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| 2. |
- Chávez de Paz, Luis Eduardo, et al.
(författare)
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Oral bacteria in biofilms exhibit slow reactivation from nutrient deprivation
- 2008
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 154, s. 1927-1938
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Tidskriftsartikel (refereegranskat)abstract
- The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone-yeast extract-glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm(-2) had increased by 30-35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.
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| 3. |
- Chávez de Paz, Luis Eduardo
(författare)
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Redefining the Persistent Infection in Root Canals : Possible Role of Biofilm Communities
- 2007
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Ingår i: Journal of Endodontics. - : Elsevier BV. - 0099-2399 .- 1878-3554. ; 33:6, s. 652-662
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Tidskriftsartikel (refereegranskat)abstract
- Current concepts suggest that persisting infections subsequent to endodontic therapy are caused by one or two bacterial species that are "too robust" to be eliminated by conventional treatment measures. As a consequence, numerous studies are exploring the characteristics of these "most" resistant organisms to define an effective treatment strategy to eradicate them from root canals. By taking an ecological perspective, the main objective of this review is to present evidence that the nature of persisting endodontic infections depends not on the robustness of the organisms in the infected site, but on their capability of adapting their physiology to the new environmental conditions set by the treatment. Changes in the environment, such as an increase in pH by calcium hydroxide or the effect of antimicrobials, are capable of triggering genetic cascades that modify the physiological characteristics of bacterial cells. Surface adherence by bacteria to form biofilms is a good example of bacterial adaptation and one that is pertinent to endodontic infections. Increasing information is now available on the existence of polymicrobial biofilm communities on root canal walls, coupled with new data showing that the adaptive mechanisms of bacteria in these biofilms are significantly augmented for increased survival. This ecological view on the persisting infection problem in endodontics suggests that the action of individual species in persisting endodontic infections is secondary when compared to the adaptive changes of a polymicrobial biofilm community undergoing physiological and genetic changes in response to changes in the root canal environment.
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| 4. |
- Chávez de Paz, Luis Eduardo, et al.
(författare)
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Response to alkaline stress by root canal bacteria in biofilms
- 2007
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Ingår i: International Endodontic Journal. - : Wiley. - 0143-2885 .- 1365-2591. ; 40:5, s. 344-355
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Tidskriftsartikel (refereegranskat)abstract
- To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. METHODOLOGY: Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. RESULTS: Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. CONCLUSIONS: In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.
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| 5. |
- Chávez de Paz, Luis Eduardo, et al.
(författare)
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The effects of antimicrobials on endodontic biofilm bacteria
- 2010
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Ingår i: Journal of Endodontics. - : Elsevier. - 0099-2399 .- 1878-3554. ; 36:1, s. 70-77
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Tidskriftsartikel (refereegranskat)abstract
- Introduction In the present study, confocal microscopy, a miniflow cell system, and image analysis were combined to test in situ the effect of antimicrobials and alkali on biofilms of Enterococcus faecalis, Lactobacillus paracasei, Streptococcus anginosus, and Streptococcus gordonii isolated from root canals with persistent infections. Methods Biofilms formed for 24 hours were exposed for 5 minutes to alkali (pH = 12), chlorhexidine digluconate (2.5%), EDTA (50 mmol/L), and sodium hypochlorite (1%). The biofilms were then characterized by using fluorescent markers targeting cell membrane integrity (LIVE/DEAD) and metabolic activity (5-cyano-2,3-ditolyl tetrazolium chloride and fluorescein diacetate). In addition, the biofilm architecture and the extent to which coating of the substrate surface with collagen influenced the resistance pattern to the chemicals were also analyzed. Results NaOCl (1%) affected the membrane integrity of all organisms and removed most biofilm cells. Exposure to EDTA (50 mmol/L) affected the membrane integrity in all organisms but failed to remove more than a few cells in biofilms of E. faecalis, L. paracasei, and S. anginosus. Chlorhexidine (2.5%) had a mild effect on the membrane integrity of E. faecalis and removed only 50% of its biofilm cells The effects were substratum-dependent, and most organisms displayed increased resistance to the antimicrobials on collagen-coated surfaces. Conclusions The biofilm system developed here was sensitive and differences in cell membrane integrity and removal of biofilm cells after exposure to antimicrobials commonly used in endodontics was discernible.
