JCM Accepts, published online ahead of print on 8 January 2014 J. Clin. Microbiol. doi:10.1128/JCM.03009-13 Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Title: High Frequency of Acinetobacter soli Among Acinetobacter Isolates Causing

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Bacteremia at a Japanese Tertiary Hospital

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Running title: High frequency of Acinetobacter soli

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Key words: A. soli, bacteremia, rpoB, 16S rRNA, gyrB

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Authors: Shiro Endo,a Hisakazu Yano,a Hajime Kanamori,a Shinya Inomata,a Tetsuji

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Aoyagi,a Masumitsu Hatta,a Yoshiaki Gu,b Koichi Tokuda,a Miho Kitagawa,a and

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Mitsuo Kakua

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Affiliations: Department of Infection Control and Laboratory Diagnostics, Internal

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Medicine, Tohoku University Graduate School of Medicine, 1-1, Seiryo-machi,

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Aoba-ku, Sendai, Miyagi 980-8574, Japana

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Department of Regional Cooperation for Infectious Diseases, Tohoku University

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Graduate School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574,

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Japanb

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Address corresponding to S. Endo, [email protected]

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ABSTRACT

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Acinetobacter baumannii is generally the most frequently isolated Acinetobacter species.

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Sequence analysis techniques allow reliable identification of Acinetobacter isolates at

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the species level.

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Forty-eight clinical isolates of Acinetobacter spp. were obtained from blood cultures at

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Tohoku University Hospital. These isolates were identified at the species level by partial

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sequencing of the ribonucleic acid (RNA) polymerase β-subunit (rpoB), 16S rRNA, and

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gyrB genes. Then further characterization was done by using the polymerase chain

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reaction for detection of OXA-type β-lactamase gene clusters, metallo-β-lactamases,

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and carO genes. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence

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typing were also performed.

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The most frequent isolate is Acinetobacter soli (27.1%). Six of the 13 A. soli isolates

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were carbapenem non-susceptible and all of these isolates produced IMP-1. PFGE

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revealed that the 13 A. soli isolates were divided into 8 clusters.

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This study demonstrated that A. soli accounted for a high proportion of Acinetobacter

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isolates causing bacteremia at a Japanese tertiary hospital. Non-A. baumannii species

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were identified more frequently than A. baumannii and carbapenem non-susceptible

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isolates were found among the non-A. baumannii strains. These results emphasize the

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importance of performing epidemiological investigations of Acinetobacter species.

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3

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INTRODUCTION

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Acinetobacter species are strictly aerobic, gram-negative coccobacillary rods that are

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non-fermentive. During the last 20 years, Acinetobacter spp. have been reported to

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cause pneumonia, sepsis, wound infection, and various opportunistic infections, and

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these bacteria have been emerging as a cause of nosocomial infections. Because

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Acinetobacter are resistant to various antimicrobial agents, treatment of these infections

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can be challenging (1).

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A. baumannii generally represents the most clinically important and frequently

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isolated of the Acinetobacter spp. (2). However, according to a recent report from

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Norway, Acinetobacter nosocomialis was the most frequent isolate from blood cultures

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(3). Since the biochemical characteristics of different Acinetobacter spp. are almost

52

identical, accurate identification at the species level is difficult with routine laboratory

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methods. Recently, some molecular techniques have been reported for identification at

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the species level, such as sequencing of the 16S ribosomal ribonucleic acid (16S rRNA)

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gene, gyrB gene, recA gene, or RNA polymerase β-subunit (rpoB) gene (4–7). These

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molecular methods have recently led to progress in the accurate identification of

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Acinetobacter species.

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There is the possibility of different clinical outcomes due to infection with different

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genospecies since a difference in antibiotic susceptibility between A. baumannii and

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non-A. baumannii has been reported, as well as differences of antibiotic resistance

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mechanisms among Acinetobacter spp. (8–10). Accordingly, when Acinetobacter spp.

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are isolated from a patient with a severe infection such as sepsis, it is very important to

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accurately identify the isolate at the species level. Clinically and epidemiologically

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valuable information is provided by investigating the prevalence, antimicrobial 3

4

65

susceptibility patterns and resistance mechanism of pathogens.

