The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host-B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

The Role of Phosphatidylinositol Mannosides in the Serological Diagnosis of Mycobacterial Infections.

Accurate diagnosis of mycobacterial infections, such as bovine tuberculosis and paratuberculosis, remains challenging. Available direct diagnostic tests aimed at detecting the pathogen are highly specific but lack sensitivity, depending on the stage of infection and the prevalence of infection in a population. The sensitivity of indirect diagnostic assays that measure the host immune response to infection is similarly affected by disease characteristics. The choice of antigen used to detect a host response to infection has a critical impact on test sensitivity and specificity. Many indirect tests rely on crude antigen preparations and cell-free extracts, of which the production is poorly standardized. Moreover, these preparations contain ample uncharacterized cross-reactive compounds. To enhance serological test specificity, existing assays depend on the pre-treatment of samples and a relatively high cut-off value, that in turn influences test sensitivity. Research therefore focuses on the identification of more specific, defined antigens to improve diagnostics. In the current study, we extracted phosphatidylinositol mannosides (PIMs) and investigated their potential use in antibody-based tests. Our results demonstrate that specific IgG class antibodies are generated against PIMs in cows, but this is unrelated to tuberculosis or paratuberculosis infection status, making these antigens unsuitable for diagnostic applications. In addition, we demonstrate that PIMs are widely present in crude antigen preparations and in serum pre-absorption buffer. Our results indicate that PIMs are cross-reactive compounds with immunodominant B cell epitopes that could impair serological test specificity.

Application of Transcriptomics to Enhance Early Diagnostics of Mycobacterial Infections, with an Emphasis on Mycobacterium avium ssp. paratuberculosis.

Mycobacteria cause a wide variety of disease in human and animals. Species that infect ruminants include M. bovis and M. avium ssp. paratuberculosis (MAP). MAP is the causative agent of Johne's disease in ruminants, which is a chronic granulomatous enteric infection that leads to severe economic losses worldwide. Characteristic of MAP infection is the long, latent phase in which intermittent shedding can take place, while diagnostic tests are unable to reliably detect an infection in this stage. This leads to unnoticed dissemination within herds and the presence of many undetected, silent carriers, which makes the eradication of Johne's disease difficult. To improve the control of MAP infection, research is aimed at improving early diagnosis. Transcriptomic approaches can be applied to characterize host-pathogen interactions during infection, and to develop novel biomarkers using transcriptional profiles. Studies have focused on the identification of specific RNAs that are expressed in different infection stages, which will assist in the development and clinical implementation of early diagnostic tests.

From Cell Death to Metabolism: Holin-Antiholin Homologues with New Functions.

Programmed cell death in bacteria is generally triggered by membrane proteins with functions analogous to those of bacteriophage holins: they disrupt the membrane potential, whereas antiholins antagonize this process. The holin-like class of proteins is present in all three domains of life, but their functions can be different, depending on the species. Using a series of biochemical and genetic approaches, in a recent article in mBio, Charbonnier et al. (mBio 8:e00976-17, 2017, https://doi.org/10.1128/mBio.00976-17) demonstrate that the antiholin homologue in Bacillus subtilis transports pyruvate and is regulated in an unconventional way by its substrate molecule. Here, we discuss the connection between cell death and metabolism in various bacteria carrying genes encoding these holin-antiholin analogues and place the recent study by Charbonnier et al. in an evolutionary context.

YsbA and LytST are essential for pyruvate utilization in Bacillus subtilis.

The genome of Bacillus subtilis encodes homologues of the Cid/Lrg network. In other bacterial species, this network consists of holin- and antiholin-like proteins that regulate cell death by controlling murein hydrolase activity. The YsbA protein of B. subtilis is currently annotated as a putative antiholin-like protein that possibly impedes cell death, whereas YwbH is thought to act as holin-like protein. However, the actual functions of YsbA and YwbH in B. subtilis have never been characterized. Therefore, we examined the impact of these proteins on growth and cell death in B. subtilis. We did not find a connection to the regulation of programmed cell death, but instead, our experiments reveal that YsbA and its two-component regulator LytST are essential for growth on pyruvate. Moreover, deletion of ysbA and lytS significantly reduces pyruvate consumption. Our findings suggest that LytST induces ysbA transcription in the presence of pyruvate, and that YsbA is involved in pyruvate utilization presumably by functioning as pyruvate uptake system. We show that B. subtilis excretes pyruvate as overflow metabolite in rich medium, indicating that pyruvate could be a common nutrient in the environment. Hence, YsbA and LytST might play a major role in environmental growth of B. subtilis.

Bacillus subtilis attachment to Aspergillus niger hyphae results in mutually altered metabolism.

Interaction between microbes affects the growth, metabolism and differentiation of members of the microbial community. While direct and indirect competition, like antagonism and nutrient consumption have a negative effect on the interacting members of the population, microbes have also evolved in nature not only to fight, but in some cases to adapt to or support each other, while increasing the fitness of the community. The presence of bacteria and fungi in soil results in various interactions including mutualism. Bacilli attach to the plant root and form complex communities in the rhizosphere. Bacillus subtilis, when grown in the presence of Aspergillus niger, interacts similarly with the fungus, by attaching and growing on the hyphae. Based on data obtained in a dual transcriptome experiment, we suggest that both fungi and bacteria alter their metabolism during this interaction. Interestingly, the transcription of genes related to the antifungal and putative antibacterial defence mechanism of B. subtilis and A. niger, respectively, are decreased upon attachment of bacteria to the mycelia. Analysis of the culture supernatant suggests that surfactin production by B. subtilis was reduced when the bacterium was co-cultivated with the fungus. Our experiments provide new insights into the interaction between a bacterium and a fungus.