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| 6. |
- Fröjd, Victoria, 1986, et al.
(författare)
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In situ analysis of multispecies biofilm formation on customized titanium surfaces
- 2011
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Ingår i: Molecular Oral Microbiology. - : John Wiley & Sons. - 2041-1006 .- 2041-1014. ; 26:4, s. 241-252
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Tidskriftsartikel (refereegranskat)abstract
- Many studies to identify surfaces that enhance the incorporation of dental implants into bone and soft-tissue have been undertaken previously. However, to succeed in the clinical situation, an implant surface must not support development of microbial biofilms with a pathogenic potential. As a first step in investigating this, we used two-species and three-species biofilm models with 16S ribosomal RNA fluorescence in situ hybridization and confocal laser scanning microscopy to examine the effect of surface characteristics on biofilm formation by species that can colonize titanium implants in vivo: Streptococcus sanguinis, Actinomyces naeslundii and Lactobacillus salivarius. Surfaces blasted with Al(2) O(3) (S(a) = 1.0-2.0 μm) showed a seven-fold higher bacterial adhesion after 2 h than turned surfaces (S(a) = 0.18 μm) whereas porous surfaces, generated by anodic oxidation (S(a) = 0.4 μm), showed four-fold greater adhesion than turned surfaces. Hence, increased roughness promoted adhesion, most likely through protection of bacteria from shear forces. Chemical modification of the blasted and oxidized surfaces by incorporation of Ca(2+) ions reduced adhesion compared with the corresponding non-modified surfaces. After 14 h, biofilm growth occurred in the three-species model but not in the two-species consortium (containing S. sanguinis and A. naeslundii only). The biofilm biovolume on all surfaces was similar, suggesting that the influence of surface characteristics on adhesion was compensated for by biofilm development.
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| 7. |
- Jaramillo, David E., et al.
(författare)
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Decreased Bacterial Adherence and Biofilm Growth on Surfaces Coated with a Solution of Benzalkonium Chloride
- 2012
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Ingår i: Journal of Endodontics. - : Elsevier. - 0099-2399 .- 1878-3554. ; 38:6, s. 821-825
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Tidskriftsartikel (refereegranskat)abstract
- Introduction Secondary biofilm formation by oral bacteria after breakdown/fracture of temporary or permanent restorations imposes a challenge to the outcome of root canal treatment. This study focuses on benzalkonium chloride (BAK) coating on dentin or polystyrene surfaces and its influence on the early adhesion and biofilm formation by oral and root canal bacteria. Methods Microbial adhesion and biofilm growth on surfaces coated with BAK were analyzed qualitatively with a dentin disk model and quantitatively with a mini-flow cell biofilm model. Cell viability and total biovolume were analyzed by the LIVE/DEAD technique. The repelling effect of surfaces coated with BAK was compared with NaOCl. Uncoated surfaces were used as controls. Results Scanning electron microscope images in the dentin disk model revealed that very sparse biofilms were formed on NaOCl- and BAK-coated dentin surfaces. In contrast, biofilms formed on uncoated dentin were clearly visible as numerous irregularly distributed aggregates of rods and cocci. In the mini-flow cell system, confocal laser scanning microscope analysis confirmed that biofilms formed on NaOCl- and BAK-coated surfaces showed significantly less adhesion (2 hours) and biovolume accumulation (24 and 96 hours) compared with the uncoated controls (P < .01). Furthermore, cell viability assessments showed that on uncoated controls the viability measurements were high (>89%) as well as on BAK-coated surfaces (88% viable cells). However, cell viability was significantly reduced on NaOCl-coated surfaces (59% viable cells). Conclusions This study illustrates that surface coating with a surfactant solution containing BAK does not cause cell membrane damage but might interfere with cell mechanisms of adhesion. Investigations into the clinical utility of BAK as an antibiofilm medication are warranted.
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| 8. |
- Neilands, Jessica, et al.
