1

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

2

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.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

3

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

4

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

5

(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.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

6

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.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

7

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

8

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

9

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

10

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

11

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

12

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

13

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

14

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,

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

15

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.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

16

REFERENCES

1) Bertini, A., L. Poirel, S. Bernabeu, D. Fortini, L. Villa, P. Nordmann, and A. Carattoli.

2007. Multicopy blaOXA-58 gene as a source of high-level resistance to carbapenems in

Acinetobacter baumannii. Antimicrob. Agents Chemother. 51:2324-2328.

2) Bergogne-Bérézin, E., and K. J. Towner. 1996. Acinetobacter spp. as nosocomial

pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9:

148–165.

3) Brown, S., and S. Amyes. 2006. OXA {beta}-lactamases in Acinetobacter: the story so far.

J. Antimicrob. Chemother. 57:1-3.

4) Chang, H. C., Y. F. Wei, L. Dijkshoorn, M. Vaneechoutte, C. T. Tang, and T. C.

Chang. 2005. Species-Level Identification of Isolates of the Acinetobacter calcoaceticus-

Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer

region. J. Clin. Microbiol. 43:1632-1639.

5) Coelho, J., N. Woodford, M. Afzal-Shah, and D. Livermore. 2006. Occurrence of OXA-

58-Like carbapenemases in Acinetobacter spp. collected over 10 years in three continents.

Antimicrob. Agents Chemother. 50:756-758.

6) D’Agata E.M., M.M. Gerrits, Y.W. Tang, M. Samore, J.G. Kusters. 2001. Comparison

of pulsed-field gel electrophoresis and amplified fragment-lenght polymorphism for

epidemiological investigations of common nosocomial pathogens. Infect. Control Hosp.

Epidemiol. 22:550-554.

7) Dalmastri, C., F. Alessia, A. Chiara, B. Annamaria, T. Silvia, G. Giovanni, S. Anna

Rosa, S. Lia, M. Eshwar, C. Luigi, and V. Peter. 2003. A rhizospheric Burkholderia

cepacia complex population: genotypic and phenotypic diversity of Burkholderia

cenocepacia and Burkholderia ambifaria. FEMS Microbiol. Ecol. 46:179-187.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

17

8) D’Arezzo S., A. Capone, N. Petrosillo, P. Visca. 2009. Epidemic multidrug-resistant

Acinetobacter baumannii related to European clonal types I and II in Rome (Italy). Clin.

Microbiol. Infect. 15:347-357.

9) Dijkshoorn, L., A. Nemec, and H. Seifert. 2007. An increasing threat in hospitals:

multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 5:939-951.

10) Excoffier, L., P. E. Smouse, and J. M. Quattro. 1992. Analysis of molecular variance

inferred from metric distances among DNA haplotypes: application to human mitochondrial

DNA restriction data. Genetics 131:479-491.

11) Excoffier, L., G. Laval, S. Schneider. 2005. Arlequin version 3.1: an integrated software

package for population genetics data analysis. Evolutionary Bioinformatics Online 1: 47–

50.

12) Fanci, R., B. Bartolozzi, S. Sergi, E. Casalone, P. Pecile, D. Cecconi, R. Mannino, F.

Donnarumma, A. G. Leon, S. Guidi, P. Nicoletti, G. Mastromei, and A. Bosi. 2008.

Molecular epidemiological investigation of an outbreak of Pseudomonas aeruginosa

infection in an SCT unit. Bone Marrow Transplant 43:335-338.

13) Fontana, C., M. Favaro, M.C. Bossa, G.P. Testore, F. Leonardis, S. Natoli, and C.

Favalli,. 2008. Acinetobacter baumannii in intensive care unit: a novel system to study

clonal relationship among the isolates. BMC Infect. Dis. 11:868-873.

14) Gaston, M.A., and P.R., Hunter.1989. Efficient selection of tests for bacteriological

typing schemes J. Clin. Pathol . 42: 763–766.

15) Gerner-Smidt, P., and I. Tjernberg. 1993. Acinetobacter in Denmark: II. Molecular

studies of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex. APMIS 101:

826–832.

