antibiotics Article A High-Throughput Metabolic Microarray Assay Reveals Antibacterial Effects of Black and Red Raspberries and Blackberries against Helicobacter pylori Infection Candace Goodman 1, Katrina N. Lyon 2, Aitana Scotto 2, Cyra Smith 2, Thomas A. Sebrell 2, Andrew B. Gentry 3, Ganesh Bala 1, Gary D. Stoner 2 and Diane Bimczok 2,* 1 Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA; candace.goodman@montana.edu (C.G.); b.narayanaganesh@gmail.com (G.B.) 2 Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA; KatrinaLyon@montana.edu (K.N.L.); aitanam96@gmail.com (A.S.); cyrasmith23@gmail.com (C.S.); andysebrell@montana.edu (T.A.S.); Gary.Stoner@osumc.edu (G.D.S.) 3 Bozeman Health GI Clinic, Bozeman Health Deaconess Hospital, Bozeman, MT 59715, USA; agentry@bozemanhealth.org * Correspondence: diane.bimczok@montana.edu; Tel.: +1-406-994-4928 Abstract: Helicobacter pylori infection is commonly treated with a combination of antibiotics and proton pump inhibitors. However, since H. pylori is becoming increasingly resistant to standard  antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that  black and red berries may have antibacterial properties. Therefore, we analyzed the antibacterial Citation: Goodman, C.; Lyon, K.N.; effects of black and red raspberries and blackberries on H. pylori. Freeze-dried powders and organic Scotto, A.; Smith, C.; Sebrell, T.A.; extracts from black and red raspberries and blackberries were prepared, and high-performance liquid Gentry, A.B.; Bala, G.; Stoner, G.D.; chromatography was used to measure the concentrations of anthocyanins, which are considered Bimczok, D. A High-Throughput the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, Metabolic Microarray Assay Reveals a high-throughput metabolic growth assay based on the Biolog system was developed and validated Antibacterial Effects of Black and Red Raspberries and Blackberries against with the antibiotic metronidazole. Biocompatibility was analyzed using human gastric organoids. Helicobacter pylori Infection. Antibiotics All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging 2021, 10, 845. https://doi.org/ from 0.49 to 4.17%. Antimicrobial activity was higher for extracts than powders and appeared to be 10.3390/antibiotics10070845 independent of the anthocyanin concentration. Importantly, human gastric epithelial cell viability was not negatively impacted by black raspberry extract applied at the concentration required for Academic Editor: Maria complete bacterial growth inhibition. Our data suggest that black and red raspberry and blackberry Stefania Sinicropi extracts may have potential applications in the treatment and prevention of H. pylori infection but differ widely in their MICs. Moreover, we demonstrate that the Biolog metabolic assay is suitable for Received: 7 June 2021 high-throughput antimicrobial susceptibility screening of H. pylori. Accepted: 8 July 2021 Published: 12 July 2021 Keywords: antibiotic; high-throughput assay; H. pylori; anthocyanin; berry; organoid Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Helicobacter pylori is the major cause of human gastric disease worldwide [1,2]. H. pylori is an acid-resistant, Gram-negative bacterium that persistently infects the gastric mucosa of approximately half the world’s population, leading to chronic active gastritis [1]. A proportion of infected individuals also develop peptic ulcer disease, autoimmune gastri- Copyright: © 2021 by the authors. tis or gastric adenocarcinoma, the second leading cause of cancer-related mortality [3]. In Licensee MDPI, Basel, Switzerland. This article is an open access article spite of decades of active research, no effective vaccine to prevent H. pylori-associated ill- distributed under the terms and nesses has been developed [4]. Once diagnosed, H. pylori infection is generally treated with conditions of the Creative Commons a combination of antibiotics and proton pump inhibitors. However, increased resistance to Attribution (CC BY) license (https:// two of the standard antibiotics included in H. pylori treatment regimens, clarithromycin and creativecommons.org/licenses/by/ metronidazole, has been reported in multiple studies, with resistance rates ranging from 4.0/). 