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MICR 420
Emerging and Re-Emerging
Infectious Diseases
Lecture 3:
Challenge of Antibiotic Resistance
S. aureus (MRSA), E. faecalis (VRE), A. baumannii
Dr. Nancy McQueen & Dr. Edith Porter
Overview
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Challenge of antibiotic resistance
S. aureus
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E. faecalis
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Methicillin resistance
Vancomycin resistance
A. baumannii
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MDR
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Morphology
Virulence factors
Diseases
Diagnosis
 Culture, biochemicals
 PCR: toxin production
 Antibiogram

Penicillin binding protein
2a latex agglutination
assay
Challenge of Antibiotic
Resistance
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Antibiotic resistance
Resistance genes
Transfer of resistance genes
Antibiotic Resistance
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Enzymes that degrade or modify the antibiotic
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Alteration of the antibiotic target
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Penicillin binding proteins
Funnel
connection to
an outer
membrane
channel
Inhibition of antibiotic entry
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b-lactamases
modifying enzymes like acetyltransferases
Porin mutations
LPS modifications
Efflux pumps
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Pore
Central cavit
promiscuous
site
Cell
membrane
Tetracycline efflux
Multidrug resistance efflux
Resistant bacteria are NOT more virulent but they are harder to destroy!
Antibiotic
Resistance Genes
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Plasmid and chromosomal encoded
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Spontaneous mutations
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E.g., Plasmid: beta-lactams, aminoglycosides
E.g.,Chromosomal: fluoroquinolones, tetracyclines
At low doses of antibiotics
Acquisition of resistance genes
Acquisition of Resistance
Genes
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Inherited
Horizontally
 Conjugation
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Less
efficient
Transduction
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Cell to cell contact
Virus-mediated
Transformation

Uptake of free DNA
Occurs also between distantly related genera such as between
gram-positive and gram-negative bacteria!
Major Causes for Increased
Antibiotic Resistance
Distribution of Antibiotics Manufactured in the U.S.
(Currently about 50 Million Pounds per Year)
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Human
Animal
Agriculture
Improper use in medicine
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After Stuart B. Levy in “The Challenge of Antibiotic Resistance”
Incomplete therapy
Inappropriate use for viral
infections
Unnecessary use of broadspectrum antibiotics
Low dose use in animal
husbandry to promote
animal growth
Use in agriculture leads to
unwanted distribution of
aerosols
Detection of Antibiotic
Resistance
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Conventional antibiotic disk diffusion (Kirby
Bauer) and determination of minimal
inhibitory concentrations
DNA microarrays for detection of plasmidmediated antimicrobial resistance (and
virulence factor) genes
Active Learning Exercise
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What role do transposons play in antibiotic
resistance?
Staphylococcus aureus
Staphylococcus aureus
MSA Agar Plates
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Gram positive cocci
In clusters
Facultative anaerobic
Catalase positive
Mannitol positive
Coagulase positive
Often beta-hemolytic
Normal flora (low numbers)
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S. epidermidis
Nostrils
Pharynx
Perineal
S. aureus
S. aureus: Virulence Factors
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Coagulase
Various enzymes
Protein A
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Wound Infections
Purulent infections
Binds antibodies via Fc receptor
Various toxins
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Enterotoxins
Exfoliative toxin (epidermiolysin)
Toxic shock syndrome toxin
Toxin specific diseases
S. aureus Food Intoxication
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Uptake of preformed enterotoxin
Nausea, vomiting, diarrhea
Self limited
Short duration
Staphylococcal Exfoliative
Toxin: SSSS
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Staphylococcal scalded skin syndrome
Cleaves cell adhesion molecules of
keratinocytes (desmoglein)
(http://dermatlas.med.jhmi.edu/derm/)
Toxic Shock Syndrome
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First described in
menstruating women using
certain types of tampons
High fever, rash, skin
peeling in palms, shock,
multiple organ failure
Staphylococcus TSST
