The Spectrum of Antimicrobial Activity of Cyadox against Pathogens Collected from Pigs, Chicken, and Fish in China

Cyadox has potential use as an antimicrobial agent in animals. However, its pharmacodynamic properties have not been systematically studied yet. In this study, the in vitro antibacterial activities of cyadox were assayed, and the antibacterial efficacy of cyadox against facultative anaerobes was also determined under anaerobic conditions. It was shown that Clostridium perfringens and Pasteurella multocida (MIC = 0.25 and 1 μg/mL) from pigs, Campylobacter jejuni and Pasteurella multocida from poultry, E. coli, Streptococcus spp., and Flavobacterium columnare from fish were highly susceptible to cyadox (MIC= 1 and 8 μg/mL). However, F. columnare has no killing effect for drug tolerance. Under in vitro anaerobic conditions, the antibacterial activity of cyadox against most facultative anaerobes was considerably enhanced Under anaerobic conditions for the facultative anaerobes, susceptible bacteria were P. multocida, Aeromonas spp. (including A. hydrophila, A. veronii, A. jandaei, A. caviae, and A. sobria, excluding A. punctata), E. coli, Salmonella spp. (including S. choleraesui, S. typhimurium, and S. pullorum), Proteus mirabilis, Vibrio fluvialis, Yersinia ruckeri, Erysipelothrix, Acinetobacter baumannii, and Streptococcus agalactiae (MICs were 0.25~8 μg/mL, MBCs were 1–64 μg/mL). Intermediate bacteria were Enterococcus spp. (including E. faecalis and E. faecium), Yersinia enterocolitica, and Streptococcus spp. (MICs mainly were 8~32 μg/mL, MBCs were 16~128 μg/mL). This study firstly showed that cyadox had strong antibacterial activity and had the potential to be used as a single drug in the treatment of bacterial infectious diseases.


Introduction
Cyadox is a synthetic compound belonging to quinoxaline-1,4-dioxides, which are widely used as an antibacterial agent with a broad spectrum of antimicrobial activity and growth promoters in veterinary medicine [1]. Compared with the other members of quinoxalines such as carbadox and olaquindox, the cyadox is safer [2][3][4][5] according to the long term toxicity test, a subchronic oral toxicity test, and a phototoxicity test of cyadox in previous studies [6] and can promote the growth of different animals with more obvious effects such as better growth-enhancing functions in food-producing animals including fish, goats, pigs and poultry with less toxic effects than olaquindox, when used as feed additive [7] in animal feed. Moreover, further studies have demonstrated that cyadox was better as a growth promotor if compared with carbadox and olaquindox [8]. Since carbadox and olaquindox have been banned or limited to be used in food animals due to their toxicities, making cyadox as a substitution product having a capacious prospect in animal husbandry and aquaculture. Cyadox has excellent pharmacokinetic characteristics. Previous studies have shown the distribution and metabolism of Cyadox in swine, and six major metabolites were identified as follows: Disdesoxy-Cyadox (Cy1), Cyadox 4monoxide (Cy2), N-decyanoacetyl Cyadox (Cy4), Quinocaline-2-carboxylic acid (Cy6), 11, 12-dihydro-bisdesoxycaydox (Cy9), 2-hydromethyl-quinoxaline (Cy12). To fully reflect the pharmacodynamic of cyadox, it is necessary to detect the antibacterial activity of cyadox and its metabolites.
However, there are few studies on the pharmacodynamics of cyadox at present. As a potent antimicrobial agent, Cyadox had been proved to have a wide spectrum of activity against many pathogenic bacteria of pigs, poultry, and fish [9]. In vivo, cyadox reduces diarrhea frequencies of different animals and prevents E. coli infection in piglets and broilers [10]. It has high antimicrobial activity in vitro against E. coli under anaerobic conditions. MIC values for cyadox in MHB (Mueller-Hinton broth) against E. coli were 1 to 4 µg/mL [11]. Some studies showed that cyadox could promote the growth of swine, chicken, and fish [3,12]. However, there are only limited data on the prophylactic schedule in piglets. At present, there is not a scientifically validated dosage for treating E. coli diarrhea.
However, the results of previous studies were not sufficient to explain the antimicrobial characteristics of cyadox. Hence, there is a need for a further and complete study to build up the antimicrobial spectrum using the standard method of Clinical and Laboratory Standards Institute (CLSI) approved by the FDA.
The purpose of this study was to evaluate the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of cyadox in vitro against different species of bacteria from pigs, poultry, and fishes, most of which were enteric pathogens, and compare the antimicrobial spectrum of cyadox with other commonly used antimicrobial agents. Under anaerobic conditions, the antimicrobial activity of some quinoxalines were different as compared to cyadox because cyadox exhibits much stronger activity in the absence of oxygen [10]. therefore, cyadox may be active against facultative anaerobes under anaerobic conditions. Based upon systematic toxicological and microbiological safety evaluations, cyadox shows much lower toxicity and higher safety than other well-known quinoxalines such as olaquindox and carbadox, which have been banned or strictly limited in their use in food-producing animals because of their potential toxicities [13]. However, it is hopeful that cyadox would be developed as a replacer of olaquindox and carbadox with greater safety and excellent antimicrobial activity. Based on the related guidelines and standards of the Clinical and Laboratory Standards Institute (CLSI), we determined the in vitro antibacterial activities of cyadox and established the antimicrobial spectrum of cyadox comprehensively and systematically in pathogenic bacteria from swine, chicken, and fish in the present study. The deep knowledge about the pharmacodynamics of cyadox will lay a solid foundation for the application of cyadox as a new veterinary drug.

