Beruflich Dokumente
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Ratio Diversity
Phylotype
The importance of oral flora and its signature population was presented by P.D. Marsh in 2000. He proposed the Ecological or Plaque Hypothesis, which recognized the importance of normal flora vs. pathogenic flora and the selective pressure that could influence the organism relative distribution.
Transmission
Bioburden
Disease Threshold
Health
Health Planktonic/Biofilm
Pathogenic flora
Disease
We hypothesized that the oral flora signature of selected microbes could predict or correlate with oral disease (Liability Index) and within closed families established closeness or relatedness. We utilized both non-culture (BANA, Strep. mutans) and culture techniques, recognizing that viable, but non-cultable is a premise of biofilms. We also constructed an Oral Microbial Signature (OMS) of the three viable, sentinel organisms; 2 prokaryotes and 1 eukaryote: Staph. aureus, Strep. pyogenes (Group A Beta Strep) and Candida albicans (yeast). The use of the microbial signature and integration of Marshs scheme is shown below; with emphasis on screening in the field and culturing in the laboratory by oral anatomic site. Staph. aureus was also selected as a key marker to differentiate MRSA from CAMRSA and antibiotic resistance using an international electronic database for comparison.
A) Screen (Field)
1. Strep. mutans 2. BANA
B) Culture (Lab)
Quantification of 3 sentinel markers:
Throat
The figure above describes how to establish a Familial Liability Index (viable and nonviable methods) and Oral Microbial Index (OMS) (viable methods only).
Objective: With a smear of tongue coatings or subgingival plaque, BANA test strips can detect three species of anaerobic bacteria, often associated with periodontal conditions and oral malodor: Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythus. Methods: Peptidases in certain anaerobic bacteria can hydrolyze the peptide analog Nbenzoyl-D-L-arginine-2 naphthylamide (BANA). One of the hydrolytic products of this reaction is B-naphthylamide, which reacts with a diazo reagent Fast Black K producing a permanent blue color. Only T.denticola, P. gingivalis, and T. forsythus for over 60 plaque species that have been tested, have been found to possess significant amounts of BANA-hydrolyzing enzyme (6). Occasionally some strains of Capnocytophaga and Bacteroides were weakly BANA positive, but this was an inconsistent finding (6). Blood and saliva do not hydrolyze BANA and do not interfere with this test.(2, 5) Background of instrumentation: The tongue is wiped with a cotton swab. For periodontal risk assessment, subgingival plaque is obtained with a curette from the mesio-buccal sites of the four first molars (tooth numbers 3, 14, 19, & 30) and a stimudent woodpick from the tongue dorsum. The samples are placed on the BANA test strip, which is then inserted into a slot on a small toaster-sized incubator. The incubator automatically heats the sample to 55 for 5 minutes. If P. gingivalis, T. forsythus or T. denticola are present, the test strip turns blue. The bluer it turns, the higher the concentration and the greater the number of organisms. A color guide is printed on the container. Result Interpretationss: The BANA Test is scored both in the field and at the WVU lab. The BANA is scored as follows: Positive: A blue color. This indicates that one or more of the three BANA positive species are present at levels >100,000 CFU. Weak-positive: A faint blue color. This indicates that one or more BANApositive species are present at levels >10,000 and <100,000 CFU. Negative: No blue color. This indicates that the BANA positive species are bleow the detection level of 10,000CFU in the plaque sample.
References: 1. Loesche WJ, Syed SA, Schmidt E, Morrison EC. Bacterial profiles of subgingival plaques in periodontitis. J Periodontol 1985;56:447-56. 2. Simonson LG, Robinson PJ, Pranger RJ, Cohen ME, Morton HE. Treponema denticola and Porphyromonas gingivalis as prognostic markers following periodontal treatment. J Periodontol 1992; 63:270-273. 3. Loesche, W.J., Lopatin, D.E., Stoll, J., Van Poperin, N. and Hujoel, P.P. Comparison of various detection methods for periodontopathic bacteria: Can culture be considered as the primary reference standard? J. Clin. Microbiol. 30:418-426. 1992. 4. Loesche, W.J., Bretz W.A., Kerschensteiner D., Stoll J.A., Socransky S.S., Hujoel, P.P., Lopatin D.E. Development of a diagnostic test for anaerobic periodontal infections based on plaque hydrolysis of benzoyl-DL-arginine naphthylamide. J. Clin Microbiol. 28:15511559, 1990. 5. Loesche, W.J., Lopatin, D.E., Giordano, J., Alcoforado, G. and Hujoel, P. Comparison of the benzoyl-DL-arginine-naphthylamide (BANA) test, DNA probes, and immunological reagents for ability to detect anaerobic periodontal infections due to Porphyromonas gingivalis, Treponema denticola, and Bacteroides forsythus. J. Clin. Microbiol. 30:427 433, 1992. 6. Socransky SS, Haffajee AD, Cuglini MA et al. Microbial complexes in subgingival plaque. J Dent Res 1997; 76(special issue): 302. 7. Loesche WJ, Giordano JR: Treatment Paradigms in Periodontal Disease. Compendium for Dental Education, 1997;18(3):221-232. 8. Loesche WJ, Kazor CE, Taylor GW. The optimization of the BANA test as a screening instrument for gingivitis among subjects seeking dental treatment. J Clin Perio 1997;24:718-726.