production triggered in
these tampons
TSST resorption through
vaginal mucosa
Toxic Shock Syndrome
Pathogenesis
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Superantigen mediated
Uncontrolled immune response to
staphylococcal and streptococcal toxins
Early signs
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Fever
Dizziness
Confusion
Flat red rash over large areas of the body
Shock and multi-organ failure
Superantigens
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Activate numerous T
Helper cells
simultaneously
T Helper cells release
numerous
proinflammatory
cytokines
Bacterial Superantigens
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Staphylococcal superantigens
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S. aureus
Over 20 described
TSST, exfoliatins, enterotoxins
Streptococcal
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S. pyogenes (Group A beta-hemolysing
streptococci)
Exotoxin A and C, and others
New Threats by S. aureus
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Nosocomial infections (healthcare-associated
infections, HAIs)
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15% of all isolates; 8% of all HAIs
Spread of MRSA in the community
Vancomycin resistant MRSA
Methicillin Resistance
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High level resistance encoded by MecA
Encodes alternative penicillin binding protein PBP2a
 Structural changes in transpeptidase penicillin-binding proteins
 Plays role in peptidoglycan synthesis
 Low affinity of binding to beta-lactams
Carried by staphylococcal cassette chromosome mec (SCCmec)
 Unique mobile genetic element
 Integrated into the S. aureus chromosome
 Composed of the mec gene complex encoding methicillin
resistance and the ccr gene complex that encodes recombinases
responsible for its mobility
 These elements also carry various resistance genes for nonbeta-lactam antibiotics
Spread of MRSA S. aureus in
the Community
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Adam H et al.
 Fatal case of post-influenza, community-associated MRSA
pneumonia in an Ontario teenager with subsequent familial
transmission. Can Commun Dis Rep. 2007 Feb 15;33(4):45-8.
van der Flier M et al.
 Fatal pneumonia in an adolescent due to community-acquired
methicillin-resistant Staphylococcus aureus positive for PantonValentine-leukocidin. Ned Tijdschr Geneeskd. 2003 May
31;147(22):1076-9.
Francis JS et al.
 Severe community-onset pneumonia in healthy adults caused by
methicillin-resistant Staphylococcus aureus carrying the PantonValentine leukocidin genes. Clin Infect Dis. 2005 Jan
1;40(1):100-7.
First Reports of Vancomycin
Intermediate and Resistant MRSA
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Hososaka Y et al. Nosocomial infection of betalactam antibiotic-induced vancomycin-resistant
Staphylococcus aureus (BIVR). J Infect
Chemother. 2006 Aug;12(4):181-4.
Resistance mechanism described for VISA
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Sequential point mutations in key global regulatory
genes associated predominately with cell wall
thickening and restricted vancomycin access to its site
of activity in the division septum (Howden et al., 2010)
Enterococcus faecalis
Enterococcus faecalis
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Gram positive cocci
In pairs and chains
Catalase negative
Non-hemolytic
Facultative anaerobic
Bile esculin positive
Normal flora in intestine
Often highly resistant to
antibiotics
Nosocomial infections
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Urinary tract infection
Wound infections
Endocarditis
Vancomycin resistance
observed
E. faecalis Virulence factors
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Surface protein
(ESP)
Adherence
Antiphagocytic
Variations
observed
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Escapes immune
response
Vancomycin Resistant E.
faecalis (VRE)
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Vancomycin binds to an
essential substrate at a late
stage of the biosynthetic
pathway of peptidoglycan
(Reynolds 1989)
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D-Ala-D-Ala in peptidoglycan
precursor
Vancomycin resistance is
caused by the production of
depsipeptide D-Ala-D-Lac,
which replaces D-Ala-D-Ala
(Gin and Zhanel, 1996)
Results in a more-than1,000-fold lowering of the
affinity of vancomycin for its
target (Reynolds and
Courvalin, 2005).
New Threats by E. faecalis
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Spread of VRE in the hospital setting
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Zubaidah et al. Hospital-acquired vancomycin-resistant enterococci: now
appearing in Kuala Lumpur Hospital. Med J Malaysia. 2006 Oct;61(4):487-9.
Comert et al. First isolation of vancomycin-resistant enteroccoci and spread of a
single clone in a university hospital in northwestern Turkey. Eur J Clin Microbiol
Infect Dis. 2007 Jan;26(1):57-61.