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Therefore, this study was performed to clarify the species distribution, antibiotic

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susceptibility profile, and carbapenem resistance mechanisms of 48 non-duplicate

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Acinetobacter spp. causing bacteremia at a Japanese tertiary hospital.

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METHODS

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Bacterial Strains and Identification

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Forty-eight consecutive and non-duplicate clinical isolates of Acinetobacter spp. were

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obtained from blood cultures at Tohoku University Hospital (1,200 beds) between

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January 2007 and April 2012. These clinical isolates were obtained from inpatients who

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had at least two criteria for the systemic inflammatory response syndrome (temperature

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>38°C or 90 /min, respiration rate >20 /min or partial pressure of

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carbon dioxide (PaCO2) 12,000 /mm3, 10% immature forms) (11). The isolates were identified at the genus level by

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using the Vitek-2 System (Siemens Healthcare Diagnostics Japan, Tokyo, Japan).

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Species identification of the isolates was initiated by partial sequencing of the rpoB

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gene (zone 1) according to the method of Karah et al. (3, 4). Polymerase chain reaction

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(PCR) products were purified with a QIA quick PCR Purfication Kit (Qiagen, Hilden,

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Germany), followed by DNA sequencing using an ABI BigDye Terminator v3.1 Cycle

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Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI3730xl

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Analyzer (Applied Biosystems). BLASTn (http://www.ddbj.nig.ac.jp/) was used for

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sequence. A. soli was identified by partial sequencing of the rpoB gene (zone 2), plus

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sequencing of the gyrB and 16S rRNA genes, as well as by sequencing zone 1 of rpoB

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(4–7). The 16S rRNA gene sequences were analyzed by the EzTaxon-e server 4

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(http://eztaxon-e.ezbiocloud.net/).

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Antimicrobial Susceptibility Testing

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Minimum inhibitory concentrations (MICs) were determined according to the methods

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and breakpoints defined by the Clinical and Laboratory Standards Institute (CLSI) (12).

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The agar dilution method was used to obtain MICs for the following antimicrobial

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agents: ampicillin/sulbactam (Pfizer, New York City, New York, USA), piperacillin

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(Sigma-Aldrich), piperacillin / tazobactam (Sigma-Aldrich), cefotaxime

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(Sigma-Aldrich), ceftazidime (Sigma-Aldrich), cefepime (Bristol-Myers Squibb, Tokyo,

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Japan), imipenem (Banyu Pharmaceutical, Tokyo, Japan), meropenem (Dainippon

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Sumitomo Pharma, Osaka, Japan), gentamicin (Sigma-Aldrich), amikacin

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(Sigma-Aldrich), levofloxacin (Daiichi Sankyo), and colistin (Sigma-Aldrich). The

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range of concentrations tested was 0.06 to 256 μg/mL and the quality control strains

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were Escherichia coli ATCC 25922 and E. coli ATCC 35218.

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Molecular Characterization of β-lactamases Genes

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All isolates were further characterized by employing PCR for detection of the following

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genes: OXA-51-like, OXA-23-like, OXA-24-like, OXA-58-like, OXA-143-like,

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OXA-245-like, ISAba1/blaOXA-58-like complex, ISAba2/blaOXA-58-like complex,

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ISAba3/blaOXA-58-like complex, IS18/blaOXA-58-like complex, metallo-β-lactamases

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(MBL) genes (including IMP-1, IMP-2, VIM-1, VIM-2, SIM-1, and NDM-1), and the

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carO (outer membrane protein) gene (13–17).

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Sequencing of the OXA-type β-lactamase and MBL genes was performed according to the method of Pournaras et al. (18). BLASTn was used for sequence analysis. 5

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Molecular Typing by Pulsed-field Gel Electrophoresis and Multilocus Sequence

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Typing Analysis

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The clonal relationships of A. soli isolates were determined by pulsed-field gel

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electrophoresis (PFGE) with the SmaI restriction enzyme (19). Cluster analysis was

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done with GelCompar II v.3.0 (Applied Maths, Sint-Martens-Latem, Belgium) and the

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unweighted pair-group method using arithmetic averages (UPGMA). Strains were

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considered to belong to the same cluster if they showed ≥80% similarity of the PFGE

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profile (20).

