Carbapenemase-producing Enterobacteriaceae represent a serious public health threat worldwide. Carbapenemase genes, harbored on a transferable plasmid, have been isolated globally with distinct geographical features. Klebsiella pneumoniae, included in Enterobacteriaceae, also produces carbapenemase and often shows hypervirulence. Overlapping carbapenem resistance and hypervirulence in K. pneumoniae have been reported, but such strains have not yet been found in Japan. Here, we screened 104 carbapenemase-producing K. pneumoniae isolates collected from 37 hospitals and outpatient clinics in Japan between September 2014 and July 2015. PCR and DNA sequencing demonstrated IMP-1 in 21 isolates and IMP-6 in 83 isolates, 77 of which coharbored CTX-M-2. Most of the isolates showed low MICs toward imipenem and meropenem but high MICs toward penicillin and cephalosporins. Conjugation experiments with an Escherichia coli J53 recipient showed that most of the plasmids in IMP-6 producers were transferable, whereas only one-half of the plasmids in IMP-1 producers were transferable. PCR-based replicon typing and multiplex PCR identified five isolates belonging to the CG258 non-tonB79 cluster and no isolate belonging to the CG258-tonB79 cluster or sequence type 307 (ST307). Four K1-ST23 isolates, 10 K2-ST65 isolates, and 7 K2-ST86 isolates were detected that harbored virulence genes. The resistance genes in 85 isolates were transferable, but the virulence genes were not transferred. These results demonstrate the acquisition of IMP-type carbapenemase genes and CTX-M-type genes among hypervirulence isolates in Japan, warranting further attention and countermeasures. In this study, we have determined the molecular characteristics and epidemiology of IMP-6 producers that coharbored various CTX-M genes in Japan. IMPORTANCE Carbapenems serve as a last resort for the clinical treatment of multidrug-resistant infections. Therefore, the rapid spread of carbapenemase-producing strains represents a serious public health threat, further limiting antibiotic choices. The current findings of hypervirulent carbapenemase-producing Klebsiella pneumoniae clinical isolates in Japan demonstrate the potential broad spread and transfer of these genes, necessitating close surveillance.

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Carbapenem-resistant Enterobacteriaceae (CRE) strains have become globally distributed in the past decade, resulting in concern over the control of hospital infections and antimicrobial therapies (1, 2). The majority of CRE isolates are carbapenemase-producing Enterobacteriaceae (CPE) strains, so early detection of CPE strains is essential for providing optimal antimicrobial therapies and preventing horizontal transmission. In 2015, van der Zwaluw et al. reported the carbapenem inactivation method (CIM), a new method for detecting carbapenemase producers with high sensitivity and specificity (3). However, it was unclear whether CIM can identify IMP producers with a low carbapenem MIC or non-CPE strains that are highly carbapenem resistant. In this study, we evaluated whether CIM can identify CPE strains independently of their carbapenem MICs.Were used 233 CPE and 51 non-CPE strains isolated in general hospitals across Japan from 2012 to 2016 and stocked in our laboratory. The strains were collected from blood, sputum, wounds, urine, and feces. Their antibiotic susceptibilities were determined by the agar dilution method in accordance with the recommendations of the CLSI (4). The CPE strains included 191 IMP, 20 KPC, 17 NDM, and 5 OXA-48-like (OXA-48 and OXA-244) producers. The non-CPE strains included 31 extended-spectrum beta-lactamase (CTX-M, SHV, and TEM) producers and 5 plasmid-mediated AmpC ␤-lactamase (DHA, CMY, and CFE) producers. They were identified by DNA sequence amplification as previously described (5-10). Of the non-CPE strains, 13 highly carbapenem-resistant strains were identified as producing cephalosporinases and lacking porin function by SDS-PAGE, DNA sequencing, and quantitative reverse transcription-PCR (11).The CIM was conducted as previously described (3). The isolates were cultured on Mueller-Hinton agar (MHA) plates. A full 10-l inoculation loop of each strain was suspended in 400 l of sterile distilled water, and a 10-g meropenem (MEM) susceptibilitytesting disk (E-DF85; EIKEN) was immersed in the solution. After incubation at 35°C for 2 h, the disk was removed and placed on an MHA plate inoculated with a 0.5 McFarland standard of Escherichia coli strain ATCC 25922 with a sterile cotton swab. Finally, the plate was incubated overnight at 35°C and the inhibition zone around each disk was measured. Inhibition circles Ͻ10 mm in diameter were judged to indicate CIM positivity.The CIM showed a sensitivity of 100% (233/233) for CPE strains and a specificity of 96.1% (49/51) for non-CPE strains (Table 1). The MICs of MEM for the 191 IMP producers ranged from 0.125 to 32 g/ml (MIC 50 ϭ 0.5 g/ml, MIC 90 ϭ 2 g/ml), and those of imipenem (IPM) ranged from 0.06 to 8 g/ml (MIC 50 ϭ 0.125 g/ml, MIC 90 ϭ 0.5

A 57-year old woman was admitted to our hospital with massive pericardial fluid. Culture of the pericardial fluid was negative, however, Binax NOW Streptococcus pneumoniae urinary antigen test was positive in pericardial fluid. 16S rDNA sequencing and PCR for lyt(A) gene of the pericardial fluid sample confirmed the microbiological diagnosis of S. pneumoniae. The patient was treated with surgical drainage and continuous intravenous infusion of penicillin G and its concentration in the serum and pericardial effusion was monitored. Incorporation of molecular methods such as antigen testing and nucleic acid sequencing would benefit the management of infectious diseases especially in culture negative cases.