Shigella spp have been associated with community-wide outbreaks in urban settings. We analysed a sustained shigellosis outbreak in Seattle, WA, USA, to understand its origins and mechanisms of ...antimicrobial resistance, define ongoing transmission patterns, and optimise strategies for treatment and infection control.
We did a retrospective study of all Shigella isolates identified from stool samples at the clinical laboratories at Harborview Medical Center and University of Washington Medical Center (Seattle, WA, USA) from May 1, 2017, to Feb 28, 2022. We characterised isolates by species identification, phenotypic susceptibility testing, and whole-genome sequencing. Demographic characteristics and clinical outcomes of the patients were retrospectively examined.
171 cases of shigellosis were included. 78 (46%) patients were men who have sex with men (MSM), and 88 (52%) were people experiencing homelessness (PEH). Although 84 (51%) isolates were multidrug resistant, 100 (70%) of 143 patients with data on antimicrobial therapy received appropriate empirical therapy. Phylogenomic analysis identified sequential outbreaks of multiple distinct lineages of Shigella flexneri and Shigella sonnei. Discrete clonal lineages (ten in S flexneri and nine in S sonnei) and resistance traits were responsible for infection in different at-risk populations (ie, MSM, PEH), enabling development of effective guidelines for empirical treatment. The most prevalent lineage in Seattle was probably introduced to Washington State via international travel, with subsequent domestic transmission between at-risk groups.
An outbreak in Seattle was driven by parallel emergence of multidrug-resistant strains involving international transmission networks and domestic transmission between at-risk populations. Genomic analysis elucidated not only outbreak origin, but directed optimal approaches to testing, treatment, and public health response. Rapid diagnostics combined with detailed knowledge of local epidemiology can enable high rates of appropriate empirical therapy even in multidrug-resistant infection.
None.
Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal ...surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule molecular inversion probes (smMIPs), a high-throughput sequencing technology combining multiplexed target capture with read quantification mediated by unique molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci.
We designed a 159-smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and 2 sex-linked loci. We developed methods for detecting and quantitating loci absent from 1 cell population but present in another, which could be used to sensitively identify chimeric cell populations.
Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10000 chimeric cells were demonstrated without prior knowledge of genotypes, and mixtures of up to 4 separate donors could be deconvoluted. Quantitative linearity over 4 orders of magnitude and false-positive rates <1 in 85000 events were achieved. Eleven of 11 posttransplant clinical specimens from patients with hematological malignancies testing positive for residual cancer by conventional methods had detectable chimeric populations by smMIP, whereas 11 of 11 specimens testing negative by conventional methods were low-positive for chimerism by smMIP.
smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.
Despite modern antiseptic techniques, surgical site infection (SSI) remains a leading complication of surgery. However, the origins of SSI and the high rates of antimicrobial resistance observed in ...these infections are poorly understood. Using instrumented spine surgery as a model of clean (class I) skin incision, we prospectively sampled preoperative microbiomes and postoperative SSI isolates in a cohort of 204 patients. Combining multiple forms of genomic analysis, we correlated the identity, anatomic distribution, and antimicrobial resistance profiles of SSI pathogens with those of preoperative strains obtained from the patient skin microbiome. We found that 86% of SSIs, comprising a broad range of bacterial species, originated endogenously from preoperative strains, with no evidence of common source infection among a superset of 1610 patients. Most SSI isolates (59%) were resistant to the prophylactic antibiotic administered during surgery, and their resistance phenotypes correlated with the patient's preoperative resistome (
= 0.0002). These findings indicate the need for SSI prevention strategies tailored to the preoperative microbiome and resistome present in individual patients.
Genomic chimerism represents co-existing cells with different genotypes and has diagnostic significance in transplant engraftment monitoring, residual cancer detection, and other contexts. We ...previously described an approach to chimerism detection by interrogating variably present or absent genomic loci using single-molecule molecular inversion probes (smMIPs) and next-generation sequencing, which provided ultrasensitive limits of detection (<1 in 10,000 cells) but was not reliably quantitative. Herein, smMIP testing was modified to accurately quantitate chimeric cells by incorporating copy number neutral control loci for data normalization and computationally modeling cell mixtures from individual-specific genotypes. Data demonstrate precision and accuracy over three orders of magnitude (0.01% to 50% chimerism). Seventy hematopoietic stem cell transplant specimens from single (n = 42) or double (n = 28) donors were evaluated, benchmarking smMIP against conventional variable number tandem repeat (VNTR) analysis and an unrelated, ultrasensitive polymorphism-specific quantitative PCR (PS-qPCR) assay. Quantitative concordance of all three assays was high (P < 0.0005, Pearson correlation coefficient), although smMIP correlated better with VNTR testing than PS-qPCR. smMIP and PS-qPCR collectively identified low-level chimerism in all specimens testing negative by VNTR (n = 41 and n = 45 of 48 specimens, respectively). This work demonstrates the feasibility of smMIP-based chimerism testing for quantitative and ultrasensitive measurement of genomic chimerism at practical levels approaching one in one million cells, and cross-validates the approach.
