Biology subjects

Moller, A. G.

Publications and source records attributed to Moller, A. G..

3 recordsLinked to original sources

bioRxiv2022

Development of an amplicon nanopore sequencing strategy for detection of mutations conferring intermediate resistance to vancomycin in Staphylococcus aureus strains

Staphylococcus aureus is a major cause of bacteremia and other hospital-acquired infections. The cell-wall active antibiotic vancomycin is commonly used to treat both methicillin-resistant (MRSA) and sensitive (MSSA) infections, but vancomycin intermediate S. aureus (VISA) variants can arise through de novo mutations. Here we performed pilot experiments to develop a combined PCR/long-read sequencing-based method for detection of previously known VISA-causing mutations. We amplified 16 genes (walR, walK, rpoB, graR, graS, vraF, vraG, stpI, vraR, vraS, agrA, sarA, clpP, ccpA, prsA, and yvqF) known from prior studies to be associated with mutations responsible for VISA as 10 amplicons and sequenced amplicon pools as long-reads with Oxford Nanopore adapter ligation on Flongle flow cells. We then detected mutations by mapping reads against a parental consensus or known reference sequence and comparing called variants against a database of known VISA mutations from laboratory selection. There was high (>1000x) coverage of each amplicon in the pool, no relationship between amplicon length and coverage, and the ability to detect the causative mutation (walK 646C>G) in a VISA mutant derived from the USA300 strain (N384-3 from parental strain N384). Mixing mutant (N384-3) and parental (N384) DNA at various ratios from 0 to 1 mutant suggested a mutation detection threshold of roughly the average minor allele frequency of 6.5% at 95% confidence (two standard errors above mean mutation frequency). The study lays the groundwork for direct S. aureus antibiotic phenotype inference using rapid nanopore sequencing from clinical samples. ImportanceBacteremia mortality is known to increase rapidly with time after infection, making rapid diagnostics and treatment necessary. Successful treatment depends on correct administration of antibiotics based on knowledge of strain antibiotic susceptibility. Staphylococcus aureus is a major causative agent of bacteremia and is also increasingly antibiotic resistant. In this work, we develop a method to improve detection of a complex, polygenic antibiotic resistance phenotype in S. aureus, vancomycin-intermediate resistance (VISA) through long-read genomic sequencing of amplicons representing genes most commonly mutated in VISA selection. This method both speeds up VISA determination relative to purely culture-based detection and incorporates the most comprehensive database of VISA genetic determinants known to date.

microbiology↗

bioRxiv2021

Species-scale genomic analysis of S. aureus genes influencing phage host range and their relationships to virulence and antibiotic resistance genes

Phage therapy has been proposed as a possible alternative treatment for infections caused by the ubiquitous bacterial pathogen Staphylococcus aureus. However, successful phage therapy requires knowing both host and phage genetic factors influencing host range for rational cocktail formulation. To further our understanding of host range, we searched 40,000+ public S. aureus genome sequences for previously identified phage resistance genes. We found that phage adsorption targets and genes that block phage assembly were significantly more conserved than genes targeting phage biosynthesis. Core phage resistance genes had similar nucleotide diversity, ratio of non-synonymous to synonymous substitutions, and functionality (measured by delta-bitscore) to other core genes in a set of 380 non-redundant S. aureus genomes (each from a different MLST sequence type). Non-core phage resistance genes were significantly less consistent with the core genome phylogeny than all non-core genes in this set. Only superinfection immunity genes correlated with empirically determined temperate phage resistance, accessory genome content, and numbers of accessory antibiotic resistance or virulence genes encoded per strain. Taken together, these results suggested that, while phage adsorption genes are heavily conserved in the S. aureus species, they are not undergoing positive selection, arms race dynamics. They also suggested genes classified as involved in assembly are least phylogenetically constrained and superinfection immunity genes best predict both empirical phage resistance and levels of phage-mediated HGT. ImportanceStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen that is commonly antibiotic resistant. It causes diverse diseases affecting both the skin and internal organs. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent, such as phage therapy, in which viruses specific to infecting bacteria clear infection. S. aureus phage host range not only determines whether phage therapy will be successful by killing bacteria but also horizontal gene transfer through transduction of host genetic material by phages. In this work, we comprehensively reviewed existing literature to build a list of S. aureus phage resistance genes and searched our database of almost 43,000 S. aureus genomes for these genes to understand their patterns of evolution, finding that prophages superinfection immunity correlates best with phage resistance and HGT. These findings improved our understanding of the relationship between known phage resistance genes and phage host range in the species.

microbiology↗

bioRxiv2020

Genes influencing phage host range in Staphylococcus aureus on a species-wide scale

Staphylococcus aureus is a human pathogen that causes serious diseases ranging from skin infections to septic shock. Bacteriophages ("phages") are both natural killers of S. aureus, offering therapeutic possibilities, as well as important vectors of horizontal gene transfer in the species. Here, we used high-throughput approaches to understand the genetic basis of strain-to-strain variation in sensitivity to phages, which defines the host range. We screened 259 diverse S. aureus strains covering more than 40 sequence types for sensitivity to eight phages, which were representatives of the three phage classes that infect the species. The phages were variable in host range, each infecting between 73 and 257 strains. Using genome-wide association approaches, we identified putative loci that affect host range and validated their function using USA300 transposon knockouts. In addition to rediscovering known host range determinants, we found several previously unreported genes affecting bacterial growth during phage infection, including trpA, phoR, isdB, sodM, fmtC, and relA. We used the data from our host range matrix to develop predictive models that achieved between 40 and 95% accuracy. This work illustrates the complexity of the genetic basis for phage susceptibility in S. aureus but also shows that with more data, we may be able to understand much of the variation. With a knowledge of host range determination, we can rationally design phage therapy cocktails that target the broadest host range of S. aureus strains and address basic questions regarding phage-host interactions, such as the impact of phage on S. aureus evolution. ImportanceStaphylococcus aureus is a widespread, hospital- and community-acquired pathogen, many strains of which are antibiotic resistant. It causes diverse diseases ranging from local to systemic infection and affects both the skin and many internal organs, including the heart, lungs, bones, and brain. Its ubiquity, antibiotic resistance, and disease burden make new therapies urgent. One alternative therapy to antibiotics is phage therapy, in which viruses specific to infecting bacteria clear infection. In this work, we identified and validated S. aureus genes that influence phage host range - the number of strains a phage can infect and kill - by testing strains representative of the diversity of the S. aureus species for phage host range and associating strains genome sequences with host range. These findings together improved our understanding of how phage therapy works in the bacterium and improve prediction of phage therapy efficacy based on the infecting strains predicted host range.

microbiology↗