Manuskript 1

Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis

Prüller, S., Turni, C., Blackall, P. J., Beyerbach, M., Strutzberg-Minder, K., Kaspar, H., Meemken, D., Klein, G., Kreienbrock, L. and Kehrenberg, C.

Manuskript in Vorbereitung zur Publikation

Autorenbeteiligung:

Design der Studie: SP, CK

Durchführung der Experimente: SP

Auswertung der Daten: SP, MB, LK, CK

Erstellung des Manuskripts: SP, CK

Bereitstellung der Reagenzien/Arbeitsplatz

und wissenschaftliche Diskussion: GK, CT, PJB, KSM, HK, DM

Towards a Standardized Method for Broth Microdilution Susceptibility Testing of Haemophilus parasuis

SANDRA PRÜLLER,¹ CONNY TURNI,² PATRICK J. BLACKALL,² MARTIN BEYERBACH,³ GÜNTER KLEIN,¹ LOTHAR KREIENBROCK,³ KATRIN STRUTZBERG-MINDER,⁴ HEIKE KASPAR,⁵ DIANA MEEMKEN,¹ and CORINNA

KEHRENBERG^1*

1 Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany

2The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, EcoSciences Precinct, Dutton Park, Queensland, Australia

3 Department of Biometry, Epidemiology and Information Processing, WHO Collaborating Centre for Research and Training in Veterinary Public Health, University of Veterinary

Medicine Hannover, Foundation, Germany

4Innovative Veterinary Diagnostics (IVD) Laboratory, Hannover, Germany

5Federal Office of Consumer Protection and Food Safety, Berlin, Germany

Keywords: broth microdilution, susceptibility testing, MIC values, Haemophilus parasuis, fastidious organism, TMB medium, supplemented CAMHB medium

* Corresponding author: Mailing address: Institute for Food Quality and Food Safety, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany. Phone: +49-511-856-7554. Fax: +49-511-856-7694. Electronic mail address:

corinna.kehrenberg@tiho-hannover.de

ABSTRACT

The aim of the study was to develop a broth microdilution susceptibility testing method for Haemophilus (H.) parasuis that could be performed easily by diagnostic laboratories. To achieve this, the first step was to perform growth curves in four different media: cation-adjusted Mueller-Hinton broth (CAMHB) with lysed horse blood, Veterinary Fastidious Medium (VFM), Haemophilus Test Medium (HTM) and Test Medium Broth (TMB) for three field isolates from two different serovars and one type strain. The second step was the development of a new medium, CAMHB plus reduced nicotinamide-adenine-dinucleotide (NADH) and heat-inactivated chicken serum. The suitability of the new medium and TMB for broth microdilution susceptibility testing was examined using one type strain and 46 field isolates representing 13 serovars.

The same H. parasuis isolates from the growth experiments were evaluated in five replications for both media. Data were collected after 16 to 20 and 24 to 48 hours incubation time. Incubation conditions and inoculum size was according to the CLSI-approved performance standards given in document VET01-A4. Four different QC-strains were used to examine the effect of the media to the antimicrobial agents. For statistical analysis, a model with two different variables (precondition of five identical MIC values and MIC mode accepting a deviation of ± one dilution step, respectively) was used to calculate the homogeneity of MIC values from five replicates for 21 antimicrobial agents and antimicrobial combinations. The results showed that no significant differences for both variables were seen between the two time points. A difference in homogeneity between both media was observed for the first variable however the difference

was not significant. Subsequently, 47 H. parasuis isolates were tested with the newly developed medium and the TMB medium. Statistical analysis of all isolates and antimicrobial agents showed no significant difference between MIC values obtained in supplemented CAMHB and TMB medium. Because of a simplified implementation in routine diagnostic and a lower chance of interference between medium components and antimicrobial agents, supplemented CAMHB is recommended with an incubation time of 20 or 24 hours.

INTRODUCTION

Haemophilus (H.) parasuis is an abundant colonizer of the upper respiratory tract in swine (HOLTKAMP 2007). Although in H. parasuis endemic herds, young piglets are usually protected by maternal immunity, several environmental stress factors may lead to disease as well as infections of naive populations. The acute form of the disease, Glaesser´s disease, is characterized by clinical signs of polyarthritis, polyserositis, meningitis, and pneumonia (amongst others) and can lead to sudden death of animals, especially in weaner pigs (OLIVEIRA u. PIJOAN 2004; BROCKMEIER et al. 2013).

