Intestinal Bacterial Translocation Contributes to Diabetic Kidney Disease.

BACKGROUND: In recent years, many studies have focused on the intestinal environment to elucidate pathogenesis of various diseases, including kidney diseases. Impairment of the intestinal barrier function, the "leaky gut," reportedly contributes to pathologic processes in some disorders. Mitochondrial antiviral signaling protein (MAVS), a component of innate immunity, maintains intestinal integrity. The effects of disrupted intestinal homeostasis associated with MAVS signaling in diabetic kidney disease remains unclear. METHODS: To evaluate the contribution of intestinal barrier impairment to kidney injury under diabetic conditions, we induced diabetic kidney disease in wild-type and MAVS knockout mice through unilateral nephrectomy and streptozotocin treatment. We then assessed effects on the kidney, intestinal injuries, and bacterial translocation. RESULTS: MAVS knockout diabetic mice showed more severe glomerular and tubular injuries compared with wild-type diabetic mice. Owing to impaired intestinal integrity, the presence of intestine-derived Klebsiella oxytoca and elevated IL-17 were detected in the circulation and kidneys of diabetic mice, especially in diabetic MAVS knockout mice. Stimulation of tubular epithelial cells with K. oxytoca activated MAVS pathways and the phosphorylation of Stat3 and ERK1/2, leading to the production of kidney injury molecule-1 (KIM-1). Nevertheless, MAVS inhibition induced inflammation in the intestinal epithelial cells and KIM-1 production in tubular epithelial cells under K. oxytoca supernatant or IL-17 stimulation. Treatment with neutralizing anti-IL-17 antibody treatment had renoprotective effects. In contrast, LPS administration accelerated kidney injury in the murine diabetic kidney disease model. CONCLUSIONS: Impaired MAVS signaling both in the kidney and intestine contributes to the disrupted homeostasis, leading to diabetic kidney disease progression. Controlling intestinal homeostasis may offer a novel therapeutic approach for this condition.

D-Serine inhibits the attachment and biofilm formation of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Although therapeutic agents for methicillin-resistant Staphylococcus aureus (MRSA) are clinically available, MRSA infection is still a life-threatening disease. Bacterial attachment and biofilm formation contribute significantly to the initiation of MRSA infection. Controlling MRSA's attachment and biofilm formation might reduce the frequency of MRSA infection. According to recent data, some amino acids can reduce MRSA's attachment on plates; however, their precise inhibitory mechanisms remain unclear. Therefore, we explored the effect of the amino acids on bacterial adhesion and biofilm formation in vitro and in vivo MRSA infection models. METHODS: We tested the inhibitory effect of amino acids on MRSA and Escherichia coli (E. coli) in the attachment assay. Moreover, we evaluated the therapeutic potential of amino acids on the in vivo catheter infection model. RESULTS: Among the amino acids, D-Serine (D-Ser) was found to reduce MRSA's ability to attach on plate assay. The proliferation of MRSA was not affected by the addition of D-Ser; thus, D-Ser likely only played a role in preventing attachment and biofilm formation. Then, we analyzed the expression of genes related to attachment and biofilm formation. D-Ser was found to reduce the expressions of AgrA, SarS, IcaA, DltD, and SdrD. Moreover, the polyvinyl chloride catheters treated with D-Ser had fewer MRSA colonies. D-Ser treatment also reduced the severity of infection in the catheter-induced peritonitis model. Moreover, D-Ser reduced the attachment ability of E. coli. CONCLUSION: D-Ser inhibits the attachment and biofilm formation of MRSA by reducing the expression of the related genes. Also, the administration of D-Ser reduces the severity of catheter infection in the mouse model. Therefore, D-Ser may be a promising therapeutic option for MRSA as well as E. coli infection.

The increased frequency of methicillin-resistant Staphylococcus aureus with low MIC of beta-lactam antibiotics isolated from hospitalized patients.

