Emergence of aac(6')-Ie-aph(2'')-Ia-positive enterococci with non-high-level gentamicin resistance mediated by IS1216V: adaptation to decreased aminoglycoside usage in Taiwan.

OBJECTIVES: To explore the mechanisms mediating the different levels of gentamicin resistance in enterococci. METHODS: Susceptibility testing with gentamicin and PCR of resistance determinants were performed in 149 enterococcal isolates. Genetic relatedness was characterized by MLST and PFGE analysis. Sequences of the aac(6')-Ie-aph(2'')-Ia gene and its surrounding environment were determined by Illumina sequencing. Stability assays of gentamicin resistance were carried out to evaluate the probability of loss of the high-level gentamicin resistance (HLGR) phenotype. RESULTS: A total of 17 (11.4%) aac(6')-Ie-aph(2'')-Ia-positive enterococcal isolates (2 Enterococcus faecalis and 15 Enterococcus faecium) with non-HLGR phenotype were found. MLST analysis revealed that the 2 E. faecalis belonged to ST116 and ST618, while all the 15 E. faecium belonged to clonal complex 17. Sequence analysis demonstrated that IS1216V was inserted into the 5'-end of aac(6')-Ie-aph(2'')-Ia, leading to loss of HLGR phenotype. Three IS1216V insertion types were found, and type II and III were frequently found in E. faecium. Interestingly, a total of 38 aac(6')-Ie-aph(2'')-Ia-positive E. faecium with HLGR phenotype also had type II or type III IS1216V insertion. Sequencing of the aac(6')-Ie-aph(2'')-Ia-positive HLGR E. faecium E37 revealed that an intact aac(6')-Ie-aph(2'')-Ia was located adjacent to IS1216V-disrupted aac(6')-Ie-aph(2'')-Ia. In a non-antibiotic environment, E37 tended to lose HLGR phenotype with a probability of 1.57 × 10-4, which was largely attributed to homologous recombination between the intact and disrupted aac(6')-Ie-aph(2'')-Ia. CONCLUSIONS: This is first study to elucidate that the E. faecium is capable of changing its HLGR phenotype, which may contribute to adaptation to hospital environments with decreased usage of gentamicin.

The Association of Targeted Gut Microbiota with Body Composition in Type 2 Diabetes Mellitus.

The association between body composition and gut microbiota in type 2 diabetes mellitus (DM) remains unknown. To elucidate the correlation of body composition and gut microbiota, we conducted a clinical study to enroll 179 patients with type 2 DM. Body composition of lean tissue index (LTI) and fat tissue index was measured by Body Composition Monitor. Eight pairs of 16S rRNA gene primers specific to Firmicutes, Bacteroidetes, the Clostridium leptum group, Bacteroides, Bifidobacterium, Akkermansia muciniphila, Escherichia coli, and Faecalibacterium prausnitzii were used to measure their abundance by quantitative polymerase chain reaction. The results showed that type 2 DM with higher abundance of phylum Firmicutes and a higher ratio of phyla Firmicutes to Bacteroidetes (phyla F/B ratio) had higher LTI. This significant correlation between phyla F/B ratio and LTI was especially evident in type 2 DM with high body mass index, and independent of glycemic control or dipeptidyl peptidase-4 inhibitor usage. In conclusion, our study demonstrated the positive association of LTI with the abundance of phylum Firmicutes and the phyla F/B ratio in type 2 DM.

Orai1/CRACM1 overexpression suppresses cell proliferation via attenuation of the store-operated calcium influx-mediated signalling pathway in A549 lung cancer cells.

