Article | Open | Published:

Rapid detection of tetracycline resistance in bovine Pasteurella multocida isolates by MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA)

Scientific Reportsvolume 8, Article number: 13599 (2018) | Download Citation

Abstract

Pasteurella multocida is notorious for its role as an opportunistic pathogen in infectious bronchopneumonia, the economically most important disease facing cattle industry and leading indication for antimicrobial therapy. To rationalize antimicrobial use, avoiding imprudent use of highly and critically important antimicrobials for human medicine, availability of a rapid antimicrobial susceptibility test is crucial. The objective of the present study was to design a MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) procedure for tetracycline resistance detection in P. multocida. This procedure was validated on 100 clinical isolates with MIC-gradient strip test, and a comparison with disk diffusion was made. Sensitivity and specificity of the MBT-ASTRA procedure were 95.7% (95% confidence interval (CI) = 89.8–101.5) and 100% (95% CI = 100–100), respectively, classifying 98% of the isolates correctly after only three hours of incubation. Sensitivity and specificity of disk diffusion were 93.5% (95% CI = 86.3–100.6) and 96.3% (95% CI = 91.3–101.3) respectively, classifying 95% of the isolates correctly. In conclusion, this MBT-ASTRA procedure has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

Introduction

Pasteurella multocida, a Gram-negative coccobacillus, causes many important diseases in a wide range of hosts1. In different ruminant species and pigs it is one of the most frequently isolated pathogens in infectious bronchopneumonia2,3, a disease which is the leading cause of morbidity and antimicrobial use in these species4. In more tropical regions of the world, especially Africa and Asia, P. multocida can also cause highly fatal hemorrhagic septicemia in cattle and buffaloes5. In other animal species, this bacterium has been associated with presentations like sepsis6, but also otitis7 and peritonitis8. In humans, P. multocida can cause several life-threatening conditions, like sepsis9 and endocarditis10.

Today, to control bronchopneumonia mass medication is still one of the preferred therapies in different food-producing animal species4,11,12. Given the issue of high level antimicrobial resistance in these industries and the one health initiatives founded to combat this13,14,15, preventative antimicrobial use is no longer considered appropriate and antimicrobial treatment should be targeted16. In several European countries like Belgium, Germany or the Netherlands, guidelines suggest and legislation requires sampling and susceptibility testing of animal pathogens before specific antimicrobial agents can be used17,18,19,20,21,22,23. In addition, formularies have been initiated in several countries to guide veterinarians to select appropriate antimicrobials mainly based on their clinical efficacy and importance for human medicine24,25. Especially in more intensive cattle rearing systems which rely on mass medication, antimicrobial multiresistance in P. multocida is common, hampering empiric antimicrobial therapy12,26,27,28. Multiresistant isolates, that compromise 12 antimicrobial resistance genes, are threatening the future of these production systems29.

To date, mainly disk diffusion susceptibility tests are used in veterinary practice to guide antimicrobial therapy. These take a minimum of 2 days after sampling before results are available, but in most cases, due to different practical reasons, a waiting period of 3–4 days is average. In most outbreaks of infectious bronchopneumonia postponing an appropriate therapy, until susceptibility testing results are available, is not acceptable for economic and animal welfare reasons. On the other hand initiation of an inappropriate antibiotic treatment will increase antimicrobial resistance selection pressure30. Disk diffusion susceptibility tests have shown a sensitivity of only 85.7% for the detection of tetracycline resistance in P. multocida compared to the agar dilution technique as the gold standard in the past31, which might result to therapeutic failure in practice. The agar dilution technique is however not commonly used in routine veterinary diagnostics, being a more laborious and expensive technique compared with disk diffusion. Although the antimicrobial gradient strip method is not regarded as the gold standard like the broth dilution technique currently, results achieved by both tests have a good correlation32,33,34. Other advantages of the antimicrobial gradient strip test are the ease of use in combination with reliability and interpretability, resulting in a simple, near gold standard test. Since previous studies regarding new diagnostic techniques use this method for diagnostic accuracy, this was also performed as reference test in the current study.

Guidance of antimicrobial therapy in food animals is urgently needed, especially given the dense populations and potentially high disease incidences in the current production systems. Latter results in exposure of many animals, their bacteria and environment to antimicrobial selection pressure. To support veterinarians in their decision making process for antimicrobial therapy, not only highly accurate, but especially rapid diagnostic procedures are crucial. This to avoid unnecessary production loss, animal suffering or antimicrobial selection pressure.

The MALDI Biotyper antibiotic susceptibility test rapid assay (MBT-ASTRA) method, a MALDI-TOF MS-based approach for susceptibility testing, characterized by a semi-quantitative measurement of bacterial proteins, has shown promising results in obtaining rapid antimicrobial susceptibility results for human pathogens35. For some fast growing bacteria involved in sepsis in humans only 1–4 hours are needed for susceptibility testing35,36,37,38. Also for slow growers like mycobacteria this method seems viable39. Furthermore, this technique shows a high sensitivity and specificity compared to minimum inhibitory concentration (MIC)-gradient strip test35,36,37,38. The potential of MBT-ASTRA for veterinary indications, especially those associated with mass medication, has not been explored. Furthermore applying this method on fastidious growers like P. multocida or any other Pasteurellaceae has not yet been described.

