Repositioning of the global epicentre of non-optimal cholesterol

High blood cholesterol is typically considered a feature of wealthy western countries(1,2). However, dietary and behavioural determinants of blood cholesterol are changing rapidly throughout the world(3) and countries are using lipid-lowering medications at varying rates. These changes can have distinct effects on the levels of high-density lipoprotein (HDL) cholesterol and non-HDL cholesterol, which have different effects on human health(4,5). However, the trends of HDL and non-HDL cholesterol levels over time have not been previously reported in a global analysis. Here we pooled 1,127 population-based studies that measured blood lipids in 102.6 million individuals aged 18 years and older to estimate trends from 1980 to 2018 in mean total, non-HDL and HDL cholesterol levels for 200 countries. Globally, there was little change in total or non-HDL cholesterol from 1980 to 2018. This was a net effect of increases in low- and middle-income countries, especially in east and southeast Asia, and decreases in high-income western countries, especially those in northwestern Europe, and in central and eastern Europe. As a result, countries with the highest level of non-HDL cholesterol-which is a marker of cardiovascular riskchanged from those in western Europe such as Belgium, Finland, Greenland, Iceland, Norway, Sweden, Switzerland and Malta in 1980 to those in Asia and the Pacific, such as Tokelau, Malaysia, The Philippines and Thailand. In 2017, high non-HDL cholesterol was responsible for an estimated 3.9 million (95% credible interval 3.7 million-4.2 million) worldwide deaths, half of which occurred in east, southeast and south Asia. The global repositioning of lipid-related risk, with non-optimal cholesterol shifting from a distinct feature of high-income countries in northwestern Europe, north America and Australasia to one that affects countries in east and southeast Asia and Oceania should motivate the use of population-based policies and personal interventions to improve nutrition and enhance access to treatment throughout the world.Peer reviewe

Targeting of PBP1 by β-lactams determines recA/SOS response activation in heterogeneous MRSA clinical strains.

The SOS response, a conserved regulatory network in bacteria that is induced in response to DNA damage, has been shown to be associated with the emergence of resistance to antibiotics. Previously, we demonstrated that heterogeneous (HeR) MRSA strains, when exposed to sub-inhibitory concentrations of oxacillin, were able to express a homogeneous high level of resistance (HoR). Moreover, we showed that oxacillin appeared to be the triggering factor of a β-lactam-mediated SOS response through lexA/recA regulators, responsible for an increased mutation rate and selection of a HoR derivative. In this work, we demonstrated, by selectively exposing to β-lactam and non-β-lactam cell wall inhibitors, that PBP1 plays a critical role in SOS-mediated recA activation and HeR-HoR selection. Functional analysis of PBP1 using an inducible PBP1-specific antisense construct showed that PBP1 depletion abolished both β-lactam-induced recA expression/activation and increased mutation rates during HeR/HoR selection. Furthermore, based on the observation that HeR/HoR selection is accompanied by compensatory increases in the expression of PBP1,-2, -2a, and -4, our study provides evidence that a combination of agents simultaneously targeting PBP1 and either PBP2 or PBP2a showed both in-vitro and in-vivo efficacy, thereby representing a therapeutic option for the treatment of highly resistant HoR-MRSA strains. The information gathered from these studies contributes to our understanding of β-lactam-mediated HeR/HoR selection and provides new insights, based on β-lactam synergistic combinations, that mitigate drug resistance for the treatment of MRSA infections

