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Molecular characterization of Acinetobacter isolates collected during a three-
year surveillance program in intensive care units of six hospitals in Florence
(Italy): a population structure analysis
Running title. Structure analysis of a nosocomial Acinetobacter population
Francesca Donnarummaa, Simona Sergi
a¶, Cristina Indorato
a, Giorgio Mastromei
a, Roberto
Monnannia, Pieluigi Nicoletti
b, Patrizia Pecile
b, Daniela Cecconi
b, Roberta Mannino
b, Sara
Bencinic, Rosa Fanci
c, Alberto Bosi
c, Enrico Casalone
a*
aCIBIACI and Department of Evolutionary Biology, University of Florence, Florence, Italy;
bLaboratory of Microbiology, Careggi Hospital, Florence, Italy;
cStem Cell Transplantation Unit, Department of Haematology, Careggi Hospital, University of
Florence, Florence, Italy
¶ Present address: Department of Biomedical Science and Technology Section of General
Microbiology and Virology & Microbial Biotechnologies, University of Cagliari (Italy).
*Enrico Casalone
Department of Evolutionary Biology
Via Romana 17/19
50125 Florence, Italy
Tel 0039 0552288245-Fax 0039 0552288250
enrico.casalone@unifi.it
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.01916-09 JCM Accepts, published online ahead of print on 24 February 2010
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ABSTRACT
The strain diversity and the population structure of nosocomial Acinetobacter isolated from
patients admitted to different hospitals in Florence, Italy, during a three years surveillance
program, were investigated by AFLP. The most part of isolates (84.5%) were identified as A.
baumannii, confirming this species as the most common hospital Acinetobacters. Three well
distinct A. baumannii clonal groups (A1, A2 and A3) were defined. The A1 isolates appeared to be
genetically related to the well characterized European clones EII. A2 was responsible for three
outbreaks which occurred in two intensive care units. Space/time population dynamic analysis
showed that A1 and A2 were successful nosocomial clones. Most of the A. baumannnii isolates
were imipenem resistant. The genetic determinants of carbapenem resistance were investigated by
multiplex PCR, showing that resistance, independently of hospital origin, period of isolation or
clonal group, was associated with the presence of bla OXA-58-like gene, and with ISAba2 and ISAba3
elements flanking this gene. bla OXA-58 appeared to be horizzontally transferred.
This study showed that the high discriminatory power of AFLP is useful for identification and
typing purposes of nosocomial Acinetobacters. Moreover the use of AFLP in a real-time
surveillance program allowed us the recognition of clinically relevant and widespread clones and
their monitoring in hospital settings. The correlation between clone diffusion, imipenem resistance
and the presence of the bla OXA-58-like gene is discussed.
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INTRODUCTION
Acinetobacters are a heterogeneous group of ubiquitous microorganisms. Within the Acinetobacter
genus, A. baumannii and the genomic species gen.sp. 3 and gen.sp. 13TU share common
characteristics: low prevalence in the community, rare occurrence in the environment, and the
emergence, in 1980s, as opportunistic pathogens in critically ill patients in intensive care units
(ICUs) (9). These opportunistic pathogens form, with the environmental species A. calcoaceticus,
a genetically and phenotypically similar group of microorganisms, the so called A. calcoaceticus-
A. baumannii complex (Acb complex) (15). The ecology, epidemiology, and pathology studies of
the member species of the Acb complex are hampered by the limited capability of phenotypic and
DNA-DNA hybridization analysis to correctly discriminate them (15). In the last years, to comply
with the need of a correct species discrimination (4), unsatisfactory phenotypic commercial system
for Acinetobacters identification have been substituted by genotypic methods. One of these
method is Amplified Fragment Length Polymorphism (AFLP) (24), which has been found to be
useful for the differentiation of Acinetobacter strains (4, 6, 36). The extensive application of
genotypic analysis to population studies of A. baumannii has identified three high similar, wide
spread European clones (EU I-III clones). EU clones are assumed to represent distinct, genetically
stable and well hospital-adapted clonal lineages, which only known selectively advantageous trait
is their high resistance to antimicrobial agents (9, 25, 26).