Therefore, the primary objective of the present study was to design and validate an MBT-ASTRA procedure for tetracycline resistance detection in P. multocida from bovine origin. The secondary objective was to determine diagnostic accuracy of this MBT-ASTRA procedure and classic disk diffusion compared to MIC-gradient strip testing.

Results

Determination of standard testing conditions for MBT-ASTRA

To determine the optimal growth medium and starting concentration of the bacterial suspension, one susceptible isolate (P114: MIC: 0.19 µg/mL) was incubated in two different media using three different starting concentrations for incubation times varying between 0 and 6 hours (Fig. 1). This experiment was repeated two times. No difference in growth, expressed by the area under the curve (AUC), was seen between Cation-adjusted Mueller Hinton broth (CAMHB) and Brain Heart Infusion broth (BHIB) (Fig. 1). Starting concentration clearly affected growth, reaching sufficient bacterial growth for MALDI-TOF identification after 6, 2–4 and 0 h with 1.5 × 106 CFU/mL, 1.5 × 107 CFU/mL and 1.5 × 108 CFU/mL, respectively (Fig. 1).

Figure 1
Figure 1

Comparison of CAMHB (A) and BHIB (B) medium and different P. multocida starting concentrations to optimize bacterial growth to allow identification in an MBT-ASTRA procedure. Presented spectra are representative for the repetitions made using a single P. multocida isolate (P114, MIC: 0.19 µg/mL).

Second, the antibiotic concentration allowing a clear separation between resistant and susceptible isolates was determined. To obtain this value, three susceptible (P114; MIC 0.19 µg/mL, P103; MIC 0.25 µg/mL and P113; MIC 0.5 µg/mL) and three resistant (P47; MIC 16 µg/mL, P162; MIC 24 µg/mL and P98; MIC 48 µg/mL) isolates of P. multocida were tested with tetracycline concentrations of 0, 2, 4 and 8 µg/mL and were incubated for 0, 3, 4, 5 and 6 hours. This experiment was performed 3 times independently, each using 1 resistant and 1 susceptible strain. After incubation, the growth derived from an increased protein signal of these isolates was calculated by measuring the AUC of the obtained spectrum. In the susceptible isolates, the AUC of the sample with antibiotic should be significantly reduced in comparison with the sample without antibiotic. For resistant isolates, no significant difference in AUC should be noticed. The relative growth (RG) ratio is the ratio of the AUC derived from spectra with and without antibiotic and is used to distinguish susceptible from resistant isolates according to an empirical cut-off value. A clear visual difference between susceptible and resistant isolates was achieved at a tetracycline concentration of 4 µg/mL and a RG cut-off value of 0.5 (Fig. 2). In this preliminary test, the three resistant isolates showed a RG > 0.5, while the susceptibile isolates showed a RG < 0.5. A correct classification of all strains was already possible after 3 hours of incubation with 4 µg/mL tetracycline (Fig. 2).

Figure 2
Figure 2

Area under the curve (AUC) (A) and Relative Growth (RG) (B) box plots of 3 susceptible and 3 resistant P. multocida isolates after 3 hours of incubation without antibiotic (=ZER) or with 4 µg/mL of tetracycline (=FOU). For resistant strains, no clear difference between both AUCs is noticed and RGs are high, whereas susceptible strains obtain a lower RG. The horizontal red line represents a RG cut-off value of 0.5.

Determination of the diagnostic accuracy of the MBT-ASTRA procedure and comparison with the disk diffusion method

MIC distribution of the study population

A total of 100 clinical P. multocida isolates were used to validate the MBT-ASTRA with the MIC-gradient strip test as reference test used in the current study. Of the isolates tested, 54 and 46 were classified as susceptible or resistant by MIC-gradient strip test, respectively. This experiment was conducted once. Tetracycline MIC-values for the quality control reference strains were tested twice and were in the acceptable range according to CLSI standards40, i.e. Staphylococcus aureus ATCC 29213 MIC-value 0.38 µg/mL and 0.5 µg/mL (range 0.12–1 µg/mL), Escherichia coli ATCC 25922 MIC-value 0.5 µg/mL and 1.5 µg/mL (range 0.5–2 µg/mL) and Enterococcus faecalis ATCC 29212 MIC-value 16 µg/mL (range 8–32 µg/mL). The tested isolates showed a bimodal MIC distribution, ranging between 0.094 and 48 µg/mL (Fig. 3). According to veterinary CLSI standards40, P. multocida isolates are considered responsive to treatment (=susceptible) with tetracycline in cattle when they show an MIC value ≤ 2 µg/mL.

Figure 3
Figure 3

MIC values for tetracycline of 100 recent clinical isolates of P. multocida used to determine diagnostic accuracy of an MBT-ASTRA procedure for this antimicrobial-bacterium combination. MIC values were determined by the MIC-gradient strip test. The vertical line represents the CLSI clinical breakpoint for susceptibility (≤2 µg/mL)40.