22 to 80% [5,6]. Recently, clarithromycin-resistant H. pylori was included in the WHO’s Antibiotics 2021, 10, 845. https://doi.org/10.3390/antibiotics10070845 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 845 2 of 19 high-priority pathogens list for research and development of new antibiotics [7]. Moreover, poor patient compliance with complex medication regimens contributes to decreased treat- ment success [8,9]. Therefore, eradication rates of H. pylori have dropped below 75% in several countries [10,11]. The high failure rate of traditional H. pylori therapies points to an urgent need for novel alternative treatments or preventative strategies to combat H. pylori infection [12]. A significant body of research in recent years has shown that natural dietary com- ponents, especially plants, contain many bioactive compounds—neutraceuticals—with antibacterial effects [13–15]. Multiple different berries and their products show signifi- cant antimicrobial activity in vitro and in vivo, and some promising studies suggesting effectiveness against H. pylori have been published. Thus, data by Chatterjee et al. [16] showed significant inhibition of H. pylori growth in the presence of extracts from rasp- berry, strawberry, cranberry, elderberry, blueberry and bilberry. In another recent study, extracts from unripe Korean raspberries and elm tree bark used in combination significantly suppressed H. pylori growth both in vitro and in a mouse model [17]. Amongst the mul- tiple bioactive natural compounds, anthocyanins in colored berries of the genus Rubus have attracted special attention. Anthocyanins are glycosylated, water-soluble phenolic compounds that are responsible for the red, purple and blue coloring of multiple berry species [14]. Anthocyanins are strong antioxidants that have been used successfully in cancer chemoprevention models [18] and that have been implicated in the antibacterial activities of berry preparations [19,20]. In an in vitro model of H. pylori infection, the an- thocyanin cyanidin 3-O-glucoside significantly decreased H. pylori-induced cell death [21]. Since anthocyanin-containing berry products also have proven anti-inflammatory effects and are stable under acidic conditions [22,23], their potential application in gastric H. pylori infection is particularly attractive. In our study, we developed a high-throughput metabolic assay to screen different black raspberry, red raspberry and blackberry preparations for their ability to prevent H. pylori growth in vitro. In addition, a gastric organoid model was used to evaluate the biocompatibility of black raspberry extract. Our results demonstrate that all berry powders and extracts tested caused a significant reduction in H. pylori growth in two different strains at concentrations between 0.5 and 3%. An optimum preparation of black raspberry extract used at 0.5% led to complete inhibition of H. pylori growth but did not affect the viability of primary gastric epithelial cells. These results suggest that preparations from black and red raspberries and blackberries have potential as novel antimicrobial agents to combat H. pylori infection. 2. Results 2.1. Analysis of Powders and Extracts of Black and Red Raspberries and Blackberries for Anthocyanin Content and Composition In order to study the potential antibacterial effects of black raspberry (BRB), red rasp- berry (RRB) and blackberry (BB) compounds on H. pylori, freeze-dried berry powders were purchased from different suppliers or were prepared in our laboratory from fresh-frozen berries. Organic extracts of all berry powders were then prepared using hexane/ethanol extraction. The workflow for sample preparation is shown in Figure 1A, and the different starting materials used are listed in Table 1. AAntnitbiiboitoitcisc s2022012,1 1, 01,0 x, 8F4O5R PEER REVIEW 4 o3f o2f01 9 Figure 1. Preparation and anthocyanin