Innovations in rapid Mycoplasma bovis diagnostics with MALDI-TOF MS and nanopore sequencing

Mycoplasma bovis is a leading, primary cause of pneumonia, arthritis, otitis and mastitis in cattle, resulting in impaired animal welfare and huge economic losses in all cattle sectors worldwide. This small bacterium lost its cell wall and several physiological mechanisms through evolution, whereupon it acquired inherent resistance against many conventional antimicrobials (e.g. penicillines, cephalosporines, sulfonamides, ..). Next to this natural resistance, M. bovis may acquire resistance against other antimicrobials as well. Currently, isolation and identification of M. bovis by culture takes 1-2 weeks, and subsequent antimicrobial susceptibility testing is currently not performed in routine diagnostics. No standard protocol is available and the lack of clinical breakpoints limits the translation of in vitro results to clinical outcome predictions of antimicrobial treatment. At a higher price, faster identification is possible with PCR (2 days). Although diagnostic accuracy of PCR is expected to be higher than culture, scientific information on this matter is limited. To be able to control M. bovis and start appropriate antimicrobial treatment immediately, there is a great need for rapid and reliable diagnostic tools for this pathogen. However, next to control, prevention of M. bovis spreading into/within the herd is also very important. How M. bovis is exactly transmitted, and whether there are specific M. bovis strains associated with antimicrobial resistance or sectors, has not been elucidated yet. Key factors for successful control and prevention are the formulation of specific biosecurity protocols and guidelines targeted to M. bovis. To achieve this, a rapid diagnosis of infected or carrier animals and better insights into the spread of M. bovis between herds, sectors, and countries are needed. Therefore, the general aim of this thesis was to develop new Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and nanopore sequencing based diagnostic methods for rapid identification, strain typing, and antimicrobial susceptibility testing of M. bovis, and to apply those methods on Belgian field samples, gaining better insight into the epidemiology of M. bovis. In the general introduction (Chapter 1) a literature overview is provided, presenting current state-of-the-art on disease course, risk factors and treatment of M. bovis. Subsequently, the many different diagnostic techniques available for identification, strain typing, and susceptibility testing are described. Next to the existing techniques, more innovative techniques, such as MALDI-TOF MS and nanopore sequencing and their potential as rapid diagnostic methods are explained. In the first experimental study different methods were explored to identify M. bovis cultures grown on solid medium with MALDI-TOF MS (Chapter 3.1). The most straight-forward method, being the direct transfer method, is broadly applied for most bacteria, but faced several problems for M. bovis identification. In this study, these problems were better identified and it was shown that medium-related peaks (mostly obtained from horse serum and colistin) can result in false positive Mycoplasma alkalescens and Mycoplasma arginini identification. Unfortunately, it was not possible to obtain a more reliable direct transfer protocol. Therefore, in Chapter 3.2 the identification of M. bovis with MALDI-TOF MS from liquid medium was further explored and optimized. Here it was shown that identification was possible within 24 hours after inoculation of one colony from a solid medium into liquid medium. Supplementing pleuropneumonia-like organism broth (PPLO-broth) with pyruvate prolonged the possibility of M. bovis identification to at least 120 hours after inoculation. Also, supplementation with antimicrobials prevented overgrowth with other bacteria, and did not influence the identification score. Although with the previous two methods, a step towards more rapid identification of M. bovis was set, prior isolation of M. bovis from any sample is still necessary and could easily take 5-10 days. Therefore, methods to identify M. bovis directly from bronchoalveolar lavage fluid (BALf) with MALDI-TOF MS (Chapter 3.3) and nanopore sequencing (Chapter 3.4) were developed and validated in a Bayesian latent class model on 104 and 100 BALf from calves, respectively. It was possible to identify M. bovis with MALDI-TOF MS within 2-3 days with a sensitivity and specificity of 86.6% (CI95%: 69.4-97.6%) and 86.4% (76.1-93.8%), respectively. While sensitivity and specificity of nanopore sequencing were 77.4% (58.6-92.3%) and 97.3% (91.1-99.7%), respectively. Also when 5 BALf were pooled, both methods were still reliable, and therefore very cost-effective possibilities. In addition, the in Belgium widely used selective-indicative agar method based on lipase-activity, which was never validated before using a large number of field samples, showed a sensitivity of 70.5% (52.1-87.1%) and specificity of 93.9% (85.9-98.4). All three methods are useful in routine laboratories, depending on the diagnostic needs of the applicant. Currently the prevalence of M. bovis in the Belgian dairy and beef sector is estimated at 30%, whereas 100% of the veal calf herds tested positive. Together with the high antimicrobial use in the