Problems with multi-resistant Acinetobacter spp. Kevin Towner Dept. of Clinical Microbiology Nottingham University Hospitals NHS Trust.

Problems with multi-resistant Acinetobacter spp.

Kevin Towner

Dept. of Clinical Microbiology

Nottingham University Hospitals NHS Trust

Members of the genus Acinetobacter are now recognised as significant nosocomial pathogens

• Critically-ill patients requiring mechanical ventilation in ICUs

• Wound infections (trauma patients)

• Community-acquired infections (usually in patients with co-morbidities, with most reports from tropical or sub-tropical areas)

Which Acinetobacter?

• Modern molecular-based taxonomy recognises at least 33 different genomic groups

• 18 of these have species names

• A further 28 groups have been identified that contain multiple strains, and there are at least 21 ungrouped single strains

Three major overlapping populations

• Hospitals and hospitalised patients‘multi-resistant’ isolatesA. baumannii, sp.3, sp.13TU(particularly adapted to this environment?)

• Skin (humans and animals) / foodstuffs‘sensitive’ isolatesA. johnsonii, A. lwoffii, A. radioresistens

• Soil / environment / wastewaters‘sensitive’ isolatesA. calcoaceticus, A. johnsonii

Natural habitats of other species still poorly defined

Problems in the hospital setting since 1976

• Persistence

resistant to drying and disinfectants

• Antibiotic resistance

increasing proportion of isolates are multi-resistant

(including carbapenems)

remarkable ability to acquire resistance genes

• Causes outbreaks

A Typical ICU Problem

• 41% (77/189) carriage of a multi-resistant isolate amongst ICU patients

• 71% of these were colonised in the first week on ICU

• Of those colonised in the first week, 26% (vs. 5%) developed clinically significant infection

Corbella et al. (1996) Clinical Infectious Diseases 23:329.

Where is the ‘reservoir’ for nosocomial infection with Acinetobacter baumannii ?

• Patients admitted from the community?

• Patients admitted from other hospitals?

• Within the hospital itself?

Hospital sources

• Hands of staff• Ventilators• Humidifiers• Oxygen analysers• Respirometers• Bronchoscopes• Lotion dispensers• Bed frames• Rubbish bins• Sinks

• Air supply• Jugs• Bowls• Soap• Hand cream• Plastic screens• Bed linen• Service ducts /dust• Bedside charts• Patients

Survival of Acinetobacter in the environment

• Survives in dry particles and dust for up to 10 days (7 days for S. aureus)

• Encapsulated strains survive for >4 months on PVC, ceramics, rubber, steel

• Survives exposure to chlorhexidine, gluconate and phenol-based disinfectants

• Survives exposure to radiation

What’s the problem with Acinetobacter?

• Epidemic spread among patients in hospitals, particularly in ICUs

• Patients disseminate large numbers of organisms into their environment

• Survival on numerous surfaces and inanimate objects

• Resistant to drying and disinfectants• Difficult to eradicate

How does Acinetobacter compare with MRSA in terms of

epidemiology?

• in individual hospitals?

• on a global scale?

Typing methods for Acinetobacter

• RAPD is useful for same-day typing of RAPD is useful for same-day typing of isolates at the local levelisolates at the local level

M13 DAF4

M LUH

702

0

LUH

660

9

LUH

657

1

LUH

654

7

LUH

648

8

LUH

694

0

LUH

694

6

LUH

694

7

LUH

694

8

LUH

694

9-1

LUH

694

9-2

LUH

702

0

LUH

660

9

LUH

657

1

LUH

654

7

LUH

648

8

LUH

694

0

LUH

694

6

LUH

694

7

LUH

694

8

LUH

694

9-1

LUH

694

9-2

M M M

600 bp

300 bp

100 bp

RAPD-PCR with primers M13 and DAF4

J Clin Microbiol 35: 3071-3077

Typing methods for Acinetobacter

• RAPD is useful for same-day typing of isolates at the local level

• PFGE using PFGE using ApaApaI is still the typing standard I is still the typing standard used by most central reference laboratories used by most central reference laboratories (3-5 days)(3-5 days)

Examples of PFGE gels using ApaI

Typing methods for Acinetobacter

• RAPD is useful for same-day typing of isolates at the local level

• PFGE using ApaI is still the typing standard used by most central reference laboratories (3-5 days)

• Automated AFLP analysis on a DNA Automated AFLP analysis on a DNA sequencer provides good results for sequencer provides good results for archiving in databases (5 days)archiving in databases (5 days)

AFLP for typing Acinetobacter

• DNA preparation according to Boom method• Restriction (EcoRI and MseI) and ligation adaptors•Amplification with a labelled primer• Cy-5 labelled fragment separation on an automated sequencer• Analysis by BioNumerics

• 3 widespread ‘European clones’ (lineages) have been identified using AFLP

Pearson correlation [0.0%-97.8%]

AFLP

10

0

95

90

85

80

75

70

65

AFLP

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RUH 3425

RUH 1093

RUH 1752

RUH 3428

RUH 2208

RUH 2207

RUH 2688

RUH 3414

RUH 3424

RUH 3281

RUH 3410

RUH 3423

RUH 0414

RUH 3413

RUH 3429

RUH 1486

RUH 2180

RUH 3422

RUH 0134

RUH 3245

RUH 3240

RUH 1907

RUH 3238

RUH 3247

RUH 3282

RUH 0436

RUH 0875

RUH 3242

RUH 0510

RUH 2037

RUH 3239

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~85% Grouping of 31 A. baumannii isolates

