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Microbe WarsWho Is Winning ?

Paul D. EleazerUniversity of Alabama at Birmingham

We just found Archea

Microbe 2011;6(1):26-9

More stable cell membranes = live in more harsh placesDridi et al. Anaerobe 2011

Just one family of Archaea in our oceans is so numerous that a chain of them would surround the Milky Way 5x Microbe 12-2011

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The Second Golden Age of Micro

• Previously undiscovered organisms• Newly discovered microbial causes of

familiar diseases• Microbial diversity is increasing

• The Tooth is not a stone– Many microbe-sized paths and caves

1. Know your Enemy2. Our Weapons3. Dental Unit Water

Outline

Life Forms That Dental Problems

•Bacteria•Funguses•Spirochetes•Viruses•Prions

Now we are learning about long-term diseases

Old: 350 oral species, now 700-900

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Bacteria are everywhere• 2.8 Kilometers below Antarctic ice, with

reproduction once every 300 years• 1 mile below Seattle living only on rocks and

water• Salt mines• At deep sea vents –

– very high temperature and pressure• (In autoclaves)

• Bacteria are Very Promiscuous w/ their DNA

10X more bacterial cells than cells in your bodyEnough viruses for 100 per bacterium

Bacterial Advantages

• They eat everything• They live everywhere• Their genome > 3.3 million genes

– We have 23,000

• They have been here 3.4 billion years– We have been here about 100 thousand

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Cholera (Vibrio cholerae)

• 100,000 death / year• From contaminated food or water• Only becomes virulent in body

– Needs body temperature• RNA turns on virulence gene(s) Weber, PNAS 2014

Things Change• Food-borne Listeria monocytogenes -

typical enteropathogen– New strain directs attacks heart MUSCLE

(Microbe June 2011)

• VREF• Clostridium difficile

More Emerging Threats

• E. coli – super bug emerged this decade (O157:H7)

• New E. coli superbug this year (Germany) enterohemorrhagic (EHEC) Science June 2011

• Classic MRSA• New MRSA in cows Science June 2011 –

does not register on lab tests as MRSA. Yet it is

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Funguses• Hard to kill

– Same protein metabolism as us

• Candida albicans

Actinomyces – now called a bacteria, was a “fungus”

Geriatric (root) caries

Hard to kill – 4 w. PCN or Sx +1 w. PCN

Spirochetes• Hard to culture• Most every dental sample has lots

– New research methods will help

Viruses• Bacteriophages

– Transfer DNA between bacteria

• Hepatitis B– No longer a problem

• Hepatitis C = 1.6% US population– Still a risk, but getting better– Many infected patients unaware– Liver function ?– Maybe not so many in dental aerosol

• Adam Powell’s research at UAB

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Bacterio-phage (virus) carries good or bad to bacterial cell

Bacteriophages make cover ofMicrobe

Carry lethal DNA into bacteria

Prions• Just a chain of proteins• Can duplicate itself• Autoclaving ineffective

– Alkaline soak before autoclaving

• Bind to steel

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We can fight bacteria

But, they are more evil than we thought:Biofilm

DNA bankQuorum sensingPhysical barrier

MotilityMore

E. coli w/ pili

What we have already done has changed our world

•Pasteurization•Good water•Refrigeration•Antibiotics

Endo Landmark•Kakahashi, Stanley, and

Fitzgerald, OOO 1965Germ-free rats pulp exposures healed rapidly

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Endo Bacteria• Caries = aerobes and facultatives

oxygen + sugar acid• Penetration into pulp = less oxygen

– Facultatives and Obligate anaerobes

• Endo flare-up = facultative – Metronidazole not indicated

Munson’s research• Used selected PCR primers• Found 20+ different bacteria types in av.

Endo infection

More recently Siqueira used more primers and found more, with range from 13-80 J Endo 2011

Bacteria are smaller than tubules

Chronic canal infection:

More tubules infected

Bacteria go deeper in tubules

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Pathways to the Pulp

• Bacteria are everywhere

• Anachoresis• Dentin tubules

– caries– prophy– abrasion, etc.– “long in the tooth”

• Fractures• Perio disease

• Pulp caps• Leaking Margins

Kumar’s research• Similar to Munson’s• Used different PCR segments (many

different) and found a similar # of different bacteria in perio infections

• Conclusion: There more than 20 different types of bugs in every infection we treat

Perio Bacteria Heart Disease

• Non-venereal Chlamydia• Porphyromonas gingivalis• Did the heart bacteria cause perio disease• What about antibiotic tx for heart disease ?

