Biology subjects

Satapoomin, N.

Publications and source records attributed to Satapoomin, N..

8 recordsLinked to original sources

bioRxiv2026

Gentamicin Treatment in a Murine Infection Model Suggests Extracellular Orientia tsutsugamushi Contributes to Systemic Dissemination

Orientia tsutsugamushi, an obligate intracellular bacterial pathogen, infects multiple cell types and disseminates via the blood and lymphatic system to affect various organs in human and other mammalian hosts. This bacterium replicates exclusively within living cells; however, it also exists transiently in a metabolically inactive extracellular form. While infected leukocytes are known contributors to the systemic spread of the bacterium, the role of the inactive extracellular form in dissemination has been unclear. In this study, we demonstrate that extracellular bacteria are present in the cell-free plasma fraction of infected human and murine blood. Furthermore, treatment with gentamicin, a membrane-impermeable antibiotic, significantly reduces systemic bacterial growth in a murine infection model. These findings suggest a potential role for the extracellular form of the bacterium in the spread of infection and raise the possibility of exploring the use of gentamicin to augment conventional antibiotic therapy during scrub typhus infection.

microbiology↗

bioRxiv2025

Comparative virulence analysis of seven diverse strains of Orientia tsutsugamushi reveals a multifaceted and complex interplay of virulence factors responsible for disease.

Orientia tsutsugamushi is an obligate intracellular bacterium found in Leptotrombidium mites that causes the human disease scrub typhus. A distinguishing feature of O. tsutsugamushi is its extensive strain diversity, yet differences in virulence between strains are not well defined nor well understood. We sought to determine the bacterial drivers of pathogenicity by comparing murine infections using seven strains combined with epidemiological human data to rank each strain in terms of relative virulence. Murine cytokine expression data revealed that the two most virulent strains, Ikeda and Kato, induced higher levels of IL-6, IL-10, IFN-{gamma} and MCP-1 than other strains, consistent with increased levels of these cytokines in severe human scrub typhus patients. We sought to identify the mechanistic basis of the observed differential virulence between strains by comparing their genomes, in vitro growth properties and cytokine/chemokine induction in host cells. We found that there was no single gene or gene group that correlated with virulence, and no clear pattern of in vitro growth rate that predicted disease. However, microscopy-based analysis of the intracellular infection cycle revealed that the only fully avirulent strain in our study, TA686, differed from all the virulent strains in its subcellular localisation and expression of its surface protein ScaC. We conclude that drivers of pathogenicity in Orientia tsutsugamushi are distributed throughout the genome, likely in the large and varying arsenal of effector proteins encoded by different strains, and that these interact in complex ways to induce differing immune responses and thus differing disease outcomes in mammalian hosts. Author SummaryScrub typhus is a vector-borne human disease caused by the bacterium Orientia tsutsugmushi and spread by mites. There are numerous different strains of this bacterium with some causing more severe disease in humans than others, and some that do not cause any illness at all. The factors driving these differences are not yet understood, and gaining insight into them could aid in vaccine development and help predict the severity of disease caused by new isolates. To better determine the mechanistic basis of pathogenicity in scrub typhus, we carried out experiments in which we compared seven diverse strains for virulence in animals. We measured their ability to cause disease in mice, so that we could reliably classify them as virulent or avirulent in this model. We then analysed various genomic and biological aspects to identify disease markers in both mice and humans. We report here that there is no single factor that predicts whether a strain will be pathogenic or not, but that disease in scrub typhus is a complex process resulting from the activity of multiple bacterial genes working together to drive different immune responses in the host, resulting in either clearance of the bacteria from the host, or escalating disease. Future work exploring the relationship of bacterial effector proteins will help to disentangle this complex relationship in mechanistic detail.

microbiology↗

bioRxiv2024

Improving Nitrofurantoin Resistance Prediction in Escherichia coli from Whole Genome Sequence by Integrating NfsA/B Enzyme Assays

