PmrAB, the two-component system of Acinetobacter baumannii, controls the phosphoethanolamine modification of lipooligosaccharide in response to metal ions.

Acinetobacter baumannii is highly resistant to antimicrobial agents, and XDR strains have become widespread. A. baumannii has developed resistance to colistin, which is considered the last resort against XDR Gram-negative bacteria, mainly caused by lipooligosaccharide (LOS) phosphoethanolamine (pEtN) and/or galactosamine (GalN) modifications induced by mutations that activate the two-component system (TCS) pmrAB. Although PmrAB of A. baumannii has been recognized as a drug resistance factor, its function as TCS, including its regulatory genes and response factors, has not been fully elucidated. In this study, to clarify the function of PmrAB as TCS, we elucidated the regulatory genes (regulon) of PmrAB via transcriptome analysis using pmrAB-activated mutant strains. We discovered that PmrAB responds to low pH, Fe2+, Zn2+, and Al3+. A. baumannii selectively recognizes Fe2+ rather than Fe3+, and a novel region ExxxE, in addition to the ExxE motif sequence, is involved in the environmental response. Furthermore, PmrAB participates in the phosphoethanolamine modification of LOS on the bacterial surface in response to metal ions such as Al3+, contributing to the attenuation of Al3+ toxicity and development of resistance to colistin and polymyxin B in A. baumannii. This study demonstrates that PmrAB in A. baumannii not only regulates genes that play an important role in drug resistance but is also involved in responses to environmental stimuli such as metal ions and pH, and this stimulation induces LOS modification. This study reveals the importance of PmrAB in the environmental adaptation and antibacterial resistance emergence mechanisms of A. baumannii. IMPORTANCE: Antimicrobial resistance (AMR) is a pressing global issue in human health. Acinetobacter baumannii is notably high on the World Health Organization's list of bacteria for which new antimicrobial agents are urgently needed. Colistin is one of the last-resort drugs used against extensively drug-resistant (XDR) Gram-negative bacteria. However, A. baumannii has become increasingly resistant to colistin, primarily by modifying its lipooligosaccharide (LOS) via activating mutations in the two-component system (TCS) PmrAB. This study comprehensively elucidates the detailed mechanism of drug resistance of PmrAB in A. baumannii as well as its biological functions. Understanding the molecular biology of these molecules, which serve as drug resistance factors and are involved in environmental recognition mechanisms in bacteria, is crucial for developing fundamental solutions to the AMR problem.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

A Specific Strain of Lactic Acid Bacteria, Lactobacillus paracasei, Inhibits Inflammasome Activation In Vitro and Prevents Inflammation-Related Disorders.

Some strains of lactic acid bacteria (LAB) have anti-inflammatory effects, but the mechanism underlying the alleviation of inflammation by LAB is not fully understood. In this study, we examined the inhibitory effect of a certain strain of LAB, Lactobacillus paracasei, on inflammasome activation, which is associated with various inflammatory disorders. Using bone marrow-derived macrophages from BALB/c mice, we found that L. paracasei, but not L. rhamnosus, suppressed NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1ß secretion. L. paracasei also had inhibitory effects on AIM2 and NLRC4 inflammasome activation as well as the NLRP3 inflammasome. These inhibitory effects of L. paracasei on inflammasome activation were dependent on autocrine IL-10 induced by L. paracasei-stimulated macrophages. Furthermore, IL-10 production by L. paracasei-stimulated macrophages was involved with phagocytosis and the NOD2 signaling pathway in macrophages. In addition to in vitro studies, oral administration of L. paracasei in C57BL/6 mice reduced monosodium urate crystal-induced peritoneal inflammation in vivo. Moreover, continuous intake of L. paracasei in C57BL/6 mice alleviated high fat diet-induced insulin resistance and aging-induced expression of biomarkers for T cell senescence. Taken together, we demonstrated that L. paracasei inhibits inflammasome activation in vitro and exhibits an anti-inflammatory function in vivo. These results indicate that LAB that have inhibitory effects on inflammasome activation might contribute to the alleviation of inflammation-related disorders.

