Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring

Author:

Gütgemann Franziska1,Heuvelink Annet2ORCID,Müller Anja1,Churin Yury1,Buter Rianne2ORCID,Jung Arne3ORCID,Feberwee Anneke2ORCID,Wiegel Jeanine2,Kumm Franziska1,Braun Ann Sophie1,Yue Min45ORCID,Soriano-Vargas Edgardo6,Swanepoel Stefan7,Botteldoorn Nadine8,Kirchner Miranda9ORCID,Kehrenberg Corinna1ORCID

Affiliation:

1. Institute for Veterinary Food Science, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany

2. Royal GD, Deventer, the Netherlands

3. Clinic for Poultry, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany

4. Hainan Institute of Zhejiang University, Sanya, China

5. Department of Veterinary Medicine, Institute of Preventive Veterinary Science, Zhejiang University College of Animal Sciences, Hangzhou, China

6. Centro de Investigación y Estudios Avanzados en Salud Animal, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma del Estado de México, Toluca, Mexico

7. Deltamune, Centurion, South Africa

8. DGZ Vlaanderen, Torhout, Belgium

9. Animal and Plant Health Agency, Addlestone, Surrey, United Kingdom

Abstract

ABSTRACT 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.

Funder

German Federal Ministry of Food and Agriculture

Publisher

American Society for Microbiology

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