Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB + BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms

Elucidation of the mechanism behind the potentiating activity of baicalin against <i>Burkholderia cenocepacia</i> biofilms

<div><p>Reduced antimicrobial susceptibility due to resistance and tolerance has become a serious threat to human health. An approach to overcome this reduced susceptibility is the use of antibiotic adjuvants, also known as potentiators. These are compounds that have little or no antibacterial effect on their own but increase the susceptibility of bacterial cells towards antimicrobial agents. Baicalin hydrate, previously described as a quorum sensing inhibitor, is such a potentiator that increases the susceptibility of <i>Burkholderia cenocepacia</i> J2315 biofilms towards tobramycin. The goal of the present study is to elucidate the molecular mechanisms behind the potentiating activity of baicalin hydrate and related flavonoids. We first determined the effect of multiple flavonoids on susceptibility of <i>B</i>. <i>cenocepacia</i> J2315 towards tobramycin. Increased antibiotic susceptibility was most pronounced in combination with apigenin 7-O-glucoside and baicalin hydrate. For baicalin hydrate, also other <i>B</i>. <i>cepacia</i> complex strains and other antibiotics were tested. The potentiating effect was only observed for aminoglycosides and was both strain- and aminoglycoside-dependent. Subsequently, gene expression was compared between baicalin hydrate treated and untreated cells, in the presence and absence of tobramycin. This revealed that baicalin hydrate affected cellular respiration, resulting in increased reactive oxygen species production in the presence of tobramycin. We subsequently showed that baicalin hydrate has an impact on oxidative stress via several pathways including oxidative phosphorylation, glucarate metabolism and by modulating biosynthesis of putrescine. Furthermore, our data strongly suggest that the influence of baicalin hydrate on oxidative stress is unrelated to quorum sensing. Our data indicate that the potentiating effect of baicalin hydrate is due to modulating the oxidative stress response, which in turn leads to increased tobramycin-mediated killing.</p></div

BH affects regulation of genes involved in glucarate metabolism of <i>B</i>. <i>cenocepacia</i> J2315.

<p>The reactions depicted in black are significantly (p < 0.05) upregulated (fold changes of “BH vs. Ctrl” / “TOB+BH vs TOB”). The involved enzymes are <i>gudD</i> (glucarate dehydratase), BCAM2512 (5-keto-4-deoxyglutarate dehydratase) and BCAM2514 (ɑ-ketoglutarate semialdehyde dehydrogenase). For the reactions depicted in grey no significant differential expression in either “BH vs. Ctrl” or “TOB+BH vs TOB” was observed.</p

Strains used in the present study.

<p>Strains used in the present study.</p

ROS production in <i>B</i>. <i>cenocepacia</i> J2315 biofilms after treatment with TOB alone or in combination with BH.

<p>Accumulation of ROS in <i>B</i>. <i>cenocepacia</i> J2315 biofilms, expressed as fluorescence generated after incubation with H₂DCFDA, after 24 hours treatment with TOB (4 x MIC), TOB in combination with BH (250 μM) or an untreated pH-matched control. Data presented are means, error bars are standard deviations. The experiment was conducted six times. *: Significant difference (p < 0.05) compared to treatment with TOB alone.</p

Potentiating effect of BH and other flavonoids.

<p>Data shown are percentage survival of <i>B</i>. <i>cenocepacia</i> J2315 biofilm cells treated with the combination of a flavonoid (100 μM) and TOB (4 x MIC) compared to TOB alone. The tested flavonoids were scutellarin (scut), apigenin 7-O-glucoside (api), luteolin 7-O-glucoside (lut), schaftoside (scha), myricitrin (myr) and baicalin hydrate (BH). *: statistically significant (p < 0.05) less survival compared to TOB alone. Error bars are standard deviations (SD) (n = 4).</p

Influence of BH on TOB susceptibility of <i>B</i>. <i>cenocepacia</i> K56-2 biofilms and its ΔBCAL2641 mutant.

<p>Data are averages of log(CFU/ml) surviving cells after treatment with TOB (8 x MIC) alone or in combination with BH (250 μM). Influence of BH on biofilm susceptibility was evaluated in <i>B</i>. <i>cenocepacia</i> K56-2 (wild type) and its ΔBCAL2641 deletion mutant. The MIC for TOB in both strains was 128 μg/ml. The experiment was conducted in triplicate. *: significantly different compared to the wild type (p < 0.05). **: significant difference compared to TOB alone (p < 0.05). Error bars are SD.</p

BH affects regulation of genes involved in putrescine biosynthesis of <i>B</i>. <i>cenocepacia</i> J2315.

<p>The reactions depicted in black are significantly (p < 0.05) differentially regulated (fold changes of “BH vs. Ctrl” / “TOB+BH vs TOB”). The enzymes involved the putrescine synthesis pathway are ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and agmatinase. NS: no significant change in gene expression (p > 0.05).</p

ROS production in <i>B</i>. <i>cenocepacia</i> J2315 and its triple QS mutant after treatment with TOB alone or in combination with BH.

<p>Accumulation of ROS, expressed as fluorescence (average ± SD) generated after incubation with H₂DCFDA, in planktonic cultures of <i>B</i>. <i>cenocepacia</i> J2315 and its triple QS mutant treated with TOB (4 x MIC) or the combination with BH (250 μM) and a pH-matching control after 16 hours. MIC for TOB was 256 μg/ml and 128 μg/ml for the wild type and its triple QS mutant respectively. The experiment was conducted using six biological replicates. *: statistically significant difference compared to the wild type (p < 0.05).</p