Microbiological tests of periodontal significance

3.  Introduction
 Collection and transport of specimen
 Limitations of conventional periodontal diagnosis
 Microbiological analysis
 Bacterial culturing
 Microscopic methods
 Immunodiagnostic methods
 Enzymatic methods
 Molecular biology techniques
 Conclusion
 References

4.  Diagnostic microbiology- involves the study of specimens
taken from patients suspected of having infection
Steps of a diagnostic cycle:
1) clinical request and provision of clinical information
2) collection and transport of specimen
3) laboratory analysis
4) Interpretation of the microbiology report and use of the
information

6. Request form:
 Clinical condition of the patient
 Age
 Date of onset of illness
 Recent/current antibiotic therapy
 Antibiotic allergies
 History of previous specimen

7.  Specimens should be collected before antibiotic therapy as
it will alter the quality and quantity of the organisms
 Exceptions-patients who are seriously ill, immunologically
compromised or not responding to a specific antibiotics
 Specimens should be collected following appropriate
protocol
 Specimens should be as fresh as possible- anaerobes,viruses
die..staphylococci, coliforms may multiply…at room
temperature
 Transport specimens in an appropriate medium and as fast
as possible
 Medium should be compatible with the organisms
 Transport specimen in safe, robust containers to avoid
contamination

8.  For assessing organisms in periodontal infections, an
appropriate subgingival plaque sample has to be obtained
 Obtaining it from appropriate sites and in adequate quantity
 Mombelli et al- four individual subgingival specimens from
each quadrant
 Sample collected and transported in an anaerobic
environment with minimal transport time
 Curettes vs paper points

9.  Conventional diagnosis of gingivitis depends on clinical
signs and symptoms
 Conventional diagnosis of periodontitis depends on clinical
attachment loss and radiographic bone loss
 Tells us about the extent and severity of disease
 Does not give information about the cause of the disease,
on the patients susceptibility to disease, whether the disease
is progressing, whether it is in remission, or whether the
response to therapy will be positive or negative.
 Disease process is mulitfactorial in origin in which
periodontal pathogens, host response , and genetic,
systemic, and behavioural risk factors interplay to develop
the disease process

10.  Many publications have reported that certain micro-
organisms from the subgingival microbiota , particularly
gram negative anaerobes, are the major etiologic factors of
chronic and aggressive periodontitis
 More than 300 species isolated from different individuals
 40 species from a single site

11.  According to criteria described by Socransky-
1) Strong evidence for Aa, Pg, Tf
2) Moderate evidence for campylobacter rectus,
eubacterium nodatum, fusobacterium nucleatum,
peptostreptococcus micros, prevotella intermedia,
prevotella nigrescens, streptococcus intermedius, and
spirochetes such as treponema denticola

12. Microbiological tests are useful..
1) To identify putative pathogens and supporting the
diagnosis of various forms of periodontal disease
2) To serve as indicators of disease initiation and
progression and healing
3) To determine which periodontal sites are at higher risk
for active destruction
4) To moniter periodontal therapy
5) To aid in treatment planning of patients with aggressive
or non responding periodontitis by helping the doctor in
selection of adjunctive antimicrobial therapy

13.  Microbiologic testing for the presence or absence of Pg and Aa
to distinguish subjects with aggressive periodontitis from
those with chronic periodontitis has its limitations-according
to systematic review by Mombelli et al.

14. Bacterial culturing
 Still considered gold standard when determining the
performance of new microbial diagnostic methods
 Originally used by Louis Pasteur. He used urine and
meat broth. First solid medium...cooked cut potato used
by Robert Koch
 Koch used gelatin to solidify liquid media but not
successful as gelatin liquified at 24˚c and also by many
proteolytic bacteria
 Frau Hesse suggested use of agar to solidify liquid media

15. Advantage of this method
1) Clinician can obtain relative and absolute counts of the
cultured species
2) Only in vitro method able to assess for antibiotic
susceptibility of the microbes

16. Disadvantage
1) Can only grow live bacteria. Thus strict sampling and
transport conditions are essential
2) Some putative organisms such as treponemas species
and Tf are fastidious and difficult to culture
3) Low sensitivity. Detection limits average 103 to 104
bacteria. Thus low number of specific pathogens go
undetected.
4) Requires sophisticated equipments and experienced
personnel
5) Time consuming and expensive

