COHRA MICROBIOLOGY PROCEDURE MANUAL: INSTRUMENTATION DESCRIPTIONS: BANA, DENTOCULT, & THROAT SWABS

I. MICROBIOLOGY OVERVIEW: INTRODUCTION TO FAMILIAL LIABILITY INDEX

There are four reservoirs of normal microbial flora as shown below; these reservoirs are interactive and act as a continuum for re-colonization, one site to another. Interestingly, the oral cavity (upper GI) has the greatest diversity (700 phylotypes), and hence the microbial population with the highest potential for survival. Until recently, it was the least understood/defined reservoir with potential contribution to the total health or disease of the patient. Now, its link to systemic disease is growing logarithmically via evidencebased research and observations.

4 NF Reservoirs of Bacteria/Yeast An Interactive Continuum

Human Source Bioburden
CFUs/ml Anaerobic: Aerobic

Ratio Diversity
Phylotype

GIT (Lower GI) Urogenital Oral (Upper GI) Skin

1011 108 106 106

1000:1 100:1 10:1 1:1

200 200 700 50

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.

Ecologic (Plaque) Hypothesis

Transmission

Major ecological pressure

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.

Evaluation of Oral Microbial Status

Oral Ecosystem
Stable Normal Flora Bioburden Biodiversity Anatomic Location Tongue Plaque Saliva

A) Screen (Field)
1. Strep. mutans 2. BANA

B) Culture (Lab)
Quantification of 3 sentinel markers:

1. S. aureus 2. Yeast (Candida) 3. S. pyogenes

Throat

Disease = Microbial Imbalance

The figure above describes how to establish a Familial Liability Index (viable and nonviable methods) and Oral Microbial Index (OMS) (viable methods only).

PRELIMINARY STUDY: EVIDENCE-BASED

To test our hypothesis, a feasibility study with mother-child pairs was undertaken in 2002. Objective: To develop a familial Liability Index for oral microbial status that reflects an imbalance of oral domains based on the presence of risk indicators in saliva, interproximal plaque, tongue and throat. 36 mother child-pairs from Webster and Nicholas counties, West Virginia, participated in this study. Methods: Samples were assayed using four tests: 1) the mutans streptococci (MS) test on salvia samples, 2) the BANA Test (BT) on interproximal plaque samples, 3) the BT on tongue scraping samples and 4) the throat cultures to isolate any of the three sentienal organisms (S. aureus, S. pyogenes and yeast) on throat swab samples. The corresponding thresholds for a (+) risk indicator were, 1) 105 CFUs of MS salivary levels for the MS Test, 2) >105 CFUs/mg of plaque for the BT test on plaque samples, 3) >104 and < 105 CFUs for a weak (+) BT on tongue scrapings and 4) >104 CFUs/swab for any of the sentinel markers for the throat cultures . Relative risk estimates were employed to analyze the data. Results: The mean age of mothers was 42.1 years and 14.7 years for children. 91 % of mothers and children had at least one (+) risk indicator. Overall, 76% of mother childpairs had at least one (+) concordant oral microbial risk indicator. Accordingly, the relative risk (RR) of children having concordant results with their mothers was increased 2.9 (BT-plaque), 1.25 (BT-tongue), 0.7 (sentinel organisms) and 1.75 (MS) times. Conclusions: The cumulative RR for having at least one (+) concordant indicator was 6.7. Mother-child pairs shared similarities of oral microbial risk indicators that allow for the development of a Liability Index. References 1. Corbet E, Zee-K, Lo E. Periodontal diseases in Asia and Oceania. Periodontology 2002: 29: 122-152. 2. Petersen P, et al. The global burden of oral diseases and risks to oral health. Bulletin of the World Health Organization 2005: 83: 661-669. 3. United States General Accounting Office. Oral Health: Dental Disease Is a Chronic Problem Among Low-Income Populations. 2000: 1-44. GAO/HEHS-00-72. 4. United States General Accounting Office. Oral Health: Factors Contributing to Low Use of Dental Services by Low-Income Populations. 2000: 1-44. GAO/HEHS-00149. 5. Corby P, et al. Microbial risk indicators of early childhood caries. J Clin Micro 2005: 5753-5759. 6. Aas, J. Defining the normal bacterial flora of the oral cavity. J Clin Micro 2005: 5721-5732. 7. Thomas, J. and Nakaishi, L. Managing the Complexity of a Dynamic Biofilm (Article in Importance of Antiseptic Mouthrinses in the Daily Oral Care Regimen Supplement). JADA . November 2006.

II. Viable, Non-Cultable

A.

BANAs (3-Perio Pathogens)

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.

B. Dentoicult SM (mutans Strepococci-Caries)

Objective: Today, Mutans streptococci are considered the primary bacterial cause of caries. Their presence or, even more so, their absence, is a strong indicator of high or low susceptibility (respectively) to caries. Methods: Dentocult SM Strips, mutans streptococci are selectively enriched on two kinds of plastic strips that have been treated to simulate the tooth surface. Dentocult SM Screening Strips are useful in identifying patients with mutans streptococci. The test kit's Site Strips provide more detailed information on the caries activity in progress in the patient's mouth or on a particular tooth surface. This knowledge helps the dentist design treatment strategies and individual preventive care for patients of all ages and in all situations. Background of instrumentation: Sample collection from saliva and plaque using Dentocult stripsScreening Strips and/or Sample application using Site Strips Incubation (48 to 96 hours) in selective nutrient broth provided. Interpretation using card provided with test kit. Result Interpretations: The Dentocult Test strips are scored both in the field and at the WVU lab as follows:
Test Strip Results: Result: Number of Mutans Streptococcus for each Test Strip

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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