Sensitivity surveillance of Pseudomonas aeruginosa isolates for several antibacterial agents in Chubu area (2013-2014).

We investigated the susceptibility to antibacterial agents of 186 clinical isolates of Pseudomonas aeruginosa isolated from medical facilities in Gifu, Aichi, Toyama, and Fukui prefectures from October 2013 to February 2014. MIC50/90 of piperacillin (PIPC), tazobactam/piperacillin (TAZ/PIPC), ceftazidime (CAZ), cefepime (CFPM), imipenem (IPM), meropenem (MEPM), doripenem (DRPM), aztreonam (AZT), ciprofloxacin (CPFX), levofloxacin (LVFX), amikacin (AMK) and colistin (CL) against P aeruginosa was 8/32, 4/32, 2/8, 2/16, 1/32, 0.5/8, 0.25/4, 8/32, 0.25/8, 0.5/16, 4/8 and 1/1pg/mLrespectively. Two strains of multidrug resistant P aeruginosa were isolated (1.1%). They were isolated from the respiratory tract, intra-abdominal, and urinary infection. The susceptible ratio against P aeruginosa derived from intra-abdominal infection for carbapenem was lower than those from respiratory tract and urinary infection. The susceptible ratio against P aeruginosa derived from urinary infection for penicillin, cephem, monobactam, and fluoroquinolone was lower than those from respiratory and intra-abdominal infection. It is meaningful to pay attention to the susceptibility to antibacterial agents in each clinical specimen from infected organ.

Crystal structure of IMP-2 metallo-ß-lactamase from Acinetobacter spp.: comparison of active-site loop structures between IMP-1 and IMP-2.

IMP-2, a subclass B1 metallo-ß-lactamase (MBL), is a Zn(II)-containing hydrolase. This hydrolase, involved in antibiotic resistance, catalyzes the hydrolysis of the C-N bond of the ß-lactam ring in ß-lactam antibiotics such as benzylpenicillin and imipenem. The crystal structure of IMP-2 MBL from Acinetobacter spp. was determined at 2.3 Å resolution. This structure is analogous to that of subclass B1 MBLs such as IMP-1 and VIM-2. Comparison of the structures of IMP-1 and IMP-2, which have an 85% amino acid identity, suggests that the amino acid substitution at position 68 on a ß-strand (ß3) (Pro in IMP-1 versus Ser in IMP-2) may be a staple factor affecting the flexibility of loop 1 (comprising residues at positions 60-66; EVNGWGV). In the IMP-1 structure, loop 1 adopts an open, disordered conformation. On the other hand, loop 1 of IMP-2 forms a closed conformation in which the side chain of Trp64, involved in substrate binding, is oriented so as to cover the active site, even though there is an acetate ion in the active site of both IMP-1 and IMP-2. Loop 1 of IMP-2 has a more flexible structure in comparison to IMP-1 due to having a Ser residue instead of the Pro residue at position 68, indicating that this difference in sequence may be a trigger to induce a more flexible conformation in loop 1.

[Sensitivity surveillance of Streptococcus pneumoniae isolates for several antibacterial agents in Gifu and Aichi prefectures (2011-2012)].

We investigated the susceptibility to antibacterial agents, genotype of penicillin-binding protein (PBP) genes and macrolide resistant genes, and the serotypes against 270 strains of Streptococcus pneumoniae isolated from medical facilities in Gifu and Aichi prefectures between October 2011 and April 2012. These results were compared with those against S. pneumoniae isolated in 2008-2009 and 2010-2011. The number of gPSSP with 3 normal PBP genes, gPISP with 1 or 2 normal PBP genes and gPRSP with 3 abnormal genes isolated in 2011-2012 was 15 (5.6%), 162 (60.0%) and 93 (34.4%) strains, respectively. Compared with those isolated in 2008-2009 and 2010-2011, the numbers of gPRSP were decreasing. On the other hand, the isolates with no macrolide-resistant gene, only mefA, only ermB, and both mefA and ermB were 16 (5.9%), 75 (27.8%), 153 (56.7%) and 26 (9.6%). Compared with those isolated in 2008-2009 and 2010-2011, the numbers of isolates with ermB, which was usually associated with high-level resistance, were increasing. The prevalent pneumococcal serotypes in children were type 3 (14.4%), following by type 15 and 19F (9.3%). The coverages of 7-valent pneumococcal conjugate vaccine (PCV7) and 13-valent pneumococcal conjugate vaccine (PCV13) were calculated as 22.9% and 49.2%, respectively. The coverages of PCV7 and PCV13 in gPRSP isolated from children were 47.7% (21/44 strains) and 72.7% (32/44 strains). The MIC90 of each antibacterial agent was as follows; 0.125pg/mL for imipenem, panipenem and garenoxacin, 0.25 µg/mL for meropenem and doripenem, 0.5 µg/mL for cefditoren, moxifloxacin and tosufloxacin, 1 µg/mL for amoxicillin, clavulanic acid/amoxicillin, cefteram, cefcapene and ceftriaxone, 2 µg/mL for benzylpenicillin, ampicillin, sulbactam/ampicillin, piperacillin, tazobactam/piperacillin and levofloxacin, 4 µg/mL for cefdinir, flomoxef and pazufloxacin, 16 µg/mL for minocycline, > 64 µg/mL for clarithromycin and azithromycin, and these MIC90s were about the same as those in 2010-2011.

