This article is part of the research theme “Antimicrobial use, antimicrobial resistance and the microbiome of food animals”. View all 13 articles
       Organic acids continue to be in high demand as additives to animal feed. To date, the focus has been on food safety, particularly reducing the incidence of foodborne pathogens in poultry and other animals. Several organic acids are currently being studied or are already in commercial use. Among the many organic acids that have been extensively studied, formic acid is one of them. Formic acid is added to poultry diets to limit the presence of Salmonella and other foodborne pathogens in the feed and in the gastrointestinal tract after ingestion. As the understanding of the efficacy and impact of formic acid on the host and foodborne pathogens grows, it is becoming clear that the presence of formic acid can trigger specific pathways in Salmonella. This response can become more complex when formic acid enters the gastrointestinal tract and interacts not only with Salmonella already colonizing the gastrointestinal tract but also with the gut’s own microbial flora. The review will examine the current results and prospects for further research on the microbiome of poultry and feed treated with formic acid.
       In both livestock and poultry production, the challenge is to develop management strategies that optimize growth and productivity while limiting food safety risks. Historically, the administration of antibiotics at subtherapeutic concentrations has improved animal health, welfare, and productivity (1–3). From a mechanism of action perspective, it has been proposed that antibiotics administered at subinhibitory concentrations mediate host responses by modulating gastrointestinal (GI) flora and, in turn, their interactions with the host (3). However, ongoing concerns about the potential spread of antibiotic-resistant foodborne pathogens and their potential association with antibiotic-resistant infections in humans have led to the gradual withdrawal of antibiotic use in food animals (4–8). Therefore, the development of feed additives and improvers that meet at least some of these requirements (improved animal health, welfare, and productivity) is of great interest from both an academic research and commercial development perspective (5, 9). A variety of commercial feed additives have entered the animal food market, including probiotics, prebiotics, essential oils and related compounds from various plant sources, and chemicals such as aldehydes (10–14). Other commercial feed additives commonly used in poultry include bacteriophages, zinc oxide, exogenous enzymes, competitive exclusion products, and acidic compounds (15, 16).
       Among existing chemical feed additives, aldehydes and organic acids have historically been the most widely studied and used compounds (12, 17–21). Organic acids, particularly short-chain fatty acids (SCFAs), are well-known antagonists of pathogenic bacteria. These organic acids are used as feed additives not only to limit the presence of pathogens in the feed matrix but also to exert active effects on gastrointestinal function (17, 20–24). In addition, SCFAs are produced by fermentation by intestinal flora in the digestive tract and are thought to play a mechanistic role in the ability of some probiotics and prebiotics to counteract pathogens ingested in the gastrointestinal tract (21, 23, 25).
       Over the years, various short-chain fatty acids (SCFAs) have attracted much attention as feed additives. In particular, propionate, butyrate, and formate have been the subject of numerous studies and commercial applications (17, 20, 21, 23, 24, 26). While early studies focused on the control of foodborne pathogens in animal and poultry feed, more recent studies have shifted their focus to the overall improvement of animal performance and gastrointestinal health (20, 21, 24). Acetate, propionate, and butyrate have attracted much attention as organic acid feed additives, among which formic acid is also a promising candidate (21, 23). Much attention has been focused on the food safety aspects of formic acid, in particular the reduction of the incidence of foodborne pathogens in livestock feed. However, other possible uses are also being considered. The overall objective of this review is to examine the history and current status of formic acid as a livestock feed improver (Figure 1). In this study, we will examine the antibacterial mechanism of formic acid. In addition, we will take a closer look at its effects on livestock and poultry and discuss possible methods to improve its effectiveness.
       Fig. 1. Mind map of the topics covered in this review. In particular, the following general objectives were focused on: to describe the history and current status of formic acid as a livestock feed improver, the antimicrobial mechanisms of formic acid and the impact of its use on animal and poultry health, and potential methods to improve efficacy.
       The production of feed for livestock and poultry is a complex operation involving multiple steps, including physical processing of grain (e.g., milling to reduce particle size), thermal processing for pelleting, and the addition of multiple nutrients to the diet depending on the specific nutritional needs of the animal (27). Given this complexity, it is not surprising that feed processing exposes grain to a variety of environmental factors before it reaches the feed mill, during milling, and subsequently during transportation and feeding in compound feed rations (9, 21, 28). Thus, over the years, a very diverse group of microorganisms has been identified in feed, including not only bacteria but also bacteriophages, fungi, and yeasts (9, 21, 28–31). Some of these contaminants, such as certain fungi, can produce mycotoxins that pose health risks to animals (32–35).
       Bacterial populations can be relatively diverse and depend to some extent on the respective methods used for isolation and identification of microorganisms as well as the source of the sample. For example, the microbial composition profile may differ prior to heat treatment associated with pelleting (36). Although classical culture and plate plating methods have provided some information, the recent application of the 16S rRNA gene-based next-generation sequencing (NGS) method has provided a more comprehensive assessment of the forage microbiome community (9). When Solanki et al. (37) examined the bacterial microbiome of wheat grains stored for a period of time in the presence of phosphine, an insect control fumigant, they found that the microbiome was more diverse after harvest and after 3 months of storage. Furthermore, Solanki et al. (37) (37) demonstrated that Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomyces were the dominant phyla in wheat grains, Bacillus, Erwinia, and Pseudomonas were the dominant genera, and Enterobacteriaceae constituted a minor proportion. Based on taxonomic comparisons, they concluded that phosphine fumigation significantly altered bacterial populations but did not affect fungal diversity.