Recently, theory is under debate

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Invaders are mixed,Anaerobes soon take over

• Fusobacterium• Peptostreptococcus• Peptococcus• Bacteroides

– Bacteroides– Prevotella– Porphyromonas

• Lactobacillus

• Eubacterium• Capnocytophagia• Actinomyces• Campylobacter• Selenomonas• Veillonella• Wolinella• Propionibacterium

Biofilm Problem• Can form outside root • Disinfectant (H2O2) for surgery• Quorum sensing• Persister cell (like submarine)• Problem for Implants

– Penile implants ?– Breast implants OK

Biofilm challenges our therapy

Costerton says 1000 X antibiotic concentration needed to kill all biofilm bacteria

Endo-Perio –Will not respond to endo and antibiotics

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Antibiotics and vaccines

185/100,000/y

To 35/100,000/y

Now creeping up

1990’s: Lyme disease discovered• Unrecognized and untreated: dire result• Tetracycline tx = AOK

Many Viral Diseases persist

Unquestionably• We will identify more diseases as microbial• Natural selection will lead to more

resistance• Some bugs will always be susceptible

•Unquestionably, microbeswill win eventually

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Extent of the Problem• Poor recording – we all know of

examples• CDC 2011: $20 Billion in excess

healthcare costs, 8 Billion days sick

• IOM 2010: 63,000 additional deaths per year (25,000 in Europe)

David Norrington’s Research

• Grew biofilm on dentin • Add an antibiotic

Ampicillin, Doxycycline, Clindamycin, Azithromycin, Metronidazole

• Biofilm unchanged at 8 days

OOO Apr 2008

Biofilm = Genetics changes Bacteria• Planktonic form =

free swimming– What we grow in

lab• When attached,

they choose different genes from their DNA and thus behave verydifferently Party Time !

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One Bacteria Survives on Another’s Waste

Much of change we see is actually already there

That is:Bacterial adaptability is less

from mutation than from gene expression

Biofilm• Attaches w/ holdfasts• Slime = extracellular food storage • Tumbleweed-like mats• Outer layer = protection • Quorum sensing• Gene swapping

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How do Obligate Anaerobes survive from one site to the

next ?

•Biofilm–Core cells survive

•Cell membrane shuts pores•Metabolism stops

Antibiotics Prior to Endo Appointment ?

• Yes, if infected• Will not help pulpitis without necrosis

Dental microbes cause brain abscess / implant infection

Sullivan, 1990, J Bone Joint Surg

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Can bacteria go thru vessel wall(Can one tooth’s infection spread to another)?

Lemierre’s Disease = Fusobacterium necrophorum from neck infection spreads into jugular vein to cause a clot. Also spreads into mediastinum (lungs)

Can infection in one tooth invade vessels of adjacent tooth???

Lemierre’s Disease

- Acute lung infections-DentalSource ?

Most infections stay putMorse, Clinical Endodontology, 1974

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Antibiotics are indicated for active infections

Danger – Up or Down

Morse, Clinical Endodontology, 1974

Canine Space Swelling

Morse, Clinical Endodontology, 1974

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Canine Space Swelling into Cavernous Sinus

35% mortality

Morse, Clinical Endodontology, 1974

Mandibular Anterior Spaces

Morse, Clin. Endodontology, 1974

Sublingual - Submandibular

• Sublingual Space– Above mylohyoid muscle, displaces tongue

• Submandibular space– Below mylohyoid muscle, into neck

• Mylohyoid has no posterior boundary, infection can get into neck easily

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Buccal space –maxillary molar can look like mandibularMorse, Clinical Endodontology, 1974

Active Tuberculosis•Patients cannotbe treated in your office

Clinical Warning:

•New strains of Mycobacterium tuberculosis are resistant to all antibiotics

•Tuberculosis bacteria are suspended in aerosols for hours

•Dental treatment creates these aerosols

Others

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1. Know your Enemy

2. Our Weapons3. Dental Unit Water

Outline

Our Weapons•Techniques•Disinfectants•Antimicrobial drugs•Probiotics•Culture and Sens. Test

Our techniques•Handwashing is 10 X

better than disinfectant hand rub (Clinton, unpublished)

•Change gloves after rubber dam placed (Luckey)

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Jeff Luckey’s Study• J Endodontics, 2006, p.646• Our exam gloves in box = very low

bacteria count, even after a week in the operatory

• Very high bacteria count after exam, anesthesia

• Should change to new gloves after rubber dam placed and tooth disinfected

Endo Technique•We really should use better sterile technique for endo–Barrier for x-ray machine ?–Film packet to darkroom ?