The great capacity of A. baumannii to survive in dry conditions on the patient skin (20) can
contribute to its carriage in the community and to the spreading of epidemic strains in the hospital
environment. As a demonstration of its survival capability, A. baumanni is often recovered from
various sites in the patients’ environment, including bed curtains, furniture and hospital equipment
(35). Currently, A. baumannii ranks among the most important nosocomial pathogens (9), the most
frequent infections being bloodstream infections and ventilator-associated pneumonia; in ICUs,
these infections are often associated with poorly prognostic clinical manifestation, whereas urinary
tract infections are more benign (9). To make the situation worse, A. baumannii, already
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intrinsically resistant to a number of commonly used antibiotics (5), can acquire antimicrobial
genes conferring resistance to broad-spectrum β-lactams, aminoglycosides, fluoroquinolones and
tetracyclines, becoming multidrug resistant (MDR) (2). Of particular concern is the worldwide
emergence of the resistance to carbapenems (5, 23), that have been the most important agents for
the treatment of infections caused by MDR A. baumannii in the last years (5). bla OXA genes
encoding carbapenem-hydrolysing β-lactamases (carbapenemases), belonging to molecular class D
(OXA enzymes), have emerged globally as the main mechanism responsible for this resistance
(38). The bla OXA genes of Acinetobacter spp. can be divided into four phylogenetic subgroups:
bla OXA-51-like gene, intrinsic to A. baumannii (3), is normally expressed at low level but can be
over-expressed as a consequence of the insertion of an ISAba1 sequence upstream of the gene
(33); in contrast, bla OXA-23-like, bla OXA-24-like and bla OXA-58-like genes were consistently associated
with resistance or, at least, with reduced susceptibility (38).
In this study, with the aim to investigate species and strain diversity and to determinate the
population structure of nosocomial Acinetobacter, we have analyzed, by AFLP, the genotypic
similarities between isolates collected from patients admitted to different hospitals in Florence,
Italy. Furthermore, to obtain a comprehensive view of the emergence of carbapenem resistance
among clinical Acinetobacter isolates, the susceptibility to carbapenems and the presence of genes
linked to carbapenem resistance were determined.
MATERIALS AND METHODS
Surveillance system, specimen collection and phenotypical analysis of bacterial isolates.
From July 2006 to December 2008, during a surveillance program of nosocomial infections (31),
71 isolates were identified as A. baumannii and tested for antimicrobial susceptibility, according to
the recommendations of the Clinical and Laboratory Standards Institute (CLSI), at the Laboratory
of Microbiology of Careggi hospital (Florence, Italy), using the automated Vitek2 System
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(BioMerieux, Marcy l’Etoile, France). The isolates were collected from 64 patients admitted to 21
high-risk wards (15 adult ICUs, 5 neonatal ICUs, 1 bone marrow transplantation unit) of six public
hospitals, located in the district of Florence, Italy: a University Hospital (H1, 1700 beds), a
Children University Hospital (H2, 180 beds), and four non-university hospital (H3, 161 beds; H4,
246 beds; H5, 104 beds; H6, 263 beds). All patients admitted to the monitored hospital wards, in
the reported time period, and resulting positive to A. baumannii were included in the study. A.
baumannii strains from the same patient, independently of specimen, and showing the same AFLP
pattern (see below), were considered duplicates. For further analysis, only one strain for duplicate
was used.
The surveillance system consists of the Laboratory Information System (LIS; Dianoema, Bologna,
Italy), connected to the real-time epidemiological information system Vigi@ct (Biomerieux, Las
Balmas, France). Vigi@ct points out presumed hospital acquired infections (HAI) and outbreak
events and prints documents of pathogen detection to be sent, with a clinical questionnaire, to the
corresponding hospital ward. Ward clinicians define the event as infection or colonization and in
case confirm the outbreak. An outbreak, as defined by the Centers for Disease Control and
Prevention (USA), involved at least two infected patients. Infections, defined on the basis of a
positive culture from a presumed infected site, or a body-fluid or other sterile sites, were classified
as HAI or community acquired infection (CAI) when obtained > 48 hours or < 48 hours after
hospital admission, respectively; colonisation (C) was defined as a positive culture from a
specimen in the absence of clinical signs or symptoms of infection. Once compiled, clinical
questionnaires should be returned to the Laboratory of Microbiology of the Careggi Hospital for
the updating of the Vigi@ct systems.
