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Research in Microbiology 155 (2004) 105–112 www.elsevier.com/locate/resmic

Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals Helke van Dessel a , Lenie Dijkshoorn b , Tanny van der Reijden b , Nancy Bakker b , Armand Paauw a , Peterhans van den Broek b , Jan Verhoef a , Sylvain Brisse a,∗ a Eijkman-Winkler Center for Microbiology, Infectious Diseases and Inflammation, University Medical Center Utrecht, AZU G04.614, P.O. Box 85500,

3508 GA, Utrecht, The Netherlands b Department of Infectious Diseases C5-P, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands

Received 15 July 2003; accepted 2 October 2003 First published online 7 October 2003

Abstract The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.  2003 Elsevier SAS. All rights reserved. Keywords: Acinetobacter; Epidemic clone; Antimicrobial resistance; Ribotyping; Genetic characterization

1. Introduction Members of the genus Acinetobacter are increasingly important nosocomial pathogens, and numerous well-documented hospital outbreaks have been described, mostly caused by multiresistant Acinetobacter baumannii [1,4,7,9, 17,30,35]. In addition, several studies have shown the geographically widespread occurrence of multiresistant A. baumannii strains, which are genotypically and phenotypically highly similar, suggesting a clonal relatedness of these strains. A study of A. baumannii strains from several un* Corresponding author. Present address: Unité Biodiversité des Bac-

téries Pathogènes Emergentes, INSERM U389, Institut Pasteur, 25–28, rue du Docteur Roux, 75724 Paris cédex 15, France. E-mail address: [email protected] (S. Brisse). 0923-2508/$ – see front matter  2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.10.003

related hospital outbreaks in northwestern Europe revealed the existence of two major clones (clone I and II) [11], while Vila et al. [38] described one widespread amikacin-resistant clone in Spanish hospitals. Furthermore, phenotypic and genotypic typing of 103 clinical Acinetobacter isolates from the Czech Republic led to the recognition of two predominant groups (group A and B), corresponding at least in part to the northwestern outbreak clones I and II [28]. The origin of these widespread clones is unknown. One possibility is that they have been spreading from hospital to hospital in the course of time, but it is also possible that they originate from yet unknown reservoirs in the community or environment, whilst antibiotic usage acts as a selective force favouring the emergence of those strains that have the genetic potential to develop antibiotic resistance. It is not yet known whether the emergence of A. baumannii worldwide is

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Fig. 1. AFLP fingerprints obtained for the 50 strains under study. The UPGMA dendrogram was obtained after cluster analysis using the Pearson coefficient and a 2% optimization parameter. On the dendrogram scale, the Pearson coefficient was converted to percent values by the software BioNumerics (Applied Maths). Strain codes, AFLP types, PFGE types, genotypes based on combined PFGE and AFLP data, ribotypes (RT) and geographic origin are given. The dotted line corresponds to the cut-off level of 90% used to delineate AFLP types.

due to the expansion of the clonal lineages described in Europe or of other clonal lineages. One study of blood isolates in the USA did not indicate any interhospital spread [40], but clonal spread of carbapenem-resistant A. baumannii was reported recently [29]. Of the various molecular typing methods applied to Acinetobacter, ribotyping, pulsed-field gel electrophoresis (PFGE) and AFLP have proven valuable to distinguish strains in hospital outbreak investigation and epidemiological studies [4,5,11,16,20,36,37], while AFLP also appeared to be a key method to delineate the northwestern European clones I and II. In a previous study [5], automated ribotyping was performed on 159 Acinetobacter isolates that were collected from 18 European and South African hospitals. Most (145 out of 159) of the isolates were selected because they showed decreased susceptibility to sitafloxacin, an advanced-generation fluoroquinolone, and all selected strains were resistant to other classes of antibiotics. The 159 isolates were separated into 21 ribogroups, and three of these ribogroups were particularly interesting because they were

both numerically important and geographically widespread. Ribogroup 210-60-2, which includes reference strains of clone I [11] and which we now name ribogroup 1 for simplicity and clarity, included 17 isolates and was found in Italy, Poland, Spain and South Africa. Ribogroup 210-41-3, which includes reference strains of clone II [11] and which we now name ribogroup 2, included 52 isolates and was found in Belgium, France, Germany, Greece, Poland, Portugal, Spain and Turkey. Ribogroup 210-52-4 (ribogroup 3) included 23 isolates and was found in France, Italy, The Netherlands and Spain. Isolates belonging to these major ribogroups generally showed higher levels of resistance to gentamicin and imipenem compared to isolates belonging to sporadic ribogroups. While the genus Acinetobacter currently comprises 32 named and unnamed species [6,23,24] and many yet unclassified strains, it is A. baumannii that is mostly encountered among clinical isolates and is responsible for most outbreaks [1]. Commercial identification systems based on phenotypic tests are unable to identify most Acinetobacter

