Pseudomonas asiatica sp. nov., isolated from hospitalized patients in Japan and Myanmar.

A novel Gram-negative, aerobic, rod-shaped, non-spore-forming bacterial strain, RYU5T, was isolated from a stool sample of an inpatient at a hospital in Okinawa, Japan. The optimal growth temperature of RYU5T was 30 °C. Phylogenetic analysis based on the sequences of housekeeping genes, including the 16S rRNA, rpoB, rpoD and gyrB genes, showed that RYU5T was a member of the Pseudomonas putida group and was located close to Pseudomonas monteilii and P. putida. Whole-genome comparisons, using average nucleotide identity and digital DNA-DNA hybridization, confirmed that strain RYU5T should be classified as a novel species of Pseudomonas. Phenotypic characterization tests showed that utilization of d-mannose, d-serine, l-arabinose and d-fructose could distinguish this strain from other related species of the genus Pseudomonas. Based on genetic and phenotypic evidence, strain RYU5T should be classified as a novel species, for which the name Pseudomonas asiatica sp. nov. is proposed. The type strain is RYU5T (=DSM 107182T, =JCM 32716T), with a DNA G+C content of 62.25 mol%.

Emergence of a carbapenem-resistant and colistin-heteroresistant Enterobacter cloacae clinical isolate in Japan.

A carbapenem-resistant and colistin-heteroresistant clinical isolate of Enterobacter cloacae was obtained from an inpatient in Okinawa, Japan. The minimum inhibitory concentrations of both imipenem and meropenem were 32 µg/mL. The isolate showed heteroresistance to colistin using the Etest method and resistance to colistin using the broth microdilution method. It had a disrupted ompC and a mutation in the promoter region of blaACT-2, but did not harbor any genes encoding carbapenemase. The disruption of ompC and the mutation in blaACT-2 was associated with the carbapenem resistance of this isolate. This isolate also had mutations in pmrAB and phoPQ encoding two-component regulatory systems, which may be associated with colistin heteroresistance.

An improved carbapenem inactivation method, CIMTrisII, for carbapenemase production by Gram-negative pathogens.

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa. We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae, Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

A carbapenem-resistant clinical isolate of Aeromonas hydrophila in Japan harbouring an acquired gene encoding GES-24 ß-lactamase.

Several species of Aeromonas produce the enzyme CphA metallo-ß-lactamase. This study describes an isolate of Aeromonas hydrophila harbouring an acquired gene encoding the carbapenemase GES-24. This isolate was obtained from an inpatient in Okinawa, Japan, with no apparent record of travelling overseas. The minimum inhibitory concentrations of carbapenems against this isolate were 8 µg ml-1 for imipenem and 16 µg ml-1 for meropenem. Recombinant GES-24 hydrolyzed all of the tested ß-lactams, including imipenem and meropenem. The genomic environment surrounding blaGES-24 was intI1-blaGES-24-aac(6')-IIc-qacEdelta1-sulI-orfX-tetR-tetE. This is the first report of A. hydrophila producing a GES-type carbapenemase.

Emergence of IncX4 plasmids encoding mcr-1 in a clinical isolate of Klebsiella pneumoniae in Japan.

The mcr-1 gene encodes a phosphoethanolamine transferase that confers resistance to colistin by transferring phosphoethanolamine to lipid A. A 33-kb IncX4 plasmid harboring mcr-1 was detected in a Klebsiella pneumoniae isolate and two Escherichia coli isolates, and a 66-kb IncI2 plasmid was detected in three E. coli isolates in hospitals in Okinawa, Japan.

Emergence of ArmA, a 16S rRNA methylase in highly aminoglycoside-resistant clinical isolates of Klebsiella pneumoniae and Klebsiella oxytoca in Okinawa, Japan.

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 µg/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.

A hemin auxotrophic Enterobacter cloacae clinical isolate with increased resistance to carbapenems and aminoglycosides.