Nitrofurantoin resistance in Escherichia coli is primarily caused by mutations damaging two enzymes, NfsA and NfsB. Studies based on small isolate collections with defined nitrofurantoin MICs have found significant random genetic drift in nfsA and nfsB making it extremely difficult to predict nitrofurantoin resistance from whole genome sequence (WGS) where both genes are not obviously disrupted by nonsense or frameshift mutations or insertional inactivation. Here we report a WGS survey of 200 E. coli from community urine samples, of which 34 were nitrofurantoin resistant. We characterised individual non-synonymous mutations seen in nfsA and nfsB among this collection using complementation cloning and assays of NfsA/B enzyme activity in cell extracts. We definitively identified R203C, H11Y, W212R, A112E, A112T and A122T in NfsA and R121C, Q142H, F84S, P163H, W46R, K57E and V191G in NfsB as amino acid substitutions that reduce enzyme activity sufficiently to cause resistance. In contrast, E58D, I117T, K141E, L157F, A172S, G187D and A188V in NfsA and G66D, M75I, V93A and A174E in NfsB, are functionally silent in this context. We identified that 9/166 (5.4%) of nitrofurantoin susceptible isolates were "pre-resistant", defined as having loss of function mutations in nfsA or nfsB. Finally, using NfsA/B enzyme activity assay and proteomics we demonstrated that 9/34 (26.5%) of nitrofurantoin resistant isolates carried functionally wild-type nfsB or nfsB/nfsA. In these cases, enzyme activity was reduced through downregulated gene expression. Our biological understanding of nitrofurantoin resistance is greatly improved by this analysis, but is still insufficient to allow its reliable prediction from WGS data.

microbiology↗

bioRxiv2023

Harvesting and amplifying gene cassettes confers cross-resistance to critically important antibiotics

Amikacin and piperacillin/tazobactam are frequent antibiotic choices to treat bloodstream infection, which is commonly fatal and most often caused by bacteria from the family Enterobacterales. Here we show that two gene cassettes located side-by-side in and ancestral integron similar to In37 have been "harvested" by insertion sequence IS26 as a transposon that is already globally disseminated among the Enterobacterales. This transposon encodes the enzymes AAC(6)-Ib-cr and OXA-1, reported, respectively, as amikacin and piperacillin/tazobactam resistance mechanisms. However, by studying bloodstream infection isolates from 769 patients from, three hospitals serving a population of 1.5 million people in South West England, we show that increased enzyme production due to mutation in an IS26/In37-derived hybrid promoter or, more commonly, transposon copy number amplification is required to simultaneously remove these two key therapeutic options; in many cases leaving only the last-resort antibiotic, meropenem. These findings may help improve the accuracy of predicting piperacillin/tazobactam treatment failure, allowing stratification of patients to receive meropenem or piperacillin/tazobactam, which may improve outcome and slow the emergence of meropenem resistance.

microbiology↗

bioRxiv2023

Hound: A novel tool for automated mapping of genotype to phenotype in bacterial genomes assembled de novo

Increasing evidence suggests that microbial species have a strong within species genetic heterogeneity. This can be problematic for the analysis of prokaryote genomes, which commonly relies on a reference genome to guide the assembly process. Any difference between reference and sample genomes can introduce errors in the detection of small insertions, deletions, structural variations and even point mutations. This phenomenon jeopardises the genomic surveillance of antibiotic-resistant bacteria, with predictions of resistance varying between laboratories. Here we present Hound, an analysis pipeline that integrates publicly available tools to locally assemble prokaryote genomes de novo, detect genes by similarity using the proteins they encode as query, and report the mutations found. Three features are exclusive to Hound: it reports relative gene copy number, retrieves sequences upstream the start codon to detect mutations in promoter regions--which allow gene expression signals to be integrated--and, importantly, can merge contigs based on a user-given query sequence to reconstruct genes that are fragmented by the assembler. To demonstrate Hound, we screened through 5,032 bacterial whole-genome sequences isolated from farmed animals and human infections, using the amino acid sequence encoded by blaTEM-1, to predict resistance to amoxicillin/clavulanate which is driven by over-expression of this gene. We believe this tool can facilitate the analysis of prokaryote species that currently lack a reference genome, and can be scaled up to build automated systems for antibiotic susceptibility prediction.

