Molecular Characterization of Acinetobacter Isolates Collected in [PDF]

JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1297–1304 0095-1137/10/$12.00 doi:10.1128/JCM.01916-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 48, No. 4

Molecular Characterization of Acinetobacter Isolates Collected in Intensive Care Units of Six Hospitals in Florence, Italy, during a 3-Year Surveillance Program: a Population Structure Analysis䌤 Francesca Donnarumma,1 Simona Sergi,1† Cristina Indorato,1 Giorgio Mastromei,1 Roberto Monnanni,1 Pieluigi Nicoletti,2 Patrizia Pecile,2 Daniela Cecconi,2 Roberta Mannino,2 Sara Bencini,3 Rosa Fanci,3 Alberto Bosi,3 and Enrico Casalone1*

Received 28 September 2009/Returned for modification 8 November 2009/Accepted 17 February 2010

The strain diversity and the population structure of nosocomial Acinetobacter isolated from patients admitted to different hospitals in Florence, Italy, during a 3-year surveillance program, were investigated by amplified fragment length polymorphism (AFLP). The majority of isolates (84.5%) were identified as A. baumannii, confirming this species as the most common hospital Acinetobacter. Three very distinct A. baumannii clonal groups (A1, A2, and A3) were defined. The A1 isolates appeared to be genetically related to the wellcharacterized European EU II clone. 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 a bla OXA-58-like gene and with ISAba2 and ISAba3 elements flanking this gene. bla OXA-58 appeared to be horizontally transferred. This study showed that the high discriminatory power of AFLP is useful for identification and typing of nosocomial Acinetobacter isolates. 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 blaOXA-58-like gene is discussed. differentiation of Acinetobacter strains (4, 6, 36). The extensive application of genotypic analysis to population studies of A. baumannii has identified three highly similar, widespread European clones (EU I to III). EU clones are assumed to represent distinct, genetically stable, clonal lineages, well adapted to hospitals, whose 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 ventilatorassociated 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, which is already intrinsically resistant to a number of commonly used antibiotics (5), can acquire antimicrobial genes conferring resistance to broadspectrum ␤-lactams, aminoglycosides, fluoroquinolones, and tetracyclines, becoming multidrug resistant (MDR) (1). Of particular concern is the worldwide emergence of the resistance to carbapenems (5, 23), which have been the most important agents for the treatment of infections caused by MDR

Acinetobacters are a heterogeneous group of ubiquitous microorganisms. Within the genus Acinetobacter, 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 the 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 (Acb) complex” (15). Studies of the ecology, epidemiology, and pathology of the member species of the Acb complex are hampered by the limited capability of phenotypic and DNA-DNA hybridization analyses to correctly discriminate them (15). In the last years, to comply with the need for correct species discrimination (4), unsatisfactory phenotypic commercial systems for Acinetobacter identification have been replaced by genotypic methods. One of these method is amplified fragment length polymorphism (AFLP) (24), which has been found to be useful for the

* Corresponding author. Mailing address: Department of Evolutionary Biology, Via Romana 17/19, 50125 Florence, Italy. Phone: 39 0552288245. Fax: 39 0552288250. E-mail: [email protected]. † Present address: Department of Biomedical Science and Technology Section of General Microbiology and Virology & Microbial Biotechnologies, University of Cagliari, Italy. 䌤 Published ahead of print on 24 February 2010. 1297

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CIBIACI and Department of Evolutionary Biology, University of Florence, Florence, Italy1; Laboratory of Microbiology, Careggi Hospital, Florence, Italy2; and Stem Cell Transplantation Unit, Department of Haematology, Careggi Hospital, University of Florence, Florence, Italy3

