Comparison of Streptococcus mutans Adhesion to Fixed Orthodontic Wires in different Types of Saliva under Laboratory Conditions

This study aimed to determine the antimicrobial activity of ethanol-extracts obtained from Ocimum gratissimum L. (clove or African basil, Lamiaceae) and O. santum L. (holy basil) against some microorganisms present in oral cavity related to either medical or dental disease. Antimicrobial properties of both ethanol-extracts of Ocimum species against Streptococcus mutans KPSK2, S. pyogenes ATCC 19615, Staphylococcus aureus ATCC 16794, and Candida albicans ATCC 10231 were primarily determined by agar disk diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal or fungicidal concentration (MBC or MFC) of these herbal extracts were further determined by broth micro-dilution method. Ethanol-extracts of O. sanctum L. and O. gratissimum L. inhibited the growth of all tested microorganisms in various degrees ranging from the strongest antimicrobial activity of O. sanctum against S. pyogenes [MIC at 0.19% (w/v); MBC at 0.78% (w/v)] to the least inhibitory activity of O. gratissimum against C. albicans [MIC at 12.5% (w/v); undetectable MFC]. The ethanol-extract of O. sanctum showed stronger antimicrobial property against the tested bacteria and fungus than O. gratissimum. The ethanol-extracts of both Ocimum species showed stronger antibacterial than antifungal activity. However, the ethanol-extract of O. gratissimum even at a high concentration of 50% (w/v) was unable to eliminate the tested fungus. Ethanol-extracts of Ocimum species contain effective antibacterial and antifungal properties that may be beneficial for further development of antimicrobial agents in medical and dental fields.

Several concentrations of 9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan (C-390), ranging from 5 to 100 micrograms/ml, were incorporated in brain heart infusion agar, MacConkey agar, and xylose-lysine-deoxycholate agar to evaluate the recovery of Pseudomonas aeruginosa from 538 sputum, 174 urine, and 22 stool samples. Seventy-six sputum samples containing P. aeruginosa grew this bacterium alone on brain heart infusion and MacConkey agars with a C-390 concentration of 25 micrograms/ml or greater. Other microorganisms present in these specimens grew only on media without C-390, and significantly less growth was observed on media with less than 20 micrograms of C-390 per ml. In few samples containing Klebsiella pneumoniae (3 of 30) and Serratia spp. (3 of 10), these organisms grew on all C-390 media and concentrations tested. The remaining sputum samples grew other bacteria and yeasts only on media without C-390. Brain heart infusion and MacConkey agars with C-390 were equally effective in recovering P. aeruginosa and suppressing the growth of a wide range of bacteria and yeasts from urine and stool samples. Xylose-lysine-deoxycholate agar with C-390 did not show a selective or suppressive advantage over xylose-lysine-deoxycholate agar alone for recovering P. aeruginosa from stool specimens. These results indicate that use of the correct medium and C-390 concentration would provide a suitable primary medium for inhibiting a wide range of bacteria and yeasts and would select the growth of P. aeruginosa from clinical specimens.

To evaluate the efficacy of 0.5%, 2.5% and 5.25% sodium hypochlorite (NaOCl) as intracanal irrigants associated with hand and rotary instrumentation techniques against Enterococcus faecalis within root canals and dentinal tubules. A total of 180 extracted human premolar teeth were infected for 21 days with E. faecalis. The specimens were divided into 12 groups, as follows: group 1: 5.25% NaOCl + Hybrid technique (Valdrighi et al. 1998); group 2: 5.25% NaOCl + nickel-titanium (NiTi) rotary technique 4 mm shorter than the apex (by FOP-UNICAMP); group 3: 5, 25% NaOCl + NiTi rotary technique (Hero 642); group 4: 2.5% NaOCl +Hybrid technique; group 5: 2.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 6: 2.5% NaOCl + NiTi rotary technique (Hero 642); group 7: 0.5% NaOCl + Hybrid technique; group 8: 0.5% NaOCl + NiTi rotary technique 4 mm shorter than the apex; group 9: 0.5% NaOCl + NiTi rotary technique (Hero 642); group 10: sterile saline solution + Hybrid technique; group 11: sterile saline solution + NiTi rotary technique 4 mm shorter than the apex; group 12: sterile saline solution + NiTi rotary technique (Hero 642). Canals were sampled before and after preparation. After serial dilution, samples were plated onto brain heart infusion (BHI) agar, and the colony forming units (CFU) that were grown were counted. The teeth were sectioned into three thirds and dentine chips were removed from the canals with conical burs. The samples obtained with each bur were immediately collected into test tubes containing BHI broth, and were incubated at 37 degrees C and plated onto BHI agar. The CFU were counted and analysed. At all depths and thirds of the root canals and for all techniques used, 5.25% NaOCl was shown to be the most effective irrigant solution tested when dentinal tubules were analysed, followed by 2.5% NaOCl. No differences among concentrations in cleaning the canals were found. Especially at higher concentrations, NaOCl, was able to disinfect the dentinal tubules, independent of the canal preparation technique used.

