Diagnostic Microbiology and Infectious Disease 75 (2013) 180–186

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Diagnostic Microbiology and Infectious Disease

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Antimicrobial Susceptibility Studies

Characterization of resistance mechanisms and genetic relatedness ofcarbapenem-resistant Acinetobacter baumannii isolated from blood, Italy☆

Roberta Migliavacca a, Paula Espinal b, Luigi Principe c, Monica Drago d, Giulia Fugazza a,1, Ignasi Roca b,Elisabetta Nucleo a, Silvia Bracco c, Jordi Vila b, Laura Pagani a, Francesco Luzzaro c,⁎a Department of Clinical Surgical Diagnostic and Pediatric Sciences, Section of Microbiology, University of Pavia, Pavia, Italyb Department of Clinical Microbiology, School of Medicine, IDIBAPS and Barcelona Centre for International Health Research, Hospital Clinic-Universitat de Barcelona, Barcelona, Spainc Laboratory of Microbiology, Ospedale Alessandro Manzoni, Via dell'Eremo, 9/11, 23900 Lecco, Italyd Laboratory of Microbiology, Niguarda Ca' Granda Hospital, Milan, Italy

☆ This work was supported by “Programmi di Ricerca2008) of the Italian Ministry for University and Resear100114).⁎ Corresponding author. Tel.: +39-341-489630; fax:

E-mail address: f.luzzaro@ospedale.lecco.it (F. Luzza1 Giulia Fugazza spent 3 months (February to April 201

which belongs to IDIBAPS (Institut d'Investigacions Biothe research counterpart of the Hospital Clinic of Barcel

0732-8893/$ – see front matter © 2013 Elsevier Inc. Alhttp://dx.doi.org/10.1016/j.diagmicrobio.2012.11.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 1 August 2012Received in revised form 11 October 2012Accepted 5 November 2012Available online 21 December 2012

Keywords:BacteremiaAntimicrobial resistanceMulti-drug resistanceOxacillinasesCarbapenemasesMolecular characterization

The aim of this study was to characterize the resistance mechanisms and genetic relatedness of 21carbapenem-resistant Acinetobacter baumannii blood isolates collected in Italy during a 1-year multicenterprospective surveillance study. Genes coding for carbapenemase production were identified by polymerasechain reaction (PCR) and sequencing. Pulsed-field gel electrophoresis (PFGE), multiplex PCRs for groupidentification, and multilocus sequence typing (MLST) were used to determine genetic relationships.Carbapenem resistance was consistently related to the production of oxacillinases, mostly the plasmid-mediated OXA-58 enzyme. Strains producing the OXA-23 enzyme (chromosomally mediated) were alsodetected. Seven PFGE clones were identified, some of which being related to international (ICL- I and ICL-II) ornational clonal lineages. Multiplex PCRs identified 4 different groups (group 2 being dominant), furtherdistinguishable in 6 sequence types by MLST. The heterogeneity of profiles highlights the diffusion ofinternational and national clonal lineages in Italy. Continuous surveillance is needed for monitoring thespread of these worrisome strains equipped with multiple drug resistance mechanisms.

di Interesse Nazionale” (PRINch and by FEMS (Fellowship-

+39-341-489601.ro).1) with Prof. Jordi Vila's group,mèdiques August Pi i Sunyer),ona, Spain.

l rights reserved.

© 2013 Elsevier Inc. All rights reserved.

1. Introduction

Acinetobacter baumannii is an important nosocomial pathogen ableto cause serious infections, especially in critical care units (Peleg et al.,2008). This pathogen may present high rates of multi-drug resistanceincluding aminoglycosides and fluoroquinolones. Carbapenems usuallyhave good potency against A. baumannii, but carbapenem resistance hasbeen increasingly reported worldwide during the last decade. Carbape-nem-hydrolysing β-lactamases (i.e., carbapenemases), belonging tomolecular class D (OXA-type enzymes), have emerged globally as themainmechanism responsible for this resistance (Poirel et al., 2010). TheblaOXA carbapenemase genes of Acinetobacter spp. can be divided into 5phylogenetic subgroups: blaOXA-51 gene, intrinsic to A. baumannii(Brown and Amyes, 2006), is normally expressed at a low level butcan be overexpressed as a consequence of the insertion of an ISAba1

