Acinetobacter baumannii - NHS Wales 07 David...Acinetobacter baumannii ‘Facts and Fiction’ Dr...

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9th Tripartite Meeting of The Celtic Microbiology Associations(Hosted by the Welsh Microbiological Association)

Cardiff 5-7th June 2009

Acinetobacter baumannii‘Facts and Fiction’

Dr David WarehamSenior Clinical Lecturer / Honorary Consultant

Queen Mary University / Barts and The London NHS Trust



Microbiology in East London

• Royal London Hospital- 1,100 bedded teaching hospital- Regional trauma centre- HEMS

• 18 bedded ITU- 18th century building

• 70 % of admissions from the trauma service- Long duration of stay

Rise of Antimicrobial Resistance

Gram positives Gram negatives

Penicillins

MacrolidesCephalosporins

Quinolones

Tetracyclines

Carbapenems

Polymyxins

Glycopeptides

1950

1960

1970

1980

1990

2000

Aminoglycosides

LinezolidStreptogramins

DaptomycinDalbavancinRetapamulin

Gram +ve Cephalosporins

????

Tigecycline

Hospital Acquired Gram-Negative Bloodstream infection at BLT (2007)

Klebsiella

Pseudomonas

Enterobacter

Morganella

E. coli

ProteusOther

Acinetobacter

n = 112

Rise in bloodstream infections due to MDR Acinetobacter in Critical Care at BLT 1998 - 2009

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

120.0%

1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Year

% susceptible only to polymyxin

Acinetobacter baumannii

• Gram-negative coccobacillus• Member of family Moraxellaciae

– Aerobic, non-fermentative, catalase +ve, oxidase -ve• Natural habitat

– Environmental ? Soil ? Plants?• Opportunistic pathogen• Very little carriage by healthy individuals

– A. baumannii found on human skin in only 0.5%• Infections

– Ventilator associated pneumoniae, – Bacteraemia, – Burn wound infection– Prosthetic device related infection

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Taxonomy of Acinetobacter

1Bouvet and Grimont

• 1990’s – 2000’s additional species described– A. parvus, A. schindleri, A. ursingii from humans,

many others from the environment– 31 species described to date 17 named

• 1980’s Acinetobacter split into 12 DNA groups1

A. baumannii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. lwoffii

• A. calcoaceticus – A. baumannii complex- Closely related and phenotypically indistinguishable- A. baumannii, Acinetobacter gemospecies 3, Acinetobacter 13TU- A. calcoaceticus - environmental

UK / European Epidemiology

• 2000 – A prevalent, multi-resistant clone identified in hospitals in South East England (SE Clone)– Majority in London– Intensive Care units

• 2003 – 2005 – expanded to 48 hospitals– OXA clones 1 / 2

• 3 European clones defined by PFGE

• 2009 - Carbapenem resistant A. baumannii Gemomic species 3 inIreland1

1Boo et al, 2009

RAPD and multiplex PCR (OXA) typing

Global Dissemination of A. baumannii 1

1Peleg et al

Countries reporting outbreaks pre 2006 red : Post 2006 yellow

Sequence based typing Schemes

1, 2 Turton, 2009

Multi-locus sequence typing7 housekeeping genes

gltA, gyrB, gdhB, recA, cpn60, gpi, rpoD

Multiplex PCR typing1

csu, OmpA and OXA-23 allellesCorresponds with major EU PFGE clones

Variable number tandem repeats2

Discrimination within PFGE types

Antimicrobial Resistance

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• Target modification• Drug destruction• Drug modification• Active drug removal• Impermeability

• Intrinsic• Acquired

– Resistance islands– AbaR1 – 45 resistance genes

of foreign origin– Class 1 integrons, transposons

• Plasmids

Antimicrobial Resistance β-lactam Resistance

• Inherent Chromosomal AmpC (ADCs)– Not inducible – regulated by ISAba1

• Class A Extended-spectrum β-lactamases– VEB-1 – clonal dissemination in France– PER 1/2 – plasmid / chromosomal – ISPa12 regulated– TEM-92/116, SHV-12, CTX-M2/43

• Carbapenemases1

– Class A not yet detected– Class B metallo-β-lactamases – IMP, VIM, SIM within class 1 integrons

• OXA- enzymes– OXA-51-like natural oxacillinase – regulated by ISAba1– OXA-23-like 2, OXA-24-like, OXA-58-like– Resistance enhanced by AdeABC efflux

• Outer membrane protein loss– CarO porin

1 Walsh, Curr Opin Infect Dis, 20082 Paton, 1993

Efflux Pumps

• Major facilitator Superfamily (MFS)– TetA - tetracycline– TetB – tetracyline, minocycline– CmlA – chloramphenicol

