Journal of Medical Microbiology Papers in Press. Published August 20, 2014 as doi:10.1099/jmm.0.077503-0 1
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An Outbreak of blaOXA-51-like- and blaOXA-66- Positive Acinetobacter baumannii ST208
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in the Emergency Intensive Care Unit
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Satomi Asai,1,2*
Kazuo Umezawa,3 Hideo Iwashita,1 Toshio Ohshima,2
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Maya Ohashi,2
Mika Sasaki,2 Hideki Hayashi,4 Mari Matsui, 5 Keigo Shibayama,5
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Sadaki Inokuchi3 and Hayato Miyachi1,2
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1. Department of Laboratory Medicine, Tokai University School of Medicine,
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2. Infection Control Division in Tokai University Hospital,
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3. Department of Critical Care and Emergency Medicine, Tokai University School of
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Medicine,
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4. Department of Bioinformatics, Education and Research Support Center, Tokai University
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School of Medicine, 143 Shimokasuya, Isehara, 259-1193, Japan.
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5. Department of Bacteriology II, National institute of infectious diseases, Tokyo, Japan.
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Running head: Drug resistance of A. baumannii
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（
）
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This work was supported by a Grant-in-Aid #23590691 for Scientific Research (C) from the
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Japan Society for the Promotion of Science, the Ministry of Education, Culture, Sports,
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Science and Technology.
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1
2
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Correspondence Satomi Asai, MD, PhD.
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Department of Laboratory Medicine, Tokai University School of Medicine,
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143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
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Phone: # +81 463 93 1121 Ex. 2450
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FAX: # +81 463 93 8607
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E-mail address: [email protected]
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Co-author's e-mail address
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Kazuo Umezawa: [email protected]
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Hideo Iwashita: [email protected]
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Maya Ohashi: [email protected]
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Hideki Hayashi: [email protected]
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Keigo Shibayama: [email protected]
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Hayato Miyachi: [email protected]
Toshio Ohshima: [email protected], Mika Sasaki: [email protected] Mari Matsui: [email protected]
Sadaki Inokuchi: [email protected]
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2
3
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ABSTRACT
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A series of clinical isolates of drug resistant Acinetobacter baumannii (DR-A. baumannii)
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with diverse drug susceptibility was detected from eight patients in the critical care and
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emergency center of Tokai University Hospital.
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2010 (A. baumannii Tokai strain 1), and subsequently, seven isolates were obtained from
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patients (A. baumannii Tokai strain 2-8) and one isolate was obtained from an air fluidized
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bed shared by five of the patients during the three months from August to November 2011.
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The isolates were classified into three types of antimicrobial drug resistance patterns (RRR,
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SRR and SSR) according to their susceptibility to IPM, AMK and CPFX, respectively.
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Genotyping of these isolates by multilocus sequence typing (MLST) revealed one sequence
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type, ST208, while that by a DiversiLabⓇ analysis revealed two subtypes.
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were positive for blaOXA-51-like and blaOXA-66, as assessed by PCR. A. baumannii Tokai strains
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1-8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC,
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as revealed by DNA sequencing. The ISAba1 upstream of blaOXA-51-like and aminoglycoside
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resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1-7 and 10
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(RRR and SRR) as assessed by PCR. Among the resistance-nodulation-division (RND)
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family pump-encoding genes (adeB, adeG and adeJ) and outer membrane porin-encoding
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genes (oprD and carO), overexpression of adeB and adeJ, and the suppression of oprD and
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carO were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by real-time
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RT-PCR.
The initial isolate was obtained in March
All the isolates
In summary, the molecular characterization of a series of isolates of DR-A. 3
4
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baumannii revealed the outbreak of ST208, and a diversity of antimicrobial drug
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susceptibilities, which was almost correlated with differential gene alterations responsible for
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each type of drug resistance.
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Key words: antimicrobials; drug resistant Acinetobacter baumannii; β-lactamase; sequence
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type.
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List of Abbreviations
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AGs, aminoglycosides; AMK, amikacin; AZT, aztreonam; CAZ, ceftazidime; CFPM,
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cefepime; CPFX, ciprofloxacin; CPZ, cefoperazone; CZOP, cefozopran; DR- A. baumannii,
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drug
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ethylenediaminetetraacetic acid; EICU, Emergency Intensive Care Unit; FOM, fosfomycin;
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FQs, fluoroquinolones; GM, gentamicin; IPM, imipenem; IPM/CS, imipenem/cilastatin; LB,
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Luria-Bertani broth; LVFX, levofloxacin; MBL, metallo-beta-lactamase; MDR- A. baumannii,
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multidrug-resistant Acinetobacter baumannii; MEPM, meropenem; MIC, minimum inhibitory
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concentration; MINO, minocycline; MLST, multi-locus sequence typing; PIPC, piperacillin;
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R, resistant; rep-PCR, repetitive-element-based polymerase chain reaction; RT-PCR, Reverse
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transcription-polymerase chain reaction; S, susceptible; SBT/ABPC, sulbactam/ampicillin; ST,
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sequence type; TAZ, tazobactam; TEIC, teicoplanin; TOB, tobramycin; VCM, vancomycin.
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RRR, IPM-resistant (R), AMK-resistant (R) and CPFX-resistant (R); SRR, IPM-susceptible
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(S), AMK-resistant (R) and CPFX-resistant (R); SSR, IPM-susceptible (S), AMK- susceptible
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(S) and CPFX-resistant (R); SSS, IPM-susceptible (S), AMK- susceptible (S) and CPFX-
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susceptible (S).
resistant
Acinetobacter
baumannii;
DRPM,
doripenem;
EDTA,
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5
6
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INTRODUCTION
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Acinetobacter baumannii (A. baumannii) is emerging as a nosocomial pathogen, particularly
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in intensive care units, including burn care units (Guzek, et al., 2013; Bayram, et al., 2013;
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Ohashi, et al., 2013). Hospitalized patients at a greater risk for Acinetobacter infections are
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those particularly very ill on a ventilator, those with a prolonged hospital stay, those who have
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open wounds and anyone with invasive devices like urinary catheters (Howard, et al., 2012,
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Wendt, et al., 1997; Wisplinghoff, et al., 2000; Ho, et al., 2010; Zheng, et al., 2013).
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A stepwise evolution in the acquisition of multidrug-resistance in clinical isolates of
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Pseudomonas aeruginosa and A. baumannii has been reported (Higgins, et al., 2010; Asai, et
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al., 2011). Elucidation of the mechanism(s) underlying the drug-resistance is important to
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prevent such resistance.
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We experienced a series of clinical isolates of DR-A. baumannii with a diverse pattern of
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drug resistance from 8 patients in the emergency intensive care unit of Tokai University
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Hospital from March 2010 to November 2011.
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resistance in the same sequence type, we studied the molecular characteristics of the clinical
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isolates and the relationship with the resistance pattern.
In order to elucidate the diversity on drug
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MATERIALS AND METHODS
Patients and clinical specimens
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The patients studied were admitted to the critical care and emergency center (57 beds,
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including three beds in the severe burn care unit) of Tokai University Hospital (total 804
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beds), because of serious burns, traffic injuries or cerebral hemorrhage.
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all patients, except two with cerebral hemorrhage, were treated alternatively with the systemic
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administration of antibiotics, including β-lactams, FQs, AGs and glycopeptides, for infections
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of wounds and/or the respiratory tract.