17.  Solid or liquid media used for bacterial or fungal growth
 Cultured cells derived from animals and humans used
for viral growth
Solid
Semi-
solid
Liquid

18.  Blood agar most widely used bacterial culture medium
 Solid media..discrete visible growth…liquid
media..diffuse growth
 Bacterial colonies

19.  Solid media..
1) Distinct colonies
2) Pigmentation or hemolysis may be present.
Identification made easy
3) Quantification of the organisms as colony forming
units (CFU)
 Agar now universally used for preparing solid media

20.  Agar-agar
 Obtained from sea weeds
 Chief constituent a long chain polysaccharide
 Also contains inorganic salts and small quantities of a
protein like substance
 No nutritive value , not affected by growth of bacteria
 Hydrolysed at high temperatures at high acid or alkaline
ph
 Melts at 98˚c and sets at 42˚c
 2% agar employed for solid media
 Manufactured in long shreds or powder form

21.  Advantages..
1) For obtaining bacterial growth from blood or water when
large volumes have to be tested
2) For preparing bulk cultures for preparation of antigens or
vaccines.
3) To test biochemical activities of bacteria for identification
purposes
4) To promote growth of small number of bacteria in
specimens contaminated with antibiotics. fluid medium
dilutes antibiotics

22.  Disadvantage of liquid media
1) Difficult to isolate different types of bacteria from
mixed populations
2) Bacteria growing in liquid media may not exhibit
characteristics for their identification

23.  Primary antibiotic tests /secondary antibiotic tests
 Simple media- eg nutrient broth
 Complex media
 Synthetic or defined media -simple peptone water
medium,1%peptone with 0.5%NaCl
 Enriched media -blood agar, chocolate agar, egg media

25.  Enrichment media- liquid in nature. Eg tetrathionate
broth.
 Selective media -solid media. Similar to enrichment
media but solid in nature. Eg desoxycholate citrate
medium
 Indicator media- sulphite in Wilson and Blair
medium, potassium tellurite in McLeods medium
 Differential media -eg MacConkeys medium.
Lactose fermenters as pink colonies and non lactose
fermenters as colourless colonies

26.  Sugar media -Any medium with fermentable substance
 Transport media- Stuarts medium for gonococci
Thyoglycolic acid as a reducing agent.
 Viral transport medium -solutions containing proteins
and balanced salts which stabilizes the virus during
transport and antimicrobials to kill bacterias
 Anaerobic media- Robertsons cooked meat medium

28.  Introduction
 Collection and transport of specimen
 Limitations of conventional periodontal diagnosis
 Microbiological analysis
 Bacterial culturing
 Microscopic methods
 Immunodiagnostic methods
 Enzymatic methods
 Molecular biology techniques
 Conclusion
 References

30. Bright field or standard microscopy
 Routinely used
 Stained smears examined with oil emersion objective(x100)
using the x10 eye piece obtaining a magnification of x1000
 Wet films are examined with a dry objective(x40)…used to
demonstrate motility of bacteria

32. Dark ground microscopy
 Specimen is illuminated obliquely by a special
condenser so that light rays do not enter objective
directly
 Instead the organisms appear bright, as the light rays hit
them, against the dark background
 Helpful in studying motile organisms
 But most putative periodontal pathogens are non
motile(Aa, Pg, Tf)

34. Phase contrast microscopy
 Rarely employed in diagnostic microbiology
 May be used to define the detailed structure of unstained
microbes

35. Fluorescence microscopy
 This method employs the principle of emission of a
different wavelength of light when light of one
wavelength strikes a fluorescent object
 UV light normally used
 Bacteria and cells stained with fluorescent dyes such as
auramine.
 Used widely in immunology – antibodies tagged with
fluorescent dyes to detect bacterial antigens

38. Electron microscopy
 Light waves replaced by a beam of electrons which
allows resolution of extremely small organisms such as
viruses
 Can be used in diagnostic virology
 Direct examination of specimens can be done but
approximately one million virus particles are needed for
such visualization.( eg rotavirus, hepatitis A virus)