       Solanki et al. (37) showed that feed sources can also contain foodborne pathogens that can cause public health problems based on the detection of Enterobacteriaceae in the microbiome. Foodborne pathogens such as Clostridium perfringens, Clostridium botulinum, Salmonella, Campylobacter, Escherichia coli O157:H7, and Listeria monocytogenes have been associated with animal feed and silage (9, 31, 38). The persistence of other foodborne pathogens in animal and poultry feed is currently unknown. Ge et al. (39) screened over 200 animal feed ingredients and isolated Salmonella, E. coli, and Enterococci, but did not detect E. coli O157:H7 or Campylobacter. However, matrices such as dry feed may serve as a source of pathogenic E. coli. In tracing the source of a 2016 outbreak of Shiga toxin-producing Escherichia coli (STEC) serogroups O121 and O26 associated with human disease, Crowe et al. (40) used whole-genome sequencing to compare clinical isolates with isolates obtained from food products. Based on this comparison, they concluded that the likely source was low-moisture raw wheat flour from flour mills. The low moisture content of wheat flour suggests that STEC can also survive in low-moisture animal feed. However, as Crowe et al. (40) note, isolation of STEC from flour samples is difficult and requires immunomagnetic separation methods to recover sufficient numbers of bacterial cells. Similar diagnostic processes may also complicate the detection and isolation of rare foodborne pathogens in animal feed. The difficulty in detection may also be due to the long persistence of these pathogens in low-moisture matrices. Forghani et al. (41) demonstrated that wheat flour stored at room temperature and inoculated with a mixture of enterohemorrhagic Escherichia coli (EHEC) serogroups O45, O121, and O145 and Salmonella (S. Typhimurium, S. Agona, S. Enteritidis, and S. Anatum) was quantifiable at 84 and 112 days and still detectable at 24 and 52 weeks.
       Historically, Campylobacter has never been isolated from animal and poultry feed by traditional culture methods (38, 39), although Campylobacter can be readily isolated from the gastrointestinal tract of poultry and poultry products (42, 43). However, feed still has its advantages as a potential source. For example, Alves et al. (44) demonstrated that inoculation of fattened chicken feed with C. jejuni and subsequent storage of the feed at two different temperatures for 3 or 5 days resulted in the recovery of viable C. jejuni and, in some cases, even their proliferation. They concluded that C. jejuni can certainly survive in poultry feed and, therefore, may be a potential source of infection for chickens.
       Salmonella contamination of animal and poultry feed has received much attention in the past and remains a focus of ongoing efforts to develop detection methods specifically applicable to feed and to find more effective control measures (12, 26, 30, 45–53). Over the years, many studies have examined the isolation and characterization of Salmonella in various feed establishments and feed mills (38, 39, 54–61). Overall, these studies indicate that Salmonella can be isolated from a variety of feed ingredients, feed sources, feed types, and feed manufacturing operations. The prevalence rates and predominant Salmonella serotypes isolated also varied. For example, Li et al. (57) confirmed the presence of Salmonella spp. It was detected in 12.5% ​​of 2058 samples collected from complete animal feeds, feed ingredients, pet foods, pet treats, and pet supplements during the 2002 to 2009 data collection period. Additionally, the most common serotypes detected in the 12.5% ​​of Salmonella samples that tested positive were S. Senftenberg and S. Montevideo (57). In a study of ready-to-eat foods and animal feed by-products in Texas, Hsieh et al. (58) reported that the highest prevalence of Salmonella was in fishmeal, followed by animal proteins, with S. Mbanka and S. Montevideo as the most common serotypes. Feed mills also present several potential points of feed contamination during mixing and adding ingredients (9, 56, 61). Magossi et al. (61) were able to demonstrate that multiple points of contamination can occur during feed production in the United States. In fact, Magossi et al. (61) found at least one positive Salmonella culture in 11 feed mills (12 sampling locations total) in eight states in the United States. Given the potential for Salmonella contamination during feed handling, transportation, and daily feeding, it is not surprising that significant efforts are being made to develop feed additives that can reduce and maintain low levels of microbial contamination throughout the animal production cycle.
       Little is known about the mechanism of the specific response of Salmonella to formic acid. However, Huang et al. (62) indicated that formic acid is present in the small intestine of mammals and that Salmonella spp. are capable of producing formic acid. Huang et al. (62) used a series of deletion mutants of key pathways to detect the expression of Salmonella virulence genes and found that formate can act as a diffusible signal to induce Salmonella to invade Hep-2 epithelial cells. Recently, Liu et al. (63) isolated a formate transporter, FocA, from Salmonella typhimurium that functions as a specific formate channel at pH 7.0 but can also function as a passive export channel at high external pH or as a secondary active formate/hydrogen ion import channel at low pH. However, this study was performed on only one serotype of S. Typhimurium. The question remains whether all serotypes respond to formic acid by similar mechanisms. This remains a critical research question that should be addressed in future studies. Regardless of the results, it remains prudent to use multiple Salmonella serotypes or even multiple strains of each serotype in screening experiments when developing general recommendations for the use of acid supplements to reduce Salmonella levels in feed. Newer approaches, such as the use of genetic barcoding to encode strains to distinguish different subgroups of the same serotype (9, 64), offer the opportunity to discern finer differences that may impact conclusions and interpretation of differences.