[Analysis of extended-spectrum CTX-M-14 beta-lactamase (ESBLs) producing Escherichia coli isolates in the same ward over the long term].

We bacteriologically and genetically analysed 30 cephalosporin-resistant Escherichia coli strains isolated from specimens from 19 neurology-ward inpatients at our hospital over the 3 years from April 2006 to March 2009, surveying and comparing subjects' backgrounds. Of the 30, 19 (63%) were urine, 6 (20%) sputum, and 3 (10%) blood. We tested extended-spectrum beta-lactamase (ESBLs) production, found in all samples. PCR and gene sequencing showed that 25 strains (83%) were CTX-M-14 and 5 (17%) CTX-M-2. Among CTX-M-14 strains, two cluster groups I and II, were obtained using pulsed-field gel electrophoresis (PFGE). Cluster group I in particular, continued to be detected for 18 months in the same hospital room. The detection rate was high at 13 (68%) in subjects with urinary catheters and morbidity was high in those with a history of cerebrovascular disease, diabetes, and hypertension. Our findings suggest that genetically identical strains may become established and spread in hospitals possibly due to inadequate contact prevention, subjects' immune status, and risk factor existence.

Practical disk diffusion test for detecting group B streptococcus with reduced penicillin susceptibility.

Although group B streptococcus (GBS) has been considered to be uniformly susceptible to beta-lactams, the presence of GBS with reduced penicillin susceptibility (PRGBS) was recently confirmed genetically. We developed a feasible and reliable method for screening PRGBS in clinical microbiology laboratories using a combination of ceftibuten, oxacillin, and ceftizoxime disks.

Change in the prevalence of extended-spectrum-beta-lactamase-producing Escherichia coli in Japan by clonal spread.

INTRODUCTION: In the early 2000s, there was a rapid increase in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in hospital settings throughout Japan. The reasons for this rapid increase are unclear. METHODS: Between 2002 and 2003, 142 clinical isolates of E. coli suspected of producing ESBL were obtained from 37 hospitals and commercial clinical laboratories in geographically distinct regions throughout Japan. They were tested for ESBL types and further subtyped for serogroups, fimH single nucleotide polymorphism, pulsed-field gel electrophoresis patterns and multilocus sequence type (MLST). Representative isolates were also subjected to plasmid analysis. RESULTS: Of 142 E. coli isolates suspected of producing ESBL, 130 were confirmed as harbouring blaCTX-M by PCR analysis and sequencing. Of these, 84 (65%) harboured CTX-M-9-group blaCTX-M. Two serogroups O25 and O86 accounted for 41% of the 130 blaCTX-M-positive E. coli. All O86 serogroup strains belonged to ST38 by MLST and they formed 18% of all the blaCTX-M-positive E. coli. Serogroup O25 strains belonged to ST131 and ST73, and formed 21% and 1% of blaCTX-M-positive E. coli, respectively. Seven characterized plasmids carrying blaCTX-M genes belonged to three distinct incompatibility groups: IncF, IncN and IncI1. CONCLUSIONS: In this study, clonally related strains of E. coli accounted for a large proportion of blaCTX-M-positive E. coli. This high proportion of clonal groups identified in different regions of Japan suggests their recent spread by mechanisms other than healthcare-associated transmission. These observations imply that restricting antimicrobial use in human clinical settings may have limited impact on the spread of ESBL-producing E. coli.

Additional effects of pranlukast in salmeterol/fluticasone combination therapy for the asthmatic distal airway in a randomized crossover study.