Diagnostic accuracy of MBT-ASTRA and disk diffusion

The MBT-ASTRA procedure was conducted for 100 clinical isolates with an incubation time of 3 hours, with and without a tetracycline concentration of 4 µg/mL in CAMHB. This test was performed once. Relative growth values were determined for all tested isolates. Receiver operating characteristics (ROC) curve analysis showed a relative growth value of 0.5 to be the optimal cut-off to differentiate resistant from susceptible isolates for this MBT-ASTRA method (Fig. 4).

Figure 4
Figure 4

Scatter plot of minimum inhibitory concentrations (MIC) obtained with the MIC-gradient strip test with MBT-ASTRA relative growth (RG) values of 100 recent bovine clinical strains of P. multocida. MBT-ASTRA testing conditions were 3 hours of incubation with a concentration of tetracycline of 4 µg/mL. The horizontal and vertical line represent the clinical breakpoint of ≤2 µg/mL and the RG cut-off value of 0.5, respectively. Two isolates with an MIC value of 6 µg/mL are considered susceptible with MBT-ASTRA, causing 2 false susceptible results.

For the disk diffusion tests, quality control reference strains were included and inhibition zones (in mm) were in the acceptable range according to CLSI standards40, i.e. Staphylococcus aureus ATCC 25923 inhibition zone 24 mm (range 24–30 mm) and Escherichia coli ATCC 25922 inhibition zone 22 mm (range 18–25 mm). The disk diffusion method with quality control reference strains included was performed once.

Table 1 shows classification of the test results of both MBT-ASTRA and disk diffusion compared to the MIC-gradient strip test. At a RG cut-off value of 0.5, a correct classification of all 54 susceptible strains and 95.7% (44/46) of resistant strains was achieved. All susceptible isolates showed RG values smaller than 0.5. All resistant isolates had RG values above 0.5, except two isolates (P33, P106) with an MIC value of 6 µg/mL that showed a RG value of 0.34 and 0.25 respectively, causing 2 false susceptible results (Fig. 4).

Table 1 2 × 2 Contingency table showing tetracycline resistance testing results of MBT-ASTRA and disk diffusion compared to MIC-gradient strip test for 100 recent clinical P. multocida isolates derived from cattle.

Diagnostic accuracy of the disk diffusion and MBT-ASTRA method was calculated using the MIC-gradient strip test as reference (Table 2). Compared to MIC-gradient strip testing, the MBT-ASTRA method achieved a sensitivity and specificity of 95.7% and 100%, respectively. Sensitivity and specificity of disk diffusion were 93.5% and 96.3%, respectively. The essential agreement of MBT-ASTRA with the MIC-gradient strip test is 98%, due to 2% very major errors (Table 2). In contrast, the disk diffusion method obtained an essential agreement of 95%, including 3% very major errors and 2% major errors (Table 2).

Table 2 Diagnostic accuracy of disk diffusion and MBT-ASTRA for tetracycline susceptibility testing in 100 clinical P. multocida isolates from cattle compared to the MIC-gradient strip test.

Discussion

This study aimed at developing a MALDI-TOF MS-based approach for rapid tetracycline susceptibility testing in P. multocida and to compare this technique and the current standard test in practice (disk diffusion) with the MIC-gradient strip test. The most widely accepted gold standard for susceptibility testing in bacteria is the broth dilution technique41,42. In order to compare the obtained data with previous studies, which commonly used the MIC-gradient strip test, the authors also opted for MIC-gradient strip testing35,36,37,38. MIC-gradient strip test results have a good correlation with MIC values achieved by broth dilution technique, and is often considered as a near gold standard test32,33,34. Therefore, the authors believe that current results based on the MIC-gradient strip test will hardly differ from broth dilution results. Further research can include latter technique as gold standard for determining the accuracy of the MBT-ASTRA method.

Another limitation was that no intermediate results were obtained from neither techniques. In practice, to avoid therapeutic failure, intermediate results are often regarded as resistant, and another antimicrobial is selected. As the MBT-ASTRA method is currently unable to correctly classify intermediate susceptibility isolates for a given antibiotic, including isolates with intermediate results on the reference test would have been an added value. Unfortunately, no isolates classified as intermediate by MIC-gradient strip test were obtained in this study. Also in previous work, this has not been accounted for36,37. Modifications to this method that would allow classification of intermediate include correlations between the relative growth and MIC value and by using a serial dilution of the given antibiotic to define an MBT-ASTRA minimal breakpoint37. However, classification as intermediate is contra productive for clinical decision making and may lead to more inappropriate treatments.

