A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis.

Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP) can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25) by immunohistochemistry and periodic acid-Schiff staining (PAS). The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

A novel antifungal is active against Candida albicans biofilms and inhibits mutagenic acetaldehyde production in vitro.

The ability of C. albicans to form biofilms is a major virulence factor and a challenge for management. This is evident in biofilm-associated chronic oral-oesophageal candidosis, which has been shown to be potentially carcinogenic in vivo. We have previously shown that most Candida spp. can produce significant levels of mutagenic acetaldehyde (ACH). ACH is also an important mediator of candidal biofilm formation. We have also reported that D,L-2-hydroxyisocaproic acid (HICA) significantly inhibits planktonic growth of C. albicans. The aim of the present study was to investigate the effect of HICA on C. albicans biofilm formation and ACH production in vitro. Inhibition of biofilm formation by HICA, analogous control compounds or caspofungin was measured using XTT to measure biofilm metabolic activity and PicoGreen as a marker of biomass. Biofilms were visualised by scanning electron microscopy (SEM). ACH levels were measured by gas chromatography. Transcriptional changes in the genes involved in ACH metabolism were measured using RT-qPCR. The mean metabolic activity and biomass of all pre-grown (4, 24, 48 h) biofilms were significantly reduced after exposure to HICA (p<0.05) with the largest reductions seen at acidic pH. Caspofungin was mainly active against biofilms pre-grown for 4 h at neutral pH. Mutagenic levels (>40 µM) of ACH were detected in 24 and 48 h biofilms at both pHs. Interestingly, no ACH production was detected from D-glucose in the presence of HICA at acidic pH (p<0.05). Expression of genes responsible for ACH catabolism was up-regulated by HICA but down-regulated by caspofungin. SEM showed aberrant hyphae and collapsed hyphal structures during incubation with HICA at acidic pH. We conclude that HICA has potential as an antifungal agent with ability to inhibit C. albicans cell growth and biofilm formation. HICA also significantly reduces the mutagenic potential of C. albicans biofilms, which may be important when treating bacterial-fungal biofilm infections.

Acetaldehyde production and microbial colonization in oral squamous cell carcinoma and oral lichenoid disease.

OBJECTIVE: The main aim of this prospective study was to explore the ability of the oral microbiome to produce acetaldehyde in ethanol incubation. STUDY DESIGN: A total of 90 patients [30 oral squamous cell carcinoma (OSCC); 30 oral lichenoid disease (OLD); 30 healthy controls (CO)] were enrolled in the study. Microbial samples were taken from the mucosa using a filter paper method. The density of microbial colonization was calculated and the spectrum analyzed. Microbial acetaldehyde production was measured by gas chromatography. RESULTS: The majority (68%) of cultures produced carcinogenic levels of acetaldehyde (>100 µM) when incubated with ethanol (22 mM). The mean acetaldehyde production by microbes cultured from smoker samples was significantly higher (213 µM) than from non-smoker samples (141 µM) (P=.0326). CONCLUSIONS: The oral microbiota from OSCC, OLD patients and healthy individuals are able to produce carcinogenic levels of acetaldehyde. The present provisional study suggests smoking may increase the production of acetaldehyde.

The cdr1B efflux transporter is associated with non-cyp51a-mediated itraconazole resistance in Aspergillus fumigatus.

OBJECTIVES: Recent increases in triazole resistance in Aspergillus fumigatus have been attributed primarily to target site (cyp51A) mutations. A recent survey of resistant isolates in Manchester showed that >50% of resistant isolates had no mutation in cyp51A or its promoter. We investigated the mechanisms of resistance in clinical azole-resistant isolates without cyp51A mutations. METHODS: Twelve azole-resistant isolates, 10 of which were itraconazole resistant, were studied. Bioinformatic comparisons between Candida albicans efflux genes and A. fumigatus genome data identified 20 putative azole transporter genes. Basal and azole-induced expression of these genes and cyp51A was quantified using RT-PCR with comparison with clinical azole-susceptible isolates. Function of high basal or itraconazole-induced expression transporters was tested by gene knockout in azole-susceptible and azole-resistant isolates. RESULTS: All susceptible strains showed minimal basal expression of cdr1B compared with 8 of 10 azole-resistant strains with high basal expression of this gene (>5-fold), 3 of which showed >30-fold increased expression. Knockout of this gene resulted in a 4-fold reduction in itraconazole, posaconazole and voriconazole MICs for a susceptible clinical isolate and a 4-fold reduction in itraconazole susceptibility in a clinical resistant isolate. One strain showed a >500-fold induction of cyp51A. No increase in basal expression or expression after induction was seen for the 18 remaining putative transporters. CONCLUSIONS: The reasons behind the shift away from target site mutation in azole-resistant isolates from Manchester are unknown. The modest change in expression of cdr1B in azole-susceptible strains implies that only study of resistant isolates will lead to further understanding of resistance mechanisms in A. fumigatus.