16) Giannouli M., F. Tomasone, A. Agodi, H. Vahaboglu, Z. Daoud, M. Triassi, A. Tsakris,

R. Zarrilli. 2009. Molecular epidemiology of carbapenem-resistant Acinetobacter

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

18

baumannii strains in intensive care units of multiple Mediterranean hospitals. J. Antimicrob.

Chemother. 63:828-830.

17) Giordano, A., P. Varesi, A. Bertini, L. Villa, A. Dionisi, M. Venditti, P. Carfagna, I.

Luzzi, C. Mancini, and A. Carattoli A. 2007. Outbreak of Acinetobacter baumannii

producing the carbapenem-hydrolyzing oxacillinase OXA-58 in Rome, Italy. Microbial

Drug Resist. 13: 37-43

18) Hu, W. S., S.-M. Yao, C.-P. Fung, Y.-P. Hsieh, C.-P. Liu, and J.-F. Lin. 2007. An OXA-

66/OXA-51-like carbapenemase and possibly an efflux pump are associated with resistance

to imipenem in Acinetobacter baumannii. Antimicrob. Agents Chemother. 51:3844-3852.

19) Janssen, P., K. Maquelin, R. Coopman, I. Tjernberg, P. Bouvet, K. Kersters, and L.

Dijkshoorn. 1997. Discrimination of Acinetobacter genomic species by AFLP

fingerprinting. Int. J. Syst. Bacteriol. 47:1179-1187.

20) Jawad, A., H. Seifert, A. M. Snelling, J. Heritage, and P. M. Hawkey. 1998. Survival of

Acinetobacter baumannii on dry surfaces: comparison of outbreak and sporadic isolates. J.

Clin. Microbiol. 36:1938-1941.

21) Lee Y.T., J.F. Turton, T.L. Chen, R.C. Wu, W.C. Chang, C.P. Fung, C.P. Chen, W.L

Cho., L.Y. Huang, L.K. Siu. 2009. First identification of blaOXA-51-like in non-

baumannii Acinetobacter spp. J. Chemother. 21:514-520.

22) Linton, C. J., A. M. Borman, G. Cheung, A. D. Holmes, A. Szekely, M. D. Palmer, P.

D. Bridge, C. K. Campbell, and E. M. Johnson. 2007. Molecular identification of

unusual pathogenic yeast isolates by large ribosomal subunit gene sequencing: 2 years of

experience at the United Kingdom mycology reference laboratory. J. Clin. Microbiol.

45:1152-1158.

23) Marqué, S., L. Poirel, C. Heritier, S. Brisse, M. D. Blasco, R. Filip, G. Coman, T. Naas,

and P. Nordmann. 2005. Regional occurrence of plasmid-mediated carbapenem-

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

19

hydrolyzing oxacillinase OXA-58 in Acinetobacter spp. in Europe. J. Clin. Microbiol.

43:4885-4888.

24) Nemec, A., T. De Baere, I. Tjernberg, M. Vaneechoutte, T. J. K. van der Reijden, and

L. Dijkshoorn. 2001. Acinetobacter ursingii sp. nov. and Acinetobacter schindleri sp. nov.,

isolated from human clinical specimens. Int. J. Syst. Evol. Microbiol. 51:1891-1899.

25) Nemec, A., L. Dijkshoorn, and T. J. K. van der Reijden. 2004. Long-term predominance

of two pan-European clones among multi-resistant Acinetobacter baumannii strains in the

Czech Republic. J. Med. Microbiol. 53:147-153.

26) Nemec, A., L. Křízova, M. Maixnerova, L. Diancourt, T.J. van der Reijden, S. Brisse,

P. van den Broek, and L. Dijkshoorn. 2008 Emergence of carbapenem resistance in

Acinetobacter baumannii in the Czech Republic is associated with the spread of multidrug-

resistant strains of European clone II. J. Antimicrob. Chemother. 62:484-489.

27) Peleg, A. Y., J. M. Bell, A. Hofmeyr, and P. Wiese. 2006. Inter-country transfer of Gram-

negative organisms carrying the VIM-4 and OXA-58 carbapenem-hydrolysing enzymes. J.

Antimicrob. Chemother. 57:794-795.

28) Poirel, L., S. Marqué, C. Heritier, C. Segonds, G. Chabanon, and P. Nordmann. 2005.

OXA-58, a novel class D {beta}-lactamase involved in resistance to carbapenems in

Acinetobacter baumannii. Antimicrob. Agents Chemother. 49:202-208.

