SEARCH

SEARCH BY CITATION

Keywords:

  • Acinetobacter baumannii;
  • carbapenemases;
  • molecular epidemiology;
  • multilocus sequence typing;
  • pulsed-field gel electrophoresis

Abstract

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

Clin Microbiol Infect 2011; 17: 197–201

Abstract

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 [14]. 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) [17]; 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 [18]. 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 [1]; 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 [6]. Eighteen PFGE types (A–R) and three PFGE subtypes (F1, F2 and P) were identified (Table 1).

Table 1.   Epidemiological, phenotypic and genotypic data of the Acinetobacter baumannii isolates included in the study
StrainHospitalYearPatientsaPFGE typeMLST3LST groupCarbapenem resistance
AlleleSTIMP MICCHDLMBL
cpn60fusAgltApyrGrecArpIBrpoB
  1. CA-Naples, Cardarelli Hospital, Naples; CT-Naples, Cotugno Hospital, Naples; F-Naples, Federico II University Hospital, Naples; M-Naples, Monaldi Hospital, Naples; SG-Beirut, Saint Gorge Hospital, Beirut; SJ-Beirut, Saint Joseph Hospital, Beirut; GR, Greece; IT, Italy; LB, Lebanon; TK, Turkey; CHDL, class D carbapenem-hydrolysing oxacillinase; IMP, imipenem; MBL, class B metallo-β-lactamase; MLST, multilocus sequence typing; PFGE, pulsed-field gel electrophoresis; ST, sequence type; 3LST, trilocus sequence-based typing.

  2. aNumber of patients from whom an isolate with each particular PFGE type was detected. Strain 3237 from Beirut, strain 3933 from Naples, strain 2977 from Agrigento and strains 3869, 3871, 3872 and 3875 from Turkey were single isolates in different hospitals.

700F-Naples/IT199981A1111511120.5  
3891Thessaloniki/GR20003B11115111216OXA-58
3886Athens/GR20054B11115111216OXA-58
3887Serres/GR20065C11115111232OXA-58VIM-4
2979Agrigento/IT200214D31115112020.5 
3130SG-Beirut/LB200417E311151120216OXA-58 
2105F-Naples/IT200243F22222222116OXA-58
2638M-Naples/IT200342F22222222116OXA-58
3892Thessaloniki/GR200323F22222222116OXA-58
3893Larissa/GR200448F22222222116OXA-58
3894Serres/GR200619F22222222132OXA-58VIM-1
4245Pozzuoli/IT20094F22222222116OXA-58
2735M-Naples/IT20042F122222222164OXA-58
3858Catania/IT200427F22222222212 
3889Athens/GR20054G22222222116OXA-58 
4026SJ-Beirut/LB20075H22222222116OXA-58
4030SG-Beirut/LB20066I22222222116OXA-58
4009Genoa/IT20074J22222222132OXA-23
3237SG-Beirut/LB20041K3322313331 
4025SG-Beirut/LB20053K33223133316OXA-58 
3890Thessaloniki/GR200312L332472425416OXA-58
3865Kocaeli/TK200547M332472425464OXA-23 OXA-58
4190M-Naples/IT20093N332472425464OXA-72
3909M-Naples/IT200755O253622812978616OXA-58
3696CT-Naples20074O253622812978616OXA-58
3933CA-Naples/IT20071O25362281297862 
4175CA-Naples/IT20092O253622812978616OXA-58 
3868Izmir/TK20032P668235415516OXA-58
3869Istanbul/TK20031P668235415516OXA-58
3871Istanbul/TK20031P16682353084516OXA-58
3872Izmir/TK20031P16682353084516OXA-58
3875Trabzon/TK20031P16682353084516OXA-58
2978Agrigento/IT20013Q2832144482 1 
2977Agrigento/IT20011Q2832144482 16OXA-58 
3866Kayseri/TK20032R2642291483 16OXA-58

MLST analysis was performed as previously described [14] (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 [7], 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).

image

Figure 1.  Geographical distribution and genotypic characterization of Acinetobacter baumannii strains included in the study. Black dots indicate the location of hospitals in which A. baumannii strains were isolated. Cities are indicated by the following letters: G, Genoa; N, Naples; AG, Agrigento; C, Catania; L, Larissa; T, Thessaloniki; AT, Athens; S, Serres; IS, Istanbul; IZ, Izmir; KO, Kocaeli; TR, Trabzon; B, Beirut. Coloured pie charts indicate the prevalence of A. baumannii isolates assigned to different sequence types (STs) in each country. The size of circles is related to the number of patients per countries as indicated. Carbapenem-hydrolysing enzymes isolated in each city are also indicated.