Staphylococcus aureus generates biofilms during many chronic human infections, which contributes to its growth and persistence in the host. Multiple genes and pathways necessary for S. aureus biofilm ...production have been identified, but knowledge is incomplete, and little is known about spontaneous mutations that increase biofilm formation as infection progresses. Here, we performed
selection of four S. aureus laboratory strains (ATCC 29213, JE2, N315, and Newman) to identify mutations associated with enhanced biofilm production. Biofilm formation increased in passaged isolates from all strains, exhibiting from 1.2- to 5-fold the capacity of parental lines. Whole-genome sequencing identified nonsynonymous mutations affecting 23 candidate genes and a genomic duplication encompassing
Six candidate genes significantly impacted biofilm formation as isogenic transposon knockouts: three were previously reported to impact S. aureus biofilm formation (
,
, and
), while the remaining three (
,
, and
) were newly implicated by this study. Plasmid-mediated genetic complementation of
,
, and
transposon mutants corrected biofilm deficiencies, with high-level expression of
and
further enhancing biofilm formation over basal levels. This work recognizes genes not previously identified as contributing to biofilm formation in S. aureus and reveals genetic changes able to augment biofilm production by that organism.
Inhaled aztreonam is increasingly used for chronic
suppression in patients with cystic fibrosis (CF), but the potential for that organism to evolve aztreonam resistance remains incompletely explored. ...Here, we performed genomic analysis of clonally related pre- and posttreatment CF clinical isolate pairs to identify genes that are under positive selection during aztreonam therapy
We identified 16 frequently mutated genes associated with aztreonam resistance, the most prevalent being
and
, and 13 of which increased aztreonam resistance when introduced as single gene transposon mutants. Several previously implicated aztreonam resistance genes were found to be under positive selection in clinical isolates even in the absence of inhaled aztreonam exposure, indicating that other selective pressures in the cystic fibrosis airway can promote aztreonam resistance. Given its potential to confer plasmid-mediated resistance, we further characterized mutant
alleles and performed artificial evolution of
for maximal activity against aztreonam. We found that naturally occurring
mutants conferred variably increased resistance to aztreonam (2- to 64-fold) and other β-lactam agents but that its maximal evolutionary capacity for hydrolyzing aztreonam was considerably higher (512- to 1,024-fold increases) and was achieved while maintaining or increasing resistance to other drugs. These studies implicate novel chromosomal aztreonam resistance determinants while highlighting that different mutations are favored during selection
and
, show that
has a high maximal potential to hydrolyze aztreonam, and provide an approach to disambiguate mutations promoting specific resistance phenotypes from those more generally increasing bacterial fitness
.
While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages ...experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in
and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the
efflux system and the target of aztreonam,
While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased
growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of
(
and
) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in
isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of
aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.
Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with
airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.
Adaptation of
to host microenvironments during chronic infection involves spontaneous mutations, yet changes underlying adaptive phenotypes remain incompletely explored. Here, we employed artificial ...selection and whole-genome sequencing to better characterize spontaneous chromosomal mutations that alter two pathogenicity phenotypes relevant to chronic infection in
: intracellular invasiveness and intracellular cytotoxicity. We identified 23 genes whose alteration coincided with enhanced virulence, 11 that were previously known and 12 (52%) that had no previously described role in
pathogenicity. Using precision genome editing, transposon mutants, and gene complementation, we empirically assessed the contributions of individual genes to the two virulence phenotypes. We functionally validated 14 of 21 genes tested as measurably influencing invasion and/or cytotoxicity, including 8 newly implicated by this study. We identified inactivating mutations (
,
, and a hypothetical membrane protein) and gain-of-function mutations (
Thr182Ile,
Thr74Ile, and Asp486Glu in a hypothetical peptidase) in previously unrecognized
virulence genes that enhance pathogenesis when introduced into a clean genetic background, as well as a novel activating mutation in the known virulence regulator gene
(Ala106Thr). Investigation of potentially epistatic interactions identified a
mutation (Ala271Val) that enhances virulence only in the context of purine operon repressor gene (
) inactivation. This project reveals a functionally diverse range of genes affected by gain- or loss-of-function mutations that contribute to
adaptive virulence phenotypes. More generally, the work establishes artificial selection as a means to determine the genetic mechanisms underlying complex bacterial phenotypes relevant to adaptation during infection.
Abstract
Background
Genetic manipulation of Staphylococcus aureus (both methicillin sensitive S. aureus, MSSA, and methicillin resistant S. aureus, MRSA) poses a technical challenge due to poor ...transformation efficiency, limited endogenous DNA repair activity, lineage-specific methylation patterns and intrinsic resistance to common selectable markers.
Methods
To address transformation efficiency we have optimized electrocompetent cell preparation and electroporation protocols for staphylococci. Further, we have improved a CRISPR counterselection platform that delivers a heterologous ssDNA recombinase and an inducible Cas9 endonuclease. When used for recombineering and counterselection, this strategy allows minimization of the number of elements necessary to transform in a single electroporation event. The Cas9 delivery platform has been modified to include a range of selectable markers including resistance to apramycin, erythromycin, kanamycin, nourseothricin, spectinomycin or trimethoprim.
Results
Overall electroporation efficiency increased by multiple orders of magnitude (> 100×) using the optimized cell preparation protocol. The CRISPR delivery platform can be stably maintained in a repressed state for multiple generations and induced with anhydrotetracycline. We have introduced targeted mutations in multiple loci using this system with an average turnaround time of 12 days.
Conclusion
This improved dual-plasmid CRISPR platform is robust and allows the investigator to rapidly and specifically alter the genomes of staphylococci. These tools will facilitate the study of how specific genetic polymorphisms contribute to various phenotypes in S. aureus, including the virulence of MRSA.
Disclosures
All Authors: No reported disclosures
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