Study Drug and Susceptibility Testing
Cyadox powder (purity percent is ≥98%) was synthesized by the Institute of Veterinary Pharmaceutics (Huazhong Agricultural University, Wuhan, China). For the preparation of the working solution for MIC determination desired amount of cyadox was dissolved in dimethyl sulfoxide (DMSO) at the concentration of 1280 µg/mL as a stock solution. For MIC (Minimum inhibitory Concentration) determination, each bacterial strain was cultured to a logarithmic phase to obtain the turbidity of the 0.5 McFarland standard and then was diluted 100 times with MH broth to obtain a density of 1 × 10 6 CFU/mL which was used as the inoculum suspension. MIC was defined as the minimum concentration of compound that resulted in no visible growth. MIC determination was performed by the microbroth dilution method according to the CLSI (Clinical and Laboratory Standards Institute, formerly NCCLS) guidelines. The test was performed in a 96-well microtiter plate in a final volume of 100 µL. Each well was inoculated with serially diluted antimicrobial agents and the inoculum suspension (1:1 v/v). Different inoculation conditions for different bacteria isolated from livestock and poultry were used for MIC determination. Nonfastidious bacteria (Escherichia coli, Salmonella spp., Yersinia spp. Proteus mirabilis, Pseudomonas spp., Staphylococcus aureus, Enterococcus spp.) were cultured in CAMHB (cation-adjusted Mueller-Hinton broth) at 37°C for 16-20 h according to the CLSI guidelines. Fastidious organisms (Pasteurella spp., Streptococcus spp., and Erysipelothrix spp.) were cultured in the media of CAMHB+LHB (cation-adjusted Mueller-Hinton broth supplemented with 2.5% lysed horse blood) for 18-20 h at 37 • C. Microaerophilic bacteria Campylobacter jejuni were cultured in CAMHB+LHB at 42 • C for 24 h under 10% CO 2 . Anaerobic bacteria, such as Clostridium perfringens were cultured in Brucella broth under 80% N 2 -10% CO 2 -10% H 2 at 37 • C for 24 h. The inoculation conditions for the bacteria isolated from fish were set according to the CLSI guidelines at a temperature of 28 • C. Vibrio fluvialis was cultured in CAMHB with 1% NaCl for 24 h; E. ictaluri was cultured in CAMHB for 48 h; Flavobacterim columnare was cultured in CAMHB diluted for 24 h; Streptococcus agalactiae was cultured in CAMHB supplemented with 2.5% lysed horse blood for 24 h. E. coli, F. columnare, Aeromonas spp. (including A. hydrophila, A. veronii, A. jandaei, A. caviae, A. sobria, and A. punctata), V. fluvialis, A. baumannii, and Y. ruckeri isolated from fish were cultured in CAMHB for 24 h. Mycobacterium tuberculosis was cultured on Lowenstein-Jensen medium (LJ) solidified by coagulation at 83 • C for 40 min and incubated at 37 • C [14]. Quality control was monitored using Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, Campylobacter jejuni ATCC 33560, Bacteroides fragilis ATCC 25285, and Bacteroides thetaiotaomicron ATCC 29741.
MBC (Minimal Bactericidal Concentration) was determined according to the document M26-AE of CLSI. The lowest concentration of antimicrobial agent that killed ≥99.9% of the starting inoculum was defined as the MBC endpoint. The double diluted inoculum suspension and 10 µL broth from 96-well with no visible growth above the MIC after 24 h incubation on MH agar, incubated for one or two nights and counted for colony, respectively, and calculated for the MBC further.
MICs of the facultative anaerobes tested under anaerobic conditions were determined according to the defined methodology of CLSI with little change in the anaerobic environement (80% N 2 -10% CO 2 -10% H 2 ). Bacteria for colony counting and MBC testing were cultured under aerobic condition.
All the experiments were performed in 3 replicates along with the quality control strains to ensure the accuracy of results.