40. Immunoelectron microscopy
 Immunoelectron microscopy - Clumps of viral particles
can be obtained by reacting the sample with antiviral
antibody .Useful for some viruses such as hepatitis A
virus
 Immunoferritin test -Ferritin can be conjugated with
antibody,and such a labelled antibody reacting with an
antigen can be visualised under electron microscope
 Immunoenzyme test -Enzymes such peroxidase, glucose
oxidase, phosphatases and tyrosinase can be conjugated
with antibodies and such a labelled antibody reacting
with an antigen can be seen under electron microscope

41. Gram stain technique
 Dry film heat fixed by gently passing through a flame
 Then flood with crystal violet for 15 seconds and wash
excess
 Flood with Lugol’s iodine for 30 seconds to fix stain and
wash excess
 Decolorize with acetone or alcohol for about 5 sec
 Wash with water

42.  Counterstain with dilute carbolfuchsin for 30 seconds
 Wash with water and blot dry
 Gram positive bacteria- They retain the violet stain by
resisting decolourization and are stained blue
 Gram negative bacteria- They loose violet stain during
decolourization and are therefore counterstained with
pink

44. Ziehl –Neelsen technique
 Organisms are exposed to hot concentrated carbolfuschin for
5 min, decolourized with acid and finally counterstained with
methylene blue or malachite green
 Bacilli will stain red against a blue background
 Useful for tubercle bacilli, which are difficult to stain with
gram stain due to thick cell wall

46.  An antigen has been defined as any substance which, when
introduced parenterally into the body, stimulates the
production of an antibody with which it reacts specifically and
in an observable manner
 Immunoglobulins- Proteins of animal origin endowed with
known antibody activity and for certain other proteins related
to them by chemical structure.The definition includes besides
antibody globulins, the abnormal proteins found in myeloma,
macroglobulinemia, cryoglobulinemia and naturally occuring
subunits of immunoglobulins

47.  Synthesised in plasma cells and lymphocytes
 All antibodies are immunoglobulins but all
immunoglobulins may not be antibodies
 IgA, IgG, IgM, IgD, IgE
 Advantage-stringent sampling and transport
methodology not required
 Higher specificity and sensitivity than bacterial culturing

48.  Disadvantage-Limited to the number of antibodies tested
 Antibiotic sensitivity cannot be studied
 Lack of validity of well controlled clinical studies
 Use of monoclonal antibodies for high specificity

51. Immunofluorescence
 Fluorescence is the property of absorbing light rays of one
particular wavelength and emitting rays with a different
wavelength
 Fluorescent dyes show up brightly under ultraviolet light
as they convert ultraviolet into visible light
 Coons and his colleagues (1942)showed that fluorescent
dyes can be conjugated to antibodies and such labelled
antibodies can be used to locate or identify antigens in
tissues

52. Direct immunofluorescence test
 Can be used for identification of bacteria, viruses or
other antigens
 Antibodies specific for the particular antigen are labelled
with a fluorescent dye
 Eg- Used for diagnosing rabies by detection of rabies
virus antigen in brain smears
 Pathogens can be identified and quantified by this
method by visualizing under a microscope
 Disadvantage- Separate fluorescent conjugates have to
be prepared against each antigen to be tested
 This disadvantage is overcome by the indirect
immunofluorescence test

53. Indirect immunofluorescence test
 In this method a secondary fluorescein-conjugated
antibody is used that reacts with the primary antigen-
antibody complex
 This method also useful for identification and
quantification of the pathogen

56.  Eg- Fluorescent treponemal antibody test for the diagnosis of
syphilis
 A drop of test serum is placed on a smear of T pallidum on a
slide
 After incubation slide washed to remove all free serum
leaving behind only the antibody globulin, if present , coated
on the surface of the treponemas
 Smear then treated with fluorescent labelled anitserum to
human gammaglobulin
 The fluorescent conjugate reacts with with antibody globulin
bound to treponemas
 Treponemas seen as bright objects against dark background
under ultraviolet illumination

57.  VDRL-Non treponemal test in which cardiolipin,
lecithin and cholesterol mixture is used as antigen
 Clumping of cardiolipin occurs in presence of antibodies
to treponema pallidum
 Not a confirmatory test
 Both tests used as T pallidum does not grow in
laboratory media