sequenceupstreamof the gene (Turton et al., 2006); in contrast, blaOXA-23,blaOXA-24/40, blaOXA-58, and blaOXA-143 determinants are consistentlyassociated with resistance or, at least, with reduced susceptibility(Higgins et al., 2009; Woodford et al., 2006). The significantcontribution of OXA carbapenemases in A. baumannii has beenemphasized, particularly when blaOXA genes are accompanied byISAba1-2 and -3 sequences (Marque et al., 2005). It is worth notingthat A. baumannii strains can acquire resistance determinants viaplasmids and class 1 and 2 integrons that carry multiple gene cassettes(Poirel et al., 2010). In particular, aminoglycoside resistance genes suchas acetyltransferase (aac), phosphotransferase (aph), and adenylyl-transferase (aad) contribute to the enzymatic inactivation of amino-glycoside antibiotics and are typically located in gene cassettes (Roca etal., 2012). The increasing number of fluoroquinolone-resistant A.baumannii strains further limits the choice of antimicrobial agents.Fluoroquinolone resistance is primarily mediated by spontaneousmutations in the quinolone resistance determining regions (QRDRs)of either the gyrA or parC gene. However, alterations of outermembrane lipopolysaccharides and fluoroquinolone efflux at theinner membrane should also be considered as factors determiningmulti-drug resistance (Roca et al., 2012).

Hospital outbreaks caused by carbapenem-resistant Acinetobacterbaumannii (CRAB) isolates have been described worldwide (Durante-Mangoni and Zarrilli, 2011) and are also increasing in Italy in the last

181R. Migliavacca et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 180–186

years (Carretto et al., 2011; D'Arezzo et al., 2011; Mendes et al., 2009).Recent data indicate that several successful epidemic A. baumanniiclones circulate in Europe, and a better understanding of theiremergence and diversity within the species is useful (Nemec et al.,2004; Spence et al., 2004). Various genotypic methods have beendeveloped for the typing of acinetobacters, includingmacrorestrictionanalysis by pulsed-field gel electrophoresis (PFGE) (Seifert et al.,2005), multilocus sequence typing (MLST) (Bartual et al., 2005), andmultiplex polymerase chain reactions (PCRs) for identification of theompA, csuE, and blaOXA-51-like sequence groups (Turton et al., 2007).This approach, associated with other methods like PFGE and MLST,may prove helpful in identifying the genotypes that are most likely tocause problems in hospitals.

Here we report on the resistance mechanisms and geneticrelatedness of 21 carbapenem-resistant A. baumannii obtained duringa prospective multicenter surveillance study conducted in Italy duringa 1-year period beginning in April 2007 (Luzzaro et al., 2011).

2. Materials and methods

2.1. Bacterial isolates and participating hospitals

Bacterial isolates were obtained during a multicenter prospectivestudy that involved 20 hospital microbiology laboratories predominant-ly located in northern and central Italy. Participating hospitals werelocated in the following towns: Milan (2 hospitals), Varese, Legnano,Novara, Verona, Bergamo, Brescia, Torino, San Giovanni Rotondo(Foggia), Ancona, Lucca, Genova, Firenze, Padova, Vicenza, Treviso,Sassari,Modena, andBologna. The surveywas conductedduringa1-yearperiod beginning inApril 2007. Theoverall results of the studyhavebeenpreviously published (Luzzaro et al., 2011).With a focus onA. baumanniistrains, 89 nonreplicate isolates were obtained from significant bloodcultures. Based on the study design, 51 of themwere sent to a referencemicrobiology laboratory (Niguarda Ca' Granda Hospital, Milan) wherebacterial isolates were re-identified to species level using the Vitek 2(bioMérieux, Marcy l'Etoile, France) automated instrument followed bydetection of blaOXA-51-like alleles (Héritier et al., 2005). Antimicrobialsusceptibility to a variety of drugs (including ceftazidime, cefepime,imipenem, meropenem, ciprofloxacin, levofloxacin, gentamicin, amika-cin, trimethoprim–sulfamethoxazole, and colistin)was evaluated by theEtest method. Interpretive breakpoints used were those recommendedby theClinical andLaboratory Standards Institute (CLSI)guidelines (CLSI,2010). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC27853 were used as quality control strains.