• Resistance Nodulation Division (RND) family– AdeABC – β-lactams, aminoglycosides,

macrolides, Chloramphenicol– AdeIJK – Above + fusidic acid, trimethoprim

• Multi-Drug and Toxic Drug Exclusion (MATE) family– AdeM – quinolones, gentamicin, triclosan

AdeC

AdeB

AdeA

Aminoglycosides

• Inactivation of aminoglycosides by– Phosphorylation – APH enzymes– Acetylation – AAC enzymes– Adenylation – ANT enyzmes

• Pan-resistance to aminoglycosides– 16S rDNA methyltransferases– ArmA – plasmid mediated / transposon

• Efflux – AbeM (MATE family pump)

Quinolone Resistance

• DNA gyrase mutations– GyrA, GyrB, ParC, ParE – high level

resistance• Efflux

– Most quinolones are substrates for AdeABC and AdeM

– Ciprofloxacinases– aac(6’)-Ib-cr - variant

Mechanisms of MDR in A. baumannii OXA-23 clone 1

• β-lactams– TEM-1 pencillinase– Overexpression of chromosomal AmpC– OXA-51 and OXA-23 carbapenemase

• Aminoglycosides– aac(3) -Ia, aac(3)-IIa, ant(2’)-Ia, strB, aac(6’)-Ib-cr– armA – 16S rRNA methytransferase

• Quinolones– QRDR GyrA/B, ParC/E mutations, AdeABC efflux

• Trim / Sul– sul1

• Tetracyclines– tetB– adeABC – multidrug efflux pump

• Polymyxin ?– Difficult to detect– Heteroresistance described

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Pathogenesis

Clinical Manifestations / Associations

• Hospital Acquired Pneumonia– Commonest site of isolation is respiratory tract colonisation v infection ?– US study – 5-10% of all ICU HAP / VAP

• Community Acquired Pneumonia– Fulminant pneumonia with bacteraemia and septic shock– Alcoholics in Tropical Australia and SE Asia– Really A. baumannii – identification problems ?

• Bloodstream Infection– Usually ICU associated, mean time of onset 26 days post admission– 2ndry to lines, pneumonia, UTI or wounds

• Wound Infections– ICU acquired SSTI and burn wound infections– Commonest isolate from traumatic military injuries – Iraq / Afghanistan

• Neurosurgical meningitis– EVD associated ventriculitis

Evasion of Host Defences

Stages in Microbial Pathogenesis

Attachment and Colonisation

Dissemination and Systemic Disease

Exit the Host

Complicated Interaction between host and organism derived factors

P. aeruginosa

P. aeruginosa

Pili

Mucoid PolysaccharideCapsule

Alginate

Quorum sensing▲Pyocyanin▼Phospolipase C◄Alkaline protease■ Exotoxin A● LPS▬Elastase

TTSSExoS, ExoT, ExoU, ExoY

Target host cell

Flagella

Toxins and proteases

Pathogenesis of A. baumannii infection

• Siderophores– Expression highly strain dependent– Antibodies in sera of patients with A. baumannii

bacteraemia

• Biofilm formation– Readily adheres to inanimate objects– Pilus biogenesis involving csu operon

Immunoreactivity of human serum v A. baumannii siderophores11Smith 1991

• Comparative genomics A. baumannii v A. baylyiType IV secretion system, pilus biogenesis, Fe uptake and metabolism

Interactions with Respiratory Epithelial Cells

• Adheres to but does not invade epithelial cells

• EU clone II > EU clone I• Elicits a cytopathic effect • Kills cells by apoptosis

• Secretion of Omp38 implicated• Stimulates a strong IL-8 response• Activation of TLR-4 pathway

• Microarray data

0

1000

2000

3000

4000

5000

6000

7000

8000

C ontrol OXA 23-1MOI 1

OXA-23-1MOI 10

Type s trainMOI 1

Type s trainMOI 10

A ci n eto b a cter A B34

0100020003000400050006000700080009000

10000

1 Hours 2 Hours 4 Hours 6 Hours 20 Hours

T im e

IL-8

secr

etio

n

MOI 10 :1

MOI 1 :1

A ci n eto b a cter A B34

0100020003000400050006000700080009000

10000

1 Hours 2 Hours 4 Hours 6 Hours 20 Hours

T im e

IL-8

secr

etio

n

MOI 10 :1

MOI 1 :10

5000

10000

15000

20000

25000

30000

35000

0 4 8 12 16 20 24Viability of A549 cells following bacterial co-culture. Numbers of A549 cells were determined by a direct cell count following 4, 8 and 24 hrs co-culture with () media alone; A.baumannii type strain at MOI of 1 () and 10 () and the oxa23 strain MOI of 1 (X) and 10 ().