58.  A single antihuman globulin fluorescent conjugate can be
employed for detecting human antibody to any antigen
 Fluorescent dyes may also be conjugated with complement
 Complements can be employed for the detection of antigen
or antibody
 Antigens also take up fluorescent labelling but not as well as
antibodies do
 for detection of antibodies by immunofluorescence the
“sandwich” technique can be used
 Commenly used fluorescent dyes- fluorescein isothiocynate
and lissamine rhodamine exhibiting blue-green and orange-
red fluorescence respectively

59.  Immunofluorescent assay has been mainly used to detect
Aa and Pg
 IFA comparable to bacterial cultures in its ability to
identify periodontal pathogens in subgingival dental
plaque samples- Zambon et al
 IFA microscopy does not require viable bacterial cells
 Sensitivity of these assays range from 82% to 100% for
detection of Aa and from 91% to 100% for detection of
Pg
 Specificity values are 88% to 92% and 87% to 89%
respectively

60. Flow cytometry /cytofluorography
 Used for rapid identification of oral bacteria
 Involves labelling of bacterial cells from patient plaque
samples with both species specific antibodies and a
second fluorescein conjugated antibody
 The suspension is then introduced into the flow
cytometer, which separates the bacterial cells into an
almost single cell suspension by means of a laminar
flow through a narrow tube
 Highly sophisticated instrument
 Very costly

62. Radioimmunoassay
 Radio isotopes used as labels
 Binder-ligand assay
 Substance whose concentration is to be determined is
termed as analyte or ligand
 The binding protein which binds to the ligand is called
the binder
 First described by Berson and Yallow in 1959 for which
they were awarded Nobel Prize in 1977

63.  Permits the measurement of analytes upto
picogram
 Can be used for quantitation of hormones, drugs,
tumour markers, viral antigens and IgE
 It is a competitive binding assay in which fixed
amounts of antibody and radiolabelled antigen
react in the presence of unlabelled antigen
 The concentration of the test antigen can be
calculated from the ratio of bound and total
antigen labels, using a standard dose response
curve

64. Enzyme immunoassay
 Enzymes used as labels
 First introduced in 1966
 Very versatile, sensitive, simple and economic procedure
 Absence of radiation hazard
 Newer automatic machines have been introduced
 Test kits available
 Two basic types 1)homogeneous 2)heterogeneous

65. Homogeneous EIA
 No need to separate the bound and free fractions
 The test can be completed in one step with all the
reagents added simultaneously
 Used only for haptens such as drugs
 Eg-Enzyme multiplied immunoassay technique (EMIT)
to detect drug molecules like opiates, cocaine,
barbiturates, amphetamine in serum

66. Heterogeneous EIA
 Requires separation of free and bound fractions either by
centrifugation or by absorption on solid surfaces and
washing
 Multistep procedure
 Reagents added sequentially
 Eg- enzyme linked immunosorbent assay (ELISA)

67. ELISA
 Involves use of an immunosorbent
 An absorbing material specific to the antigen or antibody
 Hence the name enzyme linked immunosorbent assay
 Eg-1) Particulate-cellulose, agarose
2) Solid phase- polystyrene, polyvinyl, polycarbonate
tubes or microwells
3) Membranes or discs -polyacrylamide, paper, plastic
 Done using 96-well microtitre plates suitable for automation

71.  Always use positive and negative controls
 Examples described above are of simple non competitive
sandwich ELISA
 Competitive ELISA - The test can be made more specific by
making serum antibody and enzyme labelled antibody
compete for the binding sites on the antigen
 Immunoglobulin specific elisa -eg IgM specific ELISA
 The intensity of colour produced depends on the
concentration of the antigen or antibody being detected and
is usually read photometrically for optimal quantification

72.  Simple modification of ELISA
 Each specimen tested in a separate disposable casette
 Test is rapid taking only 10 minutes compared to 2-4 hrs
taken by microplate ELISA
 No need for microplate washers or readers
 Result read visually

73.  Inbuilt positive and negative controlls
 Used for detection of HIV type 1 and 2 antibodies
 Also known as membrane immunoassay
 Useful for chairside clinical diagnosis
 Eg- Evalusite. Designed to detect Aa and Pg with105 and
106 detection limit respectively

74. Method
 Specific type 1 and 2 antigens are immobilised at separate
fixed sites on nitrocellulose membrane in cassette
 Test serum added on membrane and allowed to filter into
absorbent material placed below it in the cassette base
 Antibody if present will bind to the appropriate antigen
 After washing, enzyme labelled antihuman
immunoglobulin antibody is added
 After further washing, a substrate yielding a coloured
product is added
 positive result indicated by a coloured spot