Morbidity and mortality vary from 5-10 % on conventional farms to 75 % in specific pathogen free (SPF)-herds, resulting in high economic losses on pig farms all over the world (BAUMANN u. BILKEI 2002; TURNI u. BLACKALL 2007; NEDBALCOVÁ u.

KUČEROVÁ 2013; CHEN et al. 2015). At least 15 different H. parasuis serovars were identified so far with the most common serovars being 4 and 5 with 4 being moderately

pathogenic and 5 being highly pathogenic (KIELSTEIN 1992; RAPP-GABRIELSON u.

GABRIELSON 1992).

As antimicrobial agents are used for the treatment of disease, a reliable method for susceptibility testing of the pathogen is essential. H. parasuis is a fastidious organism which requires specific ingredients or supplements to achieve visible growth in broth and, hence, media which are used to test rapidly growing bacteria are not adequate (ZHANG et al. 2012; DAYAO et al. 2014). As a consequence, no validated and standardized method is currently available for testing of H. parasuis (SCHWARZ et al. 2010; DAYAO et al. 2014). However, a few studies investigated the susceptibility status of H. parasuis isolates quantitatively by using media such as Veterinary fastidious medium (VFM) or Haemophilus Test Medium (HTM) for broth microdilution susceptibility testing ((DEROSA et al. 2000; AARESTRUP et al. 2004; DE LA FUENTE et al. 2007; SAN MILLAN et al.

2007; ZHOU et al. 2010; XU et al. 2011; LI-LI GUO et al. 2012; NEDBALCOVÁ u.

KUČEROVÁ 2013; DAYAO et al. 2014). The VFM is recommended in CLSI standard VET01-A4 for susceptibility testing of Actinobacillus (A.) pleuropneumoniae and Histophilus somni (CLINICAL AND LABORATORY STANDARDS INSTITUTE 2013a), while HTM and cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 2.5 % to 5 % lysed horse blood (LHB) are CLSI-approved broth media (CLSI-document M07-A9) for the testing of fastidious organisms, with HTM medium being recommended for Haemophilus (H.) influenzae and Haemophilus parainfluenzae (CLINICAL AND LABORATORY STANDARDS INSTITUTE 2012a). As it has been shown that VFM and HTM are not suitable for many H. parasuis isolates, a broth microdilution susceptibility testing method with Test Medium Bouillon (TMB) has recently been proposed (DAYAO et al. 2014).

Therefore, this study aimed at (i) comparatively analyzing the suitability of different broth media for susceptibility testing of preferably a wide range of different H. parasuis serovars and (ii) identifying a broth medium for a harmonized routine diagnostic approach. For this, a CAMHB broth medium with a reduced content of supplements was developed and used for testing of 46 H. parasuis field isolates including representatives of 13 serovars in comparison to TMB medium. Subsequently, statistical analysis was performed to examine the homogeneity of MIC data and to investigate the difference between supplemented CAMHB and TMB medium. Hence, this study might contribute to establish a standardized broth microdilution susceptibility testing method for H. parasuis, one of the most important pathogens in pig production.

MATERIALS AND METHODS

Bacterial isolates

H. parasuis type strain DSM 21448 (Leibniz-Institute DSMZ, German Collection of Microorganism and Cellcultures, Braunschweig, Germany) and 46 H. parasuis field isolates were used for the present study. The isolates originated from diseased pigs and were collected at pig farms from different geographic regions in Germany between 2011 and 2013. All field isolates were provided by diagnostic laboratories in Germany and included 25 serotyped isolates of serovar 1 (n=2), 2 (n=2), 4 (n=2), 5 (n=2), 6 (n=2), 8 (n=2), 9 (n=1), 10 (n=2), 11 (n=2), 12 (n=2), 13 (n=2), 14 (n=2) and 15 (n=2). Isolates were cultured on chocolate agar with 10 % defibrinated horse blood in ambient air at an incubation temperature of 35° C ± 1° C for 24 to 48 hours. For a H. parasuis species

confirmation, a PCR assay amplifying a 821 bp internal fragment of the 16S small subunit ribosomal RNA gene was used as described earlier (OLIVEIRA et al. 2001).

