Danae M. Suchan

Septic arthritis (SA) is a cause of lameness in cattle attributed to bacterial infections. Mycoplasmopsis bovis is the best known and characterized etiological agent of SA; however, cases caused by diverse bacteria have been reported. Accordingly, we surveyed bacteria associated with septic and healthy joints from animals in western Canadian feedlots. Microbial community profiling showed that M. bovis was the most frequently detected pathogen in septic joints, followed by Metamycoplasma alkalescens and Trueperella pyogenes . In most cases, disease was ostensibly caused by a single pathogen, though polymicrobial infections and complex communities were also observed in DNA isolated from septic joints. The application of enhanced metagenomics by target DNA hybridization capture sequencing (CapSeq) provided more robust pathogen detection and characterization. CapSeq revealed resistance determinants that escaped detection using a conventional shotgun metagenomic approach. Notably, a series of nucleotide polymorphisms to M. bovis rrs , rrl , gyrA , and parC gene sequences were observed that confer resistance to macrolides and oxytetracycline-resistant T. pyogenes were also apparent in the CapSeq data. Complementary pathogen isolation, whole-genome sequencing, and phenotyping efforts were focused on the two most prominent pathogens, M. bovis and M. alkalescens , and corroborated metagenomic data sets. IMPORTANCE: Informed antimicrobial use for the treatment of septic arthritis (SA) has been limited by overlooking the potential diversity of causative agents and our knowledge of their antimicrobial resistance (AMR) profiles. This survey begins to provide epidemiological insights, offering renewed appreciation of Metamycoplasma alkalescens as an etiological agent of SA and highlighting the prominence of important AMR determinants. Finally, the survey suggests that our knowledge of even the identities of the causative agents of SA is incomplete.

Antimicrobial resistance (AMR) is a global threat. The identification and characterization of novel resistance genes is integral to AMR surveillance. The erm (55) gene was originally identified through whole genome sequencing of macrolide-resistant strains of Mycobacterium chelonae . The gene was annotated as a ribosomal methyltransferase, but its role as a determinant of macrolide resistance was not formally demonstrated. Three erm (55) alleles have now been documented. The plasmid-borne erm (55) ^P , the transposon-associated erm (55) ^T , and the chromosomal encoded erm (55) ^C exhibit ≈82% amino acid sequence identity. Here, we confirm that, when expressed from plasmids in a macrolide-susceptible strain of Escherichia coli , all three erm (55) variants confer resistance to azithromycin and clarithromycin. IMPORTANCE: Macrolide antibiotics are often the only oral treatment option for infections with rapidly growing mycobacteria such as Mycobacterium abscessus and Mycobacterium chelonae . We previously identified three variants of a newly predicted macrolide resistance gene, erm (55), in M. chelonae , including the first case of a plasmid-mediated macrolide resistance in mycobacteria. The present study provides experimental evidence that the three erm (55) variants confer macrolide resistance and that each variant is unique in the degree to which it reduces susceptibility to clinically relevant macrolides.

The diffusible toxin ColIb produced by Salmonella enterica serovar Typhimurium SL1344 is a potent inhibitor of Escherichia coli growth. To identify and parameterize metabolic cross-feeding in states of competition, we established defined communities in which E. coli was the only species able to access a sole carbon source, lactose. Although ColIb was predicted to undermine cross-feeding by killing the lactose-converting E. coli, S. enterica populations thrived in co-culture. We discovered that ColIb caused the release of active β-galactosidase from E. coli cells, which induced galactose uptake by S. enterica. Although iron limitation stimulates ColIb production and makes E. coli more sensitive to the toxin, ColIb killing in iron-limited conditions did not enhance iron acquisition or siderophore scavenging by S. enterica. Also unexpected was the rapid rate at which resistance to ColIb evolved in E. coli through spontaneous mutation of the ColIb receptor gene cirA or horizontal acquisition of the S. enterica colicin immunity gene imm. Mathematical modelling effectively predicted the growth kinetics of E. coli and S. enterica populations, revealing a tractable system in which ColIb can shrink a competitor population while simultaneously amplifying the metabolic contributions of the suppressed population.

Ignavigranum ruoffiae is a rare human pathogen. Strain CPL 242382-20 was isolated in Manitoba, Canada, from a breast cyst. Whole-genome sequencing was performed with the Oxford Nanopore Technologies MinION and Illumina MiSeq platforms. The circular chromosome is 1,949,382 bp with 39.68% G+C content and 1,765 protein-coding genes.