AFLP analysis.
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DNA was extracted from bacterial cells, killed in a 50% isopropanol solution, using the Wizard
Genomic DNA Purification Kit TM (Promega, Wisconsin, USA), following manufacturer's
instructions, and spectrophotometrically quantified by Biophotometer (Eppendorf AG, Germany).
AFLP was performed as described by Vos et al. (37) and Nemec et al. (24) with slight
modification (12). Briefly, purified DNA was digested by simultaneous use of EcoRI and MseI;
afterwards EcoRI and MseI adaptors were ligated. PCR was done with EcoRI-A (6-
carboxyfluorescein-5’-GACTGCGTACCAATTCA) and MseI-G (5’-
GATGAGTCCTGAGTAAG) primers, with a selective A and G nucleotide at 3’ end, respectively.
Amplified fragments were separated by capillary electrophoresis on ABI 310 bioanalyzer (Life
Technologies, California) and fragment-size was determined by using the internal standard 50-400
bp (Rox 400HD). Only fragments of 60–380 bp were considered in profile analysis performed by
GeneMapper 4.0 software (Applied Biosystems). Cluster analysis of the profiles was performed
using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method with the
Numerical Taxonomy and Multivariate Analysis System NTsys-pc v.2 software (Exeter Software,
USA). The percentage of similarity between profiles was calculated using the Dice correlation
coefficient.
AFLP genomic fingerprinting was used to classify strains to both species and subspecies level
(26). An isolate was identified as belonging to a defined species when it grouped with the
corresponding type strain at a level of similarity ≥59% (29); isolates grouping at ≥78% level of
similarity were considered to belong to the same clone. Reference strains for AFLP identification
were: A. baumannii, ATCC 19606; A. calcoaceticus, DSMZ30006; gen.sp. 3, DSMZ9343; gen.sp.
13TU, DSMZ30010 whereas European clones EI-III were kindly supplied by Dr. L. Dijkshoorn
(University Medical Center of Leiden, The Netherlands).
Reproducibility of AFLP analysis was assessed by comparing different profiles obtained with
replicates of the reference ATCC strains. To determine the discriminatory power of AFLP,
Simpson’s index of diversity (D) (14) with 95% confidence intervals was calculated.
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OXA gene detection
The presence of the genes encoding the OXA-23-like, OXA-24-like, OXA-51-like and OXA-58-
like class carbapenemases, was determined by Multiplex-PCR amplification. The PCR experiment
was performed on a One Personal thermocycler (Euroclone, Italy), with 50 ng of genomic DNA in
a final volume of 25 µl and Illustra Hot Start Master Mix (GE Healthcare) using already described
cycling conditions and four pairs of primers, each one specific for a different OXA gene (38).
Amplified fragments were separated on 1.5% (w/v) agarose gel. All the carbapenem resistant
isolates have been investigated by PCR for the presence of the promoter sequences ISAba1 (30),
and for that of ISAba2 and ISAba3, located 5’- and 3’-, respectively, of bla OXA-58-like gene (28; 16).
The sequence (934 bp) of the blaOXA58-like gene was determined by Sanger’s method on ABI prism
310 CE system sequencer (Life Technologies, California) using the OXA-58A (5-
TTATCAAAATCCAATCGGC-3) and OXA58B (5- TAACCTCAAACTTCTAATTC-3) primers.
Principal component analysis (PCA)
AFLP patterns were subjected to principal component analysis (PCA) to describe the general
pattern of variation between isolates of each species. This method provides an ordination of
isolates belonging to different clusters, which are plotted in two dimensions based on the scores in
the first two principal components, without imposing any grouping or phylogenetic criteria on the
results. Any isolate is recovered as a distinct point in the PCA ordinations (22). PCA was
calculated using NTsys-pc v.2 software (Exeter Software, USA).
Analysis of molecular variance (AMOVA)
AMOVA method was applied to estimate the genetic differences among A. baumannii clonal
groups. Internal variability of each clonal group was calculated using the Euclidean distances
between all possible combinations of AFLP patterns taken in pairs (10). The genetic structure of
bacterial populations was investigated by an analysis of the variance framework as reported by
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Dalmastri et al. (7). A. baumannii isolates were sorted in different populations (corresponding to
AFLP clusters) and AMOVA computes the differences among populations and among individuals
within populations. Pairwise Fsts (39) and their corresponding p-values were calculated to
quantify the differentiation between all pairs of populations. AMOVA and Fsts value generation
were performed using Arlequin ver. 3.1 software (11).