0-1 2 0 1 2 3 3
< 100,000 MS/ml saliva 100,000 1,000,000 MS/ml saliva > 1,000,000 MS/ml saliva
References:
1. Fields, Helen. Beyond drillings and fillings. U.S. News & World Report 135 (2003): 48. 20 Sept. 2004. 2. Gold, Olga G., et al. A selective medium for Streptococcus mutans. Archives of Oral Biology 18 (1973): 1357-1364. 3. Mahon, Connie R. and George Manuselis. Textbook of Diagnostic Microbiology. Philadelphia: W.B. Saunders Company, 2000. 4. Mass, Diana and Galen Robert. The Predictive Value Theory Redefines Quality Assurance. Am J Med Tech 47 (1981): 965-970. 5. Orion Diagnostica. Dentocult SM Strip Mutans. Package Insert. 6. T. Oho, et al. Simple and rapid detection of Streptococcus mutans and Streptococcus sobrinus in human saliva by polymerase chain reaction. Oral Microbiology and Immunology 15 (2000): 258-262. 7. Rosenwald, Michael. A Buggy Cavity Fix. Popular Science 262 (2004): 39. 20 Sept. 2004. 8. Rupf S, et al. Comparison of different techniques of quantitative PCR for determination of Streptococcus mutans counts in saliva samples. Oral Microbiology and Immunology 18 (2003): 50-53.
III. Cultable A. THROAT CULTURES: Staph. aureus, Beta Strep and Candida albicans.
Objective: The intent of collecting throat culture data from the COHRA population is to establish either a similarity or dissimilarity in the microbial signature of three organisms within a distinct population; in this case within A) family groups or B) geographic location. Methods: Throat cultures are obtained by wiping the back of the throat and the tonsils with a sterile swab. The swab is then removed gently without touching the teeth, gums, or tongue. It is then placed in a sterile tube containing a holding media and sent to the laboratory for organism A) detection and B) quantification. Background of instrumentation: Once cultures are received at the lab, the sterile cotton swab is rubbed across of a blood agar plate. Using a sterile loop, the plate is streaked into 3 additional quadrants. A Taxo A disc (for Beta Strep screening) is placed on the agar on top of the initial inoculation of the culture and the plate is allowed to incubate at 37 for 48 hours. After 48 hours the plate is removed from the incubator and checked for growth of organisms on the blood agar plate. Although there are numerous bacteria that can grow on the agar, we are only interested in 3 different organisms: Group A Beta Strep, C. albicans and Staph. aureus (MRSA or CA-MRSA). Results: Using the following secondary testing methods below, count the number of quadrants (14) the bacteria grew on. Group A Beta Strep. is a Gram Positive cocci, that demonstrates beta hemolysis on a 5% sheep blood agar plate. o Group A Streptococcus is sensitive to small amounts of Bacitracin while beta Strep of other serological groups are resistant. Group A Streptococcus is differentiated from other groups of beta-hemolytic Strep by the formation of a zone of inhibition around a disc impregnated with 0.04 units of Bacitracin. Candida albicans (Yeast) o Potential yeast colonies should be observed with potassium hydroxide (KOH) under a microscope for the presence of budding or pseudo-hyphae forms. Staph aureus is a Gram Positive cocci, that is coagulase positive o Potential Staph aureus colonies should be tested for coagulase. o Place 1-2 isolated colonies into 1 mL of coagulase plasma and incubate in a 37 degree incubator for 1 hour. After an hour, check to see if the plasma is coagulated; the indication of a positive test.
Optional Methicillin (Oxicillin) Resistant Staph. aureus (MRSA) is a very significant pathogen is hospitalized patients. Until recently, it was assumed that prior hospitalization was a risk factor for colonization of MRSA. Now it can, be acquired from the community. Simultaneously, CA-MRSA (with a unique cassette of virulence factors is a growing concern.
References: 1. "Throat Culture." In Illustrated Guide to Diagnostic Tests, ed. J. A. Lewis. Springhouse, PA: Springhouse Corp. 1994. 2. Perkins, A. "An Approach to Diagnosing the Acute Sore Throat." American Family Physician 55 (Jan. 1997): 131-137.
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