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Project Title: Comparative genomics and transcriptomics to identify genetic
mechanisms underlying the emergence of carbapenem resistant Acinetobacter
baumannii (CRAb)
Significance:
Acinetobacter baumannii is a difficult to treat pathogen that has an array of interactions
with the human host from asymptomatic colonization and carriage in the intestinal tract,
respiratory tract and skin to invasive infection and bacteremia, which can result in death. A.
baumannii has emerged in last two decades as an increasingly prevalent nosocomial pathogen,
and more recently as a pathogen that can rapidly develop antibiotic resistance (14, 24, 26). The
development of multi-drug resistant A. baumannii (MDRAb) and the rapid transmission and
subsequent infection of patients has become a major concern in hospitals and health care
facilities (8). A. baumannii strains have now been identified that are resistant to all known
antimicrobials (26, 37). The one exacerbating factor in addition to the rapid spread of
antimicrobial resistance is the observed high A. baumannii transmission rate in the hospital and
health care setting (23, 27, 35). These factors have prompted research into the identification of
antibiotic resistance and subsequent treatment of nosocomial infections (recently reviewed in
(12)). This proposal will address the following specific gaps in the A. baumannii knowledge
base: there is a lack of knowledge of A. baumannii genomic diversity at the level of the health
care institution, as well as, at the level of the individual patient. This proposal will address both.
Additionally, there has only been a single global transcriptional study completed on this
important pathogen. This proposal will address the transcriptional response to antibiotics by A.
baumannii, an organism that develops resistance rapidly, in an effort to understand the
transcriptional mechanism(s) involved in the development of resistance.
Antimicrobial resistance of A. baumannii has been attributed to a number of mechanisms,
such as enzymatic inactivation of β-lactamases, aminoglycosides, quinolones, tetracyclines, as
well as antibiotic efflux by outer membrane porins (8, 26). Often the resistance genes are
associated with mobile elements such as genomic islands, insertion sequence (IS) elements, or
encoded on plasmids (8, 26). One such A. baumannii genomic island identified has been
designated the “resistance island (RI)”, and has been demonstrated to contain as many as 45
genes associated with antimicrobial resistance (11). This RI is not present in all multi-drug
resistant A. baumannii (MDRAb) isolates; and neither is every isolate that contains the RI
resistant to multiple antibiotics (11), suggesting that some of the antibiotic resistant phenotype
may be linked to transcriptional and regulatory differences. Previously identified A. baumannii
isolates exhibited sensitivity to carbapenems (13-15); however, with the increased use of
carbapenems for treatment of A. baumannii infections, there has been an observed
increase in the prevalence carbapenem resistance, which is now often used as a marker
of MDRAb (13-15, 24, 29). Additionally, a recent study demonstrated that A. baumannii exhibit
increased tolerance of antibiotics in the presence of monovalent cations such as NaCl, which
was associated with the increased expression of a number of genes including many drug efflux
pumps (16). This proposal will examine the transcriptomic alteration of A. baumannii in
response to various stimuli using unbiased global methods. These studies will provide novel
insights into the transcription of A. baumannii.
A. baumannii is a relatively recently identified pathogen; however new molecular
approaches for identification and characterization are being rapidly adopted. Multi-locus
sequence typing (MLST) of A. baumannii and closely-related Acinetobacter spp. demonstrated
that A. baumannii isolates are a distinct subgroup from other closely related species (7). A.
baumannii, and A. calcoaceticus (7), along with two relatively undefined genomic groups,
Acinetobacter genomic species 13TU and Acinetobacter genomic species 3, are termed the A.
baumannii-A. calcoaceticus complex as they are difficult to distinguish phenotypically (24, 26).
Within this complex there are multiple clonal groups that have been identified and have been
linked to distinct geographic distributions. The majority of isolates that have been examined to
date are from outbreaks or isolated nosocomial infections. While it has been demonstrated that
the majority of A. baumannii outbreaks are attributed to isolates that belong to the European
clones I, II, and III, the antibiotic resistant A. baumannii strains isolated from health care centers
can include other A. baumannii lineages (6, 7, 9, 36, 39). The proposed study herein represents
a hospital acquired isolate collection that will contain representatives of each of these three
major lineages, as well as isolates outside these dominant clades (Figure 1). These studies
highlight the diversity within the A. baumannii complex using broad phylogenetic tools. The
genome sequencing and subsequent comparison is anticipated to reveal additional biomarkers
that can be used for the rapid identification and characterization of A. baumannii.
