Cystic fibrosis

John R W GovanUniversity of Edinburgh Medical School

• What is cystic fibrosis?

• Significance of respiratory infections

• Problem pathogens and new technologies

Cystic Fibrosis• Cystic Fibrosis is the most common, life-threatening,

recessively inherited disease of Caucasian populations • Carrier rate of 1 in 25 and incidence rate of I in 2500

live births

• UK ~ 7500 babies, children and young adults.

• Cause. Mutations in gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) gene which encodes an epithelial chloride channel.

• Survival. Progressive lung disease secondary to respiratory infection is the main cause of morbidity and mortality.

CFTR, defective mucocilary clearance and salt-sensitive antimicrobial peptides (defensins)

Bacterial Pathogens in CF

• Limited spectrum

• Age related sequence– Haemophilus influenzae– Staphylococcus aureus– Pseudomonas aeruginosa– Burkholderia cepacia complex– S. maltophilia/A. xylosoxidans

‘Burkholderia cepacia’ – the pathogen

• Swamp foot in US marines

• Nosocomial infections due to contaminated fluids. ICUs

• Chronic granulomatous disease.

Inherited loss of neutrophil oxidative killing leading to fatal lung infection

• ‘Cepacia syndrome’ in cystic fibrosis – fatal unexpected pneumonia with bacteraemia in 20-30% of infected patients

Edinburgh – 1986 Glass & Govan J Infect 1986; 13:157-158.

Fatal cepacia syndrome in 9-year-old CF female.

No transfer to CF sibling.

Genomovar III (B. cenocepacia)

J415, cblA and bcesm –ve

Transmission of B. cepacia Govan et al. Lancet 1993

• Spread within and between CF clinics includes social contacts.

• Outcome fatal but unpredictable

• Leper-like infection control -- segregation and anxiety!

• Virtually untreatable

‘Burkholderia cepacia’ how resistant?

• Biodegradation of synthetic herbicides and petroleum oxidants.

• Panresistance to antibiotics. Can utilise penicillin as nutrient!

• Resistance to natural antimicrobial peptides – (defensins) of amoebae, insects, animals and humans – explains CGD.

B. cepacia as an animal pathogen Berriatua et al 2000. J Clin Microbiol 2001;39: 990-4

• Untreatable outbreak of ovine mastitis

• No environmental source identified

• Genomovar III responsible

Bacterial hybrids or new taxons? Simpson et al. J Antimicrob Chemother1994; 34: 353-61

‘Burkholderia cepacia complex ’

• Evolving taxonomy– At least 10 distinct genomovars recognised by

polyphasic taxonomy and RecA PCR

• All genomovars isolated from CF patients

• However, gvr II (B. multivorans) and III (B. cenocepacia) account for 90% of isolates and most transmission – includes the ET12 lineage

‘B. cepacia’ AMMDParke & Gurion-Sherman J Phyt 2002

The biopesticide issue -update

• ‘There is no evidence that CF patients acquire B. cepacia from the environment – CF isolates are different’

- biopesticide developer 1999

• Not true – even clonal

- Govan, Vandamme & Balandreau ASM News 2000;66:124

• New use rule. Fed Reg 2002

Antibiotics in trouble!

Evolution of bacterial resistance and pathogenesis – a two way process

• Common perception:

Bacterial pathogens accumulate resistance to antibiotics -- rendering them virtually untreatable . MRSA and vancomycin-resistant Enterococcus faecium

• What’s also happening:

Inherently resistant environmental bacteria acquire opportunistic virulence for plant, human and animal hosts. Burkholderia cepacia complex

Bacteriology IDGBacteriology IDG

Collaborations

Richard TitballPetra Oyson

Ty Pitt

Eshwar Mahenthiralingam

John Govan

B. pseudomalleiB. cepacia

C.A. Hart

Peter Vandamme

Mahidol UniversitySirirurg Songsivillai

B. cepacia - general features J2315 - Edinburgh ET12 isolate

0

0

914 bp

80.9 %

82

62.8 %

92,661 bp

plasmid

2

1 x

16S-23S-5S

982 bp

86.6 %

773

66.9 %

875,977 bp

Chr. 3

73566tRNA

66.9 %67.3 %66.7 %G+C content

6 x

16S-23S-5S

971 bp

87.0 %

7,226

8,055,782 bp

Total

1x

16S-23S-5S

4 x

16S-23S-5S

rRNA

995 bp950 bpAv. gene length

87.8 %86.6 %Coding density

2,8403,531Genes

3,217,062 bp3,870,082 bpSize

Chr. 2Chr. 1

B. cepacia – chromosome 2 – LHF analysis

drug efflux,catabolism,regulators

phospholipases

fatty-acid metabolism + transport

plasmid conjugation

system

chemotaxis,helicase,

drug efflux?

phage,haemolysin

polysaccharide biosynthesis

phage

haemolysin-relatedprotein

cell-surfaceprotein

rRNA

B. cepacia and the neutrophil:dose dependent and synergistic inflamation

• Lipopeptide toxin (haemolysin) induces apoptosis at low concentrations and degranulation at high concentrations.

• B. cepacia LPS induces potent TNF and IL-8 response and primes neutrophil to a damaging response to other microbes and ‘procedures’.

Looking ahead

• Are all B. cepacia complex potentially virulent and transmissible? Transplantation, segregation and biopesticides issues

• The promise of genomics and proteomics

• ‘B. cepacia’ identification at local level. False positives 10%; False negatives 30%.

Identification of B. cepacia complex

• Variable colonial morphology

• Selective media not selective enough: Mast Cepacia medium or BCSA (Henry et al 1997).

• Accuracy of commercial identification kits?

ID of B. cepacia complex by API20NE, Vitek NFC/GNI and BBL Crystal

• Accuracy varies from 64-92 %. API20NE most accurate.

• None of the systems identified genomovar VI

• Only API20NE identified the Glasgow epidemic strain (gvr II, B. multivorans) and the epidemic ET12 lineage (gvr III, B. cenocepacia).

Some people’s idea of microbiologists?

Every CF clinic needs a friendly microbiologist!!Every CF clinic needs a friendly microbiologist!!

IBCWG (http://go.to/cepacia/)

“…as a forum for clinicians and scientists interested in advancing knowledge of B. cepacia

infection and colonisation in persons with CF through the collegial exchange of information and

promotion of coordinated approaches to

research…”

“B. cepacia”: other bacteria

• Many other bacteria are (mis)identified as B. cepacia• Known species:

– P. aeruginosa– S. maltophilia– R. pickettii– A. xylosoxidans– B. hinzii– Brevundimonas spp.– Chryseobacterium spp.– Enterobacteriaceae