Biology subjects

Buckner, M. M. C.

Publications and source records attributed to Buckner, M. M. C..

4 recordsLinked to original sources

bioRxiv2025

Strain-dependent contribution of the AcrAB-TolC efflux pump to Klebsiella pneumoniae physiology

Klebsiella pneumoniae is a prominent opportunistic pathogen increasingly associated with multidrug resistance and virulence. One of the main mechanisms of antimicrobial resistance in K. pneumoniae is active efflux, primarily mediated by the Resistance-Nodulation-Division (RND) family of pumps. AcrAB-TolC is the key RND efflux pump in K. pneumoniae, regulated by the transcriptional activator RamA and its repressor RamR. Although overexpression of AcrAB-TolC has been linked to drug resistance in various clinical strains, its physiological roles in K. pneumoniae remain insufficiently studied. In this study, we generated isogenic deletions of acrB and ramR in both the genetically tractable K. pneumoniae Ecl8 and the virulent ATCC 43816 strains. We examined the phenotype of the {Delta}acrB and {Delta}ramR mutants by assessing antimicrobial susceptibility, biofilm formation, growth under infection-related conditions, and both in vitro and in vivo infection models. Loss of acrB increased susceptibility to drugs, decreased biofilm formation, and reduced in vitro virulence in both Ecl8 and ATCC 43816. However, only in Ecl8 was the loss of AcrB found to diminish growth under infection-like conditions and decrease in vivo virulence in the Galleria mellonella infection model. In contrast, in ATCC 43816, it had no effect. Our findings suggest that AcrAB-TolC exhibits strain-specific physiological functions, highlighting its dual role in antimicrobial resistance and pathogenicity, and thereby broadening our understanding of efflux-mediated adaptations in K. pneumoniae. Exploring the broader functions of RND efflux pumps in K. pneumoniae can provide insights into the potential effects of targeting them with inhibitor molecules. ImportanceInfections caused by multidrug-resistant bacterial pathogens are among the most urgent global public health challenges. Specifically, Klebsiella pneumoniae is classified by the World Health Organisation as a critical priority pathogen for the development of new treatments. Resistance-Nodulation-Division (RND) efflux pumps significantly contribute to multidrug resistance and virulence, making them promising targets for drug development. In this study, we demonstrated that the RND efflux pump AcrAB-TolC in K. pneumoniae is essential for drug efflux, biofilm formation, and in vitro virulence. Notably, we identified strain-specific roles for AcrAB-TolC in supporting growth under infection-related conditions and virulence in the Galleria mellonella infection model. Our findings highlight that the function of RND efflux pumps can vary between strains within a species. This implies that targeting RND efflux pumps with inhibitors may yield different effects depending on the strain background.

microbiology↗

bioRxiv2024

Natural products from food sources can alter the spread of AMR plasmids in Enterobacterales

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum {beta}-lactam antibiotics in Gram-negative bacteria is a major impediment for the treatment of infections. Genes responsible for resistance to these antibiotics are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and/or the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the transmission of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based screen we found that the natural products were more active in reducing transmission of the IncK plasmid pCT in Escherichia coli ST131, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII plasmid pKpQIL. Furthermore, we found that the natural product rottlerin was more active in K. pneumoniae than in E. coli. Importantly, rottlerin was also associated with a reduced number of transconjugant bacteria in a clinical K. pneumoniae isolate harbouring a blaNDM-1 plasmid. Together, these results demonstrate the potential of natural products as promising anti-plasmid agents.

microbiology↗

bioRxiv2023

Metal complexes and conjugation: Harnessing the power of cobalt complexes to curtail plasmid transfer