genomics↗

bioRxiv2022

Identification and characterisation of Klebsiella pneumoniae and Pseudomonas aeruginosa clinical isolates with atypical β-lactam susceptibility profiles using Orbitrap liquid chromatography-tandem mass spectrometry

There is significant interest in the possibility of predicting antibacterial drug susceptibility directly though the analysis of bacterial DNA or protein. We report the use of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii transformants to define baseline predictive rules for the {beta}- lactam susceptibility profiles of {beta}-lactamase positive clinical isolates. We then deployed a robust and reproducible shotgun proteomics methodology to identify {beta}-lactamase positivity and predict {beta}-lactam susceptibility by reference to our baseline predictive rules both in cultured bacteria and in extracts of culture-positive blood. Proteomics and whole genome sequencing then allowed us to characterise K. pneumoniae and P. aeruginosa isolates that differed from the expected {beta}-lactam susceptibility profile, iteratively expanding our predictive rules. Proteomics added considerable value over and above the information generated by whole genome sequencing, allowing for gene expression, not just gene presence to be considered. Specifically, in K. pneumoniae, we identified key differences between acrR and ramR regulatory mutations and compared the effects of OmpK36 Aspartate-Threonine or Glycine-Aspartate dipeptide porin insertions on susceptibility to cefepime and carbapenems. In P. aeruginosa, we identified differences in the gene expression effects of mexR versus nalC mutations and related these to differences in {beta}-lactam MICs against isolates hyper-producing AmpC {beta}-lactamase and or producing a metallo-{beta}-lactamase.

microbiology↗

bioRxiv2021

Klebsiella pneumoniae mutants resistant to ceftazidime/avibactam plus aztreonam, imipenem/relebactam, meropenem/vaborbactam and cefepime/taniborbactam.

Using modified Klebsiella pneumoniae clinical isolates, we show that ramR plus ompK36 mutation together with production of the V239G variant KPC-3 confirs resistance to ceftazidime/avibactam plus aztreonam, imipenem/relebactam and meropenem/vaborbactam, but not cefepime/taniborbactam. This is because the V239G variant does not generate collateral {beta}-lactam susceptibility as do many other KPC-3 variants associated with ceftazidime/avibactam resistance. Additional mutation of ompK35 and carriage of a plasmid expressing the OXA-48-like carbapenemase OXA-232 was required to confer cefepime/taniborbactam resistance.

microbiology↗

bioRxiv2021

OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired β-Lactamases.

Cefalexin is a widely used 1st generation cephalosporin, and resistance in Escherichia coli is caused by Extended-Spectrum (e.g. CTX-M) and AmpC {beta}-lactamase production and therefore frequently coincides with 3rd generation cephalosporin resistance. However, we have recently identified large numbers of E. coli isolates from human infections, and from cattle, where cefalexin resistance is not {beta}-lactamase mediated. Here we show, by studying laboratory selected mutants, clinical isolates, and isolates from cattle, that OmpF porin disruption or downregulation is a major cause of cefalexin resistance in E. coli. Importantly, we identify multiple regulatory mutations that cause OmpF downregulation. In addition to mutation of ompR, already known to downregulate OmpF and OmpC porin production, we find that rseA mutation, which strongly activates the Sigma E regulon, greatly increasing DegP production, which degrades OmpF, OmpC and OmpA porins. Furthermore, we reveal that mutations affecting lipopolysaccharide structure, exemplified by the loss of GmhB, essential for lipopolysaccharide heptosylation, also modestly activate DegP production, resulting in OmpF degradation. Remarkably, given the critical importance attached to such systems for normal E. coli physiology, we find evidence for DegP-mediated OmpF downregulation, gmhB and rseA loss of function mutation in E. coli isolates derived from human infections. Finally, we show that these regulatory mutations enhance the ability of group 1 CTX-M {beta}-lactamase to confer reduced carbapenem susceptibility, particularly those mutations that cause OmpC in addition to OmpF downregulation.

microbiology↗