[Molecular typing characterization of food-borne methicillin-resistant Staphylococcus aureus in China].

Objective: To analyses the antimicrobial resistance and molecular characterization of 21 MRSA isolates cultured from retail foods from different provinces in China, and evaluate the molecular typing methods. Methods: Twenty-one MRSA isolates were obtained from national foodborne pathogen surveillance network in 2012 (Chinese salad, n=3; milk, n=1; cake, n=2; rice, n=1; cold noodle, n=1; spiced beef, n=1; dumpling, n=1; packed meal, n=1; salad, n=1; raw pork, n=9). The antimicrobial resistance of 21 strains to 12 antimicrobial agents was tested by broth dilution method. Polymerase chain reaction (PCR) and DNA sequencing were performed to obtain the genetic types of MLST (ST) and spa typing. The clonal complex (CC) was assigned by eBURST soft and the MLVA type (MT) and MLVA complex (MC) were identified via the database of the MLVA website (http://www.mlva.net). SmaI pulsed-field gel electrophoresis (SmaⅠ-PFGE) was also carried out to obtain the PFGE patterns of 21 strains. The genetic diversity and discriminatory power of typing were calculated by the Simpson's index of diversity (diversity index, DI) to find out the best genotyping method for MRSA. Results: All MRSA isolates showed multi-drug resistance(MDR), and were resistant to oxacillin, benzylpenicillin, clindamycin and erythromycin, and 71.4% (15/21), 47.6% (10/21), 42.9% (9/21) and 28.6% (6/21) of the MRSA isolates were resistant to tetracycline, ciprofloxacin, trimethoprim/sulfamethoxazole and gentamicin, respectively. Moreover, one strain was found to be resistant to all three antimicrobials of levofloxacin, moxifloxacin and rifampicin. Great diversity was found in these food-associated MRSA (6 STs, 7 spa types, and 9 MTs). PFGE patterns were more diverse than those of other three molecular typing methods (19 pulse types). The index of diversity (DI) of PFGE, MLVA, spa typing and MLST was 0.99, 0.80, 0.73, and 0.61, respectively. Among the MRSA isolates, CC9-ST9-t899-MT929-MC2236 (PFGE Cluster Ⅴ) was the most prevalent clone, which were all cultured from raw pork (9 isolates). Besides, two MRSA were identified as CC59-ST338-t437-MT621-MC621 (PFGE Cluster Ⅳ). Different clone had their own resistance spectrum profiles. Conclusion: The food-borne MRSA isolates were all MDR in this study. Different clones had their own resistance spectrum profiles. MLVA represented a promising tool for molecular epidemiology tracing of MRSA in foodborne disease events.

[Antimicrobial susceptibility and drug-resistance genes of Yersinia spp. of retailed poultry in 4 provinces of China].

Objective: To monitor the antimicrobial resistance and drug-resistance genes of Yersinia enterocolitis, Y. intermedia and Y. frederiksenii recovered from retailed fresh poultry of 4 provinces of China. Methods: The susceptibility of 25 isolated Yersinia spp. to 14 classes and 25 kinds of antibiotics was determined by broth microdilution method according to CLSI (Clinical and Laboratory Standards Institute). The antibiotic resistance genes were predicted with antibiotic resistance genes database (ARDB) using whole genome sequences of Yersinia spp. Results: In all 22 Y. enterocolitis tested, 63.7% (14 isolates), 22.8% (5 isolates), 4.6% and 4.6% of 1 isolates exhibited the resistance to cefoxitin, ampicillin-sulbactam, nitrofurantoin and trimethoprim-sulfamethoxazole, respectively. All the 25 isolates were multi-drug resistant to more than 3 antibiotics, while 64.0% of isolates were resistant to more than 4 antibiotics. A few Y. enterocolitis isolates of this study were intermediate to ceftriaxone and ciprofloxacin. Most Yersinia spp. isolates contained antibiotic resistance genes mdtG, ksgA, bacA, blaA, rosAB and acrB, and 5 isolates recovered from fresh chicken also contained dfrA1, catB2 and ant3ia. Conclusion: The multi-drug resistant Yersinia spp. isolated from retailed fresh poultry is very serious in the 4 provinces of China, and their contained many kinds of drug-resistance genes.

