Effect Found in the Combination of Kitasamycin with Other Antibiotics Used against Actinobacillus pleuropneumoniae

Background: Combinations of antibiotics are used to increase the therapeutic options via the simultaneous activities of two compounds. The goals of combination therapy are to resolve the bacterial infection while reducing the treatment cost. The use of kitasamycin is used frequently and in addition to other antibiotics, the desired combinations were not found. Methods: Fields strains: we use serotypes 1, 3, 5 y 7. Microbial culturing: The serotypes 1, 3, 5 y 7 was identified. Antibiotics and Reagents: The antibiotics used were kitasamycin; enrofloxacin and oxolinic acid. The following combinations were tested: a) kitasamycin with enrofloxacin; b) kitasamycin with norfloxacin; and c) kitasamycin with oxolinic acid. Preparation of bacterial suspensions: The cultures were adjusted to a concentration of 5 colony-forming units per ml (CFU/ml) using the same sterile culture medium as the diluent. Determination of the fractional inhibitory concentrations (FICs): To test each combination and their interactions and to calculate the fractional inhibitory concentration (FIC) of each antibiotic. Calculation of the index of the fractional minimum inhibitory concentration (FMIC): The FMIC index was calculated by applying the following formula: FIC index = FIC of antibiotic A/FIC of antibiotic B. Results: What was found showed that: A combination of kitasamycin and norfloxacin has been shown to have an indifferent effect. A combination of kitasamycin and enrofloxacin had antagonistic effects against all of the Actinobacillus pleuropneumoniae serotypes studied. Finally, a combination of kitasamycin and oxolinic acid had antagonist effects against serotypes 1, 3 and 5 and a synergistic effect against serotype 7. Conclusion: The “in vitro” combination Kitasamycin-Norfloxacin showed an indifferent effect, and combination Kitasamycin and Enrofloxacin demonstrated an antagonistic interaction between these two antibiotics. A combination of Kitasamycin and Oxolinic Acid had synergistic effect against serotype 7.


Introduction
Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia (PP), a severe and frequently fatal disease that affects pigs of any age [1]. A variety of antibiotics have been used to control PP, including penicillins, tetracyclines, sulfonamides, erythromycin and new antibiotics such as tiamulin, spectomycin [2] [3] [4]. In recent years, kitasamycin has produced very good results (Asahi Vet. S.A. & Asahi Chemical Industry Co. LTD., 1995), as have first-generation quinolones, such as nalidixic and oxolinic acid, and fluoroquinolones, such as ciprofloxacin and enrofloxacin [5]. In the antimicrobial therapy of clinical cases of PP on farms, the use of a series of antibiotics and their possible combinations have been reported to find the desired effect. Three types of interactions can result from the combination of two antibiotics: synergism, antagonism and indifference. Parenteral antibiotic treatment has proven to be the most effective strategy to prevent outbreaks of the disease; however, the cost of these treatments is high. The addition of antibiotics to food is of little value during a pleuropneumonia outbreak, as the animals become anorexic, making it difficult for them to consume a therapeutic dose that delivers the minimum inhibitory concentration (MIC) required to eliminate the microorganism; therefore, treatment by this route does not prevent infection, and it can only reduce the severity of the clinical disease [6]. Antibiotic resistant A. pleuropneumoniae strains have been isolated, especially in serotypes 1, 3, 5 and 7 [7] [8]. Combinations of antibiotics can be used to increase therapeutic options, and a common goal is to identify formulas that can resolve bacterial infections while reducing treatment costs [1] [9]. When two antimicrobial agents are combined, three types of interactions can be observed: synergistic, indifferent and antagonistic. Synergism and antagonism are difficult to predict because the effects vary depending on the microorganism and can occur only within a narrow range of drug concentrations. However, knowledge of the different modes of action of antimicrobial agents allows for the prediction of the effects of various treatments [10] [11].
The results of antibiotic tests can be plotted in graphs known as isobolograms to facilitate the data interpretation [12]. In this study, the in vitro interactions between kitasamycin and three different quinolones-enrofloxacin, floxacin, and oxolinic acid were evaluated against A. pleuropneumoniae strains belonging to serotypes 1, 3, 5 and 7.

Determination of the Fractional Inhibitory Concentrations (FICs)
stant volumes of the bacterial suspension were placed in each well of the plate.
Well H1 was left as the culture control, and well H12 was used as the medium control. The plates were then incubated at 37˚C for 20 h with gentle shaking. The interpretation of the results was performed as follows: 1) Each well was examined with a light source, and their turbidities were compared with that of well A1, which had a high turbidity due to bacterial growth. All of the wells that showed

Calculation of the Index of the Fractional Minimum Inhibitory Concentration (FMIC)
The

Trial Registration
The manuscript does not contain clinical studies or patient data; the samples used in this study were voluntarily provided by veterinarians and pig farmers.