Growth curves

To compile growth curves, type strain DSM 21448 of H. parasuis and three field isolates of serovars 5 and 14 were used. Growth curves were performed in cation-adjusted Mueller-Hinton broth (CAMHB) (Oxoid, Wesel, Germany) with lysed horse blood, HTM, VFM and TMB broth media, as these media have been proven suitable for susceptibility testing of fastidious organisms (BLACKALL 1988; BARRY et al. 1993; JORGENSEN 2000; AARESTRUP et al. 2004; DE LA FUENTE et al. 2007; DAYAO et al. 2014). In brief, isolates were grown on chocolate agar for 24 to 48 hours at 35°C ± 1°C in ambient air, colonies were suspended in 0.9 % saline and the suspension adjusted to 0.5 McFarland standard. To achieve an initial concentration of approximately 10³ to 10⁴ cfu/ml, a volume of 100 µl of the diluted cultures was transferred in 10 ml 0.9 % saline and 50 µl of this bacterial suspension was transferred to a conical flask containing pre-warmed broth medium. Isolates were cultured for 48 hours at 35°C in ambient air without shaking and the cfu/ml was determined by culture enumeration every 4 hours (up to hour 24) and 8 hours (up to hour 48), respectively. The weighted average was calculated from agar plates containing between 5 and 200 cfu/ml.

Susceptibility testing

For broth microdilution susceptibility testing, customized 50 µl volume/well microtiter plates were used (Sensititre, Trek Diagnostic Systems, East Grinstead, UK). Testing included 21 antimicrobial agents and antibiotic combinations (Table 1), four of which are currently not licensed for food producing animals but were included for comparison

reasons (cefotaxime, ciprofloxacin, imipenem, nalidixic acid). Inoculum preparation and density, incubation conditions and MIC end point determinations followed the recommendations given in CLSI-documents M07-A9 and VET01-A4 (CLSI, Wayne, PA, USA) (CLINICAL AND LABORATORY STANDARDS INSTITUTE 2013a). Reading of microtiter plates was done visually by the same person after 16 to 20 hours and 24 hours incubation of plates in an ambient air incubator, whereby the 24 hour incubation time was extended up to 48 hours for extremely slow growing H. parasuis isolates. An inoculum density of approximately 5 x 10⁵ cfu/ml was verified by inoculum control as laid down in document M07-A9. The quality control strains Escherichia (E.) coli (ATCC 25922), Enterococcus (E.) faecalis (ATCC 29212), Staphylococcus (S.) aureus (ATCC 29213) and A. pleuropneumoniae (ATCC 27090) served for quality control procedures.

According to requirements, broth microdilution susceptibility testing was performed in five replicates.

A selection of 28 H. parasuis field isolates of different serovars were used to determine visible growth in microtiter plates with and without antibiotic coating for the media of the growth determination. When comparing MIC values generated in TMB medium and supplemented CAMHB, all together 47 H. parasuis isolates were used.

Development of a modified broth medium for H. parasuis

As TMB medium is not commercially available and to provide an easier to handle medium for routine diagnostic, a new medium based on CAMHB (Oxoid, Wesel, Germany) was developed for broth microdilution susceptibility testing of H. parasuis. For this purpose, it was evaluated which of the supplements of the TMB medium were essential to provide sufficient growth of preferably all H. parasuis isolates and serovars.

CAMHB broth was taken as a basis since it is the CLSI-approved medium of choice for

susceptibility testing of commonly isolated pathogens. For supplementation of CAMHB, concentrations of supplements were equal to concentrations used in TMB medium. TMB medium includes the supplements O-A complex (oleic acid: Sigma-Aldrich, Seelze, Germany and albumin fraction V: Carl Roth, Karlsruhe, Germany), heat inactivated chicken serum (30 minutes at 56°C) (Sigma-Aldrich, Seelze, Germany), thiamine (Sigma-Aldrich, Seelze, Germany) and reduced nicotinamide-adenine-dinucleotide (NADH disodium salt, grade II) (Roche Diagnostic, Mannheim, Germany). The necessity of the supplements was tested against a set of 15 H. parasuis isolates in 96-well microtiter plates and the visible growth in the wells was determined.

To verify the NADH and chicken serum supplements from different companies, broth microdilution susceptibility testing was performed with type strain DSM 21448 and the three field isolates with 21 antimicrobial agents in two batches of NADH (β-NAD, reduced form, disodium salt) (Armin Baack, Schwerin, Germany and Sigma-Aldrich, Seelze, Germany) and chicken serum (Bio&Sell, Feucht, Germany and Pan-Biotech, Aidenbach, Germany).