Current status of A. baumannii genomics and transcriptomics:
Genomic studies have investigated the diversity of A. baumannii associated with nosocomial
infection and A. baumannii strains exhibiting multi-drug resistance (1, 2, 11, 18, 31, 34). These
seminal studies have examined a limited number of A. baumannii isolates obtained from the
same hospital and few have detailed the development of antibiotic resistance. One study
utilized genomics to chronicle the emergence of resistance to tigecycline in A. baumannii
isolated from an individual patient receiving antibiotic therapy (17). Another study compared the
genomes of four A. baumannii clinical isolates from the same hospital over time, demonstrating
diversity in the resistance
phenotypes and resistance genes
(1). These were strains isolated
at different times ranging from 1
month to over a year apart (May
2003-August 2004). These
studies are not nearly
comprehensive enough to identify
any evolutionary trends in a
hospital setting. The
advancements of high-throughput
sequencing technologies and the
ability to rapidly sequence and
analyze large numbers of
bacterial genomes in recent
years, enables a prospective
study such as the one outlined in
this proposal to be a possibility.
Fig 1. Phylogenomic analysis of a conserved region present in the genomes
of all A. baumannii, and select other Acinetobacter spp. genomes that have
been made publicly available as of March 2012. A. baumannii that were
sequenced by us at IGS are green, and genomes that are Acinetobacter spp.
other than A. baumannii are purple.
There are currently 42 A.
baumannii genomes that have
been released into the public
domain, including seven
genomes that were sequenced by
authors of this proposal
(highlighted in red and green in
Fig. 1). Phylogenomic analysis of
the A. baumannii genomes
including 12 genomes of nonbaumannii Acinetobacter species
(Figure 1, purple) demonstrates there is extensive diversity among Acinetobacter species.
Interestingly, 26 of the 42 A. baumannii genomes, sequenced to date fall into two groups; nine
genomes form a group that contains isolates identified by MLST as the European clonal lineage
I (EC I), while 17 form a group that contains isolates of the European clonal lineage II (EC II).
Of the seven genomes sequenced at IGS, three (UMB001, UMB002, UMB003) were isolated
from the University of Maryland Medical Center (UMMC) cohort study [described in (20)], and
each occupies a distinct lineage of the whole-genome phylogeny. These three A. baumannii
and detailed molecular analysis of a subset of patient isolates from samples of the same cohort
have demonstrated that we will encounter this type of diversity in the proposed isolates and that
we have the expertise to complete the comparative genomics.
Novel whole genome transcriptional studies of A. baumannii exposed to antibiotics will be
completed using a method known as RNA-seq. RNA-seq is an unbiased high-throughput
sequencing approach used to capture the total global transcriptional response of an organism or
biological system (38). To our knowledge there has been no investigation of the response
of A. baumannii on the transcriptional level to exposure to antibiotics. Since its
introduction, RNA-Seq has been used to examine the global transcriptome of several
pathogenic bacteria (22, 25, 28, 32, 33); however to date, Acinetobacter-based transcriptional
studies have primarily employed the use of qRT-PCR or microarrays. Microarray and qRT-PCR
studies are limited by the number of targets that can be feasibly examined, as well as being
biased through the a priori selection of targets. These previous studies have investigated the
limited transcriptional response of A. baumannii during exponential growth, stationary phase
incubation, growth in human serum (19), under iron limiting conditions (10), and growth with
NaCl (16) with these limited methodologies. A recent study utilized RNA-Seq to determine what
genes of A. baumannii may be involved in pathogenesis upon exposure to ethanol (5) (Note:
this study was co-authored by Dr. Vincent Bruno who is a faculty member at the Institute for
Genome Sciences and a funded investigator on the GSCID contract).
