ORIGINAL ARTICLE 10.1111/j.1469-0691.2008.02010.x Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme H. Wisplinghoff 1, C. Hippler 1, S. G. Bartual 2, C. Haefs 1, D. Stefanik 1, P. G. Higgins 1 and H. Seifert 1 1 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany and 2 Division de Microbiología, Campus de San Juan, Universidad Miguel Hernandez, San Juan de Alicante, Alicante, Spain ABSTRACT To further expand the limited multilocus sequence typing (MLST) database for Acinetobacter baumannii, 53 clinical isolates from various outbreaks in Europe and the USA, collected between 1991 and 2004, plus the A. baumannii reference strain ATCC 19606 T and 20 clinical Acinetobacter genomic species 13TU isolates from the same period, were analyzed using a new MLST scheme based on fragments of the glta, gyrb, gdhb, reca, cpn60, gpi and rpod genes. Data were compared with typing results generated using pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD)-PCR. In total, 50 sequence types (STs) were distinguished among the A. baumannii isolates investigated, and the MLST data were in high concordance with the PFGE and RAPD-PCR results. Only five clonal complexes were identified by eburst analysis, including the 21 STs listed in a previous study, suggesting high diversity among the A. baumannii isolates. With one exception, there was no relatedness among isolates from outbreaks in different countries (Europe) or regions (USA). No intercontinental spread was revealed. Acinetobacter genomic species 13TU isolates could also be analyzed using the A. baumannii MLST scheme (18 different STs) and could be distinguished from A. baumannii isolates according to characteristic sequences. It was concluded that the MLST scheme provides a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii and Acinetobacter genomic species 13TU. Keywords Acinetobacter baumannii, Acinetobacter genomic species 13TU, epidemiology, multilocus sequence typing, pulsed-field gel electrophoresis, random amplified polymorphic DNA, typing Original Submission: 5 July 2007; Revised Submission: 18 January 2008; Accepted: 18 February 2008 Edited by D. Jonas Clin Microbiol Infect 2008; 14: 708 715 INTRODUCTION Acinetobacter baumannii is a significant nosocomial pathogen that especially affects patients with impaired host defences in intensive care units, and that has been implicated in severe nosocomial infections, including bloodstream infection, pneumonia and meningitis, with mortality rates as high as 64% [1]. Similar to methicillin-resistant Staphylococcus aureus, major epidemiological features of these organisms include their propensity for Corresponding author and reprint requests: H. Seifert, Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstr. 19 21, 50935 Cologne, Germany E-mail: harald.seifert@uni-koeln.de clonal spread, their involvement in hospital outbreaks, and their resistance to multiple antimicrobial agents [2 6]. Many recent outbreaks have been caused by multidrug-resistant (MDR) strains of A. baumannii and have occurred in intensive care units where the extensive use of antibiotics may have contributed to the selection of highly resistant strains [7,8]. Currently, 10 33% of A. baumannii isolates are MDR [9,10], with increasing resistance to the carbapenems being seen in the last decade [8,11,12], leaving only limited therapeutic options. A. baumannii, together with three other closely related Acinetobacter spp., Acinetobacter calcoaceticus and Acinetobacter genomic species 3 and 13TU, have been grouped together in the A. calcoaceticus A. baumannii Journal Compilation Ó 2008 European Society of Clinical Microbiology and Infectious Diseases
Wisplinghoff et al. Acinetobacter baumannii MLST 709 (Acb) complex because they are very similar phenotypically and are often impossible to differentiate [13]. A. baumannii and Acinetobacter genomic species 13TU are responsible for most nosocomial infections, while Acinetobacter genomic species 3 is implicated less often in clinical disease [14], and A. calcoaceticus is an environmental organism that has been isolated rarely from clinical specimens. To investigate the molecular epidemiology of A. baumannii, a variety of typing systems have been developed, including ribotyping [13,15], genome analysis by selective amplified fragment length polymorphism [4], pulsed-field gel electrophoresis (PFGE) [15], randomly amplified polymorphic DNA (RAPD) analysis [16] and infrequent-restriction-site PCR [17]. Another possible approach involves the use of multilocus sequence typing (MLST). MLST is a highly discriminative typing method that is based on the sequence comparison of internal fragments of housekeeping genes. It has been applied to a variety of bacterial pathogens, including Neisseria meningitidis [18], Streptococcus pneumoniae [19], S. aureus [20], Staphylococcus epidermidis [21], Enterococcus faecium [22] and, most recently, A. baumannii [23]. MLST provides a powerful tool for global epidemiological studies and studies of the population biology of bacterial species. The MLST scheme for A. baumannii is based on 305 513-bp sequences of the conserved regions of seven housekeeping genes, i.e., glta, gyrb, gdhb, reca, cpn60, gpi and rpod. The present study was undertaken to further evaluate the MLST scheme for A. baumannii, using epidemiologically characterized A. baumannii isolates, and to investigate whether the scheme could also be used to type isolates belonging to the closely related Acinetobacter genomic species 13TU. MATERIALS AND METHODS Bacterial strains A. baumannii and Acinetobacter genomic species 13TU isolates were collected between 1985 and 1998 from various hospitals in Germany and other European countries, e.g. Belgium, Denmark and the UK. In addition, A. baumannii blood culture isolates recovered from patients in 49 medical institutions of various sizes throughout the USA between 1995 and 1998 (SCOPE Project [24]) were analyzed. Epidemiological details of the 74 isolates investigated have been described previously [6,15,25] and are summarized in Table 1. Identification of Acinetobacter spp. to the genus level was confirmed by the transformation assay of Juni [26]. Speciation was performed according to the simplified identification scheme of Bouvet and Grimont [27], and was then confirmed by ribotyping and amplified 16S rdna restriction analysis [13,28]. On the basis of preliminary typing results obtained with RAPD-PCR and PFGE (see below), the 74 clinical Acinetobacter isolates (i.e. 53 A. baumannii isolates, the A. baumannii reference strain ATCC 19606 T, and 20 isolates belonging to Acinetobacter genomic species 13TU) were selected to represent outbreak-related (one isolate outbreak) and sporadic isolates from different countries in Europe and from different regions in the USA. Epidemiological typing The relatedness of all isolates was assessed using RAPD-PCR analysis with two different primers (ERIC-2 and M13) and Ready-to-Go RAPD Analysis beads (Pharmacia Biotech, Freiburg, Germany) as described previously [16]. Analysis was confirmed using PFGE analysis of genomic DNA digested with ApaI as described previously [6]. Fingerprint patterns were compared visually and by Molecular Analyst software (Bio-Rad Laboratories, Munich, Germany). Isolate relatedness was assessed in accordance with published criteria [29], and isolates for further analysis were selected on the basis of a unique fingerprint pattern; i.e. all duplicate isolates with an identical PFGE pattern were excluded. MLST analysis was performed as described previously [23]. In brief, the sequences of internal fragments of the citrate synthase (glta), DNA gyrase subunit B (gyrb), glucose dehydrogenase B (gdhb), homologous recombination factor (reca), 60-kDa chaperonin (cpn60), glucose-6-phosphate isomerase (gpi) and RNA polymerase r70 factor (rpod) genes were determined. PCR conditions comprised 2 min at 94 C, followed by 30 cycles of 94 C for 1 min, 55 C for 1 min and 72 C for 2 min, followed by 2 min at 72 C. Sequencing of internal fragments of c. 450 bp was performed using an ABI Prism 3700 DNA sequencer (Applied Biosystems, Foster City, CA, USA) with BigDye fluorescent terminators and the primers described previously [23]. Sequences were analyzed using the VectorNTI Software package and were compared with the A. baumannii database at the MLST website (http://pubmlst.org/abaumannii/). In addition, all sequences were analyzed using the Sequence Output and MEGA programs available from the MLST website (http://www.mlst.net). For the current analysis, all A. baumannii strains described previously by Bartual et al. [23] were included. The relationship between A. baumannii sequence types (STs) was assessed using the eburst program available from the MLST website. Sequences of Acinetobacter genomic species 13TU isolates were also analyzed using Sequence Output and eburst. The similarities between the allelic profiles were analyzed by the unweighted pair-group method with arithmetic averages (UPGMA), available in MEGA suite program v. 2.1 (http://www.megasoftware.net). To obtain the index of association (Ia), the Sequence Type analysis and recombinational tests software package was used (START v. 1.05, http://outbreak.ceid.ox.ac.uk/software.htm) [30]. RESULTS MLST results were obtained for all the A. baumannii and all Acinetobacter genomic species 13TU isolates included in this study. Since the original
