Purpose:
The goal of this preliminary study is to describe the vaginal microbiome of transgender and gender nonbinary (TGNB) individuals using nonculture-based techniques. TGNB individuals may ...undergo gender-affirming surgical procedures, which can include the creation of a neovagina. Little is known about microbial species that comprise this environment in states of health or disease.
Methods:
In this pilot study, vaginal swabs were self-collected from 15 healthy self-identified TGNB participants (age 26–69 years) and 8 cisgender comparator participants (age 27–50 years) between 2017 and 2018. Next-generation 16S ribosomal RNA sequencing was used to profile individual bacterial communities from all study samples.
Results:
The TGNB cohort demonstrated significantly higher intraindividual (alpha) diversity than the cisgender group (
p
=0.0003). Microbial species commensal to the gut and skin were identified only in specimens from TGNB participants. Although
Lactobacillus
species were dominant in all cisgender comparator samples, they were found at low relative abundance (≤3%) in TGNB samples.
Conclusion:
In this study, specimens collected from neovaginas showed increased alpha diversity and substantially different composition compared with natal vaginas. In contrast to natal vaginas, neovaginas were not dominated by
Lactobacillus
, but were hosts to many microbial species. Studies that help to improve our understanding of the neovaginal microbiome may enable clinicians to differentiate between healthy and diseased neovaginal states.
Abstract
Objectives
Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including Stenotrophomonas maltophilia. Cefiderocol resistance remains uncommon and incompletely ...understood. We selected for cefiderocol-resistant S. maltophilia in vitro and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials.
Methods
We selected cefiderocol resistance in three clinical strains of S. maltophilia by serial passage in escalating concentrations of cefiderocol. Emergent cefiderocol-resistant isolates were subjected to repeat susceptibility testing against a panel of relevant antimicrobials. Isolates with confirmed MIC changes were whole genome sequenced.
Results
Each parent strain was initially susceptible to cefiderocol (MICs of 0.03125, 0.03125 and 0.125 mg/L), and one initially tested susceptible to ceftazidime/avibactam (MIC 4 mg/L). We recovered evolved isolates achieving cefiderocol resistance at MICs of 8–32 mg/L from each parental strain. Some cefiderocol resistant isolates reverted following one to four drug-free passages. Ceftazidime/avibactam MICs of passaged isolates repeatedly increased to ≥256 mg/L, and while other MICs were largely unchanged, trimethoprim/sulfamethoxazole MICs declined 4-fold in two strains. WGS revealed one evolved isolate carrying six coding mutations, while four were isogenic mutants of tonB, tolQ, smf-1 and the smeT promoter. Mutation of the smeT promoter downregulated the smeDEF efflux pump and reduced susceptibility to penicillins but increased susceptibility to several other classes including sulphonamides. Other mutations occurred in genes putatively involved in iron metabolism including smlt1148 and cirA.
Conclusions
S. maltophilia strains evolved cefiderocol resistance through different genetic pathways, but often involved iron transport. Future work is required to fully understand the role(s) of other genes in cefiderocol resistance.
Abstract
Background
Tedizolid (TDZ) is an oxazolidinone antimicrobial with broad-spectrum activity against Gram-positive bacteria including methicillin-resistant S. aureus (MRSA). Resistance to TDZ ...is uncommon but mutations in the 23S rRNA target as well as in the transferable rRNA methyltransferase gene cfr, which also mediate resistance to linezolid and chloramphenicol have been implicated. The objective of this study was to determine whether other TDZ resistance pathways exist in MRSA.
Methods
Using a well-characterized MRSA strain, N315, we selected for TDZ resistance by serial passage in escalating concentrations of TDZ in Mueller Hinton broth (MHB) starting with 0.5× the MIC. Once visible growth was achieved a sample of the broth was diluted 1:1,000 into fresh MHB with twice the previous concentration of TDZ until an isolate with an MIC of ≥4 mg/mL was recovered. This MIC was selected since it is 1 dilution above the breakpoint for resistance ≥2 mg/L). This isolate was subjected to whole genome sequencing (WGS) and MICs to other antimicrobials were assessed. Homology modeling was performed to evaluate the potential impact of the mutation on target protein function.
Results
After 10 days of serial passage we recovered a stable mutant with a TDZ MIC of 4 mg/L. WGS revealed a single nucleotide variant (A1345G) in the rpoB gene corresponding to an amino acid substitution at D449N. The following table and figure summarize the changes in drug susceptibility between the parent and evolved strain and reveals the location of the amino acid substitution relative to the TDZ binding site.
Conclusion
We have identified a novel mutation in the RNA polymerase gene, rpoB, that mediates oxazolidinone and chloramphenicol resistance. This variant lies outside of the rifampin resistance determinant clusters of rpoB that span from 1,384 to 1,464 and 1,543 to 1,590, and as expected did not affect rifampin susceptibility. The underlying molecular mechanism by which this single nucleotide variant confers TDZ resistance remains unclear but may involve transcriptional modulation by altered sigma factor binding.
Disclosures
All authors: No reported disclosures.
ABSTRACT Staphylococcus aureus is an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report ...the development of a system for the facile and high-throughput genomic engineering of S. aureus using single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureus genome. We found that EF2132 can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureus strains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureus without permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureus genome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds. IMPORTANCE Engineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus. In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureus genome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.
Clinical targeted sequencing panels are important for identifying actionable variants for patients with cancer; however, existing approaches do not provide transparent and rationally designed ...clinical panels to accommodate the rapidly growing knowledge within oncology.
We used the Clinical Interpretations of Variants in Cancer (CIViC) database to develop an Open-Sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP provides methods to identify variants within the CIViC database, build probes for variant capture, use probes on prospective samples, and link somatic variants to CIViC clinical relevance statements. OpenCAP was tested using a single-molecule molecular inversion probe (smMIP) capture design on 27 cancer samples from 5 tumor types. In total, 2,027 smMIPs were designed to target 111 eligible CIViC variants (61.5 kb of genomic space).
When compared with orthogonal sequencing, CIViC smMIP sequencing demonstrated a 95% sensitivity for variant detection (n = 61 of 64 variants). Variant allele frequencies for variants identified on both sequencing platforms were highly concordant (Pearson's
= 0.885; n = 61 variants). Moreover, for individuals with paired tumor and normal samples (n = 12), 182 clinically relevant variants missed by orthogonal sequencing were discovered by CIViC smMIP sequencing.
The OpenCAP design paradigm demonstrates the utility of an open-source and open-access database built on attendant community contributions with peer-reviewed interpretations. Use of a public repository for variant identification, probe development, and variant interpretation provides a transparent approach to build dynamic next-generation sequencing-based oncology panels.
ABSTRACT 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 Pseudomonas aeruginosa 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 mexAB-oprM efflux system and the target of aztreonam, ftsI. 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 in vitro 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 mexAB-oprM (mexR and nalD) 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 P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes. IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa 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.
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