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During this period,
Routine microbial examinations were performed on a weekly basis on clinical specimens from the patients' sputum, urine (via catheter), venous blood, wounds and so on.
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Bacteriological surveillance of environmental surfaces was performed for the medical
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equipment shared by the patients in order to seek a possible reservoir of the pathogen, such as
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an air fluidized bed.
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SSS) was used as a control.
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the possible transmission route using information on the clinical care of the patients and
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detection of A. baumannii.
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(13R-036).
A drug-sensitive strain of A. baumannii (A. baumannii Tokai strain 9, The epidemiological investigation was performed to elucidate
The study was approved by the review board of Tokai University
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Growth conditions and antibiotic susceptibility testing
Bacteria were cultured at 37 Co., Ltd., Tokyo, Japan).
℃ in Luria-Bertani broth (Kyokuto Pharmaceutical Industrial
The criteria for MDR- A. baumannii was resistance to IPM (MIC 7
8
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> 16 µg/mL), AMK (MIC > 32 µg/mL) and CPFX (MIC > 4 µg/mL), and DR- A. baumannii
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was defined as resistance to one or two of the drugs according to the Japanese National
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Guideline Concerning the Prevention of Infections and Medical Care for Patients with
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Infections.
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Biochemical identification and susceptibility testing of the isolates was performed to obtain
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MIC values using a microdilution method according to the Clinical Laboratory Standards
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Institute (CLSI, 2009) and MicroScan WalkAway-96 SI system (Siemens Japan K.K., Tokyo,
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Japan). The antibiotics used in this study were obtained as follows: AMK and IPM were
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from Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan), AZT from Eizai Co., Ltd. (Tokyo,
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Japan), CAZ from Glaxo SmithKline Co., Ltd. (Tokyo, Japan), CFPM from Bristol-Myers
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Squibb Co. (Tokyo, Japan), CPFX from Bayer HealthCare (Osaka, Japan), CZOP from
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Takeda Pharmaceutical Co., Ltd. (Osaka, Japan), DRPM from Shionogi & Co., Ltd. (Osaka,
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Japan), FOM from Meiji Seika Co., Ltd. (Tokyo, Japan), GM from Schering-Plough, Co., Ltd.
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(Tokyo, Japan), LVFX from Daiichi-Sankyo Pharmaceutical Co., Ltd. (Tokyo, Japan), MEPM
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from Daiichi-Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), MINO from Wyeth &
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Takeda Pharmaceutical Co., Ltd. (Tokyo, Japan), PIPC from Taisho Toyama Pharmaceutical
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Co., Ltd. (Tokyo, Japan) and TOB was from Towa Pharmaceutical Co., Ltd. (Osaka, Japan).
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Molecular typing
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9
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DNA templates were extracted using the ZR-Duet DNA/RNA MiniPrep Kit (Zymo
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Research, CA, USA). MLST was performed as described previously (Fu, et al., 2010;
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Bartual, et al., 2005).
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sequence type database (http://pubmlst.org/abaumannii/) to determine the alleles and
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sequence types (ST).
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rep-PCR DiversiLab Microbial Typing SystemⓇ (SYSMEX BioMérieux Co., Ltd. Tokyo,
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Japan), which
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genomes, as described previously (Higgins, et al., 2012; Carretto, et al., 2008).
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annealing temperature of the PCR (polymerase chain reaction) amplification used in this
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study was 55°C for gltA, gyrB, recA and cpn60, and 50°C for gdhB, gpi and rpoD. The
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amplification products were purified with a Qiagen DNA purification kit (Qiagen Inc., Tokyo,
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Japan).
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(Applied Biosystems, Life Technologies Japan Ltd., Tokyo, Japan).
MLST sequences were uploaded into the A. baumannii MLST
A. baumannii isolates were screened for gene homology by the
amplified the regions between the noncoding repetitive sequences in bacterial The
The DNA sequencing was performed using an ABI3500xL Genetic Analyzer
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Evaluation of the mechanisms of resistance
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Screening for MBL
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A. baumannii isolates were screened for the production of MBL by a double-disk synergy
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test with disks containing sodium mercaptoacetic acid, as described previously (Arakawa, et
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al., 2000). 9
10
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PCR assay for β-lactamase and armA
The following resistance genes were examined by PCR: blaIMP-1, blaVIM, blaOXA-23-like,
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blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaOXA-66, and ISAba1 genes, as described previously
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(Woodford, et al., 2006; Turton, et al., 2006, Lee, et al., 2010).
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encodes 16S rRNA methylases and confers high resistance to AGs, was screened by PCR
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using primers that were described previously (Yamane, et al., 2005).
The armA gene, which
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Sequencing of OXA-type β-lactamase, and gyrA and parC
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Sequencing of OXA-type β-lactamase was performed as described previously (Endo, et al.,
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2012). The quinolone resistance-determining regions (QRDRs) of gyrA and parC were
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amplified and analyzed as described previously (Liu, et al., 2012).
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amplified DNA products was performed using an ABI3500xL Genetic Analyzer (Applied
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Biosystems, Life Technologies Japan Ltd., Tokyo, Japan).
DNA sequencing of the
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Quantitative RT-PCR (qRT-PCR) RNA templates were extracted by the ZR-Duet DNA/RNA MiniPrep Kit (Zymo Research, CA, USA).
The expression of three different resistance-nodulation-division (RND) family
10
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pump-encoding genes (adeB, adeG, adeJ) and two outer membrane porin-encoding genes
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(oprD and carO) were analyzed by quantitative RT-PCR using the StepOnePlusTM Real-Time
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PCR System (Applied Biosystems, Life Technologies Japan Ltd., Tokyo, Japan)
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al., 2013; Fernando & Kumar, 2012; Peleg, et al., 2008). The primers used for the analysis
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are listed in Table 1.
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2012; Srinivasan, et al., 2011; Coyne, et al., 2010). Reactions (20 L) were set up using 400
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nM primers and 2 L of the cDNA template (diluted 1:10) with SYBR R Premix Ex TaqTM II
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(Tli RNase H Plus), and ROX plus (Takara Bio Inc., Shiga, Japan).
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carried out using the StepOne™ software program.
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normalized based on the level of the 16S rRNA mRNA gene and was expressed as a relative
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rate compared to that in the susceptible isolate of each pair (the expression of A. baumannii
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Tokai strain 9 was taken as 1.0).
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independently, and all reactions were carried out in triplicate.
(Zander, et
The housekeeping gene, 16S rRNA, was used as a control (Hou, et al.,
μ
μ
○
The data analysis was
The expression of each target gene was
Experiments were conducted at least three times
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RESULTS
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Bacterial strains and antibiotic susceptibility
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The characteristics of the A. baumannii Tokai strains are shown in Table 2.
In March
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2010, a drug resistant isolate of A. baumannii Tokai strain 1 was initially detected from the
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wound of a patient with a severe burn injury.
After one and a half years, during a period of 11
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three months from August to November 2011, another seven clinical isolates of DR-A.
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baumannii strains from patients (A. baumannii Tokai strains 2-8) were obtained.
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A. baumannii Tokai strains were classified into three types according to their susceptibility to
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three drugs (IPM, AMK and CPFX), as RRR, SRR or SSR (Tables 2 and 3).
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obtained from sputum, wounds and bile drains.
