Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase.

Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results demonstrate that L. monocytogenes has a fully functional chitinolytic system. Both chitinases show substrate degradation rates similar to those of the nonprocessive endo-chitinase SmChiC from Serratia marcescens. Compared to the S. marcescens LPMO chitin-binding protein CBP21, LmLPMO10 shows a similar rate but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium. The chitinases on the other hand are both abundantly secreted in the presence of chitin. Although LmLPMO10 is shown to promote chitin degradation in tandem with the chitinases in vitro, the secretome and transcription data question whether this is the primary role of LmLPMO10 in vivo.

A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galß1-4GlcNAc) and LacdiNAc (GalNAcß1-4GlcNAc) motifs found on vertebrate and insect cells.

There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galß1-4GlcNAcß-tetramethylrhodamine (LacNAc-TMR (Galß1-4GlcNAcß(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcß1-4GlcNAcß-TMR), LacNAc-TMR, and LacNAcß1-6LacNAcß-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the ß1-6 bond in LacNAcß1-6LacNAcß-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.

Bacterial chitinases and chitin-binding proteins as virulence factors.

Bacterial chitinases (EC 3.2.1.14) and chitin-binding proteins (CBPs) play a fundamental role in the degradation of the ubiquitous biopolymer chitin, and the degradation products serve as an important nutrient source for marine- and soil-dwelling bacteria. However, it has recently become clear that representatives of both Gram-positive and Gram-negative bacterial pathogens encode chitinases and CBPs that support infection of non-chitinous mammalian hosts. This review addresses this biological role of bacterial chitinases and CBPs in terms of substrate specificities, regulation, secretion and involvement in cellular and animal infection.