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

Penicillin 1928The Miracle Behind the Miracle Drug

• Friday’s flaw // Monday’s 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. Mary’s hospital) he observed the lysis of S. aureus colonies near the growth of a contaminating mold (Penicillium sp.)

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

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

“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

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

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…”

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

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

Dominance of Bacteria in Humans

The human body has 1013 human cells and 1014 bacterial cells

It’s an Accolade for the Bugs When We Use Initials

MRSAVISA

GISAVRSA

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

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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http://content.nejm.org/content/vol340/issue7/images/large/04f3.jpeg

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

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 >1024

• 4 cases through 8/1/05

The New Chief of StaphCommunity Acquired MRSA

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

Community-Acquired MRSA Wound Infections

• Los Angeles County Prison• MSM in Massachusetts• The St. Louis Rams football team (NEJM 2005. 352:468)• Necrotizing fasciitis in Los Angeles• High school and college football and wrestling• Military recruits• Pediatric infections

MMWR. 2003;52(5):88.

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

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.2005:40.562-573

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, 74%of CA Staphylococci have been MRSA

adapted from: Deresinski,S. CID.2005:40.562-573

The Future

• CA-MRSA will replace HA-MRSA

• Increased virulence

• Increased susceptibility

• Staphylococci will outlive the human race

0

20

40

60

%R

Oxacillin

Overall Oxacillin Results for Staphylococcus aureus by Patient Status,

1996-2004

All

Inpatient

Inpatient-ICU

Outpatient

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

N=1,101,670N=506,880

N=108,497

N=486,293

VRE

EnterococciIntrinsic Resistance

• Cephalosporins

• Aminoglycosides (low to moderate level)

• Clindamycin

• Trimethoprim/sulfamethoxazole

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

vanA and vanB Phenotypes

vanA vanB

Vancomycin MIC >64 4-1024

Teicoplanin MIC 16-512 0.5

Usual species faecium, faecalis faecium, faecalis

Acquired Yes Yes

Transferable Yes Yes

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

0

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60

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Ampicillin Vancomycin

Summary Results Among Enterococci for Ampicillin and Vancomycin,

1997-2004

E. faecalis

E. faecium

Enterococcus spp.

TSN® Database - Focus Technologies, Herndon, VA

N=192,598

N=55,914

N=402,022 N=204,746

N=61,392

N=428,324

Beta-Lactamasesor

How Bacteria Defend Themselves Against Antibiotics

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

The Original beta-lactamases

• TEM– named after patient (Temoniera, Greece, 1960’s)– Related enzymes named numerically

• TEM-2, TEM-3

– Activity against penicillins and narrow spectrum cephalosporins

– TEM related ESBL’S discovered in France in 1984, US in1988

• SHV– related to TEM, but named SHV because sulfhydral reagents

had a variable effect on substrate specificity

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.2005.352:380-391.

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

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– >20 plasmid mediated, most not inducible– resistance to cephamycins, oxyimino beta-lactams and not inhibited

by clavulanic acid• More common in Enterobacter, Serratia, and Citrobacter

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)– >17 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

Nasty Beyond NastyKPC Beta-Lactamases

• Class A Beta-lactamases with carbapenem-hydrolyzing 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

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

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

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

Quinolone Resistance

• Pseudomonas aeruginosa

• Shigella sp.• Neisseria gonorrheae• Does the future hold

another Darth Vader?• Quinolones are

important antibiotics: “choose wisely”

Levofloxacin Resistance in Common Gram Negatives 2003–2004

TSN Data

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

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

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(22%non-

responders, 28% relapse) Musher et al. CID,2005, 40:1586

• 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

Miracle BugsRespiratory Pathogens

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

High Level Penicillin-Resistance inS. pneumoniae: 1979–20031–5

1Spika JS, et al. J Infect Dis. 1991;163:1273-1278; 2Jorgensen JH, et al. Antimicrob Agents Chemother. 1990;34:2075-2080; 3Doern GV, et al. Antimicrob Agents Chemother. 2001;45:1721-1729; 4Doern GV, Brown SD. J Infect. 2004;48:56-65; 5PROTEKT Study 2002/2003.

1979-871 1988-892 1990-913 1994-953 1997-983 1999-003 2000-014

Resistant (MIC 2 mg/L)

5589 487 524 1527 1601 1531 10,103 10,894 35 15 17 30 34 33 206

1980s 1990s 2000s

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Multidrug-Resistant S pneumoniae (MDRSP)

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Alexander Project. Strains resistant to >3 inc penicillin agents, 25.8%; 4 drugs, 15.6%; 5 drugs, 7.0%.

Jacobs, et al. J Antimicrob Chemother. 2003;52:229-246.

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. 40:1288-97.2005

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

Antimicrobial Resistance with Moraxella catarrhalis

• 98% -lactamase +• 34% TMP-SMXR

• usually susceptible to:- amox/clav- macrolides- tetracyclines- fluoroquinolones- telithromycin- chicken soup- herbal teaGram stain from patient with

recurrent otitis media

Moraxella catarrhalis

Changing Lifestyles

• Time and Travel– SARS

• Aging populations

• Pressure to prescribe/treat

• Antibiotics in animal feed

• Stashing our children

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?

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

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

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