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The DR-
They were
Since the interval between the first patient and others long (>18 months), the environment of
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the ward was suspected to be a possible reservoir of the pathogen. Based on the results of
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the bacteriological surveillance of environmental surfaces, A. baumannii Tokai strain 10 was
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isolated from the cracks of a rubber frame and a lump of beads in the air fluidized bed, which
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was used by five patients during their hospitalization (A. baumannii Tokai strains 1, 2, 4, 5
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and 6).
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Molecular typing
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The molecular genotyping of isolates by a MLST analysis revealed a sequence type ST208
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for A. baumannii Tokai strains 1-8 and 10 (gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD:1, 3,
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3, 2, 2, 97 and 3) and another type for A. baumannii Tokai strain 9 (gltA, gyrB, gdhB, recA,
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cpn60, gpi and rpoD: 15, 48, 58, 42, 36, 54 and 41).
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by the rep-PCR by DiversiLab Microbial Typing SystemⓇ showed the same pattern (more
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than 97% similarity) as one type for eight of the isolates (A. baumannii Tokai strain1-7 and
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10) (Fig. 1), and the other isolates (A. baumannii Tokai strain 8 and 9) had different patterns
The molecular genotyping of isolates
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(85% and less than 70% similarity, respectively) .
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Expression of resistance-related genes The MBL assay of the clinical isolated A. baumannii Tokai strains revealed no apparent
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MBL production, and all isolates showed expression of OXA-51-like carrying OXA-66
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β-lactamase (Table 4). The expression of IMP-1, VIM, OXA-23-like, OXA-24-like and
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OXA-58-like was negative.
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baumannii Tokai strains 1-7 and 10. The DNA sequencing of gyrA and parC revealed that
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Serine-83 (TCA) was changed to TTA (Leucine) and that Ser-80 (TCG) was changed to TTT
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(Phenylalanine) or TTG (Leucine) in A. baumannii Tokai strains 1-8 and 10.
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The expression of ISAba1 and armA were found in A.
Our analysis of RND pump-encoding genes included an analysis of the expression of three
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previously characterized genes, adeB, adeG and adeJ, which encode the RND pump in the
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adeABC, adeFGH and adeIJK operons, respectively.
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Tokai strain 1,2, 8 and 9 as a representative strain from each group with the same
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susceptibility pattern was shown in Table 5.
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in A. baumannii Tokai strains 2. The expression of oprD was decreased in A. baumannii
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Tokai strains 2 and 8.
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Tokai strains 1, 2 and 8.
The result of Acinetobacter baumannii
The overexpression of adeB and adeJ was seen
The underexpression of carO was seen in isolates with A. baumannii
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DISCUSSION
We experienced a series of clinical isolates of DR-A. baumannii ST208 in the emergency
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intensive care unit of Tokai University Hospital.
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drug resistance patterns in the same sequence type in these isolates, we studied the molecular
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characteristics of these isolates and their relationship with the resistance pattern.
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In order to elucidate the diversity of the
A. baumannii Tokai strains 1-7 and 10 were positive for OXA-51-like and OXA-66
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β-lactamase and ISAba1. A. baumannii strains with resistance to AMK (A. baumannii Tokai
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strains 1-7 and 10) were positive for armA. These results are consistent with the idea that
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ISAba1 regulates the expression of OXA-51-like carrying OXA-66 β-lactamase, and that
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armA is related to AGs resistance.
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and 6) could have been derived from the same source and/or transmitted horizontally, because
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the same air fluidized bed had been used by those patients. Among them, A. baumannii
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Tokai strains 2, 4 and 6 showed multidrug resistance (RRR). These patients were treated
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with carbapenem (MEPM or DRPM) prior to sampling for at least one week, which may have
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played a role in the overexpression of adeB and adeJ in A. baumannii Tokai strain 2. A.
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baumannii Tokai strain 10 was detected from the cracks of the rubber frame and a lump of
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beads in the air fluidized bed, even though the bed had been cleaned and disinfected every
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time after use.
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reported (Yamada & Suwabe, 2013; Suzuki, et al., 2013), an outbreak of A. baumannii ST208
The five isolates (A. baumannii Tokai strains 1, 2, 4, 5
Although a few of nosocomial outbreaks of A. baumannii ST2 have been
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has not been reported previously in Japan.
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Since the pattern of the rep-PCR and sequence type of MLST in the eight isolates was the
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same as that in the initial case, it was suggested that the strain survived for a year and a half in
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the environmental reservoir.
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environmental cleaning and disinfection in order to reduce the risk of transmission is
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suggested. A. baumannii Tokai stain 8 (SSR) was also ST208, but had a different pattern by
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the rep-PCR.
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transposon or the insertion of a different plasmid might have led to the different pattern.
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During the period of an outbreak, A. baumannii with different drug susceptibility patterns
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appeared depending on the various resistance mechanisms.
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As infection control procedures, careful attention to
During the transmission from the same original organism, the presence of a
Nine isolates (A. baumannii Tokai stain1-8, and 10) had resistance to CPFX, which can be
261
explained by the mutations of gyrA and parC. Another major factor contributing to the
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resistance of this organism was the overexpression of the RND pumps (Zander, et al., 2013;
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Amin, et al., 2013; Fernando & Kumar., 2012). Our analysis of RND pump-encoding genes
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included the expression of three previously characterized genes, adeB, adeG and adeJ, which
265
encode the RND pumps in the adeABC, adeFGH and adeIJK operons, respectively.
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pumps such as AdeABC have been reported to be involved in multidrug resistance (Hou, et
267
al., 2012; Vila, et al., 2007).
268
overexpression of adeB and adeJ.
269
some β-lactams (CAZ, CFPM and CZOP) than that of A. baumannii Tokai strain 9 (SSS).
Efflux
In our study, A. baumannii Tokai strain 2 (RRR) showed the A. baumannii Tokai strain 8 showed better sensitivity to
15
16
270
This phenomenon might be associated with underexpression of adeB.
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adeB and adeJ, were related to the acquisition of multidrug-resistance. As for porins, the
272
overexpression of genes encoding RND pumps and the downregulation of genes encoding
273
porins is known to be common in clinical isolates of Acinetobacter species (Fernand, et al.,
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2013).
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without oprD does not result in resistance to carbapenem in A. baumannii Tokai strains 1 and
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8 (SRR and SSR).
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decrease in porins among Acinetobacter strains is not associated with resistance to
278
carbapenems in the presence of β-lactamases
279
Two pumps, such as
Our findings also suggest that the underexpression of carO in combination with or
This observation is consistent with previous findings showing that a
（Rumbo, et al., 2013; Singh, et al., 2013）.
In conclusion,c Furthermore, we demonstrated that drug resistance is associated with the
280
expression of ISAba1 and armA and mutations in gyrA and parC and that the overexpression
281
of adeB and adeJ plays a role in the multidrug resistance of A. baumannii Tokai strain ST208.
282
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Ho, P.L., Ho, A.Y., Chow, K.H., Lai, E.L., Ching, P. & Seto, W.H. (2010). Epidemiology
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Howard, A., O’Donoghue, M., Feeney, A. & Sleator, R.D. (2012). Acinetobacter
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Liu, Y.H., Kuo, S.C., Lee, Y.T., Chang , I.C., Yang, S.P., Chen, T.L. & Fung, C.P. (2012).