2.2. PCR analysis and sequencing

Amplification of class B, C, and D β-lactamases was carried out usingspecific primers designed to identify blaIMP-type, blaVIM-type, blaGIM-1,blaSIM-1, blaNDM-1, blaSPM-1, blaADC-like, blaOXA-23-like, blaOXA-24-likeblaOXA-51-like, and blaOXA-58-like genes (Espinal et al., 2011). Insertionsequence (IS) elements; class 1 integron variable regions; aminoglyco-side resistance determinants aacA4, aph6A, and armA; and thequinolone resistance determining regions (QRDRs) of gyrA and parCgenes were also searched for, as previously described (Doi andArakawa, 2007; Marti et al., 2008; Mendes et al., 2007; Ruiz et al.,2007; Solé et al., 2011).

PCR products were purified using Wizard SV gel and a PCR cleanupsystem (Promega, Madrid, Spain), bi-directionally sequenced, and sent toMacrogen (Seoul, Korea). The nucleotide sequences were analyzed usingthe online BLAST web server (http://www.ncbi.nlm.nih.gov/BLAST/).

2.3. Macrorestriction analysis by PFGE

Genomic DNA was digested with ApaI restriction enzyme, andfragments were separated on a CHEF-DR II apparatus (Bio-Rad, Milan,

Italy) for 20 h at 14 °C. Bacteriophage λ concatamers were used asDNA size markers. DNA restriction patterns of scanned gel pictureswere interpreted following cluster analysis with the Fingerprinting IIversion 3.0 software (Bio-Rad) using the unweighted pair-groupmethod with arithmetic averages (UPGMA). The Dice correlationcoefficient was used with a 1.2% position tolerance to analyze thesimilarities of the banding patterns. Only bands larger than 48 kbwereconsidered for the analysis. Described criteria were used for definingdifferent PFGE clones (Zarrilli et al., 2007). A. baumannii RUH875 andRUH134 were used as reference strains representative of theinternational clonal lineages I (ICL-I) and II (ICL-II), respectively(Dijkshoorn et al., 1996; Nemec et al., 2004).

2.4. Multiplex PCRs for identification of sequence type groups

Identification of PCR-based sequence groups was conducted byusing 2 multiplex PCRs designed to selectively amplify group 1 orgroup 2 alleles of the gene encoding outer-membrane protein A(ompA), the gene encoding part of a pilus assembly system requiredfor biofilm formation (csuE), and the intrinsic blaOXA-51 carbapene-mase gene of A. baumannii. Reactions were performed as described(Turton et al., 2007). Identification of a strain as a member of group 1or group 2 required the amplification of all 3 fragments in thecorresponding multiplex PCR and the absence of any amplification bythe other multiplex PCR. Group 3 isolates were defined by theamplification of only the ompA fragment in the group 2 PCR and by theamplification of only the csuE and blaOXA-51-like fragments in thegroup 1 PCR. Identification of a strain as a member of groups 4, 5, 6,and 7 was defined according to additional combinations of amplicons(Towner et al., 2008).

2.5. Multilocus sequence typing

PCRs for 7 housekeeping genes, including gltA, gyrB, gdhB, recA,cpn60, gpi, and rpoD, were performed as described (Bartual et al.,2005). PCR products were purified with a QIAquick PCR Purificationkit (QIAGEN, Hilden, Germany) and sequenced with PCR forward orreverse primers by using the BigDye® Terminator 3.1 CycleSequencing Kits (Applied Biosystems, Foster City, CA, USA) on anABI-PRISM 3730 Genetic Analyser (Applied Biosystems). The resultingsequences were analyzed using the online BLAST web server (http://www.ncbi.nlm.nih.gov/BLAST/) and the MultAlin software (http://bioinfo.genopole-toulouse.prd.fr/multalin/). Analysis of allele se-quences and sequence type (ST) assignment were performed usingthe Oxford Acinetobacter baumannii website (http://pubmlst.org/abaumannii/).