Biodegradation of orthodontic appliances. Part I. Biodegradation of nickel and chromium in vitro

Enterohemolysin production of Beutin's washed sheep blood agar plate has been used as an epidemiological marker for selective screening of Verocytotoxin-producing/Enterohemorrhagic Escherichia coli (VTEC/EHEC). In this study, we examined about the component of Beutin's washed sheep blood agar for further improvement of hemolysin production media. The following items were studied: the numbers of washings of sheep blood with phosphate buffered saline (PBS) (pH 7.2), concentration of sheep blood, kind and concentration of divalent metal ion (Ca2+, Mg2+) and basal medium. Twenty-seven strains of VT-producing E. coli of 7 different O-serotypes, 74 strains of VT-nonproducing E. coli of 24 different O-serotypes and one strain of O157 coded Escherichia hermanii were used for this basic study. In comparison of washing times of sheep blood with PBS, 5 times washing was better than 3 times, the original. In sheep blood concentration, supplement with 4% sheep blood was best for hemolysis observation. In experiment of addition of 2 divalent metal ions, Ca2+ and Mg2+, supplement with Ca2+ was more suitable than Mg2+ for hemolysis, and the supplement with 10 mM CaCl2, the original, was the best concentration. On the basal medium used in Beutin's sheep washed blood agar, 4 kinds of media were compared. In addition to Soybean-Casein Digest (SCD) agar, the original, Nutrient agar, Heart Infusion (HI) agar and Brain Heart Infusion (BHI) agar were examined, HI agar was the best blood agar base in the four media. As the above experimental results, the composition of the better Beutin's washed sheep blood agar may be summarize as follows: Heart Infusion agar 'Eiken' used for blood agar base, supplemented with 10 mM CaCl2 and 4% defibrinated sheep blood (Japan biosupp. Center) washed five times in phosphate-buffered saline, pH 7.2.

The virulence factors of Vibrio vulnificus are not yet well understood. So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism. The present research was carried out in order to study the presence of some of these enzymes in 133 V. vulnificus strains isolated from 45 seafood samples. The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99.2% for caseinolytic protease, 96.9% for DNase, 65.4% for mucinase and 46.6% for elastase. None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells. In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59.4% of strains showed opaque morphology on BHI agar and 57.9% on nutrient agar, 10.5% presented translucent morphology on both agars and 30.1 and 31.6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively. None of the translucent colonies was virulent to mice. Therefore, opacity was a useful marker for potential virulence. Of 45 food samples contaminated with V. vulnificus, 29 (64.4%) presented strains lethal to adult mice.

Capillary GC with flame ionization detection (FID) was used to determine the cellular fatty acid (CFA) profiles of six species in the new genus Cronobacter (Enterobacter sakazakii). The six different species are C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and C. genomospecies. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane-methyl tert-butyl ether. A data set for 57 strains of Cronobacter species was prepared using fatty acid profiles from two or three replicates prepared on different days. Major fatty acids of the Cronobacter strains evaluated in this study were straight-chain C12:0, C14:0, C16:0, and unsaturated C18:1, omega7c, summed C16:1 omega7c/C16:1 omega6c, and summed C14:0 3-OH/iso-C16:1, and C17:0 omega cyclo 7-8. The CFA profiles for the Cronobacter species are similar, but there are several fatty acids-C12:0, C14:0, C16:0, C18:1 omega7c, and summed C16:1 omega7c/ C16:1 omega6c--that differ significantly among these six species. Analysis of FAMEs from Cronobacter strains grown on BHI agar by a rapid GC-FID method is a sensitive procedure for the identification of these organisms, and this analytical method provides a procedure for the differentiation of strains from closely related Cronobacter species.

