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Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the M. feriruminatoris chassis confirmed the corresponding loss of H2O2 production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent Mycoplasma capricolum subsp. capripneumoniae was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.

Mycoplasma hyopneumoniae is the etiological agent of enzootic pneumonia (EP), an economically important chronic respiratory disease in pigs. M. hyopneumoniae impacts the mucociliary clearance system by disrupting the cilia and modulates the immune response, resulting in intermittent dry non-productive cough. For progressive control of EP in Switzerland, a corresponding programme was fully implemented in 2004. It is based on total depopulation strategies of affected fattening farms as well as partial depopulation in breeding farms. Surveillance of EP status in Switzerland is mainly based on real-time PCR of nasal swabs from coughing animals or suspicious lungs and thereby sporadic cases are still observed every year. In order to obtain information on the seroprevalence, serum samples of 5021 sows from 968 farms collected in 2018 at eight different slaughterhouses were analyzed for the presence of M. hyopneumoniae-specific antibodies using a commercial ELISA kit. The overall seroprevalence was low with 0.98% of sows testing positive and these seropositive animals could be allocated to 3.92% of farms tested. Most seropositive farms presented weakly positive singleton reactors and only one farm showed several strongly seropositive animals. In conclusion, the serological status mirrors the successful progressive control of M. hyopneumoniae in the Swiss domestic pig population over the years. The current study underlines the added value of serological testing in the surveillance of EP in a country with low prevalence and confirms the sustained benefit of strategic control programmes.

We report the complete 2,783,931 bp circular genome sequence of the human methicillin-resistant 28 Staphylococcus aureus strain 17Gst354 isolated from a nasal swab. The strain possessed an additional 29 4,397 bp plasmid. Moreover, we induced and sequenced its temperate phage Staphylococcus phage 30 vB_StaphS-IVBph354, which has a circular genome of 41,970 bp.

Background: Contagious bovine pleuropneumonia (CBPP) and contagious caprine pleuropneumonia (CCPP) are major infectious diseases of ruminants caused by mycoplasmas in Africa and Asia. Beyond their obvious impact on animal health, CBPP and CCPP negatively impact the livelihood and well being of a substantial proportion of livestock-dependent people affecting their culture, economy, trade and nutrition. The current vaccines used for disease control consist of a live attenuated CBPP vaccine and a bacter in vaccine for CCPP, which were developed in the 1960s and 1980s, respectively. Both of these vaccines have limitations, so better vaccines are urgently needed to improve control of these diseases. Methods: The research community met in 2018 for a workshop entitled 'Contagious bovine and caprine pleuropneumonia, an update on the current knowledge base', in Bern Switzerland. The exchange of information resulted in a number of prioritized biomedical research needs related to challenge models, vaccine target discovery and immune responses. Results: The outcome of this event highlighted the research gaps that will need to be overcome to develop safer and more effective vaccines for progressive disease control. This does not cover vaccine delivery, although we acknowledge that vaccine delivery is an important component of vaccine campaigns, especially in animals with a relatively low asset value. Conclusion: After rigorous scientific discussions, our suggested priority actions have been listed and should provide a frame for concerted action and funding. We believe these approaches will guide development of better vaccines that can be used as tools in the control of these two important pathogens.

A novel Gram-stain-positive bacterium was isolated from a purulent bovine milk sample, the bovine placenta from an abortion, the udder secretion of a heifer and the lung of a pig that had succumbed from suppurative bronchopneumonia in Switzerland from 2015 to 2019. The strains grew best under aerobic conditions with 5 % CO2 and colonies were non-haemolytic and greyish-white. They were non-motile and negative for catalase and oxidase. The genomes of the four strains 19M2397T, 15A0121, 15IMD0307 and 19OD0592 were obtained by sequencing. The results of phylogenetic analyses based on the 16S rRNA gene grouped them within the genus Trueperella in the family Arcanobacteriaceae. The genomes had DNA G+C contents of 61.2-62.2 mol% and showed digital DNA-DNA hybridization (dDDH) values of 21.4-22.8 % and average nucleotide identity (ANI) values of approximately 77 % to their closest relatives Trueperella pyogenes and Trueperella bernardiae. With respect to the presence in different livestock species we propose the name Trueperella pecoris sp. nov. The type strain is 19M2397T (=CCOS 1952T=DSM 111392T), isolated from the udder secretion of a heifer diagnosed with summer mastitis in 2019.

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications., Author summary Mycoplasmas belong to a class of cell-wall deficient bacteria characterized by minimal genomes acquired through regressive evolution. Historically, they were thought to lack many of the common bacterial virulence traits including classical exotoxins and toxin-antitoxin (TA) systems. In this work, we confirmed the presence of different functional TA systems in several isolates of the caprine pathogen Mycoplasma mycoides subsp. capri. Our data also indicate that TA systems are widespread in other mycoplasma species of veterinary importance. This work paves the way for the investigation of the biological role of TA systems during mycoplasma chronic infections as they are likely to contribute to the parasitic lifestyle of mycoplasmas, persistence in their hosts as well as the buildup of antimicrobial resistance, as recently observed. The availability of synthetic genomics tools to modify a range of Mycoplasma pathogens and well-established challenge models will foster future research and shed the light on the importance of TA systems in mycoplasmas.

Mycoplasma feriruminatoris is a fast-growing and genetically tractable mycoplasma species. We sequenced the Swiss strain IVB14/OD_0535, isolated from an Alpine ibex. This strain has a circular genome of 1,027,435 bp with a G+C content of 24.3%. It encodes 835 open reading frames (ORFs), 2 rRNA operons, and 30 tRNAs