710 Clinical Microbiology and Infection, Volume 14 Number 7, July 2008 MLST scheme was evaluated for A. baumannii, no database comparison was possible for the Acinetobacter genomic species 13TU isolates. To assess the possibility of using the MLST scheme for Acinetobacter genomic species 13TU, separate analyses were performed for A. baumannii and Acinetobacter genomic species 13TU isolates. Overall, MLST analysis revealed 50 different STs among 54 A. baumannii isolates and 18 different STs among 20 Acinetobacter genomic species 13TU isolates. In total, 65 novel STs were detected, 47 for A. baumannii (designated ST-22 to ST-68) and 18 for Acinetobacter genomic species 13TU isolates (designated ST-I to ST-XVIII). The MLST data for all isolates were in high concordance with the epidemiological typing results generated by PFGE and RAPD-PCR fingerprinting (Table 1). However, some isolates with closely related PFGE patterns, e.g. A. baumannii CGN-09 and ST-1650 [23], CGN-06 and ST-17093 [23], and Acinetobacter genomic species 13TU isolates CGN-42 and CGN-50, had different, although closely related, STs (singlelocus variants). No isolates with identical STs had different PFGE patterns. According to eburst analysis, including the 21 STs described previously [23], only five clonal complexes (CCs) were identified among A. baumannii isolates from different outbreaks, with the 55 singletons (Table 2) suggesting high diversity among the isolates. In addition, a founder could only be identified for one of the CCs. The population structure of A. baumannii, based on the sample of isolates investigated, was calculated using eburst with modified settings. The Ia was used to test for linkage disequilibrium among alleles at the seven housekeeping genes. For all A. baumannii isolates, the Ia value was 0.716 (expected variance = 0.655). Using only one representative isolate for each ST, this value was reduced to 0.546 (expected variance = 0.645). Acinetobacter genomic species 13TU isolates were investigated separately. In addition to 14 singletons, two CCs were identified. No founder could be predicted for any of the CCs (data not shown). The population structure of Acinetobacter genomic species 13TU isolates was analyzed using the same eburst settings as those for the analysis of A. baumannii. When eburst was used to analyse A. baumannii and Acinetobacter genomic species 13TU isolates together, separation of the species, as expected, was not possible. However, there were no close relationships (e.g. single-locus variants, double-locus variants, or triple-locus variants) between A. baumannii and Acinetobacter genomic species 13TU STs, and no CC contained isolates of both species (data not shown). With one exception, i.e. obviously identical strains from Cologne, Germany (CGN-37) and Nottingham, UK (CGN-77), no apparent relatedness among isolates from different outbreaks was revealed. In addition, no relationships among isolates from different countries (Europe) or regions (USA) were revealed, and no intercontinental spread was revealed. Upon comparison of the sequences of the individual alleles of A. baumannii and Acinetobacter genomic species 13TU isolates, certain parts of the sequence were characteristic for Acinetobacter genomic species 13TU and could be used for species identification (Fig. 1). Acinetobacter genomic species 13TU isolates also clustered separately from the A. baumannii isolates. Clustering based on the combined sequences of all genes is shown in Fig. 2 (generated using the UPGMA and Kimura pairwise distance method). DISCUSSION A. baumannii has a particular propensity for nosocomial cross-transmission, and outbreaks of MDR A. baumannii are being reported increasingly [5,31]. The current study is the first to apply the new MLST scheme for A. baumannii to the epidemiological typing of isolates from a wide range of hospitals and countries. This MLST scheme was originally designed to investigate the molecular epidemiology of A. baumannii, and