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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 (bioMe´rieux, Marcy l’Etoile, France). The isolates were collected from 64 patients admitted to 21 high-risk wards (15 adult ICUs, 5 neonatal ICUs, and 1 bone marrow transplantation unit) of six public hospitals, located in the district of Florence, Italy: a university hospital (H1; 1,700 beds), a university children’s hospital (H2; 180 beds), and four nonuniversity hospitals (H3, 161 beds; H4, 246 beds; H5, 104 beds; and H6, 263 beds). All patients admitted to the monitored hospital wards, in the reported time period, and positive for 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 per 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 (bioMe´rieux, 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 cases confirm the outbreak. An outbreak, as defined by the U.S. Centers for Disease Control and Prevention, 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 h or ⬍48 h after hospital admission, respectively; colonization was defined as a positive culture from a specimen in the absence of clinical signs or symptoms of infection. Once compiled, clinical questionnaires are meant to be returned to the Laboratory of Microbiology of the Careggi Hospital for updating of the Vigi@ct system. AFLP analysis. DNA was extracted from bacterial cells, killed in a 50% isopropanol solution, using the Wizard genomic DNA purification kit (Promega, Madison, WI), following the 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 selective A and G nucleotides at the 3⬘ end, respectively. Amplified fragments were separated by capillary electrophoresis on an ABI 310 bioanalyzer (Life Technologies, CA), and fragment size was determined by using a 50- to 400-bp internal standard (Rox 400HD). Only fragments of 60 to 380 bp were considered in profile analysis

performed by GeneMapper 4.0 software (Applied Biosystems). Cluster analysis of the profiles was performed by using the unweighted pair group method with arithmetic mean (UPGMA) with Numerical Taxonomy and Multivariate Analysis System NTsys-pc v.2 software (Exeter Software). 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 levels (26). An isolate was identified as belonging to a defined species when it grouped with the corresponding type strain at a level of similarity of ⱖ59% (29); isolates grouping at a ⱖ78% level of similarity were considered to belong to the same clone. The following reference strains were used for AFLP identification: A. baumannii, ATCC 19606; A. calcoaceticus, DSMZ30006; gen. sp. 3, DSMZ9343; and gen. sp. 13TU, DSMZ30010. EU clones I to III were kindly supplied by L. Dijkshoorn (University Medical Center of Leiden, 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. 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 ␮l 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% (wt/vol) agarose gel. All of the carbapenem-resistant isolates have been investigated by PCR for the presence of the promoter sequence ISAba1 (30) and for those of ISAba2 and ISAba3, located 5⬘ and 3⬘, respectively, of the blaOXA-58-like gene (16, 28). The sequence (934 bp) of the blaOXA-58-like gene was determined by Sanger’s method on an ABI prism 310 CE system sequencer (Life Technologies, CA) using the OXA-58A (5⬘-TTATCAAAATCCAATCGGC-3⬘) and OXA-58B (5⬘TAACCTCAAACTTCTAATTC-3⬘) primers. 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). AMOVA. The analysis of molecular variance (AMOVA) method was applied to estimate the genetic differences among A. baumannii clonal groups. The 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 Dalmastri et al. (7). A. baumannii isolates were sorted in different populations (corresponding to AFLP clusters), and AMOVA was used to compute the differences among populations and among individuals within populations. Pairwise genetic distance (Fst) values (39) and their corresponding P values were calculated to quantify the differentiation between all pairs of populations. AMOVA and Fst value generation were performed using Arlequin version 3.1 software (11).

RESULTS Patient data and bacterial isolates. During the study period, 71 strains, identified as A. baumannii by the 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 ⱖ65 years of age. The specimens 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 answers to a clinical questionnaire for infection/ colonization data, we have 25 infections, 27 colonizations, and 19 results of “unanswered” (Fig. 1). AFLP analysis of Acinetobacter strains. Reproducibility of AFLP analysis was higher than 90% (data not shown). AFLP

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A. baumannii in the last years (5). blaOXA genes encoding carbapenem-hydrolyzing ␤-lactamases (carbapenemases), belonging to molecular class D (OXA enzymes), have emerged globally as the main mechanism responsible for this resistance (38). The blaOXA genes of Acinetobacter spp. can be divided into four phylogenetic subgroups. The blaOXA-51-like gene, intrinsic to A. baumannii (3), is normally expressed at a low level but can be overexpressed as a consequence of the insertion of an ISAba1 sequence upstream of the gene (33). In contrast, blaOXA-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 of investigating species and strain diversity and determining 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.