The main conclusion of the present study is that the MBT-ASTRA method allows for a tetracycline susceptibility testing result for P. multocida in only three hours of incubation with high accuracy. This incubation time is in the same range compared to the MBT-ASTRA method for fast growing bacteria involved in sepsis in humans35,36,37,38. Although P. multocida is considered a fastidiously growing microorganism by CLSI40 and tetracycline is considered bacteriostatic, which might cause a longer incubation time to obtain a clear difference in AUC with or without antibiotic. Due to this short incubation period, the MBT-ASTRA method makes it possible to provide the results of the susceptibility test on the same day of identification, avoiding unnecessary production loss or animal suffering. Sensitivity (95.7%) and specificity (100%) of the presented MBT-ASTRA procedure were excessive, in the range of and even higher than compared to previous findings applying MBT-ASTRA for other species under different conditions35,36,37,38. In contrast, the disk diffusion method evaluated in this study had lower sensitivity (93.5%) and specificity (96.3%). A previous study on P. multocida isolates showed lower sensitivity (85.7%) and higher specificity (99.1%) of disk diffusion for tetracycline resistance detection31. A possible explanation for this difference could be that at that time disks with higher tetracycline contents (80 µg) were used and clinical breakpoints differed with those obtained in the current study. Classification of the 100 isolates with disk diffusion was performed using the interpretative criteria of the manufacturer’s guidelines. Currently, no P. multocida-specific clinical breakpoints for tetracycline in cattle are available for disk diffusion, while they are described for MIC testing40. Zone diameters (mm) were measured and analysed. A clear difference between susceptible (zone diameter ranging from 24–32 mm) and resistant (zone diameter ranging from 8–10 mm) isolates was noticed. Due to these considerable differences in zone diameter, modifying the clinical breakpoints would have little to no effect on the results.

Compared to the disk diffusion method as used under the current circumstances, MBT-ASTRA shows less discrepancies to the MIC-gradient strip test method. However, in 2% of the isolates tested a very major error was present, potentially resulting in treatment of the animal with tetracycline when the isolate is resistant. Both of these isolates had an MIC value of 6 µg/mL. According to CLSI standards, an MIC value of 4 µg/mL is classified as intermediate and an MIC value ≥ 8 µg/mL is classified as resistant. Considering these clinical breakpoints, both strains with an MIC value of 6 µg/mL can be classified as (borderline) resistant, resulting in 2% very major errors and a sensitivity of 95.7% with the MBT-ASTRA method. However, the clinical outcome of treatment with tetracycline of P. multocida isolates with an MIC value of 6 µg/mL might be difficult to distinctly predict.

Given its rapid results and high diagnostic accuracy, this MBT-ASTRA technique has high potential to be used in the field as a decision aid to direct antimicrobial use in food animals. In order to fulfil this potential, the possibilities of obtaining a full susceptibility test (as many antimicrobials tested at once as in disk diffusion) need to be explored, and the practical issue of availability of a MALDI-TOF MS in the regional laboratories needs to be overcome. However, in recent years the use of MALDI-TOF MS has been advanced to a standard method in clinical microbiology laboratories, due to its rapid and low-cost characteristics43. Considering the simple setup and short incubation time, the MBT-ASTRA method implements an early intervention of adequate antibiotic therapy, resulting in a cost-and –labour efficient tool like the disk diffusion method. Whether the current MBT-ASTRA settings are also applicable to other Pasteurellaceae of clinical importance in veterinary medicine, like Mannheimia haemolytica or Histophilus somni, needs to be determined.

In conclusion, the MBT-ASTRA method developed in the present study, has all the potential to fulfil the need for a rapid and highly accurate tetracycline susceptibility testing in P. multocida. This is essential to rationalize antimicrobial use in outbreaks of bronchopneumonia in cattle or other clinical presentations across species.

Methods

Determination of the standard conditions for MBT-ASTRA

Selection of the optimal growth medium and starting concentration of P. multocida

BHIB (Difco, BD Diagnostic Systems, Sparks, Md.) and CAMHB (Difco, BD Diagnostic Systems, Sparks, Md.) were used as growth media in this study. One susceptible strain of P. multocida (P114, MIC 0.19 µg/mL) was inoculated in 10 mL of BHIB or 10 mL of CAMHB at different starting concentrations. Starting concentrations tested in both media were 1.5 × 106 CFU/mL, 1.5 × 107 CFU/mL and 1.5 × 108 CFU/mL. No antibiotics were added to the medium. All tubes were placed in a shaking incubator for 0, 1, 2, 3, 4 and 6 hours at a temperature of 37 °C and an atmosphere enriched with 5% CO2. After each incubation period, 1 mL samples of each tube were transferred to Eppendorf tubes.

Selection of the optimal tetracycline concentration and incubation time

To determine the optimal concentration of tetracycline and the shortest incubation time necessary to differentiate between susceptible and resistant isolates, three susceptible isolates (P114; MIC 0.19 µg/mL, P103; MIC 0.25 µg/mL, P113; MIC 0.5 µg/mL) and three resistant isolates (P47; MIC 16 µg/mL, P162; MIC 24 µg/mL and P98; MIC 48 µg/mL) were used. Tetracycline concentrations of 0, 2, 4 and 8 µg/mL were tested (Sigma-Aldrich, Germany) at incubation periods of 0–3–4–5 and 6 hours.