       The chemical nature and dissociation form of formate may also be important. In a series of studies, Beyer et al. (65–67) demonstrated that inhibition of Enterococcus faecium, Campylobacter jejuni, and Campylobacter coli was correlated with the amount of dissociated formic acid and was independent of pH or undissociated formic acid. The chemical form of formate to which the bacteria are exposed also appears to be important. Kovanda et al. (68) screened several Gram-negative and Gram-positive organisms and compared the minimum inhibitory concentrations (MICs) of sodium formate (500–25,000 mg/L) and a mixture of sodium formate and free formate (40/60 m/v; 10–10,000 mg/L). Based on MIC values, they found that sodium formate was inhibitory only against Campylobacter jejuni, Clostridium perfringens, Streptococcus suis, and Streptococcus pneumoniae, but not against Escherichia coli, Salmonella typhimurium, or Enterococcus faecalis. In contrast, a mixture of sodium formate and free sodium formate was inhibitory against all organisms, leading the authors to conclude that free formic acid possesses most of the antimicrobial properties. It would be interesting to examine different ratios of these two chemical forms to determine whether the range of MIC values ​​correlates with the level of formic acid present in the mixed formula and the response to 100% formic acid.
       Gomez-Garcia et al. (69) tested combinations of essential oils and organic acids (such as formic acid) against multiple isolates of Escherichia coli, Salmonella, and Clostridium perfringens obtained from pigs. They tested the efficacy of six organic acids, including formic acid, and six essential oils against the pig isolates, using formaldehyde as a positive control. Gomez-García et al. (69) determined the MIC50, MBC50, and MIC50/MBC50 of formic acid against Escherichia coli (600 and 2400 ppm, 4), Salmonella (600 and 2400 ppm, 4), and Clostridium perfringens (1200 and 2400 ppm, 2), among which formic acid was found to be more effective than all organic acids against E. coli and Salmonella. (69) Formic acid is effective against Escherichia coli and Salmonella due to its small molecular size and long chain (70).
       Beyer et al. screened Campylobacter strains isolated from pigs (66) and Campylobacter jejuni strains isolated from poultry (67) and showed that formic acid dissociates at concentrations consistent with MIC responses measured for other organic acids. However, the relative potencies of these acids, including formic acid, have been questioned because Campylobacter can utilize these acids as substrates (66, 67). The acid utilization of C. jejuni is not surprising because it has been shown to have a nonglycolytic metabolism. Thus, C. jejuni has limited capacity for carbohydrate catabolism and relies on gluconeogenesis from amino acids and organic acids for most of its energy metabolism and biosynthetic activity (71, 72). An early study by Line et al. (73) used a phenotypic array containing 190 carbon sources and showed that C. jejuni 11168(GS) can utilize organic acids as carbon sources, most of which are intermediates of the tricarboxylic acid cycle. Further studies by Wagli et al. (74) using a phenotypic carbon utilization array showed that the C. jejuni and E. coli strains examined in their study are capable of growing on organic acids as a carbon source. Formate is the major electron donor for C. jejuni respiratory energy metabolism and, therefore, the major energy source for C. jejuni (71, 75). C. jejuni is able to utilize formate as a hydrogen donor via a membrane-bound formate dehydrogenase complex that oxidizes formate to carbon dioxide, protons, and electrons and serves as an electron donor for respiration (72).
       Formic acid has a long history of use as an antimicrobial feed improver, but some insects can also produce formic acid for use as an antimicrobial defense chemical. Rossini et al. (76) suggested that formic acid may be a constituent of the acidic sap of ants described by Ray (77) nearly 350 years ago. Since then, our understanding of formic acid production in ants and other insects has increased considerably, and it is now known that this process is part of a complex toxin defense system in insects (78). Various groups of insects, including stingless bees, pointed ants (Hymenoptera: Apidae), ground beetles (Galerita lecontei and G. janus), stingless ants (Formicinae), and some moth larvae (Lepidoptera: Myrmecophaga), are known to produce formic acid as a defensive chemical (76, 78–82).
       Ants are perhaps the best characterized because they have acidocytes, specialized openings that allow them to spray a venom composed primarily of formic acid (82). The ants use serine as a precursor and store large amounts of formate in their venom glands, which are sufficiently insulated to protect host ants from the cytotoxicity of formate until it is sprayed (78, 83). The formic acid they secrete may (1) serve as an alarm pheromone to attract other ants; (2) be a defensive chemical against competitors and predators; and (3) act as an antifungal and antibacterial agent when combined with resin as part of the nest material (78, 82, 84–88). Formic acid produced by ants has antimicrobial properties, suggesting that it could be used as a topical additive. This was demonstrated by Bruch et al. (88), who added synthetic formic acid to the resin and significantly improved the antifungal activity. Further evidence of the effectiveness of formic acid and its biological utility is that giant anteaters, which are unable to produce stomach acid, consume ants containing formic acid to provide themselves with concentrated formic acid as an alternative digestive acid (89).