Intravenous antibiotics reduce the presence of Aspergillus in adult cystic fibrosis sputum.

BACKGROUND: Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-colonise the airways of patients with cystic fibrosis (CF). This study aimed to assess the impact of short-term administration of intravenous antipseudomonal antibiotics during CF exacerbations on the presence of Aspergillus. METHODS: Pre- and post-antibiotic sputum samples from 26 adult patients with CF and chronic Pseudomonas colonisation were analysed for the presence of Aspergillus by fungal culture, real-time PCR and galactomannan antigen (GM). Lung function (forced expiratory volume in 1 s and forced vital capacity % predicted) and blood levels of total IgE, specific A fumigatus IgE and specific A fumigatus IgG were measured at the start and end of antibiotics. Respiratory viral real-time PCR and bacterial community profiling using ribosomal intergenic spacer analysis (RISA) were performed to estimate concurrent changes in the lung microbiome. RESULTS: Aspergillus PCR and GM were more sensitive than culture in detecting Aspergillus species (culture 8%, GM 31%, PCR 77%). There was a significant decline in the presence of Aspergillus, measured both by PCR and GM index, following antibacterial therapy (PCR: median increase in crossing threshold 1.7 (IQR 0.5-3.8), p<0.001; GM: median fall in GM index 0.7 (IQR 0.4-1.6), p=0.016). All patients improved clinically with a significant increase in lung function (p<0.0001). RISA community analysis showed large changes in bacterial community similarity in 67% of patients following antibiotics. Viral RT-PCR demonstrated the presence of a concurrent respiratory virus in 27% of patients. CONCLUSIONS: Intravenous antibiotics targeting Pseudomonas during CF pulmonary exacerbations have a negative impact on the presence of Aspergillus in sputum samples.

Alcohol and acetaldehyde in African fermented milk mursik--a possible etiologic factor for high incidence of esophageal cancer in western Kenya.

BACKGROUND: Esophageal cancer is unusually frequent in Western Kenya, despite the low prevalence of classical risk factors such as heavy drinking and tobacco smoking. Among Kenyans consumption of fermented milk is an old tradition. Our hypothesis is that alcohol and acetaldehyde are produced during the fermentation process and that their carcinogenic potential contributes to the high incidence of esophageal cancer. METHODS: Eight samples of mursik milk starter cultures were collected from different Kalenjin families in the Rift Valley province, Western Kenya. A protocol provided by the families was used for milk fermentation. Ethanol and acetaldehyde levels were measured by gas chromatography. The microbial flora in starter cultures was identified by 16S and 18S sequencing. RESULTS: 7/8 starter cultures produced mutagenic (>100 µmol/L) levels of acetaldehyde and 4/8 starter cultures produced more than 1,000 µmol/L of acetaldehyde. The highest alcohol levels (mean 79.4 mmol/L) were detected in the four fermented milks with highest acetaldehyde production. The mean number of microbial species in the starter cultures was 5 (range 2-8). Yeasts were identified in all starter cultures (mean 1.5 species/milk) but their proportion of the total microbial count varied markedly (mean 35%, range 7%-90%). A combination of yeast and lactobacilli, especially Candida krusei with Lactobacillus kefiri, with the exclusion of other species, seemed to correlate with higher acetaldehyde and ethanol levels. CONCLUSIONS: Significant levels of ethanol and acetaldehyde were produced during mursik fermentation. IMPACT: When ingested several times daily the repeated exposure to carcinogenic levels of acetaldehyde may contribute to esophageal carcinogenesis.

Impregnation with antimicrobials has an impact on degree of conversion and colour stability of acrylic liner.