2.6. Southern blot analysis

Localization of blaOXA-23, blaOXA-51, and blaOXA-58 genes wasattempted by using genomic mapping with the S1 nucleasetransforming supercoiled plasmids into linear molecules (Barton etal., 1995). Digested genomic DNA and plasmids were separated byPFGE. Probes were marked with the PCR DIG probe synthesis kit(Roche, Barcelona, Spain), and detection was performed with an anti-digoxigenin antibody conjugated to alkaline phosphatase and CPD-Star chemiluminescence substrate (Roche).

3. Results

3.1. Resistance phenotypes and epidemiologic data

Overall, 21 of 51 blood isolates included in the study werecharacterized by a carbapenem resistance phenotype (including bothimipenem and meropenem). CRAB isolates were obtained from 10centers (Fig. 1). Of note, all of them were hospital acquired (i.e.,

PADOVA, CLONE A(2 ISOLATES, OXA-58)

(2 ISOLATES, OXA-58)

NOVARA, CLONE G(1 ISOLATE, OXA-23)

(3 ISOLATES, OXA-58)

ANCONA, CLONE A(2 ISOLATES, OXA-58)

TORINO, CLONE D(3 ISOLATES, OXA-51/ISAba1)

GENOVA, CLONE E(2 ISOLATES, OXA-23)

FIRENZE

S. G. ROTONDO, CLONE C(1 ISOLATE, OXA-51/ISAba1)

LUCCA, CLONE A(1 ISOLATE, OXA-58)

CLONE F (2 ISOLATES, OXA-58) CLONE A (2 ISOLATES, OXA-58)

MILAN, CLONE A BERGAMO, CLONE B

Fig. 1. Geographic distribution of carbapenem-resistant Acinetobacter baumannii strains detected during the 1-year multicentre prospective surveillance study.

182 R. Migliavacca et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 180–186

bacteremia occurred at least 72 h after admission). Involved patientsbelonged to different age groups: 18–39 years (n = 6), 40–64 years(n = 7), and N64 years (n = 8). Most of them were admitted tointensive care units (n = 14), whereas the remaining werefrom medical and surgical wards (3 and 4 isolates, respectively).As shown in Table 1, isolates were commonly resistant to multipledrugs, including extended-spectrum cephalosporins (ceftazidimeand cefepime), fluoroquinolones (ciprofloxacin and levofloxacin),aminoglycosides (gentamicin and amikacin), and trimethoprim–

sulfamethoxazole. All strains were susceptible to colistin (MIC≤0.5 mg/L).

3.2. Molecular detection of carbapenemases

PCR analysis and sequencing revealed that 17/21 isolates carriedacquired carbapenemase determinants, including blaOXA-58-like orblaOXA-23-like genes (14 and 3, respectively). The remaining 4 isolateswere instead positive for the presence of an ISAba1 genetic elementupstream from the resident blaOXA-51-like gene (clones C and D). Thesame ISAba1 sequence was also detected upstream of the blaOXA-23-like gene in the 3 strains belonging to clones E and G, as well as the

blaOXA-58-like gene in the 3 strains belonging to clone B. ISAba2 wasfound upstream from the blaOXA-58-like gene in 11/14 cases (clones Aand F), whereas an ISAba3 element was detected downstream from allblaOXA-58-like positive strains. Of note, Southern blot analysisdemonstrated that blaOXA-58-like genes were plasmid located in allcases (data not shown). Class B metallo-β-lactamase determinantsand blaOXA-24/40-like genes were not detected in any strain.

3.3. Molecular characterization of aminoglycosides and quinoloneresistance

Class 1 integrons were detected in 10 isolates belonging to clonesA (n = 9) and G (n = 1). Strains of both clones harbored a 2.3-kbamplicon carrying aacA4 and blaOXA-20-like genes. Strains resistant toaminoglycosides (18/21) were consistently PCR-positive for thepresence of the aacA4 gene, independently on class 1 integrondetection. The co-presence of the aph6A gene was also found in 15/18strains. The armA gene was not detected in any of the isolates. Allstrains showing high-level resistance to fluoroquinolones harbored amissense mutation within the QRDR of gyrA (leading to Ser83 toLeu83 substitution). In the case of parC, mutations originated different

Table 1Phenotypic and genotypic features of carbapenem-resistant Acinetobacter baumannii blood isolates.