Data Processing
For analytical purposes, the bacteria were grouped into species or genus groups. The calculation included in MIC 50 (MBC at which 50% of the strains are inhibited), MBC 90 (MBC at which 90% of the strains are inhibited), MBC 50 (MBC at which 50% of the strains are killed), MBC 90 (MBC at which 90% of the strains are killed), and the MBC/MIC ratios were calculated to determine the presence or absence of tolerance. MIC50, MBC50, MIC90, and MBC90 were calculated by using SPSS software. The breakpoint was set in present study as follow: susceptible, MIC 90 ≤ 8 µg/mL; intermediate, 16 µg/mL ≤ MIC 90 ≤ 32 µg/mL; resistant, MIC 90 ≥ 64 µg/mL. Tolerance was defined as an MBC/MIC ratio of ≥32 or an MBC/MIC ratio of ≥16 when the MBC was greater than or equal to the MIC resistance breakpoint.

Susceptibility of Pig Pathogens to Cyadox
Under CLSI standard conditions, the MIC and MBC of cyadox against Clostridium perfringen were 0.5~1 µg/mL, which were more susceptible and stronger than that of other antibacterial agents. The cyadox was much more effective against Pasteurella multocida, Salmonella choleraesui, Erysipelothrix, and Streptococcus than olaquindox but weaker than chlortetracycline. Streptococcus were found to be resistant to chlortetracycline. Under anaerobic conditions for facultative anaerobes, the antibacterial activity of cyadox was enhanced by 4-6 times in E. coli, Pasteurella multocida, Salmonella choleraesuis, and Erysipelothrix. Compared with controls, the antibacterial activity of cyadox was stronger than that of other antibacterials against Escherichia coli; the actions of cyadox were stronger than or similar to that of olaquindox and weaker than that of chlortetracycline against other bacteria (Table 1).

Susceptibility of Poultry Pathogens to Cyadox
Following CLSI standards conditions, the most susceptible bacteria forcyadox were C. jejuni and C. perfringen with the MICs and MBCs were 0.25~1 µg/mL and 1 µg/mL, respectively. While E. faecalis and E. faecium were resistant against cyadox. Under anaerobic conditions for facultative anaerobes, the antibacterial activity of cyadox was enhanced by 4~16 times in S. pullorum, E. coli, and Enterococcus spp., which indicated an inclined effect of cyadox against these bacteria. Compared with controls, under the two incubating conditions, the antibacterial actions of cyadox were stronger than that of other antibacterial agents against E. coli and C. perfringen, and the action of cyadox was stronger than or similar to that of olaquindox but weaker than that of chlortetracycline against other bacteria ( Table 2).

Susceptibility of Fish Pathogens to Cyadox
E. coli showed a susceptible effect to cyadox with the MIC and MBC was 1 µg/mL and 16 µg/mL, respectively. For F. columnare, cyadox and sulfadimidine showed only an inhibitory effect but not a bactericidal effect. Under anaerobic conditions for facultative anaerobes, the antibacterial activity was enhanced by 8~256 times in Aeromonas spp. (included A. hydrophila, A. veronii, A. jandaei, A. caviae, and A. sobria, excluding A. punctata), V. fluvialis, A. baumannii, and Y. ruckeri. MICs and MBCs of Aeromonas spp. (excluded Aeromonas punctata), V. fluvialis, and Y. ruckeri were declined to 0.5~2 µg/mL and 1~8 µg/mL. Compared with controls, the antibacterial activity of cyadox were stronger than that of other antibacterial agents against E. coli. For Mycobacterium tuberculosis, the action of cyadox was stronger or similar to sulfadimidine but weaker than that of chlortetracycline against other bacteria except for A. baumannii (Table 3). Note: (1) The lower symbol " S " denotes the result was under CLSI condition, and " N " denotes the result was under anaerobic condition. (2) Values in brackets are the multiple drop of MIC under anaerobic conditions from aerobic conditions. "+" denotes gram-positive and "−" denotes gram-negative.   Table 3. Antimicrobial susceptibility of cyadox and controls against common pathogens isolated from fishes (unit: µg/mL).

Susceptibility of Other Pathogens to Cyadox
The results of antimicrobial susceptibility of cyadox against pathogenic bacteria isolated from humans and animals were listed in (Table 4). The antibacterial action of cyadox was stronger than that of other antibacterial agents against S. typhimurium, Y. enterocolitica, and P. mirabilis which was stronger than sulfonamide but weaker than chlortetracycline. Under anaerobic conditions, the antibacterial activity was enhanced by 8 times in Proteus mirabilis.