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infectious diseases and can be life-threatening in healthcare-settings. MRSA is classified into health-care associated (HA)-MRSA strains and community acquired (CA)-MRSA strains based on genotype and phenotype. CA-MRSA has been reported to show the lower minimal inhibitory concentration (MIC) of some antibiotics as compared to HA-MRSA. Recently, the prevalence of CA-MRSA has been increased in worldwide. CA-MRSA is isolated not only from the healthy individuals in a community but also from the patients in healthcare settings. However, the changing trend in frequency of HA-MRSA and CA-MRSA in the hospital setting is not clear. Therefore, we analyzed the trend of MIC to speculate the frequency of HA-MRSA and CA-MRSA in the facility. Moreover, gene mutations were evaluated on resistant gene loci with next generation sequencer. The frequency of strains with low MIC of beta-lactam antibiotics was gradually increased in isolated MRSA strains from the hospitalized patients. Whole genome analysis revealed the frequency of gene mutation was also decreased in some resistant loci, such as blaZ and blaR1. These findings highlight the changing trend of MRSA strains isolated from hospitalized patients.

Collagen adhesion gene is associated with bloodstream infections caused by methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) causes hospital- and community-acquired infections. It is not clear whether genetic characteristics of the bacteria contribute to disease pathogenesis in MRSA infection. We hypothesized that whole genome analysis of MRSA strains could reveal the key gene loci and/or the gene mutations that affect clinical manifestations of MRSA infection. METHODS: Whole genome sequences (WGS) of MRSA of 154 strains were analyzed with respect to clinical manifestations and data. Further, we evaluated the association between clinical manifestations in MRSA infection and genomic information. RESULTS: WGS revealed gene mutations that correlated with clinical manifestations of MRSA infection. Moreover, 12 mutations were selected as important mutations by Random Forest analysis. Cluster analysis revealed strains associated with a high frequency of bloodstream infection (BSI). Twenty seven out of 34 strains in this cluster caused BSI. These strains were all positive for collagen adhesion gene (cna) and have mutations in the locus, those were selected by Random Forest analysis. Univariate and multivariate analysis revealed that these gene mutations were the predictor for the incidence of BSI. Interestingly, mutant CNA protein showed lower attachment ability to collagen, suggesting that the mutant protein might contribute to the dissemination of bacteria. CONCLUSIONS: These findings suggest that the bacterial genotype affects the clinical characteristics of MRSA infection.

[Relationship between Clostridium difficile ITS-PCR Type and Pathogenicity].

Clostridium difficile (C. difficile) causes antibiotic-associated diarrhea and nosocomial infection. The PCR of internal transcribed spacer regions (ITS) is easily conductible in a relatively short time. The purpose of the current study is to classify C. difficile by PCR electrophoresis pattern of ITS (ITS-PCR type) and estimate the relationship of the ITS-PCR type of C. difficile with its pathogenicity. We examined 77 strains which were obtained in our hospital from March 2012 to August 2013. Toxin genes were detected by PCR using toxin gene specific primers. Antimicrobial sensitivities were measured by E-test. Pseudomembrane formation and severity of the illness in clinical patients were investigated based on the medical records. The strains were classified into the 33 ITS-PCR types. Among them, most of strains in 18 PCR types were not associated with any toxin genes. Strains with toxin A(+)/B(+)genes were classified into 14PCR types. The 3 strains with toxin B (+) strains and the two strains with toxin A(+)/B(+)/binary toxin(+) genes were classified into 1 PCR type, type 17, and type 16, respectively. 6 strains in 13 strains of type 33, and 5 strains in 11 strains of type 2 were detected from the same ward, presuming nosocomial infection. Minimum inhibitory concentrations (MICs) of vancomycin and metronidazole were ≤ 2 µg/mL, distribution of MICs were not correlated with ITS-PCR type. The pseudomembrane forming and severity of the illness were not obviously related to ITS-PCR pattern. Thus, the typing of C. difficile by ITS-PCR pattern is considered to be useful for early detection of nosocomial infection, and assessment of toxigenicity.

