Selective medium for culture of Mycoplasma hyopneumoniae

The fastidious porcine respiratory pathogen Mycoplasma hyopneumoniae has proven difficult to culture since it was first isolated in 1965. A reliable solid medium has been particularly challenging. Moreover, clinical and pathological samples often contain the fast-growing M. hyorhinis which contaminates and overgrows M. hyopneumoniae in primary culture. The aim of this study was to optimise the culture medium for recovery of M. hyopneumoniae and to devise a medium for selection of M. hyopneumoniae from clinical samples also containing M. hyorhinis. The solid medium devised by Niels Friis was improved by use of Purified agar and incorporation of DEAE-dextran. Addition of glucose or neutralization of acidity in liquid medium with NaOH did not improve the final yield of viable organisms or alter the timing of peak viability. Analysis of the relative susceptibility of M. hyopneumoniae and M. hyorhinis strains to four antimicrobials showed that M. hyopneumoniae is less susceptible than M. hyorhinis to kanamycin. This was consistent in all UK and Danish strains tested. A concentration of 2 μg/ml of kanamycin selectively inhibited the growth of all M. hyorhinis tested, while M. hyopneumoniae was able to grow. This forms the basis of an effective selective culture medium for M. hyopneumoniae.(Résumé d'auteur

Evaluation of formalin-fixed and FFPE tissues for spatially resolved metabolomics and drug distribution studies

Fixation of samples is broadly used prior to the histological evaluation of tissue samples. Though recent reports demonstrated the ability to use fixed tissues for mass spectrometry imaging (MSI) based proteomics, glycomics and tumor classification studies, to date comprehensive evaluation of fixation-related effects for spatially resolved metabolomics and drug disposition studies is still missing. In this study we used matrix assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) MSI to investigate the effect of formalin-fixation and formalin-fixation combined with paraffin embedding on the detectable metabolome including xenobiotics. Formalin fixation was found to cause significant washout of polar molecular species, including inorganic salts, amino acids, organic acids and carnitine species, oxidation of endogenous lipids and formation of reaction products between lipids and fixative ingredients. The slow fixation kinetics under ambient conditions resulted in increased lipid hydrolysis in the tissue core, correlating with the time-dependent progression of the fixation. Paraffin embedding resulted in subsequent partial removal of structural lipids resulting in the distortion of the elucidated biodistributions

Optimal experimental design for efficient toxicity testing in microphysiological systems: A bone marrow application

Introduction: Microphysiological systems (MPS; organ-on-a-chip) aim to recapitulate the 3D organ microenvironment and improve clinical predictivity relative to previous approaches. Though MPS studies provide great promise to explore treatment options in a multifactorial manner, they are often very complex. It is therefore important to assess and manage technical confounding factors, to maximise power, efficiency and scalability.Methods: As an illustration of how MPS studies can benefit from a systematic evaluation of confounders, we developed an experimental design approach for a bone marrow (BM) MPS and tested it for a specified context of use, the assessment of lineage-specific toxicity.Results: We demonstrated the accuracy of our multicolour flow cytometry set-up to determine cell type and maturity, and the viability of a “repeated measures” design where we sample from chips repeatedly for increased scalability and robustness. Importantly, we demonstrated an optimal way to arrange technical confounders. Accounting for these confounders in a mixed-model analysis pipeline increased power, which meant that the expected lineage-specific toxicities following treatment with olaparib or carboplatin were detected earlier and at lower doses. Furthermore, we performed a sample size analysis to estimate the appropriate number of replicates required for different effect sizes. This experimental design-based approach will generalise to other MPS set-ups.Discussion: This design of experiments approach has established a groundwork for a reliable and reproducible in vitro analysis of BM toxicity in a MPS, and the lineage-specific toxicity data demonstrate the utility of this model for BM toxicity assessment. Toxicity data demonstrate the utility of this model for BM toxicity assessment

Targeted desorption electrospray ionization mass spectrometry imaging for drug distribution, toxicity, and tissue classification studies

With increased use of mass spectrometry imaging (MSI) in support of pharmaceutical research and development, there are opportunities to develop analytical pipelines that incorporate exploratory high-performance analysis with higher capacity and faster targeted MSI. Therefore, to enable faster MSI data acquisition we present analyte-targeted desorption electrospray ionization–mass spectrometry imaging (DESI-MSI) utilizing a triple-quadrupole (TQ) mass analyzer. The evaluated platform configuration provided superior sensitivity compared to a conventional time-of-flight (TOF) mass analyzer and thus holds the potential to generate data applicable to pharmaceutical research and development. The platform was successfully operated with sampling rates up to 10 scans/s, comparing positively to the 1 scan/s commonly used on comparable DESI-TOF setups. The higher scan rate enabled investigation of the desorption/ionization processes of endogenous lipid species such as phosphatidylcholines and a co-administered cassette of four orally dosed drugs—erlotininb, moxifloxacin, olanzapine, and terfenadine. This was used to enable understanding of the impact of the desorption/ionization processes in order to optimize the operational parameters, resulting in improved compound coverage for olanzapine and the main olanzapine metabolite, hydroxy-olanzapine, in brain tissue sections compared to DESI-TOF analysis or matrix-assisted laser desorption/ionization (MALDI) platforms. The approach allowed reducing the amount of recorded information, thus reducing the size of datasets from up to 150 GB per experiment down to several hundred MB. The improved performance was demonstrated in case studies investigating the suitability of this approach for mapping drug distribution, spatially resolved profiling of drug-induced nephrotoxicity, and molecular–histological tissue classification of ovarian tumors specimens

Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis

Streptococcus suis causes disease in pigs worldwide and is increasingly implicated in zoonotic disease in East and South-East Asia. To understand the genetic basis of disease in S. suis, we study the genomes of 375 isolates with detailed clinical phenotypes from pigs and humans from the United Kingdom and Vietnam. Here, we show that isolates associated with disease contain substantially fewer genes than non-clinical isolates, but are more likely to encode virulence factors. Human disease isolates are limited to a single-virulent population, originating in the 1920 s when pig production was intensified, but no consistent genomic differences between pig and human isolates are observed. There is little geographical clustering of different S. suis subpopulations, and the bacterium undergoes high rates of recombination, implying that an increase in virulence anywhere in the world could have a global impact over a short timescale.Peer reviewe

Transposon mutagenesis in Mycoplasma hyopneumoniae using a novel mariner-based system for generating random mutations.

Mycoplasma hyopneumoniae is the cause of enzootic pneumonia in pigs, a chronic respiratory disease associated with significant economic losses to swine producers worldwide. The molecular pathogenesis of infection is poorly understood due to the lack of genetic tools to allow manipulation of the organism and more generally for the Mycoplasma genus. The objective of this study was to develop a system for generating random transposon insertion mutants in M. hyopneumoniae that could prove a powerful tool in enabling the pathogenesis of infection to be unraveled. A novel delivery vector was constructed containing a hyperactive C9 mutant of the Himar1 transposase along with a mini transposon containing the tetracycline resistance cassette, tetM. M. hyopneumoniae strain 232 was electroporated with the construct and tetM-expressing transformants selected on agar containing tetracycline. Individual transformants contained single transposon insertions that were stable upon serial passages in broth medium. The insertion sites of 44 individual transformants were determined and confirmed disruption of several M. hyopneumoniae genes. A large pool of over 10 000 mutants was generated that should allow saturation of the M. hyopneumoniae strain 232 genome. This is the first time that transposon mutagenesis has been demonstrated in this important pathogen and could be generally applied for other Mycoplasma species that are intractable to genetic manipulation. The ability to generate random mutant libraries is a powerful tool in the further study of the pathogenesis of this important swine pathogen

Correlating mass spectrometry imaging and liquid chromatography-tandem mass spectrometry for tissue-based pharmacokinetic studies

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a standard tool used for absolute quantification of drugs in pharmacokinetic (PK) studies. However, all spatial information is lost during the extraction and elucidation of a drugs biodistribution within the tissue is impossible. In the study presented here we used a sample embedding protocol optimized for mass spectrometry imaging (MSI) to prepare up to 15 rat intestine specimens at once. Desorption electrospray ionization (DESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) were employed to determine the distributions and relative abundances of four benchmarking compounds in the intestinal segments. High resolution MALDI-MSI experiments performed at 10 µm spatial resolution allowed to determine the drug distribution in the different intestinal histological compartments to determine the absorbed and tissue bound fractions of the drugs. The low tissue bound drug fractions, which were determined to account for 56−66% of the total drug, highlight the importance to understand the spatial distribution of drugs within the histological compartments of a given tissue to rationalize concentration differences found in PK studies. The mean drug abundances of four benchmark compounds determined by MSI were correlated with the absolute drug concentrations. Linear regression resulted in coefficients of determination (R2) ranging from 0.532 to 0.926 for MALDI-MSI and R2 values ranging from 0.585 to 0.945 for DESI-MSI, validating a quantitative relation of the imaging data. The good correlation of the absolute tissue concentrations of the benchmark compounds and the MSI data provides a bases for relative quantification of compounds within and between tissues, without normalization to an isotopically labelled standard, provided that the compared tissues have inherently similar ion suppression effects

Development of a self-replicating plasmid system for Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a prevalent swine respiratory pathogen that is a major cause of economic loss to pig producers. Control is achieved by a combination of antimicrobials, vaccination and management practices, but current vaccines offer only partial control and there is a need for improved preventative strategies. A major barrier to advances in understanding the pathogenesis of M. hyopneumoniae and in developing new vaccines is the lack of tools to genetically manipulate the organism. We describe the development and optimisation of the first successful plasmid-based system for the genetic manipulation of M. hyopneumoniae. Our artificial plasmids contain the origin of replication (oriC) of M. hyopneumoniae along with tetM, conferring resistance to tetracycline. With these plasmids, we have successfully transformed M. hyopneumoniae strain 232 by electroporation, generating tetracycline resistant organisms. The persistence of extrachromosomal plasmid and maintenance of plasmid DNA over serial passages shows that these artificial plasmids are capable of self-replication in M. hyopneumoniae. In addition to demonstrating the amenability of M. hyopneumoniae to genetic manipulation and in optimising the conditions necessary for successful transformation, we have used this system to determine the minimum functional oriC of M. hyopneumoniae. In doing so, we have developed a plasmid with a small oriC that is stably maintained over multiple passages that may be useful in generating targeted gene disruptions. In conclusion, we have generated a set of plasmids that will be valuable in studies of M. hyopneumoniae pathogenesis and provide a major step forward in the study of this important swine pathogen