       The practical use of formic acid in agriculture has been considered and studied for many years. In particular, formic acid can be used as an additive to animal feed and silage. Sodium formate in both solid and liquid form is considered safe for all animal species, consumers and the environment (90). Based on their assessment (90), a maximum concentration of 10,000 mg formic acid equivalents/kg feed was considered safe for all animal species, while a maximum concentration of 12,000 mg formic acid equivalents/kg feed was considered safe for pigs. The use of formic acid as an animal feed improver has been studied for many years. It is considered to have commercial value as a silage preservative and an antimicrobial agent in animal and poultry feed.
       Chemical additives such as acids have always been an integral element in silage production and feed management (91, 92). Borreani et al. (91) noted that to achieve optimum production of high quality silage it is necessary to maintain forage quality while retaining as much dry matter as possible. The result of such optimization is the minimization of losses at all stages of the ensiling process: from the initial aerobic conditions in the silo to subsequent fermentation, storage and re-opening of the silo for feeding. Specific methods for optimizing field silage production and subsequent silage fermentation have been discussed in detail elsewhere (91, 93-95) and will not be discussed in detail here. The main problem is oxidative deterioration caused by yeasts and molds when oxygen is present in the silage (91, 92). Therefore, biological inoculants and chemical additives have been introduced to counteract the adverse effects of spoilage (91, 92). Other considerations for silage additives include limiting the spread of pathogens that may be present in silage (e.g., pathogenic E. coli, Listeria, and Salmonella) as well as mycotoxin-producing fungi (96–98).
       Mack et al. (92) divided acidic additives into two categories. Acids such as propionic, acetic, sorbic, and benzoic acids maintain the aerobic stability of silage when fed to ruminants by limiting the growth of yeasts and molds (92). Mack et al. (92) separated formic acid from other acids and considered it a direct acidifier that inhibits clostridia and spoilage microorganisms while maintaining the integrity of silage protein. In practice, their salt forms are the most common chemical forms to avoid the corrosive properties of the acids in the non-salt form (91). Many research groups have also studied formic acid as an acidic additive for silage. Formic acid is known for its rapid acidifying potential and its inhibitory effect on the growth of detrimental silage microorganisms that reduce the protein and water-soluble carbohydrate content of silage (99). Therefore, He et al. (92) compared formic acid with acidic additives in silage. (100) demonstrated that formic acid could inhibit Escherichia coli and lower the pH of silage. Bacterial cultures producing formic and lactic acid were also added to silage to stimulate acidification and organic acid production (101). In fact, Cooley et al. (101) found that when silage was acidified with 3% (w/v) formic acid, the production of lactic and formic acids exceeded 800 and 1000 mg organic acid/100 g sample, respectively. Mack et al. (92) reviewed the silage additive research literature in detail, including studies published since 2000 that focused on and/or included formic acid and other acids. Therefore, this review will not discuss individual studies in detail but will simply summarize some key points regarding the effectiveness of formic acid as a chemical silage additive. Both unbuffered and buffered formic acid have been studied and in most cases Clostridium spp. Its relative activities (carbohydrate, protein, and lactate uptake and butyrate excretion) tend to decrease, while ammonia and butyrate production decrease and dry matter retention increases (92). There are limitations to the performance of formic acid, but its use as a silage additive in combination with other acids appears to overcome some of these issues (92).
       Formic acid can inhibit pathogenic bacteria that pose a risk to human health. For example, Pauly and Tam (102) inoculated small laboratory silos with L. monocytogenes containing three different dry matter levels (200, 430, and 540 g/kg) of ryegrass and then supplemented with formic acid (3 ml/kg) or lactic acid bacteria (8 × 105/g) and cellulolytic enzymes. They reported that both treatments reduced L. monocytogenes to undetectable levels in the low dry matter silage (200 g/kg). However, in the medium dry matter silage (430 g/kg), L. monocytogenes was still detectable after 30 days in the formic acid-treated silage. The reduction in L. monocytogenes appeared to be associated with lower pH, lactic acid, and combined undissociated acids. For example, Pauly and Tam (102) noted that lactic acid and combined undissociated acid levels were particularly important, which may be the reason why no reduction in L. monocytogenes was observed in formic acid-treated media from silages with higher dry matter contents. Similar studies should be conducted in the future for other common silage pathogens such as Salmonella and pathogenic E. coli. More comprehensive 16S rDNA sequence analysis of the entire silage microbial community may also help to identify changes in the overall silage microbial population that occur at different stages of silage fermentation in the presence of formic acid (103). Obtaining microbiome data may provide analytical support to better predict the progress of silage fermentation and to develop optimal additive combinations to maintain high silage quality.
       In grain-based animal feeds, formic acid is used as an antimicrobial agent to limit pathogen levels in various grain-derived feed matrices as well as certain feed ingredients such as animal by-products. The effects on pathogen populations in poultry and other animals can be broadly divided into two categories: direct effects on the pathogen population of the feed itself and indirect effects on pathogens that colonize the gastrointestinal tract of animals after consuming the treated feed (20, 21, 104). Clearly, these two categories are interrelated, as a reduction in pathogens in the feed should result in a reduction in colonization when the animal consumes the feed. However, the antimicrobial properties of a particular acid added to a feed matrix can be influenced by several factors, such as the composition of the feed and the form in which the acid is added (21, 105).
