Transcript Multiresistant outbreaks of Acinetobacter: how long before

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’ 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
‘sensitive’ isolates
A. 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
isolates at the local level
RAPD-PCR with primers M13 and DAF4
M13
M
DAF4
M
M
M
600 bp
300 bp
100 bp
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 ApaI is still the typing standard
used by most central reference laboratories
(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
sequencer provides good results for
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
~85%
Pearson correlation [0.0%-97.8%]
100
95
90
85
80
75
70
65
AFLP
AFLP
Grouping of 31 A. baumannii isolates
.
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 .
(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 typing) – produces clusters equivalent to
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 carbapenemresistant 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 carbapenemresistant 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