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Amino acid substitutions of quinolone resistance determining regions in GyrA and ParC
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Ohashi, M., Asai, S., Umezawa, K., Kenmochi, I., Sasaki, M., Iwashita, H., Hasunuma,
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Martínez-Martínez, L., Vila, J., Pachón, J., Cisneros, J. M., Rodríguez-Baño, J.,
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Pascual, A., Bou, G. & Tomás, M. (2013). Contribution of efflux pumps, porins, and
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β-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii.
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Antimicrob Agents Chemother 57, 5247-5257.
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Singh, H., Thangaraj, P., Chakrabarti, A. (2013). Acinetobacter baumannii: A brief account
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of mechanisms of multidrug resistance and current and future therapeutic management. J Clin
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Diagn Res 7, 2602-2605.
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Srinivasan, V.B., Rajamohan, G., Pancholi, P., Marcon, M. & Gebreyes, W.A. (2011).
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Molecular cloning and functional characterization of two novel membrane fusion proteins in
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conferring antimicrobial resistance in Acinetobacter baumannii. J Antimicrob Chemother 66,
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499–504.
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Sung, J.Y., Koo, S.H., Cho, H.H. & Kwon, K.C. (2012). AbaR7, a genomic resistance
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island found in multidrug-resistant Acinetobacter baumannii isolates in Daejeon, Korea. Ann
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Lab Med 32, 324-330.
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Suzuki, M., Matsui, M., Suzuki, S., Rimbara, E., Asai, S., Miyachi, H., Takata, T., Hiraki,
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Y., Kawano, F. & Shibayama, K. (2013). Genome sequences of multidrug-resistant
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Acinetobacter baumannii strains from nosocomial outbreaks in Japan. Genome Announc. 18,
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1(4). pii: e00476-13. doi: 10.1128/genomeA.00476-13.
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Turton, J.F., Ward, M.E., Woodford, N., Kaufmann, M.E., Pike, R., Livermore, D.M. &
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Pitt, T.L. (2006). The role of ISAba1 in expression of OXA carbapenemase genes in
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Acinetobacter baumannii. FEMS Microbiol Lett 258, 72-77.
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Vila, J., Martı´, S. & Sa´nchez-Ce´spedes, J. (2007). Porins, efflux pumps and multidrug
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resistance in Acinetobacter baumannii. J Antimicrob Chemother 59, 1210–1215.
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Wendt, C., Dietze B., Dietz, E. & Rüden, H. (1997). Survival of Acinetobacter baumannii
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on dry surfaces. J Clin Microbiol 35, 1394 –1397.
22
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Wisplinghoff, H., Edmond, M.B., Pfaller, M.A., Jones, R.N., Wenzel, R.P. & Seifert, H.
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(2000). Nosocomial bloodstream infections caused by Acinetobacter species in United States
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hospitals: clinical features, molecular epidemiology and antimicrobial susceptibility. Clin
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Infect Dis 31, 690–697.
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Woodford, N., Ellington, M.J., Coelho, J.M., Turton, J.F., Ward, M.E., Brown, S.,
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Amyes, S.G. & Livermore, D.M. (2006). Multiplex PCR for genes encoding prevalent OXA
389
carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 27, 351-353.
390
Yamada, Y. & Suwabe, A. (2013). Diverse carbapenem-resistance mechanisms in 16S
391
rRNA methylase-producing Acinetobacter baumannii. J Medical Microbiol 62, 618-622.
392
Yamane, K., Wachino, J., Doi, Y., Kurokawa, H. & Arakawa, Y. (2005). Global spread of
393
multiple aminoglycoside resistance genes. Emerg Infect Dis 11, 951-953.
394
Zander, E., Chmielarczyk, A., Heczko, P., Seifert, H. & Higgins, P.G. (2013) Conversion
395
of OXA-66 into OXA-82 in clinical Acinetobacter baumannii isolates and association with
396
altered carbapenem susceptibility. J Antimicrob Chemother 68, 308–311.
397
Zheng, W., Yuan, S. & Li, L. (2013). Analysis of hospital departmental distribution and
398
antibiotic susceptibility of Acinetobacter isolated from sputum samples. Am J Infect Control
399
41, e73-76.
400 23
24
401
FIGURE LEGENDS
402
Figure 1. The results of the rep-PCR DiversiLab Microbial Typing SystemⓇ analysis and
403
sequence type
404
（ST） in clinical isolates of A. baumannii.
A. baumannii Tokai strains 1 to 7 and 10 showed an identical pattern and a homologous rate
405
of identity > 97%.
Two isolates, A. baumannii Tokai strains 8 and 9, had different patterns
406
(85% and less than 70% similarity, respectively).
407
analysis revealed that A. baumannii Tokai strains 1 to 8 and 10 were of the same sequence
408
type (ST208).
The sequence type from the MLST
24
25
Table 1. Primer sequence of quantitative RT-PCR Primer adeB
adeG
adeJ
oprD
carO
16S rRNA
Primer sequence
Product size (bp)
Source
forward
5´-ACC GGA GGT ATC TGT TGT CG-3´
122
Fernando & Kumar., 2012
reverse
5´-GTG CGA CTT ATC CTG GTG CT-3´
forward
5´-ATC GCG TAG TCA CCA GAA CC-3´
92
Fernando & Kumar., 2012
reverse
5´-CGT AAC TAT GCF GTG CTC AA-3´
forward
5´-CAT CGG CTG AAA CAG TTG AA-3´
109
Fernando & Kumar., 2012
reverse
5´-GCC TGA CCA TTA CCA GCA CT-3´
forward
5´-CCA GCT CAG TTG CTC AAT CA-3´
134
this study
reverse
5´-CAT TTG GTT TCC AGC GTT TT-3´
forward
5´-GGT TAT AAC GGC GGT GAC AT-3´
115
this study
reverse
5´-CCA AGG ACG AAT TTC AGC AT-3´
forward
5´-CGT AAG GGC CAT GAT GAC TT-3´
150
Fernando & Kumar., 2012
reverse
5´-CAG CTC GTG TCG TGA GAT G-3´
25
26
Table 2. Information of cases and Acinetobacter baumannii Tokai strain. Strain Disease
Ward
No. 1.
The detected day
Source
from hospitalization 74% TBSA Burn
Burn center
Day 31
85% TBSA Burn
Burn center
(Ohashi, et al., 2013) 3. 40% TBSA Burn
Burn center
(Ohashi, et al., 2013) 4. 70.5% TBSA Burn
Burn center
(Ohashi, et al., 2013) 5. Traffic injury
EICU
Day 9
Sputum
Wound
Wound
Wound
EICU
Day 13
Bile drain
EICU
abscess 8. Subcortical
Day 45
Wound
Day 240
ER-b
Yes.
IPM-R, AMK-R,
ER-c
No.
IPM-R, AMK-R,
ER-c
Yes.
IPM-S, AMK-R,
ER-d
Yes
IPM-R, AMK-R,
ER-d
Yes.
OP
No.
ER-d
No.
CPFX-R Sputum
(November 15, 2011) EICU
IPM-R, AMK-R,
CPFX-R
(October 26, 2011) 7. Iliopsoas muscle
Yes.
CPFX-R
(September 23, 2011) 6. Traffic injury
ER-a
CPFX-R
(September 19, 2011) Day 44
fluidized bed
CPFX-R
(September 19, 2011) Day 7
IPM-S, AMK-R,
Use of the air
CPFX-R
(August 29, 2011) Day 33
Dr. team
of IPM, AMK and CPFX
(March 3, 2010) 2.
