Biology subjects

Kirmaier, A.

Publications and source records attributed to Kirmaier, A..

3 recordsLinked to original sources

bioRxiv2025

Poloxamer dilution as an on-demand alternative to agar dilution-based antimicrobial susceptibility testing

Agar dilution is a reference susceptibility testing method uniquely or preferentially recommended for certain antimicrobials. However, the effort required to pour individual agar plates spanning a doubling dilution range precludes its practical implementation in hospital clinical laboratories. Here, we describe an on-demand replacement for agar dilution (AD), specifically substituting poloxamer 407 (also known as Pluronic F-127) for Bacto Agar as the solidifying agent. Notably, 20% poloxamer 407 solutions (e.g., with Mueller-Hinton broth) remain liquid at refrigerated temperatures, but solidify upon warming, enabling facile set-up of poloxamer dilution testing (PD) in Petri dish or microwell format. For fosfomycin susceptibility testing, PD and reference agar dilution (AD) showed excellent categorical (CA) and essential (EA) agreement for E. coli (100% and 87%, respectively, n=31). For other Enterobacterales, excluding Klebsiella spp; CA and EA were both 82% (n=17, respectively). For P. aeruginosa, CA and EA were 60% and 100% (n=10), respectively, with the lower CA reflecting the large number of strains tested with MICs near categorical breakpoints. There were no very major errors, while major errors were only observed for Klebsiella spp. Additionally, PD substantially reduced the number of skipped dilutions 6-fold for E. coli (P < 0.0001) and inhibited swarming of Proteus species. We conclude that PD and AD, an imperfect gold standard, have essentially equivalent practical performance and that PD can therefore serve as an on-demand alternative testing methodology in clinical laboratories for fosfomycin testing of gram-negative pathogens. A broader exploration of PDs utility is thus warranted. ImportanceAccurate antibiotic susceptibility testing is essential for guiding treatment of bacterial infections. For the antibiotic fosfomycin, used to treat Escherichia coli urinary tract infections, the most reliable testing method requires solid media prepared by hand for each antibiotic concentration, which is too time consuming for most clinical laboratories to perform. Our study shows that replacing agar with an alternative temperature-sensitive gelling agent called poloxamer enables laboratories to prepare solid test plates rapidly without special equipment. This approach, which is essentially identical to traditional agar dilution, provides a practical alternative that can be implemented by hospital clinical microbiology laboratories for accurate fosfomycin testing near the point of patient care. This strategy may also be applicable to other drugs for which agar dilution is the preferred testing method, supporting expedited testing to inform treatment decisions for bacterial infections.

microbiology↗

bioRxiv2023

Contrasting Effects of SARS-CoV-2 Vaccination vs. Infection on Antibody and TCR Repertoires

Antibodies and helper T cells play important roles in SARS-CoV-2 infection and vaccination. We sequenced B- and T-cell receptor repertoires (BCR/TCR) from the blood of 251 infectees, vaccinees, and controls to investigate whether features of these repertoires could predict subjects SARS-CoV-2 neutralizing antibody titer (NAbs), as measured by enzyme-linked immunosorbent assay (ELISA). We sequenced recombined immunoglobulin heavy-chain (IGH), TCR{beta} (TRB), and TCR{delta} (TRD) genes in parallel from all subjects, including select B- and T-cell subsets in most cases, with a focus on their hypervariable CDR3 regions, and correlated this AIRRseq data with demographics and clinical findings from subjects electronic health records. We found that age affected NAb levels in vaccinees but not infectees. Intriguingly, we found that vaccination and infection have an effect on non-productively recombined IGHs, suggesting an effect that precedes clonal selection. We found that repertoires binding capacity to known SARS-CoV-2-specific CD4+ TRBs performs as well as the best hand-tuned approximate or "fuzzy" matching at predicting a protective level of NAbs, while also being more robust to repertoire sample size and not requiring hand-tuning. The overall conclusion from this large, unbiased, clinically well annotated dataset is that B- and T-cell adaptive responses to SARS-CoV-2 infection and vaccination are surprising, subtle, and diffuse. We discuss methodological and statistical challenges faced in attempting to define and quantify such strong-but-diffuse repertoire signatures and present tools and strategies for addressing these challenges.

immunology↗

bioRxiv2020

Species-specific differences in antagonism of APOBEC3 proteins by HIV-2 and SIVsmm Vif proteins

SIVsmm infecting sooty mangabeys has been transmitted to humans on at least nine independent occasions, giving rise to HIV-2 groups A to I. SIVsmm isolates replicate in human T cells and seem capable of overcoming major human restriction factors without adaptation. However, only groups A and B are responsible for the HIV-2 epidemic in Sub-Saharan Africa and it is largely unclear whether adaptive changes were associated with significant spread in humans. To address this, we examined the sensitivity of infectious molecular clones (IMCs) of five HIV-2 strains (4 group A and one AB recombinant) and representatives of five different SIVsmm lineages to inhibition by type I interferon (IFN) and various APOBEC3 proteins. We confirmed that SIVsmm strains replicate in primary human CD4+ T cells. However, SIVsmm replication was highly variable, typically lower relative to HIV-2 isolates and almost entirely prevented by type I IFN treatment. Viral propagation was generally dependent on intact vif genes, highlighting the need for efficient counteraction of APOBEC3 proteins. On average, SIVsmm strains were significantly more susceptible to inhibition by human APOBEC3D, F, G and H than HIV-2 IMCs. For example, human APOBEC3F reduced infectious virus yield of SIVsmm by [~]80% but achieved only [~]40% in the case of HIV-2. Functional and mutational analyses of human, sooty mangabey and rhesus macaque derived alleles revealed that an R128T polymorphism in APOBEC3F is important for species-specific counteraction by HIV-2 and SIVsmm Vif proteins. In addition, we found that changes of Y45H and T84S in SIVsmm Vif increase its ability to antagonize human APOBEC3F. Altogether, our results show that SIVsmm Vifs show some intrinsic activity against human ABOBEC3 proteins, but HIV-2 Vifs acquired adaptive changes to efficiently clear this barrier in the human host. AUTHOR SUMMARYSIVs infecting African monkey species do not infect humans, with one notable exception. SIVsmm from sooty mangabeys managed to cross the species barrier to humans on at least nine independent occasions. This is because SIVsmm strains seem capable of overcoming many innate defense mechanisms without adaptation and that their Vif proteins are active against human APOBEC3 proteins. Here, we show that replication of SIVsmm is highly variable in human CD4 T cells and more sensitive to interferon inhibition compared to HIV-2. While different lineages of SIVsmm were capable of counteracting human APOBEC3 proteins in a Vif-dependent manner, they were significantly more susceptible to inhibition by APOBEC3D/F/G/H compared to HIV-2. Mutational analyses revealed an R128T substitution in APOBEC3F and a T84S change in Vif are relevant for species-specific counteraction by HIV-2 and SIVsmm. Altogether, our results support that HIV-2 group A adapted to humans prior to or during epidemic spread.

immunology↗