(L. Dijkshoorn, ENEMTI Study, 2002)

Typing methods for Acinetobacter

• RAPD is useful for same-day typing of isolates at the local level

• PFGE using ApaI is still the typing standard used by most central reference laboratories (3-5 days)

• Automated AFLP analysis on a DNA sequencer provides good results for archiving in databases (5 days)

• Sequence-based typing (MLST, PCR-based Sequence-based typing (MLST, PCR-based sequence typing)sequence typing) – produces clusters equivalent to – produces clusters equivalent to those obtained using PFGE those obtained using PFGE

PCR-based sequence typing

• based on sequence analysis of three genes from strains in clusters identified by PFGE

• uses two multiplex PCRs with primers targeting different sequences of the 3 genes

• ompA • csuE• blaOXA-51-like

(Turton et al., 7th International Symposium on the Biology of Acinetobacter, 2006; Clin Microbiol Infect, in press)

PCR-based sequence typing (rapid form of MLST for local use)

Developing epidemiology of A. baumannii in the UK

• A survey in 1999-2001 identified 34 different RAPD genotypes in 46 UK hospitals

• These were shown to belong to 10 different AFLP clusters

• In general, particular strains were characteristic of particular hospitals

(J Clin Microbiol 42: 832-834)

• Between 2003 and 2006, two carbapenem-resistant A. baumannii lineages (SE clone and OXA-23 clone) became prevalent in over 40 hospitals each; susceptible only to colistin and tigecycline (J Clin Microbiol 44: 3623-3627)

• More recently, a further lineage (the Northwest strain) has become prevalent in several hospitals in the northern/midlands of the UK

Are specific carbapenem-resistant clones spreading in European hospitals?

• As part of the ARPAC project, 169 hospitals in 32 countries provided data concerning multiresistant isolates of Acinetobacter spp.

• 130 reported encountering carbapenem-resistant isolates of Acinetobacter, ranging from rare sporadic isolates to an endemic/epidemic situation

• Diverse clusters identified by RAPD, PFGE and PCR-based sequence typing in European hospitals (more than just 2 or 3 clones!)

• As in the UK, multiple isolates from a single hospital generally belonged to the same clone (some exceptions)

• Isolates belonging to sequence group 1 (European ‘clone II’ lineage) found in hospitals in Czech Republic, Germany, Greece, Italy, Poland, Spain, UK (and Argentina and Taiwan!)

• Isolates belonging to sequence group 2 (European ‘clone I’ lineage) found in hospitals in Bulgaria, Croatia, Germany, Greece, The Netherlands, Norway, Poland, Slovenia (and Argentina and Taiwan!)

• Isolates belonging to sequence group 3 (European ‘clone III lineage) found in France, Germany, The Netherlands and Spain

• At least 14 other lineages identified in European hospitals and worldwide

Acinetobacter baumannii has become a major cause of hospital-acquired infections because of its remarkable ability to survive and spread in the hospital environment and to rapidly acquire resistance determinants to a wide range of antibacterial agents

• Are we seeing worldwide spread of multiresistant lineages selected primarily on the basis of the resistance genes that they carry?

• Or is there something special about certain lineages that confers epidemic potential?

Acinetobacter – the Gram-negative MRSA?

How does the epidemiology stack-up?

• it infects the ill• it is multidrug-resistant• it prolongs hospitalisation• it causes outbreaks• it persists• multiple isolates from the same hospital usually belong

to the same clone• particular epidemic lineages are spreading globally

So what’s special about Acinetobacter?

• Perhaps by accident, it has evolved a range of its own special resistance genes (particularly carbapenemases) and the capacity to over-express them in response to antibiotic challenge

• A range of expression mechanisms (provision of promoters on insertion sequences) enables ‘foreign’ resistance genes to be expressed

What’s really special about Acinetobacter?

• It has evolved molecular mechanisms to capture (and express) resistance genes from other organisms

• Sequence analysis of a multiresistant strain, combined with comparative genomics, has revealed an 86-kb ‘resistance island’ which contains a cluster of 45 different resistance genes

PLoS Genet 2(1): e7• (analogous to SCCmec)

• Largest resistance island identified to date• Contains 88 predicted ORFs, with 45 identified

resistance genes (including 19 putative resistance genes not previously described in Acinetobacter) and 22 ORFs encoding transposases or mobility associated proteins

(? 39 ORFs from Pseudomonas spp., 30 from Salmonella spp., 15 from E. coli)

• Includes three class I integrons, two operons associated with heavy metal resistance, and genes encoding efflux pumps

• Analysis of a ‘sensitive’ isolate revealed a 20-kb ‘island’ devoid of resistance markers, but with mobility associated genes

What treatment options remain? (may be useful in individual patients, but resistance

has already appeared)

• Polymyxin (colistin)

• Sulbactam combinations

• Rifampicin/amikacin combinations

• Tigecycline

• Synthetic peptides (in development)

Acinetobacter baumannii has become a major cause of hospital-acquired infections because of its remarkable ability to survive and spread in the hospital environment and to rapidly acquire resistance determinants to a wide range of antibacterial agents

It is the ability to ‘switch’ its genomic structure, combined with variable gene expression, that probably explains the unmatched speed at which A. baumannii can respond to selection pressure from antimicrobial agents, and the main reason why outbreaks caused by this organism are rapidly becoming unmanageable