Susceptibility pattern
IPM-S, AMK-R, CPFX-R
Sputum
IPM-S, AMK-S
26
27
hemorrhage 9. Subarachnoid
(November 15, 2011) EICU
hemorrhage 10. Air fluidized bed
Day 50
CPFX-R Sputum
(November 17, 2011) EICU
(November 20, 2011)
IPM-S, AMK-S
NS
No.
CPFX-S Beads
IPM-S, AMK-R, CPFX-R
F, female; M, male; EICU, Emergency Intensive Care Unit; ER, Critical Care and Emergency Medicine; NS, Neurosurgery; OP, Orthop edics; R, resistant; S, susceptible; %TBSA, %total body surface area. One hundred-fifty nurses and 5 nurse-aids worked in the EICU and Burn center, and they were not fixed as a team.
27
28
Table 3. Susceptibility pattern of Acinetobacter baumannii Tokai strains
Strain
MIC (μg/mL)
β-Lactams No.
IPM PIPC CAZ
AGs
CFPM
S/C
AZT
MEPM
CZOP
GM
TOB
FQs
AMK
Other agents
LVFX
CPFX
MINO
FOM
ST
1,5,7,10
2
>64
>16
>16
8
16
>8
>8
>32
4
>2
4
>16
>2
2,3,4,6
>8
>64
>16
16
16
>8
16
>8
>8
>32
>4
>2
≦2
>16
>2
8
≦1
≦8
≦2
2
≦2
>16
≦2
9
≦1
≦8
4
16
≦2
1
An Outbreak of blaOXA-51-like- and blaOXA-66- Positive Acinetobacter baumannii ST208
2
in the Emergency Intensive Care Unit
3
Satomi Asai,1,2*
Kazuo Umezawa,3 Hideo Iwashita,1 Toshio Ohshima,2
4
Maya Ohashi,2
Mika Sasaki,2 Hideki Hayashi,4 Mari Matsui, 5 Keigo Shibayama,5
5
Sadaki Inokuchi3 and Hayato Miyachi1,2
6
7
1. Department of Laboratory Medicine, Tokai University School of Medicine,
8
2. Infection Control Division in Tokai University Hospital,
9
3. Department of Critical Care and Emergency Medicine, Tokai University School of
10
Medicine,
11
4. Department of Bioinformatics, Education and Research Support Center, Tokai University
12
School of Medicine, 143 Shimokasuya, Isehara, 259-1193, Japan.
13
5. Department of Bacteriology II, National institute of infectious diseases, Tokyo, Japan.
14
15
Running head: Drug resistance of A. baumannii
16
（
）
17
This work was supported by a Grant-in-Aid #23590691 for Scientific Research (C) from the
18
Japan Society for the Promotion of Science, the Ministry of Education, Culture, Sports,
19
Science and Technology.
20
1
2
21
22
Correspondence Satomi Asai, MD, PhD.
23
Department of Laboratory Medicine, Tokai University School of Medicine,
24
143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
25
Phone: # +81 463 93 1121 Ex. 2450
26
FAX: # +81 463 93 8607
27
E-mail address: [email protected]
28
Co-author's e-mail address
29
Kazuo Umezawa: [email protected]
30
Hideo Iwashita: [email protected]
31
Maya Ohashi: [email protected]
32
Hideki Hayashi: [email protected]
33
Keigo Shibayama: [email protected]
34
Hayato Miyachi: [email protected]
Toshio Ohshima: [email protected], Mika Sasaki: [email protected] Mari Matsui: [email protected]
Sadaki Inokuchi: [email protected]
35
2
3
36
ABSTRACT
37
A series of clinical isolates of drug resistant Acinetobacter baumannii (DR-A. baumannii)
38
with diverse drug susceptibility was detected from eight patients in the critical care and
39
emergency center of Tokai University Hospital.
40
2010 (A. baumannii Tokai strain 1), and subsequently, seven isolates were obtained from
41
patients (A. baumannii Tokai strain 2-8) and one isolate was obtained from an air fluidized
42
bed shared by five of the patients during the three months from August to November 2011.
43
The isolates were classified into three types of antimicrobial drug resistance patterns (RRR,
44
SRR and SSR) according to their susceptibility to IPM, AMK and CPFX, respectively.
45
Genotyping of these isolates by multilocus sequence typing (MLST) revealed one sequence
46
type, ST208, while that by a DiversiLabⓇ analysis revealed two subtypes.
47
were positive for blaOXA-51-like and blaOXA-66, as assessed by PCR. A. baumannii Tokai strains
48
1-8 and 10 (RRR, SRR and SSR) had quinolone resistance-associated mutations in gyrA/parC,
49
as revealed by DNA sequencing. The ISAba1 upstream of blaOXA-51-like and aminoglycoside
50
resistance-associated gene, armA, were detected in A. baumannii Tokai strains 1-7 and 10
51
(RRR and SRR) as assessed by PCR. Among the resistance-nodulation-division (RND)
52
family pump-encoding genes (adeB, adeG and adeJ) and outer membrane porin-encoding
53
genes (oprD and carO), overexpression of adeB and adeJ, and the suppression of oprD and
54
carO were seen in isolates of A. baumannii Tokai strain 2 (RRR), as assessed by real-time
55
RT-PCR.
The initial isolate was obtained in March
All the isolates
In summary, the molecular characterization of a series of isolates of DR-A. 3
4
56
baumannii revealed the outbreak of ST208, and a diversity of antimicrobial drug
57
susceptibilities, which was almost correlated with differential gene alterations responsible for
58
each type of drug resistance.
59
60
Key words: antimicrobials; drug resistant Acinetobacter baumannii; β-lactamase; sequence
61
type.
4
5
62
List of Abbreviations
63
AGs, aminoglycosides; AMK, amikacin; AZT, aztreonam; CAZ, ceftazidime; CFPM,
64
cefepime; CPFX, ciprofloxacin; CPZ, cefoperazone; CZOP, cefozopran; DR- A. baumannii,
65
drug
66
ethylenediaminetetraacetic acid; EICU, Emergency Intensive Care Unit; FOM, fosfomycin;
67
FQs, fluoroquinolones; GM, gentamicin; IPM, imipenem; IPM/CS, imipenem/cilastatin; LB,
68
Luria-Bertani broth; LVFX, levofloxacin; MBL, metallo-beta-lactamase; MDR- A. baumannii,
69
multidrug-resistant Acinetobacter baumannii; MEPM, meropenem; MIC, minimum inhibitory
70
concentration; MINO, minocycline; MLST, multi-locus sequence typing; PIPC, piperacillin;
71
R, resistant; rep-PCR, repetitive-element-based polymerase chain reaction; RT-PCR, Reverse
72
transcription-polymerase chain reaction; S, susceptible; SBT/ABPC, sulbactam/ampicillin; ST,
73
sequence type; TAZ, tazobactam; TEIC, teicoplanin; TOB, tobramycin; VCM, vancomycin.