MBT-ASTRA sample preparation

Eppendorf tubes, containing 1 mL samples grown for the indicated incubation time, were centrifuged at 21130 × g for 5 minutes at room temperature. After centrifugation, the supernatant was carefully aspirated and 700 µL of 70% ethanol in high performance liquid chromatography (HPLC) graded water was added to the cell pellet and vortexed. A second centrifugation and aspiration of the supernatant was performed as mentioned above. After air drying for a minimum of 10 minutes, the cell pellets were stored at −20 °C for up to a maximum of 3 days. After storing, 20 µL 70% formic acid (in HPLC graded water) was added to the cell pellet and mixed carefully. Samples were incubated for five minutes at room temperature. In a last step, 20 µL of acetonitrile was added and vortexed. Before adding acetonitrile to the cell pellet, an internal standard35 (Bruker Daltonik GmbH, Bremen, Germany, suspended in 25 µL HPLC water) was added to acetonitrile (ratio of 1/100, using 0.2 µL of internal standard in 20 µL of acetonitrile per sample) for spectra acquisition and facilitating the semi-quantitative analysis of the acquired spectra to quantify the difference in biomass corresponding to the growth between different setups. A third centrifugation step (21130 × g for 2 minutes) was performed to clarify the lysates.

MALDI-TOF MS analysis

One µL of the protein extraction was spotted in triplicate on the target plate (MSP 96 target polished steel BC). After air drying of the spots, 1 µl of matrix (10 mg/ml of α-cyano-4-hydroxy-cinnamic acid [α-HCCA] in 50% acetonitrile - 47.5% water - 2.5% trifluoroacetic acid; Bruker Daltonik GmbH, Bremen, Germany) was placed on each spot. External calibration was included in each measurement using a bacterial test standard (BTS, Bruker Daltonik GmbH, Bremen, Germany). Analysis was performed with an Autoflex III smartbeam MALDI-TOF MS instrument (Bruker Daltonik GmbH, Bremen, Germany), recording the mass range between 2.000–20.000 Da using standard settings. Automated data analysis was performed with the MBT-ASTRA software prototype written in the software package R35. A mass range of 2.500–13.500 was used for analysis. AUC and relative growth rate were calculated automatically for each setup. The duration of MALDI-TOF analysis is around 10–15 minutes for one sample. This duration is not cumulative when analysing multiple samples.

The obtained optimal test conditions were subsequently used during assay validation, analysing 100 recent clinical P. multocida isolates to determine the cut-off value of the relative growth ratio that allows a differentiation between susceptible and resistant isolates.

Determination of diagnostic accuracy of the MBT-ASTRA procedure and comparison with the disk diffusion method

Bacterial isolates and cultivation

In this experiment, 100 isolates of P. multocida from calves with bronchopneumonia collected in Belgium between 2014 and 2017 were analysed. Sampling of these calves was performed using a deep nasopharyngeal swab or a broncho-alveolar lavage as previously described44. All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University (EC 2014/164, EC 2016/20). Identification of P. multocida isolates was first achieved by standard biochemical tests45 and subsequently confirmed with MALDI-TOF MS using the direct transfer protocol as previously described46. Bacteria were cultivated overnight at 37 °C and an atmosphere enriched with 5% CO2 on Columbia agar (Oxoïd, UK) supplemented with 5% sheep blood.

Three susceptibility testing methods were performed on all isolates, namely MIC-gradient strip test, which is comparable to the standard dilution test32,33,34, the MBT-ASTRA procedure as described above and disk diffusion as the current standard test in practice.

MIC-gradient strip test

The MIC values of tetracycline were determined using MIC-gradient strips. Briefly, MIC test strips (Liofilchem, Italy) were placed on Mueller Hinton agar plates enriched with 5% defibrinated sheep blood (BD Diagnostics Systems), shortly after the plates had been uniformly inoculated via polyester swabs with 0.5 McFarland suspensions of the pure isolates. Incubation was performed for 18–24 hours under aerobic conditions at 35 °C. Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 were used as quality control reference strains. MIC values were determined according to the manufacturer’s instructions. Determination of susceptibility was performed according to the current CLSI standards for bovine P. multocida (i.e. susceptible ≤2 µg/mL, intermediate 4 µg/mL, resistant ≥8 µg/mL)40. Used media, incubation conditions, quality control strains and used interpretive criteria were applied according to CLSI standards40. Inoculation method was performed according to manufacturer’s guidelines.

MBT-ASTRA procedure

All isolates were incubated for 3 hours with and without a tetracycline concentration of 4 µg/mL in CAMHB with a starting concentration of 1.5 × 107CFU/mL. MBT-ASTRA sample preparation and MALDI-TOF MS analysis were performed immediately after incubation as mentioned above.

Disk diffusion test

Susceptibility testing of P. multocida and tetracycline was performed in accordance with CLSI standards40. Briefly, Mueller Hinton agar (Oxoïd, UK) plates, supplemented with 5% sheep blood, were uniformly inoculated via polyester swabs with 0.5 McFarland suspensions of the pure isolates. Then, tetracycline disks (30 µg/mL, Rosco, Neosensitabs, Taarstrup, Denmark) were placed on Mueller Hinton agar (Oxoïd, UK) supplemented with 5% sheep blood, shortly after the plates had been uniformly inoculated via polyester swabs with 0.5 McFarland suspensions of the pure isolates. Incubation was performed for 18–24 hours at 35 °C in ambient air. Analysis of inhibition zones (in mm) was performed and interpretative criteria were according to manufacturer’s guidelines. Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 were used as quality control reference strains.

