An Intrinsic Strain of Colistin-resistant Acinetobacter Isolated from a Japanese Patient

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1 CASE REPORT An Intrinsic Strain of Colistin-resistant Acinetobacter Isolated from a Japanese Patient Etsuko Takizawa 1, Koichi Yamada 2, Ken-ichi Oinuma 3, Kanako Sato 4, Mamiko Niki 3, Hiroki Namikawa 2, Hiroki Fujimoto 2,4, Kazuhisa Asai 4, Yukihiro Kaneko 3 and Hiroshi Kakeya 2 Abstract We herein report the first domestic case of bacteremia caused by an intrinsic strain of colistin-resistant Acinetobacter. The Acinetobacter species was detected in the hemocultures in a febrile patient. The patient was a 65-year-old-man who was admitted to our hospital for laparotomic gastrostomy. The patient s antimicrobial susceptibility patterns were atypical; they were colistin resistant but not multiple drug resistant. A sequence analysis of rpob identified the bacterium as an Acinetobacter genomic species 13BJ/14TU, which had only been previously reported in South Korea. He had never traveled to South Korea but frequently had contact with the South Korean community. We therefore demonstrated that infection with this species could occur in domestic cases. Key words: Acinetobacter, colistin-resistant, Korea, rpob () () Introduction Species of the genus Acinetobacter are gram-negative bacteria which are present in the natural environment, including the soil. In addition, they have often been isolated from medical environments and clinical materials, and are the main causative microorganisms of nosocomial infections in immunocompromised hosts (1-3). Acinetobacter baumannii is currently a problematic bacterial species for nosocomial infection control because of its multidrug resistance. It is a causative organism in many refractory infections (4). In other countries, the following three species are reported to have acquired multidrug resistance: Acinetobacter baumannii, Acinetobacter genomic species (GS) 13TU, and Acinetobacter GS 3 (5, 6). However, in identifications based on biochemical properties or those which used commercial automatic bacterial identification systems, all of the aforementioned species were identified as Acinetobacter baumannii complex, and were therefore impossible to distinguish from each other. Antimicrobial susceptibility tests have shown that most colistin-resistant species are multidrugresistant Acinetobacter (MDRA) (7). We herein report our experience with a case in which colistin-resistant Acinetobacter GS 13BJ/14TU was isolated from the hemoculture of a patient with no history of colistin treatment. Case Report A 65-year-old man was admitted to our hospital with fever and chills. He had been diagnosed with epilepsy at 3 years of age and had a cataract in his right eye from 35 years of age. He had smoked 30 cigarettes per day for 15 years before quitting smoking 4 months before his admission to hospital. Up to 3 years before admission, the patient had worked in a South Korean shop in Osaka; he had contact with South Korean individuals until up to 6 months before admission; he had never traveled to South Korea. Department of Laboratory Medicine, Osaka City University Hospital, Japan, Department of Infection Control Science, Graduate School of Medicine, Osaka City University, Japan, Department of Bacteriology, Graduate School of Medicine, Osaka City University, Japan and Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Japan Received for publication November 8, 2015; Accepted for publication November 18, 2015 Correspondence to Dr. Hiroshi Kakeya, kakeya-ngs@umin.ac.jp 2301