74
RRR, IPM-resistant (R), AMK-resistant (R) and CPFX-resistant (R); SRR, IPM-susceptible
75
(S), AMK-resistant (R) and CPFX-resistant (R); SSR, IPM-susceptible (S), AMK- susceptible
76
(S) and CPFX-resistant (R); SSS, IPM-susceptible (S), AMK- susceptible (S) and CPFX-
77
susceptible (S).
resistant
Acinetobacter
baumannii;
DRPM,
doripenem;
EDTA,
78
5
6
79
INTRODUCTION
80
Acinetobacter baumannii (A. baumannii) is emerging as a nosocomial pathogen, particularly
81
in intensive care units, including burn care units (Guzek, et al., 2013; Bayram, et al., 2013;
82
Ohashi, et al., 2013). Hospitalized patients at a greater risk for Acinetobacter infections are
83
those particularly very ill on a ventilator, those with a prolonged hospital stay, those who have
84
open wounds and anyone with invasive devices like urinary catheters (Howard, et al., 2012,
85
Wendt, et al., 1997; Wisplinghoff, et al., 2000; Ho, et al., 2010; Zheng, et al., 2013).
86
A stepwise evolution in the acquisition of multidrug-resistance in clinical isolates of
87
Pseudomonas aeruginosa and A. baumannii has been reported (Higgins, et al., 2010; Asai, et
88
al., 2011). Elucidation of the mechanism(s) underlying the drug-resistance is important to
89
prevent such resistance.
90
We experienced a series of clinical isolates of DR-A. baumannii with a diverse pattern of
91
drug resistance from 8 patients in the emergency intensive care unit of Tokai University
92
Hospital from March 2010 to November 2011.
93
resistance in the same sequence type, we studied the molecular characteristics of the clinical
94
isolates and the relationship with the resistance pattern.
In order to elucidate the diversity on drug
95
96
97
MATERIALS AND METHODS
Patients and clinical specimens
6
7
98
The patients studied were admitted to the critical care and emergency center (57 beds,
99
including three beds in the severe burn care unit) of Tokai University Hospital (total 804
100
beds), because of serious burns, traffic injuries or cerebral hemorrhage.
101
all patients, except two with cerebral hemorrhage, were treated alternatively with the systemic
102
administration of antibiotics, including β-lactams, FQs, AGs and glycopeptides, for infections
103
of wounds and/or the respiratory tract.
104
105
During this period,
Routine microbial examinations were performed on a weekly basis on clinical specimens from the patients' sputum, urine (via catheter), venous blood, wounds and so on.
106
Bacteriological surveillance of environmental surfaces was performed for the medical
107
equipment shared by the patients in order to seek a possible reservoir of the pathogen, such as
108
an air fluidized bed.
109
SSS) was used as a control.
110
the possible transmission route using information on the clinical care of the patients and
111
detection of A. baumannii.
112
(13R-036).
A drug-sensitive strain of A. baumannii (A. baumannii Tokai strain 9, The epidemiological investigation was performed to elucidate
The study was approved by the review board of Tokai University
113
114
115
116
Growth conditions and antibiotic susceptibility testing
Bacteria were cultured at 37 Co., Ltd., Tokyo, Japan).
℃ in Luria-Bertani broth (Kyokuto Pharmaceutical Industrial
The criteria for MDR- A. baumannii was resistance to IPM (MIC 7
8
117
> 16 µg/mL), AMK (MIC > 32 µg/mL) and CPFX (MIC > 4 µg/mL), and DR- A. baumannii
118
was defined as resistance to one or two of the drugs according to the Japanese National
119
Guideline Concerning the Prevention of Infections and Medical Care for Patients with
120
Infections.
121
Biochemical identification and susceptibility testing of the isolates was performed to obtain
122
MIC values using a microdilution method according to the Clinical Laboratory Standards
123
Institute (CLSI, 2009) and MicroScan WalkAway-96 SI system (Siemens Japan K.K., Tokyo,
124
Japan). The antibiotics used in this study were obtained as follows: AMK and IPM were
125
from Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan), AZT from Eizai Co., Ltd. (Tokyo,
126
Japan), CAZ from Glaxo SmithKline Co., Ltd. (Tokyo, Japan), CFPM from Bristol-Myers
127
Squibb Co. (Tokyo, Japan), CPFX from Bayer HealthCare (Osaka, Japan), CZOP from
128
Takeda Pharmaceutical Co., Ltd. (Osaka, Japan), DRPM from Shionogi & Co., Ltd. (Osaka,
129
Japan), FOM from Meiji Seika Co., Ltd. (Tokyo, Japan), GM from Schering-Plough, Co., Ltd.
130
(Tokyo, Japan), LVFX from Daiichi-Sankyo Pharmaceutical Co., Ltd. (Tokyo, Japan), MEPM
131
from Daiichi-Sumitomo Pharmaceutical Co., Ltd. (Osaka, Japan), MINO from Wyeth &
132
Takeda Pharmaceutical Co., Ltd. (Tokyo, Japan), PIPC from Taisho Toyama Pharmaceutical
133
Co., Ltd. (Tokyo, Japan) and TOB was from Towa Pharmaceutical Co., Ltd. (Osaka, Japan).
134
135
Molecular typing
8
9
136
DNA templates were extracted using the ZR-Duet DNA/RNA MiniPrep Kit (Zymo
137
Research, CA, USA). MLST was performed as described previously (Fu, et al., 2010;
138
Bartual, et al., 2005).
139
sequence type database (http://pubmlst.org/abaumannii/) to determine the alleles and
140
sequence types (ST).
141
rep-PCR DiversiLab Microbial Typing SystemⓇ (SYSMEX BioMérieux Co., Ltd. Tokyo,
142
Japan), which
143
genomes, as described previously (Higgins, et al., 2012; Carretto, et al., 2008).
144
annealing temperature of the PCR (polymerase chain reaction) amplification used in this
145
study was 55°C for gltA, gyrB, recA and cpn60, and 50°C for gdhB, gpi and rpoD. The
146
amplification products were purified with a Qiagen DNA purification kit (Qiagen Inc., Tokyo,
147
Japan).
148
(Applied Biosystems, Life Technologies Japan Ltd., Tokyo, Japan).
MLST sequences were uploaded into the A. baumannii MLST
A. baumannii isolates were screened for gene homology by the
amplified the regions between the noncoding repetitive sequences in bacterial The
The DNA sequencing was performed using an ABI3500xL Genetic Analyzer
149
150
Evaluation of the mechanisms of resistance
151
Screening for MBL
152
A. baumannii isolates were screened for the production of MBL by a double-disk synergy
153
test with disks containing sodium mercaptoacetic acid, as described previously (Arakawa, et
154
al., 2000). 9
10
155
156
157
PCR assay for β-lactamase and armA
The following resistance genes were examined by PCR: blaIMP-1, blaVIM, blaOXA-23-like,
158
blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaOXA-66, and ISAba1 genes, as described previously
159
(Woodford, et al., 2006; Turton, et al., 2006, Lee, et al., 2010).
160
encodes 16S rRNA methylases and confers high resistance to AGs, was screened by PCR
161
using primers that were described previously (Yamane, et al., 2005).
The armA gene, which
162
163
Sequencing of OXA-type β-lactamase, and gyrA and parC
164
Sequencing of OXA-type β-lactamase was performed as described previously (Endo, et al.,
165
2012). The quinolone resistance-determining regions (QRDRs) of gyrA and parC were
166
amplified and analyzed as described previously (Liu, et al., 2012).