has been validated for this purpose [23]. However, as A. baumannii and Acinetobacter genomic species 13TU share the same clinical and epidemiological characteristics and are closely related phenotypically, 20 Acinetobacter genomic species 13TU isolates were also included to address the question of whether the current MLST scheme could also be used for studying the epidemiology of Acinetobacter genomic species 13TU. Other species from the Acb complex were not included because of their lower clinical significance. The present study identified 65 novel STs, which can be explained, in part, by the fact that this was the first study to use the scheme for a broad range of isolates in the clinical setting,
Wisplinghoff et al. Acinetobacter baumannii MLST 711 Table 1. Comparison of results obtained by multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA (RAPD) for the Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates used in this study Origin Isolate Original designation Species Outbreak (Y N) Year Country PFGE or RAPD profile ST Allelic profile CGN-03 U-11432 AB Y 1991 Cologne, Germany G a, XXIb b 16 10-12-4-11-11-9-5 CGN-04 St-16706 AB Y 1991 Cologne, Germany I, XXVIIIa 18 10-13-4-11-12-11-5 CGN-5a St-19001-I AB Y 1992 Cologne, Germany I, XXVIIIb 18 10-13-4-11-12-11-5 CGN-5b St-19001-II AB Y 1992 Cologne, Germany I, XXVIIIc 18 10-13-4-11-12-11-5 CGN-77 Multi 6 AB Y Nottingham, UK K, c c 19 1-14-3-2-2-9-3 CGN-37 V-11135 AB Y 1991 Cologne, Germany K, c c 19 1-14-3-2-2-9-3 CGN-06 M-8489 II AB Y 1991 Cologne, Germany L1, XXXIIIa 22 1-15-13-12-4-12-37 CGN-07 St-17108 II AB Y 1991 Cologne, Germany L1, XXXIIIa 22 1-15-13-12-4-12-37 CGN-81 Schneider 040 AB Y 1996 Cologne, Germany NT UP 23 1-1-1-1-1-28-10 CGN-18 PGS 9771 AB N 1990 Naestved, Denmark XI 24 1-1-22-1-4-20-16 CGN-48 M-6334 AB N 1991 Cologne, Germany NT UP 25 1-1-41-6-23-31-26 CGN-35 ATCC 19606 AB N 1911 Delft, The Netherlands XIII 26 1-10-36-22-20-4-24 CGN-09 V-16319 AB Y 1991 Cologne, Germany N1, IXa 27 1-12-15-2-2-3-3 CGN-55 Berlin 1-3809 AB Y 1996 Berlin, Germany NT UP 28 1-12-3-2-2-35-4 CGN-52 PGS 50853-82 AB N Denmark VII 29 1-12-3-28-2-29-4 CGN-56 Berlin 1-3740 AB Y 1996 Berlin, Germany NT UP 30 1-12-3-29-2-34-4 CGN-39 Remscheid AB Y 1998 Remscheid, Germany NT UP 31 1-12-39-25-21-3-25 CGN-44 M-7968 AB N 1991 Cologne, Germany XX 32 1-12-4-11-4-28-2 CGN-46 M-13546 AB N 1991 Cologne, Germany XIX 33 1-12-4-11-4-29-2 CGN-23 St-17306 AB N 1991 Cologne, Germany XXXIV 34 1-15-13-12-4-49-37 CGN-71 SCOPE 31 AB Y 1995 New York, NY, USA S-k d 35 1-15-2-28-1-41-32 CGN-74 SCOPE 37 AB Y 1995 Orlando, FL, USA S-l 36 1-15-2-28-1-44-32 CGN-15 V-12277 AB N 1991 Cologne, Germany XXXVI 37 1-19-21-17-1-18-14 CGN-19 PGS 10074 AB N 1990 Vejle, Denmark XII 38 1-25-23-6-4-21-17 CGN-20 W-8834 AB N 1991 Cologne, Germany XVII 39 1-26-24-1-4-3-10 CGN-53 PGS 10086 AB N 1990 Vejle, Denmark NT 40 1-3-6-1-4-34-10 CGN-30 M-12665 AB N 1991 Cologne, Germany NT UP 41 1-31-31-24-4-10-21 CGN-54 Schneider I-03 AB N 1996 Cologne, Germany NT UP 42 1-34-14-28-22-35-2 CGN-82 Multi 2 AB Y Ghent, Belgium B 43 1-42-49-10-1-48-23 CGN-68 SCOPE 2 AB N 1995 Miami, FL, USA S-I 44 10-12-1-33-4-29-31 CGN-29 PGS 10508 AB N 1990 Denmark XVIII 45 10-12-30-24-4-9-20 CGN-79 Neustrelitz 1 AB Y 1996 Neustrelitz, Germany NT UP 46 10-12-4-11-4-29-2 CGN-76 Multi 1 AB Y Freiburg, Germany A 47 10-12-4-11-4-29-35 CGN-51 PGS 14554 AB N Denmark XXIV 48 10-12-4-6-4-33-2 CGN-10 V-12334 AB N 1991 Cologne, Germany XXIII 49 10-19-17-16-4-9-2 CGN-43 St-12084 AB N 1991 Cologne, Germany XXXII 50 10-34-40-26-22-27-2 CGN-45 V-4316 AB N 1991 Cologne, Germany XXII 51 11-12-4-11-1-28-2 CGN-01 PGS 14544 AB Y Denmark XIV 52 12-17-16-1-14-14-7 CGN-13 U-10651 AB N 1991 Cologne, Germany NT UP 53 13-12-19-6-1-9-2 CGN-31 222 98 AB N 1998 Cologne, Germany NT UP 54 13-12-32-24-4-9-22 CGN-36 St-20421 AB N 1991 Cologne, Germany X 55 13-33-37-24-4-11-2 CGN-14 M-3789 AB N 1991 Cologne, Germany XXXI 56 14-23-20-1-16-17-13 CGN-16 M-3317 AB N 1991 Cologne, Germany XXVI 57 15-24-4-18-17-19-15 CGN-26 M-2184 AB N 1991 Cologne, Germany NT UP 58 18-17-28-22-4-4-18 CGN-28 M-12225 I AB N 1991 Cologne, Germany NT UP 59 19-30-29-23-18-24-19 CGN-65 SCOPE 47 AB Y 1996 Las Vegas, NV, USA S-f 60 2-21-12-32-26-39-2 CGN-33 Berlin 5-8734 AB Y 1996 Berlin, Germany NT UP 61 20-12-34-24-1-9-2 CGN-34 Multi 25 AB N Freiburg, Germany NT UP 62 21-31-35-24 1-25-23 CGN-47 St-18748 AB N 1991 Cologne, Germany XVI 63 23-35-3-27-23-30-19 CGN-49 V-17215 AB N 1991 