1. Introduction
Antimicrobial drugs or antibiotics were discovered about a century ago and have been used widely in human and animal medicine, as well as in animal production. Antimicrobial growth promoters are antibiotics administered at low and subtherapeutic doses, which can enhance disease resistance and growth of animals [
1,
2]. However, there are growing concerns for the development of antimicrobial resistance and the potential transmission of antibiotic resistance genes in bacteria from livestock production to human beings. In the United States, therefore, the application of antibiotics as growth promoters was completely banned in the livestock industry starting January 2017 [
3]. The urgent need for developing nutritional strategies or exploring bioactive compounds that may partially or completely replace antibiotics as growth promoters for food-producing animals has remarkably increased in the livestock industry.
Organic acids and salts of acids have been widely used in animal feed as acidifiers to modify the intestinal environment as well as to enhance nutrient digestibility [
4]. The most commonly used acids include formic acid, citric acid, benzoic acid, carboxylic acids, and salts of short chain fatty acids (SCFAs) [
5,
6]. Recently, combinations of organic acids and medium chain fatty acids (e.g., lauric acid) have also demonstrated synergistic benefits on animal intestinal health and performance, compared with the individual products [
7]. In general, antimicrobial activity has been claimed or suggested as one of the primary mechanisms of action through which organic acids could enhance animal health [
8,
9,
10]. It is theorized that organic acids in their un-dissociated and uncharged state are capable of bypassing bacterial cell membranes due to their lipophilic nature [
11]. Upon entering the more alkaline interior of a bacterium, the anion and proton from organic acids may have deleterious effects on the bacterium by increasing osmotic stress and disrupting important biomolecule synthesis, which finally causes bacterial death [
12,
13,
14].
Although significant health benefits have been identified on SCFAs in vitro, direct addition of them in animal feed is limited because of their pungent odor and unpalatable flavor [
15,
16]. Therefore, SCFAs have been further processed as salt forms in combination with calcium or sodium, or as esterified forms before addition to animal feed [
17,
18,
19]. Naturally, these products are more stable and/or pleasant compared with SCFAs [
20]. An additional advantage of esterified SCFAs is that they could escape gastric digestion before reaching the small intestine of animals [
21]. Many other organic acid derivatives require further investigation because they may exhibit antimicrobial activities and therefore could be added to animal feed as alternatives to antibiotics. Thus, the objective of the current study was to determine in vitro antimicrobial activity of several organic acids and their derivatives against Gram-positive (G+) and Gram-negative (G−) bacteria that were specifically selected due to their importance in the livestock industry.
3. Discussion
Results in the current study indicated that butyric acid, valeric acid, and ProPhorce (the mixture of sodium formate and free formic acid, 40:60 w/v) had the strongest in vitro antimicrobial effects against E. coli and Salmonella strains, followed by the glyceride esters of SCFAs and lauric acid. However, sodium formate did not exhibit inhibitory effects on E. coli and Salmonella strains at the highest tested doses. Different trends were detected in the antimicrobial activities of the tested compounds against Campylobacter strains, as follows: butyric acid, valeric acid, and ProPhorce > sodium formate, valerate glycerides, monolaurin > propionate glycerides and butyrate glycerides. In addition, the strongest antimicrobial activities against G+ bacteria were observed in monolaurin, ProPhorce, butyric acid, and valeric acid. The weakest antimicrobial activities against G+ bacteria were observed in propionate glycerides and sodium formate, whereas valerate glycerides and butyrate glycerides were in the middle.