[Molecular epidemiology of the Methicillin-resistant Staphylococcus aureus(MRSA) by the internal transcribed spacer PCR (ITS-PCR) method and the phage open reading frame typing (POT) method].

Methicillin-resistant Staphylococcus aureus (MRSA) is the most common causative bacteria of hospital acquired infection, and should be rapidly identified for infection control. For this purpose, in our hospital, the PCR electrophoresis patterns of spacer regions (ITS: internal transcribed spacers) (ITS-PCR) are combined with a toxigenicity assay to establish a strain identification method for outbreak surveillance. In the present study, the usefulness of this method was evaluated in comparison with the POT (phage-open reading frame typing) method. One hundred MRSA strains isolated from inpatients in our hospital between April 2011 and March 2012 were classified into 25 patterns using the ITS-PCR method combined with a toxigenicity assay. The strains could be classified into 46 patterns using the POT method. ITS-PCR type 22 strain producing enterotoxin C and toxic shock syndrome toxin-1 could be further classified into 7 patterns using the POT method. In the outbreak of the type 22 strain, cross-infection could be excluded by additional analysis using the POT method, providing more precise information on strain identification. We identified that some strains of the same POT type consisted of different ITS-PCR types or toxigenicities. Therefore, these results suggest that the combination of ITS-PCR method plus toxigenicity assay with POT method may be a useful technique of MRSA typing.

[Trends for the etiology of infective endocarditis at our hospital these 5 years].

The aim of this study is to evaluate characteristics of infective endocarditis for 5 years at Kanazawa University Hospital. Retrospectively, we investigated 39 patients diagnosed as infective endocarditis at our hospital from 2006 to 2010 based on blood culture and/or rejected cardiac specimens. Of 39 patients with infective endocarditis, 27 were male and 12 were female. Mean age was 55.4 years and 69% patients were older than 50 years. The frequent underlying presumed diseases were cardiac diseases. Vegetation was mainly observed at mitral valve and aortic valve. Streptococcus species [14 cases (36%)] and Staphylococcus species [12 cases (31%)] were common pathogens. In Streptococcus species, the critical cause was mostly presumed to be associated with dental procedure and oral cavity. In Staphylococcus species, intravascular device and soft tissue infection were also frequently presumed. Frequency of chronic kidney disease and infection around valve were higher in Staphylococcus species than those observed in Streptococcus species [12 cases (100%) vs. 7 cases (50%); p < 0.05, 6 cases (50%) vs. 1 case (7%); p < 0.05]. Our results suggested that the etiology of patients with Staphylococcus species infection increased in number among patients suffering from infective endocarditis at our hospital.

Rapid identification of gram-negative bacteria with and without CTX-M extended-spectrum ß-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis.

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum ß-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.

Mutational analysis of human heat-shock transcription factor 1 reveals a regulatory role for oligomerization in DNA-binding specificity.

HSF (heat-shock transcription factor) trimers bind to the HSE (heat-shock element) regulatory sequence of target genes and regulate gene expression. A typical HSE consists of at least three contiguous inverted repeats of the 5-bp sequence nGAAn. Yeast HSF is able to recognize discontinuous HSEs that contain gaps in the array of the nGAAn sequence; however, hHSF1 (human HSF1) fails to recognize such sites in vitro, in yeast and in HeLa cells. In the present study, we isolated suppressors of the temperature-sensitive growth defect of hHSF1-expressing yeast cells. Intragenic suppressors contained amino acid substitutions in the DNA-binding domain of hHSF1 that enabled hHSF1 to regulate the transcription of genes containing discontinuous HSEs. The substitutions facilitated hHSF1 oligomerization, suggesting that the DNA-binding domain is important for this conformational change. Furthermore, other oligomerization-prone derivatives of hHSF1 were capable of recognizing discontinuous HSEs. These results suggest that modulation of oligomerization is important for the HSE specificity of hHSF1 and imply that hHSF1 possesses the ability to bind to and regulate gene expression via various types of HSEs in diverse cellular processes.

