An Overview of Antibiotic Resistance (How Miracle Drugs Created Miracle Bugs)

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An Overview of Antibiotic Resistance(How Miracle
Drugs Created Miracle Bugs)

• Dr. Stephen M. Brecher
• Director of Microbiology
• VA Boston Healthcare System
• Assistant Professor of Microbiology
• Boston University School of Medicine

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Penicillin 1928The Miracle Behind the Miracle
Drug

• Fridays flaw // Mondays discovery
• Fleming was going through old plates that were
  left over the weekend in the work area
• these should have been soaked in detergent and
  discarded
• The prepared mind
• Fleming had been working on lysozyme so was
  familiar with lysis
• On the now famous plate (preserved in the
  archives of St. Marys hospital) he observed the
  lysis of S. aureus colonies near the growth of a
  contaminating mold (Penicillium sp.)

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Penicillin 10 years LaterFlorey and Chain

• Fleming was unable to concentrate the substance
  from the mold due to lack of chemical
  assistance
• Howard Florey and Ernst Chain
• first looked at lysozyme
• renewed interest following the success of
  sulfonamides
• decided to look at penicillin
• Learned to extract, concentrate and stabilize
  penicillin
• miraculous results obtained in a mouse model

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WW II, Football, Fire and The Miracle Drug

• In 1941 Florey came to America to try to convince
  the government to back large scale production of
  penicillin
• WW II Most of the penicillin went to the DOD
• November 28, 1942 Holy Cross Upsets Boston
  College 55-12
• That evening, the players as well as many
  prominent Bostonians went to celebrate at the
  Cocoanut Grove nightclub in the South End
• A fire broke out and 492 people died

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The Fire That Made Penicillin Famous

• Survivors were treated with sulfadiazine for
  infections and with plasma (a 4 year old
  technology) for dehydration and shock
• With permission from the government, Merck
  (Rahway, NJ) rushed a 32 liter supply of the
  fluid culture medium from Penicillium mold to be
  used for victims with S.aureus skin infections
• Sheehan,J. and Ross, R.N. Yankee Magazine, pp.
  125-203. 1982

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The Fire That Made Penicillin Famous

• Preceding the fire, less than 100 Americans had
  been treated with penicillin
• After the fire, the media brought national
  attention to the miracle drug
• The pharmaceutical companies began large scale
  production of penicillin
• ..the Americans improved the methods of
  production so that on D-Day there was enough
  penicillin for every wounded man who needed it..
  Fleming, 1945

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Early Warning

• Fleming was working with mutants of S. aureus
  that could be grown in the presence of increasing
  concentrations of penicillin
• He was concerned that if patients did not take a
  full course of treatment, resistant strains would
  appear
• Another concern an oral form of penicillin was
  produced and was available without prescription
• the greatest possibility of evil in
  self-medication is the use of too small doses so
  that instead of clearing up infection, the
  microbes are educated to resist penicillin

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Dominance of Bugs over Drugs

• Bacteria are the dominant species on the earth
• rapid multiplication rate
• natural mutation rate
• ability to transfer or move genes via
  transformation, conjugation, transduction and
  transposition
• Collectively, these properties allow bacteria to
  survive, change and eventually flourish under
  intense selection pressure

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Evolution The Short Course

• 3.85 billion years old Bacteria
• 210 million years old Real Mammals
• 60 million years old Human-like Mammals
• 30 million years old Monkeys
• 2.5 million years old Direct Ancestors
• 0.2 million years old Neanderthals
• 0.125 million years old Homo Sapiens
• 63 years old Antibiotics

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Dominance of Bacteria in Humans

• The human body has 1013 human cells and 1014
  bacterial cells

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Its an Accolade for the Bugs When We Use Initials
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MRSA VISA GISA VRSA

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Miracle BugsS. aureus

• Penicillin
• moments to get first resistant strain
• 2004 97 resistant to penicillin
• Methicillin 2 years to get first resistant
  strain (1961)
• Original strains were nosocomial
• Infection rates 30-70 resistant of all S.aureus
  are MRSA
• New strains CA-MRSA
• Vancomycin good run, but trouble looming
• 1995 VISA in France
• 1996 VISA in Japan
• VRSA 4 US cases

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The Emergence of VISA

• France 1995 2 year old girl with leukemia and a
  central line associated bacteremia
• Treated with surgical drainage and
  quinupristin-dalfopristin (survived)
• Japan 1996 4 month old that was treated for 29
  days with vancomycin. Initial isolate was
  susceptible, subsequent isolate had vancomycin
  MIC 8 ug/ml
• Treated successfully with aberkacin and Unasyn
• United States 8 confirmed cases.
• Most in dialysis patients

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VRSA So When?