The PI’s are aware of a currently funded GSCID genome sequencing project at the J. Craig
Venter Institute (JCVI) in collaboration with Walter Reed Army Institute of Research (WRAIR)
focused on sampling the overall genomic diversity of A. baumannii by sequencing of up to 75
isolates, for the purpose of developing an A. baumannii microarray
(http://gsc.jcvi.org/projects/gsc/a_baumannii/index.php). We feel that these proposals are
complementary rather than over-lapping, in that we are focusing on an aspect of A. baumannii
biology that does not appear to be addressed by the other currently funded proposals. Specific
unique areas covered in this proposal include the investigation of the genomic diversity of
MDRAb compared to non-MDRAb isolated from patients receiving treatment in the UMMC
ICUs, the genomic diversity in patients colonized/infected for long periods of time, and the
innovative use of transcriptional profiling to address the development of antimicrobial resistance.
Additionally, Dr. Rasko has an existing and ongoing collaboration with WRAIR and has worked
with Dr. Fouts, the leader of the Acinetobacter project at JCVI.
Rationale for strain selection:
The A. baumannii strains that we propose to sequence were isolated from a cohort study at
the UMMC that was conducted from September 1, 2001 to the present (20). This cohort
combines specimen and patient information stored in an Oracle-based central data repository
that has been used in over 30 publications. This study involves adult patients that were
receiving treatment in the medical ICU (MICU) or surgical ICU (SICU). These patients had perianal swabs upon admission, weekly, and discharge from the ICU. Peri-anal swabs have been
cultured on selective media for several pathogenic bacteria including Acinetobacter spp (3).
There were a total of 2,220 Acinetobacter isolates from 985 patients isolated from June 2005 to
July 2009 and May 2011 to February 2012. With this massive cohort of isolates we have
decided to focus on the issue of antibiotic resistance, the development of resistance and the
transcriptional events associated with resistance in this clinically important pathogen. To
accomplish these goals we will complete the following Aims which will provide answers to
unanswered scientific questions in the area of A. baumannii:
Aim 1: Characterize the genomic diversity of carbapenem resistant A. baumannii (CRAb)
compared to non-CRAb from isolates obtained over a 7 year time period.
We have identified prospective A. baumannii strains for genome sequencing based on their
antimicrobial susceptibility profiles and associated patient data. In this proposal we will define
carbapenem resistant A. baumannii as exhibiting resistance to one or more carbapenems,
specifically, imipenem or meropenem. This criteria is utilized as these are the carbapenems that
are used most frequently in clinical settings. Resistance to carbapenems can be conferred by a
number of diverse mechanisms including metallo-β-lactamases, oxacillinases, and porin
modification (29).
Aim 1a: Characterize the genomic
diversity and antibiotic resistance gene
pool of CRAb and non-CRAb strains
isolated from the UMMC ICU from 20052012. The scope of work will include
generating the draft genomes of 393 A.
May 2011- March 2012
baumannii (246 CRAB and 147 nonperi-anal
155
95
60
CRAB) that were isolated from patients
sputum
131
91
40
receiving treatment in the UMMC ICU from
Totals
393
246
147
2005-2012. The Illumina platform will be
utilized due to the ability to multiplex large numbers of isolates. While the number of proposed
isolates is daunting, through multiplexing there will be a need for only two sequencing runs on
the Illumina2000 platform. The distribution of human body isolation site (rectal or sputum), the
antibiotic resistance profile and the temporal aspects of when the isolates were obtained are
presented in Table 1. There have been 246 CRAb and 147 non-CRAb isolates identified for
sequencing in this proposal. The isolates include a total of 286 A. baumannii isolated from
patients receiving treatment in the last calendar year (May 2011-March 2012). These will be
compared to 107 A. baumannii (60 CR and 47 non-CR) isolated from ICU patients that received
treatment between June 2005 to March 2009.
