Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac.

BACKGROUND: Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. METHODS: Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. RESULTS: Growth of S. aureus strain COL with 80 µg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. CONCLUSIONS: The results of this study suggest that diclofenac influences antibiotic susceptibility in S. aureus, in part, by altering the expression of regulatory and structural genes associated with cell wall biosynthesis/turnover and transport.

Reevaluation of the effect of dietary restriction on different recombinant inbred lines of male and female mice.

Dietary restriction (DR) was reported to either have no effect or reduce the lifespan of the majority of the 41-recombinant inbred (RI) lines studied by Liao et al. (Aging Cell, 2010, 9, 92). In an appropriately power longevity study (n > 30 mice/group), we measured the lifespan of the four RI lines (115-RI, 97-RI, 98-RI, and 107-RI) that were reported to have the greatest decrease in lifespan when fed 40% DR. DR increased the median lifespan of female RI-115, 97-RI, and 107-RI mice and male 115-RI mice. DR had little effect (<4%) on the median lifespan of female and male 98-RI mice and male 97-RI mice and reduced the lifespan of male 107-RI mice over 20%. While our study was unable to replicate the effect of DR on the lifespan of the RI mice (except male 107-RI mice) reported by Liao et al. (Aging Cell, 2010, 9, 92), we found that the genotype of a mouse had a major impact on the effect of DR on lifespan, with the effect of DR ranging from a 50% increase to a 22% decrease in median lifespan. No correlation was observed between the changes in either body composition or glucose tolerance induced by DR and the changes observed in lifespan of the four RI lines of male and female mice. These four RI lines of mice give the research community a unique resource where investigators for the first time can study the anti-aging mechanism of DR by comparing mice in which DR increases lifespan to mice where DR has either no effect or reduces lifespan.

Litter expansion alters metabolic homeostasis in a sex specific manner.

Nutritional manipulations early in life have been shown to influence growth rate and elicit long lasting effects which in turn has been found to impact lifespan. Therefore, we studied the long-term effects of pre-weaning dietary restriction implemented by litter expansion (4, 6, 8, 10, and 12 pups per dam: LS4, LS6, LS8, LS10, LS12) on male and female C57BL/6J mice. After weaning, these mice were fed ad libitum a commercial lab chow for the 15-month duration of the study. The male mice from large litter size (LS12) were significantly leaner and had reduced total fat mass compared to the normal size litters (LS 6) starting from weaning through to 15 months of age. Male LS10 & 12 mice also showed significant reduction in their fat depot masses at 15 months of age: gonadal, subcutaneous, and brown fat whereas the females did not mimic these findings. At 9 months of age, only male LS12 mice showed improved glucose tolerance and male LS12 mice also showed improved insulin tolerance starting at 5 months of age. In addition, we found that the male LS8, 10 & 12 mice at 15 months of age showed significantly reduced IGF-1 levels in the serum and various other organs (liver, gastrocnemius and brain cortex). Interestingly, the female LS8, 10, 12 mice showed a different pattern with reduced IGF-1 levels in serum, liver and gastrocnemius but not in the brain cortex. Similarly, the litter expanded mice showed sex specific response to levels of FGF21 and adiponectin with only the male mice showing increased FGF21 and adiponectin levels at 15 months of age. In summary, our data show that, litter expansion results in long-lasting metabolic changes that are age and sex dependent with the male mice showing an early and robust response compared to female mice.

Calorie restriction prevents age-related changes in the intestinal microbiota.

The effect of calorie restriction (CR) on the microbiome, fecal metabolome, and colon transcriptome of adult and old male mice was compared. Life-long CR increased microbial diversity and the Bacteroidetes/Firmicutes ratio and prevented the age-related changes in the microbiota, shifting it to a younger microbial and fecal metabolite profile in both C57BL/6JN and B6D2F1 mice. Old mice fed CR were enriched in the Rikenellaceae, S24-7 and Bacteroides families. The changes in the microbiome that occur with age and CR were initiated in the cecum and further modified in the colon. Short-term CR in adult mice had a minor effect on the microbiome but a major effect on the transcriptome of the colon mucosa. These data suggest that CR has a major impact on the physiological status of the gastrointestinal system, maintaining it in a more youthful state, which in turn could result in a more diverse and youthful microbiome.

