Antimicrobial Activity of Sorghum Phenolic Extract on Bovine Foodborne and Mastitis-Causing Pathogens

Antimicrobial resistance in bacterial pathogens associated with bovine mastitis and human foodborne illnesses from contaminated food and water have an impact on animal and human health. Phenolic compounds have antimicrobial properties and some specialty sorghum grains are high in phenolic compounds, and the grain extract may have the potential as a natural antimicrobial alternative. The study’s objective was to determine antimicrobial effects of sorghum phenolic extract on bacterial pathogens that cause bovine mastitis and human foodborne illnesses. Bacterial pathogens tested included Escherichia coli, Salmonella Typhimurium, Campylobacter jejuni, Campylobacter coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Klebsiella oxytoca, Staphylococcus aureus, and Enterococcus faecalis. Antibacterial activities of sorghum phenolic extracts were determined by agar-well diffusion assay. Sorghum phenolic extract was added to the wells in concentrations of 0, 100, 200, 500, 1000, or 4000 µg/mL. The control wells did not receive phenolic extract. Plates were incubated for 18–24 h, and the diameter of each zone of inhibition was measured. The results indicated that sorghum phenolic extract had inhibitory effects on Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, and Campylobacter coli.


Introduction
Increased antimicrobial resistance due to agricultural use has become a major issue around the world [1]. An increase in resistance to medically important antimicrobials, such as macrolides, third-generation and higher cephalosporins, fluoroquinolones, tetracyclines, and glycopeptides, led to increased scrutiny in the use of antibiotics in food animals with emphasis on antibiotic stewardship [2][3][4].
Zoonotic foodborne illnesses are a major public health concern, and food-producing animals are a major reservoir for many of the foodborne pathogens. The three major foodborne bacterial pathogens that food animals harbor are Shiga toxin-producing Escherichia coli, Salmonella enterica, and Campylobacter species, particularly jejuni and coli. The Center for Disease Controls recognizes the threat of these pathogens to One Health, spanning both human, animals, and the environment [5]. Brown et al. reported that 21% of 176 Salmonella foodborne illness outbreaks in the United Stated between 2003 and 2012 were resistant to at least one antibiotic [6]. Furthermore, Europe had 319 foodborne outbreaks linked to Campylobacter in 2019, which was 23% of the total number of outbreaks caused by bacteria in Europe [7]. Likewise, Campylobacter has been related to foodborne illness outbreaks in the United States and China [8,9]. In 2017, the CDC reported 23 Campylobacter outbreaks, 4% of the total number of outbreaks caused by bacteria in the United States [10]. Furthermore, illnesses involving E. coli O157 can lead to hemolytic uremic syndrome [11]. In 2019, 42 Shiga toxin-producing E. coli outbreaks were reported in Europe, which was 3% of the total number of outbreaks caused by bacteria in Europe [7]. Likewise, the United States reported that Shiga toxin-producing E. coli caused 3% of the total number of bacterial outbreaks in 2017 [10]. Hemolytic uremic syndrome (HUS) is a severe illness involving the kidneys that leads to acute renal failure and is more often seen in children and immunocompromised adults [12,13]. Furthermore, 87,923 human salmonellosis cases were reported in Europe in 2019 [7]. Antimicrobial resistance coupled with foodborne illness outbreaks among Campylobacter, Escherichia coli O157, and Salmonella enterica are a growing public health threat [6,14,15].
Furthermore, mastitis is an important production disease of dairy cattle. Mastitis is typically caused by various bacterial pathogens, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella, and causes reduced milk yield and economic losses to producers [16][17][18][19]. Ohnishi et al. [20] demonstrated that mastitis cases associated with P. aeruginosa can lead to acute systemic infections that are moderate to severe. Additionally, P. aeruginosa is known to be multidrug-resistant and, therefore, difficult to treat [20][21][22][23]. Tenhagen et al. [24] reported that 631 isolates of S. aureus from various cattle production areas were resistant to at least one antimicrobial. Isolates of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa have also shown resistance to antimicrobials [25]. These findings demonstrate the need to find antimicrobial alternatives for food animal medicine, either for treatment or for prevention of diseases.
Antimicrobial alternatives are substitutions to existing antibiotics where bacterial resistance has emerged [26]. There are three major categories of antimicrobial alternatives: natural, synthetic, and biotechnology-based [27]. Plant-based antimicrobial compounds are part of the naturally occurring antimicrobial alternative category [27,28]. These compounds are typically secondary metabolites derived from plants with antioxidant and antimicrobial activity [29,30]. The mode of actions for antimicrobial activity to combat these pathogens are believed to be the reduction of pH, disruption of the permeability of the bacterial membrane, or through manipulation of bacterial efflux pumps [29].
Sorghum phenolic extract is a collection of plant-based compounds and a potent natural antimicrobial alternative [31,32]. Likewise, it has been shown to have many health and pharmaceutical benefits [33]. Genetic diversity offers some sorghum specialty lines with high tannin content and/or high phenolic compounds that demonstrate high antioxidant activity [34]. Also, the increased concentrations of phenolic compounds have been associated with higher antioxidant activities, anticarcinogenic effects, and antimicrobial activities [31,35,36]. Kil et al. [31] reported that a cultivar of sorghum phenolic extract had minimum inhibitory concentrations (MIC) for S. aureus, Bacillus subtilis, Salmonella Typhimurium, K. pneumoniae, and E. coli of 250, 250, 500, 500, and 250 µg/mL, respectively. Furthermore, Villalobos et al. [32] demonstrated antimicrobial activity of soy flour phenolic extract against Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis. Additionally, a sorghum extract with phenol present in high amount has been shown to attenuate replication of Legionella pneumophila in mouse macrophage cells [37]. The current study's objective was to investigate the antimicrobial activity of phenolic compounds extracted from sorghum bran against foodborne and mastitis-causing pathogens associated with cattle. We hypothesized that extracts from high phenolic sorghum grain would significantly inhibit pathogens in a dose-dependent manner.