[Serotype distribution and antimicrobial resistance of Salmonella isolates from retail chicken carcasses in six provinces of China].

Objective: To obtain the serotype diversity and antimicrobial resistance of Salmonella isolates recovered from retail chicken carcasses for sale in six regions of China. Methods: From August 2010 to March 2012, each month 20 retail chicken carcasses including freshly slaughtered, chilled and frozen samples were collected from supermarkets and farmer's markets in 7 monitoring sites in Beijing, Jilin province, Inner Mongolia Autonomous, Shanxi province, Jiangsu province and Guangdong province, respectively. Samples were routinely collected for 12 months for each site. 1 680 chicken carcasses were collected in total and 2 629 Salmonella strains were isolated by PCR and biochemical method. Luminex xMAP method and classical slide agglutination method were carried out to determine isolates' serotypes. Minimal inhibitory concentrations (MICs) of 10 classes of antimicrobials including 14 agents were determined using broth micro-dilution method. Mocular methods were used to determine antimicrobial resistance genes of CIP-CTX-CT co-resistant isolates. Results: In all, 2 629 Salmonella isolates, there were 17 seorgroups and 58 serotypes, B and D1 were the dominant serogroups with rates of 34.7% (n=913) and 31.0% (n=815), Enteritidis (30.8%, n=810), Indiana (17.6%, n=463), Infantis (10.6%, n=278) were the top three serovars. We found 224 CIP-CTX co-resistant S. Indiana containing 3 colistin resistant strains, one of them carrying mcr-1 gene and being ESBLs positive, which demonstrated a nine multi drug resistance against 11 antimicrobials tested. Conclusion: These data began to describe the complicated serovar diversity and heavy antimicrobial resistance of Salmonella isolates recovered from retail chicken carcasses in six regions of China. The findings highlight the emergence of ciprofloxacin and cefotaxime co-resistant S. Indiana and also a mcr-1 positive S. Indiana with heavy multi drug resistance.

Mitochondrial Perturbation in Alzheimer's Disease and Diabetes.

Mitochondria are well-known cellular organelles that play a vital role in cellular bioenergetics, heme biosynthesis, thermogenesis, calcium homeostasis, lipid catabolism, and other metabolic activities. Given the extensive role of mitochondria in cell function, mitochondrial dysfunction plays a part in many diseases, including diabetes and Alzheimer's disease (AD). In most cases, there is overwhelming evidence that impaired mitochondrial function is a causative factor in these diseases. Studying mitochondrial function in diseased cells vs healthy cells may reveal the modified mechanisms and molecular components involved in specific disease states. In this chapter, we provide a concise overview of the major recent findings on mitochondrial abnormalities and their link to synaptic dysfunction relevant to neurodegeneration and cognitive decline in AD and diabetes. Our increased understanding of the role of mitochondrial perturbation indicates that the development of specific small molecules targeting aberrant mitochondrial function could provide therapeutic benefits for the brain in combating aging-related dementia and neurodegenerative diseases by powering up brain energy and improving synaptic function and transmission.

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease.

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.

Extinguishing Egr-1-dependent inflammatory and thrombotic cascades after lung transplantation.

Hypoxic induction of the early growth response-1 (Egr-1) transcription factor initiates proinflammatory and procoagulant gene expression. Orthotopic/isogeneic rat lung transplantation triggers Egr-1 expression and nuclear DNA binding activity corresponding to Egr-1, which leads to increased expression of downstream target genes such as interleukin-1b, tissue factor, and plasminogen activator inhibitor-1. The devastating functional consequences of Egr-1 up-regulation in this setting are prevented by treating donor lungs with a phosphorothioate antisense oligodeoxyribonucleotide directed against the Egr-1 translation initiation site, which blocks expression of Egr-1 and its gene targets. Post-transplant graft leukostasis, inflammation, and thrombosis are consequently diminished, with marked improvement in graft function and recipient survival. Blocking expression of a proximal transcription factor, which activates deleterious inflammatory and coagulant effector mechanisms, is an effective molecular strategy to improve organ preservation.

Lipopolysaccharide activation of the MEK-ERK1/2 pathway in human monocytic cells mediates tissue factor and tumor necrosis factor alpha expression by inducing Elk-1 phosphorylation and Egr-1 expression.