75. Immunoelectroblot techniques(western blot test)
 They combine the sensitivity of enzyme immunoassay, with
much greater specificity
 Used for identification of proteins
Three steps
 1) Separation of ligand-antigen component by
polyacrylamide gel electrophoresis
 2)Blotting of electrophorised ligand fraction on nitrocellulose
membrane strips
 3)Identification by radio labelled or enzyme labelled
antibodies as probes

76.  Confirmatory test for the diagnosis of HIV antibodies in
sera
 A similar technique for analysis of DNA is known as
southern blot technique and for analysis of RNA is known
as northern blot technique

77. Agglutination
Slide agglutination
Latex agglutination
 Latex beads coated with species-specific antibodies
 Agglutination when contact with antigen in 2-5 minutes

79.  Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg),
Treponema denticola (Td), and Capnocytophagia species
share a common enzymatic profile
 Trypsinlike enzyme
 The activity of this enzyme can be measured wiyh the
hydrolysis of the colourless substrate N-benzoyl-d L-
arginine-2-naphthylamide(BANA)
 Release of chromophore ß-naphthylamide on its
hydrolysis, which turns orange red when a drop of fast
garnet is added to the solution

80.  Perioscan
 Loesche proposed the use of this BANA reaction in
subgingival plaque samples to detect any of the above
mentioned periodontal pathogens and thus serve as a
marker of disease activity
 Loesche showed that shallow pockets exhibited only
10% positive BANA reactions, whereas deep pockets
(7mm) exhibited 80 % to 90% positive BANA reactions
 Beck used the BANA test as a risk indicator for
periodontal attachment loss
 Positive results indicate that one or more of Tf, Pg, Td
bacteria are present at the sampled sites

81. Perioscan

82.  Potential difficulties- May be positive at clinically
healthy sites
 Not proven whether this test can detect sites undergoing
periodontal destruction
 It only detects a very limited number of periodontal
pathogens and hence its negative result does not rule out
the presence of other important periodontal pathogens

83.  The principles of molecular biology techniques reside in
the analysis of DNA, RNA and the structure or function of
protein
 Genetic material of a bacterium is composed of a
chromosomal DNA and transferring RNA (tRNA),
ribosomal RNA (rRNA), and messanger RNA (mRNA)
 Chromosomal DNA is dispersed in the bacterial cell
without a membrane envelope

86.  Diagnostic assays employing molecular biology
techniques require specific DNA fragments that recognize
complementary-specific bacterial DNA sequences from
target microorganisms
 Thus such techniques require the ability to extract
bacterial DNA from the plaque samples and amplify the
specific DNA sequence of the target periodontal
pathogens

87. Nucleic acid probes
 Probe -A known nucleic acid molecule (DNA or RNA) from
a specific microorganism artifically synthesized and labeled
for its detection when placed with a plaque sample
 These probes use segments of a single stranded nucleic acid,
labelled with an enzyme or radioisotope that is able to
“hybridize” to the complementary nucleic acid sequence and
thus detect the presence of target microorganisms
 Hybridization - Refers to the pairing of complementary DNA
strands to produce a double-stranded nucleic acid. The
nucleotide base pair relationship is so specific that the strands
cannot anneal unless the respective nucleotide strand
sequences are complementary

88. Nucleic acid probe

89.  All hybridization methods use radiolabeled or
fluorescence labeled DNA probes that bind to the target
DNA of interest, thus allowing visualization
 DNA probes may target whole genomic DNA or
individual genes
 Whole genomic DNA is more likely to cross react with
non target microorganisms because of the presence of
homologous sequences between different bacterial
species
 Currently most of the probes used are oligonucleotides,
ranging from 20 to 30 nucleotides

92.  Whole genomic probes for detection of Aa, Pg, Td have
been developed and tested- eg DMDx, Omnigene
 Sensitivity of 96% and specificity of 86% for Aa
and sensitivity of 60% and specificity of 82% for Pg in
pure laboratory isolates when compared to culture
according to Van Steenberghe et al
 Sensitivity and specificity reduced significantly in
clinical specimen, suggesting cross reactivity with
unknown bacteria in subgingival plaque samples
 Oligonucleotide probes complementary to variable
regions of the 16S rRNA bacterial gene