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Using seven housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD),

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multilocus sequence typing (MLST) was performed for all A. baumannii isolates

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according to the method of Bartual et al. (21). DNA sequence variations were analyzed

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by using an MLST database for A. baumannii (http://pubmlst.org/abaumanni/).

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RESULTS

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Identification of Acinetobacter Species

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Among the 48 non-duplicate Acinetobacter spp. clinical isolates causing bacteremia, 8

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distinct Acinetobacter genospecies were detected. The isolates included A. soli (13,

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27.1%), A. nosocomialis (12, 25%), A. baumannii (9, 18.8%), A. ursingii (8, 16.7%), A.

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pittii (3, 6.3%), A. genomic species "close to 13TU" (1, 2.1%), A. junii (1, 2.1%) and A.

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guillouniae (1, 2.1%). The 13 A. soli isolates showed 99–100% similarity to

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CCUG59023 (GenBank accession number HQ148175) with respect to the sequences of

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zones 1 and 2 of the rpoB gene. These isolates also showed 99.5–100% similarity to A.

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soli strain B1 (T) (GenBank accession number EU290155) with respect to the 16S 6

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rRNA gene sequence and ≥97.5% similarity to JCM15062 (accession number;

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JQ411222) for the gyrB gene sequence.

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Antimicrobial Susceptibility

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Antimicrobial non-susceptibility rates are displayed in Table 1. None of the isolates

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showed reduced susceptibility to three or more of the antimicrobial agents tested. Of the

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Acinetobacter spp., only one strain of A. nosocomialis was resistant to both

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aminoglycosides and quinolones, while one strain of A. ursingii was resistant to both

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aminoglycosides and carbapenems. In addition, 4 A. soli strains were resistant to both

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carbapenems and quinolones.

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Molecular Characterization by Polymerase Chain Reaction and Sequencing

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The distribution of carbapenem resistance genes among Acinetobacter spp. (imipenem

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non-susceptible isolates) is show in Table 2. PCR amplification and sequencing revealed

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that A. soli and A. ursingii possessed blaIMP-1 and had no carO gene. Four A. soli strains

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and 1 A. ursingii strain possessed blaOXA-58-like, but blaOXA-58-like was not linked to

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ISAba1, ISAba2, ISAba3, or IS18. Carbapenem-susceptible non-A. baumannii species

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had no blaOXA-51-like genes. Among isolates that were susceptible to imipenem, only

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one A. ursingii strain possessed blaOXA-58-like, but blaOXA-58-like was not linked to

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ISAba1, ISAba2, ISAba3, or IS18.

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Molecular Typing

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PFGE indicated that the 13 A. soli isolates were divided into 8 clusters (Fig. 1). In

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addition, the 12 A. nosocomialis isolates were divided into 12 clusters, the 9 A. 7

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baumannii isolates into 9 clusters, and the 8 A. ursingii isolates into 8 clusters. When

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characterized by MLST, the A. baumannii isolates were divided into ST 80, 86, 144, 161,

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163, 175, 262, and 308.

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DISCUSSION

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Acinetobacter spp. can cause severe infections in hospitalized patients with

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compromised immune systems, including those who are dependent on medical devices.

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During the past few years, Acinetobacter spp. have emerged as a cause of nosocomial

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infection in Japan. However, there have been few reports about Acinetobacter from

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Japan, despite many reports from other countries.

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In general, the most frequent clinical isolate among Acinetobacter spp. has been

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reported to be A. baumannii (2). Current molecular methods have revealed 27 valid

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names within the Acinetobacter genus (22). In the present study, we used molecular

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techniques to investigate the frequency of Acinetobacter isolates causing bacteremia at a

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Japanese tertiary hospital. As with reports from other countries, this study showed that

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clinical isolates of Acinetobacter spp. from blood cultures included a high percentage of

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non-A. baumannii. The present study also revealed a high isolation rate for A. soli,

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although there have been few reports about this microbe elsewhere in the world. A. soli

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was first reported by Kim et al. in 2008 (4). In the present study, we identified A. soli by

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sequence analysis of the rpoB gene (zones 1 and 2), 16S rRNA gene, and gyrB gene.