BACKGROUND: Orai1/CRACM1 is a principal component of the store-operated calcium channels. Store-operated calcium influx is highly correlated with inflammatory reactions, immunological regulation, and cell proliferation. Epidermal growth factor (EGF), which plays an important role in the regulation of cell proliferation, can activate store-operated calcium channels. However, the consequences of Orai1/CRACM1 overexpression in EGF-mediated lung cancer cells growth are not known. METHODS: To investigate the role of Orai1/CRACM1 in EGF-mediated lung cancer cell proliferation, Orai1/CRACM1 plasmids were transfected into cells by lipofection. A cell proliferation assay, immunofluorescence staining, flow cytometry, and real-time polymerase chain reaction were employed to monitor cell proliferation. The calcium influx signals were investigated using a fluorescent-based calcium assay. RESULTS: Transfection of Orai1/CRACM1 plasmids resulted in the inhibition of EGF-mediated cell proliferation. ERK1/2 and Akt phosphorylation were inhibited by Orai1/CRACM1 overexpression. Expression of the cell cycle modulator p21 was induced in the Orai1/CRACM1-overexpressing cells, whereas the expression of cyclin D3 was reduced. Flow cytometry revealed that overexpression of Orai1/CRACM1 resulted in G0/G1 cell cycle arrest. Importantly, Orai1/CRACM1 overexpression significantly attenuated EGF-mediated store-operated calcium influx. In addition, application of 2-APB, a store-operated calcium channel inhibitor, resulted in the inhibition of EGF-mediated cancer cell proliferation. CONCLUSIONS: We conclude that Orai1/CRACM1 overexpression attenuates store-operated Ca(2+) influx that in turn blocks EGF-mediated proliferative signaling and drives cell cycle arrest.

2-hydroxy-4'-methoxychalcone inhibits proliferation and inflammation of human aortic smooth muscle cells by increasing the expression of peroxisome proliferator-activated receptor gamma.

Chalcone is a class of flavonoid compounds that are widely biosynthesized in plants. Epidemiological studies suggest that increased intake of flavonoids from fruits and vegetables reduces the risk of cardiovascular disease. However, the effect of chalcone on cardiovascular diseases has not been fully investigated. The aims of this study were to evaluate the antiatherosclerotic effect of 2-hydroxy-4'-methoxychalcone (AN07, a synthetic chalcone derivate) and to investigate its potential pharmacological mechanisms. Oxidized low-density lipoprotein (Ox-LDL) has been reported to stimulate proliferation of human aortic smooth muscle cells and that is one of the mechanisms resulting in atherosclerosis. In this study, we demonstrate that AN07 significantly inhibits the Ox-LDL-induced proliferation of human aortic smooth muscle cells. This effect is mediated via the inhibition of p44/42 mitogen-activated protein kinase and E-twenty six 1 phosphorylations. In the effect of anti-inflammation, AN07 decreases the Ox-LDL-stimulated upregulation of interleukin (IL) 1ß and IL-6. In addition, AN07 acts synergistically with rosiglitazone and pioglitazone to inhibit the Ox-LDL-induced proliferation of human aortic smooth muscle cells and upregulation of cyclin D1, cyclin D3, IL-1ß, and IL-6. These effects are a result of an increase in peroxisome proliferator-activated receptor gamma mRNA and protein expression stimulated by AN07 in human aortic smooth muscle cells. In conclusion, the chalcone derivate AN07 has versatile therapeutic potential against atherosclerosis by acting as peroxisome proliferator-activated receptor gamma inducer, p44/42 mitogen-activated protein kinase inhibitor, and cell cycle blocker.

Interrelationship of Gut Microbiota, Obesity, Body Composition and Insulin Resistance in Asians with Type 2 Diabetes Mellitus.