This study investigated the impact of impregnation of a poly(ethyl methacrylate) /tetrahydrofurfuryl methacrylate (PEM/THFM) polymer with chlorhexidine or fluconazole on the degree of conversion (DC) and colour stability (ΔE). The DC of uncured (0 h) and cured (24 h) samples was analysed by Fourier transform infrared spectroscopy (FTIR) and colour stability was analysed colorimetrically. The DC percentage of the control samples was significantly greater than those containing chlorhexidine and fluconazole (p≤0.05). The control discs exhibited only slight colour change compared to the impregnated discs which showed marked colour change (p≤0.05). A strong negative correlation between the extent of colour change and the degree of conversion was detected (r=0.97). The DC and colour stability were influenced by the addition of chlorhexidine or fluconazole. However, the final values were comparable to other commonly used acrylic liners and within acceptable ranges. PEM/THFM can be considered as a biocompatible drug delivery system.

Impregnation with antimicrobials challenge bonding properties and water sorption behaviour of an acrylic liner.

OBJECTIVES: To investigate the effect of impregnation of poly(ethyl methacrylate) and tetrahydro-furfuryl methacrylate (PEM/THFM) polymeric delivery system with chlorhexidine or fluconazole on its shear bond strength (SBS) and water sorption. METHODS: For SBS testing, 16 PEM/THFM discs impregnated with chlorhexidine (CHX), pure fluconazole (FLUp) or fluconazole from capsules (FLUc) and 16 drug-free control discs were prepared and bonded to heat-cured acrylic blocks. All discs were allowed to set for 24h at room temperature. After setting, half the discs (n=8) were tested immediately (Group 1). The other half was further incubated in water for 28 days at 37 °C before testing (Group 2). To evaluate water uptake, five PEM/THFM discs impregnated with CHX, FLUp or FLUc and five drug-free control discs were prepared and incubated in water. Mass changes were measured up to six months. RESULTS: The mean SBS for control, FLUp, CHX and FLUc discs were 4.01, 3.85, 3.29 and 2.26 MPa, respectively for Group 1. Group 2 showed significantly lower SBS (P≤0.05). All failures were adhesive. The percentage mass change due to water sorption ranged significantly from 12% for control to 27% for FLUc (P≤0.05). A strong negative correlation between the extent of water absorption and the SBS was detected (R=0.94, P=0.05). SIGNIFICANCE: Impregnation with antimicrobials presents a challenge to the physical and mechanical properties of a polymer. However, despite increased water uptake SBS remained acceptable for a temporary lining material and comparable to drug-free long-term lining materials. Moreover, the enhanced water uptake could contribute to improved leaching.

Fungicidal amounts of antifungals are released from impregnated denture lining material for up to 28 days.

OBJECTIVES: The aim of this study was to investigate the efficacy of a polymeric delivery system impregnated with chlorhexidine or fluconazole against Candida species. METHODS: Self-cure poly-ethyl methacrylate and tetrahydro-furfuryl methacrylate (PEM/THFM) discs impregnated with pure fluconazole substance (FLUp), fluconazole powder from capsules (FLUc) or chlorhexidine powder (CHX) were incubated in water for up to 28 days at 37 °C. The water was replaced at 24h and 3, 7, 14, 21, 28 days. The amount of released drugs and antifungal activity of the leachates was measured by bioassay. The minimal inhibitory concentration (MIC) of each drug for 46 Candida isolates was determined and compared to the released concentrations. RESULTS: A total of 53.0% of CHX, 38.5% of FLUc and 13.2% of FLUp impregnated into the discs was leached during the 28-day incubation. Of the total amount leached, 71.8% of CHX, 75.1% of FLUc and 70.5% of FLUp was released during the first week of incubation. Antifungal activity was confirmed for up to 28 days. CONCLUSION: Both chlorhexidine and fluconazole become readily leached from PEM/THFM polymer up to four weeks and that the polymerisation of the acrylic does not affect the antimicrobial activity of the agents. Importantly, the amount of drugs released exceeded the MICs of most isolates also during the fourth week of incubation. CLINICAL SIGNIFICANCE: These findings indicate the feasibility of this treatment modality for oral candidal infections, especially denture stomatitis. But further in vivo work is warranted to determine its clinical relevance and applicability.

Azole antifungal resistance today: focus on Aspergillus.