RESULTS
Patient data and bacterial isolates
During the study period, 71 strains, identified as A. baumannii by Vitek2 system, were isolated
from 64 patients admitted to different hospitals (see Materials and Methods for the selection
criteria of patients and bacterial isolates). The majority of isolates were from patients ≥65 years of
age. The specimen from which the isolates were obtained were bronchoaspirates (47.8%), blood
(12.7%), pharyngeal swabs (11.3%), urine (7%), sputum (5.6%), central venous catheter (7%) and
others (8.4%). Based on the answer to clinical questionnaire for infection (IN)/colonization (C)
data, we have 25 IN, 27 C, and 19 unanswered (Figure 1).
AFLP analysis of Acinetobacter strains
Reproducibility of AFLP analysis was higher than 90% (data not shown). AFLP analysis, used for
species identification, confirmed that all the isolates belong to the genus Acinetobacter (similarity
> 40%) (29), but only sixty isolates (84.5 %) were identified as A. baumannii, confirming Vitek2
results, whereas four isolates (5.6 %) were identified as gen.sp. 3. None of the isolates belonged to
gen.sp. 13TU and to A. calcoaceticus, and seven isolates could not be assigned to any species of
the Acb-complex (non Acb-complex isolates) (Figure 1). AFLP typing analysis grouped 60 % of
the 60 A. baumannii isolates in three groups with a cut off value ≥78%: clonal group A1 (9
isolates), clonal group A2 (24 isolates), clonal group A3 (3 isolates); the remaining 24 isolates were
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ungrouped (isolates with unique AFLP profile). A.baumannii belonging to the clonal group A1
(Figure 1) resulted to be genetically related to the EUII clone, with a level of similarity of 78%
(clone delineation level).
The genetic relatedness of A. baumannii isolates, evaluated by Simpson’s index of diversity (D),
shows that AFLP has a good discriminatory power (D = 88%, with a confidence interval of 84% to
92%) (not shown).
Antibiotic resistance of A. baumannii isolates
All Acinetobacter isolates belonging to the clonal groups A1, A2 and A3 showed a similar
antimicrobial resistance profile for β-lactam antibiotics, aminoglycosides, and quinolones;
whereas, ungrouped isolates showed a higher variability in the antimicrobial resistance profiles
(data not shown). In particular, 65 % of A. baumannii (39 isolates) were resistant to imipenem (IR),
whereas the remaining 21 were susceptible (IS); all the other isolates were I
S, except for two non
Acb-complex isolates (Figure 1). The IR phenotype was expressed in isolates of all three A.
baumannii clonal groups: A1 (7/9), A2 (16/24), A3 (3/3), and among single AFLP profile isolates
(13/24). Interestingly, whereas IR A1 strains were isolated continuously from September 2006 to
May 2008, IR A2 strains were restricted to May 2007-December 2008; in the same period, only
one IS A2 strain was isolated, whereas the remaining seven I
S A2 isolates were from July to
December 2006. The three IR A3 strains were isolated in October 2008.
Isolates were also investigated for the identity of the genetic determinants of imipenem resistance.
The presence of bla OXA genes encoding OXA-51-like, OXA-58-like, OXA-23-like, OXA-24-like
enzymes, and that of IS elements able to promote the expression of these genes, was evaluated by
multiplex PCR. bla OXA-23-like and bla OXA-24-like genes, as well as the ISAba1 sequence, were never
detected, but all A. baumannii and three non Acb-complex isolates, independently from imipenem
resistance, were positive for intrinsic bla OXA-51-like gene. All and only the IR isolates (39 A.
baumannii and two non Acb-complex), independently of hospital origin, period of isolation or
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clonal group, were positive for bla OXA-58-like gene (Figure1); this gene was always flanked by
ISAba3 element at the 3’end and by ISAba2 at the 5’ end, except in one A1 strain and in two
ungrouped strains that lack this last element (not shown).
The determination of the nucleotide sequence of the entire bla OXA-58-like gene in representatives of
the clonal groups A1, A2 and A3, shows the identity of the gene sequences among the clonal
groups and with the bla OXA-58 sequence in the GenBank database (FJ492877).