       Historically, the use of formic acid and other related acids has focused primarily on the direct control of Salmonella in animal and poultry feed (21). The results of these studies have been summarized in detail in several reviews published at different times (18, 21, 26, 47, 104–106), so only some of the key findings from these studies are discussed in this review. Several studies have shown that the antimicrobial activity of formic acid in feed matrices depends on the dose and time of exposure to formic acid, the moisture content of the feed matrix, and the bacterial concentration in the feed and the animal’s gastrointestinal tract (19, 21, 107–109). The type of feed matrix and the source of animal feed ingredients are also influencing factors. Thus, a number of studies have shown that Salmonella levels Bacterial toxins isolated from animal by-products may differ from those isolated from plant by-products (39, 45, 58, 59, 110–112). However, differences in response to acids such as formic acid may be related to differences in serovar survival in the diet and the temperature at which the diet is processed (19, 113, 114). Differences in serovar response to acid treatment may also be a factor in contamination of poultry with contaminated feed (113, 115), and differences in virulence gene expression (116) may also play a role. Differences in acid tolerance may in turn affect the detection of Salmonella in culture media if feed-borne acids are not adequately buffered (21, 105, 117–122). The physical form of the diet (in terms of particle size) may also influence the relative availability of formic acid in the gastrointestinal tract (123).
       Strategies to optimize the antimicrobial activity of formic acid added to feed are also critical. Higher concentrations of the acid have been suggested for high-contamination feed ingredients prior to feed mixing to minimize potential damage to feed mill equipment and problems with animal feed palatability (105). Jones (51) concluded that Salmonella present in feed prior to chemical cleaning are more difficult to control than Salmonella in contact with feed after chemical treatment. Thermal treatment of feed during processing at the feed mill has been suggested as an intervention to limit Salmonella contamination of feed, but this depends on feed composition, particle size, and other factors associated with the milling process (51). The antimicrobial activity of acids is also temperature dependent, and elevated temperatures in the presence of organic acids may have a synergistic inhibitory effect on Salmonella, as observed in liquid cultures of Salmonella (124, 125). Several studies of Salmonella-contaminated feeds support the notion that elevated temperatures increase the effectiveness of acids in the feed matrix (106, 113, 126). Amado et al. (127) used a central composite design to study the interaction of temperature and acid (formic or lactic acid) in 10 strains of Salmonella enterica and Escherichia coli isolated from various cattle feeds and inoculated into acidified cattle pellets. They concluded that heat was the dominant factor influencing microbial reduction, along with acid and the type of bacterial isolate. The synergistic effect with acid still predominates, so lower temperatures and acid concentrations can be used. However, they also noted that synergistic effects were not always observed when formic acid was used, leading them to suspect that volatilization of formic acid at higher temperatures or buffering effects of feed matrix components were a factor.
       Limiting the shelf life of feed before feeding to animals is one way to control the introduction of foodborne pathogens into the animal’s body during feeding. However, once the acid in the feed has entered the gastrointestinal tract, it may continue to exert its antimicrobial activity. The antimicrobial activity of exogenously administered acidic substances in the gastrointestinal tract may depend on a variety of factors, including the concentration of gastric acid, the active site of the gastrointestinal tract, the pH and oxygen content of the gastrointestinal tract, the age of the animal, and the relative composition of the gastrointestinal microbial population (which depends on the location of the gastrointestinal tract and the maturity of the animal) (21, 24, 128–132). In addition, the resident population of anaerobic microorganisms in the gastrointestinal tract (which becomes dominant in the lower digestive tract of monogastric animals as they mature) actively produces organic acids through fermentation, which in turn may also have an antagonistic effect on transient pathogens entering the gastrointestinal tract (17, 19–21).
       Much of the early research focused on the use of organic acids, including formate, to limit Salmonella in the gastrointestinal tract of poultry, which has been discussed in detail in several reviews (12, 20, 21). When these studies are considered together, several key observations can be made. McHan and Shotts (133) reported that feeding formic and propionic acid reduced the levels of Salmonella Typhimurium in the cecum of chickens inoculated with the bacteria and quantified them at 7, 14, and 21 days of age. However, when Hume et al. (128) monitored C-14-labeled propionate, they concluded that very little propionate in the diet may reach the cecum. It remains to be determined whether this is also true for formic acid. However, recently Bourassa et al. (134) reported that feeding formic and propionic acid reduced the levels of Salmonella Typhimurium in the cecum of chickens inoculated with the bacteria, which were quantified at 7, 14, and 21 days of age. (132) noted that feeding formic acid at 4 g/t to broiler chickens during the 6-week growth period reduced the concentration of S. Typhimurium in the cecum to below the detection level.
       The presence of formic acid in the diet may have effects on other parts of the poultry gastrointestinal tract. Al-Tarazi and Alshavabkeh (134) demonstrated that a mixture of formic acid and propionic acid could reduce Salmonella pullorum (S. PRlorum) contamination in the crop and cecum. Thompson and Hinton (129) observed that a commercially available mixture of formic acid and propionic acid increased the concentration of both acids in the crop and gizzard and was bactericidal against Salmonella Enteritidis PT4 in an in vitro model under representative rearing conditions. This notion is supported by in vivo data from Bird et al. (135) added formic acid to the drinking water of broiler chickens during a simulated fasting period prior to shipping, similar to the fasting broiler chickens undergo prior to transport to a poultry processing plant. Addition of formic acid to the drinking water resulted in a reduction in the number of S. Typhimurium in the crop and epididymis, and a reduction in the frequency of S. Typhimurium-positive crops, but not in the number of positive epididymis (135). Development of delivery systems that can protect organic acids while they are active in the lower gastrointestinal tract may help improve efficacy. For example, microencapsulation of formic acid and its addition to the feed has been shown to reduce the number of Salmonella Enteritidis in cecal contents (136). However, this may vary depending on the animal species. For example, Walia et al. (137) did not observe a reduction in Salmonella in the cecum or lymph nodes of 28-day-old pigs fed a mixture of formic acid, citric acid, and essential oil capsules, and although Salmonella excretion in feces was reduced at day 14, it was not reduced at day 28. They showed that horizontal transmission of Salmonella between pigs was prevented.