Small-colony variants (SCVs) were obtained from an Enterobacter cloacae clinical isolate in Okinawa, Japan. One variant showed auxotrophy for hemin with a deletion of 20 365 nucleotides, dosC-ydiK-mmuP-mmuM-tauA-tauB-tauC-tauD-hemB-yaiT-yaiV-ampH-yddQ-sbmA-yaiW-yaiY-yaiZ, including hemB, and was more resistant to aminoglycosides and carbapenems, but more susceptible to aztreonam, than the parent strain.

A Modified Carbapenem Inactivation Method, CIMTris, for Carbapenemase Production in Acinetobacter and Pseudomonas Species.

The carbapenem inactivation method (CIM) and modified CIM (mCIM) are simple and economical phenotypic screening methods for detecting carbapenemase production in Gram-negative bacteria. Although the mCIM has been recommended by the Clinical and Laboratory Standards Institute, both the CIM and mCIM have limitations. This study describes another modified CIM, called CIMTris, in which carbapenemase was extracted from bacteria with 0.5 M Tris-HCl (pH 7.6) buffer. The ability of the CIMTris to detect carbapenemase production was examined in Acinetobacter and Pseudomonas species. The CIMTris had an overall sensitivity of 97.6% and an overall specificity of 92.6%, whereas the mCIM had a sensitivity of 45.1% and a specificity of 100% for the isolates tested. These findings indicate that the CIMTris is useful for detecting carbapenemase production in Acinetobacter and Pseudomonas species.

Emergence of a colistin-resistant Escherichia coli clinical isolate harboring mcr-1 in Japan.

The mcr-1 is a gene encoding a phosphoethanolamine transferase, which confers resistance to colistin by transferring phosphoethanolamine to lipid A. We describe here the emergence of a colistin-resistant Escherichia coli clinical isolate harboring plasmid-mediated mcr-1 in Japan. The isolate belonged to ST5702 and is suspected to come from livestock and transmitted to human. This is the first report of a clinical isolate harboring mcr-1 in Japan.

[Laboratory-based evaluation of PVL-RPLA "Seiken" to detect Panton-Valentine leukocidin produced by Staphylococcus aureus].

It is well known that some isolates of Staphylococcus aureus produce pathogenic toxin, Panton-Valentine leukocidin (PVL), and that the toxin has been reported to be highly associated with community acquired methicillin resistant S. aureus (CA-MRSA). Currently, the PCR method using specific primers for the PVL gene (LukS-PV-lukF-PV) have been widely used to detect PVL. In this study, we evaluated the PVL-RPLA "Seiken", diagnostic reagent based on a reserved passive latex agglutination reaction with a specific monoclonal antibody for detecting PVL. A total of 630 clinical isolates were used. PCR method detected 34 PVL-positive (28 MRSA and 6 MSSA), and, of these, PVL-RPLA "Seiken" read positive for 32 isolates (27 MRSA and 5 MSSA), the result indicating two false negative occurrences. The concordance rate was 99.7%. In addition the recommended BHI broth, CCY medium, Dolman broth and Todd-Hewitt broth were applied for toxin preparation media. Toxin concentration produced in CCY medium was significantly higher than those in the remaining culture medium (p < 0.05). PVL-RPLA "Seiken" is a method for detecting the PVL in the culture broth by antigen antibody reaction after an overnight shaking culture. This method does not require any expensive equipments or facilities. Thus this reagent provides us with rapid, easy-to-perform, less expensive test method to detect PVL in clinical microbiology laboratories.

[Decrease in number of venipuncture tubes enables us to shorten turnaround times of blood-based testing in clinical laboratories].

