Beta-Lactamase Repressor BlaI Modulates Staphylococcus aureus Cathelicidin Antimicrobial Peptide Resistance and Virulence.

BlaI is a repressor of BlaZ, the beta-lactamase responsible for penicillin resistance in Staphylococcus aureus. Through screening a transposon library in S. aureus Newman for susceptibility to cathelicidin antimicrobial peptide, we discovered BlaI as a novel cathelicidin resistance factor. Additionally, through integrational mutagenesis in S. aureus Newman and MRSA Sanger 252 strains, we confirmed the role of BlaI in resistance to human and murine cathelidicin and showed that it contributes to virulence in human whole blood and murine infection models. We further demonstrated that BlaI could be a target for innate immune-based antimicrobial therapies; by removing BlaI through subinhibitory concentrations of 6-aminopenicillanic acid, we were able to sensitize S. aureus to LL-37 killing

Metagenomic water quality monitoring with a portable laboratory

We describe the technical feasibility of metagenomic water quality analysis using only portable equipment, for example mini-vacuum pumps and filtration units, mini-centrifuges, mini-PCR machines and the memory-stick sized MinION of Oxford Nanopore Technologies, for the library preparation and sequencing of 16S rRNA gene amplicons. Using this portable toolbox on site, we successfully characterized the microbiome of water samples collected from Birtley Sewage Treatment Plant, UK, and its environs. We also demonstrated the applicability of the portable metagenomics toolbox in a low-income country by surveying water samples from the Akaki River around Addis Ababa, Ethiopia. The 16S rRNA gene sequencing workflow, including DNA extraction, PCR amplification, sequencing library preparation, and sequencing was accomplished within one working day. The metagenomic data became available within 24e72 h, depending on internet speed. Metagenomic analysis clearly distinguished the microbiome of pristine samples from sewage influenced water samples. Metagenomic analysis identified the potential role of two bacterial genera not conventionally monitored, Arcobacter and Aeromonas, as predominant faecal pollution indicators/waterborne hazards. Subsequent quantitative PCR analysis validated the high Arcobacter butzleri abundances observed in the urban influenced Akaki River water samples by portable next generation sequencing with the MinION device. Overall, our field deployable metagenomics toolbox advances the capability of scientists to comprehensively monitor microbiomes anywhere in the world, including in the water, food and drinks industries, the health services, agriculture and beyond

Spatiotemporal variation in urban wastewater pollution impacts on river microbiomes and associated hazards in the Akaki Catchment, Addis Ababa, Ethiopia

In Addis Ababa and its environs, most urban wastewater is discharged into rivers without treatment. This study related urban wastewater characteristics to the prevalence of faecal, antibiotic resistant, and potentially pathogenic bacteria in rivers of the Akaki catchment across six locations, for the dry and wet season. Spatiotemporal variation in bacterial hazards across the catchment was up to 6 log10 units. Cooccurrence of sewage pollution marker gene HF183 in all river samples testing positive for the Vibrio cholerae marker gene ompW, and high levels of these two genes in untreated wastewater, identified human sewage as the likely source of Vibrio cholerae hazards in the catchment. Levels of the marker genes rodA for E. coli, HF183 for human host associated Bacteroides, ciaB for Arcobacter, and ompW for Vibrio cholerae were all higher in the dry season than in the wet season. Marker gene gyrB for Pseudomonas aeruginosa was not detected in the samples. From the sequencing data, notable bacterial genera in the dry season included wastewater pollution indicators Arcobacter and Aeromonas, whereas soil erosion may explain the greater prominence of Legionella, Vicinamibacter, and Sphingomonas during the wet season. Except for the most upstream location, all faecal coliform (FC) counts exceeded WHO standards of 1000 CFU/100 mL for unrestricted irrigation. Concerningly, 0.6–20% of FC had ESBL producing antimicrobial resistance traits. In conclusion, multiple bacterial hazards were of concern for river water users in the Akaki catchment, and elevated in the dry season, when the river water is being used for irrigation of vegetable fields that supply the markets of Addis Ababa. This reflects inadequate treatment and limited dilution of urban wastewater by the natural river flows during periods of low rainfall

Use of Indigenous informed epistemologies can inform intervention models to fight COVID-19 in Africa

The coronavirus disease 2019 (COVID-19) has put the world in unprecedented health and economic crisis threatening human existence and livelihoods. The pandemic has brought the world to a standstill and, has infected over 15 million and killed over 620 thousand people globally1 . The socioeconomic impact of the pandemic is also expected to be higher. Reports indicate that in the USA alone, over 38 million people have lost their jobs due to the pandemic. Reports from Europe and other parts of the world also depict a grim picture. The pandemic has affected other regions such as Australasia where significant loss of jobs and livelihoods, and changes in societal resilience manifesting in increased mental health problems and domestic violence were reported

Potential mechanisms of cathelicidin resistance.

