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

Franziska Gütgemann; Annet Heuvelink; Anja Müller; Yury Churin; Rianne Buter; Arne Jung; Anneke Feberwee; Jeanine Wiegel; Franziska Kumm; Ann Sophie Braun; Min Yue; Edgardo Soriano-Vargas; Stefan Swanepoel; Nadine Botteldoorn; Miranda Kirchner; Corinna Kehrenberg

doi:10.1128/jcm.01011-23

. 2024 Feb 16;62(3):e01011-23. doi: 10.1128/jcm.01011-23

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

Franziska Gütgemann ^1,^#, Annet Heuvelink ^2,^#, Anja Müller ¹, Yury Churin ¹, Rianne Buter ², Arne Jung ³, Anneke Feberwee ², Jeanine Wiegel ², Franziska Kumm ¹, Ann Sophie Braun ¹, Min Yue ^4,⁵, Edgardo Soriano-Vargas ⁶, Stefan Swanepoel ⁷, Nadine Botteldoorn ⁸, Miranda Kirchner ⁹, Corinna Kehrenberg ^1,^✉

Editor: Alexander J McAdam¹⁰

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

²Royal GD, Deventer, the Netherlands

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

⁴Hainan Institute of Zhejiang University, Sanya, China

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

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

⁷Deltamune, Centurion, South Africa

⁸DGZ Vlaanderen, Torhout, Belgium

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

¹⁰Boston Children's Hospital, Boston, Massachusetts, USA

^✉

Address correspondence to Corinna Kehrenberg, Corinna.kehrenberg@vetmed.uni-giessen.de

Franziska Gütgemann and Annet Heuvelink contributed equally to this article. Author order was determined in order of increasing seniority.

The authors declare no conflict of interest.

Contributed equally.

Roles

Franziska Gütgemann: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing

Annet Heuvelink: Conceptualization, Data curation, Formal analysis, Methodology, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing

Anja Müller: Formal analysis, Investigation, Methodology, Validation, Writing – review and editing

Yury Churin: Investigation, Methodology, Writing – review and editing

Rianne Buter: Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

Arne Jung: Investigation, Methodology, Resources, Writing – review and editing

Anneke Feberwee: Formal analysis, Investigation, Methodology, Writing – review and editing

Jeanine Wiegel: Investigation, Methodology, Writing – review and editing

Franziska Kumm: Investigation, Methodology, Writing – review and editing

Ann Sophie Braun: Investigation, Methodology, Writing – review and editing

Min Yue: Methodology, Writing – review and editing

Edgardo Soriano-Vargas: Investigation, Resources, Writing – review and editing

Stefan Swanepoel: Investigation, Resources, Writing – review and editing

Nadine Botteldoorn: Investigation, Resources, Writing – review and editing

Miranda Kirchner: Investigation, Methodology, Resources, Writing – review and editing

Corinna Kehrenberg: Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing

Alexander J McAdam: Editor

PMCID: PMC10935639 PMID: 38363142

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.

KEYWORDS: Avibacterium paragallinarum, broth microdilution, antimicrobial susceptibility testing, MIC values, whole-genome sequencing, CAMHB + CS + NADH, antimicrobial resistance genes, VFM

INTRODUCTION

Avibacterium (Av.) paragallinarum (formerly Haemophilus paragallinarum) is the causative agent of infectious coryza (IC) in chickens (1 –3). The fastidious pathogen causes an acute respiratory disease associated with airsacculitis, conjunctivitis, swollen head, mucopurulent or serous nasal discharge, and rhinitis (3). Disease caused by Av. paragallinarum can result in a mortality of up to 10% as well as a significant reduction in fattening performance due to lower feed conversion and a decrease in laying performance of 10%–80% (3, 4). This phenomenon is further enhanced by secondary infections with other pathogens (e.g., Mycoplasma spp.) or poor housing conditions (3, 5, 6). As a result, IC consistently leads to marked economic losses in the chicken and egg industries worldwide (3).

Antimicrobials such as macrolides, sulfonamides, and tetracyclines have been used to treat IC in poultry for years (3, 7). However, as there is no agreed method for standardized antimicrobial susceptibility testing (AST) for Av. paragallinarum, which is included in standards so far, different methods were used to determine minimal inhibitory concentration (MIC) values for Av. paragallinarum. Nevertheless, it is known that test parameters (e.g., inoculum size, medium, incubation conditions) can influence the results of MIC testing (8 –10), which may affect the evaluation of the results and thus the selection of the most appropriate antibiotic in case of treatment of disease. In addition, as Av. paragallinarum is highly fastidious and requires complex media containing sodium chloride (1.0%–1.5%) and for most isolates NAD (V-factor) (3, 11), common methods for AST of fast-growing bacteria of animal origin (such as Escherichia coli) according to CLSI supplement VET01S are not suitable for Av. paragallinarum.

In two recent studies, a broth medium, cation-adjusted Mueller-Hinton broth (CAMHB) plus 0.0025% NADH (β-NAD, reduced form, disodium salt) plus 1% heat-inactivated chicken serum (CAMHB + CS + NADH), was used to determine the susceptibility status of Av. paragallinarum (12, 13). This medium was accepted by the CLSI for AST of Glaesserella parasuis and inclusion in the next CLSI guidance document VET06 (personal communication). Furthermore, this medium has recently been recommended for harmonized AST of the closely related pathogen Av. gallinarum (14). However, since the suitability of this and other broth media for standardized MIC testing of Av. paragallinarum has not been previously demonstrated, this study aimed to investigate the suitability of this and other broth media and test parameters for standardized broth microdilution susceptibility testing of Av. paragallinarum. The method was subsequently used to examine its suitability for field isolates from different geographic areas (America, Europe, and southern Africa) and to determine the MIC values of the isolates. In addition, whole-genome sequencing (WGS) analysis was performed to investigate the presence of antimicrobial resistance genes (ARGs) in Av. paragallinarum.

MATERIALS AND METHODS

Collection of Av. paragallinarum field isolates and reference strains, culturing, and species confirmation

A total of 83 Av. paragallinarum were collected for this study, including 76 field isolates isolated between 1994 and 2022 (Table 1). The collection included 15 Av. paragallinarum, which were used for method development as their epidemiological relatedness was investigated by macrorestriction analysis as described below (Fig. 1). Of these, 14 field isolates (no. 1–14) originated from chickens and were isolated in 2009 (n = 6), 2016 (n = 2), 2018 (n = 1), 2019 (n = 2), 2020 (n = 2), and 2022 (n = 1) from poultry flocks in Germany (n = 8) and the Netherlands (n = 6). In addition, the Av. paragallinarum type strain CCUG 12835^T (synonym IPDH 2403), which was isolated in Germany in 1973 (15) and obtained from the Culture Collection University of Gothenburg (CCUG, Gothenburg, Sweden), was used for comparative purposes. The additional 68 Av. paragallinarum from Royal GD (Deventer, the Netherlands) were used to test the suitability of the developed method. These included 62 field isolates collected between 1994 and 2021 from poultry flocks in various countries in Europe (Belgium, England, Germany, the Netherlands, Spain; n = 31), southern Africa (South Africa, Zimbabwe; n = 16), and North and South America (Guadeloupe, Ecuador, Mexico, Peru; n = 15). The collection also included six Av. paragallinarum reference strains (HP14, E-3C, 2671, 221, SA-3, 0222) originating from five different continents.

TABLE 1.