Oral triazole therapy is well established for the treatment of invasive aspergillosis (IPA), allergic aspergillosis (ABPA), and chronic pulmonary aspergillosis (CPA), and is often long-term. Resistance to triazole azole antifungal drugs in Aspergillus fumigatus is now a major clinical problem in a number of European locations, in China, Canada and the USA with particularly high frequencies from the north-west of the UK, and The Netherlands. A number of centers are reporting the continuing increasing frequency and evolution of resistance mechanisms in A. fumigatus, in both azole-naïve and patients treated with azoles. The increasing rate of resistance is of concern. A number of resistance mechanisms have been found. The biofilm modality of Aspergillus growth may have a number of therapeutic implications for aspergillosis, including antifungal resistance. Microbiological diagnosis of aspergillosis is limited by poor culture yield, leading to uncertainty about the frequency of triazole resistance. Direct resistance testing in culture-negative clinical samples may add additional insights into the prevalence of azole resistance in A. fumigatus.

Oral candidosis--clinical challenges of a biofilm disease.

This review summarizes the impact of biofilms in oral candidosis with special emphasis on medically compromised patients. The concept of oral candidosis as a mixed candidal-bacterial biofilm infection has changed our understanding of its epidemiology and diagnosis as well as approach to its treatment. Candida albicans is the most common causative agent of oral candidosis although Candida species other than C. albicans are often seen in medically compromised patients with a history of multiple courses of azole antifungals. Although C. albicans is usually susceptible to all commonly used antifungals when tested in vitro, their biofilm form are highly resistant to most antifungals. Therefore, treatment consists of mechanical destruction of the biofilm in combination with topical drugs. Azole antifungals should be avoided for patients suffering from recurrent oral yeast infections due to a risk of selection and enrichment of resistant strains within the biofilm. Oral candidosis can also be a symptom of an undiagnosed or poorly controlled systemic disease such as HIV infection or diabetes. If the response to appropriate treatment is poor, other causes of oral mucositis should be excluded. Oral candidosis arises from the patient's mixed candidal-bacterial biofilm, i.e., dental plaque, whereby good self-care is important for successful therapy.

ADH1 expression inversely correlates with CDR1 and CDR2 in Candida albicans from chronic oral candidosis in APECED (APS-I) patients.

Expression of the alcohol dehydrogenase gene ADH1, which converts ethanol into carcinogenic acetaldehyde, significantly inversely correlated with the expression of CDR1 and CDR2, genes linked to azole resistance in Candida albicans isolated from chronic oral candidosis in autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED, APS-I) patients. This is a novel link between candidal two-carbon metabolism genes and azole resistance.

Commercial mouthwashes are more effective than azole antifungals against Candida albicans biofilms in vitro.

OBJECTIVE: The aim of this study was to evaluate and compare the activity of prescription and over-the-counter antimicrobial compounds against planktonic and biofilm forms of Candida albicans isolated from cases of oral candidiasis in vitro. STUDY DESIGN: The efficacy of azoles, polyenes, an echinocandin, and 4 over-the-counter mouthwashes were tested against C. albicans-derived planktonic and biofilm cells. RESULTS: Planktonic cells were shown to be highly sensitive to all of the antifungal agents tested. Sessile cells were highly resistant to azoles (≥128 mg/L) but equally sensitive to caspofungin and short treatments with Corsodyl, Listerine, and Oraldene. CONCLUSIONS: Although C. albicans is sensitive to azole antifungal agents in planktonic form, it is highly resistant within the biofilm. The good efficacy of the over-the-counter mouthwashes against candidal biofilms in vitro suggests that clinical trials should now be designed to establish their role in the clinical management of oral candidal infections.

Is dental treatment of an infected tooth a risk factor for locally invasive spread of infection?

PURPOSE: To determine the impact of antecedent dental procedures and dental health on the course of odontogenic maxillofacial infections requiring hospital care. PATIENTS AND METHODS: In this retrospective cohort study in a referral center, we evaluated medical records and panoramic radiographs of all patients admitted because of odontogenic maxillofacial infection (n = 84). The predictor variables were preceding dental treatment, antimicrobial therapy, and dental health. The outcome variables comprised infection parameters, length of stay, need for intensive care, and management during hospitalization. RESULTS: The mean age of the patients was 43.2 ± 16.5 years and 60% were men. Dental procedure preceded the spread of the infection in 49 cases (58%): endodontic treatment (n = 22), tooth extraction (n = 19), and minor first aid (n = 8). Twenty-seven patients had not received any dental or antimicrobial treatment in the recent past. Antimicrobial treatment alone had been given to 8 patients. Patients without preceding treatment had the highest C-reactive protein levels on admission and at maximum (P = .020 and P = .011) and the highest white blood cell counts on admission (P = .011). Their length of stay was also longer, and they needed intensive care more often than the other patients. Maximum C-reactive protein levels and white blood cell counts between treatment groups did not significantly differ from each other. CONCLUSIONS: The systemic response to the infection was strongest and the course of the infection most severe in the absence of preceding dental treatment and in patients with poor dental health. All types of dental treatment contributed to a less severe course of infection.

