These authors contributed equally to this work.
Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme
Version of Record online: 28 APR 2010
© 2010 The Authors. Journal Compilation © 2010 European Society of Clinical Microbiology and Infectious Diseases
Clinical Microbiology and Infection
Volume 17, Issue 2, pages 197–201, February 2011
How to Cite
Di Popolo, A., Giannouli, M., Triassi, M., Brisse, S. and Zarrilli, R. (2011), Molecular epidemiological investigation of multidrug-resistant Acinetobacter baumannii strains in four Mediterranean countries with a multilocus sequence typing scheme. Clinical Microbiology and Infection, 17: 197–201. doi: 10.1111/j.1469-0691.2010.03254.x
- Issue online: 20 JAN 2011
- Version of Record online: 28 APR 2010
- Original Submission: 25 February 2010; Revised Submission: 16 April 2010; Accepted: 19 April 2010 Editor: R. Cantón Article published online: 28 April 2010
- Acinetobacter baumannii;
- molecular epidemiology;
- multilocus sequence typing;
- pulsed-field gel electrophoresis
Clin Microbiol Infect 2011; 17: 197–201
Thirty-five multidrug-resistant Acinetobacter baumannii strains, representative of 28 outbreaks involving 484 patients from 20 hospitals in Greece, Italy, Lebanon and Turkey from 1999 to 2009, were analysed by multilocus sequence typing. Sequence type (ST)2, ST1, ST25, ST78 and ST20 caused 12, four, three, three and two outbreaks involving 227, 93, 62, 62 and 31 patients, respectively. The genes blaoxa-58, blaoxa-23 and blaoxa-72 were found in 27, two and one carbapenem-resistant strain, respectively. In conclusion, A. baumannii outbreaks were caused by the spread of a few strains.
Acinetobacter baumannii epidemics have been recently described in Europe, and are caused worldwide by a limited number of genotypic clusters of strains [1–14]. Major A. baumannii outbreak clones were initially named European clones I to III, but are now regarded as international . Multilocus sequence typing (MLST) is the standard method for defining the clonal structure of bacterial species, and has defined clones 1–3 as clonal complexes (CCs) 1–3 [14–16]. The aims of the present study were: (i) using MLST, to define the genetic identity of A. baumannii strains associated with outbreaks in Mediterranean hospitals; (ii) to compare MLST data with those obtained using pulsed-field gel electrophoresis (PFGE) and trilocus sequence-based typing (3LST) ; and (iii) to identify the genes for carbapenem-hydrolysing β-lactamases involved in these outbreaks.
Thirty-five A. baumannii strains isolated during 28 outbreaks that occurred in 20 hospitals in Greece, Italy, Lebanon and Turkey from 1999 to 2009 were included in the study. Nearly all strains were representative of cross-transmission episodes, and were isolated with identical PFGE types from more than two patients of the same or different institutions (Table 1). Antimicrobial susceptibilities were determined by a reference microdilution method . Although A. baumannii strains were not a priori selected because of a multidrug resistance phenotype, all of the strains were resistant to more than two of five antimicrobial classes and were considered to be multidrug-resistant ; 29 of 35 strains exhibited an imipenem MIC ≥16 mg/L and were considered to be carbapenem-resistant (Table 1). PFGE analysis and interpretation of PFGE profiles were performed as reported previously . Eighteen PFGE types (A–R) and three PFGE subtypes (F1, F2 and P) were identified (Table 1).
