ANTIMICROBIAL SUSCEPTIBILITY OF PASTEURELLA MULTOCIDA ISOLATED FROM SHEEP WITH FIBRINOUS PNEUMONIA

Ovine respiratory complex is a signi ﬁ cant cause of death in sheep ﬂ ocks, where Pasteurella multocida is the most frequent microorganisms isolated from animals with pneumonia. There is an urgent need to re ﬁ ne the use of different antimicrobials to avoid the problem of antimicrobial resistance and optimize the control of this disease in ovine livestock. The ﬁ rst step in approaching this problem is gaining an insight into the antimicrobial susceptibility of ovine pathogens. This study evaluated the in vitro activity of tildipirosin, gamithromycin, oxytetracycline, and dano ﬂ oxacin against Pasteurella multocida strains isolated from sheep with ﬁ brinous pneumonia. The strains were incubated following Clinical and Laboratory Standards Institute (CLSI) standard conditions and also with a modi ﬁ ed method by 25% supplementation with sheep serum. Minimum inhibitory concentrations (MIC) were determined using the broth microdilution technique. The lowest MIC 90 under standard conditions and by supplementation with sheep serum was obtained with tildipirosin. Sheep serum signi ﬁ cantly reduced tildipirosin, gamithromycin, and dano ﬂ oxacin MIC values for Pasteurella multocida strains. In brief, the potency of tildipirosine, gamithromycin, and dano ﬂ oxacin against Pasteurella multocida increases when sheep serum is added to the culture media.


INTRODUCTION
The ovine respiratory complex is a common respiratory disease in sheep, with a wide prevalence worldwide [1,2].It represents a real problem in many sheep fl ocks in most countries with intensive sheep production.It is the leading cause of economic losses due to high morbidity, treatment costs, and negative impact on lamb weight gain and growth [3].The main agents of this complex are Mannheimia haemolytica and Pasteurella multocida [4].Immunosuppression of animals due to stressful handling and adverse climatic conditions predisposes to bacterial colonization of the respiratory system [5], where Pasteurella multocida is most frequently isolated from respiratory samples in sheep with pneumonia [6][7][8].This agent has become an emerging respiratory disease in sheep, mainly in tropical and temperate climate zones [9].This respiratory complex is usually treated with broad-spectrum antibiotics such as tetracyclines, macrolides, and fl uoroquinolones, often prescribed extra-label or offlabel in sheep [10].There are few studies on this subject in sheep, with some results achieved in different species, like pigs or cattle, that have been extrapolated for use in sheep [4].Antimicrobial resistance is currently a worldwide concern, therapeutic options may be challenging, and the knowledge of antimicrobial susceptibility is essential when implementing rational and effective therapy [11,12].Antimicrobial resistance is a prominent issue requiring an interdisciplinary approach to provide and assure effective treatments to the animal and human population to prevent the spreading of antimicrobial drug resistance to other animals, people, and the environment.Currently, one of the approaches to reduce inappropriate antimicrobial use for treating animals or humans is to apply pharmacokinetic-pharmacodynamic principles to calculate therapeutic regimens where the exact determination of the minimum inhibitory concentration is essential for this approach [13].Therefore, studies that describe antibacterial susceptibility in veterinary medicine are valuable because they allow early recognition of the appearance of antibiotic resistance as well as information for PK-PD integration studies.
The pharmacokinetic properties of new macrolides (tildipirosin and gamithromycin), such as rapid absorption, extensive distribution in pulmonary tissues, and long halflives after parenteral administrations can make these antimicrobials a suitable option as antibiotic therapy against Pasteurella multocida in sheep [12,14,15].Moreover, some experiments suggest that the antimicrobial activity of some macrolides may be undervalued if we only consider the obtained MIC by CLSI methods.For example, a study showed that MIC values of Actinobacillus pleuropneumoniae against tildipirosin incubated according to standard methods was higher than those obtained when adding serum in different proportions to the culture medium (5%, 10%, 25%, and 50%) [15].In another study, the potency of tildipirosin and gamithromycin against susceptible caprine mastitis pathogens (M.agalactiae, E. coli, S. aureus, Streptococcus spp., and coagulasenegative Staphylococci) increased when serum (25%) was present in culture media [16].Therefore, determining macrolide dosages for therapeutic use should be derived from pharmacodynamic data obtained from biological fl uids because in vitro measurement of MIC in broth, performed following international recommended methods, may be misleading for estimating the in vivo potency of these antibiotics.Thus, the aim of the study was to evaluate the in vitro activity of tildipirosin, gamithromycin, oxytetracycline, and danofl oxacin against Pasteurella multocida incubated on CLSI conditions and with a deviation from CLSI methods by 25% supplementation with sheep serum.

Animals and sampling procedure
The animals used for this study were sheep slaughtered at Municipal abattoirs in the Cartagena Region of Murcia in Spain.Lungs with lesions suggestive of fi brinous pneumonia, such as pulmonary congestion, fi brin deposits on the serosa, pulmonary edema, or pleural effusion, were selected to obtain swabs tissues.A total of 50 lungs with pneumonic lesions were sampled through an aseptic incision in the trachea to introduce the swab for bronchial mucosa sampling.The samples were deposited in Stuart medium and maintained at 4 °C until transferred at the Microbiology laboratory.