Xylitol inhibits carcinogenic acetaldehyde production by Candida species.

Acetaldehyde is a highly toxic and mutagenic product of alcohol fermentation and metabolism which has been classified as a Class I carcinogen for humans by the International Agency for Research on Cancer of the World Health Organisation (WHO). Many Candida species representing oral microbiota have been shown to be capable of marked acetaldehyde production. The aim of our study was to examine the effects of various sugar alcohols and sugars on microbial acetaldehyde production. The study hypothesis was that xylitol could reduce the amount of acetaldehyde produced by Candida. Laboratory and clinical isolates of seven Candida species were selected for the study. The isolates were incubated in 12 mM ethanol and 110 mM glucose, fructose or xylitol at 37°C for 30 min and the formed acetaldehyde was measured by gas chromatography. Xylitol significantly (p < 0.0001) reduced the amount of acetaldehyde produced from ethanol by 84%. In the absence of xylitol, the mean acetaldehyde production in ethanol incubation was 220.5 µM and in ethanol-xylitol incubation 32.8 µM. This was found to be mediated by inhibition of the alcohol dehydrogenase enzyme activity. Coincubation with glucose reduced the amount of produced acetaldehyde by 23% and coincubation with fructose by 29%. At concentrations that are representative of those found in the oral cavity during the intake of proprietary xylitol products, xylitol was found to reduce the production of carcinogenic acetaldehyde from ethanol by Candida below the mutagenic level of 40-100 µM.

Persistent Candida albicans colonization and molecular mechanisms of azole resistance in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) patients.

OBJECTIVES: Patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED, APS-I) suffer from chronic candidosis caused mainly by Candida albicans, and repeated courses of azole antifungals have led to the development of resistance in the APECED patient population in Finland. The aim of our study was to address whether the patients are persistently colonized with the same or genetically closely related strains, whether epidemic strains are present and which molecular mechanisms account for azole resistance. METHODS: Sets of C. albicans (n = 19) isolates from nine APECED patients reported with decreased susceptibility to fluconazole isolated up to 9 years apart were included. The strains were typed by multilocus sequence typing. CDR1/2, MDR1 and ERG11 mRNA expression was analysed by northern blotting and Cdr1, Cdr2 and Mdr1 protein expression by western blotting, and TAC1 and ERG11 genes were sequenced. RESULTS: All seven patients with multiple C. albicans isolates analysed were persistently colonized with the same or a genetically closely related strain for a mean of 5 years. All patients were colonized with different strains and no epidemic strains were found. The major molecular mechanisms behind the azole resistance were mutations in TAC1 contributing to overexpression of CDR1 and CDR2. Six new TAC1 mutations were found, one of which (N740S) is likely to be a gain-of-function mutation. Most isolates were found to have gained multiple TAC1 and ERG11 point mutations. CONCLUSIONS: Despite clinically successful treatment leading to relief of symptoms, colonization by C. albicans strains is persistent within APECED patients. Microevolution and point mutations occur within strains, leading to the development of azole-resistant isolates.

Patterned macroarray plates in comparison of bacterial adhesion inhibition of tantalum, titanium, and chromium compared with diamond-like carbon.

Staphylococcus aureus device-related infection is a common complication in implantology. Bacterial adhesion on implant surfaces is the initial step in the infective process. The aim was to develop a method suitable for quantitative bacterial adherence studies and to test a new diamond-like carbon (DLC) coating against commonly used metallic biomaterials with regards to Staphylococcus aureus adhesion. Patterned silicon chips with spots of tantalum, titanium, chromium, and DLC were produced using ultraviolet lithography and physical vapor deposition. These patterned chips were used as such or glued to array plates, pretreated with serum and exposed to S. aureus (S-15981) for 90 min, followed by acridine orange staining and fluorescence microscopy. An adhesion index showed that the ranking order of the biomaterials was titanium, tantalum, chromium, and DLC, with the DLC being clearly most resistant against colonization with S. aureus. Micropatterned surfaces are useful for quantitative comparison of bacterial adherence on different biomaterials. In the presence of serum, DLC is superior in its ability to resist adhesion and colonization by S. aureus compared with the commonly used biomaterial metals tantalum, titanium, and chromium.