Origin and number of Avibacterium paragallinarum field isolates (n = 76) and reference or type strains (n = 7) used in this study

Origin			Quantity of Av. paragallinarum
Continent	Country	Year of isolation	Field isolates	Strains^h (strain designation)
Africa	South Africa	1994	1
		1998	2
		2002	1
		2008	1
		2011	1
		2012	4
		2014	2
		2015	3
		Unknown		1 (SA-3^*,^a)
	Zimbabwe	2006	1
America	Brazil	Unknown		1 (E-3C^*,b)
	Ecuador	2002	1
		2005	1
	Guadeloupe	Unknown	1
	Mexico	2007	3
		2008	2
		2011	1
		2015	2
		2017	1
		2019	1
		Unknown	1
	Peru	2012	1
	USA	Unknown		1 (0222^{*, c})
Asia	Japan	Unknown		1 (221^{*, d})
Europe	Belgium	2020	1
	England	2008	1
		2010	4
		2011	1
		2012	6
	Germany	1973		1 (CCUG 12835^T, ^g)
		2015	1
		2016	6
		2018	5
		2019	3
		2020	2
		2022	1
		Unknown		1 (2671^{*, e})
	Netherlands	2009	6
		2010	1
		2017	1
		2018	2
		2020	2
		2021	1
	Spain	2017	1
Oceania	Australia	Unknown		1 (HP14^{*, f})

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^{^a}

Strain SA-3, South Africa, 1982 provided by Rick Rimler (58).

^{^b}

Strain E-3C, Sao Paulo, Brazil, 1979 (59).

^{^c}

Strain 0222, California, USA, 1960 (60).

^{^d}

Strain 221, Japan, 1961 (61).

^{^e}

Strain 2671, Germany, 1979 (62).

^{^f}

Strain HP14, Australia, 1988 (63).

^{^g}

Type strain.

^{^h}

Strain is defined here as a very well-characterized and studied organism that is publicly available (typically from a strain collection).

Fig 1 — Genetic similarity and origin of 15 *Avibacterium paragallinarum* used for method development.^aThis isolate was used to compile growth curves; ^bThis isolate was selected for method validation steps.

For culturing the Av. paragallinarum isolates, tryptic soy agar plates (Merck KGaA, Darmstadt, Germany) supplemented with 10% (vol/vol) chicken serum (heat-inactivated at 56°C for 30 min) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 0.025% (vol/vol) NADH (β-NAD, reduced form, disodium salt) (Roche Diagnostic, Mannheim, Germany) (TSA+) was used. However, chocolate agar plates containing 10% (vol/vol) defibrinated horse blood (Oxoid Limited, Basingstoke, UK) were used for colony count determinations in the growth experiments, as most Av. paragallinarum showed more easily countable colonies on this agar. After 24–48 h at 37°C in an atmosphere containing 5% CO₂, transparent colonies were visible on the agar plates. In addition, Haemophilus medium (HM), according to the specifications of the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), was used for culturing isolates in broth for macrorestriction analyses. Species confirmation of Av. paragallinarum for method development was performed by a previously described species-specific PCR assay (16), modified by Muhammad and Sreedevi (17). For this, DNA was isolated from colonies selected from a 24- to 48-h-old culture on TSA+ agar plates by boiling (99°C; 15 min). In addition, the species assignment of all isolates from Royal GD Av. paragallinarum collection was done by using a real-time PCR assay as described previously by Feberwee et al. (18).

Macrorestriction and pulsed-field gel electrophoresis analyses

For selecting unrelated isolates for method validation steps, macrorestriction digests followed by pulsed-field gel electrophoresis (PFGE) were used to determine the clonal relationship of the 15 Av. paragallinarum isolates collected first (although more modern methods e.g., whole-genome sequencing with SNP analysis or cgMLST are available today). The method was based on a previously described PulseNet protocol for E. coli O157:H7, Salmonella, and Shigella with minor modifications regarding the run time and restriction enzyme used (19). In short, DNA was digested using the restriction enzyme KpnI (Thermo Fisher Scientific Inc, Vilnius, Lithuania). For Salmonella Typhimurium LT2, which served as a size marker in the analyses, the restriction enzyme XbaI (Thermo Fisher Scientific Inc, Vilnius, Lithuania) was used. DNA fragments were separated in gels at 6 V using the CHEF DR II system (BioRad, Munich, Germany), with a pulse time increasing over 20 h from an initial time of 2 s to a final extension time of 18 s. For fragment pattern analysis (UPGMA), the BioNumerics software (version 7.6; Applied Maths, Sint-Martens-Latem, Belgium) was used. The Dice coefficient was set with a position tolerance of 1% and an optimization of 0.5%.

Visible growth of Av. paragallinarum in six broth media

To select a suitable medium for susceptibility testing, preliminary growth experiments were performed with 15 Av. paragallinarum in different media to investigate whether the isolates show visible growth in microtiter plates. For this, the following six broth media were included: CAMHB (Becton Dickinson and Company, Sparks, USA), CAMHB plus 2.5% lysed horse blood (vol/vol) (Oxoid Limited) (CAMHB + LHB), CAMHB plus 0.0025% NADH (vol/vol) (Roche Diagnostics GmbH) plus 1% heat-inactivated chicken serum (vol/vol) (Sigma-Aldrich Chemie GmbH) (CAMHB + CS + NADH), as well as the Haemophilus Test Medium (HTM), Mueller-Hinton fastidious broth medium with yeast extract (MHF-Y), and Veterinary Fastidious Medium (VFM) produced according to CLSI guidelines (20 –22). In case of the VFM medium, however, the availability of one of the ingredients is discontinuous, so it is not always possible to produce it. While CAMHB without supplements is the medium included in the CLSI standard for testing rapidly growing bacteria like Enterobacterales (22), CAMHB + LHB, HTM, MHF-Y, and VFM are approved media for testing fastidious organisms (e.g., Actinobacillus pleuropneumoniae, Histophilus somni, or Haemophilus influenzae) (20 –22). The CAMHB + CS + NADH was previously used for MIC testing of Av. paragallinarum (12, 13) and was recently recommended for standardized testing of the fastidious pathogen Glaesserella parasuis (23). This medium, soon included in CLSI guidance document VET06 for broth microdilution testing of G. parasuis, was also recently recommended for standardized AST of the closely related pathogen Av. gallinarum (14).

For the tests, Av. paragallinarum colonies from a 24-h-old culture on TSA+ plates were suspended into a sterile 0.9% saline solution (Merck KGaA) to achieve a suspension with a density corresponding to that of a McFarland standard of 0.5 (approximately 10⁸ cfu/mL) using the McFarland densitometer DEN-1B (Biosan SIA, Riga, Latvia). The dilution steps to obtain an initial inoculum of approximately 5 × 10⁵ cfu/mL, the microtiter plates and their inoculation as well as the controls were performed as described in a previous publication (14). After incubation in ambient air at 35 ± 2°C, the wells were checked for visible growth at five time points (18, 20, 22, 24, and 48 h, respectively).

Compiling multi-day growth curves

Based on the data obtained from the preliminary growth test, two media (CAMHB + CS + NADH and VFM) were selected for compiling growth curves. For this purpose, two independent repetitions of growth experiments were performed on different days, determining the number of cfu/mL of four epidemiologically unrelated Av. paragallinarum (Av. paragallinarum CCUG 12835^T and field isolates nos. 3, 7, and 13) in the media. Preparation of the inoculum for the growth curves was performed as in a study on Av. gallinarum (14). During a 48-h period, a volume of 100 µL was taken from the broth medium at each of the time points mentioned (0, 4, 8, 12, 16, 20, 24, 32, and 48 h) and 10-fold serial dilutions were prepared with sterile 0.9% saline. Subsequently, the dilutions (50 µL from each dilution step) were spread in duplicate on pre-dried chocolate agar plates. After incubation of the agar plates at 37°C for 24–48 h in a microaerobic atmosphere (5% CO₂), colonies were counted, and statistical average values and standard deviations were calculated as previously described (14).