Lipopolysaccharide (LPS) induces human monocytes to express many proinflammatory mediators, including the procoagulant molecule tissue factor (TF) and the cytokine tumor necrosis factor alpha (TNF-alpha). The TF and TNF-alpha genes are regulated by various transcription factors, including nuclear factor (NF)-kappaB/Rel proteins and Egr-1. In this study, the role of the MEK-ERK1/2 mitogen-activated protein kinase (MAPK) pathway in LPS induction of TF and TNF-alpha gene expression in human monocytic cells was investigated. The MAPK kinase (MEK)1 inhibitor PD98059 reduced LPS induction of TF and TNF-alpha expression in a dose-dependent manner. PD98059 did not affect LPS-induced nuclear translocation of NF-kappaB/Rel proteins and minimally affected LPS induction of kappaB-dependent transcription. In contrast, PD98059 and dominant-negative mutants of the Ras-Raf1-MEK-ERK (extacellular signal-regulated kinase) pathway strongly inhibited LPS induction of Egr-1 expression. In kinetic experiments LPS induction of Egr-1 expression preceded induction of TF expression. In addition, mutation of the Egr-1 sites in the TF and TNF-alpha promoters reduced expression of these proinflammatory genes. It was demonstrated that LPS induction of the Egr-1 promoter was mediated by 3 SRE sites, which bound an LPS-inducible complex containing serum response factor and Elk-1. LPS stimulation transiently induced phosphorylation of Elk-1 and increased the functional activity of a GAL4-Elk-1TA chimeric protein via the MEK-ERK1/2 pathway. The data indicate that LPS induction of Egr-1 gene expression is required for maximal induction of the TNF-alpha and TF genes in human monocytic cells.

Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice.

Receptor for advanced glycation end-products (RAGE), and two of its ligands, AGE and EN-RAGEs (members of the S100/calgranulin family of pro-inflammatory cytokines), display enhanced expression in slowly resolving full-thickness excisional wounds developed in genetically diabetic db+/db+ mice. We tested the concept that blockade of RAGE, using soluble(s) RAGE, the extracellular ligand-binding domain of the receptor, would enhance wound closure in these animals. Administration of sRAGE accelerated the development of appropriately limited inflammatory cell infiltration and activation in wound foci. In parallel with accelerated wound closure at later times, blockade of RAGE suppressed levels of cytokines; tumor necrosis factor-alpha; interleukin-6; and matrix metalloproteinases-2, -3, and -9. In addition, generation of thick, well-vascularized granulation tissue was enhanced, in parallel with increased levels of platelet-derived growth factor-B and vascular endothelial growth factor. These findings identify a central role for RAGE in disordered wound healing associated with diabetes, and suggest that blockade of this receptor might represent a targeted strategy to restore effective wound repair in this disorder.

Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

A pathway leading to hypoxia-induced vascular fibrin deposition.

Hypoxemia has long been associated with vascular fibrin formation leading to thrombosis. This review describes a pathway through which mononuclear phagocytes and vascular smooth muscle cells upregulate tissue factor under hypoxic conditions. Increased expression of tissue factor triggers events leading to vascular fibrin deposition, providing insight into a novel mechanism potentially underlying thrombosis in ischemic vasculature.

The biology of the receptor for advanced glycation end products and its ligands.

Receptor for advanced glycation end products (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands includes advanced glycation end products (AGEs), amyloid fibrils, amphoterins and S100/calgranulins. The overlapping distribution of these ligands and cells overexpressing RAGE results in sustained receptor expression which is magnified via the apparent capacity of ligands to upregulate the receptor. We hypothesize that RAGE-ligand interaction is a propagation factor in a range of chronic disorders, based on the enhanced accumulation of the ligands in diseased tissues. For example, increased levels of AGEs in diabetes and renal insufficiency, amyloid fibrils in Alzheimer's disease brain, amphoterin in tumors and S100/calgranulins at sites of inflammation have been identified. The engagement of RAGE by its ligands can be considered the 'first hit' in a two-stage model, in which the second phase of cellular perturbation is mediated by superimposed accumulation of modified lipoproteins (in atherosclerosis), invading bacterial pathogens, ischemic stress and other factors. Taken together, these 'two hits' eventuate in a cellular response with a propensity towards tissue destruction rather than resolution of the offending pathogenic stimulus. Experimental data are cited regarding this hypothesis, though further studies will be required, especially with selective low molecular weight inhibitors of RAGE and RAGE knockout mice, to obtain additional proof in support of our concept.