Metabolic syndrome (MS) has been an important health issue in the world, and insulin resistance (IR) is one of the characteristics of MS, increasing the risk for the onset and poor prognosis of type 2 diabetes mellitus (T2D). However, the interactional effect of obesity or abnormal body composition on the correlation between gut microbiota and IR in T2D patients is not well-explored. This cross-sectional study used a body composition monitor to evaluate lean tissue mass and fat tissue mass. IR was calculated using homeostatic model assessment-insulin resistance (HOMA-IR). Eight pairs of 16S rRNA gene primers specific to Firmicutes, Bacteroidetes, Clostridium leptum group, Faecalibacteriumprausnitzii, B acteroides, Bifidobacterium, Akkermansia muciniphila, and Escherichia coli were utilized to measure their abundance by qPCR. One hundred and fifty-four T2D patients were enrolled and stratified by the median HOMA-IR (2.5) and body mass index (BMI) of 25 kg/m2. A lower abundance of A. muciniphila was found in T2D patients with high HOMA-IR and BMI respectively. HOMA-IR and BMI had a synergistic effect on the reduction of the abundance of A. muciniphila. After adjusting metabolic factors, the low abundance of A. muciniphila significantly increased the risk for greater severity of IR. Furthermore, the negative correlation between A. muciniphila and IR was only found in T2D patients with high lean tissue. In conclusion, decreased abundance of fecal A. muciniphila enhanced the severity of IR in Asians with T2D, especially those having lean mass, and this significant relationship was independent of obesity.

A polymorphism of the ORAI1 gene is associated with the risk and recurrence of calcium nephrolithiasis.

PURPOSE: Store-operated calcium entry has been considered an important factor to regulate inflammatory reactions in nonexcitable cells. However, the effects of genetic polymorphisms of ORAI1, a main component of store-operated calcium channels, on nephrolithiasis and stone recurrence remain unclear. We investigated the association between calcium containing nephrolithiasis and genetic variants of ORAI1 gene in Taiwanese patients. MATERIALS AND METHODS: A case-control study was performed in 136 patients with nephrolithiasis and 500 controls. Five tagging single nucleotide polymorphisms of ORAI1 were selected for genotyping. ORAI1 genotypes were determined by TaqMan® assay. Hardy-Weinberg equilibrium in cases and controls was assessed, and genetic effects were evaluated by the chi-square test and sliding window haplotype analysis. Subset analysis was done according to family history. RESULTS: Two single nucleotide polymorphisms (rs12313273 and rs6486795) of the ORAI1 gene were associated with the risk of nephrolithiasis. The C allele carrier for rs12313273 was strongly related to recurrent stone forming in patients. On sliding window analysis the results of the 2 (rs12313273 and rs7135617) and the 3 (rs12313273, rs7135617 and rs6486795) single nucleotide polymorphism haplotypes had more significant effects on the risk of nephrolithiasis than the single nucleotide polymorphism rs12313273. CONCLUSIONS: To our knowledge this is the first study identifying the novel polymorphisms of the ORAI1 gene, which may predispose to the risk of calcium nephrolithiasis and disease recurrence.

Gut Microbiota and Non-Alcoholic Fatty Liver Disease Severity in Type 2 Diabetes Patients.

INTRODUCTION: Non-alcoholic fatty liver disease (NAFLD) remains an important health issue worldwide. The increasing prevalence of NAFLD is linked to type 2 diabetes (T2D). The gut microbiota is associated with the development of NAFLD and T2D. However, the relationship between gut microbiota and NAFLD severity has remained unclear in T2D patients. The aim of this study was to evaluate the relationship of gut microbiota with the severity of NAFLD in T2D patients. METHODS: This cross-sectional study used transient elastography (FibroScan) to evaluate the severity of hepatic steatosis. We utilized qPCR to measure the abundance of Bacteroidetes, Firmicutes, Faecalibacterium prausnitzii, Clostridium leptum group, Bacteroides, Bifidobacterium, Akkermansia muciniphila, and Escherichia coli. RESULTS: Of 163 T2D patients, 83 with moderate to severe NAFLD had higher abundance of bacteria of the phylum Firmicutes with respect to 80 patients without NAFLD or with mild NAFLD. High abundance of the phylum Firmicutes increased the severity of NAFLD in T2D patients. A positive correlation between NAFLD severity and the phylum Firmicutes was found in T2D male patients with body mass index ≥24 kg/m2 and glycated hemoglobin <7.5%. CONCLUSION: Enrichment of the fecal microbiota with the phylum Firmicutes is significantly and positively associated with NAFLD severity in T2D patients. The gut microbiota is a potential predictor of NAFLD severity in T2D patients.