• VRSA had been made in the laboratory by
  transconjugation of the vanA gene from E.
  faecalis into S. aureus
• Noble et al. FEMS Microbiol. Lett. 93 195-198.
  1992
• When would this happen in humans?
• NOW

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VRSA

• July 5, 2002 MMWR S. aureus fully resistant to
  vancomycin
• 40 yo female w/ diabetes, PVD and renal failure
• First isolate to naturally acquire the vanA gene
  from E. faecalis. The patient had both VRE and
  VRSA
• MIC was gt1024
• 4 cases through 8/1/05

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The New Chief of StaphCommunity Acquired MRSA
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Community Acquired MRSA

• 4 deaths, 200 documented cases of MRSA infections
  in children (ages 1-13) in the community setting
• In the fatal cases, the initial isolates were
  resistant to oxacillin and other beta-lactam
  antibiotics
• Most isolates were susceptible to other common
  antibiotics
• Multiple, global reports of CA-MRSA (see
  www.promedmail.org for outbreak reports)
• MMWR48(32) 707-710. 1999

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Community-Acquired MRSA Wound Infections

• Los Angeles County Prison
• MSM in Massachusetts
• The St. Louis Rams football team (NEJM 2005.
  352468)
• Necrotizing fasciitis in Los Angeles
• High school and college football and wrestling
• Military recruits
• Pediatric infections

MMWR. 200352(5)88.
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Properties of CA-MRSA

• SCCmec IV or V (rather than I-III)
• Smaller
• More mobile
• Many isolates express Panton-Valentine Leukocidin
  (PVL)
• Potent toxin associated with furunculosis
• No known health care risk factors
• Skin and soft tissue infection common, occasional
  necrotizing pneumonia
• Susceptible to more antibiotics than HA-MRSA

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Characteristics of SCC types I-V
mec type Size (kb) R genes origin PVL
I 34 AG H -
II 53 ermA H -
III 67 AG, tetK H -
IV 21-24 C
V 28 C ?
adapted from Deresinski,S. CID.200540.562-573
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Epidemiological Success and Fit

• Smaller size
• Less genes
• May replicate more rapidly than HA MRSA
• May eventually displace HA MRSA
• Virulence genes
• 19 putative virulence genes found in a strain
  associated with fatality in ND
• Houston Pediatric Hospital
• since 2001, 74of CA Staphylococci have been MRSA
• adapted from Deresinski,S. CID.200540.562-573

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The Future

• CA-MRSA will replace HA-MRSA
• Increased virulence
• Increased susceptibility
• Staphylococci will outlive the human race

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N108,497
N506,880
N1,101,670
N486,293
TSN Database - Focus Technologies, Inc.,
Herndon,VA
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VRE
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EnterococciIntrinsic Resistance

• Cephalosporins
• Aminoglycosides (low to moderate level)
• Clindamycin
• Trimethoprim/sulfamethoxazole

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Emergence of VRE

• Initial reports
• England and France (1988)
• United States (1989)
• Seen in both E. faecalis and E. faecium
• There are now at least 7 phenotypes
• vanA through vanG
• The corresponding genotypes are
• vanA through vanG

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vanA and vanB Phenotypes
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The Net Result

• Enterococci that acquire the vanA phenotype are
  highly resistant to vancomycin and to teicoplanin
• Enterococci can pass the vanA gene cluster to S.
  aureus

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N55,914
N61,392
N402,022
N428,324
N192,598
N204,746
TSN Database - Focus Technologies, Herndon, VA
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Beta-LactamasesorHow Bacteria Defend Themselves
Against Antibiotics
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Beta-LactamasesAn Overview

• Enzymes that open the beta-lactam ring,
  inactivating the antibiotic
• Most likely evolved from penicillin binding
  proteins over 2 billion years ago
• 2 major sub-groups
• Serine residue at active site(Groups A,C and D)
• Metalloenzymes requiring Zn as a co-factor
  (Group B)
• Now about 500 known beta-lactamases

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The Original beta-lactamases

• TEM
• named after patient (Temoniera, Greece, 1960s)
• Related enzymes named numerically
• TEM-2, TEM-3
• Activity against penicillins and narrow spectrum
  cephalosporins
• TEM related ESBLS discovered in France in 1984,
  US in1988
• SHV
• related to TEM, but named SHV because sulfhydral
  reagents had a variable effect on substrate
  specificity

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The New Beta-Lactamases

• TEM-type ESBLs (class A)
• AA substitutions at the active site allow access
  to oxyimino-beta-lactams
• Over 130 varieties
• SHV-type ESBLs (class A)
• 68 AA similarity with TEM
• AA substitutions at active site
• Over 50 varieties
• Jacoby and Munoz-Price.The New Beta-Lactamases.
  NEJM.2005.352380-391.