<p>(A) Surface charge of <i>S</i>. <i>aureus</i> Newman and MRSA252 strains was compared by poly-L-lysine binding. The <i>S</i>. <i>aureus</i> strain Sa113 and an isogenic <i>mprF</i> mutant with increased negative surface charge were used as controls. (B, C) Hydrophobicity was measured using a modified version of the MATH (microbial adhesion to hydrocarbons) assay. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001; n.s., not significant. (D) Proteolytic activity of Newman WT + pDC123, the <i>blaI</i> mutant + pDC123 and the <i>blaI</i> mutant complemented with pBlaI on skim milk agar plates. Clearance zones around colonies indicate secreted protease activity. (E) Degradation of CRAMP or LL-37 by overnight cell-free supernatants analyzed by SDS-PAGE.</p

BlaI contributes to survival in human whole blood and virulence <i>in vivo</i>.

<p>(A) <i>S</i>. <i>aureus</i> Newman WT with empty complementation vector pDC123, the <i>blaI</i> mutant with pDC123, and the complemented <i>blaI</i> mutant strain were incubated for 1 h in human whole blood and CFU numbers enumerated. Samples were run in triplicate and data were plotted as the average percentage ± SD for each strain as compared to the initial inocula. A representative experiment of three performed is shown. **, <i>p</i><0.01. (B) CD-1 mice (<i>n</i> = 8) were injected subcutaneously on one flank with <i>S</i>. <i>aureus</i> Newman WT and on the opposite flank with <i>blaI</i> mutant bacteria, and lesion sizes were monitored for 7 days. The lesions for each individual mouse at Day 7 are plotted and the average value indicated. Overall, the <i>blaI</i> mutant lesions were significantly smaller compared to the WT (<i>p</i><0.04; paired t-test). (C-D) Survival of CD-1 mice (<i>n</i> = 10) after intraperitoneal infection with (C) 1 x 10⁶ CFU of <i>S</i>. <i>aureus</i> Newman WT or Newman <i>blaI</i> mutant or (D) 6 x 10⁸ CFU of MRSA252 or MRSA252 <i>blaI</i> mutant. Survival was monitored for 3 days. The survival for Newman or MRSA252 <i>blaI</i> mutant strain infected mice was significantly higher than for the WT infected strains as assessed by log-rank (Mantel Cox) test; the <i>p</i> values are shown in the respective graphs.</p

Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases

<div><p>Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the β3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved β3-lysine was essential for phosphoryl transfer, but our findings show that the β3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics.</p></div

MinION nanopore sequencing accelerates progress towards ubiquitous genetics in water research

In 2014, Oxford Nanopore Technologies (ONT) introduced an affordable and portable sequencer called MinION. We reviewed emerging applications in water research and assessed progress made with this platform towards ubiquitous genetics. With >99% savings in upfront costs as compared to conventional platforms, the MinION put sequencing capacity into the hands of many researchers and enabled novel applications with diverse remits, including in countries without universal access to safe water and sanitation. However, to realize the MinION’s fabled portability, all the auxiliary equipment items for biomass concentration, genetic material extraction, cleanup, quantification, and sequencing library preparation also need to be lightweight and affordable. Only a few studies demonstrated fully portable workflows by using the MinION onboard a diving vessel, an oceanographic research ship, and at sewage treatment works. Lower nanopore sequencing read accuracy as compared to alternative platforms currently hinders MinION applications beyond research, and inclusion of positive and negative controls should become standard practice. ONT’s EPI2ME platform is a major step towards user-friendly bioinformatics. However, no consensus has yet emerged regarding the most appropriate bioinformatic pipeline, which hinders intercomparison of study results. Processing, storing, and interpreting large data sets remains a major challenge for ubiquitous genetics and democratizing sequencing applications