Outbreak investigation
Three A. baumannii outbreaks occurred in two intensive care units of hospital H1 and H3, during
the study period; Table 1 lists all the isolates collected from infected patients during each outbreak
and those isolates that, in the same ward and period, were collected from colonized patients or
patients for which no infection/colonization data are available. The first two outbreaks took place
at H3-W1 in 2006, at a distance of two months each other, and involved two patients each; both
outbreaks lasted for approximately one month. In both cases, the corresponding A. baumannii
isolates belonged to the clonal group A2 and were IS. The third outbreak occurred at H1-W14 in
2008 and involved four patients in a period of one month, and also in this case the isolates
belonged to clonal group A2 but, differently from the previous two outbreaks, they all were IR.
The genetic relatedness among isolates from the same outbreak was always very high (88 % <
similarity < 95 %) (Figure1).
Hospital measures, such as the intensification of standard cross-infections precautions, including
cleaning and disinfection of patients’ room, were introduced to control cross contamination of
patients.
Space/time distribution of A. baumannii isolates
The dissemination of the three clonal groups in hospital settings was also investigated (Table 2). It
was found that the A1 clonal group was prevalently restricted to H1 hospital, whereas the A2
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clonal group spreads in different hospitals, and the three isolates belonging to clonal group A3
were collected in three different wards of two hospitals. The 24 isolates with unique profile were
from five of the six monitored hospitals. As a matter of fact, even if highly similar strains have
been found at different wards and at different time points, no epidemiological links between them,
except for the outbreak isolates, have been found.
A1 and A2 clones were persistent (isolated from July 2006 to the end of 2008) and, especially A2,
widespread (isolated from different hospitals and wards). However, a time succession of these two
clonal groups was observed: A2 was predominant in 2006 and in 2008, whereas A1 was
predominant in 2007 (Figure 2). Isolates belonging to A3 were collected only at the end of 2008.
Ungrouped isolates spread during all the study period, with a significant increase in the second
half of 2008.
Principal component analysis
In order to better represent the relatedness of isolates belonging to the three clonal groups, AFLP
profiles were subjected to PCA (Figure 3). The first two principal components accounted for 19.3
% of the total variation observed; the three clonal groups were clearly separated by the first
principal component (11.6 % of variation) supporting the results of the cluster analyses. Moreover,
we observed that A2 isolates were resolved along the second principal component (7.7 % of
variation) and in some cases A2 isolates coming from the same ward were more close each other
(data not shown). Unlike A2, A1 and A3 clonal groups showed a lower variability within group.
Because of the low total variance contained in the first two components, PCA did not provide a
clear separation of ungrouped isolates, but they were most clearly separated, essentially by the first
component, from isolates belonging to A2 than from A1 and A3 isolates.
Genetic variability among A. baumannii isolates
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AFLP data were used to evaluate the genetic differences among the three clonal groups by means
of AMOVA method. The result shows that, although most molecular variance was due to intra-
group differences (percentage of variance: 59.6 %), a statistically significant percentage of it was
also attributable to inter-group genetic differences (percentage of variance: 40.4 %). When the Fst
statistic was calculated to examine the level of genetic divergence among pairs of clonal groups,
the following percentages of pairwise difference were obtained: A1 vs A2, 38 %; A1 vs A3, 52 %;
A2 vs A3, 42 %. Overall, these results indicate a high genetic difference between groups,
confirming the existence of three well distinguished A. baumannii clones that spread in the
monitored wards.
DISCUSSION
In many papers, A. baumannii is used in a broader sense to accommodate also gen.sp 3 and gen.sp.
13TU. In this study, with the aim to differentiate the contribution of A. baumannii, gen.sp 3 and
gen.sp. 13TU to nosocomial population of Acinetobacter, we used AFLP to overcome the problems
due to uncertain phenotypic identification by Vitek2 System. Our results showed a high
discriminatory power of AFLP and confirmed the capability of AFLP to identify correctly
Acinetobacter isolates at the species level (19; 24). A. baumannii isolates were predominant (84.5
%), followed by gen.sp. 3 (5.6 %), nor gen.sp. 13TU neither A. calcoaceticus isolates were
identified; the remaining seven isolates did not belong to the Acb-complex. The levels of similarity
of the species clusters were in agreement with observations made in previous works in which
AFLP (29) or 16S-23S rRNA gene intergenic spacer sequence (4) analysis were used. Overall,
these data highlight the usefulness of AFLP in elucidating the significance of different
Acinetobacter species in the hospital environment, and specifically the role of A. baumannii (9).
