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Sunday, September 22, 2019

Convergent evolution driven by rifampin exacerbates the global burden of drug-resistant Staphylococcus aureus.

Mutations in the beta-subunit of bacterial RNA polymerase (RpoB) cause resistance to rifampin (Rifr), a critical antibiotic for treatment of multidrug-resistantStaphylococcus aureus.In vitrostudies have shown that RpoB mutations confer decreased susceptibility to other antibiotics, but the clinical relevance is unknown. Here, by analyzing 7,099S. aureusgenomes, we demonstrate that the most prevalent RpoB mutations promote clinically relevant phenotypic plasticity resulting in the emergence of stableS. aureuslineages, associated with increased risk of therapeutic failure through generation of small-colony variants (SCVs) and coresistance to last-line antimicrobial agents. We found eight RpoB mutations that accounted for 93% (469/505) of the total number of Rifrmutations. The most…

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Sunday, September 22, 2019

Plasmid-encoded transferable mecB-mediated methicillin resistance in Staphylococcus aureus.

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors…

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Sunday, September 22, 2019

Characterization and heterologous expression of the neoabyssomicin/abyssomicin biosynthetic gene cluster from Streptomyces koyangensis SCSIO 5802.

The deep-sea-derived microbe Streptomyces koyangensis SCSIO 5802 produces neoabyssomicins A-B (1-2) and abyssomicins 2 (3) and 4 (4). Neoabyssomicin A (1) augments human immunodeficiency virus-1 (HIV-1) replication whereas abyssomicin 2 (3) selectively reactivates latent HIV and is also active against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Structurally, neoabyssomicins A-B constitute a new subtype within the abyssomicin family and feature unique structural traits characteristic of extremely interesting biosynthetic transformations.In this work, the biosynthetic gene cluster (BGC) for the neoabyssomicins and abyssomicins, composed of 28 opening reading frames, was identified in S. koyangensis SCSIO 5802, and its role in neoabyssomicin/abyssomicin biosynthesis…

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Sunday, September 22, 2019

The impact of Staphylococcus aureus genomic variation on clinical phenotype of children with acute hematogenous osteomyelitis.

Children with acute hematogenous osteomyelitis (AHO) have a broad spectrum of illness ranging from mild to severe. The purpose of this study is to evaluate the impact of genomic variation of Staphylococcus aureus on clinical phenotype of affected children and determine which virulence genes correlate with severity of illness.De novo whole genome sequencing was conducted for a strain of Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA), using PacBio Hierarchical Genome Assembly Process (HGAP) from 6 Single Molecule Real Time (SMRT) Cells, as a reference for DNA library assembly of 71 Staphylococcus aureus isolates from children with AHO. Virulence gene annotation…

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Sunday, September 22, 2019

Adaptation of Pseudomonas aeruginosa to phage PaP1 predation via O-antigen polymerase mutation.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis.…

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Sunday, September 22, 2019

Genomic comparison of highly virulent, moderately virulent, and avirulent strains from a genetically closely-related MRSA ST239 sub-lineage provides insights into pathogenesis.

The genomic comparison of virulent (TW20), moderately virulent (CMRSA6/CMRSA3), and avirulent (M92) strains from a genetically closely-related MRSA ST239 sub-lineage revealed striking similarities in their genomes and antibiotic resistance profiles, despite differences in virulence and pathogenicity. The main differences were in the spa gene (coding for staphylococcal protein A), lpl genes (coding for lipoprotein-like membrane proteins), cta genes (genes involved in heme synthesis), and the dfrG gene (coding for a trimethoprim-resistant dihydrofolate reductase), as well as variations in the presence or content of some prophages and plasmids, which could explain the virulence differences of these strains. TW20 was positive for…

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Sunday, September 22, 2019

Prevalence and genomic structure of bacteriophage phi3 in human derived livestock-associated MRSA from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for…

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Sunday, September 22, 2019

Complete genome sequence and characterization of linezolid-resistant Enterococcus faecalis clinical isolate KUB3006 carrying a cfr(B)-transposon on its chromosome and optrA-plasmid.

Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic…

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Sunday, September 22, 2019

SKA: Split Kmer Analysis Toolkit for Bacterial Genomic Epidemiology

Genome sequencing is revolutionising infectious disease epidemiology, providing a huge step forward in sensitivity and specificity over more traditional molecular typing techniques. However, the complexity of genome data often means that its analysis and interpretation requires high-performance compute infrastructure and dedicated bioinformatics support. Furthermore, current methods have limitations that can differ between analyses and are often opaque to the user, and their reliance on multiple external dependencies makes reproducibility difficult. Here I introduce SKA, a toolkit for analysis of genome sequence data from closely-related, small, haploid genomes. SKA uses split kmers to rapidly identify variation between genome sequences, making it…

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Sunday, September 22, 2019

Conjugative transfer of a novel Staphylococcal plasmid encoding the biocide resistance gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and…

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Sunday, September 22, 2019

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous…

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Sunday, September 22, 2019

Chemical Synergy between Ionophore PBT2 and Zinc Reverses Antibiotic Resistance.

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer’s and Huntington’s disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to…

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Saturday, September 21, 2019

Functional analysis of the first complete genome sequence of a multidrug resistant sequence type 2 Staphylococcus epidermidis.

Staphylococcus epidermidis is a significant opportunistic pathogen of humans. The ST2 lineage is frequently multidrug resistant and accounts for most of the clinical disease worldwide. However, there are no publically available, closed ST2 genomes and pathogenesis studies have not focused on these strains. We report the complete genome and methylome of BPH0662, a multidrug resistant, hospital adapted, ST2 S. epidermidis, and describe the correlation between resistome and phenotype, as well as demonstrate its relationship to publically available, international ST2 isolates. Furthermore, we delineate the methylome determined by the two type I restriction modification systems present in BPH0662 through heterologous expression…

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Saturday, September 21, 2019

A flexible and efficient template format for circular consensus sequencing and SNP detection.

A novel template design for single-molecule sequencing is introduced, a structure we refer to as a SMRTbell template. This structure consists of a double-stranded portion, containing the insert of interest, and a single-stranded hairpin loop on either end, which provides a site for primer binding. Structurally, this format resembles a linear double-stranded molecule, and yet it is topologically circular. When placed into a single-molecule sequencing reaction, the SMRTbell template format enables a consensus sequence to be obtained from multiple passes on a single molecule. Furthermore, this consensus sequence is obtained from both the sense and antisense strands of the insert…

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