Center Strain MIC values (mg/L) as evaluated by the Etest methoda Amino acidchangesb

AGLRc CHDLd Typing

Ceftazidime Cefepime Imipenem Meropenem Ciprofloxacin Levofloxacin Gentamicin Amikacin Trimethoprim–

sulfamethoxazoleColistin gyrA parC Clone Sequence

groupSequencetype

Milan C01-268 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Milan C01-581 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Ancona C10-058 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Ancona C10-230 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Lucca C12-187 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4 OXA-58 A 1 4

Firenze C14-275 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Firenze C14-336 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Padova C15-207 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Padova C15-035 N16 N16 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

aacA4/aph6A

OXA-58 A 1 4

Bergamo C07-007 N16 N16 16 N32 4 2 N256 N32 N4 ≤0.5 aacA4/aph6A

OXA-58 B 5 20

Bergamo C07-025 N16 N16 16 N32 4 2 N256 N32 N4 ≤0.5 aacA4/aph6A

OXA-58 B 5 20

Bergamo C07-031 N16 N16 16 N32 2 2 N256 N32 N4 ≤0.5 aacA4/aph6A

OXA-58 B 5 20

S.G.Rotondo

C08-011 N16 N16 16 8 N32 N32 N256 8 N4 ≤0.5 Ser83 →Leu

Ser80 →Leu

aacA4 C 2 109

Torino C11-069 N16 N16 32 N32 N32 N32 16 4 0.25 ≤0.5 Ser83 →Leu

Ser80 →Leu

D 2 109

Torino C11-580 N16 N16 32 N32 N32 N32 16 8 0.25 ≤0.5 Ser83 →Leu

Ser80 →Leu

D 2 109

Torino C11-622 N16 N16 32 N32 N32 N32 16 8 0.25 ≤0.5 Ser83 →Leu

Ser80 →Leu

D 2 109

Genova C13-279 N16 N16 N32 16 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

Glu84 →Lys

aacA4/aph6A

OXA-23 E 2 95

Genova C13-373 N16 N16 N32 16 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

Glu84 →Lys

aacA4/aph6A

OXA-23 E 2 95

Firenze C14-1275 N16 8 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

Ser80 →LeuGlu84 →Lys

aacA4/aph6A

OXA-58 F 6 197

Firenze C14-1315 N16 8 N32 8 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

Ser80 →LeuGlu84 →Lys

aacA4/aph6A

OXA-58 F 6 197

Novara C06-397 N16 N16 N32 N32 N32 N32 N256 N32 N4 ≤0.5 Ser83 →Leu

Ser80 →Leu

aacA4 OXA-23 G 2 196

a According to the Etest method, MICs for trimethoprim–sulfamethoxazole are shown as trimethoprim values.b Amino acid changes involved in high-level resistance to fluoroquinolones.c AGLR = Resistance to aminoglycosides by aacA4/aph6A.d CHDL = Class D carbapenem-hydrolyzing β-lactamase.

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al./Diagnostic

Microbiology

andInfectious

Disease

75(2013)

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184 R. Migliavacca et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 180–186

amino acid changes including Ser80 to Leu80 (clones C, D, and G),Glu84 to Lys84 (clone E), or both (clone F).