Mastitic Pathogens
Overall, sorghum phenolic extract did not have inhibitory effects on P. aeruginosa, K. pneumoniae, and K. oxytoca (Table 1). However, significant inhibitory effects were observed between different concentrations of sorghum phenolic extract against P. aeruginosa, K. pneumoniae, and K. oxytoca (Tables 1 and 2). There were statistical differences seen for the following comparisons of within and between sorghum phenolic extract concentrations when tested against P. aeruginosa and K. pneumoniae (p < 0.0001). The concentrations of sorghum phenolic extract with the largest zones of inhibition when used against K. pneumoniae were 100 µg/mL and 200 µg/mL, both with zones of inhibition of 1.3 mm. The zones of inhibition from sorghum phenolic extract tested against K. oxytoca demonstrated statistical differences for the following concentration comparisons: 0 µg/mL (control) vs. 1000 µg/mL, 100 µg/mL vs. 4000 µg/mL, 200 µg/mL vs. 4000 µg/mL, and 500 µg/mL vs. 4000 (p = 0.05). The concentration that sorghum phenolic extract had the greatest zone of inhibition for K. oxytoca was 4000 µg/mL, with a zone of inhibition of 2.4 mm.

Minimum Inhibitory Concentrations of Antibiotics and Sorghum Phenolic Compounds
MIC was performed for the two most commonly used antibiotics in cattle production systems for prophylaxis and therapeutic purposes. Antimicrobial susceptibility testing was done for 4 bacterial isolates (Enterococcus faecalis, Staphylococcus aureus, Campylobacter coli and C. jejuni), which showed weak-to-moderate effects with sorghum phenolic compound.
Campylobacter coli and C. jejuni isolates were susceptible to erythromycin and tetracycline antibiotics. The C. coli isolate had an MIC value of 6.25 µg/mL and 1.56 µg/mL for erythromycin and tetracycline, respectively. The C. jejuni isolate had an MIC value of 3.12 µg/mL and 1.56 µg/mL for erythromycin and tetracycline, respectively. Both E. faecalis and S. aureus isolates were resistant to erythromycin and tetracycline antibiotics (>100 µg/mL). Consistent with the well diffusion assay, the sorghum phenolic compound was found inhibitory against all four bacteria by microbroth dilution procedure with an MIC value of 12.5 µg/mL, 12.5 µg/mL, 12.5 µg/mL, and 6.25 µg/mL for Campylobacter coli, C. jejuni, E. faecalis and S. aureus isolates, respectively.