veal sector, the question has been raised whether there is a possible reservoir of multi-resistant and sector-specific M. bovis strains in this sector, as previously shown for other respiratory bacteria. To better understand the molecular epidemiology and genetic relatedness of different M. bovis isolates, the full genome of 100 Belgian M. bovis isolates collected from dairy, beef and veal herds was obtained using nanopore sequencing (Chapter 4). A single nucleotide polymorphism (SNP) analysis was performed and the phylogenetic tree showed five separate genomic clusters of M. bovis isolates and one outlier circulating in Belgium between 2014 and 2019. No sector-specific isolates and no association with spatial location in Belgium were identified. At world-scale, the Belgian M. bovis isolates clustered together with European, American and Israeli strains. These results contribute to emphasizing the importance of purchase protocols and biosecurity to prevent M. bovis from entering the country or herd. In Chapter 5.1, antimicrobial susceptibility testing of 141 M. bovis isolates retrieved from Belgian dairy, beef and veal calf herds was performed with broth microdilution. Minimum inhibitory concentration values were used to establish the epidemiological cut-off (ECOFF) with visual and statistical methods to distinguish the population in wild type M. bovis and those with acquired antimicrobial resistance (non-wild type). The results showed high percentages of acquired resistance for macrolides (tilmicosin, tylosin, and gamithromycin), but no acquired resistance for tetracyclines (oxytetracycline, doxycycline). Only little acquired resistance was observed for florfenicol, gentamicin, and tiamulin, while there was limited acquired resistance to enrofloxacin. Only M. bovis isolates from beef cattle or the third genomic cluster had a significantly higher change to have acquired resistance against gamithromycin than those collected from other sectors or genomic clusters. These results support the current national formulary for respiratory disease associated with M. bovis, recommending florfenicol as first choice, and oxytetracycline and macrolides as second choice. Possibly, a small remark for gamithromycin is needed, as higher risk for acquired resistance for this antimicrobial was seen in beef cattle. In vitro susceptibility testing results should be interpreted carefully, as the association with in vivo efficacy has not confirmed yet, due to the lack of clinical breakpoints. Finally, in Chapter 5.2, upgraded genomes derived from Chapter 4 and the susceptibility data from Chapter 5.1 were combined to compare genotype and phenotype antimicrobial susceptibility of M. bovis isolates. A genome wide association study to reveal genetic markers for antimicrobial resistance in M. bovis and verifying the ECOFF values obtained by the previously used different methods was executed. Many point mutations were associated with antimicrobial resistance against the critically important antibiotics of the macrolide (A2058G in the 23S rRNA gene, Gln83His in the L22 protein) and fluoroquinolone classes. For enrofloxacin the combination of different mutations in the GyrA and ParC gene showed the step-wise acquired resistance. Also previously described mutations for tilmicosin (G478A mutation in 23S rRNA alleles), and new markers for gentamicin (A1408G and G1488A in 16S rRNA) were identified. The visual estimation of de ECOFF showed to be a reliable method, although statistical methods can help when step-wise resistance results in difficult to interpret “tailing”. Even when phenotypical resistance is not yet obtained, in case of first-step mutations it should be discouraged to use fluoroquinolones as antimicrobial therapy, as selection pressure will eventually result in phenotypical resistance as well. In the general discussion (Chapter 6), the innovations in M. bovis diagnostics achieved with this thesis are discussed. In the second part, practical recommendations for diagnostics in M. bovis outbreak management, purchase policy, and eradication or herd status certificates are proposed. In this thesis new methods to identify, strain type, and access the antimicrobial susceptibility of M. bovis were developed. When rapid identification of M. bovis with MALDI-TOF MS (Chapter 3.3) is followed by the determination of antimicrobial resistance with nanopore sequencing (Chapter 5.2) it is now possible to obtain identification, strain typing and an antibiogram for critically important antibiotics within 3-5 days. This is a major step towards better control of M. bovis in clinical outbreaks and prevent herd infection when purchasing animals. Together with these new methods, also substantial epidemiological information came to light, showing the importance of a more national approach for the prevention of introducing M. bovis into the herd and country

Mycoplasma bovis peritonitis and omental bursitis after C-section in two Belgian Blue cows

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Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS

Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing >= 2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1%Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use

Risk factors for antimicrobial use in food-producing animals : disease prevention and socio-economic factors as the main drivers?

