AUTHOR=Elken Emad Mohammed , Tan Zi-ning , Wang Qian , Jiang Xiu-yun , Wang Yu , Wang Yi-ming , Ma Hong-xia 

TITLE=Impact of Sub-MIC Eugenol on Klebsiella pneumoniae Biofilm Formation via Upregulation of rcsB

JOURNAL=Frontiers in Veterinary Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.945491

DOI=10.3389/fvets.2022.945491

ISSN=2297-1769

ABSTRACT=<p>The Rcs phosphorelay system is present in many members of the <italic>Enterobacteriaceae</italic>. The aim of this study was to illustrate the possible mechanisms of eugenol on ultimate targets of <italic>Klebsiella pneumoniae</italic> (<italic>K. pneumoniae</italic>) Rcs phosphorelay, <italic>rcsB</italic>, and impact on biofilm formation. The minimum inhibitory concentration (MIC) of eugenol against <italic>K. pneumoniae</italic> KP1 and KP1 Δ<italic>rcsB</italic> strain was determined using the 2-fold micro-dilution method. Biofilm was measured by crystal violet staining. Transcriptome sequencing was performed to investigate sub-MIC eugenol on <italic>K. pneumoniae</italic>, and gene expression at mRNA level was analyzed by RT-qPCR. <italic>In vitro</italic> biofilm formation test and molecular docking were used to evaluate the effect of eugenol and to predict potential interactions with RcsB. MicroScale Thermophoresis (MST) was conducted for further validation. MIC of eugenol against <italic>K. pneumoniae</italic> KP1 and KP1 Δ<italic>rcsB</italic> strain was both 200 μg/ml. Transcriptome sequencing and RT-qPCR results indicated that <italic>rpmg, degP, rnpA</italic>, and <italic>dapD</italic> were downregulated, while <italic>rcsB, rcsD, rcsA, yiaG</italic>, and <italic>yiaD</italic> were upregulated in the eugenol-treated group. Δ<italic>rcsB</italic> exhibited a weakened biofilm formation capacity. Additional isopropyl-β-<sc>d</sc>-thiogalactoside (IPTG) hinders biofilm formation, while sub-MIC eugenol could promote biofilm formation greatly. Docking analysis revealed that eugenol forms more hydrophobic bonds than hydrogen bonds. MST assay also showed a weak binding affinity between eugenol and RcsB. These results provide significant evidence that <italic>rcsB</italic> plays a key role in <italic>K. pneumoniae</italic> biofilm formation. Sub-MIC eugenol facilitates biofilm formation to a large extent instead of inhibiting it. Our findings reveal the potential risk of natural anti-biofilm ingredients at sub-MIC to treat drug-resistance bacteria.</p>