Discussion
Antimicrobial alternatives to control foodborne illness and bovine mastitis-causing pathogens are crucial for researchers to explore. Foodborne illness outbreaks caused by Escherichia coli O157, Salmonella Typhimurium, and Campylobacter cause economic impacts on communities due to decreased worker production and, in some cases, they can cause serious disease capable of resulting in kidney failure and death [11,[38][39][40]. Furthermore, mastitis causes economic losses to milk producers and is a major health concern for lactating cows [20,41]. Our findings suggest that sorghum phenolic extract has antimicrobial effects against Campylobacter, but not E. coli O157 or S. Typhimurium. We also observed antimicrobial effects against Staphylococcus aureus, a Gram-positive mastitiscausing bacterium, and Enterococcus faecalis, a Gram-positive bacterium, a major nosocomial pathogen, particularly in severely immunocompromised individuals. Sorghum phenolic extract had no observed antimicrobial effect on Gram-negative bacteria studied, except for Campylobacter, while it had antimicrobial effects on both Gram-positive bacteria tested. Campylobacter is a microaerophilic organism, and this could explain why it was sensitive to the sorghum phenolic extract. Other high antioxidant compounds have been demonstrated to inhibit Campylobacter as well [42,43]. For example, Duarte et al. [42] reported coriander essential oil and linalool to have zones of inhibition that were about 85 mm. Resistance of the rest of the Gram-negative bacteria could be due to their lipopolysaccharide outer member protecting them from penetration of sorghum phenolic extract [32]. A limitation of this study was that the screening of sorghum was not exhaustive and instead represents a screening of sorghums with known high phenolics. Questions of which specific compound(s) in sorghums has efficacy, and the representation of those compound(s) in the diversity of sorghum was not in the scope of this project. Furthermore, a second limitation was that only two species of Gram-positive bacteria were tested, but the findings are similar to other researchers [32,44]. Akogou et al. [44] demonstrated no antimicrobial activity of red, phytoalexin-rich sorghum extract against E. coli O157. Likewise, Villalobos et al. [32] demonstrated that Gram-positive bacteria were more susceptible to natural phenolic extract from soybeans. However, the previous authors did show inhibitory effects on E. coli and Salmonella at 1250 and 1500 µg/mL [32]. Furthermore, Kil et al. [31] demonstrated sorghum phenolic extract to have minimum inhibitory concentrations of less than 1000 µg/mL for S. aureus, Salmonella Typhimurium, Klebsiella pneumonia, and Escherichia coli for four different sorghum lines. These results are different than our findings but could be due to different methods used to determine antimicrobial activity or, more likely, differences in the sorghum cultivars used. Furthermore, an upward dose-dependent trend of antimicrobial activity was seen for C. jejuni, C. coli, E. faecalis, and S. aureus with 100 ug/mL having the lowest zone of inhibition and 4000 ug/mL having the highest zone of inhibition (Figure 1). extract from soybeans. However, the previous authors did show inhibitory effects on E. coli and Salmonella at 1250 and 1500 µg/mL [32]. Furthermore, Kil et al. [31] demonstrated sorghum phenolic extract to have minimum inhibitory concentrations of less than 1000 µg/ mL for S. aureus, Salmonella Typhimurium, Klebsiella pneumonia, and Escherichia coli for four different sorghum lines. These results are different than our findings but could be due to different methods used to determine antimicrobial activity or, more likely, differences in the sorghum cultivars used. Furthermore, an upward dose-dependent trend of antimicrobial activity was seen for C. jejuni, C. coli, E. faecalis, and S. aureus with 100 ug/ mL having the lowest zone of inhibition and 4000 ug/mL having the highest zone of inhibition (Figure 1).  Apart from Campylobacter, Gram-negative foodborne illness-causing bacteria used in this study were not inhibited by sorghum phenolic extract. However, sorghum grain could still be used as an antimicrobial alternative to these bacteria. Kumar et al. [45] engineered silver glyconanoparticles using sweet sorghum syrup that were shown to have antimicrobial effects on E. coli, P. aeruginosa, and Klebsiella planticola, as well as some Gram-positive bacteria. Furthermore, Halder et al. [46] identified a glycine and proline-rich protein in sorghum that has demonstrated antimicrobial activity against Bacillus subtilis, Rhodococcus fascians, and Escherichia coli with minimum inhibitory concentrations of 7.5, 22.4, and 75 µg/mL, respectively. Together, these findings demonstrate how sorghum grain can be used in different ways to develop antimicrobial alternatives and how further research should be conducted on sorghum antimicrobial effects.