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species [2,3]. Amplified ribosomal DNA analysis (ARDRA) is one of the few genotypic methods that has been validated using a large set of reference strains to identify species of the genus Acinetobacter [13]. In the present study, we aimed to verify the provisional identification of the isolates belonging to the three widespread ribogroups 1, 2 and 3 that was made based on automated ribotyping [5]. AFLP cluster analysis was performed to investigate the correspondence between ribogroups and the previously described northwestern European clones, defined primarily based on AFLP [11]. In addition, as ribotyping with EcoRI was shown to be less discriminatory then PFGE and AFLP for A. baumannii [31], we used these more discriminatory typing methods in order to more finely delineate possible groups of indistinguishable isolates, some of which may correspond to subsets of isolates with closer clonal relationships.

2. Materials and methods 2.1. Strains A total of 50 strains were selected for this study. These included strains RUH 875 and RUH 134, which are reference strains of the northwestern European clones I and II, respectively [11], and 48 clinical isolates from a distinct patient each. These clinical isolates belonged to three widespread ribogroups from a previous study [5] and were selected to be geographically representative of their respective ribogroup. The selection was performed as follows: at least one isolate of each ribogroup was included from each centre in which these ribogroups were found. If two or more isolates of one of these ribogroups were found in a centre, then approximately half of the total number of isolates of this ribogroup from this centre were selected randomly for inclusion. Thus, 10 isolates of ribogroup 1, previously called ribogroup 60-S-2 [5], were selected out of 17 previously identified isolates; 23 isolates of ribogroup 2, previously called ribogroup 41-S-3 [5], were selected out of 52 previously identified isolates; and 15 isolates of ribogroup 3, previously called ribogroup 52-S-4 [5], were selected out of 23 previously identified isolates [5]. The clinical and geographical origins of the strains are given in Fig. 1 and Table 1. The minimum inhibitory concentrations of 16 different antimicrobial agents (or combinations thereof ) were determined previously [5]. 2.2. ARDRA The method was carried out as described earlier [13] with minor modifications [25]. Briefly, amplified 16S rDNA was obtained by PCR and separate aliquots were digested with five restriction endonucleases (CfoI, AluI, MboI, RsaI, and MspI). The fragments obtained with each enzyme were separated by 2.5% agarose gel electrophoresis. Species identification was achieved by comparing the restriction

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profiles to those of a library of strains of described (genomic) species [13]; the reference profiles are available at http://allserv.rug.ac.be/~mvaneech/ARDRA/Acinetobacter. html. 2.3. AFLP fingerprinting The method was performed according to Nemec et al. [23]. Briefly, purified DNA was digested using EcoRI and MseI, while ligation of EcoRI and MseI adapters was performed simultaneously. PCR was done with a Cy5-labelled EcoRI + A primer (Cy5-GAC-TGCGTACCAATTCa-3; a = selective A base), and a MseI + C primer (5 -GATGAGTCCTGAGTAAc-3 ; c = selective C base). The ALF express™ DNA analysis system (Amersham Biosciences) was used for fragment separation. The peak patterns obtained were converted to TIFF files and fragments of 50–500 bp were subjected to cluster analysis using the BioNumerics 3.0 software package (Applied Maths, Sint-Martens-Latem, Belgium). The Pearson product moment coefficient (r) was used as a measure of similarity, and the unweighted pair-group arithmetic average clustering method (UPGMA) was used for grouping, using an optimization parameter of 2% and after maskingout the portions that were not relevant at the two extremities of the patterns. For species identification, the strains were grouped with strains of all described (genomic) species, using a cut-off level of 50% for species delineation [25]. AFLP genotypes were defined using a 90% clustering level, which in our hands has proven appropriate to define outbreaks. 2.4. PFGE The pellet from an overnight broth culture was resuspended in 75 mM NaCl–25 mM EDTA (pH 7.5) to a density equivalent to 0.5 McFarland. Suspensions were mixed with equal volumes of melted 2% agarose (Bio-Rad Laboratories, Richmond, USA), dispensed into wells of a plug mold, and allowed to solidify. The plugs were incubated overnight in lysis solution (6 mM Tris, 100 mM EDTA, 1 M NaCl, 0.5% w/v Brij 58 (polyoxyethylene 20 cetyl ether), 0.2% w/v sodium deoxycholate, 0.5% w/v N -lauryl sarcosine, 1 mM MgCl2 ; pH 7.5) at 55 ◦ C. Subsequently, the plugs were incubated overnight in proteolysis buffer (1% w/v N -lauryl sarcosine, 0.5 M EDTA; pH 9.5) at 55 ◦ C and digested with 20 U of ApaI restriction enzyme (Boehringer, Mannheim, Germany) at 30 ◦ C overnight. Electrophoresis was performed at 200 V constant voltage by the CHEF-DR II system (Bio-Rad) with pulse times ramping from 5 to 35 s for 24 h. Gels were stained with ethidium bromide and photographed under UV light. We defined PFGE types as sets of strains with at least four different bands among their PFGE profiles. Bands were defined after visual correction of the automatic band search algorithm of the BioNumerics package.

