- Email: [email protected]
Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan
J Infect Chemother xxx (2017) 1e3
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Case Report
Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan Kohei Uechi a, b, Tatsuya Tada c, d, Kayo Shimada d, Isamu Nakasone e, Tetsu Sonozaki f, Teruo Kirikae c, d, *, Jiro Fujita b a
Division of Clinical Laboratory and Blood Transfusion, University Hospital of the Ryukyus, Okinawa, Japan Department of Infectious Diseases, Respiratory, and Digestive Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japan d Department of Infectious Disease, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan e Control and Prevention of Infectious Disease, University Hospital of the Ryukyus, Okinawa, Japan f Department of Dermatology, University Hospital of the Ryukyus, Okinawa, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2017 Received in revised form 22 August 2017 Accepted 8 September 2017 Available online xxx
This study describes highly aminoglycoside-resistant Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates obtained from an inpatient in Okinawa, Japan, with no known record of traveling overseas. The minimum inhibitory concentrations of amikacin and arbekacin against these strains were >1024 mg/ml. Whole-genome sequencing analysis revealed that these isolates harbored armA, which encodes a 16S rRNA methylase, ArmA, that confers pan-aminoglycoside resistance. This is the second report of K. pneumoniae harboring armA and the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan. © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Aminoglycoside resistance 16S rRNA methylase ArmA Klebsiella pneumoniae Klebsiella oxytoca
1. Introduction The methylation of 16S rRNA in Gram-negative pathogens confers high resistance to all clinically important aminoglycosides [1,2]. In 2003, clinical isolates of Klebsiella pneumoniae producing ArmA, a 16S rRNA methylase, and Pseudomonas aeruginosa producing RmtA, another 16S rRNA methylase, were identified in France [3] and Japan [4], respectively. Since then, 16S rRNA methylase-producing Gram-negative pathogens have been isolated in other parts of the world, including Asian countries such as Afghanistan, Bangladesh, China, Hong Kong, India, Japan, Korea, Nepal, Oman, Pakistan, Taiwan, and Vietnam [1,4,5]. To date, 10 plasmid-associated 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH and npmA, have been identified in clinical and veterinary isolates [1,2].
* Corresponding author. Department of Microbiology, Juntendo University School of Medicine, Tokyo, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. E-mail address: [email protected] (T. Kirikae).
Although the isolation frequency of Gram-negative pathogens producing 16S rRNA methylases remains low in Japan, clinical isolates producing several types of 16S rRNA methylases have been detected [6e9]. A prospective study in Japan in 2001 found that, of 903 clinical isolates of P. aeruginosa, four (0.4%) harbored rmtA [6]. Another prospective study in 2004 showed that, of 87,626 clinical isolates of Gram-negative pathogens, 26 (0.03%) harbored armA, rmtA, rmtB, or rmtC, with most of these pathogens isolated from patients on the main island of Japan [7]. Our previous study in 2012 revealed that, of 16,343 clinical isolates of Acinetobacter spp. obtained from medical settings throughout Japan, 49 (0.3%) harbored armA [8]. Several isolates of Gram-negative pathogens harboring 16S rRNA methylase endocing genes were recently detected in Japan, but these isolates have not been detected in Okinawa, the southwestern most point of the Japanese archipelago [6e9]. To our knowledge, however, only one isolate in K. pneumoniae [7] and no isolate in K. oxytoca harboring a 16S rRNA methylase endocing gene has been reported in Japan. This is the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan and the
https://doi.org/10.1016/j.jiac.2017.09.006 1341-321X/© 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006
2
K. Uechi et al. / J Infect Chemother xxx (2017) 1e3
first report of Gram-negative pathogens harboring a 16S rRNA methylase-encoding gene in Okinawa.