BackgroundAntimicrobial resistance genes (ARG), such as extended spectrum {beta}-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes on the conjugative transfer of plasmids in Escherichia coli and Klebsiella pneumoniae. MethodsLiquid broth and solid agar conjugation assays were used to measure complex activity on four plasmids in E. coli. Additionally, the effect of cobalt complexes was tested on the transmission of the fluorescently tagged extended spectrum {beta}-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. ResultsAntimicrobial susceptibility testing of cobalt complexes revealed no antibacterial activity. The cobalt complexes significantly reduced conjugative transfer of RP4, R6K, and R388 plasmids on solid agar in E. coli and pKpQILgfp transmission in K. pneumoniae. None affected conjugative transfer of pKM101 or transmission of fluorescently tagged pCT in E. coli. The cobalt complexes had no effect on plasmid persistence, suggesting that they target conjugation rather than plasmid prevalence. ConclusionsTo the best of our knowledge, this is the first study to report reduced transmission of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as conjugation inhibitors to reduce prevalence of AMR and/or virulence genes in animals and humans. SignificanceAntimicrobial resistance is a growing problem that poses a significant threat to modern medicine. Some of the most problematic resistance genes are carried on genetic elements, called plasmids, that can spread between bacteria. While our understanding of the mechanisms and drivers of gene transfer amongst bacteria is increasing, we lack effective tools to slow down/control these processes. Here we demonstrate for the first time that novel cobalt-based compounds have anti-plasmid activity on a subset of E. coli plasmids, and are extremely potent in K. pneumoniae carrying a clinical carbapenem-resistance plasmid, without impacting plasmid maintenance. This finding forms the foundations of a potential strategy to control the transfer of genes within Gram-negative bacteria, which has implications for AMR and virulence.

microbiology↗

bioRxiv2023

Growth in a biofilm promotes conjugation of a blaNDM-1-bearing plasmid between Klebsiella pneumoniae strains

Antimicrobial resistance (AMR) is a growing problem, especially in Gram-negative Enterobacteriaceae such as Klebsiella pneumoniae. Horizontal transfer of conjugative plasmids contributes to AMR gene dissemination. Bacteria such as K. pneumoniae commonly exist in biofilms, yet most studies focus on planktonic cultures. Here we studied the transfer of a multidrug resistance plasmid in planktonic and biofilm populations of K. pneumoniae. We determined plasmid transfer from a clinical isolate, CPE16, which carried four plasmids, including the 119-kbp blaNDM-1-bearing F-type plasmid pCPE16_3, in planktonic and biofilm conditions. We found that transfer frequency of pCPE16_3 in a biofilm was orders-of-magnitude higher than between planktonic cells. In 5/7 sequenced transconjugants multiple plasmids had transferred. Plasmid acquisition had no detectable growth impact on transconjugants. Gene expression of the recipient and a transconjugant was investigated by RNA-sequencing in three lifestyles: planktonic exponential growth, planktonic stationary phase, and biofilm. We found that lifestyle had a substantial impact on chromosomal gene expression, and plasmid carriage affected chromosomal gene expression most in stationary planktonic and biofilm lifestyles. Furthermore, expression of plasmid genes was lifestyle-dependent, with unique signatures across the three conditions. Our study shows that growth in biofilm greatly increased the risk of conjugative transfer of a carbapenem resistance plasmid in K. pneumoniae without fitness costs and minimal transcriptional rearrangements, thus highlighting the importance of biofilms in the spread of AMR in this opportunistic pathogen. ImportanceCarbapenem-resistant K. pneumoniae is particularly problematic in hospital settings. Carbapenem resistance genes can transfer between bacteria via plasmid conjugation. Alongside drug resistance, K. pneumoniae can form biofilms on hospital surfaces, at infection sites and on implanted devices. Biofilms are naturally protected and can be inherently more tolerant to antimicrobials than their free-floating counterparts. There have been indications that plasmid transfer may be more likely in biofilm populations, thus creating a conjugation hotspot. However, there is no clear consensus on the effect of the biofilm lifestyle on plasmid transfer. Therefore, we aimed to explore the relationship between plasmid transfer and biofilms, and the impact of plasmid acquisition on the host bacterial cell. Our data show resistance plasmid transfer is greatly increased in a biofilm versus planktonic growth, which may be a significant contributing factor to the rapid dissemination of resistance plasmids in K. pneumoniae.

microbiology↗