), decreased expression of the class 3 porin as well as decreased affinity of PBP-1 may contribute to higher level resistance of N. meningitidis to penicillin G and other beta-lactam compounds.

The pilus-induced Ca2+ flux triggers lysosome exocytosis and increases the amount of Lamp1 accessible to Neisseria IgA1 protease.

The IgA1 protease secreted by the pathogenic Neisseriae cleaves Lamp1, a major integral membrane glycoprotein of lysosomes, and significantly reduces its steady-state levels in an infected cell. IgA1 protease hydrolysis of Lamp1 is inefficient at the low pH of lysosomes, strongly suggesting that the enzyme is unlikely to reduce Lamp1 levels within lysosomes to any appreciable extent. We therefore explored the possibility that the protease may reach Lamp1 through an alternative route. We demonstrate that Neisseria pili induce a transient increase in the levels of cytosolic free Ca2+ in A431 human epithelial cells, as demonstrated previously for ME180 cells. This Ca2+ flux triggers lysosome exocytosis, quickly altering the cellular distribution of Lamp1 and increasing surface Lamp1 levels. Finally, we demonstrate that surface Lamp1 is cleaved by IgA1 protease secreted by adherent bacteria. We conclude that the pilus-induced Ca2+ flux increases the amount of Lamp1 that is cleavable by the IgA1 protease.

Transfer of penicillin resistance between Neisseriae in microcosm.

Horizontal gene transfer between commensal and pathogenic Neisseriae is the mechanism proposed to explain how pathogenic species acquire altered portions of the penA gene, which encodes penicillin binding protein 2. These changes resulted in a moderately penicillin-resistant phenotype in the meningococci, whose frequency of isolation in Spain increased at the end of the 1980s. Little has been published about the possibility of this gene transfer in nature or about its simulation in the laboratory. We designed a simple microcosm, formed by solid and liquid media, that partially mimics the upper human respiratory tract. In this microcosm, penicillin-resistant commensal strains and the fully susceptible meningococcus were co-cultivated. The efficiency of gene transfer between the strains depended on the phase of bacterial growth and the conditions of culture. Resistance of penicillin was acquired in different steps irrespective of the source of the DNA. The presence of DNase in the medium had no effect on gene transfer, but it was near zero when nicked DNA was used. Cell-to-cell contact or membrane blebs could explain these results. The analysis of sequences of the transpeptidase domain of PBP2 from transformants, and from donor and recipient strains demonstrated that the emergence of moderately resistant transformants was due to genetic exchange between the co-cultivated strains. Finally, mechanisms other than penA modification could be invoked to explain decreased susceptibility.

Microbiologic activity in laser resurfacing plume and debris.

BACKGROUND AND OBJECTIVE: With the increasing use of laser resurfacing, concerns have arisen about the biological hazards associated with the procedure. This study analyzed the potential bacterial and viral exposure to operating room personnel as a result of the laser smoke plume in CO2 laser resurfacing. STUDY DESIGN/MATERIALS AND METHODS: Thirteen consecutive patients underwent CO2 laser resurfacing. A HEPA filter in the smoke evacuator was used to collect specimens of the laser plume smoke for cultures. The study was controlled by a second filter exposed to room air. RESULTS: The 13 patients each had one bacterial, one viral, and one control culture (total, 39 specimens). In the control group, none of the 13 specimens had any growth. No viral growth has been found to date. Of 13 bacterial cultures, 5 resulted in growth of coagulase-negative Staphylococcus. Of these five positive specimens, one also had growth of Corynebacterium and one had growth of Neisseria. CONCLUSION: The potential exists for operating personnel to be exposed to viable bacteria during laser resurfacing.

Analysis of pH-driven disruption of oral microbial communities in vitro.

Previously, a mixed culture chemostat system was used to demonstrate that the pH generated from carbohydrate metabolism, rather than carbohydrate availability per se, was responsible for the shifts observed in the oral microflora which are associated with high carbohydrate diets and the development of dental caries. The aim of this study was to determine more accurately the microbially generated pH at which such shifts occurred. Nine oral bacteria were grown in three independent chemostats, and pulsed with glucose on 10 consecutive days. In one chemostat, pH control was discontinued for 6 h, and the pH fall was restricted to a minimum value of pH 5.5; the pH fall was arrested in the other two chemostats at either pH 5. 0, or at pH 4.5. When the pH was allowed to fall, the numbers and proportions of Streptococcus mutans and Lactobacillus rhamnosus increased; this increase was directly related to the magnitude of the pH fall. Veillonella dispar was the most numerous organism following all glucose pulsing regimes, especially at low pH. The increase in proportions of acidogenic bacteria was accompanied by a fall in the proportions of acid-sensitive species (Fusobacterium nucleatum, Prevotella nigrescens, Streptococcus gordonii and Streptococcus oralis). The counts of these species were relatively stable between pH 5.5 and 4.5, but were markedly reduced when the pH fell below pH 4.5; Neisseria subflava could not persist in the culture at pH 4.5 or below. The data suggest that the disruption of communities associated with glucose metabolism and low pH can be explained in terms of a two-stage process. A fall in pH to a value between pH 5.5 and 4.5 may allow the enrichment of potentially cariogenic species, whilst permitting species associated with health to remain relatively unaffected. A further reduction in pH (