content analysis of black raspberries, red raspberries and blackberries. (A) Workflow Fifgour rbee r1r. yPrperpeapraartaiotino nanadn danatnhaolcyysaisn.in(B c)oRnteepnrte asennatlaytsiivse oLf Cbl-aMckS rsapsepcbterurrmieso, freadb rearsrypbperrerpieasr aatnidon b.laMckabjoerrrpieesa.k (sAr)e Wproesrken- t flcoywa nfoidr ibne-r3r-yO p-grelupcaorasitdioen, caynadn aidnianly-3s-iOs. -(rBu)t Rineopsriedsee,natnatdivaec LoCm-MbinSa stpioenctroufmcy oafn aid bienr-r3y- Opr-xepylaorsaytilorunt. iMnoasjoidr epaeankdsc ryeapnreidseinn-t3 - cyOa-nsiadminb-u3b-Oio-sgilduec.o(sCid)eT, octyaalnaindtihno-3c-yOa-nriunticnoonstiednet, (aTnAdC a) cinomsubsinpaetniodne dofp ocywadneidrsina-n3d-Oe-txhyalonsoyllerxuttrinacotssidoef balnadc kcyraasnpidbienr-r3ie- s O-sambubioside. (C) Total anthocyanin content (TAC) in suspended powders and ethanol extracts of black raspberries (BRB), red raspberries (RRB) and blackberries (BB) determined by LC-MS. Individual data points, mean and SD are shown. (BRB), red raspberries (RRB) and blackberries (BB) determined by LC-MS. Individual data points, mean and SD are shown. (D(D) )TATAC Cini npopwowdedresr asnadn dexetxratrcatcs tosfo BfRBBR,B R, RRBR Banadn dBBB Bpuprucrhcahsaesde dasa fsrefrsehs-hfr-ofrzoezne nbebrerriersie osro arsa fsrefreezeez-ed-rdierdie dbebreryrr ypopwowdedre. r. PPoooloelded ddataat afrforomm aalll lbbeerrrireies;s ;ininddivivididuuaal lddaatata ppooinintst,s ,mmeeaann ±± SDSD araer eshsohwown.n (.E()E )CConocnecnetnrtartaiotinosn sofo fmmajaojro rananththocoycaynainnisn sinin sususpspenenddeded ppowowdderesr soof fBBRRBB, R, RRBB anandd BBB. S. tSattaitsitsitciaclallyl ysisgingnifiificacnant tddififfefreernencecse sasa sddeteetremrminineded bbyy (C(C,D,D) )StSutuddenent’ts’ st tetsets t(E(E) )oro r twtwo-ow-waya yAANNOOVVAA aarere sshhoowwnn aass ** pp << 00.0.055, ,** *pp << 0.00.10 1anandd ***** *p p< <0.0.010.1 . Table 1. Total concentrations of Tanotthaolc aynanthinosciynabnlianc kcoandternetd (rTaAspCb)e rwryaasn cdalbcluaclkabteedrr ybyp oawddeinrsga undp etxhtera cotsndcetnetrrmaitnioedns of by LC–MS. all detected anthocyanins (Figure 1C and Table 1). Overall, large variations in anthocyanin concentrations were observed for berries from different sources and with different pro- cessing techniques. Interestingly, lyophilized but otherwise untreatHedPL bCe–rMryS p*owder from Sample SourRceRB and BB coCnotuanintreydo sfigOnriifgiicnantly hMigahteerri aalmToyupents of anBtehrorycyPaonwindse rthan thEex wtractterT/AetCha- nol extracts prepared in our laboratory (Figure 1C). ThiTsA wCa(sm lgik/1e0l0y gd)ue to (amng i/n10e0ffgic)i*ent VirginExtracts, rFeocoodvseSruyp oerf, anthocyanins in extracts prepared from fresh-frozen berries that were lyoph-VE-BRB Bradford, PA, U Unknown Freeze-dried powder 2945 2885ilizedS Ain-house (Figure 1D, p < 0.001, Student’s t test), because anthocyanin recovery was BH-BRB BerriHealth, BehrriigPhreord iuf cetsxtracts werUeS pArepared frFormee zceo-dmrimederpcoiwald beerrry pow1d1,e4r5s5 (Figure 1D). 1In1,d10iv9idual LLC, Corbett, OR, USA data for cyanidin-3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-xylosylruti- noside are presented in Figure 1E and Table 2. Notably, BRB, RRB and BB powders con- Antibiotics 2021, 10, 845 4 of 19 Table 1. Cont. HPLC–MS * Sample Source Country of Origin Material Type Berry Powder Extract TAC TAC (mg/100 g) (mg/100 g) * Dr. S. Hecht, University of UMN-BRB Minnesota, Minneapolis, USA Ethanol extract N/A 4540 MN, USA Great Value. Wal-Mart USA-RRB Stores Inc. Bentonville, AR, USA Whole frozen berries 5424 554 USA Cascadian Farms, Small CH-RRB Planet Foods, Inc., Chile Whole frozen berries 3027 336 Sedro-Woolley, WA, USA VE-BB VirginExtracts, Foods Super,Bradford, PA, USA Unknown Freeze-dried powder 6175 3465 MX-BB Western Family Foods, Inc.,Portland, OR, USA Mexico Whole frozen berries 6924 748 Cascadian Farms, Small CH-BB Planet Foods, Inc, Chile Whole frozen berries 7312 847 Sedro-Woolley, WA, USA * Absorption assumes all anthocyanins are cyanidin-3-glucoside equivalents (sum of three major