Long-read sequencing for reliably calling the mompS allele in Legionella pneumophila sequence-based typing.

Sequence-based typing (SBT) of Legionella pneumophila is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires' disease. In the L. pneumophila SBT scheme, mompS2 is one of seven genes that determine the sequence type (ST). The Legionella genome typically contains two copies of mompS (mompS1 and mompS2). When they are non-identical it can be challenging to determine the mompS2 allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 L. pneumophila genomes, there were 62 STs, 18 of which carried non-identical mompS copies. Using short-reads, the mompS2 allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried mompS1 allele 7 and mompS2 allele 15, were assigned an incorrect mompS2 allele and/or mompS gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical mompS copies, short-read assemblers occasionally failed to resolve the structure of the mompS-region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the mompS2 allele from short-reads, assess if other short-read methods were able to resolve the mompS-region, and investigate the possibility of using long-reads to obtain the mompS alleles, and thereby perform L. pneumophila SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the mompS-region and thus SBT from short-reads, but no method consistently solved the mompS2 allele. By using Oxford Nanopore Technology (ONT) sequencing together with Trycycler and Medaka for long-read assembly and polishing we were able to resolve the mompS copies and correctly identify the mompS2 allele, in accordance with Sanger sequencing/EQA results for all tested isolates (n=35). The remaining six genes of the SBT profile could also be determined from the ONT-only reads. The STs called from ONT-only assemblies were also consistent with hybrid-assemblies of Illumina and ONT reads. We therefore propose ONT sequencing as an alternative method to perform L. pneumophila SBT to overcome the mompS challenge observed with short-reads. To facilitate this, we have developed ONTmompS (https://github.com/marithetland/ONTmompS), an in silico approach to determine L. pneumophila ST from long-read or hybrid assemblies.

Legionella pneumophila in Municipal Shower Systems in Stavanger, Norway; A Longitudinal Surveillance Study Using Whole Genome Sequencing in Risk Management.

Following an incidence of Legionnaires disease (LD) in 2007, where a municipal shower system was the likely source of infection, Stavanger municipality initiated a surveillance program for Legionella as part of establishing internal risk evaluation and prevention routines. More than 250 shower systems were examined for cultivatable Legionella pneumophila. The prevalence and diversity of serogroups (sg) and sequence types (STs) of L. pneumophila were mapped using available typing techniques over a period of more than 10 years (2010-2021). The surveillance showed an overall reduction in the L. pneumophila colonisation rate in municipal systems from 11 to 4.5% following prevention measures during the period, with the highest colonisation rate in complex systems (e.g., larger nursing homes and sports complexes). Further, an approximately even distribution between sg1 and 2-14 was seen. Whole genome sequencing (WGS) revealed that only a limited number of STs were detected, and they were consistent at specific locations over time. This study showed that environmental surveillance data in combination with available typing techniques and WGS can give the municipality a better tool for risk management and an overview of ST distributions that can be a valuable asset in future source investigations.

Necessity and effect of combating Legionella pneumophila in municipal shower systems.

The objective was to obtain research-based, holistic knowledge about necessity and effect of practiced measures against L. pneumophila in municipal shower systems in Stavanger, Norway. The effects of hot water treatment and membrane-filtering were investigated and compared to no intervention at all. The studies were done under real-world conditions. Additionally, a surveillance pilot study of municipal showers in Stavanger was performed. The validity of high total plate count (TPC) as an indication of L. pneumophila was evaluated. A simplified method, named "dripping method", for detection and quantification of L. pneumophila was developed. The sensitivity of the dripping method is 5 colony-forming units of L. pneumophila/ml. The transference of L. pneumophila from shower water to aerosols was studied. Interviews and observational studies among the stakeholders were done in order to identify patterns of communication and behavior in a Legionella risk perspective. No substantial effects of the measures against L. pneumophila were demonstrated, except for a distally placed membrane filter. No significant positive correlation between TPC and L. pneumophila concentrations were found. L. pneumophila serogroup 2-14 was demonstrated in 21% of the 29 buildings tested in the surveillance pilot. Relatively few cells of L. pneumophila were transferred from shower water to aerosols. Anxiety appeared as the major driving force in the risk governance of Legionella. In conclusion, the risk of acquiring Legionnaires' disease from municipal shower systems is evaluated as low and uncertain. By eliminating ineffective approaches, targeted Legionella risk governance can be practiced. Risk management by surveillance is evaluated as appropriate.

Development of a co-culture model for in vitro toxicological studies in Atlantic salmon.

In vitro assays are needed in order to assess the effects on environmental contaminants in animals. In farmed fish, fatty fish species such as the Atlantic salmon are known to accumulate relatively high levels of persistent organic pollutants. Primary cultures consisting of cells isolated directly from a tissue or organ have traditionally been used in toxicological assessments; however, environmentally unrealistic high doses are often required in order to get a response using fish primary cells. It has been suggested that that the sensitivity of in vitro systems can be significantly improved by adding other cell types to the culture. The aim of this study was therefore to develop and test an in vitro co-culture system consisting of Atlantic salmon hepatocytes and monocytes as a potentially more sensitive model than the mono-cultures of hepatocytes used today. Monocytes isolated from blood were cultured together with primary hepatocytes. Dioxins (2,3,7,8,-TCDD and 1,2,3,7,8-PCDD) were selected as model toxicants and RT-qPCR was used to examine if the co-culture system offered improved sensitivity studying the transcription of important biotransformation and xenobiotic genes. Co-cultivating salmon hepatocytes with monocytes altered the response at the gene transcription level for CYP1a, UGT and bcl-x compared to the conventional hepatocyte mono-culture, indicating that co-culture models are promising models that should be evaluated closer for future in vitro toxicological assessments in fishes.