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According to the results of our genetic analyses, 13 isolates showed an extremely high

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similarity to A. soli (HQ148175, EU29015, JQ411222), which strongly suggested that

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they were A. soli strains. The 13 A. soli strains isolated from blood cultures in the

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present study were divided into 8 clusters according to PFGE (Fig. 1). Strains No. 8

9

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10397 and 11584 (cluster A) were both isolated from patients in the intensive care unit,

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but were isolated at different times and there was no interaction between the two

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patients, so common hospital equipment and/or staff need to be considered in examining

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the potential relationship between the two cluster A strains isolated from the ICU.

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For cluster B, the timing of isolation and the department involved differed among each

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strain. Thus, it was impossible to determine solely from PFGE analysis that any of the A.

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soli strains in clusters A or B was responsible for an outbreak.

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On the other hand, some of the A. soli strains isolated from patients in the intensive

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care unit harbored IMP-1 in the present study. Unlike the chromosomal blaOXA-51-like

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genes, mobilization of IMP metallo-β-lactamase gene via plasmids may have resulted in

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dissemination of IMP to non-A. baumannii. For example, IMP-1 producing

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Acinetobacter spp. were first reported among carbapenem-resistant strains carrying the

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blaIMP-1 gene in a Brazilian hospital in 1998, and such resistance genes were detected

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among all carbapenem-resistant Acinetobacter spp. by 2001 (23). Previously, Yamamoto

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et al. reported on Acinetobacter isolates from certain areas of Japan and stated that

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IMP-19 was the predominant IMP in Acinetobacter spp. (24). In the present study, on

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the other hand, IMP-1 was predominant, and this difference between the two studies

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suggests that the predominant IMP type may differ from region to region and that a

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certain IMP type may easily disseminate via plasmids within a region. Furthermore, it

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has been reported that the prevalence of antimicrobial resistance varies widely among

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non-A. baumannii (25). This means that it is necessary to maintain surveillance for

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carbapenem non-susceptible non-A. baumannii, including A. soli.

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We found that the predominant Acinetobacter species isolated from blood cultures changed over time (data not shown). A. nosocomialis was most frequently isolated from 9

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blood cultures in 2007 and then gradually became less common, while A. soli was first

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isolated in 2008 and its isolation rate has since increased significantly. Epidemiology

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trends must continue to be tracked.

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A. baumannii is generally reported to be the carbapenem-resistant Acinetobacter spp.

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that causes problematic infections in many patients (9, 10). However, imipenem

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non-susceptible A. baumannii was not isolated in the present study. The reason for this

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may be that the number of isolates investigated was small. In addition, the prevalence of

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imipenem-non-susceptible A. baumannii seems to be lower in Japan (26) than in other

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countries, and the A. baumannii isolates assessed in the present study were not clustered

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into clonal complex 92. Therefore, further research on a larger number of Acinetobacter

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strains isolated throughout Japan is needed.

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We previously reported that imipenem non-susceptible A. baumannii in Japan

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possessed the OXA-51-like carbapenemase gene (26), while this study showed that

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imipenem non-susceptible non-A. baumannii possesses metallo-β-lactamase and carO

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gene. Thus, the possibility of a different mechanism of non-susceptibility to imipenem

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in A. baumannii and non-A. baumannii can be suggested.

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In conclusion, this is the first report describing a high rate of A. soli among

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Acinetobacter spp. causing bacteremia. In addtition, the present results suggest that the

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distribution of Acinetobacter spp. may show regional differences. Clinical microbiology

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services need to supplement identification of Acinetobacter genomic species by

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automated systems in order to accurately identify species for prevalence tracking and

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for clinical treatment of patients, however there is no reliable automated systems

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practically. Accordingly, our findings emphasize the importance of performing further 10

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epidemiological investigation of Acinetobacter species.

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ACKNOWLEDGMENTS

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We would like to thank the laboratory staff for their assistance.

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FUNDING

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This work was supported by a Grant-in-Aid for Scientific Research (C) [24590675]

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from the Japan Society for the Promotion of Science; The Ministry of Education,

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Culture, Sports, Science and Technology.