Antimicrobial susceptibility testing of Av. paragallinarum

For broth microdilution susceptibility testing, three different customized microtiter plate layouts (Sensititre, Trek Diagnostic Systems, East Grinstead, UK; virgin polystyrene plates) were used per isolate. This resulted in the testing of 24 antimicrobial agents or antimicrobial combinations, which are listed in detail in the Results section. Microtiter plates coated with vacuum-dried antibiotics were inoculated with 50 µL of bacteria-supplemented CAMHB + CS + NADH, the broth medium selected based on the previous experiments. To assess the suitability of the broth medium and incubation conditions for susceptibility testing, five epidemiologically unrelated isolates (Av. paragallinarum CCUG 12835^T, field isolates nos. 3, 4, 7, and 11) were tested in five independent experiments. The selection of isolates was based on differences in their growth behavior, with isolates 4 and 11 being characterized by difficult culturing and particularly small colonies. The performance of broth microdilution (inoculum preparation, incubation temperature, and conditions) was done according to CLSI guidelines for testing bacteria isolated from animals (22), and the procedure was also briefly described for Av. gallinarum in a previous study by Gütgemann et al. (14). The inoculum density of approximately 5 × 10⁵ cfu/mL was verified by performing two inoculum controls as described in CLSI standard M07 and supplement VET01S (21, 24). The quality control (QC) strain E. coli ATCC 25922 was used for quality control purposes. Reading of MICs was done visually after 18, 20, 22, 24, and 48 h (±10 min) of incubation, respectively. Subsequently, the exact MIC agreements (percentage of MICs with a precondition of five identical MICs) and the essential MIC agreements (percentage of MICs that allow a deviation of the MIC mode of ±1 log₂ dilution steps) were calculated to determine the homogeneity of MICs.

After evaluating the results, the remaining Av. paragallinarum field isolates and reference strains were tested on site at Royal GD and MIC₅₀ and MIC₉₀ values (indicating the MICs required to inhibit 50% and 90% of the isolates, respectively) were calculated.

Control of medium and supplements from different producers

To investigate whether susceptibility testing with medium and supplements from different producers resulted in comparable MICs of Av. paragallinarum isolates, additional testing of the five isolates used for method validation was performed using CAMHB (Sigma-Aldrich Chemie GmbH) and the supplements chicken serum (BioWest SAS, Nuaillé, France) and NADH (Sigma-Aldrich Chemie GmbH) instead of CAMHB (Becton Dickinson and Company), chicken serum (Sigma-Aldrich Chemie GmbH), and NADH (Roche Diagnostics GmbH). The resulting MICs were then compared with the previously determined MICs collected in the fivefold tests and the exact and essential MIC agreements were calculated.

Analyses of antimicrobial resistance genes

Isolates with elevated MIC values compared to the other isolates of the Av. paragallinarum collection were tested for the presence of ARGs to assess the concordance of resistance phenotypes and genotypes. For this purpose, PCR assays were performed on 14 of the 15 Av. paragallinarum collected first, which showed higher MICs for at least one antimicrobial agent compared to the other isolates tested. The PCR assays were carried out as described previously (14, 24). Details on the tested resistance genes and references for the PCR assays are given in Table S1 in the Supplementary data.

For 62 out of 68 Av. paragallinarum from the Royal GD collection, WGS data were available from another ongoing study focussing on the typing of a large collection of Av. paragallinarum isolates. The available WGS data were used in the current study to support the phenotypical AST findings. DNA library preparation and de novo assembly were performed as previously described by Buter et al. (25). De novo contigs were used for screening for the presence of ARGs using the Find Resistance with Nucleotide DB script version 1.2 and the ResFinder Database (version 2021–02-28; minimum identity % = 60, minimum length % =60, filter overlaps = Yes) within the CLC Genomic Workbench v. 22.0.2 (Qiagen Aarhus A/S, Aarhus, Denmark). Then, consensus sequences having a ResFinder database hit were extracted from the contigs and a BLAST search in the NCBI non-redundant database was used for confirmation of the presence of sequences with a high sequence identity with AMR-associated genes. For WGS analysis, all isolates were included that showed elevated MIC values compared to other isolates against at least one antimicrobial agent, as well as 23 isolates with low MIC values for comparison purposes.

RESULTS

Clonality of 15 Av. paragallinarum used for method validation

Macrorestriction analysis and subsequent cluster analysis revealed that some of the 15 Av. paragallinarum isolates used for method validation were very diverse but some had identical band patterns (Fig. 1). Two major clusters were detected (A and B), with nine isolates belonging to group A and six isolates belonging to group B. However, within clusters A and B, the banding patterns of some isolates showed >80% similarity, allowing the isolates to be classified as epidemiologically related according to the interpretative criteria of Tenover et al. (26). The fragment patterns of the Av. paragallinarum type strain CCUG 12835^T and four field isolates (no. 3, 4, 7, and 11) differed by at least seven bands. Since these isolates are classified as epidemiologically unrelated, they were selected for method validation. These five unrelated test isolates were isolated in Germany (n = 4) or the Netherlands (n = 1) in 2020 (n = 2), 2018 (n = 1), 2009 (n = 1), or 1982 (n = 1).

Visible growth of Av. paragallinarum in different broths

Results of the preliminary growth experiments in which the visible growth of 14 Av. paragallinarum field isolates and the Av. paragallinarum type strain CCUG 12835^T was determined in six different broth media are summarized in Table 2. In general, the number of Av. paragallinarum showing visible growth increased with longer incubation times. After 48 h of incubation, all tested Av. paragallinarum showed button formations in CAMHB + CS + NADH and VFM medium. At this time point, fewer isolates showed visually readable growth in MHF-Y (n = 11), HTM (n = 8), and CAMHB + LHB (n = 6) broth. None of the isolates grew in un-supplemented CAMHB up to hour 48. The data also demonstrated significantly better readability of button formations in clear media than in the brownish-colored media (CAMHB + LHB, MHF-Y, and VFM; see Fig. S1 in the Supplemental material). Except isolate no. 3 (which showed a diffuse rather turbid growth), isolates grown in CAMHB + CS + NADH and HTM formed distinct buttons in the wells. By contrast, the buttons formed in CAMHB + LHB, MHF-Y, and VFM were less prominent and more difficult to read.

TABLE 2.

Visible growth of 15 Avibacterium paragallinarum used for method development in six broth media

Reading of plates after an incubation time of (h)	Number of isolates showing visible growth in the broth medium
Reading of plates after an incubation time of (h)	CAMHB^a	CAMHB + LHB^b	CAMHB + CS + NADH^c	HTM^d	MHF-Y^e	VFM^f
18	0	0	11	6	6	8
20	0	1	11	6	7	10
22	0	1	11	7	7	10
24	0	1	11	7	7	11
48	0	6	15	8	11	15

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^{^a}

Cation-adjusted Mueller-Hinton broth.

^{^b}

CAMHB + 2.5% lysed horse blood.

^{^c}

CAMHB + 1% chicken serum + 0.0025% NADH.

^{^d}

Haemophilus Test Medium.

^{^e}

Mueller-Hinton fastidious broth medium with yeast extract.

^{^f}

VFM, Veterinary Fastidious Medium.

