Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

Antimicrobial resistance (AMR) is an ever-increasing threat to global health. Wastewater-based surveillance is an emerging methodology that objectively enables an inclusive and comprehensive assessment of population AMR in an observed sewershed. Here we compared the resistome of two tertiary-care hospitals with two separate neighborhoods, using complimentary targeted qPCR and metagenomics of wastewater before and after selective culture enrichment for clinically important Gram negatives. In total 26 ARG-type (1225 ARG-subtypes) were found across all samples, in which beta-lactam ARG was the richest (the number of different ARG-subtypes found) followed by multidrug, fluoroquinolone, macrolide-lincosamide-streptogramin (MLS) and aminoglycoside. The composition of ARGs in wastewater differed between raw wastewater pellets and culture-enriched wastewater samples and the resistomes clustered based on the type of location (Hospitals vs neighborhoods). Hospital wastewater was found to have higher diversity and greater abundance of ARGs compared to neighborhood wastewater when the composition profiles of ARGs in both raw and culture-enriched wastewater pellets. Clinically relevant ARG (i.e., VIM, NDM metallo-beta-lactamases) were detected in culture enrichment samples that were not identified in raw samples, despite a lower targeted sequencing depth. Wastewater-based surveillance is an effective, and potentially extremely important and powerful tool that could be developed to augment hospital-based infection control and antimicrobial stewardship programs, creating a safer space for those receiving care.

Background New tools capable of dynamic assessment of the varying burden of Clostridioides difficile infections are required to mitigate increased patient morbidity, mortality, and health costs. Wastewater (WW)-based epidemiology (WBE) is an emerging science, enabling comprehensive, inclusive, and unbiased assessment of populations, spatially and temporally. We sought to detect, quantify and track C. difficile across a range of scales using WBE. Methods WW collected from two hospitals; the Rockyview General Hospital (RGH; 600 beds) and Peter Lougheed Centre (PLC; 550 beds) both based in Calgary, were compared to that from a municipal WW Treatment Plant (WWTP) in Calgary, Canada. DNA was extracted from pellets collected after WW centrifugation. A multiplexed quantitative PCR assay was used to quantify the abundance of C. difficile 16S rRNA and toxin A (tcdA) genes. These were then assessed as raw values or as normalized ratios to three fecal biomarker genes: total bacterial 16S rRNA, human 18S rRNA, and Bacteroides HF183 16S rRNA. Kruskal-Wallis and Mann-Whitney tests were performed using RStudio and GraphPad Prism (version 9.3.1). Results Eight weekly samples collected from the RGH demonstrated significant changes in the levels of total C. difficile 16S rRNA gene and tcdA over time (P=0.0004 and P=0.0005, respectively, Kruskal-Wallis). Similar trends in total C. difficile and tcdA burden over time were observed when gene copies were normalized with the three fecal biomarker genes. Over a separate 13-week comparison, C. difficile and tcdA gene target abundance was greater in hospital WW (RGH and PLC) than in community-based samples from the WWTP (P=0.048 and P=0.012, respectively, Mann-Whitney). There was no significant difference in C. difficile and tcdA gene target abundance between RGH and PLC (P=0.896 and P=0.343, respectively, Mann-Whitney). Clostridioides difficile genes in wastewater measured by quantitative PCR. C. difficile 16S rRNA and tcdA gene abundance normalized as a ratio against total bacterial load (16S rRNA) varies over time and is markedly increased in hospitals relative to a municipal wastewater treatment plant in Calgary, Canada. Conclusion WW surveillance is a powerful tool that can monitor the burden the C. difficile across a range of scales in real-time. This tool could augment infection prevention and control and antimicrobial stewardship programs to better understand factors that contribute to colonization and infection. Disclosures Thomas J. Louie, MD, Artugen: Advisor/Consultant|Artugen: Grant/Research Support|Crestone: Advisor/Consultant|Crestone: Grant/Research Support|Finch Therapeutics: Advisor/Consultant|Finch Therapeutics: Grant/Research Support|Rebiotix: Advisor/Consultant|Rebiotix: Grant/Research Support|Seres Therapeutics: Advisor/Consultant|Seres Therapeutics: Grant/Research Support|summit plc: Advisor/Consultant|summit plc: Grant/Research Support|Vedanta Biosciences: Advisor/Consultant|Vedanta Biosciences: Grant/Research Support.