Enhanced matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of bacteriophage major capsid proteins with beta-mercaptoethanol pretreatment.

Bacteriophage (phage) proteins have been analyzed previously with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). However, analysis of phage major capsid proteins (MCPs) has been limited by the ability to reproducibly generate ions from MCP monomers. While the acidic conditions of MALDI-TOF MS sample preparation have been shown to aid in disassembly of some phage capsids, many require further treatment to successfully liberate MCP monomers. The findings presented here suggest that beta-mercaptoethanol reduction of the disulfide bonds linking phage MCPs prior to mass spectrometric analysis results in significantly increased MALDI-TOF MS sensitivity and reproducibility of Yersinia pestis-specific phage protein profiles.

Necroptosis increases with age and is reduced by dietary restriction.

Necroptosis is a newly identified programmed cell death pathway that is highly proinflammatory due to the release of cellular components that promote inflammation. To determine whether necroptosis might play a role in inflammaging, we studied the effect of age and dietary restriction (DR) on necroptosis in the epididymal white adipose tissue (eWAT), a major source of proinflammatory cytokines. Phosphorylated MLKL and RIPK3, markers of necroptosis, were increased 2.7- and 1.9-fold, respectively, in eWAT of old mice compared to adult mice, and DR reduced P-MLKL and P-RIPK3 to levels similar to adult mice. An increase in the expression of RIPK1 (1.6-fold) and MLKL (2.7-fold), not RIPK3, was also observed in eWAT of old mice, which was reduced by DR in old mice. The increase in necroptosis was paralleled by an increase in 14 inflammatory cytokines, including the pro-inflammatory cytokines IL-6 (3.9-fold), TNF-α (4.7-fold), and IL-1ß (5.1-fold)], and 11 chemokines in old mice. DR attenuated the expression of IL-6, TNF-α, and IL-1ß as well as 85% of the other cytokines/chemokines induced with age. In contrast, inguinal WAT (iWAT), which is less inflammatory, did not show any significant increase with age in the levels of P-MLKL and MLKL or inflammatory cytokines/chemokines. Because the changes in biomarkers of necroptosis in eWAT with age and DR paralleled the changes in the expression of pro-inflammatory cytokines, our data support the possibility that necroptosis might play a role in increased chronic inflammation observed with age.

The effect of different levels of dietary restriction on glucose homeostasis and metabolic memory.

Over the past 50 years, dietary restriction (DR) has been shown to extend the life span of a wide variety of organisms. A hallmark feature of DR is improved glucose homeostasis resulting in increased glucose tolerance and insulin sensitivity of animals ranging from rodents to humans. In this study, we demonstrate the early effects of varying levels of DR on glucose tolerance. Within 10 days of 40% DR, glucose tolerance was significantly improved and by 120 days; 10 and 20% DR also showed enhanced glucose tolerance. All three levels of DR showed reduced adiposity, increased expression of genes involved in fat turnover, and a reduction in the expression for markers of inflammation. Studies have shown that mice fed a DR diet retained metabolic memory in terms of improved glucose tolerance even after DR is discontinued. We show that 40% DR not only has an early effect on glucose tolerance but also maintained it after DR was discontinued for 2 months. Therefore, improvement in glucose tolerance is brought about by all three levels of DR but the metabolic memory is not dose responsive.

A fish oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein response in epididymal white adipose tissue of mice.