Cologne, Germany NT UP 64 24-1-42-13-24-32-26 CGN-69 SCOPE 23 AB N 1996 New York, NY, USA NT UP 65 28-38-45-1-16-40-5 CGN-64 SCOPE 44 AB Y 1995 Las Vegas, NV, USA S-e 66 28-38-45-31-16-30-5 CGN-75 SCOPE 40 AB Y 1995 Iowa City, IO, USA NT UP 67 29-41-48-11-1-45-10 CGN-12 U-9560 AB N 1991 Cologne, Germany XXXV 68 3-22-18-1-1-16-12 CGN-02 PGS 353 13TU Y 1985 Odense, Denmark III I 1-18-1-15-15-15-8 CGN-22 PGS 4419 13TU N 1991 Denmark I II 1-28-26-20-15-13-1 CGN-73 SCOPE 8 13TU N 1995 Savannah, GA, USA NT UP III 1-37-44-34-15-43-34 CGN-42 PGS 10716 13TU N 1990 Denmark V IV 10-29-14-21-15-26-6 ACI-19 St-11681 13TU Y 1991 Cologne, Germany IV V 11-16-14-14-13-13-6 ACI-20 St-7961 13TU Y 1991 Cologne, Germany IV V 11-16-14-14-13-13-6 CGN-08 St-20045 90 13TU Y 1990 Cologne, Germany IV V 11-16-14-14-13-13-6 CGN-80 Multi 4 13TU Y Odense, Denmark d VI 11-20-14-20-15-47-6 CGN-67 SCOPE 59 13TU N 1996 Akron, OH, USA NT UP VII 11-27-14-21-15-36-6 CGN-60 SCOPE 81 13TU Y 1996 Richmond, VA, USA S-b VIII 11-27-14-21-15-38-6 CGN-78 Schneider I-08 13TU Y 1996 Cologne, Germany NT UP IX 11-27-14-21-15-46-36 CGN-50 PGS 12112 13TU N 1990 Denmark Va X 11-29-14-21-15-26-6 CGN-72 SCOPE 7 13TU N 1995 Savannah, GA, USA NT UP XI 11-40-47-30-27-42-33 CGN-21 PGS 53937 bb 13TU N Denmark VI XII 16-27-25-19-15-22-19 CGN-24 Schneider VI-10 13TU N 1996 Cologne, Germany NT UP XIII 17-29-27-21-15-23-6 CGN-32 PGS 9894 13TU N 1991 Denmark II XIV 17-32-33-20-19-13-6 CGN-57 SCOPE 74 13TU Y 1996 Richmond, VA, USA S-a XV 25-27-14-21-15-34-27 CGN-58 SCOPE 79 13TU Y 1996 Richmond, VA, USA NT UP XVI 26-36-43-20-25-36-28
712 Clinical Microbiology and Infection, Volume 14 Number 7, July 2008 Table 1. (Continued) Origin Isolate Original designation Species Outbreak (Y N) Year Country PFGE or RAPD profile ST Allelic profile CGN-59 SCOPE 80 13TU Y 1996 Richmond, VA, USA NT UP XVII 26-36-43-29-25-37-28 CGN-61 SCOPE 83 13TU Y 1996 Richmond, VA, USA NT UP XVIII 27-37-44-30-25-38-29 a Capital letters designate PFGE patterns described by Bartual et al. [23]. b Roman numerals designate PFGE patterns described by Seifert and Gerner-Smidt [15]. c Lower-case letters designate RAPD patterns described by Grundmann et al. [16]. d With prefix S- for SCOPE isolates described by Wisplinghoff et al. (S-x; [25]); NT UP, not typed in direct comparison to all other strains used in this study, or unique pattern. AB, A. baumannii; 13TU, Acinetobacter genomic species 13TU (allele numbers and STs are numbered separately). STs numbered 22 and above are novel. For the Acinetobacter genomic species 13TU STs, Roman numerals are used to avoid confusion. All Acinetobacter genomic species 13TU STs are considered to be novel. ST, sequence type; PFGE or RAPD pattern. Table 2. eburst results for the Acinetobacter baumannii isolates used in this study (isolates described previously [23] are marked in bold) ST CC FREQ SLV DLV TLV Average distance ST bootstrap group (%) Subgroup (%) 32 1 1 1 1 1 2.00 0 0 33 1 1 2 1 0 1.33 27 0 46 1 1 2 1 0 1.33 25 0 47 1 1 1 1 1 2.00 0 0 20 2 2 1 1 0 1.50 0 0 22 2 2 2 0 0 1.00 30 0 34 2 1 1 1 0 1.50 0 0 21 3 2 1 0 0 1.00 27 3 1 1 0 0 1.00 35 4 1 1 0 0 1.00 36 4 1 1 0 0 1.00 6 5 1 1 0 0 1.00 10 5 1 1 0 0 1.00 Singletons: 1 2 3 4 5 7 8 9 11 12 13 14 15 16 17 18 19 23 24 25 26 28 29 30 31 37 38 39 40 41 42 43 44 45 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 ST, sequence type; CC, clonal complex; FREQ, frequency; SLV, single-locus variant; DLV, double-locus variant; TLV, triple-locus variant. rather than in a specific set of strains selected for validation purposes only. Also, there is currently no established MLST database for Acinetobacter genomic species 13TU; thus, all 18 MLST profiles generated for Acinetobacter genomic species 13TU isolates were considered to be novel. As observed previously for A. baumannii [23], the MLST results were highly concordant with PFGE and RAPD- PCR typing results, both for A. baumannii and for Acinetobacter genomic species 13TU isolates. Therefore, the current MLST scheme appears to be useful for typing of both species. However, proper identification to the species level is required, and both species should be analyzed separately by MLST if an algorithm such as eburst is used, because the differences among species would not be visible in the eburst dataset and could lead to false conclusions. As suspected from the validation study [23], the population structure of A. baumannii seems to be highly diverse; however, because of the