Evaluation of efflux pump inhibitors of MexAB- or MexXY-OprM in Pseudomonas aeruginosa using nucleic acid dyes.

BACKGROUND: Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes. OBJECTIVE: We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen. METHODS: Escherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine ß-naphthylamide [PAßN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]). RESULTS: Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAßN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used. CONCLUSIONS: Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.

Lactobacillus paracasei KW3110 Prevents Inflammatory-Stress-Induced Mitochondrial Dysfunction in Mouse Macrophages.

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects, including the prevention of blue light exposure induced retinal inflammation and ageing-related chronic inflammation in mice. The mechanism involves the promotion of anti-inflammatory cytokine interleukin (IL)-10 production by KW3110, leading to reduced pro-inflammatory cytokine IL-1ß production. Although various stress-induced mitochondrial damages are associated with excessive inflammatory responses, the effect of KW3110 on inflammatory-stress-induced mitochondrial damage remains unknown. In this study, we investigated the effect of KW3110 on inflammatory stress-induced mitochondrial damage using the murine macrophage-like cell line J774A.1. KW3110 treatment suppressed lipopolysaccharide (LPS)-induced mitochondrial dysfunction, including downregulation of membrane potential, induction of reactive oxygen species, and respiratory dysfunction. In addition, KW3110 prevented LPS-induced disruption of mitochondrial morphology including cristae structures. IL-10 treatment also ameliorated LPS-induced mitochondrial dysfunction and morphology disruption. These results suggest that KW3110 prevents LPS-induced mitochondrial dysfunction, potentially via promoting IL-10 production in mouse macrophages. We are the first to reveal a suppressive effect of lactic acid bacteria on mitochondrial morphology disruption in inflammatory-stressed macrophages. Our findings contribute to understanding inflammatory-stress-induced mitochondrial damage and developing food ingredients with preventive effects on mitochondrial-damage-derived inflammatory conditions.

Lactobacillus paracasei KW3110 Suppresses Inflammatory Stress-Induced Premature Cellular Senescence of Human Retinal Pigment Epithelium Cells and Reduces Ocular Disorders in Healthy Humans.

Lactobacillus paracasei KW3110 (KW3110) has anti-inflammatory effects and mitigates retinal pigment epithelium (RPE) cell damage caused by blue-light exposure. We investigated whether KW3110 suppresses chronic inflammatory stress-induced RPE cell damage by modulating immune cell activity and whether it improves ocular disorders in healthy humans. First, we showed that KW3110 treatment of mouse macrophages (J774A.1) produced significantly higher levels of interleukin-10 as compared with other lactic acid bacterium strains (all p < 0.01). Transferring supernatant from KW3110- and E. coli 0111:B4 strain and adenosine 5'-triphosphate (LPS/ATP)-stimulated J774A.1 cells to human retinal pigment epithelium (ARPE-19) cells suppressed senescence-associated phenotypes, including proliferation arrest, abnormal appearance, cell cycle arrest, and upregulation of cytokines, and also suppressed expression of tight junction molecule claudin-1. A randomized, double-blind, placebo-controlled parallel-group study of healthy subjects (n = 88; 35 to below 50 years) ingesting placebo or KW3110-containing supplements for 8 weeks showed that changes in critical flicker frequency, an indicator of eye fatigue, from the week-0 value were significantly larger in the KW3110 group at weeks 4 (p = 0.040) and 8 (p = 0.036). These results suggest that KW3110 protects ARPE-19 cells against premature senescence and aberrant expression of tight junction molecules caused by chronic inflammatory stress, and may improve chronic eye disorders including eye fatigue.

Spodiobacter cordis gen. nov. sp. nov., a member of the family Flavobacteriaceae isolated from patients with infective endocarditis.