3.4. Epidemiologic typing by PFGE, multiplex PCRs, and MLST

Macrorestriction analysis by PFGE revealed the presence of 7different major clones of CRAB isolates that corresponded to specificantimicrobial susceptibility patterns (Table 1). Fingerprints werecompared to those of strains representative of ICL-I and ICL-II as wellas of clones circulating in Italy (Fig. 2). The widely spread clone A,found in 5 Italian hospitals, was found to be related to ICL-II and to thepreviously described national clone B (D'Andrea et al., 2006). On thecontrary, clone C was related to ICL-I. Clone F was related to the SMALclone (belonging to sequence group 6), previously described in Italy(Nucleo et al., 2009). Two multiplex PCR schemes designed to detectallele variations in the ompA, csuE, and blaOXA-51-like genes identified4 different sequence groups as follows: group 1 (n=9), group 2 (n=7), group 5 (n= 3), and group 6 (n= 2). According to MLST analysis,6 different sequence types (ST) were found, including ST4, ST20, ST95,ST109, ST196, and ST197. Of them, ST4 was the most frequent beingrelated to the widespread clone A. Multiplex PCR andMLST results aresummarized in Table 1 together with phenotypic and PFGE data.

4. Discussion

This study describes the resistance mechanisms and geneticrelatedness of 21 CRAB blood isolates collected in different Italianhospitals during a 1-year multicenter prospective surveillance studybeginning in April 2007.

Actually, 3 epidemic ICLs are responsible for the majority of A.baumannii infections worldwide (Peleg et al., 2008). Strains belongingto ICL-II are widespread throughout Europe, with a number ofepidemiologic studies reporting the high frequency of OXA-58producers and the recent emergence of OXA-23 producers withinthis lineage (Corvec et al., 2007; D'Arezzo et al., 2011; Di Popolo et al.,2011). In our study, conducted in 2007–2008, strains producing theOXA-58 carbapenemase and belonging to ICL-II were detected in 5centers, thus appearing widespread in Italy in that period. Aprogressive change from blaOXA-58 to blaOXA-23 gene carriage wasobserved among ICL-II CRAB isolates responsible for intensive careunit outbreaks in the main hospitals in central Italy, with A. baumannii

100

90

8070

60

B National

Clone

ISAbNV SGR

CarStrain

International clone I

D

International clone II

A

NegRUH 875

ISAbC11-069

NegRUH 134

ISAbC15-207

G

E

B

C

ISAbC06-397

ISAbC13-279

ISAbC07-025

ISAbC08-011

F

SMAL Neg65 SM 01

ISAbC14-1275

Fig. 2. Dendrogram and computer-generated image of PFGE profiles of ApaI-digested genInternational clonal lineages I and II are shown for comparison together with previously de

strains producing OXA-23 appearing in Italy in 2007 (D'Arezzo et al.,2011; Di Popolo et al., 2011; Mendes et al., 2009).

Of note, 2 strains obtained in our study (clone E) were isolated in2007 in Genova, probably in the same epidemic context caused byOXA-23 producer strains described by Di Popolo et al. (2011) in thatperiod. In addition, clone G (i.e., the other strain producing the OXA-23enzyme) was isolated in the same geographic area. These strains werenot related to ICL-II and could represent some of the sources for thespread of OXA-23 carbapenemase in northern Italy. A geneticmechanism causing gene slippage or replacement of a single clone byanother could be taken into account. In fact, the ISAba1-associatedblaOXA-23 gene is usually carried by a transposable element irrespectiveof its chromosomal or plasmid location (Minandri et al., 2012). Theincreased circulation of the OXA-23–positive isolates in central Italycould be due to the emergence of different clones which displaced thepre-existing OXA-58–positive population (Minandri et al., 2012). Theacquisition of the blaOXA-23 determinant by the new epidemic lineagecould have provided A. baumannii with a selective advantage byincreasing resistance to both imipenem and meropenem (D'Arezzo etal., 2011). In this scenario, the epidemiologic evolution of A. baumanniistrains resulted in the dissemination in northern Italy of an epidemicmultidrug-resistant clone, closely related to ICL-II, carrying bothblaOXA-23 and armA genes, as a consequence of their ability to acquireresistance determinants (Brigante et al., 2012).

Concerning the chromosomal blaOXA-51-like gene (which isintrinsic to A. baumannii), it has been shown to confer carbapenemresistance when an ISAba1 element is inserted upstream of the gene(Turton et al., 2006). In our study, a variety of insertion sequences(ISAba1, ISAba2, and ISAba3) have been found at different positions(upstream or downstream from carbapenemase genes) This variabil-ity in the insertion sequences confirms the ability of A. baumannii toacquire mobile genetic elements from external environment (Turtonet al., 2006).