During three years of a real-time epidemiological surveillance program of nosocomial infections, a
constantly updated databank of Acinetobacter AFLP profiles has been established. This databank
has been demonstrated to be highly reliable in AFLP profiles similarity analysis over time, and had
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made possible to study the population structure of Acinetobacter isolates and to define the present,
detailed space/time distribution of A. baumannii clones in different wards and hospitals of the city
of Florence. AFLP analysis identified three well distinct clonal groups of A. baumannii (A1, A2
and A3), representing 60 % of all Acinetobacter isolates. The space/time dynamic analysis, we
performed at a city-scale, has shown that A1 and A2 were time persistent, inter-hospital diffused
clones, and could be defined successful hospital clones.
A. baumannii clusters of highly similar strains, which are assumed to represent distinct clonal
lineages, occur at different location and at different time points in Europe (25; 26). These
European clones (EU I–III) have been isolated in Italy (36); and also recently, A. baumannii
hospital isolates genetically related to EU I and E II clones were identified in Rome and other
Italian cities (8; 16). Accordingly, in this study, we have identified a clonal group (A1) with high
similarity to EU II.
Despite clonal variation in antibiotic susceptibility, the European clones are usually highly
resistant to antibiotics, and it is conceivable that carbapenem resistance has substantially
contributed to their spread in Europe (26).
In this study, most A. baumannii isolates (65 %) were resistant to imipenem (IR). The incidence of
the IR phenotype increased to 68 % if only one isolate per outbreak was considered. Interestingly,
the A2 clone, as the EU clones (26), shows relevant variation in antibiotic susceptibility; A2
isolates were IS in 2006, and only in 2008 I
R A2 strains appeared. A2 established itself as a
hospital successful strain well before IR acquisition; in fact in 2006, I
S A2 isolates had already
spread in different wards of different hospitals and they were associated with two outbreaks in H3-
W1. However, after IR acquisition, A2 became the dominant A. baumannii clone, almost
completely undermining the A1 clone, and a third outbreak event by IR A2 clone occurred in 2008.
As reported for other A. baumannii clones (32), A2 ability to cause outbreaks is likely to be
multifactorial and related to particular traits other than the simple possession of antibiotic
resistance mechanisms. Nevertheless, the acquisition of certain antibiotic determinants may favour
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the selection of specific strains in hospital settings (32). The higher genetic variability of A2
respect to A1 and A3 clonal groups, showed by PCA and AMOVA analyses, did not seem
correlated to the presence of the imipinem resistance trait. The intraclonal variation may result
from ongoing diversification in space and time (25), and it could result from a relatively frequent
horizontal acquisition or loss of resistance genes, as well as from differential expression of
intrinsic genes. Further analysis should elucidate other possible traits that make this clone
successful in nosocomial settings. In the future, it will be interesting to follow the dynamic of
these and other A. baumannii clones in the monitored hospitals to verify the occurrence of clone
succession and its dependence by the IR trait.
The presence of blaOXA51 gene in non A. baumannii strains, isolated in this work, is in agreement
with recent report from Lee et al. (21), questioning the usefulness of its detection for identification
purpose as previously suggested (34, 38). As the ISAba1 sequence is never present in the IR
isolates, the resistant phenotype did not appear to be associated to blaOXA51-like gene (18, 33);
conversely, it was consistently associated with blaOXA58-like gene, and ISAba2 and ISAba3 elements
flanking it. These results confirm the emergence of blaOXA58-like gene as a worldwide responsible of
carbapenem resistance in A. baumannii (1, 5, 17, 23, 27), at least partially explained by its plasmid
location (28). Accordingly, IR A2 could be arised from the already widespread I
R A1 clone by
horizontal gene transfer of a plasmid-resident resistance gene. The appearance of the IR A3 clone
in 2008 could be similarly explained. The identity of the sequence of the blaOXA58 gene from
representative of the A1, A2 and A3 clonal groups is in accordance with horizontal transfer of the
gene between strains sharing the same hospital environment. The horizontal transfer of blaOXA58-like
gene has been already demonstrated (40, 41).