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Table 1 Overview of the 50 strains under study with typing data, antimicrobial susceptibility and geographic origin Strain code

Other code

Ribotype

AFLP

PFGE

Combined

SIT

18A418 18A410 18A518 18C075 18C103 18C144 18C132 09A242 18A350 17C003 36D042 15A250 16E057 16E027 16C085 16A502 16C024 16A528 16D086 06A102 16D042 22D035 RUH 134 14C003 10C070 18D047 06A201 12A133 04C048 06A225 16D025 12A126 18A155 18D001 18C005 18A025 17C085 18D017 16D083 17C078 36C058 11C096 11E057 11A221 11A352 11D078 RUH 875 11 COOS 11A018 14C052

LUH 6040 LUH 6039 LUH 6041 LUH 6043 LUH 6044 LUH 6045 LUH 5865 LUH 6011 LUH 6038 LUH 6034 LUH 6051 LUH 6023 LUH 6033 LUH 6032 LUH 6027 LUH 6024 LUH 6026 LUH 6025 LUH 6031 LUH 5868 LUH 6029 LUH 6049 RUH 134 LUH 6021 LUH 6012 LUH 6048 LUH 5874 LUH 5875 LUH 6009 LUH 6010 LUH 6028 LUH 6020 LUH 6037 LUH 6046 LUH 6042 LUH 6036 LUH 6035 LUH 6047 LUH 6030 LUH 5881 LUH 6050 LUH 6017 LUH 6019 LUH 6014 LUH 6015 LUH 6018 RUH 875 LUH 6016 LUH 6013 LUH 6022

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 2 3 3 4 4 4 4 4 4 4 4 4 4 4 5 5 5 6 7 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 10 10 10 10 10 11 12 13 14

1 1 1 1 1 1 1 6 1 1 5 5 2 2 2 3 2 3 2 9 3 4 8 7 10 12 10 10 10 10 10 10 10 10 10 10 10 10 11 13 14 17 15 16 16 17 18 19 16 20

1 1 1 1 1 1 1 2 3 3 6 6 4 4 4 5 4 5 4 7 5 8 10 9 11 12 13 13 13 13 13 13 13 13 13 13 13 13 14 22 23 17 15 16 16 17 18 19 20 21

2 2 2 2 2 4 2 0.5 2 2 0.12 1 2 4 2 2 2 4 2 2 2 0.06 – 2 2 2 2 4 2 8 2 2 4 4 2 2 4 2 2 2 2 2 2 2 2 2 – 2 4 2

CIP

P/T

CTR

CAZ

IMI

GEN

AMIK

COT

City, country

> 16 > 128 > 16 > 128 > 16 > 128 > 16 > 128 > 16 > 128 > 16 > 128 > 16 > 128 > 16 32 > 16 > 128 > 16 > 128 2 16 > 16 128 > 16 64 > 16 > 128 > 16 128 > 16 64 > 16 64 > 16 64 > 16 > 128 > 16 16 > 16 > 128 0.5 > 128 – – > 16 32 > 16 16 > 16 > 128 > 16 4 > 16 > 128 > 16 4 > 16 0.12 > 16 64 > 16 > 128 > 16 64 > 16 32 > 16 32 > 16 64 > 16 > 128 > 16 64 > 16 > 128 > 16 16 > 16
0.06 > 16 128 > 16 16 > 16 > 128 > 16 16 > 16 > 128 – – > 16 > 128 > 16 64 > 16 32