Download figure to PowerPoint

eBURST analysis [19] 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 [14]. ST84 formed a novel CC with ST15, as these two STs differ by a single allelic mismatch. ST82 was placed in CC10 [14] 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 [17] were concordant with MLST data (Table 1). No novel 3LST group was assigned to strains 2977, 2978 and 3866, because they were microepidemic strains [8]. Overall, the present data show that A. baumannii strains circulating between 1999 and 2009 represent a highly structured population. The MLST scheme adopted herein [14] 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 [6]. 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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

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.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References

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.

References

  1. Top of page
  2. Abstract
  3. Acknowledgements
  4. Transparency Declaration
  5. References
  • 1
    Peleg ΑΥ, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008; 21: 538582.
  • 2
    Zarrilli R, Giannouli M, Tomasone F, Triassi M, Tsakris A. Carbapenem resistance in Acinetobacter baumannii: the molecular epidemic features of an emerging problem in health care facilities. J Infect Develop Ctries 2009; 3: 335341.
  • 3
    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: 15191525.
  • 4
    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: 105112.
  • 5
    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: 36233627.
  • 6
    Zarrilli R, Casillo R, Di Popolo A et al. Molecular epidemiology of a clonal outbreak of multidrug-resistant Acinetobacter baumannii in a university hospital in Italy. Clin Microbiol Infect 2007; 13: 481489.
  • 7
    Nemec A, Krizova L, Maixnerova M et al. 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 2008; 62: 484489.
  • 8
    Giannouli M, Tomasone F, Agodi A et al. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii strains in intensive care units of multiple Mediterranean hospitals. J Antimicrob Chemother 2009; 63: 828830.
  • 9
    Mendes RE, Spanu T, Deshpande L et al. Clonal dissemination of two clusters of Acinetobacter baumannii producing OXA-23 or OXA-58 in Rome, Italy. Clin Microbiol Infect 2009; 15: 588592.
  • 10
    Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 2009; 65: 233238.
  • 11
    Giannouli M, Cuccurullo S, Crivaro V et al. Molecular epidemiology of multi-drug resistant Acinetobacter baumannii in a tertiary care hospital in Naples, Italy, shows the emergence of a novel epidemic clone. J Clin Microbiol 2010; 48: 12231230.
  • 12
    Mugnier P, Poirel L, Naas T, Nordmann P. Worldwide dissemination of the blaOXA-23 carbapenemase gene of Acinetobacter baumannii. Emerg Infect Dis 2010; 16: 3540.
  • 13
    Fu Y, Zhou J, Zhou H et al. Wide dissemination of OXA-23-producing carbapenem-resistant Acinetobacter baumannii clonal complex 22 in multiple cities of China. J Antimicrob Chemother 2010; 65: 644650.
  • 14
    Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS ONE 2010; 5: e10034.
  • 15
    Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodrìguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 2005; 43: 43824390.
  • 16
    Wisplinghoff H, Hippler C, Bartual SG et al. Molecular epidemiology of clinical Acinetobacter baumannii and Acinetobacter genomic species 13TU isolates using a multilocus sequencing typing scheme. Clin Microbiol Infect 2008; 14: 708715.
  • 17
    Turton JF, Gabriel SN, Valderrey C, Kaufmann ME, Pitt TL. Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin Microbiol Infect 2007; 13: 807815.
  • 18
    Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; eighteenth informational supplement.Approved Standard M100-S18. Baltimore, MD: CLSI, 2008.
  • 19
    Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 2004; 186: 15181530.
  • 20
    Spratt BG, Feil EJ, Smith NH. Population genetics of bacterial pathogens. In: SussmanM, ed. Molecular medical microbiology. London: Academic Press, 2001; 445484.