167
amplified DNA products was performed using an ABI3500xL Genetic Analyzer (Applied
168
Biosystems, Life Technologies Japan Ltd., Tokyo, Japan).
DNA sequencing of the
169
170
171
172
Quantitative RT-PCR (qRT-PCR) RNA templates were extracted by the ZR-Duet DNA/RNA MiniPrep Kit (Zymo Research, CA, USA).
The expression of three different resistance-nodulation-division (RND) family
10
11
173
pump-encoding genes (adeB, adeG, adeJ) and two outer membrane porin-encoding genes
174
(oprD and carO) were analyzed by quantitative RT-PCR using the StepOnePlusTM Real-Time
175
PCR System (Applied Biosystems, Life Technologies Japan Ltd., Tokyo, Japan)
176
al., 2013; Fernando & Kumar, 2012; Peleg, et al., 2008). The primers used for the analysis
177
are listed in Table 1.
178
2012; Srinivasan, et al., 2011; Coyne, et al., 2010). Reactions (20 L) were set up using 400
179
nM primers and 2 L of the cDNA template (diluted 1:10) with SYBR R Premix Ex TaqTM II
180
(Tli RNase H Plus), and ROX plus (Takara Bio Inc., Shiga, Japan).
181
carried out using the StepOne™ software program.
182
normalized based on the level of the 16S rRNA mRNA gene and was expressed as a relative
183
rate compared to that in the susceptible isolate of each pair (the expression of A. baumannii
184
Tokai strain 9 was taken as 1.0).
185
independently, and all reactions were carried out in triplicate.
(Zander, et
The housekeeping gene, 16S rRNA, was used as a control (Hou, et al.,
μ
μ
○
The data analysis was
The expression of each target gene was
Experiments were conducted at least three times
186
187
RESULTS
188
Bacterial strains and antibiotic susceptibility
189
The characteristics of the A. baumannii Tokai strains are shown in Table 2.
In March
190
2010, a drug resistant isolate of A. baumannii Tokai strain 1 was initially detected from the
191
wound of a patient with a severe burn injury.
After one and a half years, during a period of 11
12
192
three months from August to November 2011, another seven clinical isolates of DR-A.
193
baumannii strains from patients (A. baumannii Tokai strains 2-8) were obtained.
194
A. baumannii Tokai strains were classified into three types according to their susceptibility to
195
three drugs (IPM, AMK and CPFX), as RRR, SRR or SSR (Tables 2 and 3).
196
obtained from sputum, wounds and bile drains.
197
The DR-
They were
Since the interval between the first patient and others long (>18 months), the environment of
198
the ward was suspected to be a possible reservoir of the pathogen. Based on the results of
199
the bacteriological surveillance of environmental surfaces, A. baumannii Tokai strain 10 was
200
isolated from the cracks of a rubber frame and a lump of beads in the air fluidized bed, which
201
was used by five patients during their hospitalization (A. baumannii Tokai strains 1, 2, 4, 5
202
and 6).
203
204
Molecular typing
205
The molecular genotyping of isolates by a MLST analysis revealed a sequence type ST208
206
for A. baumannii Tokai strains 1-8 and 10 (gltA, gyrB, gdhB, recA, cpn60, gpi and rpoD:1, 3,
207
3, 2, 2, 97 and 3) and another type for A. baumannii Tokai strain 9 (gltA, gyrB, gdhB, recA,
208
cpn60, gpi and rpoD: 15, 48, 58, 42, 36, 54 and 41).
209
by the rep-PCR by DiversiLab Microbial Typing SystemⓇ showed the same pattern (more
210
than 97% similarity) as one type for eight of the isolates (A. baumannii Tokai strain1-7 and
211
10) (Fig. 1), and the other isolates (A. baumannii Tokai strain 8 and 9) had different patterns
The molecular genotyping of isolates
12
13
212
(85% and less than 70% similarity, respectively) .
213
214
215
Expression of resistance-related genes The MBL assay of the clinical isolated A. baumannii Tokai strains revealed no apparent
216
MBL production, and all isolates showed expression of OXA-51-like carrying OXA-66
217
β-lactamase (Table 4). The expression of IMP-1, VIM, OXA-23-like, OXA-24-like and
218
OXA-58-like was negative.
219
baumannii Tokai strains 1-7 and 10. The DNA sequencing of gyrA and parC revealed that
220
Serine-83 (TCA) was changed to TTA (Leucine) and that Ser-80 (TCG) was changed to TTT
221
(Phenylalanine) or TTG (Leucine) in A. baumannii Tokai strains 1-8 and 10.
222
The expression of ISAba1 and armA were found in A.
Our analysis of RND pump-encoding genes included an analysis of the expression of three
223
previously characterized genes, adeB, adeG and adeJ, which encode the RND pump in the
224
adeABC, adeFGH and adeIJK operons, respectively.
225
Tokai strain 1,2, 8 and 9 as a representative strain from each group with the same
226
susceptibility pattern was shown in Table 5.
227
in A. baumannii Tokai strains 2. The expression of oprD was decreased in A. baumannii
228
Tokai strains 2 and 8.
229
Tokai strains 1, 2 and 8.
The result of Acinetobacter baumannii
The overexpression of adeB and adeJ was seen
The underexpression of carO was seen in isolates with A. baumannii
230
13
14
231
232
DISCUSSION
We experienced a series of clinical isolates of DR-A. baumannii ST208 in the emergency
233
intensive care unit of Tokai University Hospital.
234
drug resistance patterns in the same sequence type in these isolates, we studied the molecular
235
characteristics of these isolates and their relationship with the resistance pattern.
236
In order to elucidate the diversity of the
A. baumannii Tokai strains 1-7 and 10 were positive for OXA-51-like and OXA-66
237
β-lactamase and ISAba1. A. baumannii strains with resistance to AMK (A. baumannii Tokai
238
strains 1-7 and 10) were positive for armA. These results are consistent with the idea that
239
ISAba1 regulates the expression of OXA-51-like carrying OXA-66 β-lactamase, and that
240
armA is related to AGs resistance.
241
and 6) could have been derived from the same source and/or transmitted horizontally, because
242
the same air fluidized bed had been used by those patients. Among them, A. baumannii
243
Tokai strains 2, 4 and 6 showed multidrug resistance (RRR). These patients were treated
244
with carbapenem (MEPM or DRPM) prior to sampling for at least one week, which may have
245
played a role in the overexpression of adeB and adeJ in A. baumannii Tokai strain 2. A.
246
baumannii Tokai strain 10 was detected from the cracks of the rubber frame and a lump of
247
beads in the air fluidized bed, even though the bed had been cleaned and disinfected every
248
time after use.
249
reported (Yamada & Suwabe, 2013; Suzuki, et al., 2013), an outbreak of A. baumannii ST208
The five isolates (A. baumannii Tokai strains 1, 2, 4, 5
Although a few of nosocomial outbreaks of A. baumannii ST2 have been
14
15
250
has not been reported previously in Japan.
251
Since the pattern of the rep-PCR and sequence type of MLST in the eight isolates was the
252
same as that in the initial case, it was suggested that the strain survived for a year and a half in
253
the environmental reservoir.
254
environmental cleaning and disinfection in order to reduce the risk of transmission is
255
suggested. A. baumannii Tokai stain 8 (SSR) was also ST208, but had a different pattern by
256
the rep-PCR.
257
transposon or the insertion of a different plasmid might have led to the different pattern.