We are making efforts to reduce the number of venipuncture tubes for blood-based testing. On the reconstruction of hematology system in 2011, we planned the system to include hemoglobin A1c (HbA1c) assay and to replace the assay instrument for erythrocyte sedimentation rate (ESR) to use EDTA-2K based whole blood. Accordingly, the revised system required a single test tube for hematological testing, resulting in reduction of blood volume collected. It was estimated that the whole blood collected from outpatients in a year decreased from 143 L to 109 L. Also, the times required to complete venipuncture after outpatient accession were significantly shortened to 10(0.71 +/- 0.27) (2.75-9.55) min, and nearly 50% of outpatients experienced < 2 min of waiting. As the times required for venipuncture were shortened, the turnaround times (TATs) from outpatient accession to finally reporting the test results to physicians were also shortened in the blood-based laboratories. The TATs after outpatient accession to reporting the test results in biochemistry and serology ranged 59 to 80 min (90%-tile), indicating 8 to 16 min less when compared with those before system reconstruction. In conclusion, the decrease in number of venipuncture tubes in hematological testing enables us to reduce the blood volume collected, and to shorten (1) times required for venipuncture procedure, (2) waiting times, and (3) TATs for blood-based testing. However, as demonstrated in HbA1c, i.e., a 50%-tile of TAT for HbA1c delayed for 5 min, the configuration of assay system can greatly influence the TATs of individual test parameters.

Dissemination of panton-valentine leukocidin-positive methicillin-resistant Staphylococcus aureus in Okinawa, Japan.

Panton-Valentine leukocidin (PVL) is a pore-forming cytotoxin that is produced by Staphylococcus aureus closely associated with skin and soft-tissue infections (SSTI). PVL-positive S. aureus strains have been identified worldwide, including in the USA; however, few studies have reported the presence of these strains in Japan. In this study, we prospectively investigated the prevalence of PVL in S. aureus strains from outpatients presenting with SSTI in Okinawa and characterized the PVL-positive S. aureus strains by polymerase chain reaction (PCR) and multilocus sequence typing (MLST). From 2008-2010, 499 clinical samples were obtained from 497 people. S. aureus was identified in 274 samples, and 36% (99 of 274) were methicillin-resistant S. aureus (MRSA). Seventeen (6.2%) PVL-positive S. aureus strains were detected by PCR, and 12 of the 17 PVL-positive strains were MRSA. Most PVL-positive S. aureus caused furuncles or carbuncles. Nine of the 17 PVL-positive isolates had an ST8 MRSA genotype and most harbored SCCmec type IVa and the arcA gene of the arginine catabolic mobile element, which is identical to the USA300 clone prevalent in the USA. PVL-positive S. aureus strains were more likely to be resistant to erythromycin (65%) and levofloxacin (53%). PVL-positive S. aureus strains have emerged and are spreading as a causative pathogen for SSTI in Okinawa.

[Antimicrobial susceptibilityof clinical isolates of aerobic gram-negative bacteria in 2008].

We determined MICs of antibacterial agents against 1145 clinical strains of aerobic Gram-negative bacteria (22 species) isolated at 16 Japanese facilities in 2008. MICs were determined using mostly broth microdilution method and antibacterial activity was assessed. Strains producing extended-spectrum beta-lactamases (ESBL) accounted for 3.8% of Escherichia coli, 2.6% of Klebsiella pneumoniae, 6.8% of Klebsiella oxytoca, 5.5% of Proteus mirabilis and 1.8% of Proteus vulgaris. ESBL produced strains were 6.8% at K. oxytoca that increased compared with 3.2% and 5.5% at P. mirabilis that decreased compared with 18.8% in 2006. Among Haemophilus influenzae, 61.7% that decreased compared with 67.7% in 2006, equaled 58.7% in 2004, were strains when classified by penicillin-binding protein 3 mutation. Against Pseudomonas aeruginosa, the activity of most antibacterial agents was similar to that in 2006. Although two antibacterial agents that tobramycin showed an MIC90 of 1 microg/mL and doripenem showed an MIC90 of 4 microg/mL against P. aeruginosa have potent activity. Of all P. aeruginosa strains, 4.3% were resistant to six agents of nine antipseudomonal agents, that decreased compared to 12.2% in 2004 and 5.7% in 2006. Against other glucose-non-fermentative Gram-negative rods, the activity of most antibacterial agents was similar to that in 2006.