       Although studies of formic acid as an antimicrobial agent in animal husbandry have primarily focused on food-borne Salmonella, there are also some studies targeting other gastrointestinal pathogens. In vitro studies by Kovanda et al. (68) suggest that formic acid may also be effective against other gastrointestinal food-borne pathogens, including Escherichia coli and Campylobacter jejuni. Earlier studies have shown that organic acids (e.g., lactic acid) and commercial mixtures containing formic acid as an ingredient can reduce Campylobacter levels in poultry (135, 138). However, as previously noted by Beyer et al. (67), the use of formic acid as an antimicrobial agent against Campylobacter may require caution. This finding is particularly problematic for dietary supplementation in poultry because formic acid is the primary respiration energy source for C. jejuni. Furthermore, part of its gastrointestinal niche is thought to be due to metabolic cross-feeding with mixed acid fermentation products produced by gastrointestinal bacteria, such as formate (139). This view has some basis. Because formate is a chemoattractant for C. jejuni, double mutants with defects in both formate dehydrogenase and hydrogenase have reduced rates of cecal colonization in broiler chickens compared with wild-type C. jejuni strains (140, 141). It is still unclear to what extent external formic acid supplementation affects gastrointestinal tract colonization by C. jejuni in chickens. Actual gastrointestinal formate concentrations may be lower due to formate catabolism by other gastrointestinal bacteria or formate absorption in the upper gastrointestinal tract, so several variables may influence this. In addition, formate is a potential fermentation product produced by some gastrointestinal bacteria, which may influence total gastrointestinal formate levels. Quantification of formate in gastrointestinal contents and identification of formate dehydrogenase genes using metagenomics may shed light on some aspects of the ecology of formate-producing microorganisms.
       Roth et al. (142) compared the effects of feeding broiler chickens the antibiotic enrofloxacin or a mixture of formic, acetic, and propionic acids on the prevalence of antibiotic-resistant Escherichia coli. Total and antibiotic-resistant E. coli isolates were counted in pooled faecal samples from 1-day-old broiler chickens and in cecal content samples from 14- and 38-day-old broiler chickens. E. coli isolates were tested for resistance to ampicillin, cefotaxime, ciprofloxacin, streptomycin, sulfamethoxazole, and tetracycline according to previously determined breakpoints for each antibiotic. When the respective E. coli populations were quantified and characterized, neither enrofloxacin nor the acid cocktail supplementation altered the total numbers of E. coli isolated from the ceca of 17- and 28-day-old broiler chickens. Birds fed the enrofloxacin supplemented diet had increased levels of ciprofloxacin-, streptomycin-, sulfamethoxazole-, and tetracycline-resistant E. coli and decreased levels of cefotaxime-resistant E. coli in the ceca. Birds fed the cocktail had decreased numbers of ampicillin- and tetracycline-resistant E. coli in the ceca compared to controls and enrofloxacin-supplemented birds. Birds fed the mixed acid also had a reduction in the number of ciprofloxacin- and sulfamethoxazole-resistant E. coli in the cecum compared to birds fed enrofloxacin. The mechanism by which acids reduce the number of antibiotic-resistant E. coli without reducing the total number of E. coli is still unclear. However, the results of the study by Roth et al. are consistent with those of the enrofloxacin group. (142) This may be an indication of a reduced dissemination of antibiotic resistance genes in E. coli, such as the plasmid-linked inhibitors described by Cabezon et al. (143). It would be interesting to conduct a more in-depth analysis of plasmid-mediated antibiotic resistance in the gastrointestinal population of poultry in the presence of feed additives such as formic acid and to further refine this analysis by assessing the gastrointestinal resistome.
       The development of optimal antimicrobial feed additives against pathogens should ideally have minimal impact on the overall gastrointestinal flora, particularly on those microbiota considered beneficial to the host. However, exogenously administered organic acids can have detrimental effects on the resident gastrointestinal microbiota and to some extent negate their protective properties against pathogens. For example, Thompson and Hinton (129) observed decreased crop lactic acid levels in laying hens fed a mixture of formic and propionic acids, suggesting that the presence of these exogenous organic acids in the crop resulted in a reduction in crop lactobacilli. Crop lactobacilli are considered a barrier to Salmonella, and therefore disruption of this resident crop microbiota may be detrimental to the successful reduction of Salmonella colonization of the gastrointestinal tract (144). Açıkgöz et al. found that the lower gastrointestinal tract effects of birds may be lower. (145) No differences were found in the total intestinal flora or Escherichia coli counts in 42-day-old broiler chickens drinking water acidified with formic acid. The authors suggested that this may be due to formate being metabolized in the upper gastrointestinal tract, as has been observed by other investigators with exogenously administered short-chain fatty acids (SCFA) (128, 129).