2. Case report The patient was a 73-year-old Japanese woman living in Okinawa prefecture, who had been diagnosed with diabetes mellitus and was suspected of having bullous pemphigoid. Beginning in 2013, she required hospitalization four times, mainly for bloodstream infections. The latest hospitalization, for suspected bloodstream infection, was from December 2015 to June 2016. While hospitalized, methicillin-resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes were isolated from a blood sample, and K. pneumoniae was isolated from a urine sample. Treatment with ceftriaxone, vancomycin, and ampicillin for 17 days seemed to cure her bloodstream infection. Two weeks later, she experienced apparent acute pneumonia, bloodstream infection and urinarytract infection. Methicillin-susceptible S. aureus (MSSA) was isolated from a blood sample, K. oxytoca and P. aeruginosa from a urine sample, and MSSA and P. aeruginosa from a sputum sample. She was treated with meropenem, vancomycin and cefazolin for two weeks. Because A. baumannii was also isolated from a sputum sample, she was treated with meropenem and vancomycin for another 10 days. After recovery she was discharged from the hospital. Of the isolates obtained from this patient, two, K. pneumoniae RYU109 and K. oxytoca RYU351, were highly resistant to amikacin and arbekacin, with MICs >1024 mg/ml. The MICs of other antibiotics against RYU109 were aztreonam 2 mg/ml, cefepime 0.25 mg/ ml, cefotaxime 4 mg/ml, ceftazidime 32 mg/ml, ciprofloxacin 0.5 mg/ ml, colistin 0.063 mg/ml, gentamicin >1024 mg/ml, imipenem 0.25 mg/ml, meropenem 0.25 mg/ml, tigecycline 0.5 mg/ml and tobramycin >1024 mg/ml. The MICs against RYU351 were aztreonam 2 mg/ml, cefepime 0.25 mg/ml, cefotaxime 32 mg/ml, ceftazidime 32 mg/ml, ciprofloxacin 0.25 mg/ml, colistin 1 mg/ml, gentamicin >1024 mg/ml, imipenem 0.25 mg/ml, meropenem 0.25 mg/ml, tigecycline 0.5 mg/ml and tobramycin >1024 mg/ml.
The entire genomes of RYU109 and RYU351 were sequenced by MiSeq™ (Illumina, San Diego, CA) and searched by CLC Genomics Workbench v.5.5 (CLC bio, Tokyo, Japan) for 923 drug resistance genes, including genes encoding b-lactamases, 16S rRNA methylases and aminoglycoside-acetyl/adenyltransferases, as well as point mutations in gyrA and parC associated with quinolone resistance. The genetic environments surrounding 16S rRNA methylaseencoding genes were determined. Multilocus sequence typing (MLST) was performed by using Institut Pasteur whole genome MLST databases (http://bigsdb.pasteur.fr/) for K. pneumoniae and PubMLST databases (http://pubmlst.org/) for K. oxytoca. K. pneumoniae RYU109 belonged to sequence type (ST) 496, and K. oxytoca RYU351 to ST22. A clinical isolate of ST496 K. pneumoniae registered in the MLST database had been obtained from a human urine sample in the Middle East. A clinical isolate of ST22 K. oxytoca registered in the PubMLST databases had been obtained from a human skin swab in Austria. K. pneumoniae RYU109 harbored genes encoding b-lactamases (blaTEM-1, blaSHV-1, blaDHA-1), a 16S rRNA methylase (armA) and aminoglycoside-acetyl/adenyltransferases (aadA1, aac(60 )-Ib). K. oxytoca RYU351 harbored genes encoding b-lactamases (blaTEM-1, blaDHA-1), a 16S rRNA methylase (armA) and aminoglycoside-acetyl/ adenyltransferases (aadA1, aac(60 )-Ib). RYU109 contained three plasmids, of sizes 365, 194 and 2 kbp; whereas RYU351 contained plasmids of sizes with 365 and 97 kbp. To determine the sizes of the plasmids harboring armA, plasmid DNAs in RYU109 and RYU351 were extracted and digested with S1 nuclease. Pulsed-field gel electrophoreses and Southern hybridization were performed. Southern blot analysis revealed that the armA genes in K. pneumoniae and K. oxytoca were located on 365 kbp plasmids (Fig. 1). When the conjugation experiments were conducted using E. coli DH5a harboring blaIMP-44 in pHSG398 [10], the plasmids were not conjugated into E. coli DH5a. The genetic environment of armA in K. pneumoniae RYU109 consisted of purR-orfX-blaDHA-1-ampRsul1-ISCR1-tnpU-armA-orfY-tnpD-mel-mph2-orfZ, with orfs X, Y, and Z encoding hypothetical proteins, 15,726 bp in size (GenBank accession no. LC209172). The genetic environment of armA in K. oxytoca RYU351 was identical to that of RYU109. The nucleotide
Fig. 1. Localization of the armA genes on plasmids from Klebsiella pneumoniae RYU109 and Klebsiella oxytoca RYU351 separated by PFGE. Lanes M1, Low Range PFG Marker; M2,CHEF DNA Size Marker-S.cerevisiae, 1: RYU109, 2: RYU351; A, PFGE patterns of S1 Nuclease-digested prasmids in Klebsiella pneumoniae RYU109 and Klebsiella oxytoca RYU351; B, Southern hybridization with armA probe; C, Southern hybridization with 16S rRNA probe.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006
K. Uechi et al. / J Infect Chemother xxx (2017) 1e3
sequences of these genetic structures were more than 99.9% identical to that of the region from nt 32,843 to nt 48,551 in the plasmid pKP048, originally has been obtained from a clinical isolate of K. pneumoniae KP048 in 2006 in China [11]. 3. Discussion This case report provides us several important clues to the emergence and dissemination of drug-resistant nosocomial pathogens in Okinawa, as well as in other parts of Japan. Okinawa is geographically located in the center of East Asia and is visited by many travelers from around the world, increasing the risk of importing novel drug-resistant genes into Japan. For example, a visitor was found to have imported a strain of A. baumannii harboring armA and blaoxa-23 [12]. The results of this case report also suggest that pathogens harboring armA have been emerging in Okinawa. The analysis of the genetic environment of armA suggests that these genes are similar to those obtained from an isolate in China. The patient in this report had no known record of traveling overseas, therefore, it is necessary to conduct epidemiological studies to clarify whether the armA gene has already spread throughout medical settings in Okinawa. Funding This study was supported by grants of Okinawa Communicable Disease Research Hub Formation Promotion Project, Okinawa Prefectural Government Commissioned Projects For Fiscal Year 2016, Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) and JSPS KAKENHI Grant Number 16K19133. Contributors TK and JF have conceptualized and designed this study. KU, TT, KS and IN have analyzed the data and interpreted the results. KU, IN and TS have collected samples and screened drug-resistant isolates. All authors have read the manuscript. Conflict of interest None to declare.