Statistical analysis

Receiver operating characteristics (ROC) curve analysis was used to determine the optimal cut-off of the relative growth ratio to distinguish resistant from susceptible isolates in the MBT-ASTRA procedure (SPSS statistics vs. 24 (IBM, New York, United States)). Performance of the MBT-ASTRA and disk diffusion method compared to the MIC-gradient strip test as the gold standard was determined. Results were presented by distinguishing very major (resistant strain by the MIC-gradient strip test method misinterpreted as susceptible by the disk diffusion/MBT-ASTRA method), major (susceptible strain by the MIC-gradient strip test method misinterpreted as resistant by the disk diffusion/MBT-ASTRA method) and minor errors (intermediate result was obtained by only one method)31,47,48. Diagnostic accuracy, sensitivity, specificity, RPV and SPV were derived from 2 × 2 contingency tables using Winepiscope 2.049.

Availability of Data and Materials

The authors declare that all information obtained from this study is presented in this paper.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Quinn, P. J., Carter, M. E. & Markey, B. Pasteurella species, In: Quin, ed. Clinical Veterinary Microbiology. Mosby International Limited, 254–258 (1994).

  2. 2.

    Woolums, A. R. The bronchopneumonias (respiratory disease complex of cattle, sheep, and goats). In: Smith BL, ed. Large Animal Internal Medicine. 5th ed. St Louis, MO: Mosby Elsevier, 584–603 (2015).

  3. 3.

    Boyce, J. D., Seemann, T., Adler, B. & Harper, M. Pathogenomics of Pasteurella multocida. Current Topics in Microbiology and Immunology 361, 23–38, https://doi.org/10.1007/82-2012-203 (2012).

  4. 4.

    Pardon, B. et al. Prospective study on quantitative and qualitative antimicrobial and anti-inflammatory drug use in white veal calves. Journal of Antimicrobial Chemotherapy 67, 1027–1038, https://doi.org/10.1093/jac/dkr570 (2012).

  5. 5.

    Carter, G. R. & De Alwis, M. C. L. Haemorrhagic septicaemia. In: Adlam,, C. and Rutter, J.M. eds. Pasteurella and Pasteurellosis. Academic Press, London, 131–160 (1989).

  6. 6.

    Malhi, P. S., Ngeleka, M. & Woodbury, M. R. Septicemic pasteurellosis in farmed elk (Cervus canadensis) in Alberta. Canadian Veterinary Journal 57, 961–963, PMCID: PMC4982567 (2016).

  7. 7.

    Jensen, R. et al. Cause and pathogenesis of middle ear infection in young feedlot cattle. Journal of the American Veterinary Medical Association 182, 967–972 (1983).

  8. 8.

    Catry, B. et al. Fatal peritonitis caused by Pasteurella multocida capsular type F in calves. Journal of Clinical Microbiology 43, 1480–1483, https://doi.org/10.1128/JCM.43.3.1480-1483.2005 (2005).

  9. 9.

    Konda, M. K., Chang, S. & Zaccheo, M. Purpura fulminans and severe sepsis due to Pasteurella multocida infection in an immunocompetent patient. BMJ Case Reports, https://doi.org/10.1136/bcr-2016-214891 (2016).

  10. 10.

    Yuji, D., Tanaka, M., Katayama & Kenichirou Noguchi, I. Pasteurella multocida infective endocarditis. The Journal of Heart Valve Disease 24, 778–779 (2015).

  11. 11.

    Timmerman, T. et al. Quantification and evaluation of antimicrobial drug use in group treatments for fattening pigs in Belgium. Preventive Veterinary Medicine 74, 251–263, https://doi.org/10.1016/j.prevetmed.2005.10.003 (2006).

  12. 12.

    Persoons, D., Dewulf, J. & Smet, A. et al. Antimicrobial use in Belgian broiler production and influencing factors. In: Persoons D., Antimicrobial use and resistance in Belgian broiler production. PhD Thesis. Ghent University, 2011. ISBN 978-90-5864-246-2.

  13. 13.

    Schrijver, R. et al. Review of antimicrobial resistance surveillance programmes in livestock and meat in EU with focus on humans. Clinical microbiology and infection 24, 577–590, https://doi.org/10.1016/j.cmi.2017.09.013 (2018).

  14. 14.

    Mukerji, S. et al. Development and transmission of antimicrobial resistance among Gram-negative bacteria in animals and their public health impact. Essays Biochemistry 61, 23–35, https://doi.org/10.1042/EBC20160055 (2017).

  15. 15.

    Chantziaras, I., Boyen, F., Callens, B. & Dewulf, J. Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. Journal of Antimicrobial Chemotherapy 69, 827–834, https://doi.org/10.1093/jac/dkt443 (2013).

  16. 16.

    WHO guidelines on use of medically important antimicrobials in food-producing animals. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO. http://www.who.int/foodsafety/publications/cia_guidelines/en (2017).

  17. 17.

    RD 21 July 2016, Filip, De Block M., Borsus W. Koninklijk besluit betreffende de voorwaarden voor het gebruik van geneesmiddelen door de dierenartsen en door de verantwoordelijken van de dieren.

  18. 18.

    FDA 2000. FDA Task Force on Antimicrobial Resistance: key recommendations and report, Washington, DC. FDA, Washington, DC: http://www.fda.gov/downloads/ForConsumers/ConsumerUpdates/UCM143458.pdf (2000).