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analysis, used for species identification, confirmed that all of the isolates belong to the genus Acinetobacter (similarity, ⬎40%) (29), but only 60 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 or A. calcoaceticus, and seven iso-

lates could not be assigned to any species of the Acb complex (non Acb-complex isolates) (Fig. 1). AFLP typing analysis grouped 60% of the 60 A. baumannii isolates in three groups with a cutoff value of ⱖ78%: clonal group A1 (9 isolates), clonal group A2 (24 isolates), and clonal group A3 (3 isolates). The remaining 24 isolates were ungrouped (isolates with a

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FIG. 1. AFLP analysis dendrogram. Thick bar, Acinetobacter sp. isolates; thin bars, A1, A2, and A3 clonal groups of A. baumannii isolates. For IN/C (infection/colonization), black boxes indicate patient infection, gray boxes indicate patient colonization, and white boxes indicate that data are not available. For R/S, black boxes indicate resistance (ⱖ16), gray boxes indicate susceptibility to imipenem, and white boxes indicate that data are not available. For the OXA genes, black boxes indicate the contemporary presence of OXA-51 and OXA-58, gray boxes indicate the presence of OXA51 (gray), and white boxes indicate the absence of both OXA-51 and OXA-58. White dots at the end of the corresponding tree branches represent (from top to bottom) the EU I, EU II, and EU III clones, and black boxes represent the ATCC reference strains of A. baumannii, gen. sp. 13TU, and A. coalcaceticus, gen. sp. 3. Shaded boxes 1, 2, and 3 within the dendrogram define groups of isolates involved in outbreak events (Table 1).

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TABLE 1. Outbreak events and epidemiological characteristics of involved isolates Outbreak eventa

Hospital and ward

1

b

Sample collection date (mo/day/yr)

Infection/colonization

H3-W1

1 2 3

8/09/06 8/17/06 8/17/06

2

H3-W1

1 2 3

3

H1-W14

1 2 3 4

Imipenem resistance/sensitivity

AFLP profile

Infection Colonization Infection

Ipms

A2

11/08/06 12/04/06 12/06/06

Infection Infection Colonization

Ipms

A2

8/01/08 8/08/08 8/23/08 8/23/08

NDb Infection Infection ND

Ipmr

A2

For more details, see Fig. 1. ND, not determined (data not available).