Dirty Area on Tray

• Keep a dirty area for used items• Dixie cup of disinfectant for used

files– Protection from stick injury

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Rubber Dam• Up to 500X reduction in aerosol

(essentially zero)• Aerosols can last 30 min as microdroplets

suspended in air• Miller Proc 1st Intl Symp Aerobiol

1963,pp97-120

• Block anesthesia• Consider anatomy• Consider gravity • Cut to bone• Many need to open bone• Consider in-dwelling drain• Antibiotic ?

Incision for Drainage

Ripe to Drain

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Anesthesia for I & D• Block

Infiltration into acidic pH = poorWand = poorPressure from anesthetic = poor

• Freeze

Consider anatomy

Cut lowest part

? Insert drain

1500 B.C.

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Drain by Aspiration

Disinfectants• Kill quickly

– No problem with resistance

• Damage host cells– Keep in canal

Clorox periapically

Routine Canal Irrigants• Clorox• Chlorhexidine 2%• Iodine-potassium iodide• EDTA• MTAD

– Tetracycline, citric acid, detergent

Remove Smear Layer = get to bacteria in tubules

•Shahravan JOE 2007 Syst. Review (EDTA, various acids, Tetra.)

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Chad Christensen, JOE Apr 2008

• Clorox = pH 12• Less alkaline = more antimicrobial• More alkaline = better tissue

dissolution

Don’t Forget H2O2

• Steven Thomas, J Endod 2003• Anaerobes grown in sterile roots• Exposed to air or 3% H2O2

• Air killed anaerobes more slowly

Intracanal meds• One visit or Two ?• Phenolics ? • Calcium hydroxide

– selects for Enterococcus faecalis– pH dissipates to cementum, & with time– may help external resorption

• Ca(OH)2 + another disinfectant

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Calcium hydroxide is dangerous

Endo #31Lindgren JOM-FS 2002

Ca(OH)2 #29 Paresthesia / surgery

Ahlgren et al. OOO, 2003

Good outcome 6 mo. later

Ahlgren et al. OOO, 2003

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Sterilize Canal ?• Like oral hygiene, we cannot

achieve sterility• We should strive to reduce bacteria

as much as possible

Calcium hydroxide needs a second disinfectant

and Time is needed too

Clorox needed to dissolve tissue

Mand. 2 PM

Max. Molars

Bjorndahl and Skidmore

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Chlorhexidine ?• Substantivity w/

dentin

Clorox + Chlorhexidine • A carcinogenic,

brown-staining precipitate forms

MTAD ? Q-Mix ?• Tetracycline• Citric acid• Detergent• 5 minute soak after instrumentation

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CHX + NaOCL

Para-chloroaniline precipitate– Brown color (even @ 0.19% Clorox)– Unknown implications

• Leakage• Systemic methemoglobinemia

Basrani et al. (Toronto) JOE Aug. 2007

Laser - Radiosurgery

•Kill bacteria•Live bacteria and viruses in

smoke plume

No technique kills everything

Summary: Everything works, but nothing kills everything

Nair (TEM & LM) fins harbor many bacteria / pulp, even after 5.25% Clorox

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Antibiotics vs. Disinfectants

• Antibiotics attack only bacteria – Metabolic differences– Structural differences– Spare host cells

• Disinfectants– Kill fast (no resistance)– Host cells harmed too

Disinfectants for perio biofilm ?

•Chlorhexidine works•Listerine works•Paul Keyes

–1970’S–H2O2 + baking soda

Viable, Not Cultivable•Costerton estimates 2X as

many bacteria as grow in lab

•Some lie dormant•Some only grow

fastidiously

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Why Give Antibiotics

•Patient demand•Treat existing infection•Prevent infection (pain)

Endo Tip: Is patient swollen ? Febrile ?