Differential recognition of heat shock elements by members of the heat shock transcription factor family.

Heat shock transcription factor (HSF), an evolutionarily conserved stress response regulator, forms trimers and binds to heat shock element (HSE), comprising at least three continuous inverted repeats of the sequence 5'-nGAAn-3'. The single HSF of yeast is also able to bind discontinuously arranged nGAAn units. We investigated interactions between three human HSFs and various HSE types in vitro, in yeast cells, and in HeLa cells. Human HSF1, a stress-activated regulator, preferentially bound to continuous HSEs rather than discontinuous HSEs, and heat shock of HeLa cells caused expression of reporter genes containing continuous HSEs. HSF2, whose function is implicated in neuronal specification and spermatogenesis, exhibited a slightly higher binding affinity to discontinuous HSEs than did HSF1. HSF4, a protein required for ocular lens development, efficiently recognized discontinuous HSEs in a trimerization-dependent manner. Among four human gamma-crystallin genes encoding structural proteins of the lens, heat-induced HSF1 preferred HSEs on the gammaA-crystallin and gammaB-crystallin promoters, whereas HSF4 preferred HSE on the gammaC-crystallin promoter. These results suggest that the HSE architecture is an important determinant of which HSF members regulate genes in diverse cellular processes.

Interaction between heat shock transcription factors (HSFs) and divergent binding sequences: binding specificities of yeast HSFs and human HSF1.

The target genes of the heat shock transcription factor (HSF) contain a cis-acting sequence, the heat shock element (HSE), which consists of multiple inverted repeats of the sequence 5'-nGAAn-3'. Using data acquired in this and a previous study, we have identified the HSEs in 59 of 62 target genes of Saccharomyces cerevisiae Hsf1. The Hsf1 protein recognizes continuous and discontinuous repeats of the nGAAn unit; the nucleotide sequences and configuration of the units diverge slightly among functional HSEs. When Schizosaccharomyces pombe HSF was expressed in S. cerevisiae cells, heat shock induced S. pombe HSF to bind to various HSE types, which properly activated transcription from almost all target genes, suggesting that the S. pombe genome also contains divergent HSEs. Human HSF1 induced the heat shock response via HSEs with continuous units in S. cerevisiae cells but failed to do so via HSEs with discontinuous units. Binding of human HSF1 to the discontinuous type of HSE was observed in vitro but was significantly inhibited in vivo. These results show that human HSF1 recognizes HSEs in a slightly different way than yeast HSFs and suggest that the configuration of the unit is an important determinant for HSF-HSE interactions.

Stress-induced transcription of the endoplasmic reticulum oxidoreductin gene ERO1 in the yeast Saccharomyces cerevisiae.

Gene transcription changes dramatically in response to various stresses. This event is an obligatory step for adaptation of cells to certain environments. Endoplasmic reticulum (ER) oxidoreductin encoded by the ERO1 gene of the yeast Saccharomyces cerevisiae is essential for the formation of protein disulfide bonds in the ER and for cell viability. We show that transcription of ERO1 is regulated by two transcriptional activators in response to different stresses. In the unfolded protein response induced by the reductant dithiothreitol, transcription factor Hac1 activates ERO1 transcription through a sequence that diverges from the consensus Hac1-binding sequence. Heat shock transcription factor Hsf1 activates ERO1 in response to heat, ethanol, and oxidative stresses. Using cells containing mutations in the Hac1- and Hsf1-binding sequences of the chromosomal ERO1 promoter, we demonstrate that Hac1-regulated transcription of ERO1 confers resistance to dithiothreitol. Although mutations in the Hsf1-binding sequences do not affect the sensitivity of cells to heat, ethanol, or oxidative stresses, both the Hac1- and Hsf1-regulated pathways are critical for normal growth under complex stress conditions.