       Protecting formic acid through some form of encapsulation may help it reach the lower gastrointestinal tract. (146) noted that microencapsulated formic acid significantly increased the total short-chain fatty acid (SCFA) content in the cecum of pigs compared to pigs fed unprotected formic acid. This result led the authors to suggest that formic acid may effectively reach the lower gastrointestinal tract if it is properly protected. However, several other parameters, such as formate and lactate concentrations, although higher than those in pigs fed a control diet, were not statistically different from those in pigs fed an unprotected formate diet. Although pigs fed both unprotected and protected formic acid showed nearly a three-fold increase in lactic acid, lactobacilli counts were not altered by either treatment. The differences may be more pronounced for other lactic acid-producing microorganisms in the cecum (1) that are not detected by these methods and/or (2) whose metabolic activity is affected, thereby altering the fermentation pattern such that resident lactobacilli produce more lactic acid.
       To more accurately study the effects of feed additives on the gastrointestinal tract of farm animals, higher-resolution microbial identification methods are needed. In the past few years, next-generation sequencing (NGS) of the 16S RNA gene has been used to identify microbiome taxa and compare the diversity of microbial communities (147), which has provided a better understanding of the interactions between dietary feed additives and the gastrointestinal microbiota of food animals such as poultry.
       Several studies have used microbiome sequencing to evaluate the response of the chicken gastrointestinal microbiome to formate supplementation. Oakley et al. (148) conducted a study in 42-day-old broiler chickens supplemented with various combinations of formic acid, propionic acid, and medium-chain fatty acids in their drinking water or feed. Immunized chickens were challenged with a nalidixic acid-resistant Salmonella typhimurium strain and their ceca were removed at 0, 7, 21, and 42 days of age. Cecal samples were prepared for 454 pyrosequencing and sequencing results were evaluated for classification and similarity comparison. Overall, treatments did not significantly affect the cecal microbiome or S. Typhimurium levels. However, overall Salmonella detection rates decreased as the birds aged, as confirmed by taxonomic analysis of the microbiome, and the relative abundance of Salmonella sequences also decreased over time. The authors note that as the broilers aged, the diversity of the cecal microbial population increased, with the most significant changes in the gastrointestinal flora observed across all treatment groups. In a recent study, Hu et al. (149) compared the effects of drinking water and feeding a diet supplemented with a mixture of organic acids (formic acid, acetic acid, propionic acid, and ammonium formate) and virginiamycin on cecal microbiome samples from broiler chickens collected at two stages (1–21 days and 22–42 days). Although some differences in cecal microbiome diversity were observed among treatment groups at 21 days of age, no differences in α- or β-bacteria diversity were detected at 42 days. Given the lack of differences at 42 days of age, the authors hypothesized that the growth advantage may be due to earlier establishment of an optimally diverse microbiome.
       Microbiome analysis focusing only on the cecal microbial community may not reflect where in the gastrointestinal tract most of the effects of dietary organic acids occur. The upper gastrointestinal tract microbiome of broiler chickens may be more susceptible to the effects of dietary organic acids, as suggested by the results of Hume et al. (128). Hume et al. (128) demonstrated that most of the exogenously added propionate was absorbed in the upper gastrointestinal tract of birds. Recent studies on the characterization of gastrointestinal microorganisms also support this view. Nava et al. (150) demonstrated that a combination of a mixture of organic acids [DL-2-hydroxy-4(methylthio)butyric acid], formic acid, and propionic acid (HFP) affected the gut microbiota and increased Lactobacillus colonization in the ileum of chickens. Recently, Goodarzi Borojeni et al. (150) demonstrated that a combination of organic acid mixture [DL-2-hydroxy-4(methylthio)butyric acid], formic acid, and propionic acid (HFP) affected the gut microbiota and increased Lactobacillus colonization in the ileum of chickens. (151) studied feeding broiler chickens a mixture of formic acid and propionic acid at two concentrations (0.75% and 1.50%) for 35 days. At the end of the experiment, the crop, stomach, distal two-thirds of the ileum, and cecum were removed and samples were taken for quantitative analysis of specific gastrointestinal flora and metabolites using RT-PCR. In culture, the concentration of organic acids did not affect the abundance of Lactobacillus or Bifidobacterium, but increased the population of Clostridium. In the ileum, the only changes were a decrease in Lactobacillus and Enterobacter, whereas in the cecum these flora remained unchanged (151). At the highest concentration of organic acid supplementation, the total lactic acid concentration (D and L) was reduced in the crop, the concentration of both organic acids was reduced in the gizzard, and the concentration of organic acids was lower in the cecum. There were no changes in the ileum. With regard to short-chain fatty acids (SCFAs), the only change in the crop and gizzard of birds fed organic acids was in the propionate level. Birds fed the lower concentration of organic acid showed an almost tenfold increase in propionate in the crop, whereas birds fed the two concentrations of organic acid showed an eight- and fifteen-fold increase in propionate in the gizzard, respectively. The increase in acetate in the ileum was less than twofold. Overall, these data support the view that most of the effects of external organic acid application were evident in yield, whereas organic acids had minimal effects on the lower gastrointestinal microbial community, suggesting that fermentation patterns of the upper gastrointestinal resident flora may have been altered.