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Acknowledgments This study was approved by the Ethical Committee, University of the Ryukyus (approval number: 890), and the Biosafety Committee, National Center for Global Health and Medicine (approval number: 28-M-053). The research was supported by grants of Okinawa Communicable Disease Research Hub Formation Promotion Project, Okinawa Prefectural Government Commissioned Projects For Fiscal Year 2016, Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) and JSPS KAKENHI Grant Number 16K19133. References [1] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 2012;15:133e48. [2] Doi Y, Wachino J, Arakawa Y. Aminoglycoside resistance: the emergence of acquired 16S ribosomal RNA methyltransferases. Infect Dis Clin North Am 2016;30:523e37. [3] Galimand M, Courvalin P, Lambert T. Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother 2003;47:2565e71. [4] Yokoyama K, Doi Y, Yamane K, Kurokawa H, Shibata N, Shibayama K, et al. Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 2003;362:1888e93. [5] Tada T, Miyoshi-Akiyama T, Kato Y, Ohmagari N, Takeshita N, Hung NV, et al. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam. BMC Infect Dis 2013;13:251. [6] Yamane K, Doi Y, Yokoyama K, Yagi T, Kurokawa H, Shibata N, et al. Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 2004;48:2069e74. [7] Yamane K, Wachino J, Suzuki S, Shibata N, Kato H, Shibayama K, et al. 16S rRNA methylase-producing, Gram-negative pathogens. Jpn Emerg Infect Dis 2007;13:642e6. [8] Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. Dissemination of 16S rRNA methylase ArmA-producing Acinetobacter baumannii and emergence of OXA-72 carbapenemase coproducers in Japan. Antimicrob Agents Chemother 2014;58:2916e20. [9] Nagasawa M, Kaku M, Kamachi K, Shibayama K, Arakawa Y, Yamaguchi K, et al. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria. J Infect Chemother 2014;20:635e8. [10] Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. IMP-43 and IMP-44 metallo-b-lactamases with increased carbapenemase activities in multidrug-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2013;57:4427e32. [11] Jiang Y, Yu D, Wei Z, Shen P, Zhou Z, Yu Y. Complete nucleotide sequence of Klebsiella pneumoniae multidrug resistance plasmid pKP048, carrying blaKPC-2, blaDHA-1, qnrB4, and armA. Antimicrob Agents Chemother 2010;54:3967e9. [12] Tojo M, Mawatari M, Hayakawa K, Nagamatsu M, Shimada K, Mezaki K, et al. Multidrug-resistant Acinetobactor baumannii isolated from a traveler returned from Brunei. J Infect Chemother 2015;21:212e4.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Case Report
Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan Kohei Uechi a, b, Tatsuya Tada c, d, Kayo Shimada d, Isamu Nakasone e, Tetsu Sonozaki f, Teruo Kirikae c, d, *, Jiro Fujita b a
Division of Clinical Laboratory and Blood Transfusion, University Hospital of the Ryukyus, Okinawa, Japan Department of Infectious Diseases, Respiratory, and Digestive Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Department of Microbiology, Juntendo University School of Medicine, Tokyo, Japan d Department of Infectious Disease, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan e Control and Prevention of Infectious Disease, University Hospital of the Ryukyus, Okinawa, Japan f Department of Dermatology, University Hospital of the Ryukyus, Okinawa, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 May 2017 Received in revised form 22 August 2017 Accepted 8 September 2017 Available online xxx
This study describes highly aminoglycoside-resistant Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates obtained from an inpatient in Okinawa, Japan, with no known record of traveling overseas. The minimum inhibitory concentrations of amikacin and arbekacin against these strains were >1024 mg/ml. Whole-genome sequencing analysis revealed that these isolates harbored armA, which encodes a 16S rRNA methylase, ArmA, that confers pan-aminoglycoside resistance. This is the second report of K. pneumoniae harboring armA and the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan. © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Aminoglycoside resistance 16S rRNA methylase ArmA Klebsiella pneumoniae Klebsiella oxytoca
1. Introduction The methylation of 16S rRNA in Gram-negative pathogens confers high resistance to all clinically important aminoglycosides [1,2]. In 2003, clinical isolates of Klebsiella pneumoniae producing ArmA, a 16S rRNA methylase, and Pseudomonas aeruginosa producing RmtA, another 16S rRNA methylase, were identified in France [3] and Japan [4], respectively. Since then, 16S rRNA methylase-producing Gram-negative pathogens have been isolated in other parts of the world, including Asian countries such as Afghanistan, Bangladesh, China, Hong Kong, India, Japan, Korea, Nepal, Oman, Pakistan, Taiwan, and Vietnam [1,4,5]. To date, 10 plasmid-associated 16S rRNA methylase genes, armA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH and npmA, have been identified in clinical and veterinary isolates [1,2].
* Corresponding author. Department of Microbiology, Juntendo University School of Medicine, Tokyo, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. E-mail address: [email protected] (T. Kirikae).