  19. 19.

    Chantziaras, I., Boyen, F., Callens, B. & Dewulf, J. Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. Journal of Antimicrobial Chemotherapy 69, 827–834, https://doi.org/10.1093/jac/dkt443 (2014).

  20. 20.

    Avorn, J. L., et al Antibiotic resistance: synthesis of recommendations by expert policy groups. Alliance for the prudent use of antibiotics. World Health organization (WHO/CDS/CSR/DRS/2001.10) (2001).

  21. 21.

    No authors listed. Calls to restrict veterinary use of antimicrobials. Veterinary Record 171, 10.1136/vr.e7755 (2012).

  22. 22.

    Fairles, J. The Veterinarian’s role in antimicrobial stewardship. The Canadian Veterinary Journal 54, 207–210 (2013).

  23. 23.

    https://www.jpiamr.eu/wp-content/uploads/2017/11/Report-_JPIAMR-workshop-on-Environmental-dimensions-of-AMR-Gothenburg-Sweden-2017_FINAL.pdf (2017)

  24. 24.

    Antimicrobial Consumption and Resistance in Animals (AMCRA) 2020. Een ambitieus maar realistisch plan voor het antibioticumbeleid bij dieren tot en met 2020. Retrieved from https//www.amcra.be/sites/default/files/bestanden/AMCRA%202020%20finaal/NL%20-%20definitief_0.pdf. (2015).

  25. 25.

    de Greeff, S. C. & Mouton, J. W. Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands. Lelystad, The Netherlands; 2017. Available at: https://www.rivm.nl/dsresource?objectid=f9799644-beb0-405b-b35f-c3db66dc153b&type=pdf&disposition=inline (2017).

  26. 26.

    Catry, B. et al. Detection of tetracycline-resistant and susceptible Pasteurellaceae in the nasopharynx of loose group-housed calves. Veterinary Research Communication 30, 707–715 (2006).

  27. 27.

    Catry, B. et al. Variability in acquired resistance of Pasteurella and Mannheimia isolates from the nasopharynx of calves, with particular reference to different herd types. Microbial Drug Resistance 11, 387–94, https://doi.org/10.1089/mdr.2005.11.387 (2005).

  28. 28.

    Hendriksen, R. S., et al Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002–2004. Acta Veterinaria Scandinavia 50, https://doi.org/10.1186/1751-0147-50-28 (2008).

  29. 29.

    Michael, G. B. et al. ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: analysis of the regions that comprise 12 antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy 67, 84–90, https://doi.org/10.1093/jac/dkr406 (2012).

  30. 30.

    Marshall, B. M. & Levy, S. B. Food animals and antimicrobials: impacts on human health. Clinical Microbiology Reviews 24, 718–733, https://doi.org/10.1128/CMR.00002-11 (2011).

  31. 31.

    Catry, B., Dewulf, J. & de Kruif, A. Accuracy of susceptibility testing of Pasteurella multocida and Mannheimia haemolytica. Microbial Drug Resistance 13, 204–211, https://doi.org/10.1089/mdr.2007.721 (2007).

  32. 32.

    Huang, M. B., Baker, C. N., Banerjee, S. & Tenover, F. C. Accuracy of the E test for determining antimicrobial susceptibilities of staphylococci, enterococci, campylobacter jejuni, and gram-negative bacteria resistant to antimicrobial agents. Journal of Clinical Microbiology 30, 3243–3248 (1992).

  33. 33.

    Baker, C. N., Stocker, S. A., Culver, D. M. & Thornsberry, C. Comparison of the E-test to agar dilution, broth microdilution, and agar diffusion susceptibility testing techniques by using a special challenge set of bacteria. Journal of Clinical Microbiology 29, 533–538 (1991).

  34. 34.

    Rennie, R. P., Turnbull, L., Brosnikoff, C. & Cloke, J. First comprehensive evaluation of the MIC evaluator device compared to Etest and CLSI reference dilution methods for antimicrobial susceptibility testing of clinical strains of anaerobes and other fastidious bacterial species. Journal of Clinical Microbiology 50, 1153–1157, https://doi.org/10.1128/JCM.05397-11 (2012).

  35. 35.

    Lange, C., Schubert, S. & Jung, J. Quantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry for rapid resistance detection. Journal of Clinical Microbiology 52, 4155–4162, https://doi.org/10.1128/JCM.01872-14 (2014).

  36. 36.

    Sparbier, K., Schubert, S. & Kostrzewa, M. MBT-ASTRA: A suitable tool for fast antibiotic susceptibility testing? Methods 104, 48–54, https://doi.org/10.1016/j.ymeth.2016.01.008 (2016).

  37. 37.

    Jung, J., Hamacher, C. & Gross, B. Evaluation of a semiquantitative matrix-assisted laser desorption ionization-time of flight mass spectrometry method for rapid antimicrobial susceptibility testing of positive blood cultures. Journal of Clinical Microbiology 54, 2820–2824, https://doi.org/10.1128/JCM.01131-16 (2016).

  38. 38.