Organisms belonging to the Streptococcus milleri group (SMG) are known for their role in pyogenic infections but have recently been implicated as etiological agents of pulmonary exacerbation in adult patients with cystic fibrosis (CF). The prolonged exposure of CF patients to antibiotics prompted us to investigate the susceptibility profiles of 118 SMG isolates from the airways of CF patients to 12 antibiotics compared to 43 SMG isolates from patients with invasive infections. We found that approximately 60% of all isolates failed to grow using the standard medium for disc diffusion, Mueller-Hinton blood agar (MHBA), so we explored the usefulness of brain heart infusion (BHI) agar for susceptibility testing. Zone-of-inhibition comparisons between BHI and MHBA showed strong correlations for six antibiotics, and interpretations were similar for both medium types. For ceftriaxone and cefepime, both groups of isolates were highly susceptible. Tetracycline resistance levels were comparable between the two groups (22% in CF isolates and 17.4% in invasive isolates). However, more than half of the CF isolates were not susceptible to azithromycin, erythromycin, and clindamycin, compared to 11%, 13%, and 6.5% of invasive isolates, respectively. There were 5-fold and 8-fold increased risks of azithromycin and clindamycin resistance, respectively, for the isolates from the airways of CF patients relative to the invasive isolates. Macrolide resistance was strongly linked to chronic azithromycin therapy in CF patients. This study shows that BHI agar is a suitable alternative for antimicrobial susceptibility testing for the SMG and that SMG isolates from the airways of CF patients are more resistant to macrolides and clindamycin than strains isolated from patients with invasive infections.

Listeria monocytogenes is a Gram-positive human pathogen that is responsible for serious infections in immunocompromised individuals and pregnant women. Because of recent epidemics caused by food contaminated with L. monocytogenes, rapid methods for the detection of this pathogen in food are of interest. Capillary gas chromatography with flame ionization detection (GC-FID) was used to determine the cellular fatty acid profiles of six species of Listeria. The six different species are L. monocytogenes, L. ivanovii, L. innocua, L. welshimeri, L. seeligeri, and L. grayi. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane/methyl tert-butyl ether. A preliminary data set for 15 strains of Listeria species was prepared using fatty acid profiles from two or three replicates prepared on different days. Major fatty acids of the Listeria strains evaluated in this study were C15:0 iso, C15:0 ante iso, C16:0 iso, C16:0, C17:0 iso, and C17:0 ante iso. All of the major fatty acids differ significantly among these six species. The two fatty acids C17:0 ante iso and C15:0 ante iso showed the highest percentages, and the ratio of the two clearly showed significant differences between the human pathogen L. monocytogenes and the five nonpathogenic species. Analysis of FAMEs from Listeria strains grown on BHI agar by a GC-FID method is a sensitive procedure for identification of these organisms and differentiation between pathogenic and nonpathogenic species.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectiveMalassezia is the most abundant fungal skin commensal organism, representing 50%–80% of total fungi present on the skin. It has been associated with many skin disorders such as pityriasis versicolor (PV) and seborrheic dermatitis/dandruff (SD/D). The role of Malassezia in disease manifestation is not discerned. It is important to understand its interaction with bacterial flora such as Staphylococcus epidermidis and S. capitis in vitro. We have studied the interaction of Malassezia and Staphylococcus species isolated from skin flora.MethodsMalassezia restricta, M. globosa (n = 5) isolated from patients with SD and M. furfur (n = 5) isolated from PV were sub-cultured on Modified Dixon's agar (MDA). Staphylococcus epidermidis and S. capitis were isolated from patients with SD and sub-cultured on brain heart infusion (BHI) agar. Malassezia species requires media supplemented with lipids (MDA) for its growth. Bacteria and Malassezia were quantified on MDA and BHI agar by Miles and Mishra method to perform interaction between them. For direct interaction, suspensions (100 μl) of M. restricta, M. globose, and M. furfur were prepared in normal saline and added to wells on the plates of lawn cultures containing S. epidermidis and S. capitis (107 CFU/ml). Plates were incubated for 12 h at 35°C and observed for zone of inhibition. To investigate the release of antibacterial compounds into the extracellular environment, M. furfur was inoculated in modified Dixon's broth (MDB) and incubated at 35°C for 5 days. Supernatant was collected at 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h of incubation and evaluated for antibacterial activity by agar-well diffusion assay. Effect of cell-free supernatant of Malassezia on growth of bacteria was also monitored by growth kinetics of S. epidermidis for 24 h in the absence and presence of M. furfur supernatant using Epoch-2 microplate spectrophotometer.ResultsMDA supported the growth of bacteria at different cell densities (107-103 CFU/ml count) and incubation time of S. epidermidis and S. capitis was similar on MDA and BHI. Zone of inhibition (ZOI) was witnessed with M. restricta (20.6 ± 3 mm, 21 ± 3 mm), M. globosa (21 ±1 mm, 22.6 ±2 mm) and M. furfur isolates (16.5 ± 1 mm, 18 ± 2 mm) against S. capitis and S. epidermidis respectively by direct interaction. Inhibition of bacteria by M. furfur was noted from 48-120 h as ZOI (21.7 ±5.1 mm) was observed on bacterial lawn cultured plate. When growth kinetics of S. epidermidis was monitored in presence of M. furfur supernatant, maximum value reached upto 0.26 ± 0.019 only from 0.01 ± 0.001 at OD600 in 9 h including lag phase of 4 h (Fig. 1). However, OD600 value reached upto 0.97 ± 0.005 in 8 h including lag phase of 1.5 h in absence of supernatant. Doubling time calculated from logistic growth equation was 76.6 ± 4.4 and 65.2 ± 2.9 minutes in the presence and absence of supernatant respectively.ConclusionInhibition of bacteria by Malassezia species noted in our study has not been reported earlier. The possible production of antibacterial compounds by Malassezia might be responsible for dysbiosis leading to disease.

