Gómez-García et al. Porcine Health Management (2019) 5:32 https://doi.org/10.1186/s40813-019-0139-4 RESEARCH Open Access Antimicrobial activity of a selection of organic acids, their salts and essential oils against swine enteropathogenic bacteria Manuel Gómez-García1* , Cinta Sol2, Pedro J. G. de Nova1, Mónica Puyalto2, Luis Mesas2, Héctor Puente1, Óscar Mencía-Ares1, Rubén Miranda1, Héctor Argüello1, Pedro Rubio1 and Ana Carvajal1 Abstract Background: Accurate screening of new alternative antimicrobial compounds is essential for their use to control pathogens in swine production due to the replacement of antibiotics and zinc oxide. Most in vitro studies have separately reported the antimicrobial activity of organic acids and essential oils (EOs) using diverse methods for susceptibility testing. In addition, in vitro outcomes can help in the selection of the suitable antimicrobial compound and effective combinations of these compounds in the control of pathogens of interest in pork production. Therefore, the aim of this study is to determinate the antibacterial activity of six organic acids and six EOs against Escherichia coli, Salmonella spp. and Clostridium perfringens isolates, some of them multi-resistant to antibiotics, from swine origin. The synergistic effects between the products with higher activity for each bacteria were also calculated. Results: All products tested showed activity against at least one bacterial species, except for black pepper EO. The results showed that formic acid with the shortest chain length was the most effective against E. coli and Salmonella spp., while the sodium salt of coconut fatty acid distillates with long chain acids was the most effective against C. perfringens.The susceptibility of isolates tested to EOs was similar, a result that demonstrates a similar activity of these products against phylogenetically unrelated pathogens. In addition, an additive effect was shown for carvacrol-oregano EO for E. coli, formic acid-carvacrol and formic acid-thymol for Salmonella spp. and carvacrol-cinamaldehyde for C. perfringens. Conclusions: The susceptibility of isolates to EOs was similar, a result that demonstrates a similar activity of these products against phylogenetically unrelated pathogens in contrast to organic acids. In addition, an additive effect was shown for several combinations of these compounds. Keywords: Pig, feed additives, organic acids, Essential oils, Minimum inhibitory concentration, Minimum bactericidal concentration, Fractional inhibitory concentration, enteric pathogens Background reduce the use of antibiotics, other compounds with anti- Nowadays, demand for antibiotic-reduced or antibiotic- microbial activity could be considered to replace them free farm animals is rising globally. Its achievement can within control strategies. In this context, the use of plant only be accomplished through a combination of strategies, extracts or phytobiotics and organic acids have gained which include husbandry, biosecurity and alternatives to renewed interest because of their potential antimicrobial antibiotics [1, 2]. The swine industry is foremost in anti- activity [1, 4–7]. These compounds can be used as feed ad- microbial consumption [3]. Consequently, pig enteric path- ditives, potentially conferring benefits to health and growth ogens frequently harbour antimicrobial resistance to a to the host due to their antimicrobial activity and immune large number of compounds. As there is a requirement to response enhancement [4, 8]. Essential oils (EOs) are volatile lipophilic compounds constituted of a complex mixture of terpenoids and phe- * Correspondence: [email protected] nols [9, 10] and are one of the most interesting groups 1Department of Animal Health, Faculty of Veterinary Medicine, Universidad de León, Campus de Vegazana, s/n, 24007 León, Spain of phytobiotic compounds [11, 12]. Their antibacterial Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gómez-García et al. Porcine Health Management (2019) 5:32 Page 2 of 8 activity seems to be associated with the disruption of Table 1 Main characteristics of bacterial strains used the structure and function of bacteria cell membranes Escherichia coli Virotype Antibiotic resistance due to their hydrophobicity [12]. Organic acids have profile also antimicrobial properties based on their ability to EC 60 a STb CEF, LIN cross the cell membrane, due to the lipophilic nature EC 61 a STb LIN of their undissociated