Two gram-negative, catalase-negative, oxidase-positive strains (PAGU 1467T and PAGU 1468) isolated from patients with infective endocarditis were investigated to determine their taxonomic status. 16S rRNA gene sequence analysis indicated that the two strains were members of the Bergeyella-Chryseobacterium-Riemerella branch of the family Flavobacteriaceae. Strains PAGU 1467T and PAGU 1468 were highly related to each other (98.8% 16S rRNA gene sequence similarity). Phylogenetically closely-related species to PAGU 1467T comprised Bergeyella zoohelcum (95.0% 16S rRNA gene sequence similarity), Riemerella anatipestifer (94.3%) and Cloacibacterium normanense (94.3%). The major fatty acids of the two isolates were iso-C15:0 , iso-C17:0 3-OH and iso-C15:0 3-OH. The presence of C16:0 3-OH and iso-C15:0 2-OH allowed these isolates to be distinguished from B. zoohelcum. Menaquinone MK-6 was the only respiratory quinone in these organisms; this is a consistent characteristic of the family Flavobacteriaceae. The guanine-plus-cytosine content of the genomic DNA was 42.0%, which is higher than that of other close phylogenetic relatives. On the basis of their phenotypic properties and genetic distinctiveness, isolates PAGU 1467T and PAGU 1468 were classified within the novel genus Spodiobacter, as Spodiobacter cordis gen. nov., sp. nov., which is also the type species. The type strain of S. cordis is PAGU 1467T ( = CCUG 65564T = NBRC 109998T ).

Description of Paraclostridium bifermentans subsp. muricolitidis subsp. nov., emended description of Paraclostridium bifermentans (Sasi Jyothsna et al., 2016), and creation of Paraclostridium bifermentans subsp. bifermentans subsp. nov.

Taxonomic studies of strain PAGU 1678T , an obligately anaerobic, gram-positive, spore-forming bacterium isolated from biobreeding rat feces, were performed. This strain has been demonstrated to have the ability to exacerbate pathosis in a mouse model of dextran sulfate sodium-induced ulcerative colitis. Phylogenetic analysis based on the 16S rRNA gene showed high homology with Paraclostridium bifermentans. To clarify the correct taxonomic position of strain PAGU 1678T , a comparative taxonomic study using P. bifermentans PAGU 2008T (═JCM 1386T ) and the closely related bacterial species P. benzoelyticum PAGU 2068T (═LMG 28745T ) was carried out. Despite the close similarity of 16S rRNA gene sequences, DNA-DNA hybridization between strain PAGU 1678T and P. bifermentans PAGU 2008T was 60.03% on average, average nucleotide identity was 96.17%, and it was shown to have different genomic sequences. Biochemically, strain PAGU 1678T could be differentiated from P. bifermentans PAGU 2008T by H2 S production. Furthermore, strain PAGU 1678T was characterized by the presence of two phospholipids with different polarity on polar lipid analysis. In addition, strain PAGU 1678T differed from P. bifermentans PAGU 2008T in findings on whole-cell protein analysis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. On the basis of these biochemical and genetic characteristics, a novel subspecies of P. bifermentans with the name Paraclostridium bifermentans subsp. muricolitidis subsp. nov. is here proposed, with PAGU 1678T (═CCUG 72489T ═NBRC 113386T ) as the type strain, which automatically creates P. bifermentans subsp. bifermentans subsp. nov. JCM 1386T (═ATCC 638T ═DSM 14991T ).

Custom-made, antibiotic-loaded, acrylic cement spacers using a dental silicone template for treatment of infected hip prostheses.

PURPOSE: Antibiotic-loaded acrylic cement (ALAC) spacers are useful for treatment of infected prostheses in the course of a two-stage revision. Spacers are handmade or are made using a commercial template, with reportedly good treatment outcomes. This study aimed to confirm the usefulness of custom-made ALAC spacers shaped like bipolar hip prostheses using a dental silicone template for treatment of infected hip prostheses, and described their manufacture. METHODS: This study evaluated 10 patients who underwent two-stage revision for treatment of infected hip prostheses. Custom-made ALAC spacers were used in all patients. Templates were made with dental silicone. We investigated the following in treatment of the infected hip prostheses: bacterial pathogens; antibiotic-cement mixtures; waiting time to revision; dislocation, breakage, and migration of custom-made ALAC spacers; current hip status; progress during follow-up; presence or absence of recurrence; and walking ability. RESULTS: Dislocation, breakage, and migration were not observed in custom-made ALAC spacers. All patients recovered after two-stage revision without additional surgery and showed no recurrence during the follow-up period. CONCLUSION: Custom-made ALAC spacers shaped like bipolar hip prostheses using a template made of dental silicone may be useful for treatment of infected hip prostheses.