cyanidins). BRB: black raspberry; RRB: red raspberry; BB: blackberry; VE: VirginExtracts; BH: BerriHealth; UMN: University of Minnesota; CH: Chile; MX: Mexico; TAC: total anthocyanin content; HPLC–MS: high-performance liquid chromatography/mass spectrometry. To determine the concentration of anthocyanins, all samples were analyzed by LC– MS for the presence of keracyanin (cyanidin-3-O-rutinoside), kuromanin (cyanidin-3-O- glucoside) and cyanidin-3-O-xylosyrutinoside (Table 1 and Figure 1A,B). Because of over- lapping peaks, the cyanidin-3-O-xylosyrutinoside may include cyanidin-3-O-sambubioside, another phenolic berry compound that has a similar composition and MW as the xylosyl- rutinoside, and that is known to be present in BRB at a low concentration [24]. Total anthocyanin content (TAC) was calculated by adding up the concentrations of all detected anthocyanins (Figure 1C and Table 1). Overall, large variations in anthocyanin con- centrations were observed for berries from different sources and with different processing techniques. Interestingly, lyophilized but otherwise untreated berry powder from RRB and BB contained significantly higher amounts of anthocyanins than the water/ethanol extracts prepared in our laboratory (Figure 1C). This was likely due to an inefficient recovery of anthocyanins in extracts prepared from fresh-frozen berries that were lyophilized in-house (Figure 1D, p < 0.001, Student’s t test), because anthocyanin recovery was higher if extracts were prepared from commercial berry powders (Figure 1D). Individual data for cyanidin- 3-O-glucoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-xylosylrutinoside are presented in Figure 1E and Table 2. Notably, BRB, RRB and BB powders contained similar levels of cyanidin-3-O-sambubioside and cyanidin-3-O-glucoside, but cyanidin-3-O-rutinoside levels were significantly higher in BRBs than in RRBs and BBs, as previously described (Figure 1E, p < 0.05, mixed model ANOVA). Antibiotics 2021, 10, 845 5 of 19 Table 2. Anthocyanin composition within powdered berries and berry extracts determined by HPLC–MS. HPLC–MS * (mg/100 g) Sample Cyanidin-3-O-glucoside Cyanidin-3-O-rutinoside Cyanidin-3-O-xylosylrutinoside VE-BRB Powder 1593 7025 1352 Extract 1537 No data 1348 BH-BRB Powder 2490 7356 1609 Extract 2487 7025 1597 UMN-BRB Powder No data No data No data Extract 440 4100 2420 USA-RRB Powder 1999 1993 1432 Extract 355 98 101 CH-RRB Powder 1678 No data 1349 Extract 267 43 26 VE-BB Powder 2806 2010 1359 Extract 2113 No data 1352 MX-BB Powder 3499 2076 1349 Extract 564 121 63 CH-BB Powder 3995 1966 1351 Extract 748 60 39 * BRB: black raspberry; RRB: red raspberry, BB: blackberry; VE: VirginExtracts; BH: BerriHealth; UMN: University of Minnesota; CH: Chile; MX: Mexico; TAC: total anthocyanin content; HPLC–MS: high-performance liquid chromatography/mass spectrometry. 2.2. Development and Validation of a High-Throughput Assay to Measure H. pylori Growth A metabolic bacterial growth assay based on the Biolog system was developed to test a large number of different berry products at different concentrations. This system enables kinetic analysis of microbial growth in a 96-well format based on detection of a redox-sensitive dye by the OmniLog® incubator-reader [25]. Since optimal H. pylori growth requires microaerophilic conditions, the 96-well plates were sealed into a plastic sleeve with a CO2 Gen Compact sachet to reduce oxygen levels. As shown in Figure 2A and the Supplemental Video S1, addition of H. pylori bacteria to the plates at different dilutions resulted in a dose-dependent color change over 48 h. Growth curves had a typical appearance, with an exponential growth phase followed by a plateau phase (Figure 2B). Area under the curve measurements showed significant differences in the growth of H. pylori plated at different concentrations, which was confirmed by endpoint measurements at 590 nm in a standard ELISA reader (Figure 2C,D). These results show that H. pylori growth can be effectively analyzed in liquid cultures using a high-throughput metabolic growth assay. Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 20 Antibiotics 2021, 10, 845 6 of 19 Figure 2. Development of a high-throughput metabolic assay to measure H. pylori growth. (A) Images of 96-well plate s Figucoren t2a.i nDinegvevlaorpiomuesncto nocf ean thraigtiho-ntshroofuHg.hppyulotr im(1e0t0ab=olsitco caksssaoylu ttoio mn euasesduraet HO.D p6y0l0or=i g0r.5o)wotbhta. i(nAe)d Ibmyatghees Oomf 9n6iL-woge®ll plates continaicnuibnagto vr/arreioaudesr caotndcieffnetrreanttiotinmse opfo Hin.t spyalfoterri (p1la0t0i n=g stthoeckba scoteluritai.o(nB )uGserodw atth OcuDr6v0e0s b=a 0se.5d) onbtaabisnoerdba bnyce thate5 O62mnnmiLforg® incu- batothr/erewadelelrs oatu tdliinffeedreintp tainmeel Ap.o(iCn)tsA arfetaeru npdlaetrinthge tchuer vbeawctaesridae. t(eBrm) Ginreodwutshi ncgurGvreasp hbPaaseddP orinsm abasnodrbshaonwces asitg 5n6ifi2 cnamnt for the welldsi fofeurtelninc esdi ninH p. paynloerli Am.e t(aCb)o lAisrmeab eutnwdeenr tchuelt ucruersvwei twhadsif fdeeretenrtminiintiealdc ounscinengt rGatriaopnshoPfabda cPterriisam. ( Dan)dEn sdhpoowinst asibgsnoribfiacnacnet differ- ence(4s8 inh) Hof. apnylHor.ip myloertiacbuoltluisrme a bneatlywzeedni ncua lsttuarnedsa rwdi9th6- dwieflfleprelantte irneaitdiaerl caot 5n9c0entmra. tDioantas aorfe breapcrtesreian.t a(tDiv)e Eonf dnp=o4insitm aiblasrorbance (48 hex) poefr aimn eHn.t spwyliotrhi c5u–6lttuerceh nanicalyrzeepdli ciant eas setacnhd. aIrndd i9v6id-wuaellld paltatpeo rinetasd, emre aatn 5±90S nDma.r eDsahtao warne. r*e**ppre90%) at 0.5% (5 mg/mL). This inhibitory concentration is similar to or lower than those of Rubus extracts described in other studies [16,32,42], but several log folds higher than standard antibiotics such as amoxicillin and clarithromycin [43] or the metronidazole used in our study, which completely blocked H. pylori growth at 136 µg/mL. Since anthocyanins are considered the major active ingredients of black and red berries, we hypothesized that anthocyanins would also be responsible for the antibacterial activi- ties observed in our experiments. It was expected that correlation analysis would show a significant inverse relationship between the anthocyanin concentration and the MIC of the berry preparations. Surprisingly, no significant correlation between the concentration of any anthocyanin analyzed in our preparations and increased antibacterial activity was detected, indicating that antimicrobial activity was largely independent of anthocyanins. Anthocyanins exhibit antimicrobial activity against Gram-negative bacteria by causing damage to the cell walls, membranes, and intercellular matrix [44]. Importantly, antho- cyanins have been linked to the antimicrobial effects of berry preparations in previous studies [45–47] and are responsible for the major chemopreventive effects of blackberries and black and red raspberries [18,24,48–50]. However, our correlation analysis suggests that the antibacterial effects against H. pylori were independent of anthocyanins and thus must be caused by other active compounds. Indeed, ellagic acid, another polyphenol present in red and black berries, is known to exert antibacterial activity against H. pylori as well as other bacteria [17,51,52]. In addition, Lengsfeld et al. demonstrated that berry- derived polysaccharides can combat H. pylori infection in vivo by preventing bacterial binding to the gastric mucosa [53]. Additional studies have shown antibacterial effects for berry-derived sanguiin H-6 [35] and rubusoside [54]. Further experiments are needed to identify the raspberry and blackberry compounds that mediate antimicrobial activity against H. pylori. It remains to be tested whether BRB extract can be used to successfully treat H. pylori infection in vivo. Berry compounds have been investigated in many studies, and their phar- macokinetics and pharmacodynamics have previously been characterized [51,55]. In our study, human gastric organoids were used as model of primary human gastric epithelial cells to analyze compatibility of BRB extract with gastric epithelial cells, since