       Clearly, a more in-depth characterization of the microbiome is needed to fully elucidate microbial responses to formate throughout the gastrointestinal tract. A more in-depth analysis of the microbial taxonomy of specific gastrointestinal compartments, particularly upper compartments such as the crop, may provide further insight into the selection of certain groups of microorganisms. Their metabolic and enzymatic activities may also determine whether they have an antagonistic relationship with pathogens entering the gastrointestinal tract. It would also be interesting to conduct metagenomic analyses to determine whether exposure to acidic chemical additives during the life of birds selects for more “acid-tolerant” resident bacteria, and whether the presence and/or metabolic activity of these bacteria would represent an additional barrier to pathogen colonization.
       Formic acid has been used for many years as a chemical additive in animal feed and as a silage acidifier. One of its main uses is its antimicrobial action to limit the number of pathogens in feed and their subsequent colonization in the gastrointestinal tract of birds. In vitro studies have shown that formic acid is a relatively effective antimicrobial agent against Salmonella and other pathogens. However, the use of formic acid in feed matrices may be limited by the high amount of organic matter in feed ingredients and their potential buffering capacity. Formic acid appears to have an antagonistic effect on Salmonella and other pathogens when ingested via feed or drinking water. However, this antagonism occurs primarily in the upper gastrointestinal tract, as formic acid concentrations may be reduced in the lower gastrointestinal tract, as is the case with propionic acid. The concept of protecting formic acid through encapsulation offers a potential approach to delivering more acid to the lower gastrointestinal tract. Furthermore, studies have shown that a mixture of organic acids is more effective in improving poultry performance than administration of a single acid (152). Campylobacter in the gastrointestinal tract may respond differently to formate, as it can use formate as an electron donor, and formate is its main energy source. It is unclear whether increasing formate concentrations in the gastrointestinal tract would be beneficial to Campylobacter, and this may not occur depending on other gastrointestinal flora that can use formate as a substrate.
       Additional studies are needed to investigate the effects of gastrointestinal formic acid on non-pathogenic resident gastrointestinal microbes. We prefer to selectively target pathogens without disrupting members of the gastrointestinal microbiome that are beneficial to the host. However, this requires a more in-depth analysis of the microbiome sequence of these resident gastrointestinal microbial communities. Although some studies have been published on the cecal microbiome of formic acid-treated birds, more attention is needed to the upper gastrointestinal microbial community. Identification of microorganisms and comparison of similarities between gastrointestinal microbial communities in the presence or absence of formic acid may be an incomplete description. Additional analyses, including metabolomics and metagenomics, are needed to characterize functional differences between compositionally similar groups. Such characterization is critical to establish the relationship between the gastrointestinal microbial community and bird performance responses to formic acid-based improvers. Combining multiple approaches to more accurately characterize gastrointestinal function should enable the development of more effective organic acid supplementation strategies and ultimately improve predictions of optimal bird health and performance while limiting food safety risks.
       SR wrote this review with assistance from DD and KR. All authors made substantial contributions to the work presented in this review.
       The authors declare that this review received funding from Anitox Corporation to initiate the writing and publication of this review. The funders had no influence on the views and conclusions expressed in this review article or on the decision to publish it.
       The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
       Dr. DD would like to acknowledge support from the University of Arkansas Graduate School through a Distinguished Teaching Fellowship, as well as ongoing support from the University of Arkansas Cell and Molecular Biology Program and the Department of Food Sciences. Additionally, the authors would like to thank Anitox for initial support in writing this review.
       1. Dibner JJ, Richards JD. Use of antibiotic growth promoters in agriculture: history and mechanisms of action. Poultry Science (2005) 84:634–43. doi: 10.1093/ps/84.4.634
       2. Jones FT, Rick SC. History of antimicrobial development and surveillance in poultry feed. Poultry Science (2003) 82:613–7. doi: 10.1093/ps/82.4.613
       3. Broom LJ. Subinhibitory theory of antibiotic growth promoters. Poultry Science (2017) 96:3104–5. doi: 10.3382/ps/pex114
       4. Sorum H, L’Abe-Lund TM. Antibiotic resistance in foodborne bacteria—consequences of disruptions in global bacterial genetic networks. International Journal of Food Microbiology (2002) 78:43–56. doi: 10.1016/S0168-1605(02)00241-6
       5. Van Immerseel F, Cauwaerts K, Devriese LA, Heesebroek F, Ducatel R. Feed additives for the control of Salmonella in feed. World Journal of Poultry Science (2002) 58:501–13. doi: 10.1079/WPS20020036
       6. Angulo FJ, Baker NL, Olsen SJ, Anderson A, Barrett TJ. Antimicrobial use in agriculture: controlling the transmission of antimicrobial resistance to humans. Seminars in Pediatric Infectious Diseases (2004) 15:78–85. doi: 10.1053/j.spid.2004.01.010
       7. Lekshmi M, Ammini P, Kumar S, Varela MF. Food production environments and the evolution of antimicrobial resistance in animal-derived human pathogens. Microbiology (2017) 5:11. doi: 10.3390/microorganisms5010011
       8. Lourenço JM, Seidel DS, Callaway TR. Chapter 9: Antibiotics and gut function: history and current status. In: Ricke SC, ed. Improving gut health in poultry. Cambridge: Burley Dodd (2020). Pages 189–204. DOI: 10.19103/AS2019.0059.10
       9. Rick SC. No. 8: Feed Hygiene. In: Dewulf J, van Immerzeel F, eds. Biosecurity in Animal Production and Veterinary Medicine. Leuven: ACCO (2017). Pages 144–76.