Short-chain fatty acids are fatty acids with a chain of less than six carbon atoms, which are primarily produced by hindgut fermentation of dietary fiber [
22]. Propionic acid and butyric acid produced in the gastrointestinal tract of animals are considered particularly important metabolites that have antimicrobial effects on pathogenic bacteria [
22,
23]. The antimicrobial activities of butyric acid have been widely reported in previously published research to effectively inhibit G− and G+ bacteria, such as commensal
E. coli,
Klebsiella pneumoniae,
S. Typhimurium, and
C. perfringens [
8,
9,
14,
24,
25]. It has been also reported that the valeric acid-producing bacteria
Oscillibacter valericigenes were more abundant in the fecal samples of healthy people than people with Crohn’s disease [
26], indicating valeric acid may also benefit intestinal health. Results in the current study suggest that valeric acid has similar antimicrobial activity against G− and G+ bacteria in comparison to butyric acid. The mode of action is likely due to the ability of these acids to penetrate bacterial cell membrane and to acidify cell cytoplasm, thus inhibiting bacterial growth [
12,
13,
14]. Other mechanisms have been also proposed that organic acids could reduce ATP production by uncoupling electron transport, or they could interrupt nutrient uptake by disturbing bacterial cell membrane [
11,
27,
28].
The present study also observed that ProPhorce exhibited stronger antimicrobial activities against G− and G+ bacteria compared with sodium formate. ProPhorce is a mixture of sodium formate and formic acid; therefore, current results indicate that formic acid likely has stronger in vitro antimicrobial activity than sodium formate. To that end, formic acid is the major component responsible for antimicrobial effects of ProPhorce in vitro. These results were not surprising because the antimicrobial activity of formic acid has been confirmed and widely reported against a broad range of bacterial strains, including
E. coli,
S. Typhimurium,
Campylobacter strains, and
S. mutans in previously published research [
29,
30,
31,
32]. Formic acid is a colorless liquid with pungent odor that has been commonly used in animal feed as an organic acidifier [
6,
33,
34]. However, results from the current study suggest sodium formate has very limited antimicrobial activity in vitro.
Glyceride esters of SCFAs have several remarkable advantages compared with free SCFAs. They are more stable and have a less stringent odor compared with free SCFAs, increasing their potential as alternatives to antibiotics in animal feed. In addition, the ester forms of organic acids are digested and absorbed as lipids, which ensures they pass the low-pH stomach and successfully deliver their antimicrobial effects to the small intestine of animals. In the current study, butyrate glycerides and valerate glycerides exhibited comparable inhibitory effects on G− and G+ bacteria, although their antimicrobial activities were not as strong as their acid forms. However, propionate glycerides has weaker antimicrobial activities against G− and G+ bacteria compared with butyrate glycerides and valerate glycerides. Results of the present study were consistent with previously published research that indicated butyrate glycerides had antimicrobial effects on many
E. coli strains,
S. Typhimurium, and
C. perfringens strains in vitro [
25,
35].
Medium chain fatty acids have recently attracted increased attention due to their potential antimicrobial activities and their potential ability to suppress the development of antibiotic-resistant genes in bacteria [
21,
36,
37]. Lauric acid is a C12 fatty acid and has been indicated to have the strongest antimicrobial activity compared with other medium chain fatty acids [
38,
39,
40]. Although Schlievert and Peterson [
41] reported several G− bacteria, including
Salmonella and
E. coli strains, were not susceptible to monolaurin, results of the present study suggest monolaurin has similar or even stronger antimicrobial activity against
E. coli,
S. Typhimurium, and
C. jejuni strains compared with butyrate glycerides. This could be due to different bacterial strains that have different susceptibility. These observations are consistent with a study reported by Anacarso et al. [
35], in which 37
E. coli strains were highly susceptible to a blend containing monolaurin and butyrate glycerides, although the antimicrobial activity was not tested with individual monoglycerides in this study. In agreement with previously published research [
35,
41,
42], the present study demonstrated that G+ bacteria were more susceptible to monolaurin than G− bacteria, with MIC values from 10 to 500 mg/L against G+ bacteria and MIC values from 600 to 10,000 mg/L against G− bacteria. It has also been reported that monolaurin actively inhibited the growth of