258
During the period of an outbreak, A. baumannii with different drug susceptibility patterns
259
appeared depending on the various resistance mechanisms.
260
As infection control procedures, careful attention to
During the transmission from the same original organism, the presence of a
Nine isolates (A. baumannii Tokai stain1-8, and 10) had resistance to CPFX, which can be
261
explained by the mutations of gyrA and parC. Another major factor contributing to the
262
resistance of this organism was the overexpression of the RND pumps (Zander, et al., 2013;
263
Amin, et al., 2013; Fernando & Kumar., 2012). Our analysis of RND pump-encoding genes
264
included the expression of three previously characterized genes, adeB, adeG and adeJ, which
265
encode the RND pumps in the adeABC, adeFGH and adeIJK operons, respectively.
266
pumps such as AdeABC have been reported to be involved in multidrug resistance (Hou, et
267
al., 2012; Vila, et al., 2007).
268
overexpression of adeB and adeJ.
269
some β-lactams (CAZ, CFPM and CZOP) than that of A. baumannii Tokai strain 9 (SSS).
Efflux
In our study, A. baumannii Tokai strain 2 (RRR) showed the A. baumannii Tokai strain 8 showed better sensitivity to
15
16
270
This phenomenon might be associated with underexpression of adeB.
271
adeB and adeJ, were related to the acquisition of multidrug-resistance. As for porins, the
272
overexpression of genes encoding RND pumps and the downregulation of genes encoding
273
porins is known to be common in clinical isolates of Acinetobacter species (Fernand, et al.,
274
2013).
275
without oprD does not result in resistance to carbapenem in A. baumannii Tokai strains 1 and
276
8 (SRR and SSR).
277
decrease in porins among Acinetobacter strains is not associated with resistance to
278
carbapenems in the presence of β-lactamases
279
Two pumps, such as
Our findings also suggest that the underexpression of carO in combination with or
This observation is consistent with previous findings showing that a
（Rumbo, et al., 2013; Singh, et al., 2013）.
In conclusion,c Furthermore, we demonstrated that drug resistance is associated with the
280
expression of ISAba1 and armA and mutations in gyrA and parC and that the overexpression
281
of adeB and adeJ plays a role in the multidrug resistance of A. baumannii Tokai strain ST208.
282
16
17
283
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399
41, e73-76.
400 23
24
401
FIGURE LEGENDS
402
Figure 1. The results of the rep-PCR DiversiLab Microbial Typing SystemⓇ analysis and
403
sequence type
404
（ST） in clinical isolates of A. baumannii.
A. baumannii Tokai strains 1 to 7 and 10 showed an identical pattern and a homologous rate
405
of identity > 97%.
Two isolates, A. baumannii Tokai strains 8 and 9, had different patterns
406
(85% and less than 70% similarity, respectively).
407
analysis revealed that A. baumannii Tokai strains 1 to 8 and 10 were of the same sequence
408
type (ST208).
The sequence type from the MLST
24
25
Table 1. Primer sequence of quantitative RT-PCR Primer adeB
adeG
adeJ
oprD
carO
16S rRNA
Primer sequence
Product size (bp)
Source
forward
5´-ACC GGA GGT ATC TGT TGT CG-3´
122
Fernando & Kumar., 2012
reverse
5´-GTG CGA CTT ATC CTG GTG CT-3´
forward
5´-ATC GCG TAG TCA CCA GAA CC-3´
92
Fernando & Kumar., 2012
reverse
5´-CGT AAC TAT GCF GTG CTC AA-3´
forward
5´-CAT CGG CTG AAA CAG TTG AA-3´
109
Fernando & Kumar., 2012
reverse
5´-GCC TGA CCA TTA CCA GCA CT-3´
forward
5´-CCA GCT CAG TTG CTC AAT CA-3´
134
this study
reverse
5´-CAT TTG GTT TCC AGC GTT TT-3´
forward
5´-GGT TAT AAC GGC GGT GAC AT-3´
115
this study
reverse
5´-CCA AGG ACG AAT TTC AGC AT-3´
forward
5´-CGT AAG GGC CAT GAT GAC TT-3´
150
Fernando & Kumar., 2012
reverse
5´-CAG CTC GTG TCG TGA GAT G-3´
25
26
Table 2. Information of cases and Acinetobacter baumannii Tokai strain. Strain Disease
Ward
No. 1.
The detected day
Source
from hospitalization 74% TBSA Burn
Burn center
Day 31
85% TBSA Burn
Burn center
(Ohashi, et al., 2013) 3. 40% TBSA Burn
Burn center
(Ohashi, et al., 2013) 4. 70.5% TBSA Burn
Burn center
(Ohashi, et al., 2013) 5. Traffic injury
EICU
Day 9
Sputum
Wound
Wound
Wound
EICU
Day 13
Bile drain
EICU
abscess 8. Subcortical
Day 45
Wound
Day 240
ER-b
Yes.
IPM-R, AMK-R,
ER-c
No.
IPM-R, AMK-R,
ER-c
Yes.
IPM-S, AMK-R,
ER-d
Yes
IPM-R, AMK-R,
ER-d
Yes.
OP
No.
ER-d
No.
CPFX-R Sputum
(November 15, 2011) EICU
IPM-R, AMK-R,
CPFX-R
(October 26, 2011) 7. Iliopsoas muscle
Yes.
CPFX-R
(September 23, 2011) 6. Traffic injury
ER-a
CPFX-R
(September 19, 2011) Day 44
fluidized bed
CPFX-R
(September 19, 2011) Day 7
IPM-S, AMK-R,
Use of the air
CPFX-R
(August 29, 2011) Day 33
Dr. team
of IPM, AMK and CPFX
(March 3, 2010) 2.
Susceptibility pattern
IPM-S, AMK-R, CPFX-R
Sputum
IPM-S, AMK-S
26
27
hemorrhage 9. Subarachnoid
(November 15, 2011) EICU
hemorrhage 10. Air fluidized bed
Day 50
CPFX-R Sputum
(November 17, 2011) EICU
(November 20, 2011)
IPM-S, AMK-S
NS
No.
CPFX-S Beads
IPM-S, AMK-R, CPFX-R
F, female; M, male; EICU, Emergency Intensive Care Unit; ER, Critical Care and Emergency Medicine; NS, Neurosurgery; OP, Orthop edics; R, resistant; S, susceptible; %TBSA, %total body surface area. One hundred-fifty nurses and 5 nurse-aids worked in the EICU and Burn center, and they were not fixed as a team.
27
28
Table 3. Susceptibility pattern of Acinetobacter baumannii Tokai strains
Strain
MIC (μg/mL)
β-Lactams No.
IPM PIPC CAZ
AGs
CFPM
S/C
AZT
MEPM
CZOP
GM
TOB
FQs
AMK
Other agents
LVFX
CPFX
MINO
FOM
ST
1,5,7,10
2
>64
>16
>16
8
16
>8
>8
>32
4
>2
4
>16
>2
2,3,4,6
>8
>64
>16
16
16
>8
16
>8
>8
>32
>4
>2
≦2
>16
>2
8
≦1
≦8
≦2
2
≦2
>16
≦2
9
≦1
≦8
4
16
≦2
![[New clone of ST-187 Acinetobacter baumannii responsible for an outbreak in an intensive care unit].](/assets/img/hold.png)