Endo Causes Bacteremia• Bender’s 1950’s research • Heimdahl (J Clin Micro 1990) lysis-filtration

= found more bacteria after endo (20%)• Debelian (Endod Dent Traumatol 1995) 4

of 7 pts: endo wi/ canal had bacteremia• Leonardo (J Endo 2002) biofilm on root

surface w/ periapical area• Local Degerming

Endo tip: Endo causes bacteremias, even if confined to canal

Antibiotics Prior to Endo Appointment ?

• Yes, if infected, without drainage• Will not help pulpitis without necrosis• American Academy of Orthopedic Surgeons

2009: All joint patients should get prophylactic antibiotics before invasive dental tx. – No time limit after sx.

– Vasculature around joint is very different

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Lancet Epub Nov 19, 2014

• Slowly increasing Infective Endocarditis in England– Their government recommended cessation of

antibiotic prophylaxis in 2008– May not be Cause Effect

• Dayer et al. Incidence of IE in England 2000-13.

Antibiotics down, IE up (high and low risk individuals)

Orange = AmoxBlue = Clinda

Orange dentistsBlue = MD

Orange = mortalityBlue = incidence

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New Research on Bact. Endocarditis

• Lockhart et al. Circulation 2008;117:3118.• Lit review:

– 170 oral species in blood– 275 species caused IE– Merging lists 98 species

• N = 290 patients for dental extraction– Gr. 1 Brushing alone (extraction after study)– Gr. 2 Amox, ext. after 1 hour– Gr. 3 Ext. wo/ antibiotic

Results: IE-causing Bacteremia

• Extraction worst• Reduced

w/Antibiotic • Brushing only

Yellow = procedure

Prophylactic Antibiotics ?• Cochrane Reviews

– Sickle cell pts. w/ antibiotic = fewer pneumococcal infections

– C-sections pts. w/ antibiotics = fewer infections– CF pts. w/ antibiotics = no difference in groups– Bronchitis pts. w/ antibiotics = slightly better– Pulpitis w/ antibiotics = same pain

(Did the coverage keep the pulpitis fromprogressing into an abscess?)

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Perhaps a Single, Large antibiotic dose is what we need ???

L Buccal Space - Mild Swelling

Rapid WorseningAirway ? Headache / vision ?

Monitor Fever

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My Regimen• Monitor signs / symptoms,

including fever • Bactericidal drug, change if

no better wi/ 24 hours• Bacteriostatic drug, change

if no better wi/ 48 hours

1. Innate resistance to drug (Lactobacillus to vancomycin)2. Bacteria destroy drug (B lactamase producers) (some defeat Augmentin)3. Antibiotic pump (tetracycline resistance)4. Bacteria change their binding sites5. Patient fails to take drug6. Pt. eats something that binds drug (tetracycline)7. Problem was not bacterial8. Abscess allows poor drug penetration9. Bacterial mutation10. Bacteria pick up DNA from

a. environmentb. phage (virus) transferc. conjugation (occurs between different sp.)

11. Persister cell – maybe deep within biofilm12. Communication between bacteria (Quorum sensing)13. Bacteria can hide in cells Anaerobes did not show increased antibiotic resistance (3 year

study in Estonia) (Loivukene Anaerobe 9:57, 2002)

Mechanisms of Bacterial Resistance

Antibiotic Resistance

Pens/cephs and vancomycin work on new cell wall

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Antibiotic Resistance Rates Vary

AmpicillinClindamycin

Metronidazole

Anaerobe 2003, 9:105-111.

Resistance Varies

Metronidazole

Resistance

Clindamycin

Penicillin

Population’s # Dosed Days, Metro.

50% of antibiotics used to promote growth in livestock

• 1995 Denmark sharply limits antibiotics in pigs, chickens,…

• Less human antibiotic resistance

• Other methods keep yields up Sci Am, Apr 2011

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NEW CDC Program• Late 2011, CDC began monitoring

program for hospitals • Resistance patterns to be traced

No antibiotic kills everythingResistant forms ALWAYS flourish because they have unlimited food

Refill Antibiotic ?

• Insert art of culture tubes

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Low-Dose Antibiotics OK

• Periostat – anticollagenase = antitumor ?• Azithromycin used in sub-lethal dose =

breaks up biofilm without killing Pseudomonas

• Topical antibiotics – FDA = OK• Intracanal antibiotics ?