Egr-1, a master switch coordinating upregulation of divergent gene families underlying ischemic stress.

Activation of the zinc-finger transcription factor early growth response (Egr)-1, initially linked to developmental processes, is shown here to function as a master switch activated by ischemia to trigger expression of pivotal regulators of inflammation, coagulation and vascular hyperpermeability. Chemokine, adhesion receptor, procoagulant and permeability-related genes are coordinately upregulated by rapid ischemia-mediated activation of Egr-1. Deletion of the gene encoding Egr-1 strikingly diminished expression of these mediators of vascular injury in a murine model of lung ischemia/reperfusion, and enhanced animal survival and organ function. Rapid activation of Egr-1 in response to oxygen deprivation primes the vasculature for dysfunction manifest during reperfusion. These studies define a central and unifying role for Egr-1 activation in the pathogenesis of ischemic tissue damage.

Expression of Egr-1 in late stage emphysema.

The transcription factor early growth response (Egr)-1 is an immediate-early gene product rapidly and transiently expressed after acute tissue injury. In contrast, in this report we demonstrate that lung tissue from patients undergoing lung reduction surgery for advanced emphysema, without clinical or anatomical evidence of acute infection, displays a selective and apparently sustained increase in Egr-1 transcripts and antigen, compared with a broad survey of other genes, including the transcription factor Sp1, whose levels were not significantly altered. Enhanced Egr-1 expression was especially evident in smooth muscle cells of bronchial and vascular walls, in alveolar macrophages, and some vascular endothelium. Gel shift analysis with (32)P-labeled Egr probe showed a band with nuclear extracts from emphysematous lung which was supershifted with antibody to Egr-1. Egr-1 has the capacity to regulate genes relevant to the pathophysiology of emphysema, namely those related to extracellular matrix formation and remodeling, thrombogenesis, and those encoding cytokines/chemokines and growth factors. Thus, we propose that further analysis of Egr-1, which appears to be up-regulated in a sustained fashion in patients with late stage emphysema, may provide insights into the pathogenesis of this destructive pulmonary disease, as well as a new facet in the biology of Egr-1.

Pulmonary expression of early growth response-1: biphasic time course and effect of oxygen concentration.

Hypoxia induces complex adaptive responses. In this report, induction of early growth response-1 (Egr-1) transcripts in lungs of mice subjected to hypoxia is shown to be dose and time dependent. Within 30 min of hypoxia, Egr-1 transcripts were approximately 20-fold elevated in 6% oxygen, approximately 5.2-fold increased by 10% oxygen, and returned to the normoxic baseline by 12% oxygen. Time course studies up to 48 h showed a biphasic profile with an initial steep rise in Egr-1 transcripts after 0.5 h of hypoxia and a second elevation beginning after 20-24 h. Hypoxic induction of Egr-1 was paralleled by enhanced expression of the downstream target gene tissue factor. Egr-1 and tissue factor antigen were visualized in bronchial and vascular smooth muscle and in alveolar macrophages. Egr-1 has the capacity to modulate expression of genes involved in the remodeling of the extracellular matrix and properties of smooth muscle, thus possibly contributing to the pulmonary response to chronic hypoxia.

Protein kinase C-beta and oxygen deprivation. A novel Egr-1-dependent pathway for fibrin deposition in hypoxemic vasculature.

Fibrin deposition is a salient feature of hypoxemic vasculature and results from induction of tissue factor. Such tissue factor expression in an oxygen deficient environment is driven by the transcription factor Early Growth Response (Egr)-1. Using homozygous null mice for the protein kinase C beta-isoform gene (PKCbeta null), PKCbeta is shown to be upstream of Egr-1 in this oxygen deprivation-mediated pathway for triggering procoagulant events. Whereas wild-type mice exposed to hypoxia (6%) displayed a robust increase in tissue factor transcripts and antigen, and vascular fibrin deposition, PKCbeta null animals showed a markedly blunted response. Consistent with a central role for Egr-1 in hypoxia-induced expression of tissue factor, PKCbeta null mice subjected to oxygen deprivation displayed at most a minor elevation in Egr-1 transcripts, antigen, and intensity of the gel shift band by electrophoretic mobility shift assay, compared with normoxic animals. These data firmly establish PKCbeta as a trigger for events leading to induction of Egr-1 and tissue factor under hypoxic conditions, and provide insight into a biologic cascade whereby oxygen deprivation recruits targets of PKCbeta and Egr-1, thereby amplifying the cellular response.