In conclusion, AFLP, as already suggested by Spence et al. (32.), can be an important tool for
monitoring the distribution and the prevalence of A. baumannii genotypes in a defined space/time
context. In this study, by the establishment of an AFLP database, storing the fingerprint profiles of
a population of Acinetobacter isolates collected in three years of a hospital surveillance program,
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we were able to: 1) rapidly detect outbreak-events; 2) define the Acinetobacter population structure
at a city level of analysis; 3) to demonstrate the spread of relevant A. baumanni clones in the
hospitals under surveillance. The prompt identification of relevant clones is of great importance,
because it can lead to stop the spread of pathogens by immediate alert programmes (13).
Furthermore, the molecular analysis of the genetic determinants of imipenem resistance allowed us
to formulate a working hypothesis about the spread of the blaOXA58 gene and its role in the MDR
phenotype.
ACKOWLEDGMENTS
We are grateful to Dr. Emanuele Goti for its contribution to sizing analysis of AFLP products. We
also thank L Dijkshoorn for provision of the A. baumannii European clones I-III, and M. Giannouli
for providing us with imipenem-resistant A. baumannii strains carrying ISAba2 and ISAba3
elements. This study was supported by a grant from Regione Toscana.
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Table 1. Outbreak events (see also Figure 1) and epidemiological characteristics of involved
isolates.
a nd, data not available
Outbreak
event
Patient
Sample
collection
date
Hospital-
Ward
Infection/
Colonization
Imipenem
resistance/
sensitivity
AFLP
Profile
1 09/08/2006 IN
2 17/08/2006 C 1
3 17/08/2006
H3-W1
IN
IS A2
1 08/11/2006 IN
2 04/12/2006 IN 2
3 06/12/2006
H3-W1
C
IS A2
1 01/08/2008 nd a
2 08/08/2008 IN
3 23/08/2008 IN
3
4 23/08/2008
H1-W14
nd
IR A2 on M
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Table 2. Spatial distribution of A. baumannii isolates.
AFLP profile WARD
A1 A2 A3 / a
H1-W1 - 1 - 4
H1-W2 3 - - -
H1-W4 1 1 - -
H1-W5 1 - - -
H1-W6 2 3 1 -
H1-W10 - - - 1
H1-W12 - - - 1
H1-W13 1 - - -
H1-W14 - 4 - -
H1-W15 - 1 - -
H1-W16 - - - 1
H1-W17 - 1 1 -
H1-W18 - 2 - -
H1-W19 - - - 3
H1-W20 - - - 1
H1-W21 - - - 1
H2-W2 - 1 - -
H3-W1 1 7 - 2
H4-W1 - - - 1
H5-W1 - - - 3
H6-W1 - 3 1 6
Total 9 24 3 24
a A. baumannii isolates with unique AFLP profile
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LEGENDS TO FIGURES
Figure 1. AFLP analysis dendrogram. Bars: thick bar, Acinetobacter sp. isolates ; thin bars, A1,
A2 and A3 clonal groups of A. baumannii isolates. Boxes: IN/C, patient’s infection (black),
colonization (grey), data not available (white); R/S, resistance (R≥16, black) or susceptible (grey)
to imipenem, data not available (white); OXA genes, contemporary presence of OXA-51 and
OXA-58 (black), presence of OXA51 (grey), absence of both OXA51 and OXA58 (white). Dots at
the end of corresponding tree branches (from top to bottom): EU I, EU II, and EU III clones
(white); ATCC reference strains of A. baumannii, gen. sp. 13TU, A.coalcaceticus, gen.sp.3 (black).
Shadowed boxes within the dendrogram: 1, 2 and 3 define groups of isolates involved in outbreak
events (see also Table 1).
Figure 2. Time distribution of A. baumannii clones A1 ( ), A2 ( ), A3( ) and isolates with unique
AFLP profile ( ) in hospital settings.
Figure 3. Principal component analysis of the AFLP profiles of A. baumannii isolates: A1 ( ), A2
( ), A3 ( ) clones, and isolates with unique profile ( ).
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