Endosulfan in vitro toxicity in Atlantic salmon hepatocytes obtained from fish fed either fish oil or vegetable oil.

The composition of the feed may alter the cellular composition of an organism and thus has the potential to influence a xenobiotic response. The main aim of this study was to see if the fatty acid composition of primary hepatocytes isolated from Atlantic salmon (Salmo salar L.) obtained from fish fed either a fish oil or a vegetable oil based diet, influenced the response to endosulfan exposure in vitro. The primary cultures were exposed to six different concentrations of endosulfan (0.001, 0.01, 0.1, 1, 10 and 100 microM) for 48 h. Cell morphology as well as a molecular toolbox of 16 genes encoding stress responsive and biotransformation proteins was examined. Endosulfan exposure caused moderate cytotoxicity and steatosis in a dose-dependent manner in the hepatocytes. In general, endosulfan hepatoxicity seems to be unaffected by the fatty acid composition of the hepatocytes. Exceptions were general stress (HSP70) and markers for estrogen exposure (ZP and VTG), which appeared to be slightly less responsive in hepatocytes isolated from the vegetable oil fed fish.

Transcriptional effects of PFOS in isolated hepatocytes from Atlantic salmon Salmo salar L.

The main aim of the current in vitro experiment was to search for makers for PFOS exposure in isolated hepatocytes from Atlantic salmon Salmo salar, based on genes responding to PFOS exposure in other animals. Primary cell cultures of hepatocytes were exposed to four concentrations of PFOS (2.1-6.2-15.1-25.0 mg/L) for 24 and 48 h and the transcriptional levels of 12 genes encoding proteins known to respond to PFOS were quantified with real-time RT-PCR. The 12 examined genes were caspase 3B (apoptosis), GSH-Px and HSP70 (cellular stress), CYP1A, CYP3A, GST and UGT (P450 and phase II enzymes), acyl-CoA oxidase, PPARalpha, PPARbeta and PPARgamma (lipid metabolism) and Na(+)-K(+)-ATPase (ion regulation). Most of the studied genes responded in a dose-dependent manner to PFOS exposure, although the transcriptional differences in general where small with regard to fold change. Our results clearly suggest that PFOS exposure enhanced cellular stress in the examined cells, even though the exact mechanisms behind this stress remain unknown. The results from this in vitro experiment showed that genes known to be affected by PFOS exposure in other species also were induced in hepatocytes of Atlantic salmon, giving us the rationale to expand to testing the actual in vivo magnitude of effect in Atlantic salmon exposed to PFOS at doses usually seen in nature/diets.

Sexual dimorphic expression pattern of a splice variant of zebrafish vasa during gonadal development.

In Drosophila, the RNA helicase VASA (VAS) is required for both germ line formation and oocyte differentiation. While the murine VAS homologue is required for spermatogenesis, it is dispensable for germ line formation. The molecular basis for this apparently dual role of VAS in germ line ontogeny is, however, unclear. Recent evidence indicates that fish, like flies, employs VAS both in early and late stages of the germ line development and that there is a sex-linked differential expression of splice variants. We show here that the longer of two splice variants of zebrafish vas is transiently downregulated in the germ line around the time when the germ cells reach the developing gonad. Using transgenic vas::EGFP fish lines, which allow us to distinguish between male and female individuals, we show that the long splice variant reappears in both sexes at around day 25 and is subsequently downregulated during male gonadal development. Our data further suggest that there is a switch from maternal to zygotic expression of the long splice variant of vas as sexual dimorphic development commences.

Expression of a vas::EGFP transgene in primordial germ cells of the zebrafish.

In zebrafish, maternally produced vasa (vas) transcripts become targeted to the cleavage planes of early embryos and subsequently incorporated into the primordial germ cells (PGCs). Zygotic vas transcription occurs from the onset of gastrulation. Here, we report on the characterisation of the zebrafish vas locus. The gene consists of 27 exons, spans about 25kb, and contains two CpG-rich regions. We have used vas regulatory regions to establish transgenic zebrafish lines expressing enhanced green fluorescent protein (EGFP) in their PGCs. Maternally encoded vas::EGFP transcripts and VAS::EGFP protein segregate with the PGCs during embryogenesis. We find that the maternally deposited vas::EGFP transcripts are stable during embryogensis at least up to 50h of development. Vas::EGFP transcripts could not be detected in embryos that inherit the transgene from males, most likely due to the lack of one or more regulatory elements required for early zygotic expression. We show that vas::EGFP transcripts become enriched to the cleavage planes in early embryos, a finding that supported an RNA localisation signal localised within the vas region of these transcripts.