still limited number of isolates that have been analyzed to date (49 isolates in the database and 54 in the present study), it may be too early to reach a definitive conclusion. The Ia of 0.716 was lower than in the previous analysis (Ia = 2.592 [23]), probably reflecting the much more heterogeneous population of isolates investigated. Limiting the analysis to only one representative isolate per ST did not change the relationship (0.546 vs. 1.393). Generally, clonal populations are identified by an Ia value that differs significantly from zero [30]. The value of this analysis is somewhat limited, since isolates were selected to reflect diversity rather than as a representative cross-section of the population. It
Wisplinghoff et al. Acinetobacter baumannii MLST 713 Fig. 1. Sequences of selected Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates. Alleles of gdhb and reca are displayed as examples. Characteristic differences are displayed in bold font. Only the polymorphic sites of the 12 selected isolates are displayed. Numbers denote positions within the multilocus sequence typing sequence. 13TU, Acinetobacter genomic species 13TU; AB, A. baumannii. appears from the present data that the MLST scheme has a high discriminative power that probably exceeds the discrimination among isolates that is seen with other MLST schemes. This may reflect the fact that most of the isolates were selected on the basis of having a distinct PFGE pattern; nevertheless, isolates of methicillin-resistant S. aureus with different PFGE types are often represented by a single ST [32]. Recent data generated using PCR electrospray ionisation mass spectrometry (ESI-MS) revealed clonal relatedness among most A. baumannii isolates collected from patients treated in US military hospitals during Operation Iraqi Freedom and the well-characterized European A. baumannii hospital clones I III [33]. Although the possibility of a common ancestor was considered, no firm epidemiological link could be established. Similar to MLST, the PCR ESI-MS method uses eight PCR products generated from the conserved regions of the six housekeeping genes efp, trpe, adk, muty, fumc and ppa [33]. In contrast to MLST, the mass spectra of the PCR products, rather than the sequences, are compared, thereby making it one of the fastest systems available for typing A. baumannii. Identification of Acinetobacter genomic species 13TU is also possible; however, typing of this species by PCR ESI-MS has not yet been attempted, and further evaluation of this method is needed to determine its future potential. Apart from a single instance in which an identical outbreak strain (ST-19) was recovered in Nottingham, UK and Cologne, Germany, with no obvious epidemiological link, no apparent relatedness was revealed among isolates recovered from outbreaks in different countries in Europe or in different regions in the USA. In particular, representatives of European clones I and II (i.e. ST-12 and ST-6, respectively) were not found among the isolates investigated. However, the significance of this finding is somewhat limited by the fact that most isolates were from Germany and the USA, and comprised both hospital outbreak-related and sporadic isolates, and only a few isolates from other European countries were included. Further studies should include isolates that were found in previous reports to be associated with large multicentre outbreaks, e.g. those reported from Spain [34], France [35] and countries in northwestern Europe [4,36]. The primers described previously enabled the necessary fragments from all A. baumannii and Acinetobacter genomic species 13TU isolates tested to be amplified and sequenced, and speciesspecific homologous regions in the seven genes were revealed. Using UPGMA analysis, these differences were sufficient to differentiate unambiguously between A. baumannii and Acinetobacter genomic species 13TU isolates. In conclusion, the MLST scheme appears to provide a high level of resolution and is a promising tool for studying the epidemiology of A. baumannii. The scheme in its current form can also be used for epidemiological studies of Acinetobacter genomic species 13TU isolates, for which a novel database will need to be created. The usefulness of the MLST scheme for studying the population structure of A. baumannii, which appears to be highly diverse, remains to be determined by studies with a larger number of isolates from diverse geographical locations.