BACKGROUND: Salmeterol/fluticasone combination (SFC) therapy is used to control inflammation in the distal airway of patients with well-controlled asthma, but the efficacy of this approach is unclear. OBJECTIVES: The goal of the study was to evaluate the effect of pranlukast, a leukotriene receptor antagonist (LTRA), on distal airway inflammation and pulmonary resistance in patients with asthma that was well-controlled using SFC therapy alone. METHODS: The subjects were 32 patients with well-controlled asthma (age 61.1+/-17.8 years old, Step 3 in the GINA guidelines, Asthma Control Test score 23.2+/-1.8 points) based on use of SFC therapy alone for more than 3 months. These subjects were randomly assigned to groups receiving SFC alone or SFC+LTRA (pranlukast 450 mg daily) and then switched to the opposite group after 4 weeks in a crossover manner. Eosinophilic inflammation in induced sputum samples was assessed after each treatment period. Sputum was induced by inhalation of 10% hypertonic saline for 15 min. Impulse oscillometry parameters (R5, R20, X5 and AX) and spirometry were examined during each period. The Asthma-related Quality of Life Questionnaire (AQLQ) was also administered in each period. RESULTS: The ECP levels in late-phase sputum were significantly higher than those in early-phase sputum with SFC therapy alone (178.3+/-166.0 vs. 65.5+/-68.9 microg/l, p<0.001), whereas these values did not differ significantly with SFC+LTRA treatment (70.9+/-95.1 vs. 54.6+/-65.7, p=0.554). ECP levels in late-phase sputum with SFC therapy were also significantly higher than those with SFC+LTRA (p=0.045). The values of R5, R20, R5-R20 (kPa/(L/s)), and AX (kPa/L) all significantly improved during with SFC+LTRA treatment compared with SFC alone (median (25-75 percentile)): 0.350 (0.283-0.440) vs. 0.340 (0.280-0.378), p=0.036; 0.280 (0.233-0.365) vs. 0.270 (0.240-0.318), p=0.019; 0.050 (0.030-0.110) vs. 0.500 (0.030-0.073), p=0.032; and 0.570 (0.308-1.045) vs. 0.410 (0.263-0.820), p=0.014; respectively. Pulmonary function indexes did not differ significantly between the two treatments, but the symptom and activity limitation domains of the AQLQ were significantly improved by SFC+LTRA treatment. CONCLUSION: This study suggests that the combination of SFC and LTRA may give better control of residual eosinophilic inflammation in the distal airway compared with SFC therapy alone.

First molecular characterization of group B streptococci with reduced penicillin susceptibility.

Group B streptococci (GBS; Streptococcus agalactiae) are the leading cause of neonatal invasive diseases and are also important pathogens for adults. Penicillins are the drugs of first choice for the treatment of GBS infections, since GBS have been regarded to be uniformly susceptible to penicillins so far. Here we characterize the first strains of GBS with reduced penicillin susceptibility (PRGBS) identified in Japan. Fourteen PRGBS strains were clinically isolated from the sputa of elderly patients from 1995 to 2005; and the MICs of penicillin, oxacillin, and ceftizoxime ranged from 0.25 to 1 microg/ml, 2 to 8 microg/ml, and 4 to 128 microg/ml, respectively. Moreover, some strains were also insusceptible to ampicillin, cefazolin, cefepime, and cefotaxime. All the PRGBS isolates tested possessed a few amino acid substitutions adjacent to the conserved SSN and KSG motifs (amino acids 402 to 404 and 552 to 554, respectively) of PBP 2X, and the amino acid substitutions could be classified into two types, Q557E and V405A. Western blotting analysis of the 14 clinical isolates with anti-PBP 2X-specific serum suggested that the amount of PBP 2X among the 14 PRGBS isolates was reduced, although the 2 ATCC strains produced a significant amount of PBP 2X. The introduction of PRGBS-derived PBP 2X genes into penicillin-susceptible strains through allelic exchange elevated their penicillin insusceptibility, suggesting that these altered PBP 2X genes are responsible for the penicillin insusceptibility in PRGBS strains. In this study, we characterized for the first time PRGBS strains on a molecular basis, although several reports have so far mentioned the existence of beta-lactam-insusceptible GBS from a phenotypic standpoint.

Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor.