> 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 – 64 64 > 64 32 > 64 > 64 32 > 64 > 64 > 64 64 > 64 > 64 > 64 > 64 32 32 64 > 64 > 64 > 64 > 64 > 64 – > 64 > 64 64

> 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 > 64 16 64 32 64 32 32 32 32 64 > 64 32 > 64 – 16 16 16 32 32 16 16 32 32 32 32 16 32 > 64 16 8 32 32 > 64 > 64 > 64 > 64 > 64 – > 64 > 64 16

> 16 > 16 > 16 > 16 > 16 > 16 > 16 1 > 16 > 16 0.5 1 2 8 2 2 1 2 2 8 2 1 – 1 0.5 8 0.5 1 0.5 1 1 1 2 0.5 1 1 > 16 1 8 0.25 0.5 1 0.5 2 0.25 1 – 8 2 0.12

> 32 > 32 > 32 > 32 > 32 > 32 > 32 16 > 32 > 32 > 32 8 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 – > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 > 32 – > 32 > 32 > 32

64 > 64 > 64 4 > 64 4 2 16 > 64 > 64 64 4 > 64 > 64 > 64 > 64 > 64 > 64 > 64 64 > 64 4 – > 64 > 64 > 64 32 > 64 > 64 > 64 > 64 64 64 64 64 64 > 64 64 64 32 2 > 64 2 > 64 2 > 64 – > 64 > 64 > 64

>8 >8 >8 4 >8 8 8 4 8 >8 2 >8 >8 >8 >8 >8 >8 >8 >8 >8 0.25 >8 – >8 >8 >8 >8 >8 2 >8 >8 >8 >8 >8 >8 >8 >8 >8 4 >8 >8 >8 >8 >8 >8 >8 – >8 8 >8

Barcelona, SP Barcelona, SP Barcelona, SP Barcelona, SP Barcelona, SP Barcelona, SP Barcelona, SP Athens, GR Barcelona, SP Madrid, SP Pretoria, SA Coimbra, PT Sevilla, SP Sevilla, SP Sevilla, SP Sevilla, SP Sevilla, SP Sevilla, SP Sevilla, SP Lille, FR Sevilla, SP Ankara, TK Clone II ref. Krakow, PO Genoa, IT Barcelona, SP Lille, FR Utrecht, NL Paris, FR Lille, FR Sevilla, SP Utrecht, NL Barcelona, SP Barcelona, SP Barcelona, SP Barcelona, SP Madrid, SP Barcelona, SP Sevilla, SP Madrid, SP Pretoria, SA Rome, IT Rome, IT Rome, IT Rome, IT Rome, IT Clone I ref. Rome, IT Rome, IT Krakow, PO

SIT, sitafloxacin; CIP, ciprofloxacin; P/T, piperacillin-tazobactam; CTR, ceftriaxone; CAZ, ceftazidime; IMI, imipenem; GEN, gentamicin; AMIK, amikacin; COT, trimethoprim-sulfamethoxazole. The susceptibility data of reference strains RUH 134 and RUH 875 were not determined using the same methods and they are therefore not presented here.

3. Results 3.1. Identification of isolates as A. baumannii Except for the isolates received from Rome, all isolates had the combined profile ‘1 1 1 2 3’ or ‘1 1 1 2 1’ for

the respective enzymes CfoI, AluI, MboI, RsaI and MspI, which according to the library of profiles of reference strains identified the organisms as A. baumannii. The Rome strains (Fig. 1) had a new CfoI restriction pattern. The resulting fiveenzyme restriction profile ‘new 1 1 2 1’ was not included in the library of reference strains, which left these strains