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Extended Spectrum Beta Lactamases (ESBLs)

• Bacterial enzymes produced primarily by E. coli
  and Klebsiella species that break down the
  beta-lactam ring of third and fourth generation
  cephalosporins
• Ceftriaxone, cefotaxime, cefpodoxime, cetazidime,
  cefepime and aztreonam
• Now seen in Salmonella, Proteus species and other
  enterics
• Using traditional MIC and disk diffusion
  breakpoints, these isolates can test as
  susceptible to third and fourth generation
  cephalosporins and to aztreonam
• Anecdotal cases suggested that patients who were
  infected with these organisms failed apparently
  appropriate therapy with those antibiotics

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ampC Beta-Lactamases

• Chromosomally-mediated
• Low level constitutive beta-lactamase production
  to inducible high-level beta-lactamase production
• Induced by beta-lactam antibiotics
• Hydrolyze most cephalosporins, except cefepime
  and carbapenems
• Not inhibited by clavulanic acid
• Plasmid -Mediated
• gt20 plasmid mediated, most not inducible
• resistance to cephamycins, oxyimino beta-lactams
  and not inhibited by clavulanic acid
• More common in Enterobacter, Serratia, and
  Citrobacter

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Carbapenemases

• Serine Carbapenemases (Class A)
• inhibited by clavulanic acid
• hydrolyze cephalosporins, carbapenems,
  monobactams
• Found in enteric gram negatives, Pseudomonas and
  Acinetobacter
• Metallo-Beta-Lactamases (Class B)
• gt17 Plasmid mediated IMP type, original ones were
  chromosomal
• not inhibited by clavulanic acid
• difficult to detect in lab, use EDTA with and w/o
  imipenem

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Nasty Beyond NastyKPC Beta-Lactamases

• Class A Beta-lactamases with carbapenem-hydrolyzin
  g activity
• Exist on plasmids
• inhibited by clavulanic acid
• Originally reported in K. pneumoniae in North
  Carolina in 2001(KPC-1)
• KPC-2 and KPC-3 found in NYC
• Has subsequently been seen in K. pneumoniae,
  Salmonella sp., Enterobacter aerogenes and E.
  cloacae

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Resistance Patterns in Klebsiella pneumoniae
Typical ESBL ampC KPC
Ampicillin R R R R
Ticarcillin S R R R
Cefepime S R S R
Ceftriaxone S R R R
Ceftazadime S R R R
Cexoxitin S S R R
Pip/Tazo S S R R
Imipenem S S S R
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What to do if your hospital has these nasty
beta-lactamases

• Immediately fire your Infection Control
  personnel, Clinical Pharmacy staffs and your P
  T Committee.
• Close your medical and surgical intensive care
  units.
• Torch entire medical center including all
  equipment.
• Start over

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Miracle BugsGram Negatives in the ICU

• Pseudomonas aeruginosa 30-50 fluoroquinolone
  resistance in 15 years
• E. coli and Klebsiella species with Extended
  Spectrum Beta-Lactamases (ESBLs)
• ampC beta-lactamases in Enterobacter, Serratia,
  Citrobacter, etc.
• Stenotrophomonas maltophilia
• Acinetobacter baumanii
• Burkholderia cepacia

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Quinolone Resistance

• Pseudomonas aeruginosa
• Shigella sp.
• Neisseria gonorrheae
• Does the future hold another Darth Vader?
• Quinolones are important antibiotics choose
  wisely

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Levofloxacin Resistance in Common Gram Negatives
20032004TSN Data

• TSN Database - Focus Technologies, Inc.,
  Herndon,VA

Organism Total Susceptible Intermediate Resistant
E.cloacae 37,332 87.4 2.0 10.6
E. coli 506,525 88.5 0.3 11.3
K. pneumo 109,903 92.6 1.3 6.1
P. mirabilis 69,906 78.4 3.3 18.3
P. aeruginosa 134,635 62.5 5.9 31.6
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Clostridium difficileAssociated Diarrhea

• Increasing at an alarming rate
• All antibiotics are suspects, some more than
  others
• A previously uncommon strain that hyper-produces
  Toxins A and B (deletion in the tcdC gene) as
  well as has a newly characterized binary toxin
  and is frequently resistant to FQs has been
  reported1
• This strain is extremely virulent
• Many therapeutic failures with metronidazole
• Not due to resistance
• 50 failure rate with 7 d course of
  metronidazole(22non-responders, 28 relapse)
  Musher et al. CID,2005, 401586
• New alcohol-based hand gels do not kill spores
• 1. McDonald, LC. Emergence of an epidemic strain
  of Clostridium difficile in the United States,
  2001-4 Potential role for virulence factors and
  antimicrobial resistance traits. LB-2, IDSA2004