Blood metal ion concentrations in metal-on-metal total hip arthroplasty.

INTRODUCTION: The hip placement with a metal-on-metal (MOM) bearing has been used for both surface replacement and total hip arthroplasty (THA). Use of MOM bearing for hip replacement reduces the wear compared to conventional bearings. METHODS: We prospectively assessed 30 patients who underwent unilateral MOM THA. A control group of 30 patients who underwent metal-on-polyethylene THA using the implants as the other group, except for bearing, were accessed. Blood samples were collected preoperatively and at 3- , 6- , 9- , 12- , 15- , 18- , and 24-month intervals. Changes in mean blood metal ion concentration were compared between the MOM and metal-on-polyethylene groups. RESULTS: A statistically significant positive correlation was observed between blood cobalt and chromium concentrations in all of the patients. The mean blood ion concentrations of the MOM were significantly higher than those of the metal-on-polyethylene. A statistically significant negative correlation was found between maximum blood cobalt concentration and cup version angle. The maximum blood chromium concentrations in the patients who had larger cup version angles were more likely to decrease. CONCLUSIONS: We considered that cup version angle is one of the factors that have the greatest effect on blood metal ion concentration, and the target cup version angle that did not induce an increase in blood metal ion concentrations was approximately 20°.

Lung adenocarcinoma may be a more susceptive subtype to a dendritic cell-based cancer vaccine than other subtypes of non-small cell lung cancers: a multicenter retrospective analysis.

OBJECTIVE: The J-SICT DC Vaccine Study Group provides dendritic cell (DC) vaccines for compassionate use under unified cell production and patient treatment regimens. We previously reported beneficial effects of DC vaccines on the overall survival of 62 patients with advanced non-small cell lung cancer (NSCLC) in a single-center analysis. Here, we extended analysis to 260 patients with NSCLC who were treated at six centers. METHODS: Of the 337 patients who met the inclusion criteria, we analyzed 260 patients who received ≥5 peptide-pulsed DC vaccinations once every 2 weeks. RESULTS: The mean survival time (MST) from diagnosis was 33.0 months (95 % confidence interval [CI]: 27.9-39.2), and that from time of first vaccination was 13.8 months (95 % CI 11.4-16.8). An erythema reaction at the injection site that was ≥30 mm in diameter was correlated most strongly with overall survival from the first vaccine (≥30 vs. < 30 mm: MST 20.4 vs. 8.8 months, P < 0.001). We reported a similar finding in our previous analysis of patients with advanced pancreatic cancer. Interestingly, although such findings were common between patients with adenocarcinoma and those with other subtypes, the former group experienced significantly prolonged overall survival and a higher response rate for erythema (56.3 vs. 37.3 %, respectively, P = 0.014). CONCLUSIONS: This is the first multicenter study that suggests a possible clinical benefit of DC vaccines for patients with advanced NSCLC, especially those with adenocarcinoma. These findings suggest a specific potential responder population for DC vaccines and warrant further investigation in well-controlled prospective randomized trials.

Clinical and bacteriological characteristics of Helicobacter cinaedi infection.