    Maxson, T., Taylor-Howell, C. L. & Minogue, T. D. Semi-quantitative MALDI-TOF for antimicrobial susceptibility testing in Staphylococcus aureus. PLoS ONE 12, https://doi.org/10.1371/journal.pone.0.183899 (2017).

  39. 39.

    Ceyssens, P.-J., Soetaert, K. & Timke, M. Matrix-assisted laser desorption ionization-time of flight mass spectrometry for combined species identification and drug sensitivity testing in mycobacteria. Journal of Clinical Microbiology 55, 624–634, https://doi.org/10.1128/JCM.02089.16 (2017).

  40. 40.

    CLSI. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; Approved Standard-Fifth Edition. CLSI document VET01-S3. Wayne, PA: Clinical and Laboratory Standards Institute (2015).

  41. 41.

    Jorgensen, J. H. & Ferraro, M. J. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Medical microbiology 49, 1749–1755, https://doi.org/10.1086/647952 (2009).

  42. 42.

    Hubert, S. K., Nguyen, P. D. & Walker, R. D. Evaluation of a computerized antimicrobial susceptibility system with bacteria isolated from animals. Journal of Veterinary Diagnostic Investigation 10, 164–168, https://doi.org/10.1177/104063879801000208 (1998).

  43. 43.

    Seng, P. et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of flight mass spectrometry. Clinical Infectious Diseases 49, 543–551, https://doi.org/10.1086/600885 (2009).

  44. 44.

    Van Driessche, L., Valgaeren, B. & De Schutter, P. Effect of sedation on the intrapulmonary position of a bronchoalveolar lavage catheter in calves. Veterinary Record 179, https://doi.org/10.1136/vr.103676 (2016).

  45. 45.

    Quinn, P. J. et al. Antimicrobial agents. In: Quinn, ed. Clinical Veterinary Microbiology. Mosby International Limited, 1994, 254–258.

  46. 46.

    Kuhnert, P., Bisgaard, M. & Korczak, B. M. Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry. Journal of Microbiological Methods 89, 1–7, https://doi.org/10.1016/j.mimet.2012.02.001 (2012).

  47. 47.

    Breteler, K. B., Rentenaar, R. J., Verkaart, G. & Sturm, P. D. Performance and clinical significance of direct antimicrobial susceptibility testing on urine from hospitalized patients. Scandinavian Journal of Infectious patients 43, 771–776, https://doi.org/10.3109/00365548.2011.588609 (2011).

  48. 48.

    Rhodes, N. J. et al. Unacceptably high error rates in Vitek 2 testing of cefepime susceptibility in Extended-Spectrum-β-Lactamase-producing Escherichia coli. Antimicrobial Agents and Chemotherapy 58, 3757–3761, https://doi.org/10.1128/AAC.00041-14 (2014).

  49. 49.

    Thrusfield, M., Ortega, C., de Blas, I., Noordhuizen, J. P. & Frankena, K. WIN EPISCOPE 2.0: improved epidemiological software for veterinary medicine. Veterinary Record 148, 567–572, https://doi.org/10.1136/vr.148.18.567 (2001).

Download references

Acknowledgements

This study was funded by a PhD Fellowship of the Research Foundation - Flanders (FWO- 1S52616N). The MALDI-TOF mass spectrometer was financed by the Research Foundation Flanders (FWO-Vlaanderen) as Hercules project G0H2516N (AUGE/15/05).We thank Bruker Daltonik GmbH for providing internal standard.

Author information

Author notes

  1. Filip Boyen and Bart Pardon jointly supervised this work.

Affiliations

  1. Department of Large Animal Internal Medicine, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium

    • Laura Van Driessche
    • , Jade Bokma
    • , Linde Gille
    • , Piet Deprez
    •  & Bart Pardon
  2. National Reference Center for Tuberculosis and Mycobacteria, Scientific Institute of Public Health (WIV-ISP), Juliette Wytsmanstraat 14, 1050, Elsene, Belgium

    • Pieter-Jan Ceyssens
  3. Bruker Daltonik GmbH, Fahrenheitstr. 4, 28359, Bremen, Germany

    • Katrin Sparbier
  4. Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820, Merelbeke, Belgium

    • Freddy Haesebrouck
    •  & Filip Boyen

Authors

  1. Search for Laura Van Driessche in:

  2. Search for Jade Bokma in:

  3. Search for Linde Gille in:

  4. Search for Pieter-Jan Ceyssens in:

  5. Search for Katrin Sparbier in:

  6. Search for Freddy Haesebrouck in:

  7. Search for Piet Deprez in:

  8. Search for Filip Boyen in:

  9. Search for Bart Pardon in:

Contributions

L.V.D. designed and conducted all experiments, collected literature data, analysed data, prepared figures and prepared the paper. J.B. conducted the validation study. P.-J.C. and K.S. supervised the work and provided insights in MBT-ASTRA sample preparation and MALDI-TOF MS analysis. P.D. supervised the work. F.B. and B.P. designed the study, analysed data, prepared figures and supervised the work. All authors read and reviewed the final manuscript.

Competing Interests

The authors declare no competing interests.

Corresponding author

Correspondence to Laura Van Driessche.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41598-018-31562-8

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.