Changes in the expression of mexB, mexY, and oprD in clinical Pseudomonas aeruginosa isolates.

Changes in expression levels of drug efflux pump genes, mexB and mexY, and porin gene oprD in Pseudomonas aeruginosa were investigated in this study. Fifty-five multidrug-resistant P. aeruginosa (MDRP) strains were compared with 26 drug-sensitive strains and 21 strains resistant to a single antibiotic. The effect of the efflux inhibitor Phe-Arg-ß-naphthylamide on drug susceptibility was determined, and gene expression was quantified using real-time quantitative real-time reverse transcription polymerase chain reaction. In addition, the levels of metallo-ß-lactamase (MBL) and 6'-N-aminoglycoside acetyltransferase [AAC(6')-Iae] were investigated. Efflux pump inhibitor treatment increased the sensitivity to ciprofloxacin, aztreonam, and imipenem in 71%, 73%, and 29% of MDRPs, respectively. MBL and AAC(6')-Iae were detected in 38 (69%) and 34 (62%) MDRP strains, respectively. Meanwhile, 76% of MDRP strains exhibited more than 8-fold higher mexY expression than the reference strain PAO1. Furthermore, 69% of MDRP strains expressed oprD at levels less than 0.01-fold of those in PAO1. These findings indicated that efflux pump inhibitors in combination with ciprofloxacin or aztreonam might aid in treating MDRP infections.

Novel approach for rapid detection of extended spectrum ß-lactamase and metalloid-ß-lactamase using drug susceptibility testing microfluidic device (DSTM).

BACKGROUND/PURPOSE: Rapid detection of ß-lactamases is important in a recent situation where resistant bacteria are increasing. By using the drug susceptibility testing microfluidic device (DSTM), rapid screening of extended spectrum ß-lactamases (ESBLs) and metallo-ß-lactamases (MBLs) has become possible. METHODS: ß-lactams and ß-lactamase inhibitors were pre-fixed in the DSTM for use. A bacterial suspension in Mueller-Hinton broth (McF 0.25) was introduced into the device, and the effects of ß-lactamase inhibitor on morphological changes caused by ß-lactam were evaluated after 3 h incubation. RESULTS: Clinical isolates genetically confirmed to produce ß-lactamase were used. Of the 84 ESBL-producing strains, 80 strains (95%) turned to be ESBL positive, and five strains (6%) of them MBL were positive as well as ESBL. Four strains (5%) were negative for both ESBL and MBL. Of the 24 MBL-producing strains, 23 strains (96%) were positive for MBL. All the 43 AmpC-producing strains were negative for both ESBL and MBL. Of the 156 ESBL- and MBL-nonproducing strains, 155 strains (99%) were negative for both ESBL and MBL, and one strain was positive for ESBL. With this method, the detection sensitivity was 95% and the specificity was 100% for ESBL, whereas the detection sensitivity was 96% and the specificity was 98% for MBL. These results were not significantly different from the results of the disc diffusion method. CONCLUSION: The DSTM method allows rapid detection of ß-lactamases in 3 h and may be a useful replacement for the disc diffusion method.

Direct antibiotic susceptibility testing of blood cultures of gram-negative bacilli using the Drug Susceptibility Testing Microfluidic (DSTM) device.

Proper treatment of bloodstream infections requires rapid, early determination of appropriate antibiotic agents, emphasizing the need for more rapid drug susceptibility testing. The Drug Susceptibility Testing Microfluidic (DSTM) device represents a novel method in which a small amount of bacterial suspension is injected into the microchip-like device and cultured for 3 h. However, it remains unknown whether the DSTM method can directly determine antibiotic susceptibilities from positive blood cultures. Here, we developed a new approach to directly assess drug susceptibility, using the DSTM method for positive blood cultures. We compare the utility and accuracy of DSTM with those of conventional susceptibility testing methods. Fifty positive blood cultures identified as gram-negative bacilli were used herein. The outcomes of drug susceptibility and resistance assays for positive blood cultures were compared to those of conventional susceptibility testing methods to evaluate their utility and accuracy. Method agreement rates between DSTM and standard methods often exceed 90%, suggesting a high positive correlation with conventional methods. Furthermore, our results show that a combination of multiple drugs in the DSTM device helps identify extended-spectrum ß-lactamase (ESBL)- and AmpC-ß-lactamase (AmpC-)-producing microorganisms. In conclusion, DSTM method enables effective drug susceptibility and resistance screening within 3 h from positive blood cultures and is suitable for the rapid and personalized determination of the antimicrobial regimen.