Combination of Kitasamycin with Norfloxacin
The MICs for norfloxacin were 0.64 μg/ml for serotypes 1 and 3, 1.28 μg/ml for

Combination of Kitasamycin with Oxolinic Acid
The MIC for kitasamycin was similar to the previous value for serotypes 1 Trial registration The manuscript does not contain clinical studies or patient data; the samples used in this study were voluntarily provided by veterinarians and pig farmers.

Discussion
Porcine pleuropneumonia (PP) was first isolated by Pattison in 1957, and it has  and fatal if antibiotic treatment is not initiated after the first case in the herd has occurred; therefore, its control requires immediate parenteral antibiotic therapy  [12]. In this study, pairwise combinations of kitasamycin and three different members of the quinolone family (oxolinic acid, floxacin and enrofloxacin), all of which are commonly used for the treatment of PP produced by A. pleuropneumoniae serotypes 1, 3, 5 and 7, were tested using the "checkerboard method", which relies on microdilution to establish possible synergy.
With oxolinic acid, an effect was observed that depended on the A. pleuropneumoniae serogroup: for serotype 1, the interaction was indifferent; for serotypes 3 and 5 it was antagonistic, and for serotype 7 it was synergistic-indifferent. These differences are due to the different resistance patterns mentioned above [2] [3] [7]. In the case of the combination of kitasamycin with floxacin, the effect for all serotypes was indifference, with the exception of serotype 7, which presented a clear synergistic effect. This finding is in opposition to the antagonistic effect observed for the combination of kitasamycin with enrofloxacin against this serotype, as previously reported by Lewin and Smith [18]. It is not surprising that inhibitors of protein synthesis, such as kitasamycin, and inhibitors of RNA synthesis, such as rifamycin, have antagonistic effects on the bactericidal activity of quinolones, as the target of their action is DNA gyrase. The company Asahi (Asahi Vet. S.A. and Asahi Chemical Industry Co. LTD., 1995) performed studies on pneumonia in pigs and reported MICs for kitasamycin ranging from 1 μg/ml to 6.25 μg/ml against pathogenic organisms such as A. pleuropneumoniae.
These data agree with the results obtained in this study.
The data obtained in this study are comparable to a previously reported MIC value [4] for enrofloxacin of 0.06 μg/ml against strains of A. pleuropneumoniae from the United States. This value is 2.6 times higher than the value obtained with serotypes 1 and 7 (0.15 μg/ml), 10.4-fold higher than that for serotype 3 (0.64 μg/ml), and 5.2-fold higher than that for serotype 5 (0.31 μg/ml). These values, despite being comparatively high, represent relatively low antibiotic concentrations and thus are consistent with what has been reported for the widely used broad spectrum fluoroquinolones in clinical contexts [18].
The variations in the effects of the same antibiotic against the different A. pleuropneumoniae serotypes can be explained based on the acquisition of antibiotic resistance factors; however, it has not been fully proven whether the presence of R plasmids causes these effects in A. pleuropneumoniae serotypes, since multiresistant A. pleuropneumoniae strains have been found, suggesting that resistance is indeed mediated by plasmids [20] (although the authors of this study  [22] (that this microorganism obtains its antibiotic resistance genes via external sources. One possibility is via plasmid transfer from the Enterobacteriaceae family to susceptible A. pleuropneumoniae strains, which could be associated with the presence of a small cryptic plasmid in the recipient strain. Several plasmids have been identified in A. pleuropneumoniae strains: pVM104, pVM105 and pVM106. The pVM104 and pVM106 plasmids are identical with a molecular weight of 2.3 megadaltons, and these plasmids encode sulfonamide and streptomycin resistance; the plasmid pVM105 has a PM of 3.5 megadaltons and encodes sulfonamide and ampicillin resistance. However, none of these plasmids can promote their own transformation [21]. The combinations of antimicrobials and their probable effects on pathogenic microorganisms of veterinary interest presented here indicate that the routine use of antibiotics in veterinary medicine has led to the development of multiresistance that limits their use in the treatment of porcine respiratory syndrome involving porcine pleuropneumonia.

Conclusion
The combination of kitasamycin with enrofloxacin demonstrated an antagonistic interaction between these two antibiotics when in combination. The combination of kitasamycin with norfloxacin revealed an indifferent interaction between these antibiotics when applied in combination against all four serotypes.
Combination of kitasamycin and oxolinic acid had synergistic effect against serotype 7. The results showed an indifference effect for this combination against serotype 1, an antagonistic effect was found for serotypes 3 and 5, for serotype 7, the effect was synergistic-indifferent, with one synergistic point.