Passive Sampling of SARS-CoV-2 for Wastewater Surveillance.

The shedding of pathogens by infected humans enables the use of sewage monitoring to conduct wastewater-based epidemiology (WBE). Although most WBE studies use data from large sewage treatment plants, timely data from smaller catchments are needed for targeted public health action. Traditional sampling methods, like autosamplers or grab sampling, are not conducive to quick ad hoc deployments and high-resolution monitoring at these smaller scales. This study develops and validates a cheap and easily deployable passive sampler unit, made from readily available consumables, with relevance to the COVID-19 pandemic but with broader use for WBE. We provide the first evidence that passive samplers can be used to detect SARS-CoV-2 in wastewater from populations with low prevalence of active COVID-19 infections (0.034 to 0.34 per 10,000), demonstrating their ability for early detection of infections at three different scales (lot, suburb, and city). A side by side evaluation of passive samplers (n = 245) and traditionally collected wastewater samples (n = 183) verified that the passive samplers were sensitive at detecting SARS-CoV-2 in wastewater. On all 33 days where we directly compared traditional and passive sampling techniques, at least one passive sampler was positive when the average SARS-CoV-2 concentration in the wastewater equaled or exceeded the quantification limit of 1.8 gene copies per mL (n = 7). Moreover, on 13 occasions where wastewater SARS-CoV-2 concentrations were less than 1.8 gene copies per mL, one or more passive samplers were positive. Finally, there was a statistically significant (p < 0.001) positive relationship between the concentrations of SARS-CoV-2 in wastewater and the levels found on the passive samplers, indicating that with further evaluation, these devices could yield semi-quantitative results in the future. Passive samplers have the potential for wide use in WBE with attractive feasibility attributes of cost, ease of deployment at small-scale locations, and continuous sampling of the wastewater. Further research will focus on the optimization of laboratory methods including elution and extraction and continued parallel deployment and evaluations in a variety of settings to inform optimal use in wastewater surveillance.

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

A combined metabolomic and bioinformatic approach to investigate the function of transport proteins of the important pathogen Mycoplasma bovis.

Mycoplasma bovis is an economically important pathogen of the cattle industry worldwide, and there is an urgent need for a more effective vaccine to control the diseases caused by this organism. Although the M. bovis genome sequence is available, very few gene functions of M. bovis have been experimentally determined, and a better understanding of the genes involved in pathogenesis are required for vaccine development. In this study, we compared the metabolite profiles of wild type M. bovis to a number of strains that each contained a transposon insertion into a putative transporter gene. Transport systems are thought to play an important role in survival of mycoplasmas, as they rely on the host for many nutrients. We also performed 13C-stable isotope labelling on strains with transposon insertions into putative glycerol transporters. Integration of metabolomic and bioinformatic analyses revealed unexpected results (when compared to genome annotation) for two mutants, with a putative amino acid transporter (MBOVPG45_0533) appearing more likely to transport nucleotide sugars, and a second mutant, a putative dicarboxylate/amino acid:cation (Na+ or H+) symporter (DAACS), more likely to function as a biopterin/folate transporter. This study also highlighted the apparent redundancy in some transport and metabolic pathways, such as the glycerol transport systems, even in an organism with a reduced genome. Overall, this study highlights the value of metabolomics for revealing the likely function of a number of transporters of M. bovis.

Metabolite profiling of Mycoplasma gallisepticum mutants, combined with bioinformatic analysis, can reveal the likely functions of virulence-associated genes.

Mycoplasma gallisepticum is an economically important pathogen of commercial poultry. An improved understanding of M. gallisepticum pathogenesis is required to develop better control methods. We recently identified a number of M. gallisepticum mutants with defects in colonization and persistence in chickens using signature-tagged transposon mutagenesis. Loss of virulence was associated with mutations in a putative oligopeptide/dipeptide (opp/dpp) ATP-binding cassette (ABC) transporter (where the transposon was inserted into the MGA_0220 (oppD1) gene and two hypothetical proteins (encoded by MGA_1102 and MGA_0588), one of which (MGA_1102) contains a putative peptidase motif. To further characterise the function of these proteins, we compared the metabolome of each transposon mutant with that of wild type bacteria. Two independent LC/MS analyses revealed consistent significant decreases in the abundances of several amino acids and the dipeptide alanyl-glycine (Ala-Gly) in the MGA_0220 mutant, consistent with this protein being a peptide transporter. Similarly, lysine and Ala-Gly were significantly decreased in the MGA_1102 mutant, consistent with our bioinformatic analysis suggesting that MGA_1102 encodes a membrane-located peptidase. Few differences were observed in metabolite levels in the MGA_0588 mutant, suggesting that the disrupted protein has a non-metabolic role. Overall, this study indicates that metabolomics is a useful tool in the functional analysis of mutants.

Safety and efficacy of a Mycoplasma gallisepticum oppD knockout mutant as a vaccine candidate.

Control of the important poultry pathogen Mycoplasma gallisepticum is highly dependent on safe and efficacious attenuated vaccines. In order to assess a novel vaccine candidate we evaluated the safety and efficacy of the M. gallisepticum mutant 26-1. The oppD1 gene in this mutant has been interrupted by a signature-tagged transposon and previous studies have shown that it can colonise the respiratory tract of chickens without inducing significant disease. The capacity of the oppD1 mutant to induce protective immunity in the respiratory tract after vaccination by eye-drop was assessed by challenging vaccinated birds with an aerosol of the virulent M. gallisepticum strain Ap3AS. Vaccination with the oppD1 mutant was shown to fully protect against the lesions caused by pathogenic M. gallisepticum in the air sacs and tracheas. It also protected against the effect of infection on weight gain, and partially protected against colonisation of the trachea by virulent M. gallisepticum. These results indicate that a M. gallisepticum mutant with the oppD1 gene knocked out could be used as a live attenuated vaccine as it is both safe and efficacious when administered by eyedrop to chickens.

The oppD Gene and Putative Peptidase Genes May Be Required for Virulence in Mycoplasma gallisepticum.

Relatively few virulence genes have been identified in pathogenic mycoplasmas, so we used signature-tagged mutagenesis to identify mutants of the avian pathogen Mycoplasma gallisepticum with a reduced capacity to persist in vivo and compared the levels of virulence of selected mutants in experimentally infected chickens. Four mutants had insertions in one of the two incomplete oppABCDF operons, and a further three had insertions in distinct hypothetical genes, two containing peptidase motifs and one containing a member of a gene family. The three hypothetical gene mutants and the two with insertions in oppD1 were used to infect chickens, and all five were shown to have a reduced capacity to induce respiratory tract lesions. One oppD1 mutant and the MGA_1102 and MGA_1079 mutants had a greatly reduced capacity to persist in the respiratory tract and to induce systemic antibody responses against M. gallisepticum The other oppD1 mutant and the MGA_0588 mutant had less capacity than the wild type to persist in the respiratory tract but did elicit systemic antibody responses. Although M. gallisepticum carries two incomplete opp operons, one of which has been acquired by horizontal gene transfer, our results suggest that one of the copies of oppD may be required for full expression of virulence. We have also shown that three hypothetical genes, two of which encode putative peptidases, may be required for full expression of virulence in M. gallisepticum. None of these genes has previously been shown to influence virulence in pathogenic mycoplasmas.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo.

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.