Sorghum Plant Material
The diversified sorghum association panel (SAP) was evaluated at Manhattan, KS, and at Puerto Vallarta, Mexico, in 2014 along with other photosensitive lines. All lines were tested for total phenolic compounds concentrations and oxygen radical absorption capacity (ORAC) values. One photosensitive line PI570481 was chosen for the study as this line had ORAC values greater than 150 µM Trolex equivalent. The line PI570481 is a black-grained sorghum, selected due to its efficacy and high phenolic content, as previously published [37,47].

Total Phenolic Compounds Extraction and Quantification
A tangential abrasive dehulling device (Venables Machine Works, Saskatoon, SK, Canada) was used to remove the bran. Twenty-five mL of acidified methanol (1% HCl/ methanol v/v) were used to suspend 0.5 g of the bran sample, which was then shaken for two hours. The mixture was centrifuged for 15 min and the supernatant was transferred into 50 mL tubes. Total phenols in the sorghum bran extract were determined using the method described by Herald et al. [34]. Seventy-five µL of water were added to each well in a 96-well plate. Twenty-five µL of sample extract, Trolox standard (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), or blank, along with 25 µL Folin-Ciocalteu reagent, were then added to each well and allowed to equilibrate at room temperature for 6 min. After equilibrating, 100 µL of 7.5% Na 2 CO 3 were added to each well. The plate was placed at room temperature and left in the dark for 90 min.

Agar-Well Diffusion Assay
The antimicrobial activities of the sorghum phenolic extract were determined by a well diffusion assay [49]. Sixteen mm diameter wells were punched into MH agar (Becton Dickinson) and a small amount (approx. 30 µL) of melted MH agar was used to seal the bottom of the wells. A cotton swab was used to inoculate bacterial inoculum of the bacterial species to be tested onto MH agar with wells to obtain a lawn of growth. The wells were then filled with 100 µL of sorghum phenolic extract at concentrations of 0 (control), 100, 200, 500, 1000, or 4000 µg/mL with LB broth. Seventy percent ethanol was used to dissolve the sorghum phenolic extract and was added to wells as negative controls. The plates were incubated at 37 • C for 24 h aerobically or under microaerophilic conditions (for Campylobacter spp.). The diameter of the inhibitory zone was recorded.

Antibiotic Susceptibility Determinations
Minimum inhibitory concentrations of antibiotics were determined by the microbroth dilution method [48]. Antibiotics tested were erythromycin and tetracycline (Sigma-Aldrich, St. Louis, MO, USA). Antibiotic stock solutions were prepared as per manufacturer's guidelines to obtain a concentration of 1000 µg/mL based on potency of antibiotics. Antibiotics were tested at concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, and 0.098 µg/mL. The bacterial inocula were prepared by preparing 1:100 dilutions of cultures grown in 10 mL Mueller Hinton broth for 6 to 8 h. The antimicrobial susceptibility was performed in 96-well micro titer plates (Becton and Dickinson, Franklin Lakes, NJ, USA). Plates were incubated at 37 • C for 20-24 h under aerobic (Enterococcus faecalis and Staphylococcus aureus) and or microaerophilic conditions (Campylobacter coli and C. jejuni) and results were recorded as growth or no growth. The procedure was repeated again with different bacterial inoculum. As a reference standard, sorghum phenolic compound was also subjected for testing at the same concentration like antibiotics.

Statistical Analysis
The inhibitory activity of sorghum phenolic extract against foodborne illness and mastitis-causing bacterial pathogens was evaluated using a PROC MIXED procedure in SAS (Version 9.4, Cary, NC, USA). The statistical model included the concentrations of phenolic extract, replications (repeated measures as a random effect) and zone diameter of well diffusion assay. For the MIC data, values with >100 µg/mL were not analyzed. The mean comparisons within and between bacterial isolates against various concentrations of phenolic extract were tested using Tukey's Honest Significant Difference with an alpha of ≤0.05.

Conclusions
Our data demonstrated that phenolic extract of a sorghum cultivar exhibited antibacterial activity. Of the mastitis-causing pathogens and foodborne pathogens prevalent in cattle tested, only S. aureus, E. faecalis, C. jejuni, and C. coli were inhibited by the phenolic extracts in a dose-dependent manner. The selective inhibition of certain bacterial species, both Gram-positive and Gram-negative bacteria, is an interesting observation that needs to be investigated. Our study provides impetus to test additional cultivars of phenolic acid extracts of sorghum cultivars and explore value-added benefits of sorghum grains as natural antimicrobial compounds against bacterial pathogens.