MICR 420

Emerging and Re-EmergingInfectious Diseases

Lecture 3:Challenge of Antibiotic Resistance

S. aureus (MRSA), E. faecalis (VRE), A. baumannii

Dr. Nancy McQueen & Dr. Edith Porter

Overview

Challenge of antibiotic resistance S. aureus

Methicillin resistance

E. faecalis Vancomycin resistance

A. baumannii MDR

Morphology Virulence factors Diseases Diagnosis

Culture, biochemicals PCR: toxin production Antibiogram

Penicillin binding protein 2a latex agglutination assay

Challenge of Antibiotic Resistance

Antibiotic resistance Resistance genes Transfer of resistance genes

Antibiotic Resistance Enzymes that degrade or modify the antibiotic

-lactamases modifying enzymes like acetyltransferases

Alteration of the antibiotic target Penicillin binding proteins

Inhibition of antibiotic entry Porin mutations LPS modifications

Efflux pumps Tetracycline efflux Multidrug resistance efflux

Resistant bacteria are NOT more virulent but they are harder to destroy!

Antibiotic

Central cavity with promiscuous binding site

Pore

Funnel connection to an outer membrane channel

Cell membrane

Resistance Genes

Plasmid and chromosomal encoded E.g., Plasmid: beta-lactams, aminoglycosides E.g.,Chromosomal: fluoroquinolones, tetracyclines

Spontaneous mutations At low doses of antibiotics

Acquisition of resistance genes

Acquisition of Resistance Genes

Inherited Horizontally

Conjugation Cell to cell contact

Transduction Virus-mediated

Transformation Uptake of free DNA

Lessefficient

Occurs also between distantly related genera such as between

gram-positive and gram-negative bacteria!

Major Causes for Increased Antibiotic Resistance

Improper use in medicine Incomplete therapy Inappropriate use for viral

infections Unnecessary use of broad-

spectrum antibiotics Low dose use in animal

husbandry to promote animal growth

Use in agriculture leads to unwanted distribution of aerosols

After Stuart B. Levy in “The Challenge of Antibiotic Resistance”

Distribution of Antibiotics Manufactured in the U.S.(Currently about 50 Million Pounds per Year)

HumanAnimalAgriculture

Detection of Antibiotic Resistance

Conventional antibiotic disk diffusion (Kirby Bauer) and determination of minimal inhibitory concentrations

DNA microarrays for detection of plasmid-mediated antimicrobial resistance (and virulence factor) genes

Active Learning Exercise

What role do transposons play in antibiotic resistance?

Staphylococcus aureus

Staphylococcus aureus Gram positive cocci In clusters Facultative anaerobic Catalase positive Mannitol positive Coagulase positive Often beta-hemolytic Normal flora (low numbers)

Nostrils Pharynx Perineal

MSA Agar Plates

S. aureus

S. epidermidis

S. aureus: Virulence Factors

Coagulase Various enzymes Protein A

Binds antibodies via Fc receptor Various toxins

Enterotoxins Exfoliative toxin (epidermiolysin) Toxic shock syndrome toxin

Wound InfectionsPurulent infections

Toxin specific diseases

S. aureus Food Intoxication

Uptake of preformed enterotoxin Nausea, vomiting, diarrhea Self limited Short duration

Staphylococcal Exfoliative Toxin: SSSS

Staphylococcal scalded skin syndrome Cleaves cell adhesion molecules of

keratinocytes (desmoglein)

(http://dermatlas.med.jhmi.edu/derm/)

Toxic Shock Syndrome

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

Superantigen mediated Uncontrolled immune response to

staphylococcal and streptococcal toxins Early signs

Fever Dizziness Confusion Flat red rash over large areas of the body

Shock and multi-organ failure

Superantigens

Activate numerous T Helper cells simultaneously

T Helper cells release numerous proinflammatory cytokines

Bacterial Superantigens

Staphylococcal superantigens S. aureus Over 20 described TSST, exfoliatins, enterotoxins

Streptococcal S. pyogenes (Group A beta-hemolysing

streptococci) Exotoxin A and C, and others

New Threats by S. aureus Nosocomial infections (healthcare-associated

infections, HAIs) 15% of all isolates; 8% of all HAIs

Spread of MRSA in the community Vancomycin resistant MRSA

Methicillin Resistance 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 non-beta-lactam antibiotics

Spread of MRSA S. aureus in the Community 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 Panton-Valentine-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 Panton-Valentine leukocidin genes. Clin Infect Dis. 2005 Jan 1;40(1):100-7.

First Reports of Vancomycin Intermediate and Resistant MRSA

Hososaka Y et al. Nosocomial infection of beta-lactam antibiotic-induced vancomycin-resistant Staphylococcus aureus (BIVR). J Infect Chemother. 2006 Aug;12(4):181-4.

Resistance mechanism described for VISA 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 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

Urinary tract infection Wound infections Endocarditis

Vancomycin resistance observed

E. faecalis Virulence factors

Surface protein (ESP)

Adherence Antiphagocytic Variations

observed Escapes immune

response

Vancomycin Resistant E. faecalis (VRE)

Vancomycin binds to an essential substrate at a late stage of the biosynthetic pathway of peptidoglycan (Reynolds 1989) 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-than-1,000-fold lowering of the affinity of vancomycin for its target (Reynolds and Courvalin, 2005).

New Threats by E. faecalis Spread of VRE in the hospital setting 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.

Acinetobacter baumannii

Acinetobacter baumannii Pleomorphic gram-

negative or gram-variable coccoid rods Short, fat rods, resemble

cocci and diplococci Can be confused with

Neisseria spec.

Non-motile Non-fermenter Biofilm production

contributes to antibiotic resistance (Dallo SF, Weitao T 2010)

http://www.cyberspaceorbit.com/AB_picenh2.jpg

Threats by Acinetobacter baumannii

Increasingly isolated in hospital settings (war and natural disaster victims) Wound infections Septicemia

Increasingly resistant production of

carbapenemases (metallobetalactamases, CRAB)

Worldwide spreading of resistant strains

http://www.bioquell.com/Pictures/Acinetobacter%20map.jpg

Outbreaks of infections withcarbapenem resistant A. baumannii

World Wide Spread of Acinetobacter Infection. 2010 Apr 1. Successful treatment of three children with post-

neurosurgical 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

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 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