A Comparison of the Adaptive Response of Staphylococcus aureus vs. Streptococcus mutans and the Development of Chlorhexidine Resistance.

Antimicrobials with nonselective antibacterial efficacy such as chlorhexidine can be effective in reducing biofilm, but bear the risk of inducing resistance in specific bacteria. In clinical practice, bacteria such as Staphylococcus aureus have been found resistant to chlorhexidine, but other bacteria, including Streptococcus mutans, have largely remained susceptible to chlorhexidine despite its widespread use in oral healthcare. Here, we aim to forward a possible reason as to why S. aureus can acquire resistance against chlorhexidine, while S. mutans remains susceptible to chlorhexidine. Measurement of surface-enhanced fluorescence indicated that chlorhexidine caused gradual, but irreversible deformation to adhering green fluorescent S. aureus due to irreparable damage to the cell wall. Concurrently, the metabolic activity of adhering staphylococci was higher than of planktonic bacteria, suggesting efflux mechanisms may have been activated upon cell wall deformation, impeding the buildup of a high chlorhexidine concentration in the cytoplasm and therewith stimulating the development of chlorhexidine resistance in S. aureus. Exposure of S. mutans to chlorhexidine caused immediate, but reversible deformation in adhering streptococci, indicative of rapid self-repair of cell wall damage done by chlorhexidine. Due to cell wall self-repair, S. mutans will be unable to effectively reduce the chlorhexidine concentration in the cytoplasm causing solidification of the cytoplasm. In line, no increased metabolic activity was observed in S. mutans during exposure to chlorhexidine. Therewith, self-repair is suicidal and prevents the development of a chlorhexidine-resistant progeny in S. mutans.

Comparison of methods to evaluate bacterial contact-killing materials.

Cationic surfaces with alkylated quaternary-ammonium groups kill adhering bacteria upon contact by membrane disruption and are considered increasingly promising as a non-antibiotic based way to eradicate bacteria adhering to surfaces. However, reliable in vitro evaluation methods for bacterial contact-killing surfaces do not yet exist. More importantly, results of different evaluation methods are often conflicting. Therefore, we compared five methods to evaluate contact-killing surfaces. To this end, we have copolymerized quaternary-ammonium groups into diurethane dimethacrylate/glycerol dimethacrylate (UDMA/GDMA) and determined contact-killing efficacies against five different Gram-positive and Gram-negative strains. Spray-coating bacteria from an aerosol onto contact-killing surfaces followed by air-drying as well as ASTM E2149-13a (American Society for Testing and Materials) were found unsuitable, while the Petrifilm® system and JIS Z 2801 (Japanese Industrial Standards) were found to be excellent methods to evaluate bacterial contact-killing surfaces. It is recommended however, that these methods be used in combination with a zone of inhibition on agar assay to exclude that leakage of antimicrobials from the material interferes with the contact-killing ability of the surface. STATEMENT OF SIGNIFICANCE: Bacterial adhesion to surfaces of biomaterials implants can be life-threatening. Antimicrobials to treat biomaterial-associated infections often fail due to the bacterial biofilm-mode-of-growth or are ineffective due to antibiotic-resistance of causative organisms. Positively-charged, quaternized surfaces can kill bacteria upon contact and are promising as a non-antibiotic-based treatment of biomaterial-associated infections. Reliable methods to determine efficacies of contact-killing surfaces are lacking, however. Here, we show that three out of five methods compared, including an established ASTM, are unsuitable. Methods found suitable should be used in combination with a zone-of-inhibition-assay to establish absence of antimicrobial leaching, potentially interfering with contact-killing. Identification of suitable assays for evaluating bacterial contact-killing will greatly assist this emerging field as an alternative for antibiotic-based treatment of biomaterial-associated-infections.

Synergy of brushing mode and antibacterial use on in vivo biofilm formation.

OBJECTIVES: Orthodontic, multi-strand retention-wires are used as a generalized model for oral retention sites to investigate whether biofilm left-behind after powered toothbrushing in-vivo enabled better penetration of antibacterials as compared with manual brushing. METHODS: 2-cm multi-strand, stainless-steel retention-wires were placed in brackets bonded bilaterally in the upper arches of 10-volunteers. Volunteers used NaF-sodium-lauryl-sulphate-containing toothpaste and antibacterial, triclosan-containing toothpaste supplemented or not with an essential-oils containing mouthrinse. Opposite sides of the dentition including the retention-wires, were brushed manually or with a powered toothbrush. Health-care-regimens were maintained for 1-week, after which wires were removed and oral biofilm was collected. RESULTS: When powered toothbrushing was applied, slightly less bacteria were collected than after manual brushing, regardless whether an antibacterial-regimen was used or not. Powered-toothbrushing combined with antibacterial-regimens yielded lower biofilm viability than manual brushing, indicating better antibacterial penetration into biofilm left-behind after powered brushing. Major shifts in biofilm composition, with a decrease in prevalence of both cariogenic species and periodontopathogens, were induced after powered brushing using an antibacterial-regimen. CONCLUSION: Oral biofilm left-behind after powered brushing in-vivo enabled better penetration of antibacterials than after manual brushing. CLINICAL SIGNIFICANCE: Mechanical removal of oral biofilm is important for prevention of dental pathologies, but biofilm is always left-behind, such as in fissures, buccal pits, interproximal areas and gingival margins and around orthodontic appliances. Use of antibacterial toothpastes or mouthrinses can contribute to removal or killing of biofilm bacteria, but biofilm structure hampers antibacterial penetration. A synergy between brushing mode and antibacterial-regimen applied exists with clinically demonstrable effects.