Discussion
Clinical breakpoints for quinoxalines have not been established by CLSI yet [15]. This study defines the clinical breakpoints for cyadox according to the antibacterial activities of cyadox and the antibiogram of olaquindox. Cyadox has a good effect against E. coli in vitro (MIC 90 under anaerobic condition was 4 µg/mL in this test). The susceptible bacteria of olaquindox including gram-negative bacteria (P. multocida, E. coli, S. choleraesui, Shigella spp., and Proteus spp.) and gram-positive bacteria (Staphylococci), MIC 90 of these bacteria under anaerobic condition were 8 µg/mL in this study. In addition, isolates were considered to be tolerant to antimicrobial agents that were known to be bactericidal but that do not show a killing effect.
The antimicrobial effect of cyadox against pathogens isolated from pigs and poultry was similar in vitro. Under standard conditions, susceptible bacteria for cyadox were C. perfringen, C. jejuni, and P. multocida. Intermediate bacteria were Salmonella spp. (including S. choleraesui, S. typhimurium, S. pullorum), E. coli, Y. enterocolitica, P. mirabilis, Erysipelothrix, S. aureus, and Streptococcus spp. The susceptibility of C. perfringens against cyadox was similar to previous studies conducted by [11]. Resistant bacteria were P. pyocyanea, E. faecalis, and E. faecium. Under anaerobic conditions, susceptible bacteria were P. multocida, E. coli, Salmonella spp., P. mirabilis, and Erysipelothrix, intermediate bacteria were Y. enterocolitica, Streptococcus spp, E. faecalis, and E. faecium, while P. pyocyanea was resistant bacterium against cyadox. However, cyadox showed growth inhibition with a ≥16-fold against Salmonella spp. and Erysipelothrix spp. While compared with other drugs, Salmonella spp., Erysipelothrix spp., and Streptococcus spp. have a high tolerance against chlortetracycline, and C. perfringen has a high tolerance against olaquindox.
Compared with the source of pathogenic bacteria, the antimicrobial spectrum of cyadox against pigs and poultry in vitro was similar. The antimicrobial effect of cyadox against different serotypes or serogroup in the same species was almost similar, but the MICs of some Streptococcus isolated in recent years increased, which suggests no cross-resistance between quinoxalines except Streptococcus. The antimicrobial susceptibility of bacteria isolated from fish was different from that of bacteria from non-fish source, which mainly because the incubation temperature was different. Under aerobic conditions, the MICs and MBCs of cyadox in E. coli were the same as or higher than that of olaquindox, which means that the antibacterial effect of cyadox against E. coli is not as good as olaquindox in vitro. However, the effect of cyadox on the antibacterial activity in vitro was as good as that of olaquindox against E. coli infection demonstrated its activity under aerobic conditions [10]. Maybe it can turn to seek an answer from the bactericidal activity of cyadox and olaquindox under anaerobic conditions. The effect under anaerobic conditions is closer to that of the intestinal tract condition than that under the aerobic condition [16].
The antimicrobial activity of cyadox for most facultative anaerobes was significantly better in anaerobic conditions than in aerobic conditions, the sensitivity of control drug chlortetracycline and olaquindox were significantly improved as compared to Bacitracinzinc and sulfadimidine. In this test, the MICs of Escherichia coli and Salmonella spp. under CLSI condition and anaerobic condition were in accordance with that reported previously [11]. The difference in antibacterial activity of some quinoxalines under anaerobic and aerobic conditions may be due to some free radicals [17]. The antibacterial mechanism may be similar to quindoxin. There was no evidence has been found for binding of quindoxin to DNA [18]. It suggested that some free radicals responsible for the lethal effect of quindoxin, and the free radicals were generated always accompanied by a reduction of the drug and occurred only under anaerobic conditions [18].
Usually, the treatment effect in vivo can be predicted by the results in vitro [19]. Cyadox exhibited excellent in vitro activity across an extended spectrum of bacteria, encompassing all major pathogens with clinical relevance of intestines infections in pigs, poultry, and fish [20]. Cyadox used as an antimicrobial growth promoter has good potential for disease resistance, which needs further clinical trials to validate especially in fish production. In addition, the better antimicrobial activity under anaerobic conditions provides a new aspect of investigation for further clinical studies.
In conclusion, this study has determined MICs of cyadox against pathogens from swine, chicken, and fish and established the antibacterial spectrum of activity of cyadox. It is shown that cyadox has a good antibacterial activity which is better than other quinoxaline derivatives. Under in vitro anaerobic conditions, the antibacterial activity of cyadox against most facultative anaerobes is considerably better which demonstrated that cyadox is an active compound in anaerobic conditions, which provides a reasonable theoretical foundation for the clinical application of cyadox. The overall in vitro results provide predictive evidence that cyadox has high antibacterial activity that can be used alone even though we are hunting appropriate medications for drug combinations.