[Antimicrobial susceptibility of clinical isolates of aerobic gram-positive cocci and anaerobic bacteria in 2008].

The activity of antibacterial agents against aerobic Gram-positive cocci (25 genus or species, 1029 strains) and anaerobic bacteria (21 genus or species, 187 strains) isolated from clinical specimens in 2008 at 16 clinical facilities in Japan were studied using either broth microdilution or agar dilution method. The ratio of methicillin-resistant strains among Staphylococcus aureus and Staphylococcus epidermidis was 59.6% and 81.2%, suggesting that resistant strains were isolated at high frequency. Vancomycin (VCM), linezolid (LZD) and quinupristin/dalfopristin (QPR/DPR) had good antibacterial activity against methicillin-resistant S. aureus and methicillin-resistant S. epidermidis, with MIC90s of < or = 2 microg/mL. The ratio of penicillin (PC) intermediate and resistant strains classified by mutations of PC-binding proteins among Streptococcus pneumoniae was 92.0% that was highest among our previous reports. Cefpirome, carbapenems, VCM, teicoplanin (TEIC), LZD and QPR/DPR had MIC90s of < or = 1 microg/mL against PC-intermediate and resistant S. pneumoniae strains. Against all strains of Enterococcus faecalis and Enterococcus faecium, the MICs of VCM and TEIC were under 2 microg/mL, and no resistant strain was detected, suggesting that these agents had excellent activities against these species. 15.9% of E. faecalis strains and 1.2% of E. faecium strains showed intermediate to LZD. 17.1% of E. faecium strains showed intermediate or resistant to QPR/DPR. Against all strains of Clostridium difficile, the MIC of VCM was under 1 microg/mL, suggesting that VCM had excellent activity. Carbapenems showed good activity against Clostridiales, Bacteroides spp., and Prevotella spp., but one strain of Bacteroides fragilis showed resistant to carbapenems. And so, the susceptibility of this species should be well-focused in the future at detecting continuously.

[Evaluation of turnaround time (TAT) for outpatients from venipuncture accession to reporting test results of blood chemistry and blood cell analysis to physicians].

In response to the revision of social medical insurance policy, in which hospital clinics can additionally charge for laboratory testing when the test results are presented to an outpatient in a print-out form on a visiting day, we evaluated laboratory-spending times, so-called turnaround times (TATs). A total of 14,802 outpatients during the period from October 2010 to May 2011 were enrolled. TATs from venipuncture accession to completing blood collection revealed a log-normal distribution with 5 to 6 min of mode and 10(0.95 +/- 0.26) (4.90 to 16.2) min of mean +/- standard deviation. Order waiting time figured a half-normal distribution, 50% tile and 90%-tile being 4 and 16 min, respectively. TATs of blood collection and order waiting time were significantly influenced by days of the week and accession time. Through analysis of TATs from specimen receipt to reporting test results, it became apparent that the tests determined by immunoassay and erythrocyte sedimentation rate (ESR) required more minutes when compared to the remaining tests. Total TATs from venipuncture accession to reporting test results ranged 28 to 29 min (50%-tile) for complete blood count and hemoglobin A1c, whereas those of endocrinology and tumor markers were 65 to 73 min. In conclusion, the tests determined by immunoassay are rate-limiting for rapid reporting efforts in clinical laboratories. Secondly, TATs of blood collection are mostly influenced by order waiting time depending on days of the week and accession time. At present, there is no target value for TATs, however it is important to recognize the necessity to shorten laboratory-spending TATs.

Nosocomial outbreak of multidrug-resistant USA300 methicillin-resistant Staphylococcus aureus causing severe furuncles and carbuncles in Japan.