714 Clinical Microbiology and Infection, Volume 14 Number 7, July 2008 Fig. 2. Dendogram generated using the unweighted pair-group method with arithmetic averages (UPGMA) for combined multilocus sequence typing sequences of all Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates, including isolates described previously [23]. Upmost branches (including only A. baumannii isolates) are collapsed for easier viewing. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Strain designation: CGN-xx corresponds to isolate numbers in Table 1; 13A-19 and 13A-20 corresponds to Acinetobacter genomic sp. 13TU isolates Aci-19 and Aci-20 in Table 1; 13Cxx corresponds to Acinetobacter genomic sp. 13TU isolates CGN-xx in Table 1. ACKNOWLEDGEMENTS The authors wish to thank M. B. Edmond, P. Gerner-Smidt, H. Grundmann, K. J. Towner, M. Vaneechoutte and R. P. Wenzel for providing some of the isolates investigated. This work was presented, in part, at the 45th Interscience Conference on Antimicrobial Agents and Chemotherapy (Washington DC, 2005). TRANSPARENCY DECLARATION The authors declare that they have no conflicting interests in relation to this work. REFERENCES 1. Garcia-Garmendia JL, Ortiz-Leyba C, Garnacho-Montero J et al. Risk factors for Acinetobacter baumannii nosocomial bacteremia in critically ill patients: a cohort study. Clin Infect Dis 2001; 33: 939 946. 2. Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996; 9: 148 165. 3. Brisse S, Milatovic D, Fluit AC. Molecular surveillance of European quinolone-resistant clinical isolates of Pseudomonas aeruginosa and Acinetobacter spp. using automated ribotyping. J Clin Microbiol 2000; 38: 3636 3645. 4. Dijkshoorn L, Aucken H, Gerner-Smidt P et al. Comparison of outbreak and nonoutbreak Acinetobacter baumannii strains by genotypic and phenotypic methods. J Clin Microbiol 1996; 34: 1519 1525. 5. Landman D, Quale JM, Mayorga D et al. Citywide clonal outbreak of multiresistant Acinetobacter baumannii and Pseudomonas aeruginosa in Brooklyn, NY: the preantibiotic era has returned. Arch Intern Med 2002; 162: 1515 1520. 6. Seifert H, Schulze A, Baginski R et al. Comparison of four different methods for epidemiologic typing of Acinetobacter baumannii. J Clin Microbiol 1994; 32: 1816 1819. 7. Kraniotaki E, Manganelli R, Platsouka E et al. Molecular investigation of an outbreak of multidrug-resistant Acinetobacter baumannii, with characterisation of class 1 integrons. Int J Antimicrob Agents 2006; 28: 193 199. 8. Manikal VM, Landman D, Saurina G et al. Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New
Wisplinghoff et al. Acinetobacter baumannii MLST 715 York: citywide prevalence, interinstitutional spread, and relation to antibiotic usage. Clin Infect Dis 2000; 31: 101 106. 9. Jones ME, Draghi DC, Thornsberry C et al. Emerging resistance among bacterial pathogens in the intensive care unit a European and North American Surveillance study (2000 2002). Ann Clin Microbiol Antimicrob 2004; 3: 14. 10. Karlowsky JA, Draghi DC, Jones ME et al. Surveillance for antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from hospitalized patients in the United States, 1998 to 2001. Antimicrob Agents Chemother 2003; 47: 1681 1688. 11. Bogaerts P, Naas T, Wybo I et al. Outbreak of infection by carbapenem-resistant Acinetobacter baumannii producing the carbapenemase OXA-58 in Belgium. J Clin Microbiol 2006; 44: 4189 4192. 12. Coelho JM, Turton JF, Kaufmann ME et al. Occurrence of carbapenem-resistant Acinetobacter baumannii clones at multiple hospitals in London and Southeast England. J Clin Microbiol 2006; 44: 3623 3627. 13. Gerner-Smidt P. Ribotyping of the Acinetobacter calcoaceticus Acinetobacter baumannii complex. J Clin Microbiol 1992; 30: 2680 2685. 