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)
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)
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
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