The hydrocolloid impression material is one of the most important materials extensively used in several procedures in the dental field. It is mainly applied for diagnostic and planning in the rehabilitation of oral, orthodontic, and maxillofacial prostheses due to its biocompatibility with the oral tissues, low toxicity, ease of use, and relatively low cost. When doing the impression, the material might be contaminated with blood, saliva, and biofilm from within the patient’s mouth. In these procedures, there are high chance that the microorganisms can be transmitted from patients to the casting materials and then to the dentists or to the dental lab technicians. Several types of disinfectants have been introduced for use to disinfect dental impressions. This study aims to investigate the antimicrobial potential of vanillin-incorporated irreversible hydrocolloid impression material on Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, and Candida albicans. The hydrocolloid impression material used in this study is Alginate, Kromopan class A type I, Lascod, Italy. Different concentrations of vanillin (0.1%, 0.5%, and 1% w/w) were added to the impression powder, and the impression samples were made by mixing the alginate powder with water and pouring them on sterile plastic plates. Staphylococcus aureus ATCC 5638, Klebsiella pneumoniae (clinical isolate), Escherichia coli ATCC 11775, and Candida albicans ATCC 10231 were prepared to 105 CFU/ml suspensions in sterile normal saline solution. A total of 100 μL of each microbial suspension was evenly spread onto the surface of the impression and left for 1 min. Then, a 2 x 2 cm2 sterile Whatman filter paper was placed on the impression sample surface to make an imprint and transferred to the Brain Heart Infusion (BHI) agar plate. The number of colonies growing on the BHI agar was counted after incubation at 37°C for 24-48 h. Impression material without vanillin was used as a control. It was found that adding vanillin to the materials could significantly inhibit all tested microorganisms, and the inhibitory efficiency ranged from 12% to 98%. K. pneumoniae showed the most resistance since the inhibitory effect started at 0.5% w/w vanillin and the maximum suppression was 84% at 1% w/w vanillin. On the other hand, S. aureus appeared to be the most sensitive species, as the suppressive response started at 0.1% w/w vanillin and the percentage of inhibition was as high as 98% at 1% w/w vanillin. In conclusion, we combined different concentrations of vanillin (0.1%, 0.5%, and 1% w/w) into the impression material and it showed a significant antimicrobial potential against all tested oral bacteria and yeasts (S. aureus, K. pneumoniae, E. coli, and C. albicans). The suppressive effects were dose-dependent and ranged from 12% to 98%. This did not only disinfect the impression material from the inside but also disinfected the impression from the time it was inserted into the patient’s mouth. Using this hydrocolloid impression material incorporated with vanillin could be beneficial to eliminate cross-infection for dental personnel. Nonetheless, further studies are necessary to investigate some physical properties of this impression material, such as setting time, tensile strength, elastic recovery, and detailed reproduction.

Isolation of Histoplasma farciminosum from five horses, showing typical signs of histoplasmosis farciminosi (epizootic lymphangitis) was successfully attempted. The mycelial form of H. farciminosum was isolated on Sabouraud dextrose agar enriched with 2.5% glycerol, brain heart infusion (BHI) agar enriched with 10% horse blood and PPLO dextrose glycerol agar. The last medium proved to be the most effective, both for primary isolation and subculturing of the fungus. It was found that on primary isolation, the lag phase of the mycelial form of the fungus was relatively long, involving 4-8 weeks at 25 degrees C. Colonies of the mycelial form of H. farciminosum appeared on subculture as a yellowish, light brown to deep brown, convoluted, waxy, cauliflower-like growth tending to form scant aerial growth. Conversion of the mycelial form to the yeast form of H. farciminosum was successful by subculturing either on BHI agar with 5% blood or on Pine's medium and incubating at 35-37 degrees C. Complete conversion to the yeast form was achieved only after 4-5 repeated serial transfers onto fresh media every 8 days. The yeast colonies were flat, raised, slightly or deeply wrinkled, white to light gray to grayish brown, and were pasty in consistency.