29) Savelkoul, P. H. M., H. J. M. Aarts, J. de Haas, L. Dijkshoorn, B. Duim, M. Otsen, J.

L. W. Rademaker, L. Schouls, and J. A. Lenstra. 1999. Amplified-fragment length

polymorphism analysis: the state of an art. J. Clin. Microbiol. 37:3083-3091.

30) Segal, H., S. Garny, and B. G. Elisha. 2005. Is ISAba1 customized for Acinetobacter?

FEMS Microbiol. Letters 243:425-429.

31) Sergi S., F. Donnarumma, G. Mastromei, E. Goti, P. Nicoletti, P. Pecile, D. Cecconi, R.

Mannino, R. Fanci, A. Bosi, E. Casalone. 2009. Molecular surveillance and population

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

20

structure analysis of methicillin-susceptible and methicillin-resistant Staphylococcus aureus

in high-risk wards. J. Clin. Microbiol. 47:3246-3254.

32) Spence, R.P., T.J. van der Reijden, L. Dijkshoorn, and K.J. Towner. 2004. Comparison

of Acinetobacter baumannii isolates from United Kingdom hospitals with predominant

northern european genotypes by Amplified-Fragment Length Polymorphism analysis. J.

Clin. Microbiol. 42: 832–834.

33) Turton, J. F., M. E. Ward, N. Woodford, M. E. Kaufmann, R. Pike, D. M. Livermore,

and T. L. Pitt. 2006. The role of ISAba1 in expression of OXA carbapenemase genes in

Acinetobacter baumannii. FEMS Microbiol. Letters 258:72-77.

34) Turton, J. F., N. Woodford, J. Glover, S. Yarde, M. E. Kaufmann, and T. L. Pitt. 2006.

Identification of Acinetobacter baumannii by detection of the blaOXA-51-like

carbapenemase gene intrinsic to this species. J. Clin. Microbiol. 44:2974-2976.

35) van den Broek, P. J., J. Arends, A. T. Bernards, E. De Brauwer, E. M. Mascini, T. J.

K. van der Reijden, L. Spanjaard, E. A. P. M. Thewessen, A. van der Zee, J. H. van

Zeijl, and L. Dijkshoorn. 2006. Epidemiology of multiple Acinetobacter outbreaks in the

Netherlands during the period 1999-2001. Clin. Microbiol. Infect. 12:837-843.

36) van Dessel, H., L. Dijkshoorn, T. van der Reijden, N. Bakker, A. Paauw, P. van den

Broek, J. Verhoef, and S. Brisse. 2004. Identification of a new geographically widespread

multiresistant Acinetobacter baumannii clone from European hospitals. Res. Microbiol.

155:105-112.

37) Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. v. d. Lee, M. Hornes, A. Friters, J. Pot,

J. Paleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new technique for DNA

fingerprinting. Nucl. Acids Res. 23:4407-4414.

38) Woodford, N., M. J. Ellington, J. M. Coelho, J. F. Turton, M. E. Ward, S. Brown, S.

G. B. Amyes, and D. M. Livermore. 2006. Multiplex PCR for genes encoding prevalent

OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 27:351-353.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

21

39) Wright S. 1978. Variability within and among natural populations. In: Evolution and the

Genetics of Populations Vol. 4, University of Chicago Press, Chicago.

40) Zarrilli R., D Vitale., A. Di Popolo, M. Bagattini, Z. Daoud, A.U. Khan, C. Afif, M.

Triassi. 2008. A plasmid-borne blaOXA-58 gene confers imipenem resistance to

Acinetobacter baumannii isolates from a Lebanese hospital. Antimicrob. Agents Chemother.

52:4115-4120.

41) Zarrilli, R., M. Giannouli, F. Tomasone, M. Triassi, and A.Tsakris. 2009. Carbapenem

resistance in Acinetobacter baumannii: the molecular epidemic features of an emerging

problem in health care facilities. J. Infect. Dev. Ctries. 3:335-341.

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

22

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

arch 23, 2021 by guesthttp://jcm

.asm.org/

Dow

nloaded from

23

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

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

24

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 ( ).

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from

on March 23, 2021 by guest

http://jcm.asm

.org/D

ownloaded from