The COVID‐19 pandemic prompted a transition to remote delivery of courses that lack immersive hands‐on research experiences for undergraduate science students, resulting in a scientific research skills gap. In this report, we present an option for an inclusive and authentic, hands‐on research experience that all students can perform off‐campus. Biology students in a semester‐long (13 weeks) sophomore plant physiology course participated in an at‐home laboratory designed to study the impacts of nitrogen addition on growth rates and root nodulation by wild nitrogen‐fixing Rhizobia in Pisum sativum (Pea) plants. This undergraduate research experience, piloted in the fall semester of 2020 in a class with 90 students, was created to help participants learn and practice scientific research skills during the COVID‐19 pandemic. Specifically, the learning outcomes associated with this at‐home research experience were: (1) generate a testable hypothesis, (2) design an experiment to test the hypothesis, (3) explain the importance of biological replication, (4) perform meaningful statistical analyses using R, and (5) compose a research paper to effectively communicate findings to a general biology audience. Students were provided with an at‐home laboratory kit containing the required materials and reagents, which were chosen to be accessible and affordable in case students were unable to access our laboratory kit. Students were guided through all aspects of research, including hypothesis generation, data collection, and data analysis, with video tutorials and live virtual sessions. This at‐home laboratory provided students an opportunity to practice hands‐on research with the flexibility to collect and analyze their own data in a remote setting during the COVID‐19 pandemic. This, or similar laboratories, could also be used as part of distance learning biology courses.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are environmental contaminants of concern. We previously described biodegradation of two PFAS that represent components and transformation products of aqueous film-forming foams (AFFF), 6:2 fluorotelomer sulfonamidoalkyl betaine (6:2 FTAB) and 6:2 fluorotelomer sulfonate (6:2 FTSA), by Gordonia sp. strain NB4-1Y. To identify genes involved in the breakdown of these compounds, the transcriptomic response of NB4-1Y was examined when grown on 6:2 FTAB, 6:2 FTSA, a non-fluorinated analog of 6:2 FTSA (1-octanesulfonate), or MgSO₄, as sole sulfur source. Differentially expressed genes were identified as those with ± 1.5 log₂-fold-differences (± 1.5 log₂FD) in transcript abundances in pairwise comparisons. Transcriptomes of cells grown on 6:2 FTAB and 6:2 FTSA were most similar (7.9% of genes expressed ± 1.5 log₂FD); however, several genes that were expressed in greater abundance in 6:2 FTAB treated cells compared to 6:2 FTSA treated cells were noted for their potential role in carbon–nitrogen bond cleavage in 6:2 FTAB. Responses to sulfur limitation were observed in 6:2 FTAB, 6:2 FTSA, and 1-octanesulfonate treatments, as 20 genes relating to global sulfate stress response were more highly expressed under these conditions compared to the MgSO₄ treatment. More highly expressed oxygenase genes in 6:2 FTAB, 6:2 FTSA, and 1-octanesulfonate treatments were found to code for proteins with lower percent sulfur-containing amino acids compared to both the total proteome and to oxygenases showing decreased expression. This work identifies genetic targets for further characterization and will inform studies aimed at evaluating the biodegradation potential of environmental samples through applied genomics. Graphic

The plant growth-promoting rhizobacterium Delftia acidovorans RAY209 is capable of establishing strong root attachment during early plant development at 7 days post-inoculation. The transcriptional response of RAY209 was measured using RNA-seq during early (day 2) and sustained (day 7) root colonization of canola plants, capturing RAY209 differentiation from a medium-suspended cell state to a strongly root-attached cell state. Transcriptomic data was collected in an identical manner during RAY209 interaction with soybean roots to explore the putative root colonization response to this globally relevant crop. Analysis indicated there is an increased number of significantly differentially expressed genes between medium-suspended and root-attached cells during early soybean root colonization relative to sustained colonization, while the opposite temporal pattern was observed for canola root colonization. Regardless of the plant host, root-attached RAY209 cells exhibited the least amount of differential gene expression between early and sustained root colonization. Root-attached cells of either canola or soybean roots expressed high levels of a fasciclin gene homolog encoding an adhesion protein, as well as genes encoding hydrolases, multiple biosynthetic processes, and membrane transport. Notably, while RAY209 ABC transporter genes of similar function were transcribed during attachment to either canola or soybean roots, several transporter genes were uniquely differentially expressed during colonization of the respective plant hosts. In turn, both canola and soybean plants expressed genes encoding pectin lyase and hydrolases – enzymes with purported function in remodelling extracellular matrices in response to RAY209 colonization. RAY209 exhibited both a core regulatory response and a planthost-specific regulatory response to root colonization, indicating that RAY209 specifically adjusts its cellular activities to adapt to the canola and soybean root environments. This transcriptomic data defines the basic RAY209 response as both a canola and soybean commercial crop and seed inoculant.

We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3' end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).

Deinococcus sp. strain UR1, a resilient bacterium isolated from the surface of a stainless steel sign located on the University of Regina campus in Saskatchewan, Canada, was sequenced to 56-fold coverage to produce 73 contigs with a consensus length of 4,472,838 bp and a G+C content of 69.37%.