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

2. 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)

4. 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

5. Three major overlapping populations
Hospitals and hospitalised patients
‘multi-resistant’ isolates
A. baumannii, sp.3, sp.13TU
(particularly adapted to this environment?)
Skin (humans and animals) / foodstuffs
‘sensitive’ isolates
A. johnsonii, A. lwoffii, A. radioresistens
Soil / environment / wastewaters
A. calcoaceticus, A. johnsonii
Natural habitats of other species still poorly defined

6. 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

7. 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.

8. Where is the ‘reservoir’ for nosocomial infection with Acinetobacter baumannii ?
Patients admitted from the community?
Patients admitted from other hospitals?
Within the hospital itself?

9. 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

10. 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

11. 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

12. How does Acinetobacter compare with MRSA in terms of epidemiology?
in individual hospitals?
on a global scale?

13. Typing methods for Acinetobacter
RAPD is useful for same-day typing of isolates at the local level

14. RAPD-PCR with primers M13 and DAF4
M DAF4 M
LUH
LUH
LUH 7020
LUH 6609
LUH 6571
LUH 6547
LUH 6488
LUH 6940
LUH 6946
LUH 6947
LUH 6948
600 bp
300 bp
100 bp
Example of RAPD-PCR results in a local setting to trace epidemic strains. The blue underlined strains (profile 1 cq 5) are similar ( as well as the pairs of other underlined strains). Thus, RAPD-PCR works well in this setting.
J Clin Microbiol 35:

15. 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)

16. Examples of PFGE gels using ApaI
Here we should say that gel top right is with melted DNA
Also, the protocol of the gel bottom left was different
Furthermore, the lower part of the gel (<97 kb) cannot be used, but Herre will also talk about that.

17. 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)

18. 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

19. Grouping of 31 A. baumannii isolates
~85%
Grouping of 31 A. baumannii isolates
strains from study comparison of outbreak and non outbreak strains in NW Europe
Dots mark outbreak strains. Red = 'clone I', Green = 'clone II'.
It is currently our experience with larger numbers of strains that certain groups of strains cluster at 80-85%.
(L. Dijkshoorn, ENEMTI Study, 2002)

20. 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 typing) – produces clusters equivalent to those obtained using PFGE

21. 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)

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

23. Developing epidemiology of A. baumannii in the UK
A survey in 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: )

24. Between 2003 and 2006, two carbapenem-resistant A
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: )
More recently, a further lineage (the Northwest strain) has become prevalent in several hospitals in the northern/midlands of the UK

25. 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

26. 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)

27. 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

28. 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?

29. Acinetobacter – the Gram-negative MRSA
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

30. 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

31. 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)

32. 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

33. What treatment options remain
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)

34. 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