Growth curves of Av. paragallinarum with two broth media

Based on the preliminary growth tests, CAMHB + CS + NADH and VFM were used to compile growth curves with the Av. paragallinarum type strain CCUG 12835^T and three epidemiologically unrelated Av. paragallinarum field isolates (no. 3, 7, and 13). The field isolates no. 3 and 13 were selected because they were extremely difficult to culture and formed tiny dewdrop-shaped colonies (approximately 0.3 mm in size).

The growth curves of the three field isolates and the type strain, including the calculated standard deviations, are visualized in Fig S2 through S5 in the Supplemental material. Both media allowed sufficient growth of the isolates (about 5 × 10⁶–10⁹ cfu/mL) after 24- to 48-h incubation at 35 ± 2°C. Surprisingly, a short-term decrease in number of cfu/mL was observed for three isolates at the measurement time points after 24 or 32 h. However, number of cfu/mL then increased again, and, except for isolate no. 3, the other three Av. paragallinarum reached their highest colony counts (cfu/mL) after 40 h.

Homogeneity of Av. paragallinarum MIC values

Based on the growth data and the lack of availability of VFM (21), CAMHB + CS + NADH was selected as the test medium for AST of Av. paragallinarum. The homogeneity of MIC values obtained with CAMHB + CS + NADH was assessed by performing five independent experiments of MIC determinations with the five unrelated test isolates against a panel of 24 antimicrobials or antimicrobial combinations.

Since no Av. paragallinarum showed visible growth in either growth control after 18, 20, and 22 h, the MIC endpoints of the isolates were determined after 24 and 48 h of incubation. Although readable MICs were obtained for all test isolates at these two time points, the readability (button size) of visible growth in the wells improved after 48 h. However, to evaluate the most appropriate time point for reading MICs of Av. paragallinarum, the homogeneity of MICs obtained after 24 and 48 h was compared (Tables 3 and 4). In summary, the MICs obtained after 24 h were less homogeneous than those after 48 h. After 24 h, the MIC values were within the essential MIC agreement range for 12 antimicrobials, whereas for the other antimicrobials, deviations in MICs of up to ≥7 dilution levels from the MIC modes were observed (Table 3). Considerably better homogeneity of MICs was observed after 48 h of incubation, with deviations of ±1 or ±2 dilution steps from the MIC mode for 17 and 6 antimicrobials, respectively (Table 4). Only a single MIC of streptomycin showed a deviation of +3 dilution steps from the MIC mode, while the MICs of ceftiofur, cefotaxime, and cefquinome were 100% homogeneous. As a result, clearly better essential MIC agreements (accepting ±1 dilution step deviation) were seen after an incubation time of 48 h (96%–100%, mean value 98.83%) than after 24 h (84%–100%, mean value 96.33%). In addition, exact MIC agreements ranging between 64% and 100% were observed for both time points, with a slightly higher mean value after 48 h than after 24 h (86% vs. 84%, respectively).

TABLE 3.

Homogeneity of Avibacterium paragallinarum MIC values obtained from broth microdilution susceptibility testing in CAMHB + CS + NADH after 24 h of incubation at 35 ± 2°C

Antimicrobial agent	Deviation from MIC mode^a^,b,f													Exact MIC agreement (%)^c	Essential MIC agreement (%)^d
Antimicrobial agent	>−7	−7	−6	−5	−4	−3	−2	−1	0	1	2	3	4	Exact MIC agreement (%)^c	Essential MIC agreement (%)^d
Amoxicillin/clavulanic acid 2:1								4^e	21^e					84	100
Ampicillin								1	22^e	2				88	100
Cefoperazone									25^e					100	100
Cefotaxime									25^e					100	100
Ceftiofur									25^e					100	100
Cefquinome									25^e					100	100
Cephalothin								1^e	22^e	2				88	100
Ciprofloxacin									24^e	1				96	100
Colistin				2^e				2	16	5				64	92
Doxycycline						1^e		1	20	3				80	96
Enrofloxacin									21^e	4				84	100
Florfenicol								3^e	22					88	100
Gentamicin								3^e	19	1	2			76	92
Imipenem								3	22^e					88	100
Marbofloxacin									22^e	3				88	100
Nalidixic acid			1^e	1^e				3	19	1				76	92
Neomycin						1^e		5	17	2				68	96
Penicillin								2	22^e			1		88	96
Streptomycin	2^e							1	21^e		1			84	88
Tetracycline								3	21^e		1			84	96
Tiamulin				2^e		1		3	17	1	1			68	84
Tilmicosin		1^e	1^e					4	18	1				72	92
Trimethoprim/sulfamethoxazole 1:19								2	20^e	3				80	100
Tulathromycin				1^e	1^e			2	18	2	1			72	88

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^{^a}

The MIC mode indicates the most frequently measured MIC related to an isolate and agent.

^{^b}

Data are from testing five isolates in five independent replications.

^{^c}

Percentage of MIC values corresponding to the MIC mode of the isolates.

^{^d}

The essential MIC agreement allows a deviation of ±1 dilution step from the MIC mode.

^{^e}

The MIC value of at least one isolate was equal to, lower, or higher than the concentrations of the test range.

^{^f}

Data that fulfill the criteria of the essential MIC agreement are highlighted in gray.

TABLE 4.

Homogeneity of Avibacterium paragallinarum MIC values obtained from broth microdilution susceptibility testing in CAMHB + CS + NADH after 48 h incubation at 35 ± 2°C

Antimicrobial agent	Deviations from MIC mode^a^,b,f							Exact MIC agreement (%)^c	Essential MIC agreement (%)^d
	-3	-2	-1	0	1	2	3
	Amoxicillin/clavulanic acid 2:1			3^e	21^e	1				84	100
Ampicillin			1^e	22^e	2			88	100
Cefoperazone				24^e	1			96	100
Cefotaxime				25^e				100	100
Ceftiofur				25^e				100	100
Cefquinome				25^e				100	100
Cephalothin			3^e	22^e				88	100
Ciprofloxacin				23^e	22			92	100
Colistin			3	18	3	1		72	96
Doxycycline			3	17	5			68	100
Enrofloxacin				22^e	3			88	100
Florfenicol				23	2			92	100
Gentamicin			1	20	4			80	100
Imipenem			1	23^e	1			92	100
Marbofloxacin				21^e	4			84	100
Nalidixic acid		1	2	20	2			80	96
Neomycin			3	22				88	100
Penicillin			4^e	16	4		1	64	96
Streptomycin				24^e		1		96	96
Tetracycline			3	22				88	100
Tiamulin		1	2	20	2			80	96
Tilmicosin		1	2	21	1			84	96
Trimethoprim/sulfamethoxazole 1:19			2	21^e	2			84	100
Tulathromycin			2	19	3	1		76	96

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^{^a}

The MIC mode indicates the most frequently measured MIC related to an isolate and agent.

^{^b}

Data are from testing five isolates in five independent replications.

^{^c}

Percentage of MIC values corresponding to the MIC mode of the isolates.

^{^d}

The essential MIC agreement allows a deviation of ±1 dilution step from the MIC mode.

^{^e}

The MIC value of at least one isolate was equal to, lower, or higher than the concentration of the test range.

^{^f}

Data that fulfill the criteria of the essential MIC agreement are highlighted in gray.

Since it was previously shown that the medium CAMHB + CS + NADH did not affect MICs of quality control (QC) strains (23), it was tested here whether the extended incubation time of 48 h might influence the MICs of the QC strain E. coli ATCC 25922. However, the MICs were within the specified ranges given by CLSI guideline M23 (27) with the only exception of cephalothin, for which they were one dilution step above the QC range in three out of five independent replications.