N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression.

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N(epsilon)-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-kappaB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.

Hypoxia/Hypoxemia-Induced activation of the procoagulant pathways and the pathogenesis of ischemia-associated thrombosis.

Although oxygen deprivation has long been associated with triggering of the procoagulant pathway and venous thrombosis, blood hypoxemia and stasis by themselves do not lead to fibrin formation. A pathway is outlined through which diminished levels of oxygen activate the transcription factor early growth response-1 (Egr-1) leading to de novo transcription/translation of tissue factor in mononuclear phagocytes and smooth muscle cells, which eventuates in vascular fibrin deposition. The procoagulant response is magnified by concomitant suppression of fibrinolysis by hypoxia-mediated upregulation of plasminogen activator inhibitor-1. These data add a new facet to the biology of thrombosis associated with hypoxemia/stasis and imply that interference with mechanisms causing Egr-1 activation in response to oxygen deprivation might prevent vascular fibrin deposition occurring in ischemia without directly interfering with other pro/anticoagulant pathways.

CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke.

BACKGROUND AND PURPOSE: Neutrophil (PMN) recruitment mediated by increased expression of intercellular adhesion molecule-1 expression (ICAM-1, CD54) in the cerebral microvasculature contributes to the pathogenesis of tissue injury in stroke. However, studies using blocking antibodies against the common beta2-integrin subunit on the PMN, the counterligand for ICAM-1 (CD18), have demonstrated equivocal efficacy. The current study tested the hypothesis that mice deficient in CD18 would be protected in the setting of reperfused but not nonreperfused stroke. METHODS: Two groups of mice were studied, those whose PMNs could express CD18 (CD18 +/+) and those mice hypomorphic for the CD-18 gene (CD18 -/-). PMNs obtained from CD18 -/- or CD18 +/+ mice were fluorescently labeled and tested for binding to murine brain endothelial monolayers. Using a murine model of focal cerebral ischemia in which an occluding suture placed in the middle cerebral artery (MCA) is removed after 45 minutes (transient ischemia, reperfused stroke) or left in place (permanent ischemia, nonreperfused stroke), cerebral infarct volumes (% ipsilateral hemisphere by TTC staining), cerebral blood flow (CBF, % contralateral hemisphere by laser-Doppler flowmetry), and survival (%) were examined 24 hours after the initial ischemic event. Adoptive transfer studies used 111In-labeled PMNs (from either CD18 +/+ or CD18 -/- mice) to examine the relative accumulation of PMNs in the ischemic region. RESULTS: PMNs obtained from CD18 -/- mice exhibit reduced adhesivity (compared with CD18 +/+ PMNs) for both quiescent and cytokine-activated endothelial monolayers. CD18 -/- mice (n=14) subjected to transient focal cerebral ischemia demonstrated a 53% decrease in infarct volumes versus CD18 +/+ mice (n=26, P<0.05), improved penumbral CBF at 24 hours (1.8-fold, P=0.02), and a 3.7-fold decrease in mortality (P=0.02). However, when CD18 -/- mice (n=12) were subjected to permanent focal cerebral ischemia, no differences were noted in infarct volume, mortality, or CBF versus similarly treated CD18 +/+ mice (n=10). There was a greater accumulation of CD18 +/+ PMNs in the ischemic zone of CD18 +/+ animals than CD18 -/- animals subjected to reperfused stroke (82% increase, P=0.02), although there was no difference between groups when subjected to permanent MCA occlusion. CONCLUSIONS: Deficiency for the CD18 gene confers cerebral protection in a murine model of reperfused stroke, but this benefit does not extend to CD18-deficient animals subjected to permanent MCA occlusion. These data suggest that anti-PMN strategies should be targeted to reperfused stroke and may perhaps be used in conjunction with thrombolytic therapy that establishes reperfusion.