unique AFLP profile). A. baumannii belonging to clonal group A1 (Fig. 1) was found to be genetically related to the EU II 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%) (data 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 ␤-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 (Ipmr), whereas the remaining 21 were susceptible (Ipms); all of the other isolates were Ipms, except for two non Acb complex isolates (Fig. 1). The Ipmr phenotype was expressed in isolates of all three A. baumannii clonal groups: A1 (7/9), A2 (16/24), and A3 (3/3), and among single AFLP profile isolates (13/24). Interestingly, whereas Ipmr A1 strains were isolated continuously from September 2006 to May 2008, Ipmr A2 strains were restricted to May 2007 to December 2008; in the same period, only one Ipms A2 strain was isolated, whereas the remaining seven Ipms A2 isolates were isolated from July to December 2006. The three Ipmr A3 strains were isolated in October 2008. Isolates were also investigated for the identity of the genetic determinants of imipenem resistance. The presence of blaOXA genes encoding OXA-51-like, OXA-58-like, OXA-23-like, and OXA-24-like enzymes and that of IS elements able to promote the expression of these genes were evaluated by multiplex PCR. blaOXA-23-like and blaOXA-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 the intrinsic blaOXA-51-like gene. All of (and only) the Ipmr isolates (39 A. baumannii and two non-Acb complex), independently of hospital origin, period of isolation, or clonal group, were positive for the blaOXA-58-like gene (Fig. 1); 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 blaOXA-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 blaOXA-58 sequence in the GenBank database (accession no. FJ492877). Outbreak investigation. Three A. baumannii outbreaks occurred in two intensive care units of hospitals H1 and H3 during the study period; Table 1 lists all of 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 ward 1 of H3 (H3-W1) in 2006, at a distance of 2 months from each other, and involved two patients each; both outbreaks lasted for approximately 1 month. In both cases, the corresponding A. baumannii isolates belonged to clonal group A2 and were Ipms. The third outbreak occurred at H1-W14 in 2008 and involved four patients in a period of 1 month, and also in this case, the isolates belonged to clonal group A2, but differently from the previous two outbreaks, they all were Ipmr. The genetic relatedness among isolates from the same outbreak was always very high (88% ⬍ similarity ⬍ 95%) (Fig. 1). Hospital measures, such as the intensification of standard cross-infection 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 hospital H1, whereas the A2 clonal group spread 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

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a

Patient

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TABLE 2. Spatial distribution of A. baumannii isolates Hospital and ward

Total

A2

A3

1 3 1 1 2

Unique

4

1 3

1 1 1

1 4 1 1 1 2

1 3 1 1

1

9

1 7 3

1

2 1 3 6

24

3

24

2006 and in 2008, whereas A1 was predominant in 2007 (Fig. 2). Isolates belonging to A3 were collected only at the end of 2008. Ungrouped isolates spread during the entire 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 (Fig. 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 closer to 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. AFLP data were used to evaluate the genetic differences among the three clonal groups by the AMOVA method. The result shows that, although most molecular variance was due to intragroup differences (percentage of variance, 59.6%), a statistically significant percentage of it was also attributable to intergroup 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 versus A2, 38%; A1 versus A3, 52%; and A2 versus 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.

In many papers, the name “A. baumannii” is used in a broader sense to accommodate also gen. sp. 3 and gen. sp. 13TU. In this study, with the aim of differentiating the contribution of A. baumannii gen. sp. 3 and gen. sp. 13TU to the nosocomial population of Acinetobacter, we used AFLP to overcome the problems due to uncertain phenotypic identification by the 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%), and neither gen. sp. 13TU nor 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 was 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 3 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 similarity analysis of AFLP profiles over time and had made it 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 quite 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, interhospital-diffused clones and could be defined as successful hospital clones. A. baumannii clusters of highly similar strains, which are assumed to represent distinct clonal lineages, occur at different locations and at different time points in Europe (25, 26). These European clones (EU I to 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

FIG. 2. Time distribution of A. baumannii clones A1 (gray bars), A2 (black bars), and A3 (striped bar) and isolates with a unique AFLP profile (white bars) in hospital settings.

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H1-W1 H1-W2 H1-W4 H1-W5 H1-W6 H1-W10 H1-W12 H1-W13 H1-W14 H1-W15 H1-W16 H1-W17 H1-W18 H1-W19 H1-W20 H1-W21 H2-W2 H3-W1 H4-W1 H5-W1 H6-W1

DISCUSSION

No. of isolates with AFLP profile: A1

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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 (Ipmr). The incidence of the Ipmr phenotype increased to 68% if only one isolate per outbreak was considered. Interestingly, the A2 clone, like the EU clones (26), shows relevant variation in antibiotic susceptibility; A2 isolates were Ipms in 2006, and only in 2008 did Ipmr A2 strains appear. A2 established itself as a successful hospital strain well before Ipmr acquisition; in fact in 2006, Ipms A2 isolates had already spread in different wards of different hospitals and they were associated with two outbreaks in H3-W1. However, after Ipmr acquisition, A2 became the dominant A. baumannii clone, almost completely undermining the A1 clone, and a third outbreak event by the Ipmr A2 clone occurred in 2008. As reported for other A. baumannii clones (32), A2’s 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 favor the selection of specific strains in hospital settings (32). The higher genetic variability of A2 with respect to A1 and A3 clonal groups, as shown 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 ex-