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unidentified by ARDRA. Cluster analysis of the AFLP profiles of the isolates under study with approximately 200 reference strains representing all described genomic species showed that all strains of the study, including the Rome strains, were linked at 72% (Fig. 1), whereas they were linked with other A. baumannii strains at ∼50%, which identified all isolates as A. baumannii. 3.2. Diversity revealed by AFLP and PFGE Inspection of the dendrogram generated by cluster analysis of the AFLP profiles of the 50 study isolates led to the distinction of 14 different types at a 90% clustering level. Ribogroup 1 was subdivided into six AFLP types, ribogroup 2 into five AFLP types, and ribogroup 3 into three AFLP types (Fig. 1). Isolates of a given ribogroup clustered into a single AFLP branch (Fig. 1). Ribogroup 3 appeared more homogeneous than the two other ribogroups, based on the number of types and the overall similarity level (87%) of the isolates (Fig. 1). Strain RUH 875, which is representative of the previously described northwestern European clone I, showed a unique AFLP pattern (Fig. 1) and was grouped within the branch formed at a level of 78% by isolates of ribogroup 1. Strain RUH 134, which is representative of the previously described northwestern European clone II, clustered together with strain 22D035 from Ankara and strain 14C003 from Krakow at 92.5% and with the other isolates of ribogroup 2 at 82%. PFGE analysis led to the distinction of 21 different types, defined as showing at least four band differences. Ribogroup 1 was subdivided into eight PFGE types, ribogroup 2 into nine PFGE types, and ribogroup 3 into four PFGE types (Fig. 1). Overall, there was an excellent correspondence between PFGE and AFLP categorization into types (Fig. 1). When the PFGE types were distinct for isolates with identical AFLP types, the PFGE patterns of these distinct types were very similar. Combined AFLP and PFGE data led to the distinction of 23 different types: nine in ribogroup 1, ten in ribogroup 2 and four in ribogroup 3. 3.3. Distribution of the genotypes revealed by combined AFLP and PFGE data Table 1 shows the genotype, geographic origin and antimicrobial resistance profile of all isolates. The 23 genotypes discriminated when combining AFLP and PFGE data were distributed into 15 genotypes corresponding to single isolates and eight genotypes with at least two isolates each, representing 35 isolates in total (Table 1). Five of these multiple-isolate genotypes (genotypes 1, 4, 5, 16, 17 with, respectively, 7, 5, 3, 2, and 2 isolates; see Table 1) contained isolates from a single hospital, probably indicating spread of A. baumannii in that particular hospital. Three genotypes (genotypes 3, 6 and 13) were distributed in more than one hospital. Genotype 3 was found in two Spanish

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hospitals in Madrid and Barcelona. Genotype 6 was found in a Portuguese hospital in Coimbra and a South African hospital in Pretoria. Genotype 13 contained 12 isolates isolated from France (two centres), Spain (three centres) and the Netherlands (one centre). This genotype was isolated from five distinct patients from one Barcelona centre, suggesting clonal spread among patients. Antimicrobial profiles of the isolates of genotype 13 were homogeneous within a single centre, but differed among some centres: the isolates from France were susceptible to piperacillin/tazobactam and imipenem, but isolates from Utrecht, Sevilla and Barcelona were resistant to piperacillin/tazobactam, and the isolate from Madrid was, in addition, resistant to imipenem. Similarly, the two isolates of genotype 6 differed in susceptibility to piperacillin/tazobactam, gentamicin and amikacin. Notably, these two isolates showed decreased susceptibility to ciprofloxacin, but were susceptible to sitafloxacin. In contrast, isolates of genotype 3 were indistinguishable by their antibiogram. Notably, they were both resistant to gentamicin, amikacin, imipenem, piperacillin/tazobactam and showed decreased susceptibility to sitafloxacin. The five genotypes containing multiple isolates from a single centre all showed the same antimicrobial resistance pattern within the centre, with the exception of genotype 1, which contained three isolates that were susceptible to amikacin (Table 1).

4. Discussion The clonal spread of multiresistant bacteria is a major factor contributing to the growing problem of antimicrobial resistance. In several bacterial species, it was realized that strains that spread among patients and are responsible for major outbreaks often belong to a limited number of clones [8,21,34,39]. In A. baumannii, the comparison of sporadic and outbreak strains from northwestern European hospitals suggested that most of the investigated outbreak strains belonged to two major clones, named clone I and II [11]. In a previous study, we performed automated ribotyping with EcoRI on 145 sitafloxacin-resistant and 14 sitafloxacinsusceptible Acinetobacter isolates from 18 hospitals in 13 European countries and South Africa and identified three widespread ribogroups [5]. It is known [31] that the resolving capacity of ribotyping is relatively small as compared to AFLP and PFGE, as ribotyping only detects variation in and around ribosomal RNA operons, whereas the other two methods detect variation across the entire genome. In the present study, we investigated the correspondence between ribogroups and the previously described northwestern clones, and the possible diversity within the three major ribogroups using the high-resolution DNA fingerprinting methods PFGE and AFLP. We first sought to verify the identification of the isolates with Acinetobacter species using AFLP and ARDRA as compared to the previously performed identification based

