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.

Rare toxin A-negative and toxin B-positive strain of Clostridioides difficile from Japan lacking a complete tcdA gene.

INTRODUCTION: Clostridioides difficile (C. difficile) produces three kinds of toxins: toxin A (enterotoxin), toxin B (cytotoxin), and C. difficile transferase (CDT), a binary toxin. Some strains show positivity only for toxin B. These strains reportedly possess a gene for toxin A, tcdA. However, toxin A production is inhibited due to a mutated stop codon and/or deletion within the tcdA gene. Here for the first case in Japan, we describe toxin genomes and proteins of a strain possessing only toxin B and lacking a complete tcdA gene, along with clinical manifestations. METHODS: C. difficile was isolated from the bloody stool of a 60-year-old female patient treated with meropenem. Although a rapid detection kit of toxins (C. DIFF QUIK CHEK COMPLETE®, TechLab, Blacksburg, VA, USA) showed positivity, Western blotting detected no toxins. Therefore, we explored the strain's toxin genes and their sequences to determine whether the strain possessed a toxin. RESULTS: Polymerase chain reaction did not identify toxin genes. Whole-genome sequencing analysis showed that a gene for toxin A, tcdA, was completely deleted in the strain. Moreover, 701 mutations and some deletions/insertions were identified on the tcdB gene. CONCLUSIONS: We isolated a rare strain of C. difficile producing only toxin B and lacking a complete tcdA gene herein Japan. The possibility of a false negative needs to be considered with a genetic method for a diagnose of C. difficile infection.

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.

[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.

Leuconostoc bacteremia in three patients with malignancies.

Leuconostoc is a Gram-positive coccus characterized by its resistance to glycopeptide antibiotics. Generally, this bacterium is susceptible to ß-lactam antibiotics; however, here we present a leukemia patient who developed leuconostoc bacteremia during antimicrobial therapy with carbapenem. The appropriate choice of antibiotics at optimal doses enables leuconostoc infection to be overcome, even in compromised hosts. We report 3 cases of leuconostoc bacteremia: the leukemia case which was successfully treated, along with discussions of two other cases with malignancies.

[Clinical evaluation of rapid identification of bacteria from positive-testing blood culture bottles by internal transcribed spacer PCR].

Delays in diagnosis and initiation of treatment of severe infections such as sepsis greatly influence patient prognosis. Our laboratory introduced rapid identification of bacterial species by PCR for positive blood culture samples as a routine laboratory test since April 2008. We extracted DNA directly from positive blood culture bottles and amplified the internal transcribed spacer (ITS) region of pathogenic microorganisms by PCR in order to identify bacterial species from electrophoretic patterns of PCR products. Of 167 strains from 167 samples excluding three samples with polymicrobial organisms, 144 strains (86.2%) were correctly identified at species level and 17 strains (10.2%) at genus level. The time required between DNA extraction and bacterial identification was about one and one-half hours. In patients with MRSA sepsis, the time of initiation of treatments such as administration of anti-MRSA drugs and intravascular catheter removal has clearly become earlier with the introduction of ITS-PCR, resulting decreased mortality from 35.0% to 16.0%. Rapid identification of pathogens directly from blood culture bottles by ITS-PCR seems to be useful for appropriate treatment of severe infectious diseases.

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.

CRP regulator modulates multidrug resistance of Escherichia coli by repressing the mdtEF multidrug efflux genes.

Multidrug efflux pumps contribute to the resistance of Escherichia coli against many antibiotics and biocides. Here, we report that the CRP regulator modulates multidrug resistance in E. coli through repression of the genes encoding the MdtEF multidrug efflux pump. Screening of mutants for ability to increase beta-lactam resistance in E. coli led to the identification of a mutation in crp, which codes for the major global regulator of catabolite-sensitive operons. Deletion of crp significantly increased the resistance of the E. coli strain to oxacillin, azithromycin, erythromycin and crystal violet. The increase in drug resistance caused by crp deletion was completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Among the twenty drug efflux pumps in E. coli, quantitative real-time PCR analysis showed that CRP repressed the expression of mdtEF. Deletion of mdtEF completely suppressed CRP-modulated multidrug resistance. Therefore, in addition to its role in catabolite control, CRP contributes to multidrug resistance in E. coli. Our results indicate that the CRP regulator modulates multidrug resistance in E. coli by repressing expression of the MdtEF multidrug efflux pump.