Comparison of MIC values with medium and supplements from different manufacturers

When comparing the MIC values of the five Av. paragallinarum test isolates using CAMHB and supplements from different manufacturers, it was found that most MICs either did not deviate or deviated by a maximum of ±1 or ±2 dilution steps from the MIC modes (Table S2; Table 4). Only the MICs of enrofloxacin and tetracycline of isolate no. 7 deviated by a maximum of ±3 dilution steps, after additional testing with media components of other producers. Overall, the exact MIC ranged from 56.67% to 100% and the essential MIC agreement ranged from 93.33% to 100%, which is within the accepted range according to CLSI guideline M23 (27).

Testing of a Av. paragallinarum collection from different continents

The suitability of the method was tested with the remaining Av. paragallinarum that were available for the study. Testing the remaining Av. paragallinarum was carried out using the broth medium and incubation conditions as previously worked out. Although eight Av. paragallinarum formed speckled buttons in the wells of the microtiter plates, easy-to-read MICs were achieved for all isolates with CAMHB + CS + NADH. Two randomly chosen controls for testing the inoculum density revealed inoculum sizes between 3.8 × 10⁵ to 7.3 × 10⁵ cfu/mL, which is within the specifications according to standard M07 (28).

The MIC distributions and calculated MIC₅₀ and MIC₉₀ values of the entire Av. paragallinarum collection (n = 83) tested in this study are summarized in Table 5. As no approved breakpoints for Av. paragallinarum are available, the isolates could not be classified as resistant, intermediate, or susceptible to the tested antimicrobials. Nevertheless, for many antimicrobials (e.g., ampicillin, cefoperazone, cefotaxime, ceftiofur, cefquinome, cephalothin, florfenicol, gentamicin, tilmicosin, tulathromycin) unimodal MIC distributions or distributions comprising a maximum of six dilution steps were seen. By contrast, bimodal or multimodal MIC distributions were obtained for tetracyclines (doxycycline, tetracycline), fluoroquinolones (ciprofloxacin, enrofloxacin, marbofloxacin), nalidixic acid, penicillin, streptomycin, and for the antimicrobial combination trimethoprim/sulfamethoxazole (Table 5).

TABLE 5.

MIC values of 83 Avibacterium paragallinarum isolates (74 field isolates and 10 reference or type strains) obtained after 48 h incubation at 35 ± 2°C in CAMHB + CS + NADH^g

Antimicrobial agent^a	Number of Av. paragallinarum and their MIC^b (µg/mL)																		MIC₅₀^c (µg/mL)	MIC₉₀^d (µg/mL)
Antimicrobial agent^a	0.008	0.015	0.03	0.06	0.12	0.25	0.5	1	2	4	8	16	32	64	128	256	512	1024	MIC₅₀^c (µg/mL)	MIC₉₀^d (µg/mL)
AMC^e			9^h	4	51	18	1												0.12	0.25
AMP			10^h	21	26	20	6												0.12	0.25
CFP				72^h	8	2	1												≤0.06	0.12
CTX		81^h	1	1															≤0.015	≤0.015
CQN		54^h	20	5		2	2												≤0.015	0.06
XNL			72^h	7	2	1	1												≤0.03	0.06
CEF				46^h	18	17	1	1											≤0.06	0.25
CIP	31^h	9	9	2	1	3	6	21	1										0.03	1
CST						1	4	5	31	28	6	2	4	1	1ⁱ				4	8
DOX					1	2	20	17	13	3	5	15	7						2	16
ENRO	11^h	11	12	11	7	2		2	8	16	3								0.06	4
FFN					6^h	35	33	7		2									0.5	1
GEN					5^h	4	18	34	17	5									1	2
IPM		23^h	13	40	7														0.06	0.06
MAR	10^h	15	16	8	2	1	3	14	13	1									0.06	2
NAL				1^h		6	4	13	14	3	5	4	18	14	1				8	64
NEO					2^h		4	13	26	25	12			1					2	8
PEN		6^h	14	4	8	19	10	13	9										0.25	2
STR							1	3	12	23	2	1	3	1	2	1	2	32ⁱ	16	≥1024
TET					1^h	1	9	22	21				5	16	8				2	64
TIA				3			2	3	13	22	25	15							4	16
TIL						3		10	17	31	22								4	8
SXT^f		29^h	12	13	3	3	11	5	5	1	1								0.06	1
TUL						4	8	39	25	5	2								1	2

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^{^a}

AMC, amoxicillin-clavulanic acid 2:1 ratio; AMP, ampicillin; CFP, cefoperazone; CTX, cefotaxime; CQN, cefquinome; XNL, ceftiofur; CEF, cephalothin; CIP, ciprofloxacin; CST, colistin; DOX, doxycycline; ENRO, enrofloxacin; FFN, florfenicol; GEN, gentamicin; IPM, imipenem; MAR, marbofloxacin; NAL, nalidixic acid; NEO, neomycin; PEN, penicillin; STR, streptomycin; TET, tetracycline; TIA, tiamulin; TIL, tilmicosin; SXT, trimethoprim-sulfamethoxazole 1:19 ratio; TUL, tulathromycin.

^{^b}

MIC, minimal inhibitory concentration.

^{^c}

MIC₅₀ indicates the MIC that is required to inhibit 50% of the isolates.

^{^d}

MIC₉₀ indicates the MIC that is required to inhibit 90% of the isolates.

^{^e}

Data represent the concentration of amoxicillin.

^{^f}

Data represent the concentration of trimethoprim.

^{^g}

The tested range of the antimicrobials is visualized in the white area.

^{^h}

MIC values equal to or lower than the lowest concentrations tested.

^ⁱ

MIC values higher than the highest concentrations tested.

A total of 36 isolates were particularly noticeable, demonstrating higher MICs (compared to the other isolates) to three to five classes of antimicrobials: aminoglycosides (≥1,024 µg/mL streptomycin), β-lactams (1–2 µg/mL penicillin), tetracyclines (16–32 µg/mL doxycycline, 32–128 µg/mL tetracycline), (fluoro)quinolones (32–128 µg/mL nalidixic acid, 1–2 µg/mL ciprofloxacin, 2–8 µg/mL enrofloxacin, 1–4 µg/mL marbofloxacin), and/or folate synthesis inhibitors (0.25/4.75 – 8/152 µg/mL trimethoprim/sulfamethoxazole). A large proportion of these Av. paragallinarum came from Europe or America (Table S3).

Detection of antimicrobial resistance genes

During method development, PCR analyses for the presence of resistance genes were performed with 14 of the 15 isolates that were included in macrorestriction analysis, as these isolates showed higher MICs compared to the others. This was done to determine whether the resistance phenotypes corresponded to the genotypes. The PCR analyses demonstrated that all seven isolates with higher MICs of doxycycline (16–32 µg/mL) and tetracycline (64–128 µg/mL) harbored the tetracycline resistance gene tet(B) (Table 6). With the PCR primers used in this study, no other ARGs (a list of the tested resistance genes can be found in Table S1 in the Supplementary data) were detected in the isolates used for method development.

TABLE 6.