Exposure of clinical MRSA heterogeneous strains to β-lactams redirects metabolism to optimize energy production through the TCA cycle.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as one of the most important pathogens both in health care and community-onset infections. The prerequisite for methicillin resistance is mecA, which encodes a β-lactam-insensitive penicillin binding protein PBP2a. A characteristic of MRSA strains from hospital and community associated infections is their heterogeneous expression of resistance to β-lactam (HeR) in which only a small portion (≤ 0.1%) of the population expresses resistance to oxacillin (OXA) ≥ 10 µg/ml, while in other isolates, most of the population expresses resistance to a high level (homotypic resistance, HoR). The mechanism associated with heterogeneous expression requires both increase expression of mecA and a mutational event that involved the triggering of a β-lactam-mediated SOS response and related lexA and recA genes. In the present study we investigated the cellular physiology of HeR-MRSA strains during the process of β-lactam-mediated HeR/HoR selection at sub-inhibitory concentrations by using a combinatorial approach of microarray analyses and global biochemical profiling employing gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) to investigate changes in metabolic pathways and the metabolome associated with β-lactam-mediated HeR/HoR selection in clinically relevant heterogeneous MRSA. We found unique features present in the oxacillin-selected SA13011-HoR derivative when compared to the corresponding SA13011-HeR parental strain that included significant increases in tricarboxyl citric acid (TCA) cycle intermediates and a concomitant decrease in fermentative pathways. Inactivation of the TCA cycle enzyme cis-aconitase gene in the SA13011-HeR strain abolished β-lactam-mediated HeR/HoR selection demonstrating the significance of altered TCA cycle activity during the HeR/HoR selection. These results provide evidence of both the metabolic cost and the adaptation that HeR-MRSA clinical strains undergo when exposed to β-lactam pressure, indicating that the energy production is redirected to supply the cell wall synthesis/metabolism, which in turn contributes to the survival response in the presence of β-lactam antibiotics

Minimal inhibitory concentration (MIC) of SA13011-HeR <i>recA</i>-lacZ (LMR21) to antimicrobial agents used in the study.

<p>Minimal inhibitory concentration (MIC) of SA13011-HeR <i>recA</i>-lacZ (LMR21) to antimicrobial agents used in the study.</p

β-galactosidase activity (Miller units) of SA13011-HeR(<i>recA</i>P::<i>lacZ</i>) (LMR21) grown in the absence or presence of (1/4 MICs values) the corresponding antibiotics.

<p>The number of Miller units is expressed in fold change. Values represent the mean ± SD of three independent replicates.</p

Mutation rate analysis during OXA-induced HeR/HoR selection of parents and homogenous derivatives of SA13011, LMR25, and LMR23 strains.

<p>Mutation frequency was determined using rifampicin selection and expressed as a ratio of rifampicin resistance as a fraction of viable cells, during time points at 6, 27, and 33 h of the selection process, −/+OXA 0.5 µg/ml (*).</p

Strains, plasmids, and primers used in this study.

<p>Strains, plasmids, and primers used in this study.</p

Treatment effect using <i>Galleria mellonella</i>.

<p>Groups of larvae (10/group) were inoculated with either 10 µl of PBS (uninfected control group; A) or bacterial suspension containing 1.5×10⁶ CFU/ml of 13011-HoR (B–D) into the last proleg and incubated for 2 h at 37C. After this, 10 µl of IMP (10 mg/kg), NAF (5 mg/kg; B), CTX (10 mg/kg; C), BAL (5 mg/kg; D), or the corresponding IMP/β-lactam were administered (time 0) into the right hind-most proleg, and re-incubated for 24 h at 37°C. PBS treatment was given as a control with multiple injections. The treatment was repeated after the first 24 h incubation. Worms were checked daily and recorded for any deaths for a total of 10 days. A minimum of three independent experimental runs were performed for each experiment. Survival data were plotted using the Kaplan-Meir method.</p

Time-course analysis of growth (A) and rate of DNA replication (B) was performed in SA13011-HeR −/+ OXA (0.5 µg/ml) during β-lactam-induced HeR/HoR selection.

<p>Bacteria were grown −/+ OXA (0.5 µg/ml) overnight. Cultures without OXA (HeR) were then diluted to an optical density at 600 nm (OD₆₀₀) equivalent to the culture growing with OXA (HoR) and continued to grow at 37°C with shaking. (B) Both SA13011-HeR/-HoR cultures were supplemented with the radioactive methyl-[3H]-thymine precursor (1 µCi/ml); triplicates samples were removed at different time-intervals and collected for DNA extraction, eluted and transferred to Whatman paper filters; after drying, radioactivity was measured by liquid scintillation. Values represent the means of three biological replicates ± standard error of the mean (SEM), sampled in triplicate to minimize error by inter- and intra-samples, respectively.</p

Analysis of antibacterial efficacy against SA13011-HoR.

<p>Synergy time kill analysis was performed using Mueller-Hinton (MH) broth with 10⁶ CFU/ml inoculums at 0, 2, 4, 6, 8, and 24 h. Antibiotics were used as specified at the following concentrations: IMP: 0.12 µg/ml; FOX: 4 µg/ml; BAL: 0.5 µg/ml, and CTX: 4 µg/ml. A minimum of three independent experimental runs were performed for each IMP/β-lactam combination.</p