Not so good in livestock, What about humans?

Bacteria in Tubules• Miller 1890 - first report• Love and Jenkinson, (meta-analysis Crit

Rev Oaral Biol Med 2002) – Penetration varies widely– Greater w/ chronic infection

Clostridium difficile• Most common cause of abc. diarrhea• 1978-83, we blamed clindamycin• 1983-2003 worst was cephalosporins• Now, cephs and fluoroquinolones• Now, new super-strain• Tx.: metronidazole 250 qid or 500 tid x 10 d.• Not Vancomycin b/c VREF

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IV Bisphosphonate Patients Take Penicillin Continuously

Recent ChangesAn MRSA variant repels Vancomycin action

by buttressing its cell wallSieradzke and Tomasz Antimicrob Agents Chemo 2006; 50:527-533

Vancomycin interferes with cell wall synthesis

News from the Research Lab

• Tigecillin (Tigacil) new IV tetracycline has FDA approval

• Scripps Institute changed only one atom in vancomycin and got a much broader kill spectrum, including Vancomycin Resistant Enterococcus (VRE)

• More will follow shortly

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Erysipelas

Beta-hemolytic Streptococcus infection of skin

Usually responds to PCN

We may soon see more of this

Probiotics – Fight bad bugs, diseases w/ good ones• Fecal transplant – helped 70% of

Inflammatory Bowel Ds. patients (Pinn, Brandt, Montefiore Med Ctr. Conf., NYC, 2014)

• Salmonella and Listeria can infect tumors and aid immune (Forbes, U Mass Amherst, Microbe 2014 9:11;440)

• Lactobacillus reuteri - reverses osteoporosis in mice (Microbe 2014 9:11.437)

Prebiotics – foods that change flora (yogurt)

Dentists Culture ???

U of L – UAB systemNeedle aspirate, Inoculate culture, Shake,Divide culture in vessels w/ antibiotic disks,Incubate, Read in 24 and 48 hours

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Hospital Lab helps determine best antibiotic from cultured bacteria

Will your key bugs grow and stand out ?

Immediate inoculation – No 02

Different antibiotic in each tube

No growth = good possibility of antibiotic success

24 hours

Our System Avoids Oxygen

Anaerobe Systems

Morgan Hills, CA

408-782-7557

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Biofilm Problem• Can form outside root • Disinfectant (H2O2) for surgery• Quorum sensing• Persister cell (like submarine)• Problem for Implants

– Penile implants ?– Breast implants OK

Biofilm challenges our therapy

Costerton says 1000 X antibiotic concentration needed to kill all biofilm bacteria

Endo-Perio –Will not respond to endo and antibiotics

Biofilm = Genetics changes Bacteria• Planktonic form =

free swimming– What we grow in

lab• When attached,

they choose different genes from their DNA and thus behave verydifferently Party Time !

42

Biofilm• Attaches w/ holdfasts• Slime = extracellular food storage • Tumbleweed-like mats• Outer layer = protection • Quorum sensing• Gene swapping

How do Obligate Anaerobes survive from one site to the

next ?

•Biofilm–Core cells survive

•Cell membrane shuts pores•Metabolism stops

1. Know your Enemy2. Our Weapons3. Dental Unit Water (Our silent, dirty secret)

Outline

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Dental Waterline Biofilm

Most all dentists UAB

1/10 mL water on plate

•Form within a few days–Bacteria in city water–Backflow from patients

•Continually shed bacteria / funguses

Waterline Biofilms(Our Dirty Secret)

Biofilm Formation In DUWLSterilized tubing

3 days

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Layers formFungus at day 5

More layers - holdfastsAnother site - Day 6

More bacteria find a homeHigh power - Day 8

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Fully formed biofilmDay 14

Waterline Cleanser - immediateAreas of breakup

Waterline cleanser – 24 & 48 hoursFungal layer exposed

Almost totally clean

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Pathogens found in dental water

–Legionella–Pseudomonas–Perio bugs–Caries bugs–Many others

What does NOT work ?

• Drying lines overnight• Flushing lines w/ water• Any regimen that relies on

remembering

Summary• Bacteria are everywhere and

resistance is rising• Patients are less healthy• We must use antibiotics (wisely)• Return to scrupulous sterile technique

– More time for chemo-