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Expression, purification, crystallization and preliminary crystallographic study of FtsA from methicillin-resistant Staphylococcus aureus.

FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with ß-γ-imidoadenosine 5'-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, ß = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers.

FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo.

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.

The Effects of a 5-Year Physical Exercise Intervention with Music in Community- Dwelling Normal Elderly People: The Mihama-Kiho Follow-Up Project.

BACKGROUND: We previously reported the enhanced effects of physical exercise when combined with music (ExM) on cognitive function in community-dwelling normal elderly people compared to exercise alone. Following that study, participants voluntarily continued the ExM classes for 5 years. OBJECTIVE: To identify the effects of a 5-year ExM intervention on cognitive function in normal elderly people. METHODS: Fifty-four subjects continued the ExM classes once a week for 5 years (ExM group). Thirty-three subjects retired from the ExM class during the 5 years (Retired group). Twenty-one subjects never participated in any intervention over the 5 years (No-exercise group). Cognitive function and ADLs were assessed using neuropsychological batteries and the functional independence measure (FIM), respectively. The voxel-based specific regional analysis system for Alzheimer's disease (VSRAD) was used to investigate medial temporal lobe atrophy. RESULTS: Analyses of the raw scores after the 5-year intervention showed significant differences between the ExM and No-exercise groups in their MMSE scores, Raven's colored progressive matrices (RCPM) time, logical memory (LM)-I, as well as the total and physical exercise sub-scores of the FIM. Analysis of subjects aged 70- 79 years at the beginning of this project showed significantly quicker performance on the RCPM in the ExM compared to No-exercise groups. The correlation coefficients between the total number of ExM sessions attended and the degree of changes in physical, neuropsychological, and VSRAD scores were significant for RCPM performance time and LM-I scores. CONCLUSION: Long-term ExM intervention reinforces multifaceted cognitive function in normal elderly people, and is especially beneficial for psychomotor speed.

Double vascular ring: a case report of double aortic arch and concurrent pulmonary artery sling.

BACKGROUND: Double aortic arch (DAA) and pulmonary artery sling (PAS) are vascular ring formations that present in neonates and infants with symptoms of respiratory stenosis. CASE SUMMARY: The patient was a girl with suspected ventricular septal defect (VSD), right aortic arch (AA), left patent ductus arteriosus, and bilateral superior vena cava (SVC) on foetal echography in the first day of life. The girl was delivered at 40 weeks and 4 days of gestation. Ventricular septal defect, DAA, coarctation of the left AA, and bilateral SVC were diagnosed. Contrast-enhanced computed tomography at Day 16 revealed PAS with concurrent anomalous tracheal branching in addition to DAA. The right A2 segmental artery, which supplies the right upper pulmonary artery, showed abnormal branching from the left pulmonary artery (LPA). At 3 months of age, VSD patching, left AA resection distal to the root of the left subclavian artery, arterial ligament dissection, and LPA replacement were performed. DISCUSSION: Pulmonary artery sling coexists with anomalous branching of the trachea and abnormal branching of the right pulmonary artery (RPA). Our patient had an extremely rare case of DAA concurrent with PAS and presented with anomalous tracheal and RPA branching. We were concerned that increased pulmonary blood flow caused by the VSD would exacerbate tracheal displacement. Radical surgery at 3 months of age resulted in good postoperative progress.

Large-Scale Femtoliter Droplet Array for Single Cell Efflux Assay of Bacteria.

Large-scale femtoliter droplet array as a platform for single cell efflux assay of bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single bacterial cells, fluorescence-based detection of efflux activity at the single cell level, and collection of single cells from droplet and subsequent gene analysis are described in detail.