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on automated ribotyping [5]. All isolates were identified as A. baumannii, which corroborates the fact that this species is the Acinetobacter species most commonly encountered in hospital outbreaks. The fact that identification as A. baumannii was also reached by automated ribotyping [5] confirms the identification ability of the latter method for Acinetobacter (genomic) species included in the A. calcoaceticus– A. baumannii complex, as described before [15]. Like the species concept, the clone concept is a matter of debate [12,22,41], and there is no single definition or consensus method to delineate clones. For instance, the offspring of a single strain spreading in a hospital over a short period can be denoted as a clone of the index strain. In the early 1980s, however, it was recognized that isolates of a given species but found at different locations and times could be highly similar, and the isolates were also considered descendants of a common ancestor, i.e., belonging to the same clone [27]. The previously mentioned clones I and II, as well as the Czech groups A and B [26], were characterized by a ‘polyphasic’ approach including both genotypic and phenotypic methods, although the methods in both studies were not the same. In the original definition of clones I and II [11], strains of each of these clones were linked at a 90% cut-off level. However, the AFLP protocol used then, which included radioactive labelling and other restriction enzymes and selective primers, was different. Using the current protocol, the linkage level of clones I and II is around 80%, depending on the clone and the strain set (Dijkshoorn, unpublished data; Nemec et al., submitted). When the AFLP profiles from our study strains were compared with those of many other strains of clones I and II (Dijkshoorn, unpublished), the isolates of ribogroups 1 and 2 clustered with clones I and II, also around the level of 80%, and isolates of ribogroup 3 clustered separately. Therefore, we conclude that isolates of ribogroups 1 and 2 belong to clones I and II, respectively, whereas ribogroup 3 represents a new widespread clone, which we name clone III. Based on the current set of strains, clone III appears more homogeneous than clones I and II, as revealed both by the similarity of AFLP patterns and by the number of distinct genotypes. Clone III had a wide distribution as it was found in Spain, France and The Netherlands. Our results also imply that clones I and II are not limited to northwestern countries, as they were also isolated is Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). As these two clones are also highly prevalent in the Czech Republic ([26]; Nemec et al., submitted), they appear to be widely present in Europe but also in South Africa, suggesting that they may be found throughout the world. In the present study, we combined typing results of AFLP and PFGE to delineate genotypes. The purpose was to identify smaller subsets of strains with a close genetic relationship, which may correspond to subclones of the three defined clones. Although it is unclear how fast the PFGE patterns evolve in A. baumannii, we believe that the four-band cri-

teria used to distinguish PFGE types is stringent [26]. Three genotypes were found in different geographic locations. Two of these, genotype 3 and 6, belong to clone II. Interestingly, a group of multiresistant strains from the Czech Republic (termed group B) was also shown to be closely related to clone II [28]. These data suggest that genotypes 3 and 6 and group B strains may represent variants of clone II, as suggested for group B by Pantophlet et al. [28]. Genotype 13 appears to represent a widespread genotype that belongs to clone III. Isolates of this genotype were resistant to amikacin but susceptible to imipenem, with the exception of the isolate from Madrid. French isolates of genotype 13 showed the particularity of being susceptible to piperacillin/tazobactam. These differences in susceptibility profiles may be explained by the mobility of the resistance genes, often carried by mobile genetic elements [10,18,32], combined with different antimicrobial selective pressures according to the geographic region or hospital. Given that basic aspects of the population genetics of A. baumannii, such as the frequency of genetic exchange, are not known, it is so far unclear whether the differences among strains are predominantly the result of mutations or of genetic recombination, and the stability of A. baumannii genotypes over time is not known either. Approaches including sequence-based methods such as MLST will be needed to better delineate clones and follow their diversification over time [14,33]. Sets of strains representative of currently recognized clones and widespread genotypes thereof will be useful to validate new characterization methods and rapid identification tools. Early recognition of strains belonging to clones with high transmissibility potential would be beneficial by leading to the implementation of infection control measures in order to prevent further spread in hospitals. Study of the biological properties of widespread clones will be important to understand their evolutionary success and to determine whether they share special properties such as enhanced virulence or an increased capacity to survive in the hospital environment, or if their success is merely due to the fact that they acquired and express antimicrobial resistance genes [19].

Acknowledgements This study was supported in part by grant QLK2-200001404 from the European Union Fifth Framework Programme ‘Network for automated Bacterial Strain Fingerprinting in Europe’ (GENE project).

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