Table 1. Carbapenem resistant A. baumannii (CRAB) and nonCRAB strains isolated from the UMMC ICU
A. baumannii
Date & Specimen Types
Total
CRAB
non-CRAB
June 2005- March 2009
peri-anal
107
60
47
By comparing the isolates from these patient groups it will increase our chances of identifying
developing trends for a number of critical clinical questions:
1) Are there gross genomic differences between the CRAb and non-CRAb that
we can exploit as biomarkers? For example, are there genes or gene regions
that can be exploited for the development of PCR or other molecular assays that
will reliably separate the CRAb from non-CRAb isolates. If we can identify these
types of biomarkers and develop assays, we may be able to rapidly alter the
path of clinical treatment, especially in the cases of infection by a CRAb isolate.
2) Are there differences in the A. baumannii isolates that were obtained 7 years
ago versus the isolates that are obtained in the last year? This will address the
clinical question of the development/evolution of A. baumannii in general as well
as development of resistance.
In addition to the clinical questions that will be addressed above, comparative genomics of
CRAb and non-CRAb strains isolated from the same hospital, and thus a similar patient
population, over seven years will allow us to examine if there is genome content associated with
the emergence of antimicrobial resistance, that is in addition of the mechanisms directly
responsible for antimicrobial resistance. Also, the extensive patient and epidemiological data
will allow the identification of correlations between the genomic content and the clinical outcome
irrespective of antimicrobial resistance profile (i.e. are there genomic features associated with
isolates from patients with a poor clinical outcome (increased length of stay, mortality, etc) and
does antibiotic resistance play a role in those outcomes).
Through comparative genomics of the CRAb isolates from the UMMC ICUs, we will identify
the previously described antibiotic resistance gene pool among the CRAb genomes sequenced
and determine whether any of the non-CRAb isolates sequenced are closely-related to CRAb
isolates using whole genome phylogenetic analysis. This type of whole genome phylogenetic
analysis was pioneered at IGS. From the draft genomes we will construct whole-genome
phylogenies and extract housekeeping genes used for multi-locus sequence typing (MLST) to
determine whether any of the CRAb isolates sequenced in our study belong to previously
defined clonal groups associated with multi-drug resistance, or if these are novel MSLT
sequence types of A. baumannii (7). We will include temporal aspects in these analyses to
determine if there is a particularly virulence clone that was previously unidentified in the UMMC
ICU cohort.
Information derived from these analyses will allow us to develop primers for screening the
remaining A. baumannii isolated from this large cohort study. This screening will allow
examination of the prevalence of certain resistance-conferring genes or genomic regions. Using
whole-genome Mugsy (4) alignment, we will examine the dataset for lineage-specific
phylogenetic markers for CRAb isolates and MDRAb that can be used to develop multiplex PCR
assays or Taqman assays for the high throughput determination of the evolutionary history of
presumptive A. baumannii clinical isolates. We have previously used whole-genome Mugsy (4)
alignments to identify a marker that can be used for species identification of A. baumannii (31).
Aim 1b: Determine the genomic diversity of A. baumannii strains from a single patient
over multiple longitudinal time points, as well as the within-patient genomic diversity of
A. baumannii cultured from a single patient specimen.
From a review of our cohort patient data, we have identified 15 patients receiving treatment
in the UMMC ICUs between 2002 and 2009 that had two or more Acinetobacter positive
perianal swab cultures. Of the 15 patients that had ≥ 2 positive Acinetobacter swabs, we will
examine the population diversity of A. baumannii that is cultured from each swab collected from
four of the patients. Four such collections of patient samples were identified that represented A.
baumannii isolated from a single patient from weeks to months to years apart. These genomic
studies of these isolates will provide insight into the evolution of A. baumannii within the human
body over both short and long timeframes. One patient had A. baumannii isolated nearly every
week for three months, whereas the other three patients have isolates spanning multiple years.
From these four patients there are a total of 23 swabs that will be cultured to collect 10 isolates
per patient sample resulting in up to 230 colonies that will be isolated for genome sequencing.
These isolates from the same patient will address the unanswered questions of long-term
carriage of A. baumannii. The patient swabs will be cultured on CHROMAgar media that is
selective for Acinetobacter (3) and the 10 presumptive A. baumannii will be PCR confirmed
using the A. baumannii-specific phylogeny-based marker that was previously developed by this
group (31).