Although the isolation frequency of Gram-negative pathogens producing 16S rRNA methylases remains low in Japan, clinical isolates producing several types of 16S rRNA methylases have been detected [6e9]. A prospective study in Japan in 2001 found that, of 903 clinical isolates of P. aeruginosa, four (0.4%) harbored rmtA [6]. Another prospective study in 2004 showed that, of 87,626 clinical isolates of Gram-negative pathogens, 26 (0.03%) harbored armA, rmtA, rmtB, or rmtC, with most of these pathogens isolated from patients on the main island of Japan [7]. Our previous study in 2012 revealed that, of 16,343 clinical isolates of Acinetobacter spp. obtained from medical settings throughout Japan, 49 (0.3%) harbored armA [8]. Several isolates of Gram-negative pathogens harboring 16S rRNA methylase endocing genes were recently detected in Japan, but these isolates have not been detected in Okinawa, the southwestern most point of the Japanese archipelago [6e9]. To our knowledge, however, only one isolate in K. pneumoniae [7] and no isolate in K. oxytoca harboring a 16S rRNA methylase endocing gene has been reported in Japan. This is the first report of K. oxytoca harboring a 16S rRNA methylase encoding gene in Japan and the
https://doi.org/10.1016/j.jiac.2017.09.006 1341-321X/© 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006
2
K. Uechi et al. / J Infect Chemother xxx (2017) 1e3
first report of Gram-negative pathogens harboring a 16S rRNA methylase-encoding gene in Okinawa.
2. Case report The patient was a 73-year-old Japanese woman living in Okinawa prefecture, who had been diagnosed with diabetes mellitus and was suspected of having bullous pemphigoid. Beginning in 2013, she required hospitalization four times, mainly for bloodstream infections. The latest hospitalization, for suspected bloodstream infection, was from December 2015 to June 2016. While hospitalized, methicillin-resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes were isolated from a blood sample, and K. pneumoniae was isolated from a urine sample. Treatment with ceftriaxone, vancomycin, and ampicillin for 17 days seemed to cure her bloodstream infection. Two weeks later, she experienced apparent acute pneumonia, bloodstream infection and urinarytract infection. Methicillin-susceptible S. aureus (MSSA) was isolated from a blood sample, K. oxytoca and P. aeruginosa from a urine sample, and MSSA and P. aeruginosa from a sputum sample. She was treated with meropenem, vancomycin and cefazolin for two weeks. Because A. baumannii was also isolated from a sputum sample, she was treated with meropenem and vancomycin for another 10 days. After recovery she was discharged from the hospital. Of the isolates obtained from this patient, two, K. pneumoniae RYU109 and K. oxytoca RYU351, were highly resistant to amikacin and arbekacin, with MICs >1024 mg/ml. The MICs of other antibiotics against RYU109 were aztreonam 2 mg/ml, cefepime 0.25 mg/ ml, cefotaxime 4 mg/ml, ceftazidime 32 mg/ml, ciprofloxacin 0.5 mg/ ml, colistin 0.063 mg/ml, gentamicin >1024 mg/ml, imipenem 0.25 mg/ml, meropenem 0.25 mg/ml, tigecycline 0.5 mg/ml and tobramycin >1024 mg/ml. The MICs against RYU351 were aztreonam 2 mg/ml, cefepime 0.25 mg/ml, cefotaxime 32 mg/ml, ceftazidime 32 mg/ml, ciprofloxacin 0.25 mg/ml, colistin 1 mg/ml, gentamicin >1024 mg/ml, imipenem 0.25 mg/ml, meropenem 0.25 mg/ml, tigecycline 0.5 mg/ml and tobramycin >1024 mg/ml.
