An epidemiological study of the susceptibility and frequency of multiple-drug-resistant strains of Pseudomonas aeruginosa isolated at medical institutes nationwide in Japan

The susceptibility of 3233 strains of Pseudomonas aeruginosa, isolated primarily in 2001, as agents of infection at 37 medical institutes with various specialties in seven regions of Japan (ranging from Hokkaido to Kyushu/Okinawa), to 18 antipseudomonal agents known to be active against P. aeruginosa was evaluated, in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Of the 18 antipseudomonal agents, including some combinations of -lactamase inhibitors and antibacterial agents, ciprofloxacin had the lowest minimum inhibitory concentration (MIC)₅₀ (0.25µg/ml) against P. aeruginosa, followed by meropenem, with an MIC₅₀ of 0.5µg/ml. The MIC₅₀ of 7 of the examined antibacterial agents (ceftazidime, cefozopran, imipenem, biapenem, gentamicin, tobramycin, and levofloxacin) was between 1 and 2µg/ml. Among the antipseudomonal agents tested, tobramycin showed the lowest MIC₉₀ (2µg/ml), which was not significantly different from its MIC₅₀ (1µg/ml). The MIC₉₀ of the other antibacterial agents examined ranged from 8 to 64µg/ml and more. The susceptibility of the 3233 strains to the 12 antibacterial agents covered by the NCCLS guidelines was determined according to the standard method of the NCCLS guidelines. The frequency of strains resistant to meropenem, gentamicin, or tobramycin was relatively low (7.5%–8.3%). The frequency of strains showing intermediate to severe resistance to tobramycin was particularly low (8.0%). The frequency of strains resistant to aztreonam, imipenem, or levofloxacin was 16.7%–19.0%, about twice as high as the frequency of strains resistant to tobramycin. The susceptibility pattern of the 3233 strains (isolated from seven regions of Japan) to five antibacterial agents (ceftazidime, piperacillin, imipenem, gentamicin, and ciprofloxacin) was evaluated in relation to the regions from which they were isolated. The MIC₅₀ values of these antibacterial agents did not differ significantly among the regions. However, the MIC₉₀ values of ceftazidime and gentamicin were higher for strains isolated from the Kansai region than for strains isolated from other regions. The MIC₉₀ of ciprofloxacin was higher for strains isolated from the Tohoku, Kansai, and Kyushu/Okinawa regions than for strains isolated from other regions. Of the 3233 strains, 89 were classified as multiple-drug-resistant (imipenem, gentamicin, and ciprofloxacin) strains. Of these 89 strains, 42 were isolated from urine, 17 from sputum or pharyngeal mucus, 13 from pus, 8 from blood, 1 from cerebrospinal fluid, and 8 from other specimens. The frequency of multiple-drug-resistant strains was higher among strains isolated from the Tohoku and Kansai regions than in strains isolated from other regions.     

Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase blaCMY-4 promoted by insertion sequence ISEcp1

A Klebsiella pneumoniae strain, KU6500, which showed resistance to extended-spectrum β-lactams and produced the plasmid-encoded AmpC β-lactamase CMY-4, was identified from clinical isolates in Japan. The aim of this study was to identify the mechanism of the high-level expression of bla _CMY-4. Sequence analysis indicated that the promoter element of Citrobacter freundii was conserved, but the insertion sequence ISEcp1 coding with the putative promoter element, was inserted into the AmpR binding site. We determined the influence of the promoter on bla _CMY-4 expression and β-lactam resistance. Two recombinant plasmids containing the entire bla _CMY-4 gene, with or without the ISEcp1-mediated promoter sequences, were constructed and named pMWampC and pMWISEcp1, respectively. Escherichia coli DH5α (pMWISEcp1) was resistant to almost all β-lactams tested and E. coli DH5α (pMWampC) was susceptible to all, except for cephalothin. In addition, the activity of each promoter was measured by subcloning the element into a promoterless luciferase plasmid pGL3-Basic vector. The expression of the putative promoter of ISEcp1 was 18.9-fold higher than that of C. freundii. These results suggest that the putative promoter element of ISEcp1 is necessary for the high-level expression of bla _CMY-4 to confer resistance to extended-spectrum cephalosporins.     

New erm Gene in Staphylococcus aureus clinical isolates.