Helicobacter cinaedi was first isolated from rectal cultures from homosexual men in 1984. In the 1980s to mid 1990s, the microorganism was mainly isolated from samples from homosexual men or immunocompromised patients; however, during the last two decades, H. cinaedi has been isolated from immunocompromised and from immunocompetent individuals worldwide. In Japan, the isolation of this microorganism was first reported in 2003. Since then, many cases have been reported in hospitals across the country. Despite many reports, the etiological properties and pathogenicity of H. cinaedi remain elusive; however, we are increasingly able to recognize some of the features and the clinical relevance of infection. In particular, a long incubation period is essential for detection in an automatic blood culture system and many of the recent isolates are resistant to both macrolides and quinolones. Furthermore, there is an association between infection and severe or chronic illnesses, such as meningitis or arteriosclerosis, in addition to mild diseases such as fever, abdominal pain, gastroenteritis, proctitis, diarrhea, erysipelas, cellulitis, arthritis, and bacteremia. In this review, we introduce the current knowledge and our latest findings relating to H. cinaedi.

Long-term clinical and radiographic results of cementless total hip arthroplasty for patients with rheumatoid arthritis: minimal 10-year follow-up.

OBJECTIVES: The aim of this study was to clarify the long-term clinical and radiographic results of cementless total hip arthroplasty (THA) for patients with rheumatoid arthritis (RA). METHODS: Twenty-eight total hip arthroplasties in 24 patients with a diagnosis of RA were performed from October 1992 to October 1996. All components were titanium alloy with a circumferential porous coating. Six patients (six hips) died before the 10-year follow-up, and one patient (one hip) was lost to follow-up, leaving 21 joints of 17 patients for review at a minimum 10-year follow-up after surgery. There were 3 men and 14 women with an average age of 55.0 years. The average duration of RA at the time of the operation was 12.6 years, and the average follow-up period was 12.2 years. We evaluated the Japanese Orthopaedic Association (JOA) hip scores, radiographic changes and survivor rates of components. RESULTS: Compared with the preoperative JOA hip scores, there was significant improvement in the postoperative scores. Spot welds consistent with bone ingrowth were identified in 95.0% of the femoral components. No femoral components showed radiographic loosening or required revision for aseptic loosening, but two acetabular revisions were performed because of aseptic loosening. The 14-year survivor rates of the stem and cup with the end point of loosening were 100% and 88.2%, respectively. CONCLUSIONS: Cementless THA with this component design in patients with RA appears to be a promising treatment.

Comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of Helicobacter cinaedi.

The aim of this study was to establish a broth microdilution method for antimicrobial susceptibility testing of Helicobacter cinaedi and to assess the prevalence and mechanisms of fluoroquinolone resistance in Japanese clinical isolates. A broth microdilution method using modified Levinthal broth was developed and compared with the agar dilution method for testing susceptibility to ampicillin, gentamicin, tetracycline and ciprofloxacin. The minimum inhibitory concentrations obtained by these two methods were almost the same for all the antibiotics tested, demonstrating the broth microdilution method to be a suitable and reliable technique for antimicrobial susceptibility testing. A broth microdilution method for antimicrobial susceptibility test for H. cinaedi was established. This method is expected to help improve treatment.

Volatile organic compounds from human skin as biomarkers of menstruation phase and severity of premenstrual syndrome: An exploratory pilot study.

Background and aim: Numerous women of reproductive age experience physical or mental discomfort during their natural menstrual cycle due to paramenstrual symptoms, such as premenstrual syndrome (PMS). To date, there is no established biomarker for the diagnosis of PMS. This study investigated the relationship between skin gas composition and menstruation cycles, and evaluated the possibility of skin gas composition as a biomarker of paramenstrual symptoms. Methods: We conducted an exploratory pilot study. Healthy Japanese women (aged 20-29 years) underwent blood and skin gas analyses on 1 day corresponding to menstruation, preovulatory, middle luteal, and late luteal phases. Skin gas was collected from the cubital fossa and armpit using a Passive Flux Sampler; samples were analyzed for 65 volatile organic compounds (VOCs) by gas chromatography-mass spectrometry (GC-MS). Non-parametric statistical analysis was performed to identify VOCs related to the menstrual cycle, levels of female hormones, and severity of PMS. Results: Fourteen women participated; of those, 12 completed the study. Regarding the relationship with the menstrual cycles, seven and four VOCs were significantly and marginally changed, respectively, at the cubital fossa during menstruation. Of those 11 compounds, 10 were also correlated with the levels of serum female hormones. At the armpit, five and three compounds were significantly and marginally changed, respectively, during menstruation. Of those eight compounds, five were also correlated with the levels of serum female hormones. In the study of PMS severity, analysis of the changes in VOCs suggested that ketones and fatty acids are increased during menstruation in the severe PMS group versus the mild PMS group. Conclusions: The results of this study suggest that certain VOCs emitted in skin gas related to the menstrual cycle, levels of female hormones, and severity of PMS. These findings may advance the metabolic understanding and development of diagnostic biomarkers for menstruation-related symptoms.

Complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia.

We report the complete genome sequence of Helicobacter cinaedi strain PAGU611, isolated in a case of human bacteremia. The PAGU611 genome comprises a 2,078,348-bp chromosome and a 23,054-bp plasmid. The chromosome contains a unique genomic island, encoding a type VI secretion system and clustered regularly interspaced short palindromic repeat (CRISPR) loci.

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7.

The multiresistant taxonomic outlier Pseudomonas aeruginosa PA7 possesses the conserved efflux genes, mexXY; however these are linked to a unique gene encoding an outer membrane channel, dubbed oprA, that is absent in most P. aeruginosa strains. Using genetic knockouts and single copy chromosomal complementation, we showed that aminoglycoside resistance in PA7 is mediated in part by the MexXY-OprA pump, and intriguingly that MexXY in this strain can utilize either the OprA or OprM outer membrane channel, linked to the mexAB efflux genes. We also identified a small portion of the oprA gene immediately downstream of the mexY gene in PAO1, suggesting that non-PA7 P. aeruginosa strains might have possessed, but lost, the intact mexXY-oprA efflux pump locus. Consistent with this, most of a panel of serotype strains possessed the truncated oprA but the serotype O12 isolate had an intact mexXY-oprA locus, similar to PA7 and the related strain DSM 1128. We also showed that the mexZ repressor gene upstream of mexXY-oprA in PA7 is mutated, leading to overexpression of mexXY-oprA, using sequencing, homologous replacement and real-time quantitative reverse transcriptase PCR. Finally we assessed the contribution of MexXY and aminoglycoside modifying enzymes AAC together to resistance in PA7 and the AAC(6')-Iae-mediated amikacin-resistant clinical isolate IMCJ2.S1, concluding that the effect of the modifying enzymes is enhanced by functional efflux, especially in the presence of divalent cations, to develop high-level aminoglycoside resistance in P. aeruginosa.

Genus Enhydrobacter Staley et al. 1987 should be recognized as a member of the family Rhodospirillaceae within the class Alphaproteobacteria.

The genus Enhydrobacter, first reported as a member of the family Vibrionaceae, has been placed in the family Moraxellaceae, but as a genus incertae sedis in Bergey's Manual of Systematic Bacteriology 2nd edition. During our taxonomic investigation of Enhydrobacter-like organisms, we observed that the 16S rRNA sequences of E. aerosaccus-type strain versions NCIMB 12535(T) , ATCC 27094( T) and CCUG 58314(T) were very different from the accessible data (accession no. AJ550856). Phylogenetic analysis of our 16S rRNA sequence data revealed that these organisms were located within the family Rhodospirillaceae. The genera Inquilinus, Oceanibaculum, Skermanella and Nisaea were closely related (sequence similarities were 88.3~87.0%), but Enhydrobacter could be distinguished from these genera by growth characteristics, fatty acid profiles (C(19:0) cyclo ω8c; 38.4% C(18:1) ω7c; 32.2%, and C(16:0) ; 8.9% were major components), in being non-flagellated, and differing in enzymatic activities, including trypsin and ß-glucosidase. From these data, we conclude that the genus Enhydrobacter should be recognized as an independent genus of the family Rhodospirillaceae within the class Alphaproteobacteria.

Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein.

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.