White adipose tissue (WAT) mitochondrial dysfunction is linked to the pathogenesis of obesity driven insulin resistance. Dietary conditions that alter fat mass are known to affect white adipocyte mitochondrial function, however, the impact of high calorie diets on white adipocyte mitochondria is not fully understood. The aim of this study is to assess the effect of a diet rich in saturated or polyunsaturated fat on mitochondrial unfolded protein response (UPRmt), a retrograde signaling response that maintains mitochondrial homeostasis, in epididymal WAT (eWAT). Mice were fed a low fat diet (LFD), saturated fat diet (SFD) or fish oil (unsaturated fat diet, UFD) and assessed changes in eWAT mitochondria. Compared to mice fed a LFD, SFD-fed mice have reduced mitochondrial biogenesis markers, mitochondrial fatty acid oxidation enzymes and TCA cycle enzymes, suggesting an impaired mitochondrial function that could contribute to increased fat mass. In contrast, isocaloric UFD-fed mice have increased expression of mitochondrial uncoupling protein 1 (UCP1) and peroxisomal fatty acid oxidation enzymes suggesting that elevated mitochondrial uncoupling and peroxisomal fatty acid oxidation could contribute to the reduction in fat mass. Interestingly, expression of UPRmt-associated proteins caseinolytic peptidase (ClpP) and heat shock protein 60 (Hsp60) are induced by UFD, whereas SFD reduced the expression of ClpP. Based on our data, we propose that induction of UPRmt helps to preserve a functional mitochondria and efficient utilization of fat by UFD whereas a dampened UPRmt response might impair mitochondrial function and promote fat accumulation by SFD. Thus, our findings suggest a potential role of UPRmt in mediating the beneficial effects of fish oil.

Role of DNA methylation in the dietary restriction mediated cellular memory.

An important facet of dietary restriction (DR) that has been largely overlooked is that DR can have early effects that create a cellular memory, which persists even when DR is discontinued. The goal of this study was to determine if DNA methylation played a role in the cellular memory of DR by examining the effect of short-term DR on gene expression and DNA methylation and determining if the changes in expression and DNA methylation persist when DR is discontinued and mice returned to ad libitum (AL) feeding. We show that DR can induce substantial changes in gene expression within 1 month of its implementation in various tissues, and more interestingly, ~19-50% of these changes in gene expression persist across the tissues even when DR is discontinued. We then determined whether DR induced changes in DNA methylation in the promoter of three candidate genes identified from our gene expression analysis (Pomc, Hsph1, and Nts1) that correlated with the changes in the expression of these genes. Decreased methylation at three specific CG sites in the promoter of the Nts1 gene encompassing the distal consensus AP-1 site was correlated with increased Nts1 expression. Both the promoter hypomethylation and increased Nts1 expression persisted even after DR was discontinued and mice fed AL, supporting our hypothesis that DNA methylation could play a role in the memory effect of DR. The changes in DNA methylation in the Nts1 gene are likely to occur in intestinal stem cells and could play a role in preserving the intestinal stem cell pool in DR mice.

Draft Genome Sequence of Strain ATCC 33958, Reported To Be Elizabethkingia miricola.

We report the draft genome of Elizabethkingia strain ATCC 33958, which has been classified as Elizabethkingia miricola. Similar to other Elizabethkingia species, the ATCC 33958 draft genome contains numerous ß-lactamase genes. ATCC 33958 also harbors a urease gene cluster which supports classification as E. miricola.

Draft Genome Sequences of Vancomycin-Susceptible Staphylococcus aureus Related to Heterogeneous Vancomycin-Intermediate S. aureus.

We report the draft genome sequences of three vancomycin-susceptible methicillin-resistant Staphylococcus aureus strains. S. aureus strain MV8 is a sequence type 8 (ST-8) staphylococcal cassette chromosome mec element type IV (SCCmec IV) derivative, while the other two strains (S. aureus MM25 and MM61) are ST-5 SCCmec II strains. MM61 is also closely related to the heterogeneous vancomycin-intermediate S. aureus strain MM66.

Draft Genomes of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain MM66 and MM66 Derivatives with Altered Vancomycin Resistance Levels.

The draft genomes of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) strain MM66 and MM66 isolates demonstrating altered vancomycin resistance levels were produced in an effort to provide information on mutations contributing to the vancomycin resistance levels observed in these strains.

Draft Genome Sequences of Elizabethkingia meningoseptica.

Elizabethkingia meningoseptica is ubiquitous in nature, exhibits a multiple-antibiotic resistance phenotype, and causes rare opportunistic infections. We now report two draft genome sequences of E. meningoseptica type strains that were sequenced independently in two laboratories.