Data evaluation and statistical analysis

The primary aim of this study was the exploration of the MIC values and the investigation of differences in the MIC values depending on two different media. Of secondary interest was to compare the stability of MIC values after 16 to 20 hours and 24 hours incubation time.

To investigate whether the stability of the MIC values differed between the two incubation times, for each combination of isolate, medium, antibiotic agent and incubation time, MIC data consisting of five measurements was summarized to a dichotomous outcome. The outcome was set to 1 if all five measurements were equal and 0 otherwise. Stability of

measurements was analysed based on this outcome with a multi-factorial logistic regression model depending on isolate, medium, antibiotic agent and incubation time.

Differences in MIC values depending on incubation time were investigated with an ANOVA-model modelling, the log-transformed MIC values depending on the four factors.

Due to the distribution of MIC values being skewed to the right and a two-fold dilution series was used, data were log-transformed to the base 2. Both media were compared separately for antibiotic agents using the two-sided one-sample t-test for the differences between the two media. Due to the explorative nature of the experiments no multiple adjustments were performed and comparison-wise p-values were reported. Statistical analyses were carried out using SAS^® software, version 9.3 (SAS Institute, Cary, NC, USA).

RESULTS

Growth of H. parasuis in broth media recommended for fastidious organisms To determine the adequacy of different broth media for susceptibility testing, type strain DSM 21448 and three H. parasuis field isolates were cultured in HTM, VFM, CAMHB with LHB and TMB medium. The field isolates were of serovars 5 (two isolates) and 14 (one isolate). The isolates of serovar 5 were included as this serovar is regarded as the most pathogenic H. parasuis serovar in Germany. As well, two of the isolates (no.

8 of serovar 5 and 24 of serovar 14) were selected as they are extremely slow growing isolates and difficult to culture. The growth curves from two independent experiments revealed that all isolates showed almost consistent growth in VFM and TMB medium.

However, in CAMHB with LHB and HTM, a decrease of the isolates in cfu values over time was detected after only a few hours, except for type strain DSM 21448, for which an increase in cfu up to 10⁷-10⁸ cfu/ml was enumerated after 24 hours of incubation in HTM (Supplementary Material, Figure 1-4).

In addition, visible growth of 28 H. parasuis field isolates of various serovars was assessed in microtiter plates with and without antibiotic coating and, in agreement with results from growth determination, no growth was detected in all wells, including the growth-control wells, when HTM and CAMHB with LHB were used. With VFM, visible or evaluable growth could be detected only for seven out of 28 isolates although H. parasuis type strain DSM 21448 and all three H. parasuis field isolates showed a sufficient increase in cfu/ml during determinations of growth curves. Only for type strain DSM 21448, MIC end points could be determined in VFM broth. In contrast to these findings, visible growth of H. parasuis isolates was detected in microtiter plate wells with and without antibiotic coating while testing TMB broth medium.

Modification of the CAMHB broth medium for susceptibility testing of H. parasuis CAMHB broth medium enriched with all four supplements lead to visible growth of 15 H. parasuis isolates tested in 96-well plates without antimicrobial agents. Using only three supplements with CAMHB broth medium, tests revealed that NADH was essential for the growth of all H. parasuis isolates and omitting one of the supplements thiamin, inactivated chicken serum or O-A-complex showed no difference in visible growth. To further reduce the number of supplements, it was evaluated if growth of the H. parasuis isolates varied when only two supplements were added to the CAMHB broth medium.

Results of these experiments showed that supplementation of inactivated chicken serum or O-A-complex is necessary to obtain visible growth of H. parasuis isolates in CAMHB

broth medium. By the reason that O-A-complex is consisting of the two different components, oleic acid and bovine serum albumin (BSA), inactivated chicken serum together with NADH were chosen to supplement CAMHB medium for broth microdilution susceptibility testing of H. parasuis isolates.

MIC values from broth microdilution susceptibility testing of type strain DSM 21448 and three field isolates in two different batches of NADH and two batches of chicken serum showed comparable results after 24 hours of incubation with a maximum deviation of one dilution step.