93.  16S rRNA genes contains both- 1)Regions shared by
different bacteria and 2) Short stretches of variable
regions shared only by specific organisms of the same
species or genus
 When compared to cultures in clinical samples,
effectiveness of 100% in detecting Aa and Pi and 91% in
detecting Pg with oligonucleotide probes-Savitt et al
 In detecting pathogens in samples from periodontitis
patients : IFA>DNA probes>cultures

94.  Developed by Socransky et al for the detection and
estimation of levels of 40 bacterial species often found in
the oral cavity
 Whole genomic, digoxigenin-labeled DNA probes used
 Facilitates rapid processing of large number of plaque
samples with multiple hybridization for upto 40 oral
species in a single test
 DNA probes used in this technology are adjusted to permit
detection of 104 cells of each species

97.  Sophisticated laboratory equipment
 Expertise
 Thus assay not generalized for diagnostic purposes
 Applicable for epidemiologic research and ecologic studies
as it does not require viable bacteria and allows the
assessment of large number of plaque samples and multiple
species
 This method resulted in higher prevalence figures for half the
species tested (Pg, Pi, Tf and Fn) and statistically significant
higher bacterial counts for the majority of species when
compared to cultures for identification of subgingival
bacteria-Papapanou et al

98.  Developed in 1985
 Has emerged as the most powerful tool for the amplification of
genes and their RNA transcripts
 Used almost universally to study DNA and RNA obtained
from a variety of tissue sourses
 PCR allows large quantities of DNA to be obtained in a
simplified and automated manner

99. Steps :
 Isolation of DNA from a fresh tissue specimen
 Heating of the complementary double strands to split
DNA into single stranded forms which are intended to
act as template dictating the nucleotide sequence in vitro
 Amplification using a DNA polymerase that requires a
primer

100.  A primer is a known short oligonucleotide sequence
corresponding to the border of the region that is
amplified
 For obtaining amplified segments of constant length
and in large quantities, a second primer, complementary
to the opposed chain, must be used to bind the template
and flank the region of interest
 This amplification can be performed several times
known as cycles

101.  In each cycle, complementary chain denaturation,
primer hybridization, and primer extension by means of
the polymerase takes place
 With each cycle there is an exponential increase in the
quantity of DNA
 Temperature is critical throughout the cycle to control
the double chain denaturation and the stability of the
hybridization between the model fragment and the
primer
 In 1988, thermostable DNA polymerase isolated from
organism Thermus aquaticus known as Taq-polymerase
was developed

103.  This Taq-polymerase has allowed automatization of the
reaction using specific appliances called thermocyclers
 This sequenced DNA is then detected and visualized
through electrophoresis in agarose gel and ethidium
bromure, obtaining a qualitative signal
 Ashimoto et al developed a 16S rRNA based PCR
detection method to determine the prevalence of Aa, Tf,
Pg, Pi, Td.
 Compared to culture where detection limit is 104-105
cells, PCR can detect cells as low as 25-100

104.  Microbial tests using standard PCR are extremely
sensitive and specific
 But they provide only qualitative information
 Therefore their use for diagnostic and prognostic
purposes in clinical use is limited

105. Quantitative PCR methods
 1) Endpoint PCR
 2) Real time PCR

106.  This method demonstrated high degree of sensitivity,
specificity, and reproducable quantification
 Requires expensive laboratory equipment
 Thus limited use in routine diagnostic clinical
microbiology

107.  Textbook of Microbiology,5th edition-R.Ananthanarayan
 Essential Microbiology for Dentistry- L.P.Samaranayake
 Short Textbook of Medical Microbiology- Satish Gupte
 Carranza’s Clinical Periodontology,10th edition
 Molecular Microbial Diagnosis-Bruce Paster, Floyd Dewhirst
periodontology 2000,vol 51,2009.38-41
 Microbiological diagnostic testing in the treatment of periodontal
disease-Peter Loomer periodontology 2000,vol 34,2004,49-53
 Microbial testing in periodontics:value, limitations and future
directions-Luciana Shaddox, Clay Walker periodontology 2000,vol
50, 2009, 25-38
 Contemporary Oral Microbiology and Immunology- Slots,
Taubman.