USA300 methicillin-resistant Staphylococcus aureus (MRSA) has been attracting worldwide attention as a cause of community-associated MRSA (CA-MRSA) infections in the 21st century. Nosocomial outbreaks of CA-MRSA clones have been progressively more reported in Europe and the USA, but only one very recent report from Kyoto found in Japan. In February 2008, a severe MRSA infection occurred in one immunocompromised patient and three healthy medical staff members at the Department of Dermatology, Graduate School of Medicine, University of the Ryukyus. The epidemiological and clinical pattern of the infection prompted us to characterize the molecular features of the MRSA strain involved. The causative MRSA strain belonged to the multi-locus sequence type 8, staphylococcal cassette chromosome mec (SCCmec) type IVa, spa1 (alternatively t008), agr1 and coagulase type III, and carried the Panton-Valentine leukocidin (PVL) gene and the arginine catabolic mobile element. Pulsed-field gel electrophoresis analysis showed that the MRSA responsible for the outbreak was the USA300 clone. All of the isolated USA300 clones had multiple resistance against six non-ß-lactam antimicrobial drugs. We report here the first nosocomial outbreak of multidrug-resistant USA300 MRSA infections in Japan. This report shows that the USA300 clone can manifest severe skin infections such as furuncles and carbuncles even in healthy persons, which require drainage and i.v. treatment, and suggests that the clone can spread in hospital settings worldwide.

Comparison of drug sensitivity and genotypes of clinically isolated strains of levofloxacin-resistant Streptococcus pneumoniae obtained from Okinawa Island, the Japanese main island and Hong Kong.

The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is increasing worldwide. In the present study, a comparison of drug sensitivity and genotypes of clinically isolated strains of levofloxacin (LVFX)-resistant S. pneumoniae obtained from Hong Kong, Okinawa Island and the Japanese main island (Honshu) was performed. MICs of quinolones (LVFX, tosufloxacin, ciprofloxacin, gatifloxacin and sitafloxacin (STFX)) and other antibiotics (penicillin G, cefcapene, cefditoren, clarithromycin and azithromycin) were determined by a microdilution broth method according to the Clinical and Laboratory Standards Institute Standards. The quinolone-resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE of these strains were analyzed by PCR-based sequencing. All 40 strains tested had more than one amino-acid substitution in the QRDRs of gyrA, gyrB, parC or parE. Although there seemed to be some clonality in strains obtained from Hong Kong, there was no clonality in strains obtained from Okinawa and Japan. Strains obtained from Hong Kong, Okinawa Island and the Japanese main island were genetically different by pulsed-field gel electrophoresis analysis. The range of MIC values of STFX against isolates resistant to LVFX (MIC 4-32 mg l(-1)) was 0.12-0.5 mg l(-1), and MIC(80) values of STFX against LVFX-resistant isolates were 0.25 mg l(-1). This study suggests that LVFX-resistant S. pneumoniae is similar in all three locations and STFX is potent against LVFX-resistant S. pneumoniae with multiple mutations in QRDRs of gyrase A and topoisomerase IV.

[A simple disk diffusion test to identify beta-lactamase-negative, ampicillin-resistant Haemophilus influenzae--application of cephalexin, cefsulodin and cefaclor disks].