CRAB isolates are commonly resistant to multiple drugs. Concern-ing fluoroquinolones, the most frequent amino acid substitutionsoccur at position 83 of GyrA and at position 80 of ParC (Lee et al., 2005).Similarly, we found mutations at Ser83 of GyrA and at Ser80 or Glu84of ParC in several isolates. Eighteen of the 21 study isolates wereresistant to aminoglycosides. All of them showed the presence of theaacA4 gene encoding an AAC(6′)-Ib aminoglycoside acetyltransferase(which confers resistance to amikacin, netilmicin, and tobramycin).Interestingly, 15 of the latter strainswere also positive for the presence

20.0

0

30.0

040

.00

60.0

080

.00

100.

0012

0.00

140.

0016

0.00

180.

00

250.

00

300.

00

350.

00

400.

0045

0.00

500.

0060

0.00

700.

00

a3 OXA-58

bapenemase

ative

a1 OXA-51

ative

a3 OXA-58

a1 OXA-23

a1 OXA-23

a3 OXA-58

a1 OXA-51

ative

a3 OXA-58

omic DNA from representative carbapenem-resistant Acinetobacter baumannii strains.tected clones circulating in Italy.

185R. Migliavacca et al. / Diagnostic Microbiology and Infectious Disease 75 (2013) 180–186

of aphA6 genes. Possible association of integrons with multidrugresistancewas investigated, and class 1 integronswere detected in 10/21 isolates. Sequence analysis of the variable region showed thepresence of 2 gene cassettes: an aacA4 allele and blaOXA-20-like, a genecoding for a class D β-lactamase that confers resistance to amoxicillin,ticarcillin, oxacillin, and cloxacillin. A similar class 1 integron variableregion structure has been previously detected in A. baumannii in Italy(Zarrilli et al., 2007), thus suggesting that these elements can easily beexchanged by these strains in the hospital environments.

Seven different clones were found in our survey (even though thePFGE profile of clone G was difficult to interpret and use forepidemiologic surveys due to poor resolution). Clone A was detectedin 5 centers. This clone was related to the national clone B OXA-58producer, widespread in Italy since 2004 (Nucleo et al., 2009), and toICL-II, confirming the spread of this clone in Italy as previouslydescribed (Di Popolo et al., 2011). On the contrary, clone C (related toICL-I) and clone F (related to the SMAL clone) were found each in asingle center. Notably, clone F (which was carbapenem-resistant bythe acquisition of OXA-58 determinant) coexisted with clone A in thesame hospital in Florence, with the plasmidic blaOXA-58 determinantpossibly acquired from clone A by horizontal transfer. The remaining 4clones were heterogeneous with regard to carbapenemase produc-tion, remarking the ability of A. baumannii strains to acquire multipleresistance determinants.

PFGE and MLST analysis showed a high concordance degree.Different sequence types were usually associated with PFGE cloneswith ST4 being predominant (clonal complex 92; ICL-II). Of note, alarge percentage of strains related to clonal complex 92 have beenvery recently described in a 10-year collection of A. baumanniibloodstream isolates from the United States (Higgins et al., 2012).With regard to ST109, ST95, and ST197, they are single-locus variantsof each other and belong to clonal complex 109 (ICL-I). Of them,ST197 belonged to sequence group 6. Similarly, a recent report fromAustralian hospitals described an A. baumannii isolate ST109 belong-ing to the same sequence group (Post et al., 2012). The majority ofclones belonged to sequence group 1 or 2, which represents the 2broader lineages of multidrug-resistant A. baumannii circulating inEurope (Seifert et al., 2005). Strains belonging to sequence group 5have also been found in this study. To our best knowledge, this grouphas been found in several Turkish cities (Giannouli et al., 2009) butwas not previously detected in Italy. This heterogeneity of profileshighlights both the diffusion of ICLs and the spread of worrisomestrains equipped with multiple drug resistance mechanisms. Takentogether, our data suggest that CRAB isolates circulating in Italy aremostly related to major clusters of epidemic strains spreading inEurope, even though several minor clones appear to be distributed ata national level.

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