Antimicrobial resistance genes of 76 Av. paragallinarum detected by whole-genome sequencing or PCR analysis

Antimicrobial resistance genes	Number of isolates with resistance gene				MIC values of isolates carrying the resistance gene
	Detected by		Total	Percentage (%) among tested isolates
	WGS (n = 62)	PCR (n = 14)	Total	Percentage (%) among tested isolates
Aminoglycosides
aph(6)-Id	3	ND	3	4.84	STR: ≥1,024 µg/mL
aph(3'')-Ib	2	ND	2	3.23	STR: ≥1,024 µg/mL
β-lactams
bla_TEM-1B	2	ND	2	3.23	PEN: 1 µg/mL
Chloramphenicol
catA2	4	ND	4	6.45	ND^a
Polymyxins
mcr-like	1	ND	1	1.61	COL: 4 µg/mL
Sulfonamides
sul2	2	0	2	2.63	SXT: 0.5/9.5 – 2/38 µg/mL
Tetracyclines
tet(B)	19	7	26	34.21	DOX: 4–32 µg/mL TET: 32–128 µg/mL
tet(H)	1	0	1	1.31	DOX: 4–32 µg/mL TET: 32–128 µg/mL

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^{^a}

Chloramphenicol was not tested as it was not part of the microtiter plate layouts.

^{^b}

WGS, whole-genome sequencing; ND, not determined; COL, colistin; DOX, doxycycline; PEN, penicillin; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline.

For another 62 Av. paragallinarum, WGS data were available, which were analyzed for the presence of ARGs in the course of the present study. About one-third of these Av. paragallinarum (33.87%, n = 21) harbored at least one of the following ARGs: aph(6)-Id, aph(3'')-Ib, bla_TEM-1B, catA2, sul2, tet(B), tet(H), and/or mcr-like (Table 6). All Av. paragallinarum that had MICs of 4–32 µg/mL doxycycline and 32–128 µg/mL tetracycline harbored tet(B) or tet(H). In addition, a partial sequence of a mcr-like gene of 1,547 base pairs, showing 97% sequence identity to the phosphoethanolamine transferase gene of Moraxella osloensis strain NP7 (GenBank CP024443.2), was detected in an English field isolate, which showed a colistin MIC of 4 µg/mL, which was within the range of other Av. paragallinarum tested. Four American field isolates carried not only tet(B) but also the chloramphenicol resistance gene catA2. However, as the microtiter plate layouts used in this study did not contain chloramphenicol, the MIC values for these catA2-carrying Av. paragallinarum are unknown. Two European isolates with a MIC of 1 µg/mL penicillin and ≥1,024 µg/mL streptomycin carried the β-lactamase resistance genes bla_TEM-1B as well as the aminoglycoside resistance genes aph(6)-Id and aph(3″)-Ib. Another European field isolate carried only one aminoglycoside resistance gene (aph(6)-Id) and showed a comparatively lower MIC of 32 µg/mL streptomycin. Furthermore, there were two European field isolates that harbored the sulfonamide resistance gene sul2 and had MICs of 0.5/9.5 µg/mL and 2/38 µg/mL trimethoprim/sulfamethoxazole, respectively.

It was found that the majority of the ARG-carrying Av. paragallinarum from this study originated from Europe, and fewer from America (Fig. S6). Thus, 24 out of 43 European Av. paragallinarum tested for the presence of resistance genes had either one (n = 21), three (n = 1), four (n = 1), or six (n = 1) ARGs. In addition, 4 out of 17 American Av. paragallinarum tested carried two ARGs, catA2 and tet(B). African isolates tested showed no ARGs. Due to the low number of available Av. paragallinarum from Asia (n = 1) or Oceania (n = 1), their MIC values were not included in the comparison.

DISCUSSION

Antimicrobial resistance (AMR) in bacterial pathogens is currently an important public health problem (29, 30). Therefore, AST of bacterial pathogens is of prime importance, especially to be able to perform targeted antimicrobial treatment (30, 31). This requires accepted methods for AST, preferably with precise testing specifications published in accepted standards. This is still lacking for some fastidious pathogens, such as Av. paragallinarum. Therefore, this study aimed to develop a method for standardized broth microdilution testing of this fastidious and economically important pathogen. In addition, the current susceptibility status of field isolates was assessed using the proposed AST method.

For method development, the growth of 15 Av. paragallinarum was compared in six different broth media. As many Av. paragallinarum depend on the V-factor (3), it was not surprising that none of the isolates demonstrated growth in CAMHB without the supplementation of this growth factor. Nevertheless, this medium was included because it is the standard medium for testing fast-growing bacteria and is routinely being used in many diagnostic laboratories (22). The two media CAMHB + CS + NADH and VFM allowed the reading of the growth of all tested Av. paragallinarum in microtiter plates. But of course, it cannot be excluded that other media not tested here would also be suitable. As expected, both media also led to sufficient colony counts (approx. 5 × 10⁶–10⁹ cfu/mL) after 24–48 h as demonstrated in the growth curves. However, since the availability of VFM is not guaranteed due to the discontinued production of supplement C (20), CAMHB + CS + NADH was chosen as the broth medium for further validation steps. This decision was also supported by the fact that this medium was already used for AST of Av. paragallinarum in two previous studies (12, 13) and has recently been proposed for standardized AST of the closely related species Av. gallinarum (14), as well as for another fastidious pathogen, G. parasuis (23). The medium CAMHB + CS + NADH will also be included in the forthcoming CLSI guidance document VET06 as a test medium for G. parasuis (personal communication). Using the same medium for different bacterial species is an advantage for veterinary diagnostic laboratories and it is even possible that CAMHB + CS + NADH is also a suitable medium for standardized AST of further fastidious pathogens for which there are no approved AST methods yet. However, in a previous study on Av. paragallinarum (12), the reproducibility of the obtained MICs did not yet comply with the requirements of the CLSI. Since Av. paragallinarum is relatively slow growing, different incubation times needed to be evaluated. Although some previous studies examining MICs of Av. paragallinarum used incubation times of 20, 22, or 24 h (7, 12, 13, 32 –36), the MICs could be read at the earliest after 24 h in the course of this study. In addition, low essential MIC agreements were observed by Heuvelink et al. (12) using a 22-h incubation period and, in this study, after 24 h (77.8%–95.6% and 84%–100%, respectively), which for many antimicrobials did not meet the requirement of ≥90% for a new method according to CLSI guideline M23 (27). For this reason, an extension of the incubation period seemed necessary and indeed resulted in higher essential MIC agreements ranging between 96% and 100%, thus exceeding the criterion required by the CLSI (27). This extended incubation time is still within the times suggested in CLSI standards (24–48 h) for fastidious bacteria, such as Campylobacter jejuni (22). Since the MICs of the QC strain E. coli ATCC 25922 were within the required MIC ranges after 24 h (12, 23) and after 48 h for all tested strain-antimicrobial agent combinations except for cephalothin (this study), a complex validation of new QC strains seems not necessary. In addition, testing can be performed with media and supplements from different producers and still leads to comparable MICs, as shown by high essential MIC agreements. Hence, for suitability testing, the remaining Av. paragallinarum available for method development were subsequently tested. Although eight isolates formed scattered instead of distinct buttons in the wells of microtiter plates, which were similar to those formed by Av. gallinarum (14), easily readable MICs were obtained for all isolates. However, as mentioned above, it was not possible to classify Av. paragallinarum as resistant, intermediate, or sensitive because there are currently no accepted breakpoints for this pathogen, despite the interpretive criteria recommended by Blackall (32, 33). Nevertheless, the bimodal and/or broad MIC distributions observed for some agents (e.g., quinolones, streptomycin, tetracyclines, trimethoprim/sulfamethoxazole) indicated the existence of non-wild-type subpopulations with acquired resistance (37). Of particular note was the detection of 36 isolates with higher MICs (MIC values that were at the right edge of the distribution) against ≥3 classes of antimicrobials (aminoglycosides, β-lactams, folate synthesis inhibitors, quinolones, tetracyclines), indicating phenotypical multidrug resistance (38). Av. paragallinarum with elevated MICs against these antimicrobials has been reported previously (7, 12, 34, 35, 39). Of these, sulfonamides and tetracyclines are among the antimicrobials commonly used to control Av. paragallinarum infections in poultry (3, 7), so isolates with elevated MICs are problematic for therapy.