By sequencing the genomes of multiple isolates from a single patient we will examine intraindividual genomic diversity of A. baumannii. We will be able to compare the observed intraindividual genomic diversity to the non-CRAb and CRAb populations observed in the UMMC
ICUs over time. This approach has been undertaken previously in the ETEC GSCID project
using isolates that were obtained from a single subject at a single time point, but exhibited
diverse antigenic profiles. While these studies are still ongoing, they have lead to the novel
finding of both stable and diverse E. coli population structures within an individual (Rasko,
unpublished data). The study of the E. coli population is confounded by the fact that there are
E. coli commensal species in the gastrointestinal tract of many people, so the question of
carriage versus normal flora impacts the findings. In the current Acinetobacter proposal, there
should not be A. baumannii isolates as part of the normal flora, or at least it has not previously
been identified as part of the normal flora, and thus any isolate could be considered to have
pathogenic potential. The genomic stability of Acinetobacter is thought to be poor (34), however
this point has never been directly examined through whole genome sequencing and thus this
will be one of the novel findings in this proposal.
Aim 2: Identify genes involved in the emergence of multi-drug resistant A. baumannii
using global transcriptional analysis.
It has been previously demonstrated that not all MDR isolates of A. baumannii contain a
genomic island that has been linked to the development of resistance (1, 2, 11, 18, 30, 31). We
hypothesize that there are transcriptional differences in isolates that will result in increased
resistance to antibiotics. A recent study demonstrated differences in the transcriptional response
of E. coli carrying an IncA/C multi-drug resistance plasmid when it was exposed to different
antibiotics (21). We will examine the global transcriptional response of CRAb and non-CRAb to
several types of antibiotics including a fluoroquinolone (ciprofloxacin), a carbapenem
(imipenem), an aminoglycoside (amikacin), and a polymyxin (colistin). These are antibiotics that
are commonly used for the treatment of A. baumannii, and A. baumannii clinical isolates have
been described with resistance to one or more of these antibiotics (24, 26). We will use RNASeq to analyze the global transcriptome of A. baumannii grown under standard laboratory
conditions with and without the addition of sub-inhibitory doses of antibiotics. We will select
CRAb and non-CRAb isolates from the analysis in Aim 1a that are determined by wholegenome phylogenetic analysis to belong to the same evolutionary lineages, to minimize the
confounding factors of the results. These will include one CRAb and one non-CRAb from each
of the three commonly identified EC groups as well as newly identified lineages of MDR and
non-MDR Acinetobacter isolates.
Briefly, these representative CRAb and non-CRAb isolates will be grown in Mueller-Hinton
broth (MHB) to an OD600= 0.5 incubated at 37˚C with shaking. The RNA will be isolated using
standard methodologies in the Rasko Laboratory (32) and the transcriptomes of two biological
replicates for each CRAb and non-CRAb examined for each experimental condition will be
sequenced (Table 2). CRAb and non-CRAb strains will be grown overnight without antibiotic
then diluted 1:100 into fresh media supplemented with 25% (low) or 75% (high) of the minimum
inhibitory concentration (MIC) of each antibiotic as determined by agar plating. These
concentrations are meant to induce the mechanisms that may lead to the development of
antimicrobial resistance, and would represent the early stages of antibiotic treatment before
clinically relevant doses of antibiotic are reached in the patient.
We also propose to use RNA-Seq to determine the transcriptome of the CRAb and nonCRAb isolates exposed to the monovalent cation, NaCl. A recent study that used microarray to
investigate the transcriptional response of A. baumannii to growth with NaCl demonstrated that
A. baumannii had increased expression of genes involved in antibiotic resistance, and isolates
exhibited increased tolerance to colistin in the presence of high NaCl concentrations (16). We
will compare the transcriptomes of the CRAb and non-CRAb isolates upon exposure to NaCl, or
when they are exposed to low (25% MIC) and high (75% MIC) levels of several antibiotics, to
identify select genes that are involved in increased tolerance to antibiotics (Table 2). The
differences in expression of these target genes will be validated using qRT-PCR upon exposure
to antibiotics with and without the addition of NaCl.