|Strain||Hospital||Year||Patientsa||PFGE type||MLST||3LST group||Carbapenem resistance|
MLST analysis was performed as previously described  (http://www.pasteur.fr/mlst). Only ten different sequence types (STs) were found. Strains with PFGE profiles A–C, D and E, F–J and L–N were assigned to ST1, ST20, ST2 and ST25, respectively; strains with PFGE profiles K, O, P, P1, Q and R were assigned to ST3, ST78, ST15, ST84, ST82 and ST83, respectively. Interestingly, more than six band differences were found among PFGE profiles A, B and C, among PFGE profiles F, G, H, I and J, and among PFGE profiles L, M and N, respectively, showing that international STs ST1, ST2 and ST25 represent heterogenous genotypic entities. ST2 predominated, being identified in 12 strains from 11 hospitals in Greece, Italy and Lebanon (Table 1 and Fig. 1). Together, these 12 strains represented 12 clusters that involved 227 (46.2% of the total) infected patients. The other most frequently assigned STs were ST1, ST20, ST25 and ST78. They were identified in four, two, three and four strains, respectively, representing 93 (18.9%), 31 (6.3%), 62 (12.6%) and 62 (12.6%) patients. The results are in accordance with previous reports showing that many hospital A. baumannii strains circulating in Europe and elsewhere belonged to international clones CC1 and CC2, respectively [1,3,4,7,12,14]. Also, as in the Czech Republic , a shift towards ST2 (international clone II) was observed in Greece, Italy and Lebanon. Notably, strains assigned to ST2 and PFGE profile F were observed to progressively overtake, numerically, those assigned to ST1. Indeed, between 1999 and 2006, ST1 represented four strains (93 patients, 23% of the total over this period), whereas between 2002 and 2009, ST2 represented 12 strains (227 patients, 56% of the total over this period; Table 1 and Fig. 1). Moreover, strains assigned to ST2 with PFGE profile F were isolated in three Italian hospitals and three Greek hospitals (Table 1), thus suggesting that this clone might have encountered favourable conditions for spread within the same city or between different countries, as described for other A. baumannii epidemics [1,2,5,6]. Our data also demonstrate the diffusion of strains assigned to ST25 in Greece, Italy and Turkey, of those assigned to ST78 in Italy, and of those assigned to ST15 and ST84 in Turkey (Table 1 and Fig. 1).
eBURST analysis  of the ten STs as compared with the 82 profiles of the database showed that ST20 and ST1 are single-locus variants and belong to CC1 . ST84 formed a novel CC with ST15, as these two STs differ by a single allelic mismatch. ST82 was placed in CC10  as a single-locus variant of ST10. All other STs were singletons, i.e. differed by at least two genes from all other profiles. A. baumannii CCs can be regarded as clones [14,19,20]. Typing results generated by 3LST  were concordant with MLST data (Table 1). No novel 3LST group was assigned to strains 2977, 2978 and 3866, because they were microepidemic strains . Overall, the present data show that A. baumannii strains circulating between 1999 and 2009 represent a highly structured population. The MLST scheme adopted herein  differs from the MLST scheme used in previous publications [13,15,16]; correspondence can be established using genome sequences or reference strains.
In accordance with previous findings [1,2,9,10,12], a class D carbapenem-hydrolysing oxacillinase was found in all 29 carbapenem-resistant strains by PCR and DNA sequence experiments, performed as reported previously . The blaOXA-58 gene was identified in 27 strains assigned to 18 distinct PFGE profiles and STs. The blaOXA-23 gene was identified in an ST2 strain from Genoa, Italy, and in an ST25 strain from Kocaeli, Turkey; the blaOXA-72 gene was found in an ST25 strain from Naples, Italy. The metallo-β-lactamase-encoding genes blaVIM-1 and blaVIM-4 were detected in ST2 and ST1 strains isolated in Greece (Table 1 and Fig. 1). No acquired class D carbapenem-hydrolysing oxacillinasess or metallo-β-lactamasess were identified in the six carbapenem-susceptible strains (Table 1). In accordance with our data, the genes blaOXA-23, blaOXA-24 and blaOXA-58 were found in a few distinct clusters defined by other typing methods, some of which correspond to international clones I, II and III [7,10,12,13].
In conclusion, A. baumannii outbreaks in four Mediterranean countries were caused by the spread of strains belonging to few genotypes, in particular ST2 and, to a lesser extent, ST1, ST25 and ST78, probably favoured by the blaOXA-58, blaOXA-23 and blaOXA-72 genes. The MLST scheme utilized herein represents a useful standardized typing method for identifying important A. baumannii clones and tracking their geographical expansion.
We are grateful to all colleagues who generously provided strains included in this study. We also thank D. Vitale, CEINGE Biotecnologie Avanzate, Napoli, Italy, for technical support in DNA sequencing.
This work was supported in part by grants from Agenzia Italiana del Farmaco, Italy (AIFA2007 contract no. FARM7X9F8K) and from Ministero dell’Istruzione, dell’Universitae della Ricerca, Italy (PRIN 2008 to R. Zarrilli). Platform Genotyping of Pathogens and Public Health receives financial support from the Institut Pasteur and the Institut de Veille Sanitaire (Saint-Maurice, France). The authors declare that they have no conflicting interests in relation to this work.
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