Vonberg et al. [Prevention and control of the spread of vancomycin-resistant
enterococci : Results of a workshop held by the German Society for Hygiene
and Microbiology.] Anaesthesist. 2007 Feb;56(2):151-7.
Huang et al. Risk of acquiring antibiotic-resistant bacteria from prior room
occupants. Arch Intern Med. 2006 Oct 9;166(18):1945-51.
Bar et al. Systemic inflammatory response syndrome in adult patients with
nosocomial bloodstream infections due to enterococci. BMC Infect Dis. 2006
Sep 26;6:145.
Furtado et al. Risk factors for vancomycin-resistant Enterococcus faecalis
bacteremia in hospitalized patients: an analysis of two case-control studies. Am
J Infect Control. 2006 Sep;34(7):447-51.
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Acinetobacter baumannii
Acinetobacter baumannii
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Pleomorphic gramnegative or gramvariable coccoid rods
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Short, fat rods, resemble
cocci and diplococci
Can be confused with
Neisseria spec.
Non-motile
Non-fermenter
Biofilm production
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contributes to antibiotic
resistance (Dallo SF,
Weitao T 2010)
http://www.cyberspaceorbit.com/AB_picenh2.jpg
Threats by Acinetobacter
baumannii
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Increasingly isolated in
hospital settings (war
and natural disaster
victims)
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Wound infections
Septicemia
Increasingly resistant
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Outbreaks of infections with
carbapenem resistant A. baumannii
production of
carbapenemases
(metallobetalactamases,
CRAB)
Worldwide spreading of
resistant strains
http://www.bioquell.com/Pictures/Acinetobacter%20map.jpg
World Wide Spread of
Acinetobacter
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Infection. 2010 Apr 1. Successful treatment of three children with postneurosurgical multidrug-resistant Acinetobacter baumannii meningitis.
Ozdemir H et al (Turkey)
J Infect Dev Ctries. 2010 Mar 29;4(3):164-7. In vitro antimicrobials activity
against endemic Acinetobacter baumannii multiresistant clones. Rodriguez
CH et al. (Buonos Aires)
Scand J Infect Dis. 2010 Mar 26. The microbiological characteristics of
patients with crush syndrome after the Wenchuan earthquake. Wang T et
al. (China)
J Hosp Infect. 2010 Mar 18. Emergence of an extreme-drug-resistant (XDR)
Acinetobacter baumannii carrying bla(OXA-23) in a patient with acute
necrohaemorrhagic pancreatitis. Grosso F et al. (Portugal)
J Med Assoc Thai. 2009 Dec;92 Suppl 7:S173-80. Clonal spread of
carbapenem resistant Acinetobacter baumannii in the patients and their
environment at BMA Medical College and Vajira Hospital. Phumisantiphong
U et al. (Thailand)
Increasing Nosocomial Infections
with Drug Resistant A. baumannii
Search Terms*
1988 1998 2008
2009
AB & infection
1
27
181
172
AB & nosocomial
0
16
72
78
AB & resistant
1
23
186
226
Take Home Message
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Increasing antibiotic resistance is a major threat to
public health.
Of particular concern are MRSA, VISA and VRSA,
VRE, and A. baumannii.
Antibiotic resistance is mediated by expression of
enzymes inactivating the antibiotic, alteration of the
drug target, prevention of drug entry into the cell,
and drug efflux pumps.
Inappropriate drug use is the major cause.
Resources
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Microbiology: An Introduction, by Tortora, Funke and Case; Pearson
Prentice Hall; 9th ed, 2007, Chapter 11; pp 436t, 591, 593, 606.
633b
Current Issues: pp 62 -71
Alekshun MN and Levy SB (2007) Molecular mechanisms of
antibacterial multidrug resistance. Cell 128(6):1037-50
Emerging Infectious Diseases: pp28 – 30
http://www.microbelibrary.org
CDC fact sheets
http://dermatlas.med.jhmi.edu/derm/
Hiramatsu K, Cui L, Kuroda M, Ito T. The emergence and evolution
of methicillin-resistant Staphylococcus aureus. Trends Microbiol.
2001 Oct;9(10):486-93.
Embedded in slides