Gut Microbiota and Subclinical Cardiovascular Disease in Patients with Type 2 Diabetes Mellitus.

Type 2 diabetes (T2D) is associated with an increased risk of cardiovascular disease (CVD). The gut microbiota may contribute to the onset and progression of T2D and CVD. The aim of this study was to evaluate the relationship between the gut microbiota and subclinical CVD in T2D patients. This cross-sectional study used echocardiographic data to evaluate the cardiac structure and function in T2D patients. We used a quantitative polymerase chain reaction to measure the abundances of targeted fecal bacterial species that have been associated with T2D, including Bacteroidetes, Firmicutes, Clostridium leptum group, Faecalibacterium prausnitzii, Bacteroides, Bifidobacterium, Akkermansia muciniphila, and Escherichia coli. A total of 155 subjects were enrolled (mean age 62.9 ± 10.1 years; 57.4% male and 42.6% female). Phyla Bacteroidetes and Firmicutes and genera Bacteroides were positively correlated with the left ventricular ejection fraction. Low levels of phylum Firmicutes were associated with an increased risk of left ventricular hypertrophy. High levels of both phylum Bacteroidetes and genera Bacteroides were negatively associated with diastolic dysfunction. A high phylum Firmicutes/Bacteroidetes (F/B) ratio and low level of genera Bacteroides were correlated with an increased left atrial diameter. Phyla Firmicutes and Bacteroidetes, the F/B ratio, and the genera Bacteroides were associated with variations in the cardiac structure and systolic and diastolic dysfunction in T2D patients. These findings suggest that changes in the gut microbiome may be the potential marker of the development of subclinical CVD in T2D patients.

Gut microbiota compositions and metabolic functions in type 2 diabetes differ with glycemic durability to metformin monotherapy.

AIMS: The metabolic derangements in type 2 diabetes have been attributed to compositional changes in the gut microbiota. Metformin, the first-line treatment for type 2 diabetes, has been found to modulate the gut microbiota. However, no literature has reported the associations between the composition of the gut microbiota and glycemic durability to metformin monotherapy. METHODS: A total of 375 patients with type 2 diabetes were recruited, among which 14 and 11 patients were eligible as the metformin durable group and nondurable group, respectively. Fecal samples were collected to analyze the gut microbiota by Illumina sequencing of the 16S rRNA gene, and PICRUSt2 was adopted to infer microbial functional differences. RESULTS: Although the two groups had similar biochemical profiles and microbial metabolites, the pattern of microbiota clustering was different. The intra-group diversity was significantly reduced in the durable group. For the microbial metabolic pathways, the biosynthesis of thiamine and lipopolysaccharide was dominant in the durable group. CONCLUSIONS: There were different compositions of gut microbiota with unique microbial metabolic pathways between type 2 diabetes with and without glycemic durability to metformin monotherapy. Microbial salvage by increasing thiamine biosynthesis might be beneficial for the metformin durable group to maintain optimal glycemic control.

A Possible Role of Insertion Sequence IS1216V in Dissemination of Multidrug-Resistant Elements MESPM1 and MES6272-2 between Enterococcus and ST59 Staphylococcus aureus.

Sequence type 59 (ST59) is the dominant type of community-associated methicillin-resistant Staphylococcus aureus (MRSA) in Taiwan. Previously, we reported that ST59 MRSA harbors enterococcal IS1216V-mediated multidrug-resistant composite transposons MESPM1 or MES6272-2. The MES were found to have a mosaic structure, largely originating in enterococci and partly native to S. aureus. The current study aimed to track the origin of the MES and how they disseminated from enterococci to ST59 S. aureus. A total of 270 enterococcal isolates were analyzed, showing that two ST64 Enterococcus faecalis isolated in 1992 and 11 clonal complex 17 Enterococcus faecium harbored MESPM1-like and MES6272-2-like structures, respectively. Sequence analysis revealed that ST64 E. faecalis strain N48 acquired the MESPM1-like structure on the plasmid pEflis48. The pEflis48 harbored the enterococci-originated region (erythromycin, kanamycin, and streptomycin resistances) and the S.aureus-originated region (chloramphenicol resistance) of MESPM1 but was separated by the replication region of the plasmid. Homologous recombination between the two direct repeats of IS1216V resulted in excision of the replication region of the plasmid to regenerate MESPM1. The p4780-1 and pV19 of E. faecium carried MES6272-2-like structures with IS1216V, albeit with multiple insertions by other insertion sequences. The findings show that IS1216V plays important roles in bidirectional gene transfer of multidrug resistance between enterococci and S. aureus.

