Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of
Avibacterium paragallinarum
and resistance monitoring
Authors: Gütgemann Franziska , Heuvelink Annet , Müller Anja , Churin Yury , Buter Rianne , Jung Arne , Feberwee Anneke , Wiegel Jeanine , Kumm Franziska , Braun Ann Sophie , Yue Min , Soriano-Vargas Edgardo , Swanepoel Stefan , Botteldoorn Nadine , Kirchner Miranda , Kehrenberg Corinna 
Published: February 2024

This study aimed to develop a method for standardized broth microdilution antimicrobial susceptibility testing (AST) of
Avibacterium
(
Av
.)
paragallinarum
, the causative agent of infectious coryza in chickens. For this, a total of 83
Av. paragallinarum
isolates and strains were collected from 15 countries. To select unrelated isolates for method validation steps, macrorestriction analyses were performed with 15
Av. paragallinarum
. The visible growth of
Av. paragallinarum
was examined in six broth media and growth curves were compiled. In Veterinary Fastidious Medium and cation-adjusted Mueller-Hinton broth (CAMHB) + 1% chicken serum + 0.0025% NADH (CAMHB + CS + NADH), visible growth of all isolates was detected and both media allowed adequate bacterial growth. Due to the better readability of
Av. paragallinarum
growth in microtiter plates, CAMHB + CS + NADH was chosen for AST. Repetitions of MIC testing with five epidemiologically unrelated isolates using a panel of 24 antimicrobial agents resulted in high essential MIC agreements of 96%–100% after 48-h incubation at 35 ± 2°C. Hence, the remaining 78
Av. paragallinarum
were tested and demonstrated easily readable MICs with the proposed method. Differences in MICs were detected between isolates from different continents, with isolates from Africa showing lower MICs compared to isolates from America and Europe, which more often showed elevated MICs of aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole. PCR analyses of isolates used for method development revealed that isolates with elevated MICs of tetracyclines harbored the tetracycline resistance gene
tet
(B) but none of the other tested resistance genes were detected. Therefore, whole-genome sequencing data from 62
Av. paragallinarum
were analyzed and revealed the presence of sequences showing nucleotide sequence identity to the genes
aph(6)-Id
,
aph(3″)-Ib
,
bla
TEM-1B
,
catA2, sul2
,
tet
(B),
tet
(H), and
mcr-
like. Overall, the proposed method using CAMHB + CS + NADH for susceptibility testing with 48-h incubation time at 35 ± 2°C in ambient air was shown to be suitable for
Av. paragallinarum
. Due to a variety of resistance genes detected, the development of clinical breakpoints is highly recommended.

IMPORTANCE

Avibacterium paragallinarum
is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.