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A. baumannii Lipopolysaccharide

• A. baumannii LPS is a potent activator of TLR-4– mouse pneumonia model

• Stimulates both TLR-4 AND TLR-2 on human cells1

• Antibodies to A. baumannii are widepread in healthy individuals2

• 1Erridge, 2007• 2 Morgan and Poxton, 2009

Metabolic Diversity of Acinetobacter

• Acinetobacter loves sugar !

• Acinetobacter loves alcohol !

• Acinetobacter loves fat !

Acinetobacter and Sugar ?

• Acinetobacter burn wound infection associated with impaired glucose tolerance1

– 9.8 X increase in impaired glucose tolerance p< 0.0001 !– Independent of diabetes status– Independent of burn severity

• Why ?– Acinetobacter metalloprotease– Cleaves insulin, IGF-1 and glucagon2

• How ?– No insulin / hormone levels measured– Enzyme is periplasmic – not secreted

1 Furniss, J Burn Care 20052 Fricke, 1989

Acinetobacter and Alcohol ?

• Acinetobacter can grow in the presence of small quantities of ethyl alcohol• Exposure to alcohol makes A. baumannii pathogenic to C. elegans

Growth of MDRAB in Ethanol Supplementated Media

0

0.1

0.2

0.3

0.4

0.5

0.6

0 5 10 15 20 25 30

Time (hours)

OD 6

00

MM10%1%0.1%0.01%

Growth of MDRAB in Media Supplemented with Alcohol Based Handrubs

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

MM Only

1% Soft

alind

1% Skin

man

1% Pure

ll

1% Spir

igel

OD

600 OXA 23 clone-1

OXA 23 Clone-2South East Clone

6



1

17

25

33

48

kDa 1 2OXA-23 Clone 1

Alcohols promote secretion of OmpA

Acinetobacter and Fat !

• Lipase production well characterised in non-baumannii species e.g A. venetius• ‘Lack of subcutaneous fat in burn wound grafts’• MDRAB produce lipases

– Which are enhanced by some antibiotics...

0 2 4 6 8 10

Streptomycin

Nalidixic acid

Ampicillin

Tetracycline

Ertapenem

Imipenem

Ant

ibio

tic

Fold induction

Clinical Impact, Treatment and Control

Studies on the Clinical Impact of A. baumannii

• Dozens of studies but little consensus !• Enormous methdological heterogeneity• Prospective v retrospective• Case control v cohort

– What defines a ‘case’ – polymicrobial infections / different sites etc– What defines a ‘control’ – no A, baumannii infection, infection with

something else, colonisation v infection• Appropriate ‘Matching’ for co-morbidities• Problems with speciation• Geographical variation

– Impact of individual clones• Most studies indentify excess hospital / ITU stay• No consensus on attributable mortality !

Does A. baumannii Infection have Attributable Mortality ?

• Yes– Lortholary, 1995, infection / colonisation v not– Garcia-Garmendia, 1999 – infection / colonisation v not– Abbo, 2007 - isolation of MDRAB v not– Grupper, 2007 – A. baumannii bacteraemia v no bacteraemia– Kwon, 2007 – Imipenem S v Imipenem R infection

• No– Blot, 2003 – A. baumannii bacteraemia v no bacteraemia– Garnacho, 2003 – A. baumannii VAP v no A. baumannii infection– Albrecht, 2006 – A. baumannii infection v colonisation in burns– Loh 2006, - A. baumannii in respiratory secretions v not– Sunenshine, 2007 CDC – MDRAB v no infection

Mortality from MDR A. baumannii Bacteraemia at BLT1

Either polymyxin doesn’t work ? OR the organism has no impact on mortality ?1Wareham, 2008

Retrospective cohort study of 399 Acinetobacter spp bacteraemia (MDRAB n=78)

Impact of carbapenem R, polymyxin Rx and ITU care

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Impact of ITU Requirement on Mortality from MDRAB Bacteraemia

A. baumannii bacteraemia is a surrogate marker of mortality in ITU ?

‘Polymyxin E and Polymyxin B - Colistin’

• Colistin susceptibility testing- Disc testing is unreliable- Bactericidal in-vitro but ‘heteroresistance’ with regrowth described in time kill

assays

• Polymyxin formulations- Colistimethate sodium a prodrug metabolised to colistin- Parenteral and nebulised formulations

• Colistin sulphate- Oral and topical use

• Uncertain pharmacokinetics- Commercial preparations contain different amounts of active drug - Never subjected to fomal drug development programme

• If colistin were a new drug in 2009 would it receive a licence ?