they closely represent the architecture and cellular complexity of the human gastric mucosa [56,57]. Organoids are three-dimensional long-term cultures of primary cells maintained in a gelati- nous extracellular matrix in the presence of specific growth factors. Importantly, exposure of the organoids to BRB did not negatively impact cell viability at the concentrations tested. Moreover, previous animal studies aimed at characterizing the chemopreventive proper- ties of berries have demonstrated that berry products including powdered BRBs are well tolerated at dietary concentrations of up to 10% [58] and that supplementation of the diet Antibiotics 2021, 10, 845 14 of 19 with 5% BRB powder, equivalent to 45 g/day in humans [59], prevented esophageal, oral and colon cancer in rats and colonic polyps in mice [48,60,61]. In a phase I clinical trial, administration of 60 g of BRB powder per day had beneficial effects in colorectal cancer patients with no significant side effects except transient diarrhea or constipation [62]. Other studies have demonstrated that berry extracts have anti-Helicobacter activity in animal models. Thus, Park et al. [17] recently showed that extracts prepared from dried, unripened Korean raspberry (Rubus crataegifolius) decreased H. pylori colonization by about 4 log-fold in a murine model of infection with H. pylori strain SS1. Notably, the berry preparation used in the study by Park et al. was highly potent, with an in vitro MIC90 of 150 µg/mL [17]. In summary, we have established a high-throughput metabolic growth assay to ana- lyze antimicrobial effects of berry preparations against H. pylori. Both freeze-dried powders and ethanol extracts from BRBs, RRBs and BBs obtained from various sources significantly suppressed growth of multiple H. pylori strains in vitro. Toxicity studies with human gastric organoids demonstrated good biocompatibility over a wide range of concentra- tions, including the MIC90 determined in the growth assay. Together, our findings confirm the potential of berry products as antimicrobial agents but highlight the importance of carefully selecting specific preparations with high antibacterial activity. 4. Materials and Methods 4.1. Berry Powders and Preparation of Extracts Commercially available black raspberry (Rubus occidentalis; BRB), blackberry (Rubus fruticosus; BB) and red raspberry (Rubus idaeus; RRB) samples were either purchased as freeze-dried powders or fresh-frozen whole berries. One additional BRB extract prepared as described in previous studies in Dr. S. Hecht’ laboratory at the University of Minnesota was included for comparison [50,63,64] Suppliers and countries of origin for the berries are listed in Table 1. Fresh-frozen whole berries were processed into freeze-dried powder using a food processor (Cuisinart® Elemental, Stamfort, CT, USA) and lyophilizer (SP VirTis Genesis Pilot Lyophilizer, Warminster, PA, USA) (Figure 1A). Berry powders were either used directly in experiments after mixing the material with IF-10a media plus dye D, both Biolog, Hayward, CA, USA at a final concentration of 0.26–4.17% (w/v; powders) or were processed for hexane/ethanol extraction (extracts). Notably, berry powders contain both water soluble and insoluble materials, so that mixing of the powders with aqueous media results in a colloidal suspension. For hexane/ethanol extraction, nonpolar compounds were extracted using 3 × 100 mL hexane per 10 g powder. Samples were filtered between each extraction, and the hexane filtrate discarded. Anthocyanins and all water-soluble compounds were then extracted using an 80:20 ethanol:water mixture (3 × 100 mL per 10 g sample). This extract was dried to a syrup under reduced pressure at 30 ◦C then lyophilized to yield between 1 and 4 g per 20 g powder. All berry preparations were stored in airtight containers at −20 ◦C until use. 4.2. Analysis of Anthocyanin Content Concentrations of major active compounds, i.e., cyanidin-3-O-glucoside, cyanidin- 3-O-rutinoside, cyanidin-3-O-xylosyl-rutinoside and cyanidin-3-sambubioside, for both powder and extract samples were measured by HPLC–MS. The lyophilized samples were mixed with 80:20 (water: sample). The samples were filtered and then injected into an LC– MS system (Agilent 6538 UHD-QTOF equipped with Agilent 1290 infinity UPLC). Upon extracting the chromatograms based on the reported m/z, calculations were performed by integrating the peak to obtain the area. Anthocyanin standards were purchased from Extrasynthese S.A.S. (Lyon, France). 