Single-Cell Detection and Collection of Persister Bacteria in a Directly Accessible Femtoliter Droplet Array.

A directly accessible femtoliter droplet array as a platform for single-cell detection and collection of persister bacteria is described. Device microfabrication, femtoliter droplet array formation and concomitant enclosure of single cells, long-term culture and observation of single cells in droplets, and collection of identified persisters from single droplets are described in detail.

A Microfluidic Channel Method for Rapid Drug-Susceptibility Testing of Pseudomonas aeruginosa.

The recent global increase in the prevalence of antibiotic-resistant bacteria and lack of development of new therapeutic agents emphasize the importance of selecting appropriate antimicrobials for the treatment of infections. However, to date, the development of completely accelerated drug susceptibility testing methods has not been achieved despite the availability of a rapid identification method. We proposed an innovative rapid method for drug susceptibility testing for Pseudomonas aeruginosa that provides results within 3 h. The drug susceptibility testing microfluidic (DSTM) device was prepared using soft lithography. It consisted of five sets of four microfluidic channels sharing one inlet slot, and the four channels are gathered in a small area, permitting simultaneous microscopic observation. Antimicrobials were pre-introduced into each channel and dried before use. Bacterial suspensions in cation-adjusted Mueller-Hinton broth were introduced from the inlet slot and incubated for 3 h. Susceptibilities were microscopically evaluated on the basis of differences in cell numbers and shapes between drug-treated and control cells, using dedicated software. The results of 101 clinically isolated strains of P. aeruginosa obtained using the DSTM method strongly correlated with results obtained using the ordinary microbroth dilution method. Ciprofloxacin, meropenem, ceftazidime, and piperacillin caused elongation in susceptible cells, while meropenem also induced spheroplast and bulge formation. Morphological observation could alternatively be used to determine the susceptibility of P. aeruginosa to these drugs, although amikacin had little effect on cell shape. The rapid determination of bacterial drug susceptibility using the DSTM method could also be applicable to other pathogenic species, and it could easily be introduced into clinical laboratories without the need for expensive instrumentation.

Anti-Helicobacter pylori agents. 5. 2-(Substituted guanidino)-4-arylthiazoles and aryloxazole analogues.

To extend the SAR study of guanidinothiazoles as a structurally novel class of anti-H. pylori agents, a series of 2-(substituted guanidino)-4-arylthiazoles and some 4-aryloxazole analogues were synthesized and evaluated for antimicrobial activity against H. pylori. Some of them were also subjected to H2 antagonist and gastric antisecretory assays. Several arylthiazoles were identified as potent anti-H. pylori agents, and of these, thienylthiazole derivative 44 exhibited the strongest activity (MIC = 0.0065 microg/mL) among the compounds obtained in our guanidinothiazole studies. Although 44 was void of H2 antagonist activity, pyridylthiazole derivative 39 had both potent anti-H. pylori and H2 antagonist activities. Thiazolylthiazole derivative 46 also showed potent anti-H. pylori activity, but the H2 antagonist activity was weak. On the other hand, no attractive activities were found in pyrimidyl, oxazolyl, isoxazolyl, imidazolyl, and oxadiazolylthiazole derivatives. The anti-H. pylori activity of the aryloxazole analogues was weaker than those of the corresponding arylthiazole derivatives, though they had potent H2 antagonist activity.

[Adult sacrococcygeal teratoma: a case report].

We report a case of adult sacrococcygeal teratoma resected by an abdominosacral approach. A cystic mass 13 cm in diameter in the pelvic cavity and left hydronephrosis were detected incidentally by abdominal computed tomographic (CT) scan in a 55-year-old man. The pelvic tumor extending from the presacral area to the coccyx was resected via a combined abdominal and transsacral approach. The resected specimen weighed 700 g and the pathological diagnosis was mature teratoma. While the sacrococcygeal area is the most frequent site of teratoma in infants, it is a rare site in adults. This is to our knowledge, the 30th case report of adult sacrococcygeal teratoma in Japan. At one month after the operation the patient had no bowel dysfunction and no dysbasia, but he had mild bladder dysfunction requiring self-catheterization twice a day at twelve months. The patient had no evidence of disease at twenty months after the operation.