2 Table. The Minimum Inhibitory Concentrations (MICs) of Antimicrobial Agents for the Isolated Bacterium. Antimicrobial agents MIC (μg/ml) Determination Piperacillin >64 R Tazobactam/Piperacillin 8 Ceftazidime >16 R Cefepime 8 S Cefozopran 4 S Sulbactam/Cefoperazone 16 S Amikacin 8 S Tobramycin 4 S Gentamicin 2 S Minocycline 2 S Levofloxacin.5 S Ciprofloxacin 0.5 S Imipenem 1 Meropenem 2 S Doripenem 1 S Aztreonam >16 Colistin >4 R Sulfamethoxazole/Trimethoprim 2 S Fosfomycin >16 S: susceptible, I: intermediate, R: resistant Two months before admission, the patient had difficulty ingesting food and liquids, and a swelling was found in his neck. He therefore visited the emergency ward of a nearby hospital. CT scans of the neck revealed a tumor lesion of 7 cm in size. The tumor was found to have caused narrowing of the patient s airways, which led to his referral to our hospital. An emergency tracheotomy was carried out on the same day. Based on the biopsy results, the patient was diagnosed with hypopharyngeal cancer. Chemoradiation therapy was scheduled to begin later; however, on the 18th day of hospitalization, the patient developed a fever of approximately 39 accompanied by chills. Cefazolin was administered, however, the patient s blood pressure and platelet count were found to have both decreased the next day. The patient was treated for septic shock and disseminated intravascular coagulation (DIC), and antibiotic treatment was initiated using meropenem. The antibiotic therapy was accompanied by the administration of noradrenaline and γ- globulin. Hemoculture was performed in a BacT/ALERT 3D system using aerobic and anaerobic culture bottles (Sysmex biomérieux, Tokyo, Japan). Only the aerobic culture bottles were positive after approximately 19 hours of culture. Gram staining after culturing revealed the presence of gramnegative bacilli. Bacterial isolation and culturing were carried out at 35 in the presence of carbon dioxide, using 5% sheep blood agar, MacConkey agar, and CA sheep blood agar (Nissui Pharmaceutical, Tokyo, Japan). Based on the colony growth after one day, it was determined that the bacteria were highly likely to be glucose-nonfermenting gram-negative bacteria. Microbial identification was performed using a MicroScan Walkaway 40 system (Beckman Coulter, Tokyo, Japan), TSI agar (Eiken Chemical, Tokyo, Japan), and oxidase tests (Nissui Pharmaceutical). The results showed that the bacterium was A. baumannii/haemolyticus (biotype 41046). Subsequent testing by NF-18 (Nissui Pharmaceutical) identified the bacterium as A. baumannii (code ). Antimicrobial susceptibility tests were carried out by the broth microdilution method established by the Clinical and Laboratory Standards Institute, and the minimum inhibitory concentration (MIC) was determined (8). The strain was found to be resistant to piperacillin, ceftazidime, and colistin and susceptible to other agents, including aminoglycosides, fluoroquinolones, and carbapenems (Table). Colistin susceptibility was also confirmed by the disk diffusion method using KB disk colistin (Eiken Chemical) (9) and Mueller Hinton agar (Nippon Becton Dickinson Co., Ltd., Tokyo, Japan), and compared to the susceptibility of a reference strain, ATCC While the reference strain showed an obvious inhibition ring, the clinical isolate did not (Fig. 1). Tests to screen for the production of metallo-beta-lactamase were performed using ceftazidime, cefepime, imipenem, meropenem, and sodium mercaptoacetate (SMA), a metallobeta-lactamase inhibitor (10). The results showed no production of metallo-beta-lactamase by this bacterium. In addition, the absence of OXA-51-like genes, which members of the A. baumannii complex usually possess, was confirmed using a polymerase chain (PCR) method that was previously reported by Woodford et al. (11, 12) (Fig. 2). Because the isolate showed carbapenem susceptibility, and because the results of contrast-enhanced CT did not show any apparent portal of entry of the bacterium, the patient was continuously treated with meropenem for 14 days. The patient s fever and other bacteremia-related clinical symptoms eventually subsided. Hemocultures at this time were negative. A scheduled laparotomic