Antimicrobial susceptibility of western Canadian Brachyspira isolates: Development and standardization of an agar dilution susceptibility test method.

The re-emergence of Brachyspira-associated disease in pigs since the late 2000s has illuminated some of the diagnostic challenges associated with this genus; notably, the lack of standardized antimicrobial susceptibility testing (AST) methods and interpretive criteria. Consequently, laboratories have relied heavily on highly variable in-house developed methods. There are currently no published investigations describing the antimicrobial susceptibility of Brachyspira isolates collected from pigs in Canada. The first objective of this study was therefore to develop a standardized protocol for conducting agar dilution susceptibility testing of Brachyspira spp., including determining the optimal standardized inoculum density, a key test variable that impacts test performance. The second objective was to determine the susceptibility of a collection of western Canadian Brachyspira isolates using the standardized methodology. After assessing multiple media, an agar dilution test was standardized in terms of starting inoculum (1-2 × 108 CFU/ml), incubation temperature and time, and assessed for repeatability. The antimicrobial susceptibility of a collection of clinical porcine Brachyspira isolates (n = 87) collected between 2009-2016 was then determined. This method was highly reproducible; repeat susceptibility testing yielded identical results 92% of the time. Although most of the isolates had very low MICs to the commonly used antimicrobials to treat Brachyspira-associated infections, several isolates with elevated MICs (>32 µg/ml) for tiamulin, valnemulin, tylosin, tylvalosin, and lincomycin were identified. Overall, this study underscores the importance of establishing CLSI approved clinical breakpoints for Brachyspira to facilitate the interpretation of test results and support the evidence-based selection of antimicrobials in swine industry.

Validation of an antimicrobial susceptibility testing protocol for Brachyspira hyodysenteriae and Brachyspira pilosicoli in an international ring trial.

Brachyspira hyodysenteriae and Brachyspira pilosicoli cause economically important enteric disease in pigs. Treatment of these infections often includes antimicrobial administration, which can be most effective when therapeutic options are informed by antimicrobial susceptibility testing data. Here we describe a method for broth dilution antimicrobial susceptibility testing of these bacteria, both of which are difficult to culture in vitro. The protocol was evaluated for its fitness for use in an inter-laboratory ring trial involving eight laboratories from seven countries, and employing eleven test strains (5 Brachyspira hyodysenteriae including the type strain B78T and 6 Brachyspira pilosicoli) and six antibiotics. Overall intra- and inter-laboratory reproducibility of this method was very good (>90 % MICs at mode +/- 1 log2). Whole genome sequencing revealed good correspondence between reduced susceptibility and the presence of previously defined antimicrobial resistance determinants. Interestingly, lnu(C) was identified in B. pilosicoli isolates with elevated MICs of lincomycin, whilst tva(B) was associated with elevated MICs of pleuromutilins in this species. We designated two new control strains with MICs lying within currently tested ranges, including for the pleuromutilins, in contrast to the control strain B. hyodysenteriae B78T. These were deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH. The validation of a standard protocol and identification of new control strains facilitates comparisons between studies, establishment of robust interpretative criteria, and ultimately contributes to rational antimicrobial use when treating infected livestock.

Identification of Brachyspira species by cpn60 universal target sequencing is superior to NADH oxidase gene sequencing.

The pig colon is the habitat of diverse Brachyspira species, of which only a few are of clinical importance. Methods for identification have shifted from phenotypic to molecular testing over the last two decades. Following the emergence of B. hampsonii it became evident that relying on species-specific PCRs carries the risk of overlooking important new species. Consequently, sequencing was proposed as an unbiased alternative for identification of isolates. So far, the main target for identification across species has been the NADH oxidase gene (nox). However, multiple copies of this gene in the genome and potential lateral gene transfer reduce confidence when using this gene. This study compared identification and phylogentic relationship inferred from nox sequencing to that inferred from sequencing of the cpn60 universal target using a collection of 168 isolates from different Brachyspira species. The majority of isolates had an identical identification with both methods. There were a few outliers in the trees with uncertain assignment to a species by BLAST analysis. A few major discrepancies pertained to the pathogenic species B. hampsonii (2), B. pilosicoli (1) and B. suanatina (1). Weakly haemolytic variants of B. hyodysenteriae were assigned to the correct species by both methods. Some of the isolates identified as B. hampsonii also had a weakly haemolytic phenotype.

A review of the current state of antimicrobial susceptibility test methods for Brachyspira.

The re-emergence of swine dysentery (Brachyspira-associated muco-haemorrhagic colitis) since the late 2000s has illuminated diagnostic challenges associated with this genus. The methods used to detect, identify, and characterize Brachyspira from clinical samples have not been standardized, and laboratories frequently rely heavily on in-house techniques. Particularly concerning is the lack of standardized methods for determining and interpreting the antimicrobial susceptibility of Brachyspira spp. The integration of laboratory data into a treatment plan is a critical component of prudent antimicrobial usage. Therefore, the lack of standardized methods is an important limitation to the evidence-based use of antimicrobials. This review will focus on describing the methodological limitations and inconsistencies between current susceptibility testing schemes employed for Brachyspira, provide an overview of what we do know about the susceptibility of these organisms, and suggest future directions to improve and standardize diagnostic strategies.

Immunolocalization of arginine kinase (AK) in Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides.

Arginine kinase (AK) is a member of the phosphagen kinase family. AK plays a major role in cellular energy metabolism in invertebrates including nematodes. In the present study, we performed the direct immunofluorescence test to determine the immunolocalization of AK in different stages of the life cycle (eggs, larvae, and adult worms) of Toxocara canis, Toxocara vitulorum, and Ascaris lumbricoides. Our results indicated variable levels of expression of AK in different stages. Moreover, strong fluorescence was observed in cleaving eggs than in dormant eggs. The highest activity of the enzyme was observed in the fully developed eggs. This may be due to high expression of AK in embryonic development, which is associated with increased energy demand due to cleavage and cellular differentiation. Surprisingly, expression of AK is significantly higher in the middle part and posterior end compared to anterior end of the larvae. In addition, AK is highly concentrated in cellular and metabolically active parts of the body such as hypodermis, muscle, intestine, ovaries, oviducts, and uterus, while it is absent in noncellular areas like cuticle. The present study revealed the presence of AK in T. canis, A. lumbricoides, and T. vitulorum and that it plays a major role in energy metabolism of these nematodes. Interestingly, antiserum was prepared against the recombinant T. canis AK and reacts with the native AKs of T. canis, A. lumbricoides, and T. vitulorum. AK levels could vary in relation to maximum potential rates of ATP turnover, oxidative capacity, and energy output. Further studies on subcellular localization of AK in these important helminths provide new information for researchers to develop effective anthelmintics against the parasites of veterinary and of public health importance.