4.3. Helicobacter pylori Strains and Culture Conditions Two well-characterized cagA+, vacA s1/m1 H. pylori strains, originally isolated from human patients, were used in our experiments: the reference strain 60190 (kind gift from Dr. G. Perez-Perez, New York University, ATCC #49503 [65]), and strain PMSS1 (kind Antibiotics 2021, 10, 845 15 of 19 gift from Dr. K. Wilson, Vanderbilt University), which is widely used in murine infection experiments [66]. H. pylori strain 60190 was shown to be susceptible to metronidazole, amoxicillin, clarithromycin, levofloxacin, rifampicin and tetracycline [67], and strain PMSS1 was confirmed to be susceptible to metronidazole, amoxicillin, clarithromycin, and tetracy- cline in previous studies [68,69]. For the experiments, bacteria were grown at 37 ◦C under microaerophilic conditions on Brucella agar plates, 5% sheep blood (Becton Dickinson) for 3 days. Colonies were harvested into warm Brucella broth supplemented with 10% FBS and were then cultured in a shaking incubator for a further 18 h period prior to use in the experiments. 4.4. High-Throughput Helicobacter pylori Growth Assay High-throughput bacterial growth assays were performed in 96-well plates using an OmniLog (Biolog, Hayward, CA, USA) plate reader-incubator. Bacterial growth was visualized with a proprietary redox-sensitive tetrazolium dye [70] (dye D, Biolog). Serial dilutions of berry suspensions or extracts (0.26–4.17% w/v) or of metronidazole (8.5–136 µg/mL; Acros Organics, Fair Lawn, NJ, USA) prepared in IF-10a were added to the plates as indicated together with dye D (0.01%) and PM additive (0.05% BSA, 0.01% NaHCO3 and 0.045% glucose w/v final concentrations). Live H. pylori was resuspended in IF-10a (Biolog) to a final OD600 = 0.5, which corresponds to 3.4 × 108 bacteria/mL [71], and 20 µL of the bacterial suspension were added to the plates together with berry prepa- rations at appropriate dilutions, for a total volume of 120 µL. For analysis, loaded plates were sealed in a gas-impermeable bag with a CO2 Compact sachet (Oxoid, Nepean, ON, Canada), following the manufacturer’s instructions for culturing of microaerophilic bac- teria within the OmniLog incubator-reader. Using the bags and sachets was necessary to create microaerophilic conditions, since the OmniLog does not have a controlled CO2 atmosphere. Plates then were incubated at 37 ◦C in the OmniLog incubator for 30–48 h. Absorbance values were recorded at 562 nm every 15 min. In some instances, 10-fold serial dilutions of the H. pylori cultures were recovered from the plates and were re-streaked on Brucella agar plates to confirm growth and growth suppression. 4.5. Data Analysis for Bacterial Growth Assays To analyze H. pylori growth inhibition by berry compounds, OmniLog data were exported to Excel using the Biolog Data Converter (version 1.0) and PM Analysis Software (Microbe, version 1.20.02, all Biolog, Hayward, CA, USA). Absorption data for each sample and time point were normalized to baseline by subtracting the average of the first four absorption values from each data point. To quantitate bacterial growth over time, peak area under the curve (AUC; absorbance [562 nm] × h) was determined using GraphPad version 8.3.1 (San Diego, CA, USA). The minimum inhibitory concentration 90 (MIC90) of a berry preparation was defined as the concentration at which the AUC values for the growth curves were decreased to ≤10% of the maximum. 