Robert A. Petit

Dissecting Vancomycin-Intermediate Resistance in Staphylococcus aureus Using Genome-Wide Association

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes.

Transmission and Microevolution of USA300 MRSA in U.S. Households: Evidence from Whole-Genome Sequencing

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) USA300 is a successful S. aureus clone in the United States and a common cause of skin and soft tissue infections (SSTIs). We performed whole-genome sequencing (WGS) of 146 USA300 MRSA isolates from SSTIs and colonization cultures obtained from an investigation conducted from 2008 to 2010 in Chicago and Los Angeles households that included an index case with an S. aureus SSTI. Identifying unique single nucleotide polymorphisms (SNPs) and analyzing whole-genome phylogeny, we characterized isolates to understand transmission dynamics, genetic relatedness, and microevolution of USA300 MRSA within the households. We also compared the 146 USA300 MRSA isolates from our study with the previously published genome sequences of the USA300 MRSA isolates from San Diego (n = 35) and New York City (n = 277). We found little genetic variation within the USA300 MRSA household isolates from Los Angeles (mean number of SNPs ± standard deviation, 17.6 ± 35; π nucleotide diversity, 3.1 × 10−5) or from Chicago (mean number of SNPs ± standard deviation, 12 ± 19; π nucleotide diversity, 3.1 × 10−5). The isolates within a household clustered into closely related monophyletic groups, suggesting the introduction into and transmission within each household of a single common USA300 ancestral strain. From a Bayesian evolutionary reconstruction, we inferred that USA300 persisted within households for 2.33 to 8.35 years prior to sampling. We also noted that fluoroquinolone-resistant USA300 clones emerged around 1995 and were more widespread in Los Angeles and New York City than in Chicago. Our findings strongly suggest that unique USA300 MRSA isolates are transmitted within households that contain an individual with an SSTI. Decolonization of household members may be a critical component of prevention programs to control USA300 MRSA spread in the United States. IMPORTANCE USA300, a virulent and easily transmissible strain of methicillin-resistant Staphylococcus aureus (MRSA), is the predominant community-associated MRSA clone in the United States. It most commonly causes skin infections but also causes necrotizing pneumonia and endocarditis. Strategies to limit the spread of MRSA in the community can only be effective if we understand the most common sources of transmission and the microevolutionary processes that provide a fitness advantage to MRSA. We performed a whole-genome sequence comparison of 146 USA300 MRSA isolates from Chicago and Los Angeles. We show that households represent a frequent site of transmission and a long-term reservoir of USA300 strains; individuals within households transmit the same USA300 strain among themselves. Our study also reveals that a large proportion of the USA300 isolates sequenced are resistant to fluoroquinolone antibiotics. The significance of this study is that if households serve as long-term reservoirs of USA300, household MRSA eradication programs may result in a uniquely effective control method. USA300, a virulent and easily transmissible strain of methicillin-resistant Staphylococcus aureus (MRSA), is the predominant community-associated MRSA clone in the United States. It most commonly causes skin infections but also causes necrotizing pneumonia and endocarditis. Strategies to limit the spread of MRSA in the community can only be effective if we understand the most common sources of transmission and the microevolutionary processes that provide a fitness advantage to MRSA. We performed a whole-genome sequence comparison of 146 USA300 MRSA isolates from Chicago and Los Angeles. We show that households represent a frequent site of transmission and a long-term reservoir of USA300 strains; individuals within households transmit the same USA300 strain among themselves. Our study also reveals that a large proportion of the USA300 isolates sequenced are resistant to fluoroquinolone antibiotics. The significance of this study is that if households serve as long-term reservoirs of USA300, household MRSA eradication programs may result in a uniquely effective control method.

The single-species metagenome: subtyping Staphylococcus aureus core genome sequences from shotgun metagenomic data

In this study we developed a genome-based method for detecting Staphylococcus aureus subtypes from metagenome shotgun sequence data. We used a binomial mixture model and the coverage counts at >100,000 known S. aureus SNP (single nucleotide polymorphism) sites derived from prior comparative genomic analysis to estimate the proportion of 40 subtypes in metagenome samples. We were able to obtain >87% sensitivity and >94% specificity at 0.025X coverage for S. aureus. We found that 321 and 149 metagenome samples from the Human Microbiome Project and metaSUB analysis of the New York City subway, respectively, contained S. aureus at genome coverage >0.025. In both projects, CC8 and CC30 were the most common S. aureus clonal complexes encountered. We found evidence that the subtype composition at different body sites of the same individual were more similar than random sampling and more limited evidence that certain body sites were enriched for particular subtypes. One surprising finding was the apparent high frequency of CC398, a lineage often associated with livestock, in samples from the tongue dorsum. Epidemiologic analysis of the HMP subject population suggested that high BMI (body mass index) and health insurance are possibly associated with S. aureus carriage but there was limited power to identify factors linked to carriage of even the most common subtype. In the NYC subway data, we found a small signal of geographic distance affecting subtype clustering but other unknown factors influence taxonomic distribution of the species around the city.