Currently, beta-lactamase-negative (BLN) ampicillin-resistant (AR) strains of Haemophilus influenzae are prevalent in Japan. BLNAR strains are defined by the presence of specific mutation(s) in the ftsI gene but are not phenotypically distinguishable by ampicillin (ABPC) susceptibility. In the present study, we showed that cephalexin (CEX), cefsulodin (CFS), and cefaclor (CCL) disk diffusion tests can be effectively used to identify BLNAR strains. A total of 169 clinical isolates of BLN H. influenzae, consisting of 113 of BLNAR and 56 of BLN, ampicillin-susceptible (AS), were included. All the isolates were genetically defined by detection of the TEM gene and partial sequencing of the ftsI gene. The Clinical and Laboratory Standards Institute (CLSI) standard broth microdilution and disk diffusion tests for ABPC provided 20% and 19% false susceptible rates, respectively. Alternatively, 34 cephem agents were tested using disk diffusion. Of the agents tested, CEX, CFS, and CCL disks could effectively discriminate between BLNAR and BLNAS isolates. All the BLNAS isolates showed visible growth inhibitory zones around CEX and CFS disks, but 108 (95.6%) and 106 (93.8%) BLNAR isolates did not. The results indicated 100% predictive values (PVs) for BLNAR and PVs for BLNAS were 91.8% for CEX and 88.9% for CFS. The CLSI-based interpretations for CCL (> or =20 mm) also highly correlated with BLNAR and BLNAS, PVs for BLNAR and for BLNAS being 100% and 93.3%, respectively. With simplicity and discriminability of the test method, we recommend a CEX disk diffusion test in combination with a rapid beta-lactamase test to identify BLNAR isolates in clinical laboratories.

[Identification and characterization of Panton-Valentine leukocidin-positive Staphylococcus aureus isolated in Okinawa, Japan].

We experienced hospital-acquired infection in March 2008 that three nurses became infected with Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA). Accordingly, we performed the retrospective study to determine the prevalence of PVL-positive S. aureus in Okinawa. A total of 731 clinical isolates, consisting of 600 MRSA and 131 methicillin-susceptible isolates in Okinawa, were included. Of the isolates, 16 were positive for PVL gene (lukS-PV-lukF-PV). All the PVL-positive isolates were MRSA, and the first appeared in March 2008. The isolates from the University Hospital were characterized as staphylococcal chromosomal cassette mec type IVa. Through the analysis of pulsed-field gel electrophoresis (PFGE), 16 PVL-positive MRSA isolates were divided in three groups. One isolate (the first group) from the other hospital was less similar (< 40% similarity) when compared with the remaining 15 isolates from the University Hospital. The second group consisted of two respective paired isolates from the same department wards, and those were very similar with each other, indicating possible patient-to-patient transmission. The 11 isolates were characterized as the third group with >80% similarity. The DiversiLab system (bioMérieux) based on repetitive-sequence-based PCR typing demonstrated that the isolates of the third group were similar and indistinguishable with the strains of USA300 clone. However, the first and second groups were not determinable which USA clone was the origin. With these, we could conclude that the PVL-positive MRSA close to USA300 clone first appeared in Okinawa in 2008 and is now becoming prevalent multi-focally. Also, person-to-person transmission is already likely in a hospital setting.

[Laboratory-based evaluation of loop-mediated isothermal amplification (LAMP) to detect Cryptosporidium oocyst and Giardia lamblia cyst in stool specimens].

To establish an alternative and more sensitive test method to detect oocyst of Cryptosporidium parvum and cyst of Giardia lamblia in clinical stool specimens, loop-mediated isothermal amplification (LAMP) was evaluated. Minimum cell concentrations at which LAMP assay could detect C. parvum oocyst and G. lamblia cyst were determined as 6.25 x 10(-1) and 3.12 x 10(-1) cells/assay when the stool specimens were spiked with the respective parasites. The results indicated 400 times higher sensitivities or more when compared to the microscopic readings. Twenty and nineteen diarrhea stool specimens spiked with C. parvum oocyst or G. lamblia cyst were assayed by LAMP. The results indicated that 14 (70%) and 16 (84%) samples successfully resulted in positive readings. But the remaining 6 and 3 samples were read as negative probably due to residual stool color. However, further dilutions of DNA extraction samples and addition of bovine serum albumin to LAMP reaction mixture showed positive effects on the occurrence of false-negative readings. With these results, we can conclude that the LAMP assay provides us an accurate and highly sensitive test method to detect C. parvum oocyst and cyst of G. lamblia, in place of labor-intensive and experience-dependent microscopic examination, in clinical laboratories.