4.6. Human Gastric Organoid Culture and Viability Assay Human gastric organoid cultures (gastroids) were established and maintained as previously described [72,73]. Briefly, human gastric tissue samples were obtained with informed consent and IRB approval from patients undergoing endoscopy and biopsy at the Bozeman Health Deaconess Hospital (protocol DB050718-FC). Alternatively, tissue samples from sleeve gastrectomy surgeries were provided by the National Disease Research Interchange (protocol DB062615-EX). None of the donors were positive for active H. pylori infection, as determined by rapid urease CLO test (Halyard Health, Alpharetta, GA, USA). Gastric glands were prepared by collagenase digestion and then were plated in Matrigel. Following polymerization, matrigel was overlaid with L-WRN medium which includes Advanced DMEM/F12 (Gibco by Life Technologies, Grand Island, NY, USA) and 50% supernatant from murine L-WRN cells—which secrete Wnt3a, noggin, and R-spondin 3—and supplemented with 10% FBS (Rocky Mountain Bio, Missoula, MT, USA), 1% Antibiotics 2021, 10, 845 16 of 19 L-Glutamine, 10µM Y-27632 (Tocris Biosciences, Bristol, UK), 10 µM SB-431542 (Tocris Biosciences, Bristol, UK), and 10 mM HEPES buffer. Black raspberry extract prepared in 90% DMSO and 10% HCl was externally administered to the organoids for a 48 h treatment at 37 ◦C with 5% CO2. Control cultures were treated with dilutions of 90% DMSO/10% HCl alone. To determine cell viability, organoids were harvested by trypsinization, and single- cell suspensions stained with 7-aminoactinomycin D (7-AAD; ThermoFisher Scientific, Waltham, MA, USA) were analyzed on an LSR II flow cytometer (Becton Dickinson). 4.7. Statistical Analysis Data shown are representative of three or more replicate experiments. For OmniLog assays, 3–6 technical replicates were prepared. All data were analyzed using GraphPad version 8.3.1 (San Diego, CA, USA). Data are shown as the mean ± SD. Student’s t test or a one- or two-way ANOVA with Tukey’s or Dunnett’s multiple comparisons test were used to determine statistical significance. Differences were considered significant at p ≤ 0.05. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antibiotics10070845/s1, Video S1: Time lapse series of plate images of the H. pylori culture in Figure 2 obtained on the OmniLog™ incubator-reader shows color development consistent with H. pylori metabolism and growth over 48 h. Author Contributions: Conceptualization and methodology, C.G., G.D.S. and D.B.; investigation, C.G., K.N.L., A.S., C.S., T.A.S., A.B.G., G.B. and D.B.; formal analysis, C.G., K.N.L., A.S., G.B., G.D.S. and D.B.; writing—original draft preparation, C.G., K.N.L., G.D.S. and D.B.; writing—review and editing, all authors; funding acquisition, G.D.S., C.G. and D.B. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by grant funding from the Oregon Raspberry and Blackberry Commission (to GS and CG; https://oregon-berries.com/, accessed on 5 June 2021); Montana INBRE (NIH award P20GM103474, to DB, AS and KL; https://inbre.montana.edu/, accessed on 5 June 2021); the Montana Agricultural Experiment Station (project #1015768/MONB00450, to DB; https: //agresearch.montana.edu/maes.html, accessed on 5 June 2021) and an equipment grant from the M. J. Murdock Charitable Trust (#2016028, to DB; https://murdocktrust.org/grant-opportunities/, accessed on 5 June 2021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Institutional Review Board Statement: Human tissue sample collection was approved by the Insti- tutional Review Board of Montana State University, protocols DB050718-FC and DB062615-EX. Informed Consent Statement: Written consent was obtained from study participants unless collected materials were de-identified surgical discard materials covered under exempt protocol DB062615-EX. Data Availability Statement: Original data will be made available by the authors upon reason- able request. Acknowledgments: The collaborative support of Bozeman Health Deaconess Hospital for collecting human tissue samples is greatly appreciated. We also would like to thank Stephen Hecht, University of Minnesota, for providing BRB extract. 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