The European Union requests an urgent decrease in antimicrobial use (AMU) in food producing animals to reduce antimicrobial resistance in animals and humans and safeguard the efficacy of antimicrobials for future generations. The identification of risk factors (RFs) for AMU is essential to obtain a rapid reduction. The aim of this review was to summarize the current knowledge of RFs for AMU in veal calves, pigs and poultry. Thirty-three observational studies were included. Well-identified RFs for an increased AMU are frequent purchase of animals, herd size (large or small depending on the animal species), and a lack of selected biosecurity measures. Also in beef breed calves, more antimicrobials are used than in Holstein calves. AMU is influenced by the farmer, the veterinarian and by the integration. In general, socio-economic RFs are largely unexplored. The causal factors for AMU are multiple and complex, with possible confounding factors and unidentified interactions. Additional knowledge of socio-economic drivers appears particularly urgent to create tailor-made guidelines and awareness campaigns for each sector

Non-specific, agar medium-related peaks can result in false positive Mycoplasma alkalescens and Mycoplasma arginini identification by MALDI-TOF MS

MALDI-TOF MS is a fast and accurate tool to identify Mycoplasma species in liquid media. However, when trying to identify presumptive Mycoplasma bovis (M. bovis) colonies from solid medium (the "direct transfer method") a surprisingly high occurrence of M. arginini and M. alkalescens identification was observed. It was hypothesized that agar medium components are associated with false positive identification with Mycoplasma spp., as M. bovis colonies are very small and grow into the agar. The objective of this study was to determine whether complete modified pleuropneumonia-like organism (PPLO) agar (supplemented with horse serum, sodium pyruvate, technical yeast extract, ampicillin sodium salt and colistin) and the separate components, result in false identification as Mycoplasma spp. by MALDI-TOF MS. A total of 100 samples were examined, of which 33% of the modified PPLO agar spots were identified as M. alkalescens (16%) and M. arginini (17%)), albeit with relatively low score values ( < 1.85). No false identification of M. bovis was obtained. Several medium components (unsupplemented PPLO agar, horse serum and colistin) resulted in spectra with peaks showing close matches with peaks present in the M. alkalescens and M. arginini database spectra. This study shows that the direct transfer method should be interpreted with caution, and one should strive to pick as little as possible agar when sampling Mycoplasma-like colonies from solid medium containing PPLO agar, horse serum and/or colistin

High quality MinION and Flongle long-read nanopore genome assemblies of Mycoplasma bovis using taxon-specific training of the Bonito basecaller

Mycoplasma bovis is a major and primary bovine pathogen causing respiratory and reproductive disorders, mastitis and arthritis. Due to its persistent nature it is difficult to combat infections on farms. No effective vaccine is able to prevent M. bovis infection, leaving antimicrobials as the only first-line treatment. Nevertheless, therapy with antimicrobials is rarely efficient since increased resistance to many commercially available antimicrobials has been reported. An accurate diagnosis including antimicrobial resistance profiling is required to combat infections with working antibiotics, but this cannot be achieved fast enough with classical diagnostics. Here we developed a nanopore-based workflow allowing M. bovis species typing and Antimicrobial Resistance (AMR) Profiling applicable in point-of-care settings in control of M. bovis infections. Our new diagnostic tool was verified with 100 field strains of M. bovis for which whole genome sequencing and MIC testing was performed for practice-relevant antibiotics. Besides whole genome species typing, Single Nucleotide Polymorphism (SNP) analysis was performed to associate strain-specific genetic markers with its phenotypic AMR antibiogram. Raw fast5 outputs ranged from 5.4 Gb up to 17.2 Gb, with an average N50 of 5.5 ± 1.3 Kb per run with 11 M. bovis strains. Furthermore, including the M. bovis PG45 type strain within every run as internal control, inter-run accuracy reached up to 99.95% sequence identity. Since computation time presents a new bottleneck in this new workflow, we exploited a GPU-based bioinformatics pipeline speeding up full bioinformatics analysis to be completed within 10 hours for 11 M. bovis genomes. This new M. bovis diagnostics pipeline delivers a high accurate species identification along with an accurate genotypic antibiogram. This will be accelerated even further to facilitate proper antimicrobial therapy selection for the rapid control of bovine mycoplasmosis