form, modifying the proton and EC 67 a F18, STa, STb, EAST1 LIN, ENR, SUL associated anion concentrations in the cytoplasm [13]. EC 96 a STb AML, CEF, LIN, SUL Consequently, purine bases and essential enzymes are a – negatively affected and bacterial viability decreases EC 107 AML, CEF, LIN, SUL, DOX [14]. These acids are generally available as calcium, EC 115 a F18, EAST1 AML, LIN, SUL potassium or sodium salts to decrease odour and volatility and facilitate the manufacturing processes Salmonella spp. Serotype/Phagotype Antibiotic [15]. Despite their well-known antimicrobial proper- resistance profile ties, field results are not always succesful [16–18]. SP 11 a Typhimurium/DT 104 AML, SPC, LIN, SUL, These results demonstrate that further research is FFC needed in order to optimise concentrations, combina- SP 28 a London LIN, SUL tions and interactions of these compounds against CECT 443 Typhimurium LIN target pathogens. This information would be consid- ered of value to increase the accuracy of treatments CECT 700 Infantis AML, CEF, LIN, SUL, DOX with EOs and/or organic acids. CECT 915 Choleraesuis LIN The present study evaluates the antimicrobial activity of six organic acids and six EOs against E. coli, C. per- CECT 4300 Enteriditis LIN, SUL fringens and Salmonella spp. Interactions between the compounds with greater activity against each bacterial Clostridium perfringens Toxigenic type Antibiotic resistance profile species were also investigated. CP 3 a Type A (alpha toxin) CEF, LIN, ENR, SUL a Methods CP 34 Type A (alpha toxin) SUL Bacterial strains and growth conditions CP 52 a Type A (alpha toxin) LIN, ENR, SUL The eighteen strains used in this study belonged to the CP 89 a Type A (alpha toxin) Not detected collection of the Infectious Diseases Unit (IDUC) at the CP 99 a Type A (alpha toxin) LIN, SUL Veterinary Faculty of the University of León. The collec- CP H a Type A (alpha toxin) SUL tion included strains from the Spanish Type Culture AML amoxicillina, SPC spectinomycin, ENR enrofloxacin, SUL sulphanomides, Collection (CECT) and field isolates recovered from fae- DOX doxycycline, CEF cephalotin, LIN lincomycin, FFC florfenicol cal samples from diarrhoea outbreaks on Spanish swine a Field isolates recovered from faecal samples collected from swine farms farms. Table 1 summarises the main characteristics of in Spain strains used, including virotype for E. coli, serotype for Salmonella spp. and toxigenic type for C. perfringens.In Organic acids and essential oils addition, the antimicrobial resistance profile of each The products evaluated in this studywereprovidedbyNorel strain was determined by disc diffusion test, using break- SA (Spain). They included six organic acids: formic acid point values provided by Clinical and Laboratory Stand- (purity 85%), propionic acid (99%), sodium butyrate (98%), ard Institute [19] and the Comité de l’Antibiogramme de sodium heptanoate (95%), pelargonic acid (99%) and sodium la Société Française de Microbiologie. salt of coconut fatty acid distillates(67%),andsixEOs:cin- Frozen cultures stored at − 80 °C were revived by in- namaldehyde (97–98%), thymol (99%), carvacrol (99%), oculation onto appropriate agar plates, tryptic soy agar clove EO (eugenol 80%), oregano EO (phenols 65–75%) and (TSA) (Scharlab, Spain) for E. coli and Salmonella spp. black pepper EO (piperine 40%). Formaldehyde (40%) was and fastidious anaerobe agar (FAA) (Neogen, United also tested as a positive control. Kingdom) for C. perfringens. E. coli and Salmonella iso- The organic acids sodium butyrate, sodium heptanoate lates were incubated at 37 °C for 24 h under aerobic con- and sodium salt of coconut fatty acid distillates were ob- ditions while C. perfringens were grown at 38.5 °C for 24 tained as powder products and were resuspended in 50 to 36 h in an anaerobic workstation with an oxygen-free mM sodium phosphate buffer (pH 6.0) depending on their anaerobic gas mixture (80% N2, 10% H2 and 10% CO2). solubility. Final concentrations of these stock solutions Subsequently, the purity of the cultures was confirmed were 500,000 ppm (w/v) for sodium butyrate, 50,000 ppm for each strain by examination of colony morphology (w/v) for sodium heptanoate and 10,000 ppm (w/v) for the and Gram staining. sodium salt of coconut fatty acid distillates. Other organic Gómez-García et al. Porcine Health Management (2019) 5:32 Page 3 of 8 acids and formaldehyde were provided as liquid prepara- lowest concentration