Test ranges of quality control strains obtained with TMB and supplemented CAMHB broth medium

To investigate the influence of test media on MIC values of QC strains, broth microdilution susceptibility testing of QC strains was performed using TMB and supplemented CAMHB medium. Fivefold repeated susceptibility testing revealed that nearly all MIC values of the CLSI recommended quality control strains E. coli (ATCC 25922), A. pleuropneumoniae (ATCC 27090), S. aureus (ATCC 29213) and E. faecalis (ATCC 29212) were within acceptable ranges in TMB medium (CLINICAL AND LABORATORY STANDARDS INSTITUTE 2013a). The only exceptions for TMB were the combinations A. pleuropneumoniae / gentamicin and S. aureus / tilmicosin. In supplemented CAMHB, all MIC values of the four quality control strains were within the acceptable ranges with the only exception of A. pleuropneumoniae and the antibiotics gentamicin and penicillin.

Homogeneity of H. parasuis MIC values using two different broth media

To assess the homogeneity and the stability of MIC values obtained with supplemented CAMHB and TMB medium with two different incubation times, H. parasuis type strain and two field isolates included in growth curve determinations were used.

Since H. parasuis isolate no. 52 only showed visible growth after 48 hours in supplemented CAMHB, this isolate was not omitted in statistical analysis. The MIC determinations were repeated in five independent experiments. A panel of 21 antimicrobials / antimicrobial combinations was included in the repeated tests and two different incubation times of 16 to 20 hours and 24 hours were analyzed. A comparison of the relative frequencies from MIC mode for the combination of antimicrobial agent, bacterial strain and medium are shown in Figure 1 (16-20 hours incubation time) and Figure 2 (24 hours incubation time). Evaluation was done by taking one model with two variables (precondition of five identical MIC values and MIC mode accepting a deviation of ± one dilution step, respectively) into consideration.

The multi-factorial logistic regression analysis revealed that no influence of the different incubation times on MIC values obtained in both media was observed, neither for the first variable of five identical MIC values (p=1.0000) nor for the second variable of MIC mode

± one dilution step (p=0.4841). While for the first variable, the percentage of homogeneous MIC values was 46.83 % after 16-20 and after 24 hours of incubation, the percentages were 95.24 % after 16 to 20 hours and 96.83 % after 24 hours incubation time for the second variable. By comparing the homogeneity of MIC values obtained in both media, supplemented CAMHB showed 42.06 % compared to 51.59 % homogenous MICs for TMB medium of five identical MIC values, even though no significant difference was seen (p=0.0626). For the MIC mode ± one dilution step, 94.44 % of MIC values showed homogenous MIC for supplemented CAMHB, while 97.62 % of MIC values were

homogenous for TMB medium (p=0.1968). A significant influence on the homogeneity of MIC values for the first variable was seen for the antimicrobial agents (p=0.0118) and the bacterial strains (p=0.0016).

Results from MIC determinations of a larger set of H. parasuis field isolates

Using both media, TMB and supplemented CAMHB, there was no difficulty to determine visible growth of all 47 H. parasuis isolates in the wells of microtiter plates and reading of MIC end points was feasible. Control of inoculum density showed the required inoculum size of 5 x 10⁵ cfu/ml. For isolates, which demonstrated weak growth, the inoculum size should be verified and optionally adjusted. The MIC values of the isolates are presented in Figures 3 and 4.

Considering the results from supplemented CAMHB broth, a bimodal distribution of MICs was detected for the antimicrobial agents ampicillin, ciprofloxacin, colistin, doxycyclin, enrofloxacin, nalidixic acid, penicillin, streptomycin, tetracycline and tilmicosin, whereas a unimodal distribution was determined for the remaining antibiotics tested. However, a broad distribution of MIC values was found for the antibiotic combination trimethoprim/sulfamethoxazole. As there are no CLSI recommended breakpoints for H. parasuis isolates available, a classification of isolates as susceptible or resistant was not possible. However, the majority of isolates proved to have very low

Considering the results from supplemented CAMHB broth, a bimodal distribution of MICs was detected for the antimicrobial agents ampicillin, ciprofloxacin, colistin, doxycyclin, enrofloxacin, nalidixic acid, penicillin, streptomycin, tetracycline and tilmicosin, whereas a unimodal distribution was determined for the remaining antibiotics tested. However, a broad distribution of MIC values was found for the antibiotic combination trimethoprim/sulfamethoxazole. As there are no CLSI recommended breakpoints for H. parasuis isolates available, a classification of isolates as susceptible or resistant was not possible. However, the majority of isolates proved to have very low

Im Dokument Entwicklung von Methoden zur Empfindlichkeitstestung von Bordetella bronchiseptica und Haemophilus parasuis im Bouillon-Mikrodilutionsverfahren (Seite 55-87)