The entire genomes of RYU109 and RYU351 were sequenced by MiSeq™ (Illumina, San Diego, CA) and searched by CLC Genomics Workbench v.5.5 (CLC bio, Tokyo, Japan) for 923 drug resistance genes, including genes encoding b-lactamases, 16S rRNA methylases and aminoglycoside-acetyl/adenyltransferases, as well as point mutations in gyrA and parC associated with quinolone resistance. The genetic environments surrounding 16S rRNA methylaseencoding genes were determined. Multilocus sequence typing (MLST) was performed by using Institut Pasteur whole genome MLST databases (http://bigsdb.pasteur.fr/) for K. pneumoniae and PubMLST databases (http://pubmlst.org/) for K. oxytoca. K. pneumoniae RYU109 belonged to sequence type (ST) 496, and K. oxytoca RYU351 to ST22. A clinical isolate of ST496 K. pneumoniae registered in the MLST database had been obtained from a human urine sample in the Middle East. A clinical isolate of ST22 K. oxytoca registered in the PubMLST databases had been obtained from a human skin swab in Austria. K. pneumoniae RYU109 harbored genes encoding b-lactamases (blaTEM-1, blaSHV-1, blaDHA-1), a 16S rRNA methylase (armA) and aminoglycoside-acetyl/adenyltransferases (aadA1, aac(60 )-Ib). K. oxytoca RYU351 harbored genes encoding b-lactamases (blaTEM-1, blaDHA-1), a 16S rRNA methylase (armA) and aminoglycoside-acetyl/ adenyltransferases (aadA1, aac(60 )-Ib). RYU109 contained three plasmids, of sizes 365, 194 and 2 kbp; whereas RYU351 contained plasmids of sizes with 365 and 97 kbp. To determine the sizes of the plasmids harboring armA, plasmid DNAs in RYU109 and RYU351 were extracted and digested with S1 nuclease. Pulsed-field gel electrophoreses and Southern hybridization were performed. Southern blot analysis revealed that the armA genes in K. pneumoniae and K. oxytoca were located on 365 kbp plasmids (Fig. 1). When the conjugation experiments were conducted using E. coli DH5a harboring blaIMP-44 in pHSG398 [10], the plasmids were not conjugated into E. coli DH5a. The genetic environment of armA in K. pneumoniae RYU109 consisted of purR-orfX-blaDHA-1-ampRsul1-ISCR1-tnpU-armA-orfY-tnpD-mel-mph2-orfZ, with orfs X, Y, and Z encoding hypothetical proteins, 15,726 bp in size (GenBank accession no. LC209172). The genetic environment of armA in K. oxytoca RYU351 was identical to that of RYU109. The nucleotide
Fig. 1. Localization of the armA genes on plasmids from Klebsiella pneumoniae RYU109 and Klebsiella oxytoca RYU351 separated by PFGE. Lanes M1, Low Range PFG Marker; M2,CHEF DNA Size Marker-S.cerevisiae, 1: RYU109, 2: RYU351; A, PFGE patterns of S1 Nuclease-digested prasmids in Klebsiella pneumoniae RYU109 and Klebsiella oxytoca RYU351; B, Southern hybridization with armA probe; C, Southern hybridization with 16S rRNA probe.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006
K. Uechi et al. / J Infect Chemother xxx (2017) 1e3
sequences of these genetic structures were more than 99.9% identical to that of the region from nt 32,843 to nt 48,551 in the plasmid pKP048, originally has been obtained from a clinical isolate of K. pneumoniae KP048 in 2006 in China [11]. 3. Discussion This case report provides us several important clues to the emergence and dissemination of drug-resistant nosocomial pathogens in Okinawa, as well as in other parts of Japan. Okinawa is geographically located in the center of East Asia and is visited by many travelers from around the world, increasing the risk of importing novel drug-resistant genes into Japan. For example, a visitor was found to have imported a strain of A. baumannii harboring armA and blaoxa-23 [12]. The results of this case report also suggest that pathogens harboring armA have been emerging in Okinawa. The analysis of the genetic environment of armA suggests that these genes are similar to those obtained from an isolate in China. The patient in this report had no known record of traveling overseas, therefore, it is necessary to conduct epidemiological studies to clarify whether the armA gene has already spread throughout medical settings in Okinawa. Funding This study was supported by grants of Okinawa Communicable Disease Research Hub Formation Promotion Project, Okinawa Prefectural Government Commissioned Projects For Fiscal Year 2016, Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) and JSPS KAKENHI Grant Number 16K19133. Contributors TK and JF have conceptualized and designed this study. KU, TT, KS and IN have analyzed the data and interpreted the results. KU, IN and TS have collected samples and screened drug-resistant isolates. All authors have read the manuscript. Conflict of interest None to declare.