gastrostomy was completed on the 31st day of hospitalization. The patient did not experience a relapse, however, he did not receive any further aggressive medical treatment for his hypopharyngeal cancer. The patient was transferred to another facility on the 56th day of hospitalization (Fig. 3). Since the isolate was preliminarily identified as a minor Acinetobacter species, further identification by partial sequencing of the rpob gene (zones 1 and 2) was initiated, according to the method of La Scola et al. (13). A PCR was carried out using MightyAmp DNA Polymerase Ver. 2 (Takara Bio Inc., Shiga, Japan) and a thermal cycler (TP350; Takara Bio Inc.). The PCR products were purified with a NucleoSpin Gel and PCR Clean-up Kit (Macherey- Nagel GmbH & Co. KG, Düren, Germany), and DNA sequencing was performed using an ABI 3130xl Genetic Analyzer and an ABI BigDye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, USA). The BLASTN algorithm ( was used for a sequence alignment homology search. The sequence identities of zone 1 (bp 2915 to 3269) and zone 2 (bp 3271 to 3775) in rpob showed 99.7% (354/355) and 99.6% (503/505) homology, respectively, to Acinetobacter GS 13BJ strain LUH1720; our clinical strain was distinct from other reference strains (Fig. 4, 5). 2302

3 a b Reference strain Acinetobacter baumannii ATCC19606 Test strain Acinetobacter spp. OCU Ac7 Figure 1. Colistin susceptibility testing by the disk diffusion method using KB disk colistin. A reference strain, Acinetobacter baumannii ATCC19606 (a) showed an obvious inhibition ring. The test strain Acinetobacter spp. OCU Ac7 showed no such ring (b). M 1 2 oxa-51-like gene (353 bp) Lane M: -bp DNA ladder Lane 1: Reference strain Acinetobacter baumannii ATCC19606 Lane 2: Test strain Acinetobacter spp. OCU Ac7 Figure 2. The PCR for OXA-51-like genes. OXA-51-like genes were detected in the reference strain, Acinetobacter baumannii ATCC19606 (lane 1), but not in the test strain, Acinetobacter spp. OCU Ac7 (lane 2). The left lane (lane M) shows a -bp DNA ladder marker. Discussion In the present study, we investigated a case in which colistin-resistant Acinetobacter GS 13BJ/14TU was isolated from the hemocultures of a patient. While the number of reports on colistin resistance have gradually been increasing in other countries (14) and a report based on a study conducted in the United States showed that 5.3% of all Acinetobacter strains detected during an eight-year period were resistant to colistin (15, 16), encounters with colistin-resistant Acinetobacter species are still rare in Japan. The production and sale of colistin in Japan was only approved in Colistin resistance frequently indicates multidrug-resistance, as is seen with multidrug-resistant Pseudomonas aeruginosa ( MDRP ) and multidrug-resistant Acinetobacter (MDRA) (17). Most colistin-resistant bacteria have been detected in patients with a history of previous colistin treatment; therefore, colistin resistance is usually considered to have been acquired during treatment. However, colistin-resistant strains without multiple drug resistance have been reported in South Korea. These strains were isolated from patients who had never received colistin treatment, suggesting that the strains were intrinsically resistant to colistin (18, 19). These isolates were distinguished from the A. baumannii complex and are now classified as Acinetobacter GS 13BJ/14TU, based on partial nucleotide sequence homology. The clinical isolate reported here did not show multiple antimicrobial resistance and was colistinresistant in a patient with no history of colistin treatment. In addition, a nucleotide sequence analysis revealed that this isolate was highly similar to Acinetobacter GS 13BJ/14TU. The patient s history of exposure to the South Korean community supports the idea that this infection was likely to be related to a South Korean strain. Further study is required