• Concerns over nephrotoxicity and neurotoxicity not seen with modern use

Unorthodox Treatment Regimens v MDR strains

• In-vitro studies of colistin containing combinations– Polymyxin B + imipenem + rifampicin – Synergy– Colistin + rifampicin – Synergy– Colistin + minocycline – Synergy– Colistin + ceftazidime – Synergy– Polymyxin B + meropenem / rifampicin azithromycin – Synergy

• In-vivo studies of colistin containing combinations– Colistin + rifampacin – effective in mouse pneumonia and rat model

• Case reports– Colistin + rifampicin – ‘favourable response’

• Sulbactam– Intrinsic activity via PBP 2 inhibition– Option in isolates susceptible in-vitro ?– Some reports of clinical efficacy in non-severe infections

Tigecycline ?

• Brought to market as a Gram-positive agent for skin and soft tissue infections

• Good in-vitro activity v MDRAB• Bacteriostatic• Rapid emergence of resistance due to overexpression of efflux pumps• Case series of infections involving MDRAB treated with tigecycline1

– 68 % clinical response rate– 41 % overall mortality– Recurrent episodes of bacteraemia with development of frank

resistance in 3 cases• Very low serum levels 0.8 mg/L• Sub MIC for most strains of A. baumannii (1.5 mg/L)• BSAC breakpoints S< 1 R >2

1 Gordon, JAC 2009

Infection Control Considerations

• Environmental decontamination paramount– A. baumannii very resitant to dessication

• Antimicrobial policies– Most antibiotics implicated as risk factors for acquisition and persistance– Increased reliance on carbapenems probably implicated in

dissemination of OXA-clones• Isolation

– Successful in some instances, may be impractical• Screening / decontamination

– Patients often have enteric carriage – selective CHROMagar– Enteral polymyxins for SDD ?– Nebulised polymyxins for respiratory colonisation ?– Need RCT data

A. baumannii: Facts and Fiction

• Facts– Successful clones of A. baumannii disseminated worldwide– The organism has a formidable capacity for capturing

antimicrobial resistance genes– It has become exceptionally adapted to the hospital

• Fiction ?– The pathogenesis of A. baumannii infection is well understood– A. baumannii infection has major clinical impact– Polymyxins and tigecyline are effective treatments for sensitive

strains

• Is it the Gram-negative MRSA – or is it the Gram-negative CoNS ?

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0

50

100

150

200

250

300

350

1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

Year

Num

ber

Increase in Pubmed indexed articles on A. baumannii 1997-08 Acknowledgements

• Gordon NC, Wareham DW. Evaluation of CHROMagar Acinetobacter for the Detection of Enteric Carriage of Multi-drug Resistant Acinetobacter baumannii in Critically Ill Patients. Journal of Clinical Microbiology, 2009, In Press

• Wareham DW, Gordon NC, Casals JB, Bean DC. Reduced susceptibility of multi-drug resistant Acinetobacter baumannii to tigecycline in combination with 1-(1-naphthylmethyl)-piperazine is not a pH dependant phenomenon. Journal of Antimicrobial Chemotherapy, 2009 Mar 11. [Epub ahead of print]

• Gordon NC, Wareham DW. A Review of Clinical and Microbiological Outcomes Following Treatment of Infections Involving Multidrug Resistant Acinetobacter baumannii with Tigecycline. Journal of Antimicrobial Chemotherapy, 2009, 63:775-80.

• Bean DC, Wareham DW. Paradoxical Effect of 1-(1-naphthylmethyl)-piperazine on Resistance to Tetracyclines in Multi-drug Resistant Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy, 2009, 63: 349-352

• Wareham DW, Bean DC, Khanna P, Hennessey E, Krahe D, Ely A, Millar M . Bloodstream Infection due to Acinetobacter spp: Epidemiology, Risk Factors and Impact of Antimicrobial Resistance. European Journal of Clinical Microbiology and Infectious Disease 2008 27:607-612

• Edwards J, Patel G, Wareham DW. Action of Commercial Alcohol Handrubs on the Growth and Secretion of Extracellular Proteins from Multidrug Resistant Strains of Acinetobacter baumannii, Journal of Medical Microbiology2007, 56: 1595 - 1599

Microbiology SpRs Consultant ColleaguesNicola Gordon Michael MillarPriya Khanna Daniel KraheJustin Edwards

Queen Mary University FundingDavid Bean Joan Dawkins FoundationEnid Hennessey Hospital Infection Society

Mason Research FundPeel Trust

Acinetobacter baumannii - NHS Wales 07 David...Acinetobacter baumannii ‘Facts and Fiction’ Dr...

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