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Miracle BugsRespiratory Pathogens
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Miracle Bugs Streptococcus pneumoniae

• Same exposure to penicillin as S. aureus
• 25 years w/o resistance
• In the last 10 years rapid evolution of
  penicillin resistance and then multi-drug
  resistance
• Mechanism of penicillin resistance
  transformation of DNA from oral streptococci
• 2004 approximately 35 of isolates are
  penicillin non-susceptible, 22 of these are
  multi-drug resistant

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High Level Penicillin-Resistance inS.
pneumoniae 1979200315
Resistant (MIC ? 2 mg/L)
25
20
15
Percentage
10
5
0
Year Isolates Centers
1979-871
1988-892
1990-913
1994-953
1997-983
1999-003
2000-014
2002-035
5589 487 524
1527 1601 1531
10,103 10,894 35 15
17 30 34
33 206
1980s
1990s
2000s
1Spika JS, et al. J Infect Dis.
19911631273-1278 2Jorgensen JH, et al.
Antimicrob Agents Chemother. 1990342075-2080
3Doern GV, et al. Antimicrob Agents Chemother.
2001451721-1729 4Doern GV, Brown SD. J Infect.
20044856-65 5PROTEKT Study 2002/2003.
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Multidrug-Resistant S pneumoniae (MDRSP)

Multidrug Resistant
Year
Alexander Project. Strains resistant to gt3 inc
penicillin agents, 25.8 4 drugs, 15.6 5
drugs, 7.0.
Jacobs, et al. J Antimicrob Chemother.
200352229-246.
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Association Between Antibiotic Use and Resistance
in S. pneumoniae Isolated from Invasive Infections

• Erythromycin use was not associated with
  resistance to other classes of antibiotics
• Clarithromycin use was associated with increased
  likelihood of erythromcyin resistance
• Azithromycin use was associated with increased
  risk of resistance to macrolides, penicillins and
  TMP-SMX
• Authors concluded that their findings were
  consistent with 3 of 4 studies that showed the
  use of long half-life macrolides are associated
  with increased macrolide resistance in
  pneumococci and Group A streptococci
• Vanderkooi et al. Predicting Antimicrobial
  Resistance in Invasive Pneumococcal Infections.
  CID. 401288-97.2005

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Antimicrobial Resistance with Haemophilus
influenzae

• HIB vaccine
• Very few invasive
• H. influenzae infections
• 25-30 beta-lactamase
• ? bln ampr strains
• 30 TMP-SMXR
• 1 levofloxacin resistant strain ( MIC 32)
  recently seen in a long term care facility
  (Nazir, J. et al. ICCAC abstract C2-647.
  September, 2002)

Haemophilus influenzae
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Antimicrobial Resistance with Moraxella
catarrhalis

• 98 ?-lactamase
• 34 TMP-SMXR
• usually susceptible to
• - amox/clav
• - macrolides
• tetracyclines
• fluoroquinolones
• telithromycin
• chicken soup
• herbal tea

Moraxella catarrhalis
Gram stain from patient with recurrent otitis
media
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Changing Lifestyles

• Time and Travel
• SARS
• Aging populations
• Pressure to prescribe/treat
• Antibiotics in animal feed
• Stashing our children

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The Strain From Spain

• Iceland had no reports of resistant S.
  pneumoniae until 1988
• By 1992, 17 of all isolates were multi-drug
  resistant
• 70 of the isolates were serotype 6B
• This was one of the resistant strains that was
  common in Spain
• Why and how did it show up in Iceland?

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The Strain From Spain

• Icelandic families vacation in Spain
• Icelandic children probably picked up the strains
  from Spanish children
• High usage of tetracycline and SXT probably
  contributed to selection pressure
• The majority of resistant strains were from
  around Reykjavik which houses 57 of the total
  population of 250,000
• 80 of children, ages 2-6 attend day care centers

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Day Care CentersThe Real POOP

• Should be renamed Germ Sharing Centers
• Think about it
• Toilet training challenged populations
• Heavy droolers
• Close contact
• Sharing food and utensils
• Clonal spread of resistant strains likely

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Summary and Conclusions

• Antibiotics have been used in the United states
  for only 60 years
• We have gone from the age of miracle drugs to the
  age of miracle bugs
• The prudent use of antibiotics is essential

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