Tracking the evolution of the two successful CC59 methicillin-resistant Staphylococcus aureus clones in Taiwan: the divergence time of the two clades is estimated to be the 1980s.

Clonal complex 59 (CC59) is the dominant community-associated methicillin-resistant Staphylococcus aureus (MRSA) strain in Taiwan and includes the Asian-Pacific clone with Panton-Valentine leukocidin (PVL)-negative/staphylococcal cassette chromosome mec (SCCmec) IVg and the Taiwan clone characterised as PVL-positive/SCCmec V (5C2&5). Nevertheless, data on the evolutionary history of the two dominant CC59 MRSA clones in Taiwan are scarce. In this study, a total of 258 CC59 S. aureus strains from Taiwan were classified by multiple-locus variable-number tandem repeat analysis (MLVA), which revealed two major clusters (MT1 and MT2) with distinct mobile genetic elements (MGEs). However, sequencing and PCR mapping of the ß-lactamase-producing plasmid revealed no difference among all CC59 S. aureus strains. Bayesian evolutionary analysis of 18 of the CC59 S. aureus strains based on core genome alignment revealed two clades: (i) Clade A, which shared the samples with MT1, had the features of mainly harbouring gentamicin-resistant MES6272-2 or MES4578, φSA3 translocation in νSaß and SCCmec IVg; and (ii) Clade B, which shared the samples with MT2, had the features of mainly harbouring streptomycin-resistant MESPM1, PVL phage and SCCmec V (5C2&5). Based on the time-calibrated phylogenetic tree, the estimated time of divergence of the two clades was in the 1980s. These results suggest that the CC59 S. aureus progenitor acquired a ß-lactamase-producing plasmid and then developed the varied genetic backgrounds, which were associated with the acquisition and maintenance of distinct MGEs, leading to differences in antimicrobial susceptibility profiles and molecular virulence determinants.

Involvement of the epidermal growth factor receptor in Pb²+-induced activation of cPLA2/COX-2 genes and PGE2 production in vascular smooth muscle cells.

Lead (Pb²+) is one of the most common heavy metal pollutants, which can cause chronic cardiovascular diseases. To clarify the mechanism by which Pb²+ induces inflammatory reactions, we examined the expression of inflammatory genes including encoding cyclooxygenase-2 (COX-2), cytosolic phospholipase A2 (cPLA2), and their down stream product prostaglandin E2 (PGE2) in CRL1999 cells that is a vascular smooth muscle cell line from human aorta. The expression of COX-2/cPLA2 genes and PGE2 secretion was increased markedly after cells were exposed to 1 µM Pb²+. PD098059, a MEK inhibitor, suppressed Pb²+-mediated inflammatory reactions; this indicates the involvement of the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). Furthermore, Pb²+-induced activation of the COX-2/cPLA2 genes was inhibited by both epidermal growth factor receptor (EGFR) inhibitors (AG1478 and PD153035) and EGFR siRNA. Short-term stimulation with Pb²+ induced EGFR phosphorylation at the Tyr residue (position, 1173). Importantly, overexpression of EGFR resulted in a significant potentiation effect on Pb²+-induced gene expression. Taken together, our results indicate that 1 µM Pb²+ can induce PGE2 secretion by upregulating the transcription of COX-2/cPLA2 genes. EGFR is the key target in the plasma membrane responsible for transmitting Pb²+ signals in order to trigger downstream inflammatory cascades.