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COMPETING INTEREST

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The authors have declared that no competing interests exist.

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Chemother. 67:1623–1626.

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Figure legend Figure 1.

PFGE analysis of Acinetobacter soli and isolation date.

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Isolates were divided into 8 clusters. The cutoff value for cluster delineation was 80%

344

similarity. Restriction enzyme: SmaI. Cluster analysis: GelCompar II v.3.0 and UPGMA.

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Similarity coefficient: Jaccard. Band tolerance: 0.87%. Optimization: 0.00%.

346

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Table 1.

Non-Susceptible Acinetobacter Species Causing Bacteremia Number of non-susceptible strains (%)

Drug / Species

A. soli

A. nosocomialis

A. baumannii

A. ursingii

A. pittii

Others

(n=13)

(n=12)

(n=9)

(n=8)

(n=3)

(n=3)a

Ampicillin / Sulbactam

8 (61.5)

2 (16.7)

0

2 (25)

1 (33.3)

0

13 (27.1)

Piperacillin

9 (69.2)

9 (75)

3 (33.3)

8 (100)

3 (100)

2 (66.7)

34 (70.8)

Total

Piperacillin / Tazobactam

10 (76.9)

6 (50)

2 (22.2)

6 (75)

3 (100)

1 (33.3)

28 (58.3)

Cefotaxime

12 (92.3)

11(91.7)

4 (44.4)

7 (87.5)

3 (100)

2 (66.7)

39 (81.3)

Ceftazidime

10 (76.9)

3 (25)

0

5 (62.5)

1 (33.3)

1 (33.3)

20 (41.7)

Cefepime

11 (84.6)

3 (25)

1 (11.1)

3 (37.5)

1 (33.3)

0

19 (39.6)

Imipenem

6 (46.2)

0

0

1 (12.5)

0

0

7 (14.6)

Meropenem

6 (46.2)

1 (8.3)

0

1 (12.5)

0

0

8 (16.7)

Gentamicin

0

2 (16.7)

0

1 (12.5)

0

0

3 (6.3)

Amikacin

6 (46.2)

1 (8.3)

1 (11.1)

1 (12.5)

0

0

9 (18.8)

Levofloxacin

6 (46.2)

1 (8.3)

1 (11.1)

0

0

1 (33.3)

9 (18.8)

Colistin

4 (30.8)

10 (83.3)

4 (44.4)

0

0

2 (66.7)

20 (41.7)

348

Breakpoints according to the Clinical and Laboratory Standards Institute (12).

349

a

Other strain include A. close to 13 TU, A. junii, and A. guillouniae.

350

17

18

351

Table 2.

Characterization of Imipenem Non-Susceptible Acinetobacter spp. Causing Bacteremia MIC (μg/mL)

OXAtype-β-lactamase genes

Metallo-β-lactamase genes

Species OXA-51- OXA-23- OXA-24- OXA-58- OXA-143- OXA-245(Strain No.)

MEPM like

like

like

likea

like

like

IMP-1a IMP-2 VIM SIM-1 NDM-1

carO

A. soli (8657)

16

16

-

-

-

+b

-

-

+

-

-

-

-

-

A. soli (9266)

32

16

-

-

-

-

-

-

+

-

-

-

-

-

A. soli (10397)

16

16

-

-

-

-

A. soli (11013)

352

IPM

16

8

-

-

-

-

-

+

-

-

-

-

-

b

-

-

+

-

-

-

-

-

b

+

A. soli (11354)

16

16

-

-

-

+

-

-

+

-

-

-

-

-

A. soli (11584)

16

32

-

-

-

+b

-

-

+

-

-

-

-

-

A. ursingii (9640)

32

32

-

-

-

+b

-

-

+

-

-

-

-

-

Abbreviations: IPM, imipenem; MEPM, meropenem; MIC, minimum inhibitory concentration.

353

IPM and MEPM were determined by the agar dilution method.

354 355

a b

Sequencing of the blaOXA-58-like and blaIMP-1 genes yielded OXA-58 and IMP-1, respectively. These were not linked to ISAba1, ISAba2, ISAba3, or IS18.

18