Although only a limited number of isolates was available for this study without random selection, so that no clear conclusions can be drawn, the observed data seem to indicate that AMR depends on the geographical origin of Av. paragallinarum isolates. For example, African Av. paragallinarum showed lower MICs of fluoroquinolones and tetracycline, while a large proportion of American and European Av. paragallinarum showed comparatively higher MICs. Although this comparison must be considered with caution due to the different numbers of isolates included, these differences might be due to variations in the use of antimicrobials in the individual countries. However, it must also be taken into account that the use of antibiotics varies between different types of poultry production (40, 41).

To assess the correlation between resistance phenotypes and genotypes during method development, Av. paragallinarum with elevated MICs used for method development were subjected to PCR analysis for the presence of ARGs. In all seven isolates exhibiting elevated MICs of doxycycline (16–32 µg/mL) and tetracycline (64–128 µg/mL), the tetracycline resistance gene tet(B) was detected, demonstrating a good correlation between genotype and phenotype. By contrast, no ARGs were detected in Av. paragallinarum with elevated MICs of penicillin, quinolones, streptomycin, and/or trimethoprim/sulfamethoxazole with the PCR primers used. This might be due to the large number of ARGs conferring resistance to penicillin (42), streptomycin (43), or trimethoprim (44), of which only a smaller spectrum was tested here. In addition, chromosomal mutations may also be responsible for reduced susceptibility to quinolones, streptomycin, sulfonamides, and trimethoprim (45 –49).

To investigate the presence of non-PCR-identified ARGs, WGS data were analyzed for 62 Av. paragallinarum. Combining the PCR and WGS data, it was demonstrated that many American (4 out of 17) and European (24 out of 43) Av. paragallinarum tested in this study harbored one to four ARGs. Some of these ARGs have already been detected in Av. paragallinarum or related bacterial species (e.g., Av. gallinarum, G. parasuis) and confer resistance to aminoglycosides (aph(6)-Id), β-lactams (bla_TEM), sulfonamides (sul2), or tetracyclines (tet(B)) (14, 36, 50, 51). Nevertheless, to our knowledge, this study is the first that describes the presence of nucleotide sequences showing high sequence identity to aph(3″)-Ib, catA2, tet(H), and a mcr-like gene in Av. paragallinarum, genes that are known to mediate aminoglycoside, β-lactam, chloramphenicol, tetracycline, or colistin resistance. Of note, the isolate carrying a mcr-like gene had a colistin MIC that corresponded to the MIC₅₀ value of the isolate collection (4 µg/mL) and, thus, was not higher than the values of many isolates without a mcr-like gene. Similar observations were made with mcr-2.1-bearing E. coli and Salmonella spp. exhibiting comparable MICs ranging between 4 and 8 µg/mL (52 –55). Yet, a previous study showed that the naturally occurring mcr-like genes from Moraxella spp., to which the gene detected in our isolate showed very high similarity, conferred colistin resistance after being experimentally introduced into an E. coli host (56). However, whether our isolate should be considered colistin resistant (as would be the case for Enterobacterales) cannot be determined without clinical breakpoints. Further studies are required to clarify the functionality of the gene in Av. paragallinarum. Furthermore, since high-level resistance to colistin is often not only due to the presence of mcr genes but rather to mutations (57), similar mechanisms can be assumed for Av. paragallinarum, representing an interesting area of future research.

Conclusion

Reproducible MICs were obtained using CAMHB + CS + NADH as a medium for susceptibility testing of Av. paragallinarum by broth microdilution with an incubation time of 48 h at 35 ± 2°C in ambient air. For quality control purposes, E. coli ATCC 25922 and the QC ranges included in the CLSI standards can be used (except cephalothin). Based on the results of this study, it can be concluded that the method is suitable to be used as a standardized method for susceptibility testing of Av. paragallinarum and can be included in standards of susceptibility testing. Many of the Av. paragallinarum tested had elevated MICs for aminoglycosides, quinolones, tetracyclines, and trimethoprim/sulfamethoxazole, most of them originating from America or Europe. Using PCR and WGS, 28 out of 76 Av. paragallinarum tested were found to carry at least one nucleotide sequence showing high sequence identity to a resistance gene, aph(6)-Id, aph(3″)-Ib, bla_TEM-1B, catA2, sul2, tet(B), tet(H), and/or mcr-like. Therefore, future development of clinical breakpoints for this pathogen is strongly recommended.

ACKNOWLEDGMENTS

Special thanks to Cornelia Dürrschmidt, Jan Paulus, Karin Simon, and Claudia Walter for their excellent technical support.

This research was funded by the German Federal Ministry of Food and Agriculture (BMEL) (grant number FKZ 2818HS015).

Contributor Information

Corinna Kehrenberg, Email: Corinna.kehrenberg@vetmed.uni-giessen.de.

Alexander J. McAdam, Boston Children's Hospital, Boston, Massachusetts, USA

DATA AVAILABILITY

Raw sequence data were submitted to the sequence read archive (SRA) under BioProject number PRJNA1044294 and accession numbers SAMN38380366 to SAMN38380422. Field isolates 11 and 13, used for compilation of growth curves and method validation, can be found within this project under accession numbers SAMN38380376 (isolate 11) and SAMN38380375 (isolate 13). Genome data of test isolates 4 and 7 can be found separately under BioProject number PRJNA1048828, accession numbers SAMN38658941 (isolate 4) and SAMN38658940 (isolate 7). Genome data of type strain CCUG 12835^T can be accessed via accession number GCF_002921155.1.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jcm.01011-23.

Supplemental figures and tables. jcm.01011-23-s0001.docx.

Tables S1 to S3, Figures S1 to S6.

jcm.01011-23-s0001.docx^{(2.3MB, docx)}

DOI: 10.1128/jcm.01011-23.SuF1

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental figures and tables. jcm.01011-23-s0001.docx.

Tables S1 to S3, Figures S1 to S6.

jcm.01011-23-s0001.docx^{(2.3MB, docx)}

DOI: 10.1128/jcm.01011-23.SuF1

Data Availability Statement

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[B9] 9. Kowalska-Krochmal B, Dudek-Wicher R. 2021. The minimum inhibitory concentration of antibiotics: methods, interpretation, clinical relevance. Pathogens 10:165. doi: 10.3390/pathogens10020165 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Traczewski MM, Ambler JE, Schuch R. 2021. Determination of MIC quality control parameters for exebacase, a novel lysin with antistaphylococcal activity. J Clin Microbiol 59:e0311720. doi: 10.1128/JCM.03117-20 [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B12] 12. Heuvelink A, Wiegel J, Kehrenberg C, Dijkman R, Soriano-Vargas E, Feberwee A. 2018. Antimicrobial susceptibility of Avibacterium paragallinarum isolates from outbreaks of infectious coryza in Dutch commercial poultry flocks, 2008–2017. Vet Microbiol 217:135–143. doi: 10.1016/j.vetmic.2018.03.008 [DOI] [PubMed] [Google Scholar]