14. Horrevorts A, Bergman K, Kollee L et al. Clinical and epidemiological investigations of Acinetobacter genomospecies 3 in a neonatal intensive care unit. J Clin Microbiol 2002; 33: 1567 1572. 15. Seifert H, Gerner-Smidt P. Comparison of ribotyping and pulsed-field gel electrophoresis for molecular typing of Acinetobacter isolates. J Clin Microbiol 1995; 33: 1402 1407. 16. Grundmann HJ, Towner KJ, Dijkshoorn L et al. Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp. J Clin Microbiol 1997; 35: 3071 3077. 17. Yoo JH, Choi JH, Shin WS et al. Application of infrequentrestriction-site PCR to clinical isolates of Acinetobacter baumannii and Serratia marcescens. J Clin Microbiol 1999; 37: 3108 3112. 18. Maiden MC, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1998; 95: 3140 3145. 19. Feil EJ, Smith JM, Enright MC et al. Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data. Genetics 2000; 154: 1439 1450. 20. Enright MC, Day NP, Davies CE et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000; 38: 1008 1015. 21. Wisplinghoff H, Rosato AE, Enright MC et al. Related clones containing SCCmec type IV predominate among clinically significant Staphylococcus epidermidis isolates. Antimicrob Agents Chemother 2003; 47: 3574 3579. 22. Homan WL, Tribe D, Poznanski S et al. Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol 2002; 40: 1963 1971. 23. Bartual SG, Seifert H, Hippler C et al. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 2005; 43: 4382 4390. 24. Wisplinghoff H, Bischoff T, Tallent SM et al. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 2004; 39: 309 317. 25. Wisplinghoff H, Edmond MB, Pfaller MA et al. Nosocomial bloodstream infections caused by Acinetobacter species in United States hospitals: clinical features, molecular epidemiology, and antimicrobial susceptibility. Clin Infect Dis 2000; 31: 690 697. 26. Juni E. Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus. J Bacteriol 1972; 112: 917 931. 27. Bouvet PJM, Grimont PAD. Identification and biotyping of clinical isolates of Acinetobacter. Ann Inst Pasteur Microbiol 1987; 138: 569 578. 28. Vaneechoutte M, Elaichouni A, Maquelin K et al. Comparison of arbitrarily primed polymerase chain reaction and cell envelope protein electrophoresis for analysis of Acinetobacter baumannii and A. junii outbreaks. Res Microbiol 1995; 146: 457 465. 29. Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 2233 2239. 30. Jolley KA, Feil EJ, Chan MS et al. Sequence type analysis and recombinational tests (START). Bioinformatics 2001; 17: 1230 1231. 31. Bou G, Cervero G, Dominguez MA et al. Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacter baumannii strain with a carbapenem-hydrolyzing enzyme: high-level carbapenem resistance in A. baumannii is not due solely to the presence of beta-lactamases. J Clin Microbiol 2000; 38: 3299 3305. 32. Wisplinghoff H, Ewertz B, Wisplinghoff S et al. Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany from 1984 1998. J Clin Microbiol 2005; 43: 5445 5451. 33. Ecker JA, Massire C, Hall TA et al. Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry. J Clin Microbiol 2006; 44: 2921 2932. 34. Vila J, Ruiz J, Navia M et al. Spread of amikacin resistance in Acinetobacter baumannii strains isolated in Spain due to an epidemic strain. J Clin Microbiol 1999; 37: 758 761. 35. Naas T, Coignard B, Carbonne A et al. VEB-1 Extendedspectrum beta-lactamase-producing Acinetobacter baumannii, France. Emerg Infect Dis 2006; 12: 1214 1222. 36. van Dessel H, Dijkshoorn L, van der Reijden T et al. Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals. Res Microbiol 2004; 155: 105 112.