pression 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 Ipmr trait. The presence of the blaOXA-51 gene in non-A. baumannii strains, isolated in this work, is in agreement with a recent report from Lee et al. (21) questioning the usefulness of its detection for identification purposes, as previously suggested (34, 38). As the ISAba1 sequence is never present in the Ipmr isolates, the resistant phenotype did not appear to be associated with the blaOXA-51-like gene (18, 33); conversely, it was consistently associated with blaOXA-58-like gene, and ISAba2 and ISAba3 elements flanking it. These results confirm the emergence of blaOXA-58-like gene as being responsible worldwide for carbapenem resistance in A. baumannii (2, 5, 17, 23, 27), at least partially explained by its plasmid location (28). Accordingly, Ipmr A2 could have arisen from the already widespread Ipmr A1 clone by horizontal gene transfer of a plasmidresident resistance gene. The appearance of the Ipmr A3 clone in 2008 could be similarly explained. The identity of the sequence of the blaOXA-58 gene from representatives 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 the blaOXA-58-like gene has been already demonstrated (40, 41). In conclusion, AFLP, as already suggested by Spence et al.

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FIG. 3. Principal component analysis of the AFLP profiles of A. baumannii isolates: A1 (gray circles), A2 (black circles), and A3 (striped circles) clones and isolates with a unique profile (white circles).

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STRUCTURE OF A NOSOCOMIAL ACINETOBACTER POPULATION

ACKNOWLEDGMENTS We are grateful to Emanuele Goti for his contribution to sizing analysis of AFLP products. We also thank L. Dijkshoorn for providing the A. baumannii European clones I to 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. REFERENCES 1. Bergogne-Be´re´zin, E., and K. J. Towner. 1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9:148–165. 2. 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. 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, and J. G. Kusters. 2001. Comparison of pulsed-field gel electrophoresis and amplified fragment-length 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. 8. D’Arezzo, S., A. Capone, N. Petrosillo, and 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, and S. Schneider. 2005. Arlequin version 3.1: an integrated software package for population genetics data analysis. Evol. 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. 2009. 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.

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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. Marque´, S., L. Poirel, C. Heritier, S. Brisse, M. D. Blasco, R. Filip, G. Coman, T. Naas, and P. Nordmann. 2005. Regional occurrence of plasmidmediated carbapenem-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. Krˇízova, 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. Marque´, 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. Amplifiedfragment 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. Lett. 243:425–429. 31. Sergi, S., F. Donnarumma, G. Mastromei, E. Goti, P. Nicoletti, P. Pecile, D. Cecconi, R. Mannino, R. Fanci, A. Bosi, and E. Casalone. 2009. Molecular surveillance and population 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. Lett. 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.

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(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 3 years of a hospital surveillance program, we were able to (i) rapidly detect outbreak events, (ii) define the Acinetobacter population structure at a city level of analysis, and (iii) 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 stopping the spread of pathogens by programs that provide an immediate alert (13). Furthermore, the molecular analysis of the genetic determinants of imipenem resistance allowed us to formulate a working hypothesis about the spread of the blaOXA-58 gene and its role in the MDR phenotype.

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J. CLIN. MICROBIOL. encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J. Antimicrob. Agents 27:351–353. 39. Wright, S. 1978. Evolution and the genetics of populations, vol. 4. Variability within and among natural populations. University of Chicago Press, Chicago, IL. 40. Zarrilli, R., D Vitale., A. Di Popolo, M. Bagattini, Z. Daoud, A. U. Khan, C. Afif, and 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. Countries 3:335–341.

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