Antibiofilm and Antimicrobial-Enhancing Activity of Chelidonium majus and Corydalis cheilanthifolia Extracts against Multidrug-Resistant Helicobacter pylori

Abstract

Helicobacter pylori is a Gram-negative bacterium that colonizes the stomach of about 60% of people worldwide. The search for new drugs with activity against H. pylori is now a hotspot in the effective and safe control of this bacterium. Therefore, the aim of this research was to determine the antibacterial activity of extracts from selected plants of the Papaveraceae family against planktonic and biofilm forms of the multidrug-resistant clinical strain of H. pylori using a broad spectrum of analytical in vitro methods. It was revealed that among the tested extracts, those obtained from Corydalis cheilanthifolia and Chelidonium majus were the most active, with minimal inhibitory concentrations (MICs) of 64 µg/mL and 128 µg/mL, respectively. High concentrations of both extracts showed cytotoxicity against cell lines of human hepatic origin. Therefore, we attempted to lower their MICs through the use of a synergistic combination with synthetic antimicrobials as well as by applying cellulose as a drug carrier. Using checkerboard assays, we determined that both extracts presented synergistic interactions with amoxicillin (AMX) and 3-bromopyruvate (3-BP) (FICI = 0.5) and additive relationships with sertraline (SER) (FICI = 0.75). The antibiofilm activity of extracts and their combinations with AMX, 3-BP, or SER, was analyzed by two methods, i.e., the microcapillary overgrowth under flow conditions (the Bioflux system) and assessment of the viability of lawn biofilms after exposure to drugs released from bacterial cellulose (BC) carriers. Using both methods, we observed a several-fold decrease in the level of H. pylori biofilm, indicating the ability of the tested compounds to eradicate the microbial biofilm. The obtained results indicate that application of plant-derived extracts from the Papaveraceae family combined with synthetic antimicrobials, absorbed into organic BC carrier, may be considered a promising way of fighting biofilm-forming H. pylori.

Keywords: 3-bromopyruvate; Bioflux; Helicobacter pylori; Papaveraceae; amoxicillin; bacterial cellulose; biofilm; flow system; sertraline; synergism.

Figure 1

Graphical presentation of checkerboard assays between selected plant extracts (A4/2 and A5/1) and selected synthetic substances (amoxicillin, 3-bromopyruvate, and sertraline) against H. pylori 8064. Abbreviations: MIC, minimal inhibitory concentration; FICI, fractional inhibitory concentration index.

Figure 2

Representative light and fluorescence microscopy pictures taken at ×10 magnification of H. pylori 8064 strain treated with plant extract A4/2 and its combination with tested synthetic compounds (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)). Bacteria were treated with substances as follows: MIC of a plant extract, ¼×MIC of a plant extract + ¼ × MIC of AMX, ¼×MIC of a plant extract + ¼ × MIC of 3-BP, or ¼×MIC of a plant extract + ½×MIC of SER. The study controls were microcapillaries colonized by bacteria not exposed to any antimicrobial substances. In the case of fluorescence pictures, bacteria were stained with the LIVE/DEAD kit, in which green and yellow/orange fluorescence indicates live and damaged/dead bacteria, respectively. Scale bars show 20 µm.

Figure 3

Representative light and fluorescence microscopy pictures taken at ×10 magnification of H. pylori 8064 strain treated with plant extract A5/1 and its combination with tested synthetic compounds (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)). Bacteria were treated with substances as follows: MIC of a plant extract, ¼ × MIC of a plant extract + ¼ × MIC of AMX, ¼ × MIC of a plant extract + ¼ × MIC of 3-BP, or ¼ × MIC of a plant extract + ½ × MIC of SER. The study controls were microcapillaries colonized by bacteria not exposed to any antimicrobial substances. In the case of fluorescence pictures, bacteria were stained with the LIVE/DEAD kit, in which green and yellow/orange fluorescence indicates live and damaged/dead bacteria, respectively. Scale bars show 20 µm.

Figure 4

Degree of biofilm development of H. pylori 8064 treated with the tested plant extracts (A4/2 or A5/1) and their combination with tested synthetic compounds (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)) during the Bioflux-generated flow conditions. Bacteria were treated with substances as follows: MIC of a plant extract, ¼ × MIC of a plant extract + ¼ × MIC of AMX, ¼ × MIC of a plant extract + ¼ × MIC of 3-BP, or ¼ × MIC of a plant extract + ½ × MIC of SER. The study controls were microcapillaries colonized by bacteria not exposed to any antimicrobial substances. The p-value represented by *** is equal to <0.0001.

Figure 5

Viability of H. pylori 8064 biofilm cells treated with the tested plant extracts (A4/2 or A5/1) and their combination with tested synthetic compounds (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)) during the Bioflux-generated flow conditions. Bacteria were treated with substances as follows: MIC of a plant extract, ¼ × MIC of a plant extract + ¼ × MIC of AMX, ¼ × MIC of a plant extract + ¼ × MIC of 3-BP, or ¼ × MIC of a plant extract + ½ × MIC of SER. The study controls were microcapillaries colonized by bacteria not exposed to any antimicrobial substances. Viability was determined by measuring the green/red fluorescence ratio of bacteria stained with the LIVE/DEAD kit. The p-value represented by *** is equal to <0.0001.

Figure 6

Viability of H. pylori 8064 lawn biofilms treated with cellulose disks chemisorbed with 1 mg of the tested plant extracts (A4/2 or A5/1) or an appropriate weight combination with tested synthetic compounds (amoxicillin (AMX), 3-bromopyruvate (3-BP), or sertraline (SER)) determined on the basis of checkerboard assays (0.25 mg of a plant extract + 0.25 mg of AMX, or 0.25 mg of a plant extract + 0.25 mg of 3-BP, or 0.25 mg of a plant extract + 0.5 mg of SER). Disks with 1% DMSO were the negative control of the studies. The p-value represented by *** is equal to <0.0001.