[B13] 13. Luna-Castrejón LP, Buter R, Pantoja-Nuñez GI, Acuña-Yanes M, Ceballos-Valenzuela K, Talavera-Rojas M, Salgado-Miranda C, Heuvelink A, de Wit S, Soriano-Vargas E, Feberwee A. 2021. Identification, HPG2 sequence analysis, and antimicrobial susceptibility of Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layers in Sonora state, Mexico. Avian Dis 65:95–101. doi: 10.1637/aviandiseases-D-20-00103 [DOI] [PubMed] [Google Scholar]

[B14] 14. Gütgemann F, Müller A, Churin Y, Jung A, Kumm F, Heuvelink A, Yue M, Kehrenberg C. 2022. Proposal of a method for harmonized broth microdilution antimicrobial susceptibility testing of Avibacterium gallinarum. J Clin Microbiol 60:e0041922. doi: 10.1128/jcm.00419-22 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15. Hinz KH. 1973. Contribution to the differentiation of haemophilus-strains isolated from chickens. II: serological examinations using the plate-agglutination test. Avian Pathol 2:269–278. doi: 10.1080/03079457309353803 [DOI] [PubMed] [Google Scholar]

[B16] 16. Chen X, Chen Q, Zhang P, Feng W, Blackall PJ. 1998. Evaluation of a PCR test for the detection of Haemophilus paragallinarum in China. Avian Pathol 27:296–300. doi: 10.1080/03079459808419339 [DOI] [PubMed] [Google Scholar]

[B17] 17. Muhammad TMN, Sreedevi B. 2015. Detection of Avibacterium paragallinarum by polymerase chain reaction from outbreaks of infectious coryza of poultry in Andhra Pradesh. Vet World 8:103–108. doi: 10.14202/vetworld.2015.103-108 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18. Feberwee A, Dijkman R, Buter R, Soriano-Vargas E, Morales-Erasto V, Heuvelink A, Fabri T, Bouwstra R, de Wit S. 2019. Identification and characterization of dutch Avibacterium paragallinarum isolates and the implications for diagnostics. Avian Pathol 48:549–556. doi: 10.1080/03079457.2019.1641178 [DOI] [PubMed] [Google Scholar]

[B19] 19. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ. 2006. Standardization of pulsed-field GEL electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for pulsenet. Foodborne Pathog Dis 3:59–67. doi: 10.1089/fpd.2006.3.59 [DOI] [PubMed] [Google Scholar]

[B20] 20. CLSI . 2016. Performance standards for antimicrobial susceptibility testing. In CLSI supplement M100S, 26th ed. Clinical and Laboratory Standards Institute. [Google Scholar]

[B21] 21. CLSI . 2019. Replacement broth medium for veterinary fastidious medium and new Actinobacillus pleuropneumoniae ATCC 27090 and Histophilus somni ATCC 700025 minimal inhibitory concentration quality control ranges

[B22] 22. CLSI . 2020. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. In Clinical and laboratory standards Institute, 5th edition. Vol. supplement VET01S. [Google Scholar]

[B23] 23. Prüller S, Turni C, Blackall PJ, Beyerbach M, Klein G, Kreienbrock L, Strutzberg-Minder K, Kaspar H, Meemken D, Kehrenberg C. 2017. Towards a standardized method for broth microdilution susceptibility testing of Haemophilus parasuis. J Clin Microbiol 55:264–273. doi: 10.1128/JCM.01403-16 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24. Gütgemann F, Müller A, Churin Y, Jung A, Braun A-S, Yue M, Kehrenberg C. 2022. Development of a harmonized method for antimicrobial susceptibility testing of Bordetella avium using broth microdilution and detection of resistance genes. J Appl Microbiol 132:1775–1787. doi: 10.1111/jam.15305 [DOI] [PubMed] [Google Scholar]

[B25] 25. Buter R, Feberwee A, de Wit S, Heuvelink A, da Silva A, Gallardo R, Soriano Vargas E, Swanepoel S, Jung A, Tödte M, Dijkman R. 2023. Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping. Avian Pathol 52:362–376. doi: 10.1080/03079457.2023.2239178 [DOI] [PubMed] [Google Scholar]

[B26] 26. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field GEL electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239. doi: 10.1128/jcm.33.9.2233-2239.1995 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27. CLSI . 2018. Development of in vitro susceptibility testing criteria and quality control parameters. In CLSI guideline M23. Clinical and Laboratory Standards Institute. [Google Scholar]

[B28] 28. CLSI . 2018. Methods for dilution antimicrobial susceptibility tests for bacteria that grow Aerobically. In CLSI standard M07, 11th ed. Clinical and Laboratory Standards Institute. [Google Scholar]

[B29] 29. Antimicrobial Resistance Collaborators . 2022. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399:629–655. doi: 10.1016/S0140-6736(21)02724-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30. Gajic I, Kabic J, Kekic D, Jovicevic M, Milenkovic M, Mitic Culafic D, Trudic A, Ranin L, Opavski N. 2022. Antimicrobial susceptibility testing: a comprehensive review of currently used methods. Antibiotics (Basel) 11:427. doi: 10.3390/antibiotics11040427 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31. Fuhrmeister AS, Jones RN. 2019. The importance of antimicrobial resistance monitoring worldwide and the origins of SENTRY antimicrobial surveillance program. Open Forum Infect Dis 6:S1–S4. doi: 10.1093/ofid/ofy346 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Blackall PJ. 1988. Antimicrobial drug resistance and the occurrence of plasmids in Haemophilus paragallinarum. Avian Dis 32:742–747. doi: 10.2307/1590993 [DOI] [PubMed] [Google Scholar]

[B33] 33. Blackall PJ, Yamamoto R. 1989. Haemophilus gallinarum—a re-examination. J Gen Microbiol 135:469–474. doi: 10.1099/00221287-135-2-469 [DOI] [PubMed] [Google Scholar]

[B34] 34. Hsu YM, Shieh HK, Chen WH, Sun TY, Shiang JH. 2007. Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains. Vet Microbiol 124:209–218. doi: 10.1016/j.vetmic.2007.04.024 [DOI] [PubMed] [Google Scholar]

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PERMALINK

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

Franziska Gütgemann

Annet Heuvelink

Anja Müller

Yury Churin

Rianne Buter

Arne Jung

Anneke Feberwee

Jeanine Wiegel

Franziska Kumm

Ann Sophie Braun

Min Yue

Edgardo Soriano-Vargas

Stefan Swanepoel

Nadine Botteldoorn

Miranda Kirchner

Corinna Kehrenberg

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

MATERIALS AND METHODS

Collection of Av. paragallinarum field isolates and reference strains, culturing, and species confirmation

TABLE 1.

Fig 1.

Macrorestriction and pulsed-field gel electrophoresis analyses

Visible growth of Av. paragallinarum in six broth media

Compiling multi-day growth curves

Antimicrobial susceptibility testing of Av. paragallinarum

Control of medium and supplements from different producers

Analyses of antimicrobial resistance genes

RESULTS

Clonality of 15 Av. paragallinarum used for method validation

Visible growth of Av. paragallinarum in different broths

TABLE 2.

Growth curves of Av. paragallinarum with two broth media

Homogeneity of Av. paragallinarum MIC values

TABLE 3.

TABLE 4.

Comparison of MIC values with medium and supplements from different manufacturers

Testing of a Av. paragallinarum collection from different continents

TABLE 5.

Detection of antimicrobial resistance genes

TABLE 6.

DISCUSSION

Conclusion

ACKNOWLEDGMENTS

Contributor Information

DATA AVAILABILITY

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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