Antimicrobial resistance of Gallibacterium anatis isolated from layer poultry farms in Croatia

Gallibacterium anatis is part of the normal microflora of the lower genital tract and the upper respiratory tract in chickens. In recent years it has been considered as an important causative agent of reproductive disorders and respiratory manifestations in layers and commercial broilers, which poses a serious problem in poultry production. Simultaneous infection with other microorganisms and compromised immunological status are predisposing factors nurturing the infection of G. anatis. Although avian pathogenic Escherichia coli (APEC) isolates have been incriminated as the causative agent of laying hen peritonitis, G. anatis are frequently isolated from similar lesions. The purpose of this study was to determine the level of antimicrobial resistance in G. anatis strains isolated from poultry farms in Croatia. During this epizootiological survey, poultry carcasses were brought from farms to the Department of Poultry Diseases with Clinic where pathomorphological examination was done. During examination, swab samples were taken from trachea, liver, caeca, cloaca and oviduct. Samples were plated using a sterile microbiological loop on 5% sheep blood agar and incubated aerobically at 37oC for 24 hours. Bacterial ß-hemolytic colonies were verified as Gram- negative via Gram staining and biochemical testing was done to affirm the identification. Isolates were further confirmed as G. anatis using MALDI-TOF spectrometry method. Afterwards, they were subcultured on brain-heart infusion (BHI) broth and stored at -80oC until further testing. Antimicrobial susceptibility test was performed using disk diffusion method on 5% sheep blood agar. The antimicrobials used were cefotaxime, marbofloxacin, erythromycine, enrofloxacin, ciprofloxacin, doxycycline, sulfamethoxazole/trimethoprim, ampicillin, tetracycline, penicillin, streptomycin, amoxicillin/clavulanic acid, chloramphenicol, gentamycin, norfloxacin and sulphonamides. The subcultures were suspended in 0, 9% NaCl to an optical density equivalent to that of McFarland 0, 5 standards. Plates were incubated aerobically at 37oC for 24 hours and afterwards the interpretation was performed according to the manufacturer. The results showed high level of antimicrobial resistance of G. anatis as suspected. All isolates were multidrug resistant to at least 9 out of 16 antimicrobials used in disk diffusion test, and showed susceptibility in all isolates only to gentamycin. Due to high variety of pathological lesions in carcasses and simultaneous infection with other microorganisms as E. coli and Hafnia alvei, it is most likely an opportunistic pathogen. Such resistant strains could significantly compromise poultry production, so in that case use of autogenous vaccine should be considered.