Staphylococcus aureus viewed from the perspective of 40,000+ genomes

Low-cost Illumina sequencing of clinically-important bacterial pathogens has generated thousands of publicly available genomic datasets. Analyzing these genomes and extracting relevant information for each pathogen and the associated clinical phenotypes requires not only resources and bioinformatic skills but organism-specific knowledge. In light of these issues, we created Staphopia, an analysis pipeline, database and application programming interface, focused on Staphylococcus aureus, a common colonizer of humans and a major antibiotic-resistant pathogen responsible for a wide spectrum of hospital and community-associated infections. Written in Python, Staphopia’s analysis pipeline consists of submodules running open-source tools. It accepts raw FASTQ reads as an input, which undergo quality control filtration, error correction and reduction to a maximum of approximately 100× chromosome coverage. This reduction significantly reduces total runtime without detrimentally affecting the results. The pipeline performs de novo assembly-based and mapping-based analysis. Automated gene calling and annotation is performed on the assembled contigs. Read-mapping is used to call variants (single nucleotide polymorphisms and insertion/deletions) against a reference S. aureus chromosome (N315, ST5). We ran the analysis pipeline on more than 43,000 S. aureus shotgun Illumina genome projects in the public European Nucleotide Archive database in November 2017. We found that only a quarter of known multi-locus sequence types (STs) were represented but the top 10 STs made up 70% of all genomes. methicillin-resistant S. aureus (MRSA) were 64% of all genomes. Using the Staphopia database we selected 380 high quality genomes deposited with good metadata, each from a different multi-locus ST, as a non-redundant diversity set for studying S. aureus evolution. In addition to answering basic science questions, Staphopia could serve as a potential platform for rapid clinical diagnostics of S. aureus isolates in the future. The system could also be adapted as a template for other organism-specific databases.

Fine-scale differentiation between Bacillus anthracis and Bacillus cereus group signatures in metagenome shotgun data

Background It is possible to detect bacterial species in shotgun metagenome datasets through the presence of only a few sequence reads. However, false positive results can arise, as was the case in the initial findings of a recent New York City subway metagenome project. False positives are especially likely when two closely related are present in the same sample. Bacillus anthracis, the etiologic agent of anthrax, is a high-consequence pathogen that shares >99% average nucleotide identity with Bacillus cereus group (BCerG) genomes. Our goal was to create an analysis tool that used k-mers to detect B. anthracis, incorporating information about the coverage of BCerG in the metagenome sample. Methods Using public complete genome sequence datasets, we identified a set of 31-mer signatures that differentiated B. anthracis from other members of the B. cereus group (BCerG), and another set which differentiated BCerG genomes (including B. anthracis) from other Bacillus strains. We also created a set of 31-mers for detecting the lethal factor gene, the key genetic diagnostic of the presence of anthrax-causing bacteria. We created synthetic sequence datasets based on existing genomes to test the accuracy of a k-mer based detection model. Results We found 239,503 B. anthracis-specific 31-mers (the Ba31 set), 10,183 BCerG 31-mers (the BCerG31 set), and 2,617 lethal factor k-mers (the lef31 set). We showed that false positive B. anthracis k-mers—which arise from random sequencing errors—are observable at high genome coverages of B. cereus. We also showed that there is a “gray zone” below 0.184× coverage of the B. anthracis genome sequence, in which we cannot expect with high probability to identify lethal factor k-mers. We created a linear regression model to differentiate the presence of B. anthracis-like chromosomes from sequencing errors given the BCerG background coverage. We showed that while shotgun datasets from the New York City subway metagenome project had no matches to lef31 k-mers and hence were negative for B. anthracis, some samples showed evidence of strains very closely related to the pathogen. Discussion This work shows how extensive libraries of complete genomes can be used to create organism-specific signatures to help interpret metagenomes. We contrast “specialist” approaches to metagenome analysis such as this work to “generalist” software that seeks to classify all organisms present in the sample and note the more general utility of a k-mer filter approach when taxonomic boundaries lack clarity or high levels of precision are required.

USA300 MRSA lineages persist on multiple body sites following infection

Abstract BACKGROUND USA300 methicillin-resistant Staphylococcus aureus (MRSA) is a community- and hospital- acquired pathogen that frequently causes infections but also can survive on the human body asymptomatically as a part of the normal flora. We devised a comparative genomic strategy to track colonizing USA300 at different body sites after S. aureus infection. METHODS We sampled ST8 S. aureus from subjects at the site of a first known MRSA infection. Within 60 days of this infection and again 12 months later, each subject was tested for asymptomatic colonization in the nose, throat and perirectal region. 93 S. aureus strains underwent whole genome shotgun sequencing. RESULTS Genome sequencing revealed that 23 patients carried USA300 intra-subject lineages (ISLs), defined as having an index infection isolate (III) and closely related strains. Pairwise distance between strains in different ISLs was 48 to 162 single nucleotide polymorphisms (SNPs), whereas within the same ISL it was 0 to 26 SNPs. At the initial sampling time among 23 subjects, we isolated S. aureus from the nose, throat and perirectal sites from 15, 11 and 15 of them, respectively. Twelve months later we isolated S. aureus within the same ISL from 9 subjects, with 6, 3 and 3 strains from the nose, throat and perirectal area, respectively. The median time from initial acquisition of the S. aureus USA300 strains to culture of the index infection was estimated at 18 weeks. Strains in ISLs from the same subject differed in plasmid and prophage content, and contained deletions that removed the mecA-containing SCCmec and ACME regions. Five strains contained frameshift mutations in agr toxin-regulating genes. Persistence of an ISL was not associated with clinical or demographic subject characteristics. CONCLUSION Clonal lineages of USA300 may continue to colonize people at one or more anatomic sites up to a year after an initial infection and experience loss of the SCCmec, loss and gain of other mobile genetic elements, and mutations in the agr operon.