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Acknowledgments This study was approved by the Ethical Committee, University of the Ryukyus (approval number: 890), and the Biosafety Committee, National Center for Global Health and Medicine (approval number: 28-M-053). The research was supported by grants of Okinawa Communicable Disease Research Hub Formation Promotion Project, Okinawa Prefectural Government Commissioned Projects For Fiscal Year 2016, Research Program on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED) and JSPS KAKENHI Grant Number 16K19133. References [1] Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 2012;15:133e48. [2] Doi Y, Wachino J, Arakawa Y. Aminoglycoside resistance: the emergence of acquired 16S ribosomal RNA methyltransferases. Infect Dis Clin North Am 2016;30:523e37. [3] Galimand M, Courvalin P, Lambert T. Plasmid-mediated high-level resistance to aminoglycosides in Enterobacteriaceae due to 16S rRNA methylation. Antimicrob Agents Chemother 2003;47:2565e71. [4] Yokoyama K, Doi Y, Yamane K, Kurokawa H, Shibata N, Shibayama K, et al. Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa. Lancet 2003;362:1888e93. [5] Tada T, Miyoshi-Akiyama T, Kato Y, Ohmagari N, Takeshita N, Hung NV, et al. Emergence of 16S rRNA methylase-producing Acinetobacter baumannii and Pseudomonas aeruginosa isolates in hospitals in Vietnam. BMC Infect Dis 2013;13:251. [6] Yamane K, Doi Y, Yokoyama K, Yagi T, Kurokawa H, Shibata N, et al. Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 2004;48:2069e74. [7] Yamane K, Wachino J, Suzuki S, Shibata N, Kato H, Shibayama K, et al. 16S rRNA methylase-producing, Gram-negative pathogens. Jpn Emerg Infect Dis 2007;13:642e6. [8] Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. Dissemination of 16S rRNA methylase ArmA-producing Acinetobacter baumannii and emergence of OXA-72 carbapenemase coproducers in Japan. Antimicrob Agents Chemother 2014;58:2916e20. [9] Nagasawa M, Kaku M, Kamachi K, Shibayama K, Arakawa Y, Yamaguchi K, et al. Loop-mediated isothermal amplification assay for 16S rRNA methylase genes in Gram-negative bacteria. J Infect Chemother 2014;20:635e8. [10] Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. IMP-43 and IMP-44 metallo-b-lactamases with increased carbapenemase activities in multidrug-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 2013;57:4427e32. [11] Jiang Y, Yu D, Wei Z, Shen P, Zhou Z, Yu Y. Complete nucleotide sequence of Klebsiella pneumoniae multidrug resistance plasmid pKP048, carrying blaKPC-2, blaDHA-1, qnrB4, and armA. Antimicrob Agents Chemother 2010;54:3967e9. [12] Tojo M, Mawatari M, Hayakawa K, Nagamatsu M, Shimada K, Mezaki K, et al. Multidrug-resistant Acinetobactor baumannii isolated from a traveler returned from Brunei. J Infect Chemother 2015;21:212e4.
Please cite this article in press as: Uechi K, et al., Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan, J Infect Chemother (2017), https://doi.org/10.1016/j.jiac.2017.09.006