Capillary gas chromatography with flame ionization detection (GC‐FID) analysis of chemical components of bacterial cells has provided useful information for rapid detection and identification of bacteria in clinical and diagnostic bacteriology laboratories and currently has increased significance for both food safety and security. GC‐FID was used to determine the cellular fatty acid (CFA) profiles of closely related Yersinia species. For GC‐FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 (C for 24 h were obtained by saponification, methylation and extraction into hexane/methyl tert‐butyl ether. A data set for each Yersinia species was prepared using fatty acid profiles from five replicates prepared on different days. Major fatty acids of the 26 Yersinia strains evaluated in this study were straight‐chain 12:0, 14:0, 15:0, 16:0 and unsaturated 16:1 ω7c/16:1 ω6c, 18:1 ω7c, and 14:0 3OH/16:1 iso, and 17:0 ωcyclo 7‐8. The Yersinia species Y. enterocolitica, Y. pseudotuberculosis, Y. frederiksenii, Y. intermedii, Y. kristensenii, and Y. pestis have unique fatty acid patterns. The CFA profiles for Y. pestis and Y. pseudotuberculosis are similar, but there are several fatty acids, 16:1 ω5c, 16:0, 17:1 ω7c, 17:0 ωcyclo 7‐8, 19:0 and summed 18:2 ω6c, 9c/18:0 ante that differ significantly between these two species. Analysis of FAMEs from Yersinia strains grown on BHI agar by a rapid GC‐FID method can provide a sensitive procedure for the identification of these organisms, and this analytical method provides a procedure applicable to issues of both food safety and security.

Epidemiological relationships were investigated between 40 methicillin-resistant Staphylococcus aureus (MRSA) strains with decreased glycopeptide susceptibility isolated from November 1998 to March 1999 from 39 patients (17 infected and 22 colonized patients) in nine wards of the Broussais Hospital, Paris, France. Reduced glycopeptide susceptibility was readily detected on brain heart infusion (BHI) agar containing 6 microg of teicoplanin per ml and on gradient plates, but not by the standard disk diffusion method. The MICs of vancomycin and teicoplanin, determined on BHI agar, were 4 and 8 to 32 microg/ml, respectively (standard antibiotic dilution), and 4 to 8 and 8 to 32 microg/ml, respectively (E-test). All strains were resistant to macrolides, aminoglycosides, tetracycline, rifampin, sulfonamides, and pefloxacin, showed reduced susceptibility to fusidic acid and fosfomycin, and were susceptible to trimethoprim and chloramphenicol. Pulsed-field gel electrophoresis and lysotyping revealed that a multidrug-resistant MRSA clone with decreased susceptibility to glycopeptides has been discretely endemic since at least 1996 in our institution, where it was responsible for an outbreak in November and December 1998.

Surgical site infections (SSI) are post-surgical incisional infections in superficial or deep tissues, including organs. Due to their importance in veterinary medicine, the role of surgical blades in bacterial dissemination to internal tissues of dogs undergoing surgery was evaluated. A total of 46 dogs presented for orthopedic or soft tissue surgery in different anatomical regions were included in this study. From each animal two swab samples were collected, from the skin post-asepsis and from the scalpel blade after skin incision, for bacterial growth evaluation in Brain Heart Infusion (BHI) agar and detection of methicillin-resistant species. Results showed that 30.4% (14/46) and 28.3% (13/46) of the post-asepsis and blade samples originated positive bacterial cultures in BHI agar, respectively. However, only 10.8% (5/46) of the positive blade samples also corresponded to a positive post-asepsis sample. Nevertheless, all samples were negative for methicillin-resistant bacteria. Although no dog has developed SSI, the present report showed that the scalpel blade may act as a dissemination vehicle of potential bacterial pathogens to superficial or internal tissues of dogs undergoing surgery, potentially leading to SSI development. Therefore, it is recommended to use a single blade for skin incision and a new blade for the remaining surgical approach, reducing the potential of bacteria dissemination into deeper tissues by the first skin incision blade.