to learn whether Acinetobacter GS 13BJ/14TU existed in Japan prior to the present case or whether it has been imported; however, our study confirmed that domestic infection with this species can occur and underscores the need for clinicians to take preventive measures against the nosocomial spread of the infection. In comparison to A. baumannii complex, which is frequently isolated in severe and intractable infections, non-a. baumannii complex strains are believed to be less virulent in humans. Although non-a. baumannii complex strains account for only a small number of Acinetobacter strains, recent reports have shown that they can cause severe infections, such as urinary tract infections, ventilator-associated 2303

4 Admission Tracheotomy Day 1 Gastrostomy Discharge Day 18 Day 32 Day 56 cefazolin meropenem 1g 3 -globulin ( C) 39 noradrenalin WBC (/ L) 4,600 2,900 4,000 4, CRP (mg/dl) Plt ( 10 4 /μl) Hemoculture + - Acinetobacter spp. Figure 3. The clinical course of the present case. This patient developed septic shock and disseminated intravascular coagulation due to Acinetobacter infection. He was treated with meropenem, γ-globulin, and noradrenalin. These drugs were effective, and the meropenem injections were continued for 14 days. WBC: white blood cell, CRP: C-reactive protein, Plt: platelet OCU Ac7 GS 13BJ/14TU (KJ124842) A. gyllenbergii (EU477148) A. parvus (DQ207488) A. beijerinckii (EU477124) A. johnsonii (DQ207485) A. baumannii (DQ207471) A. nosocomialis (EU477118) A. calcoaceticus (DQ207474) A. pittii (EU477114) A. lwoffii (DQ060363) A. schindleri (DQ207490) A. bereziniae (DQ207475) A. guillouiae (DQ207476) A. haemolyticus (DQ207484) A. junii (DQ207486) A. soli (HQ148175) A. baylyi (EU477155) A. tandoii (DQ207491) A. ursingii (DQ231239) P. cryohalolentis (CP000323) 0.05 Figure 4. The phylogenetic analysis of the Acinetobacter species based on the rpob gene sequences. The dataset was composed of partial rpob gene sequences (the region from position 2915 to position 3775) of 19 representative Acinetobacter species, the strain obtained in this study (OCU Ac7), and Psychrobacter cryohalolentis as an outgroup. The tree was constructed using the neighbor-joining method in the CLUSTALW and MEGA 4 software programs. The bootstrap values were calculated from 1,000 replicates. The values of greater than 50% are shown. The GenBank accession numbers of each sequence are indicated in parentheses. The bar represents 0.05 substitutions per nucleotide position. 2304

5 GS 13BJ: 2915 AATACAAAAT CTTCGAAGAA GCAGCACGTG AACGTATTGT TCGTTTGTTG AAAGGTCAAG AATCTAACGG TGGTGGTTCA ACCAAACGTG GCGATAAACT 3014 OCU Ac7: 2915 AATACAAAAT CTTCGAAGAA GCAGCACGTG AACGTATTGT TCGTTTGTTG AAAGGTCAAG AATCTAACGG TGGTGGTTCA ACCAAACGTG GCGATAAACT 3014 GS 13BJ: 3015 TTCTGAAGAT GTATTGTCAG GTTTAGAGTT GGTTGACTTA CTTGAAATCC AGCCGACTGA TGAAGCAATC GCAGAGCGTT TAACTCAAAT TCAAGTGTTC 3114 OCU Ac7: 3015 TTCTGAAGAC GTATTGTCTG GTTTAGAGTT GGTTGACTTA CTTGAAATCC AGCCGACTGA TGAAGCAATC GCAGAGCGTT TAACTCAAAT TCAAGTGTTC 3114 ********* ******** * ********** ********** ********** ********** ********** ********** ********** ********** GS 13BJ: 3115 TTGAAAGAGA AGAGCTACGA GATTGACGAG AAGTTTGCTG AGAAGAAACG TAAACTTTCT ACAGGTGATG AATTAACAAC GGGTGTATTG AAAGTTGTTA 3214 OCU Ac7: 3115 TTGAAAGAGA AGAGCTACGA GATTGACGAG AAGTTTGCTG AGAAGAAACG TAAACTTTCT ACAGGTGATG AATTAACAAC GGGTGTATTG AAAGTTGTTA 3214 GS 13BJ: 3215 AGGTTTACCT TGCGGTGAAA CGTCGTATCC AGCCTGGTGA TAAGATGGCG GGTCGTCACG GTAACAAAGG TGTTGTATCT AACATCTTAC CGGTTGAAGA 3314 OCU Ac7: 3215 AGGTTTACCT TGCGGTGAAA CGTCGTATCC AGCCTGGTGA TAAGATGGCG GGTCGTCACG GTAACAAGGG TGTTGTATCT AACATCTTAC CGGTTGAAGA 3314 ********** ********** ********** ********** ********** ********** ******* ** ********** ********** ********** GS 13BJ: 3315 CATGCCGCAT GATGCGAATG GTGTACCAGT CGACATCGTA TTGAACCCAC TAGGTGTACC GTCACGTATG AACGTGGGTC AGATTCTTGA GACTCACTTA 3414 OCU Ac7: 3315 CATGCCGCAT