The VIM-2 metallo-beta-lactamase enzyme from Pseudomonas aeruginosa catalyzes the hydrolysis of most beta-lactam antibiotics including carbapenems, and there are currently no potent inhibitors of such enzymes. We found rac-2-omega-phenylpropyl-3-mercaptopropionic acid, phenylC3SH, to be a potent inhibitor of VIM-2. The structure of the VIM-2-phenylC3SH complex was determined by X-ray crystallography to 2.3 A. The structure revealed that the thiol group of phenylC3SH bridged to the two zinc(II) ions and the phenyl group interacted with Tyr67(47) on loop1 near the active site, by pi-pi stacking interactions. The methylene group interacted with Phe61(42) located at the bottom of loop1 through CH-pi interactions. Dynamic movements were observed in Arg228(185) and Asn233(190) on loop2, compared with the native structure (PDB code: 1KO3 ). These results suggest that the above-mentioned four residues play important roles in the binding and recognition of inhibitors or substrates and in stabilizing a loop in the VIM-2 enzyme.

[A case of sepsis caused by Enterobacter cloacae producing both type SHV-12 and type CTX-M-14 ESBLs in a post bone-marrow transplantation patient].

Third-generation cephalosporin-resistant Enterobacter cloacae was isolated from the blood culture of a 31-year-old woman after bone-marrow transplantation. Since this strain was resistant to third-generation cephalosporins, the production of extended-spectrum beta-lactamases (ESBLs) was suspected. PCR and a sequence analysis confirmed two ESBL genes, bla(SHV-12) and bla(CTX-M-14). No bacteria were detected after meropenem was administered, and symptoms abated. This is, to our knowledge, the first report in Japan of E. cloacae clinical isolates simultaneously producing both SHV-12 and CTX-M-14 ESBL. In cases where chromosomal AmpC over-production of E. cloacae concomitantly produces ESBL, caution should be exercised due to the potential development of resistance against extended-spectrum beta-lactam agents.

[Development and evaluation of novel loop-mediated isothermal amplification for rapid detection of bla(IMP-1) and bla(VIM-2) genes].

Loop-mediated isothermal amplification (LAMP) amplifies a target gene with high specificity and rapidity under isothermal conditions. LAMP assays were developed for the rapid detection of metallo-beta-lactamase (MBL) genes such as bla(IMP-1)) and bla(VIM-2). We initially designed specific primers to detect MBL genes for LAMP assays and evaluated the specificity and sensitivity of these assays. LAMP assays amplified MBL genes under a constant temperature of 63 degrees C within 1 hour, and were compared to PCR in MBL-producing strains. The results of MBL genes typing by LAMP assays agree completely with PCR results. The lower detection limits of bla(IMP-1)- and bla(VIM-2)-LAMP assays using real-time turbidimeters were 30cfu/test and 3cfu/test. After amplification, products were directly observed by the naked eye with a fluorescent detection reagent. In conclusion, LAMP assays are convenient, rapid, and fully feasible for detecting MBL genes in ordinary clinical microbiology laboratories without special apparatus.

[One case with Erysipelothrix rhusiopathiae endocarditis].

We report case of a 67-year-old alcoholic fisherman who developed infective endocarditis caused by Erysipelothrix rhusiopathiae. The initial manifestations were fever and back pain of approximately three months' duration. Auscultation of the heart revealed a loud systolic murmur at the apex and a diastolic murmur over the aortic valve area. Echocardiographic studies showed vegetations on both the aortic valve and mitral valve. Blood cultures grew Erysipelothrix rhusiopathiae, and it was sensitive to aminobenzyl penicillin. No other bacteria grew aut. A diagnosis of infective endocarditis caused by the Erysipelothnx rhusiopatniae was made, and the patient was treated with aminobenzyl penicillin 12g/day for 6 weeks. His clinical course was complicated by heart failure, multiple cerebral embolism, and renal infarctions. However, he recovered without valve replacement. Although the exact route of infection remains unknown, erosions of the skin, of his palms at the time of tho initial examination appeared to be one possible source of the systemic infection in this case. The serotype of the bacteria was Ib. To our knowledge this is the first case of serotyping of bacterium that caused endocarditis in humans.

Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.

BACKGROUND: Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. METHODS: We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. FINDINGS: An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. INTERPRETATION: Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.

Characterization of Klebsiella pneumoniae and Escherichia coli strains that produce CTX-M-2-type broad spectrum beta-lactamase isolated from a child with leukemia.

An 8-year-old girl with acute leukemia had bacteremia caused by Klebsiella pneumoniae producing CTX-M-2-type broad spectrum beta-lactamase. K. pneumoniae and Escherichia coli strains producing the same enzyme and harboring identical conjugative plasmids were recovered from stoor culture. Patients with frequent episodes of neutropenia and prophylactic administration of beta-lactams are at risk of harboring colonizing strains that produce broad spectrum beta-lactamases.