Crystal structure of FtsA from Staphylococcus aureus.

The bacterial cell-division protein FtsA anchors FtsZ to the cytoplasmic membrane. But how FtsA and FtsZ interact during membrane division remains obscure. We have solved 2.2 Å resolution crystal structure for FtsA from Staphylococcus aureus. In the crystals, SaFtsA molecules within the dimer units are twisted, in contrast to the straight filament of FtsA from Thermotoga maritima, and the half of S12-S13 hairpin regions are disordered. We confirmed that SaFtsZ and SaFtsA associate in vitro, and found that SaFtsZ GTPase activity is enhanced by interaction with SaFtsA.

Design of a large-scale femtoliter droplet array for single-cell analysis of drug-tolerant and drug-resistant bacteria.

Single-cell analysis is a powerful method to assess the heterogeneity among individual cells, enabling the identification of very rare cells with properties that differ from those of the majority. In this Methods Article, we describe the use of a large-scale femtoliter droplet array to enclose, isolate, and analyze individual bacterial cells. As a first example, we describe the single-cell detection of drug-tolerant persisters of Pseudomonas aeruginosa treated with the antibiotic carbenicillin. As a second example, this method was applied to the single-cell evaluation of drug efflux activity, which causes acquired antibiotic resistance of bacteria. The activity of the MexAB-OprM multidrug efflux pump system from Pseudomonas aeruginosa was expressed in Escherichia coli and the effect of an inhibitor D13-9001 were assessed at the single cell level.

Gallated form of tea catechin, not nongallated form, increases fecal starch excretion in rats.

This study was carried out to elucidate the structural advantage of a gallated form of tea catechin on modulating bioavailability of dietary starch in rats. Animal studies demonstrated that the addition of 0.5% (w/w) (-)-epigallocatechin gallate (EGCG) to the diet brought about a significant increase in the starch content in the feces collected for 2 d at the fourth week of feeding over that with the control diet. Of the gross starch that the rats consumed from their respective diets during the fecal collection period, 0.1% (for control diet) and 1.9% (for EGCG diet) were estimated to be excreted in the feces. However, such a significant increase in the fecal excretion of starch by the EGCG diet was lost by undergoing hydrolysis of EGCG to (-)-epigallocatechin (EGC) and gallic acid (GA). In vitro investigation also showed that EGCG inhibited porcine pancreatic α-amylase activity in a concentration-dependent fashion, whereas the hydrolyzed preparation (the mixture of EGC and GA) exhibited a lack of the inhibitory activity for α-amylase. The modification of dietary starch digestion by inhibiting intestinal α-amylase activity with EGCG may be responsible at least in part for increasing fecal output of starch in rats. Thus, the attachment of a galloyl moiety to the tea flavan-3-ol skeleton may be of key importance for reducing intestinal digestion of dietary starch in rats.

A microfluidic device for simple and rapid evaluation of multidrug efflux pump inhibitors.

Recently, multidrug-resistant pathogens have disseminated widely owing essentially to their increased multidrug efflux pump activity. Presently, there is a scarcity of new antibacterial agents, and hence, inhibitors of multidrug efflux pumps belonging to the resistance-nodulation-cell division (RND) family appear useful in the treatment of infections by multidrug-resistant pathogens. Moreover, recent progress in microfabrication technologies has expanded the application of nano/micro-devices to the field of human healthcare, such as the detection of infections and diagnosis of diseases. We developed a microfluidic channel device for a simple and rapid evaluation of bacterial drug efflux activity. By combining the microfluidic device with a fluorogenic compound, fluorescein-di-ß-D-galactopyranoside, which is hydrolyzed to a fluorescent dye in the cytoplasm of Escherichia coli, we successfully evaluated the effects of inhibitors on the RND-type multidrug efflux pumps MexAB-OprM and MexXY-OprM from Pseudomonas aeruginosa in E. coli. Our new method successfully detected the MexB-specific inhibitory effect of D13-9001 and revealed an unexpected membrane-permeabilizing effect of Phe-Arg-ß-naphthylamide, which has long been used as an efflux pump inhibitor.