Role of the AraC-XylS family regulator YdeO in multi-drug resistance of Escherichia coli.

Multi-drug efflux pumps contribute to the resistance of Escherichia coli to many antibiotics and biocides. In this study, we report that the AraC-XylS family regulator YdeO increases the multi-drug resistance of E. coli through activation of the MdtEF efflux pump. Screening of random fragments of genomic DNA for their ability to increase beta-lactam resistance led to the isolation of a plasmid containing ydeO, which codes for the regulator of acid resistance. When overexpressed, ydeO significantly increased the resistance of the E. coli strain to oxacillin, cloxacillin, nafcillin, erythromycin, rhodamine 6G and sodium dodecyl sulfate. The increase in drug resistance caused by ydeO overexpression was completely suppressed by deleting the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Quantitative real-time PCR showed that YdeO activated only mdtEF expression and none of the other drug efflux pumps in E. coli. Deletion of mdtEF completely suppressed the YdeO-mediated multi-drug resistance. YdeO enhances the MdtEF-dependent drug efflux activity in E. coli. Our results indicate that the YdeO regulator, in addition to its role in acid resistance, increases the multi-drug resistance of E. coli by activating the MdtEF multi-drug efflux pump.

The AraC-family regulator GadX enhances multidrug resistance in Escherichia coli by activating expression of mdtEF multidrug efflux genes.

Multidrug efflux pumps contribute to the resistance of Escherichia coli to many antibiotics and biocides. Here, we report that the AraC-family regulator GadX increases multidrug resistance in E. coli through activation of the MdtEF efflux pump. Screening of random fragments of genomic DNA for ability to increase beta-lactam resistance led to the isolation of a plasmid containing gadX, which codes for the regulator of acid resistance. When overexpressed, gadX significantly increased the resistance of the E. coli strain to oxacillin, cloxacillin, nafcillin, erythromycin, rhodamine 6G, and sodium dodecyl sulfate. The increase in drug resistance caused by gadX overexpression was completely suppressed by deleting the multifunctional outer membrane channel gene tolC. TolC interacts with different drug efflux pumps. Quantitative real-time polymerase chain reaction (PCR) showed that GadX activated the expression of mdtEF but none of the other drug efflux pumps in E. coli. Deletion of mdtEF completely suppressed GadX-mediated multidrug resistance. Our results indicate that the GadX regulator, in addition to its role in acid resistance, increases multidrug resistance in E. coli by activating the MdtEF multidrug efflux pump.

Catheter-related fungemia due to fluconazole-resistant Candida nivariensis.

We report on a case of fungemia due to fluconazole-resistant Candida nivariensis (MIC, > or =128 microg/ml). Internal transcribed spacer PCR followed by microchip gel electrophoresis with a blood culture that tested positive revealed a unique pattern different from those of other pathogenic yeasts.

N-acetyl-d-glucosamine induces the expression of multidrug exporter genes, mdtEF, via catabolite activation in Escherichia coli.

The expression of MdtEF, a multidrug exporter in Escherichia coli, is positively controlled through multiple signaling pathways, but little is known about signals that induce MdtEF expression. In this study, we investigated compounds that induce the expression of the mdtEF genes and found that out of 20 drug exporter genes in E. coli, the expression of mdtEF is greatly induced by N-acetyl-d-glucosamine (GlcNAc). The induction of mdtEF by GlcNAc is not mediated by the evgSA, ydeO, gadX, and rpoS signaling pathways that have been known to regulate mdtEF expression. On the other hand, deletion of the nagE gene, encoding the phosphotransferase (PTS) system for GlcNAc, prevented induction by GlcNAc. The induction of mdtEF by GlcNAc was also greatly inhibited by the addition of cyclic AMP (cAMP) and completely abolished upon deletion of the cAMP receptor protein gene (crp). Other PTS sugars, glucose and d-glucosamine, also induced mdtEF gene expression. These results suggest that mdtEF expression is stimulated through catabolite control.

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae.

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.

Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis.

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification ( approximately 1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.