Table 2. Experimental conditions for RNA-Seq analysis of CRAB and non-CRAB isolates
No Treatment
Control
MHB only
Stress agent
Carbapenem
Fluoroquinoline
Polymyxin
Aminoglycoside
NaCl
imipenem
25% MIC
75% MIC
2
2
2
2
ciprofloxacin
25% MIC
75% MIC
2
2
2
2
colistin
25% MIC
75% MIC
2
2
2
2
amikacin
25% MIC
75% MIC
2
2
2
2
CRAB EC I
non-CRAB EC I
2
2
2
2
CRAB EC II
non-CRAB EC II
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
other CRAB 1
other non-CRAB 1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
16
2
2
16
2
2
16
2
2
16
2
2
16
2
2
16
other CRAB 2
2
2
2
2
other non-CRAB 2
2
2
2
2
Totals
16
16
16
16
Total Sequencing
160
*Numbers represent biological duplicates for each isolate and experimental condition
By defining the transcriptomes of A. baumannii from multiple evolutionary lineages we will
examine whether differences in genomic diversity or transcriptional responses to antibiotic
exposure pre-dispose certain A. baumannii to becoming carbapenem or possibly multi-drug
resistant. If such lineages or genetic signatures exist that link A. baumannii with an increased
likelihood of multi-drug resistance, we will develop markers for the rapid identification of these
isolates in the clinical laboratory setting.
Summary
This proposal will attempt to evaluate both the genomic and transcriptomic diversity of a
well-characterized, existing collection of A. baumannii isolates. The genomic diversity will be
assessed by sequencing isolates obtained over the course of seven years in a single hospital
setting, but also includes both antibiotic resistant and non-resistant strains. Genomic
comparisons of resistant and sensitive isolates may provide insight into the development of
resistance in this pathogen, as well as the evolutionary processes that underlay this phenotype.
In recognition of the fact that not all phenotypic alterations will be genomic, we will also examine
the transcriptomic responses to commonly used antibiotics to A.baumannii. These studies will
enhance our understanding of the species structure in this important emerging human pathogen
and attempt to understand the development of antimicrobial resistance.
Assembled Team:
The assembled team includes both clinical and genomic investigators. This group has worked
together in the past to examine the genomic diversity of a small number of A. baumannii
isolates, resulting in a publication and the identification of a conserved genetic signature for A.
baumannii. The team is confident that they can complete the aims as outlined in the proposal.
J. Kristie Johnson is an Associate Professor in the Department of Pathology and Epidemiology
and Public Health. Dr. Johnson is a board certified clinical microbiologist. Dr. Johnson’s
research focuses on the detection, transmission, and control of antimicrobial resistant
organisms concentrating on methicillin resistant Staphylococcus aureus (MRSA) and resistant
Gram-negative bacteria to include multi-drug resistant Enterobacteriaceae (KPC, ESBLs, and
plasmid mediated AmpC), Acinetobacter baummannii, and Pseudomonas aeruginosa.
Tracy Hazen is a Postdoctoral Fellow who is co-mentored by David Rasko, J. Kristie Johnson
and Anthony Harris. She is focused on the studies of genome-scale evolution in E. coli and
Klebsiella species. Her role on this proposal will be to guide the laboratory work on the
transcriptomics and comparative genomics.
Anthony Harris is a Professor in the Department of Epidemiology and Public Health at the
University of Maryland School of Medicine. He is an infectious disease physician and the
hospital epidemiologist at the University of Maryland Medical center. He has over 110
publications in the area of antibiotic-resistance and hospital epidemiology. He has published a
number of papers on A. baumanii. He has NIH funding that created the cohort of strains that are
going to be used on this proposal. On this proposal, he will provide clinical expertise, access to
the strains and use the relational database to correlate outcomes with genomic and
transcriptional findings.
David Rasko is an Assistant Professor in the Institute for Genome Sciences and the
Department of Microbiology and Immunology at the University of Maryland School of Medicine.
His work focuses on the evolution and pathogenesis of Gram-negative human pathogens. He
has published papers relating to A. baumannii genome evolution with the other investigators on
this proposal and will direct the comparative genomic and transcriptomic analyses for this
project.
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