Antibacterial and antifungal activities of new acylated derivatives of epigallocatechin gallate.

(-)-Epigallocatechin-3-O-gallate (EGCG) has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori et al. (2008) found that addition of long acyl chains (C16-18) to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal MICs of the derivatives were also two to fourfold lower than those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate and palmitate (C16) for 17 Staphylococcus aureus strains were 4-32 µg/ml, although MIC of EGCG for these 17 strains was ≥128 µg/ml. C16 demonstrated rapid bactericidal activity against methicillin-resistant S. aureus (MRSA) ATCC43300 at ≥16 µg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane-permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported in Escherichia coli using the efflux pump AcrAB-TolC. The tolC deletion mutant exhibited higher sensitivity to EGCG and the derivatives than wild-type. Addition of long alkyl chains to EGCG significantly enhanced its activities against several bacteria and fungi, particularly against S. aureus including MRSA. C16 might potentially become under specified circumstances an alternative or supplement to antibiotics and disinfectants in the future.

Evaluation of multidrug efflux pump inhibitors by a new method using microfluidic channels.

Fluorescein-di-ß-D-galactopyranoside (FDG), a fluorogenic compound, is hydrolyzed by ß-galactosidase in the cytoplasm of Escherichia coli to produce a fluorescent dye, fluorescein. We found that both FDG and fluorescein were substrates of efflux pumps, and have developed a new method to evaluate efflux-inhibitory activities in E. coli using FDG and a microfluidic channel device. We used E. coli MG1655 wild-type, ΔacrB (ΔB), ΔtolC (ΔC) and ΔacrBΔtolC (ΔBC) harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of Pseudomonas aeruginosa. Two inhibitors, MexB-specific pyridopyrimidine (D13-9001) and non-specific Phe-Arg-ß-naphthylamide (PAßN) were evaluated. The effects of inhibitors on pumps were observed using the microfluidic channel device under a fluorescence microscope. AcrAB-TolC and analogous pumps effectively prevented FDG influx in wild-type cells, resulting in no fluorescence. In contrast, ΔB or ΔC easily imported and hydrolyzed FDG to fluorescein, which was exported by residual pumps in ΔB. Consequently, fluorescent medium in ΔB and fluorescent cells of ΔC and ΔBC were observed in the microfluidic channels. D13-9001 substantially increased fluorescent cell number in ΔBC/pABM but not in ΔBC/pXYM. PAßN increased medium fluorescence in all strains, especially in the pump deletion mutants, and caused fluorescein accumulation to disappear in ΔC. The checkerboard method revealed that D13-9001 acts synergistically with aztreonam, ciprofloxacin, and erythromycin only against the MexAB-OprM producer (ΔBC/pABM), and PAßN acts synergistically, especially with erythromycin, in all strains including the pump deletion mutants. The results obtained from PAßN were similar to the results from membrane permeabilizer, polymyxin B or polymyxin B nonapeptide by concentration. The new method clarified that D13-9001 specifically inhibited MexAB-OprM in contrast to PAßN, which appeared to be a substrate of the pumps and permeabilized the membranes in E. coli.

Studies on anti-Helicobacter pylori agents. Part 3: A novel, efficacious cephem derivative, FR193879.

The synthesis, therapeutic efficacy against H. pylori, and preliminary safety of the novel cephem derivative, FR193879 (8a) are described. Compound 8a having a (4-carbamoylmethylthiazol-2-yl)thio moiety at the 3-position and a phenylacetamido at the 7-position was found to have good safety showing a nontoxic dose of > 100 mg/kg in dogs in a 4-week repeat dose toxicity study and extremely potent therapeutic efficacy against H. pylori, showing 30 times superior activity compared to AMPC, and did not display cross-resistance with CAM or MNZ.