GATGCGAATG GTGTACCAGT CGACATCGTA TTGAATCCAC TAGGTGTACC GTCACGTATG AACGTGGGTC AGATTCTTGA GACTCACTTA 3414 ********** ********** ********** ********** ***** **** ********** ********** ********** ********** ********** GS 13BJ: 3415 GGTATGGCGG CCAAAGGTCT TGGCGATAAA ATCGAAAAAA TGTTGAAAGA ACAACGTACA GTGATTGAAC TGCGTGAATT CTTAGACAAG ATTTATAACA 3514 OCU Ac7: 3415 GGTATGGCGG CCAAAGGTCT TGGCGATAAA ATCGAAAAAA TGTTGAAAGA ACAACGTACA GTGATTGAAC TGCGTGAATT CTTAGACAAG ATTTATAACA 3514 GS 13BJ: 3515 AGGTTGGTGG TGAGCAGGAA GAGCTTGATA GCTTGACTGA TGCAGAAATC TTGGCGCTTT CAGGCAACTT ACGTGCTGGT GTTCCATTGG CAACACCTGT 3614 OCU Ac7: 3515 AGGTTGGTGG TGAGCAGGAA GAGCTTGATA GCTTGACTGA TGCAGAAATC TTGGCGCTTT CAGGCAACTT ACGTGCTGGT GTTCCATTGG CAACACCTGT 3614 GS 13BJ: 3615 ATTTGATGGT GCTGAAGAAA GCCAGATCAA AGACTTGCTT GAACTTGCTG ACATCTCACG TACTGGTCAA ACGGTATTGT TTGACGGACG TACAGGTGAA 3714 OCU Ac7: 3615 ATTTGATGGT GCTGAAGAAA GCCAGATCAA AGACTTGCTT GAACTTGCTG ACATCTCACG TACTGGTCAA ACGGTATTGT TTGACGGACG TACAGGTGAA 3714 GS 13BJ: 3715 CAGTTTGACC GTCCTGTAAC TGTAGGTTAC ATGTACATGC TCAAATTGAA CCACTTGGTT G 3775 OCU Ac7: 3715 CAGTTTGACC GTCCTGTAAC TGTAGGTTAC ATGTACATGC TCAAATTGAA CCACTTGGTT G 3775 ********** ********** ********** ********** ********** ********** * Figure 5. The comparison between the partial rpob gene sequences of Acinetobacter GS 13BJ (KJ124842) and the OCU Ac7 strain. The numbers indicate the nucleotide positions. Identical nucleotides are denoted by*. pneumonia, and bacteremia (6, 20). In addition, microbiological tests in routine laboratories only recognize a few species of Acinetobacter strains, such as A. baumannii complex, A. nosocomialis, andacinetobacter genomic species; the isolation rate of the minor strains could therefore be underestimated. Each bacterial species has been indicated to be associated with the characteristic clinical symptoms, drug susceptibility, and the mortality rate. In particular, A. baumannii infections tend to be associated with a poor prognosis, a high mortality rate, and a high rate of drug resistance in comparison to infections with other bacterial species (5, 21, 22). The accurate identification of Acinetobacter species is therefore extremely important for making therapy decisions, predicting prognoses, and for achieving a proper epidemiological understanding. A nucleotide sequence analysis is a reliable method for discriminating between minor species, but it is difficult to perform in a routine laboratory setting. Nonetheless, it should be considered in cases of isolates with atypical drug susceptibility patterns or in patients with specific clinical backgrounds (18, 23). In conclusion, we herein reported the first case of bacteremia in which a Japanese man was domestically infected with an intrinsic colistin-resistant Acinetobacter spp. that had only previously been reported in South Korea. We emphasize the need for genetic testing to identify bacterial species in cases in which routine laboratory tests detect a bacterium exhibiting atypical bacteriological features, such as an atypical drug susceptibility pattern. In addition, our report emphasizes the importance of determining the source of infection based on patient information, such as their history of international travel or their history of invasive procedures. The authors state that they have no Conflict of Interest (COI). Financial Support This research was partially supported by the Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development, a Grantin-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI ), and Pfizer academic contributions. Acknowledgement We thank Yukimi Kira and Yoriko Yabunaka (Central Laboratory of Osaka City University Medical School) for their technical support, and Hisakazu Yano (Nara Medical University) for his technical advice. References 1. Bergogone-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. 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