Staphylococcus, Streptococcus,
Bacillus, and several other G+ bacterial strains with relatively low MIC values [
37,
43]. As discussed above, the antimicrobial activities of fatty acids and their derivatives are mainly due to the disruption of bacterial cell membranes and the subsequent cell disorganization. However, the ability of medium chain fatty acids to disrupt cellular membranes has been demonstrated to vary among bacterial strains. This variation in susceptibility is likely due to the different outer membranes of the bacteria. For instance, G+ bacteria have cell walls composed of thick layers of peptidoglycan, whereas G− bacteria have a thin layer of peptidoglycan and an outer membrane that is primarily composed of lipopolysaccharides and proteins [
40,
44]. The O-side chains of lipopolysaccharides comprise an effective barrier for hydrophilic molecules, such as lipids [
40,
45]. In addition, these lipopolysaccharides are strongly connected, which makes it difficult for molecules to penetrate the outer membranes. This could be the reason that G− bacteria were less susceptible to monolaurin than G+ bacteria in the present study. The outer membrane of
Campylobacter species expresses lipooligosaccharides that lack the O-side chain [
46]; therefore, they are also more susceptible to monolaurin compared with
E. coli and
S. Typhimurium. Other mechanisms have been suggested for the antimicrobial effects of monolaurin on G+ bacteria, including the disturbance of toxin and exo-protein production at the transcriptional level or the regulation of bacterial signaling pathways that are critical for bacterial survival [
37,
41,
47].
With the purpose of understanding antimicrobial susceptibility of bacterial strains in this study, the MIC values of antimicrobial drugs were also tested on the same strains of bacteria. All tested strains of
E. coli and
Salmonella exhibited multidrug resistance (i.e., resistant to ≥3 drugs). With the limited available MIC interpretive criteria, both strains of
Campylobacter were determined resistant to ciprofloxacin, gentamicin, and tetracycline. The development of resistance to commonly used antibiotics by G− bacteria has gained increasing concern. For example, in 2012, the United States Department of Agriculture (USDA)’s national animal health monitoring system (NAHMS) isolated 1614
E. coli strains from swine production sites in 13 states that represented 91% of the U.S. pig inventory. Almost all
E. coli isolated from swine (91.2%) were resistant to tetracycline (an antimicrobial drug used to treat pneumonia, certain skin infections, etc.), and more than one-third of the isolated
E. coli were resistant to sulfisoxazole, a common sulfa antibiotic [
48]. Interestingly, F18
E. coli, one of the most dominant types of pathogenic
E. coli causing post-weaning diarrhea in piglets, was shown in this study to be susceptible in vitro to the organic acids and their derivatives, although the strain was determined resistant and intermediate resistant to multiple antimicrobial drugs. Post-weaning diarrhea accounts for 20–30% of cases of mortality in weanling pigs, causing huge economic loss in the pig industry [
49,
50]. Results of the present study suggest organic acid derivatives could be supplemented as antibiotic alternatives to prevent or control post-weaning diarrhea caused by F18
E. coli infection.
In regard to antimicrobial susceptibility of the tested G+ bacterial strains, broad resistance in
Enterococcus, multidrug resistance in
C. perfringens, and resistance to at least one drug in
Streptococcus were observed in the present study. Interestingly, monolaurin at relatively low concentrations in our study inhibited the in vitro growth of these antimicrobial resistant pathogens. Taking
C. perfringens as an example, this bacterial species is one of the most common foodborne pathogens in humans, and is also responsible for severe infections in animals, especially in poultry [
33,
51].
C. perfringens-induced necrotic enteritis may cause sudden death of broiler chickens, with mortality rates of up to 50% [
52,
53,
54]. Subclinical
C. perfringens infection also contributes to huge economic loss due to poor performance and high cost of medication and maintenance [
55]. Although organic acids (i.e., formic acid, butyric acid, etc.) have been widely reported to control necrotic enteritis and to promote performance of chickens, the utilization of their derivatives are limited [
33,
56]. In summary, our results showed promising in vitro antimicrobial effects of tested organic acids and their derivatives against tested bacterial strains that are resistant to commonly used antimicrobial drugs. In vivo animal trials are needed to evaluate the efficacy of organic acid derivatives on animal health, such as in pigs and poultry.