Isolation and identifi cation of Pasteurella multocida
Bacteria were isolated from swab lung samples on Columbia blood agar plates (bioMérieux España, Madrid, Spain) and incubated at 37 °C for 24 h.After obtaining the pure culture, Gram staining was performed.Gram-negative coccobacilli were tested for oxidase.Gram-negative and oxidase-positive isolates were biochemically identifi ed by the commercial identifi cation system API 20NE strips (bioMérieux España, Madrid, Spain) and further confi rmed by a species-specifi c PCR assay.Finally, thirty bacterial isolates were included after their characterization and maintained at the Microbiology laboratory strain collection until antimicrobial susceptibility analysis was performed.

Antimicrobial susceptibility testing
Tildipirosin, gamithromycin, oxytetracycline, and danofl oxacin (Cymit Química, Barcelona, Spain) were selected for the antimicrobial susceptibility testing.Antibiotics were dissolved in suitable solvents to make stock solutions and then diluted in sterile distilled water following the guidelines of the Clinical and Laboratory Standards Institute [17].Sheep serum was obtained from six healthy sheep.Blood samples were centrifuged at 1500 g for 10 min, and the freshly collected serum was pooled and divided into 1 ml portions, stored at -80°C, and thawed immediately before the experiment.
The minimum inhibitory concentrations of tildipirosin, gamithromicin, danofl oxacin, and oxytetracycline were determined following standard conditions of CLSI or with a modifi cation of the CLSI method by 25% supplementation of sheep serum [17].Minimum inhibitory concentration tests were performed by the microdilution broth method [17].Brief, serial two-fold dilutions of the antimicrobial agents were prepared to start from the stock solution of each antibiotic.Broth dilutions were made using cation-adjusted Mueller-Hinton broth (Merck, Madrid, Spain) with 5% of defi brinated horse blood (Thermo Fisher Scientifi c, Massachusetts, USA).Concentrations of all antibiotics ranging from 0.03 to 128 mg/L were used.Inoculums were prepared by diluting an overnight MHB culture in buffered saline solution to a 0.5 McFarland Turbidity Scale and diluting again 40-fold before testing.The U-bottomed microtiter plates were incubated at 37°C for 24 h.The MIC was defi ned as the lowest concentration of antibiotic where bacterial growth was completely inhibited.The reference strains Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were used as controls for each plate.

RESULTS
The present study provides the profi le of antibacterial susceptibility of Pasteurella multocida strains isolated from sheep with fi brinous pneumonia.Pasteurella multocida was susceptible to tildipirosin, gamithromycin, and danofl oxacin but not for oxytetracycline on standard conditions.The lowest MIC 90 values were obtained for tildipirosin on standard condition (MIC 90 = 0,5 µg/ml) and those deviated from CLSI method (MIC 90 = 0,25 µg/ml).Tildipirosin, gamithromycin and danofl oxacin showed a decrease in the MIC 90 under deviated conditions compared to standard CLSI conditions.Oxytetracycline showed no change in MIC 90 between standard condition and deviation from the CLSI method.Minimal inhibitory concentration values for the antibiotics tested against Pasteurella multocida are summarized in Table 1.
Table 1.Frequency of minimal inhibitory concentrations for tildipirosin (TIL), gamithromycin (GAM), oxytetracycline (OXY), and danofl oxacin (DAN) under standard conditions or addition of serum (+SERUM) in the culture media against Pasteurella multocida strains isolated from sheep (n=30), as well as MIC 50 and MIC 90.The addition of the serum to the culture medium was associated with an appreciable increase in the potency of tildipirosin, gamithromycin, and danofl oxacin.However, the potency of oxytetracycline was unchanged against Pasteurella multocida (Figure 1).

DISCUSSION
Extensive knowledge of antimicrobial activity for treating diseases caused by microorganisms such as Pasteurella multocida in sheep is required to achieve an effective and rational treatment that prevents the appearance of bacterial resistance.However, there are limited studies of bacterial susceptibility on isolated strains from sheep.The current work provides a brief description of the antimicrobial susceptibility of several antibiotics that could be used against Pasteurella multocida.Previous studies on sheep isolates showed lower values of MIC 90 than those obtained in the present study for danofl oxacin (MIC 90 =0,12 mg/L), oxytetracycline (MIC 90 =16 mg/L) [4] and for tetracycline and doxycycline (MIC 90 values of 32 and 16 mg/L, respectively) [18].The same occurs in the case of other fl uoroquinolones as enrofl oxacin, with MIC 90 values of 0,5 mg/L [18].Other studies in sheep also evidenced the susceptibility of Pasteurella multocida to fl uoroquinolones, mainly enrofl oxacin [2,19,20].
Previous studies showed higher MIC values for tetracyclines than fl uoroquinolones and macrolides, as was found in the present work, although the strains studied showed extensive susceptibility to these antibiotics [18,20].Concerning macrolides, there are no studies on tildipirosin and gamithromycin in sheep isolates.Previous studies of new macrolides such as tulathromycin in this species showed higher MIC 90 values (MIC 90 = 4 mg/L) than tildipirosin and gamithromicin [4].For tilmicosin the values obtained are also higher in sheep than in our work [4].Furthermore, the present study demonstrates that macrolides and danofl oxacin have markedly lower MICs against different pathogens when assayed in culture media broth supplemented with serum, compared with MHB (CLSI recommendation for in vitro susceptibility testing studies).