[IMP-1 type metalo-beta-lactamase producing Serratia marcescens strains isolated from blood culture between 1991 to 2000].

Infection caused by metallo beta-lactamase (MBL)-producing bacteria have been increasingly reported in Japan in recent years. We investigated the prevalence, clinical backgrounds and molecular epidemiology of MBL-producing Serratia marcescens strains isolated from blood cultures at our university hospital between 1991 and 2000. Forty-three S. marcescens strains were isolated in the period, and the incidence reached about 2% total bacteria detection in 1998 and 1999. There were 13 ceftazidime-resistant strains (31%), and five of them produced MBL. MICs of imipenem (IPM) were 4-16 mg/ml, of which one strain was susceptible to IPM. Of the five MBL-producers, four were obtained from a tertiary emergency ward, the underlying diseases being either serious trauma or cerebrovascular diseases. The other was isolated from a patient who underwent kidney transplantation. S. marcescens was no longer isolated from patients after administration of aztreonam (AZT), implying that AZT was clinically effective. When analyzed genetically using the AP-PCR technique, the patterns were identical among strains isolated in 1996, 1997 and 1998 and those isolated in 1999 and 2000 respectively, suggesting that the same strains may have inhabited the hospital wards for prolonged periods. Countermeasures against nosocomial infection was undertaken thereafter, resulting in reduction of isolation frequency. It cannot be excluded that the resistant bacteria spread as the amount of carbapenem consumption increased. Prudent use of antibiotics is mandatory to prevent further dissemination of such MBL producers.

Outbreak of CTX-M-3-type extended-spectrum beta-lactamase-producing Enterobacter cloacae in a pediatric ward.

A first resistant strain of Enterobacter cloacae was isolated from a blood specimen in a pediatric patient with immature teratoma-developed sepsis after combination chemotherapy. The strain produced extended-spectrum beta-lactamase (ESBL), and the same ESBL-producing strains were detected in urine samples from other patients in the pediatric ward. All strains harbored genes for bla (CTX-M-3) by polymerase chain reaction (PCR) and sequencing analysis. Analysis of pulsed-field gel electrophoresis revealed that all strains were the same clonal type. These results suggest that ESBL-producing strains might be transmitted in the ward via contact among patients or medical staff.

New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate.

Plasmid-mediated Qnr and AAC(6')-Ib-cr have been recognized as new molecular mechanisms affecting fluoroquinolone (FQ) resistance. C316, an Escherichia coli strain demonstrating resistance to various FQs, was isolated in Japan. Resistance to FQs was augmented in an E. coli CSH2 transconjugant, but PCR failed to detect qnr genes, suggesting the presence of novel plasmid-mediated FQ resistance mechanisms. Susceptibility tests, DNA manipulation, and analyses of the gene and its product were performed to characterize the genetic determinant. A novel FQ-resistant gene, qepA, was identified in a plasmid, pHPA, of E. coli C316, and both qepA and rmtB genes were mediated by a probable transposable element flanked by two copies of IS26. Levels of resistance to norfloxacin, ciprofloxacin, and enrofloxacin were significantly elevated in E. coli transformants harboring qepA under AcrB-TolC-deficient conditions. QepA showed considerable similarities to transporters belonging to the 14-transmembrane-segment family of environmental actinomycetes. The effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on accumulation of norfloxacin was assayed in a qepA-harboring E. coli transformant. The intracellular accumulation of norfloxacin was decreased in a qepA-expressing E. coli transformant, but this phenomenon was canceled by CCCP. The augmented FQ resistance level acquired by the probable intergeneric transfer of a gene encoding a major facilitator superfamily-type efflux pump from some environmental microbes to E. coli was first identified. Surveillance of the qepA-harboring clinical isolates should be encouraged to minimize further dissemination of the kind of plasmid-dependent FQ resistance determinants among pathogenic microbes.

Novel plasmid-mediated 16S rRNA m1A1408 methyltransferase, NpmA, found in a clinically isolated Escherichia coli strain resistant to structurally diverse aminoglycosides.

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

16S rRNA methylase-producing, gram-negative pathogens, Japan.

To investigate the exact isolation frequency of 16S rRNA methylase-producing, gram-negative pathogenic bacteria, we tested 87,626 clinical isolates from 169 hospitals. Twenty-six strains from 16 hospitals harbored 16S rRNA methylase genes, which suggests sparse but diffuse spread of pan-aminoglycoside-resistant microbes in Japan.