(författare)
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Chitosan Nanoparticles Affect the Acid Tolerance Response in Adhered Cells of Streptococcus mutans
- 2011
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Ingår i: Caries Research. - : S. Karger. - 0008-6568 .- 1421-976X. ; 45:6, s. 501-505
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Tidskriftsartikel (refereegranskat)abstract
- In this study we evaluated the effect of chitosan nanoparticles on the acid tolerance response (ATR) of adhered Streptococcus mutans. An ATR was induced by exposing S. mutans to pH 5.5 for 2 h and confirmed by exposing the acid-adapted cells to pH 3.5 for 30 min, with the majority of cells appearing viable according to the LIVE/DEAD® technique. However, when chitosan nanoparticles were present during the exposure to pH 5.5, no ATR occurred as most cells appeared dead after the pH 3.5 shock. We conclude that the chitosan nanoparticles tested had the ability to hinder ATR induction in adhered S. mutans.
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| 9. |
- Pihl, Maria, et al.
(författare)
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Differential effects of Pseudomonas aeruginosa on biofilm formation by different strains of Staphylococcus epidermidis.
- 2010
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Ingår i: FEMS Immunology and Medical Microbiology. - : Wiley-Blackwell. - 0928-8244 .- 1574-695X. ; 46:3, s. 439-446
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas aeruginosa and Staphylococcus epidermidis are common opportunistic pathogens associated with medical device-related biofilm infections. 16S rRNA fluorescence in situ hybridization and confocal scanning laser microscopy were used to study these two bacteria in dual-species biofilms. We have shown that fresh isolates of S. epidermidis form biofilms more avidly on polymer surfaces than laboratory strains. In dual-species biofilms the presence of P. aeruginosa reduced biofilm formation by S. epidermidis, although the different clinical isolates differed in their susceptibility to this effect. The most resistant fresh isolate coexisted with P. aeruginosa for up to 18 hours and was also resistant to the effects of culture supernatant from P. aeruginosa biofilms which caused dispersal of other S. epidermidis strains from established biofilms. Thus, different strains of S. epidermidis differed in their capacity to withstand the action of P. aeruginosa, with some fresh isolates being better equipped than laboratory strains to coexist in biofilms with P. aeruginosa. Our data suggest that where S. epidermidis and P. aeruginosa are present on abiotic surfaces such as medical devices, S. epidermidis biofilm-formation can be inhibited by P. aeruginosa through two mechanisms: disruption by extracellular products, possibly polysaccharides, and, in the later stages, by cell lysis.
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| 10. |
- Pihl, Maria, et al.
(författare)
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Effects of clinical isolates of Pseudomonas aeruginosa on Staphylococcus epidermidis biofilm formation
- 2010
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Ingår i: FEMS Immunology and Medical Microbiology. - : Wiley-Blackwell. - 0928-8244 .- 1574-695X. ; 59:3, s. 504-512
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas aeruginosa is often found in chronic infections, including cystic fibrosis lung infections and those related to chronic wounds and venous ulcers. At the latter sites, P. aeruginosa can be isolated together with Staphylococcus epidermidis, and we have therefore explored the effect of clinical isolates and laboratory strains of P. aeruginosa strains on colonisation by S. epidermidis in dual-species biofilms. Biofilm formation was assayed using 16S rRNA fluorescence in situ hybridization and confocal laser scanning microscopy. Amongst the six P. aeruginosa strains tested, one particular strain, denoted 14:2, exerted a significant inhibitory effect and even after six hours, S. epidermidis levels in dual-species biofilms were reduced by more than 85% compared to biofilms without P. aeruginosa. Interestingly strain 14:2 was found to be negative for classical virulence determinants including pyocyanin, elastase and alkaline protease. Therefore, we suggest that less virulent phenotypes of P. aeruginosa which may develop over time in chronic infections, could counteract colonisation by S. epidermidis, ensuring persistence and dominance by P. aeruginosa in the host micro-habitat. Further studies are required to explain the inhibitory effect on S. epidermidis, although, extracellular polysaccharides produced by P. aeruginosa might play a role in this phenomenon.
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