We have identified erm(Y), a novel gene class that was originally designated ermGM, from a Staphylococcus aureus strain that has a plasmid that also harbors msr(A) and mph(C), genes that encode an efflux mechanism and a putative phosphorylase, respectively. The nucleotide and deduced amino acid sequences of erm(Y) were 81 and 76% identical to those of erm(T), respectively.     

Usefulness of a novel system for measuring glucose area under the curve while screening for glucose intolerance in outpatients

Aims/Introduction

To realize the effectiveness of a novel system for measuring glucose area under the curve (AUC) using minimally invasive interstitial fluid extraction technology (MIET), outpatients undergoing oral glucose tolerance tests (OGTT) were investigated for the efficacy of screening for glucose intolerance using this system.

Materials and Methods

Fifty outpatients scheduled to undergo a 75‐g OGTT for medical reasons were recruited to the study. An area of skin on the forearm was pretreated with microneedle arrays before the application of hydrogels for interstitial fluid extraction. Plasma glucose (PG) levels were measured every 30 min for 2 h to calculate reference (actual) AUC. The AUC was predicted by MIET on the basis of glucose extracted by the hydrogel using sodium ion levels as the internal standard.

Results

Good correlation between MIET‐predicted and reference AUCs obtained using PG levels was confirmed for a wide AUC range. By introducing a threshold level for AUC to separate glucose intolerance with peak glucose ≥180 mg/dL from normal glucose tolerance, the system was demonstrated to provide better screening accuracy compared with conventional methods that use HbA1c and fasting PG levels. The results of a questionnaire‐based survey administered to the subjects suggested that this system was readily accepted by the majority as a painless monitoring method.

Conclusions

The findings suggest that our glucose AUC measurement system using MIET would be useful for screening of glucose intolerance. In the future, this system may prove to be a useful aid as a screen for glucose intolerance before performing an OGTT for diagnosis.     

Double-patch technique through right ventriculotomy for residual shunt after infarct exclusion technique for post-infarction ventricular septal perforation

We report a case in which the double-patch technique was used to repair residual shunt after infarct exclusion technique for post-infarction ventricular septal perforation (VSP). A 68-year-old man was diagnosed with post-infarction VSP and surgical interveniton was performed by infarct exclusion technique through left ventriculotomy. Residual shunt was observed by left ventriculography, and he developed cardiac failure in spite of medical therapy. Additional surgery for residual shunt was performed 5 months after the initial operation. The VSP was closed with 2 pericardial patches onto both sides of the septum through right ventriculotomy. Complete closure of the defect was accomplished, and no residual shunt was observed by post-operative left ventriculography. The patient has been doing well with no signs of cardiac failure.     

Cytotoxic T lymphocytes efficiently recognize human colon cancer stem-like cells

Cancer stem-like cells (CSCs) and tumor-initiating cells (TICs) are a small population of cancer cells that share three properties: tumor initiating ability, self-renewal, and differentiation. These properties suggest that CSCs/TICs are essential for tumor maintenance, recurrence, and distant metastasis. Here, we show that cytotoxic T lymphocytes (CTLs) specific for the tumor-associated antigen CEP55 can efficiently recognize colon CSCs/TICs both in vitro and in vivo. Using Hoechst 33342 dye staining, we isolated CSCs/TICs as side population (SP) cells from colon cancer cell lines SW480, HT29, and HCT15. The SP cells expressed high levels of the stem cell markers SOX2, POU5F1, LGR5, and ALDH1A1 and showed resistance to chemotherapeutic agents such as irinotecan or etoposide.To evaluate the susceptibility of SP cells to CTLs, we used CTL clone 41, which is specific for the CEP55-derived antigenic peptide Cep55/c10orf3_193 (10) (VYVKGLLAKI). The SP cells expressed HLA class I and CEP55 at the same level as the main population cells. The SP cells were susceptible to CTL clone 41 at the same level as main population cells. Furthermore, adoptive transfer of CTL clone 41 inhibited tumor growth of SW480 SP cells in vivo. These observations suggest that Cep55/c10orf3_193(10) peptide-based cancer vaccine therapy or adoptive cell transfer of the CTL clone is a possible approach for targeting chemotherapy-resistant colon CSCs/TICs.