A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and ...gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis.
Summer outbreaks of tularemia that occurred from 1995 through 2005 in 2 locations in Sweden affected 441 persons. We performed an epidemiologic investigation of these outbreaks using a novel ...strategy, involving high-resolution genotyping of Francisella tularensis isolates obtained from 136 patients (using 18 genetic markers developed from 6 F. tularensis genome sequences) and interviews with the patients. Strong spatial associations were found between F. tularensis subpopulations and the places of disease transmission; infection by some subpopulations occurred within areas as small as 2 km(2), indicating unidentified environmental point sources of tularemia. In both locations, disease clusters were associated with recreational areas beside water, and genetic subpopulations were present throughout the tularemia season and persisted over years. High-resolution genotyping in combination with patients' statements about geographic places of disease transmission provided valuable indications of likely sources of infection and the causal genotypes during these tularemia outbreaks.
•Mutations accumulate in specific genes during laboratory culture.•The mutational profile of a laboratory cultured strain is different from wild strain.•Low frequency mutations are amplified by ...serial passaging in the laboratory.•Biological enrichment of mutations can link bacterial culture to its source.•Highly similar bacteria can be resolved by low frequency variants in the population.
Challenges of investigating a suspected bio attack include establishing if microorganisms have been cultured to produce attack material and to identify their source. Addressing both issues, we have investigated genetic variations that emerge during laboratory culturing of the bacterial pathogen Francisella tularensis. Key aims were to identify genetic variations that are characteristic of laboratory culturing and explore the possibility of using biological amplification to identify genetic variation present at exceedingly low frequencies in a source sample. We used parallel serial passage experiments and high-throughput sequencing of F. tularensis to explore the genetic variation. We found that during early laboratory culture passages of F. tularensis, gene duplications emerged in the pathogen genome followed by single-nucleotide polymorphisms in genes for bacterial capsule synthesis. Based on a biological enrichment scheme and the use of high-throughput sequencing, we identified genetic variation that likely pre-existed in a source sample. The results support that capsule synthesis gene mutations are common during laboratory culture, and that a biological amplification strategy is useful for linking a F. tularensis sample to a specific laboratory variant among many highly similar variants.
Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed ...a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.
Sequence-based typing of
has led to insights in the evolutionary developments of tularemia. In Europe, two major basal clades of
subsp.
exist, with a distinct geographical distribution. Basal clade ...B.6 is primarily found in Western Europe, while basal clade B.12 occurs predominantly in the central and eastern parts of Europe. There are indications that tularemia is geographically expanding and that strains from the two clades might differ in pathogenicity, with basal clade B.6 strains being potentially more virulent than basal clade B.12. This study provides information on genotypes detected in the Netherlands during 2011-2017. Data are presented for seven autochthonous human cases and for 29 European brown hares (
) with laboratory confirmed tularemia. Associated disease patterns are described for 25 European brown hares which underwent post-mortem examination. The basal clades B.6 and B.12 are present both in humans and in European brown hares in the Netherlands, with a patchy geographical distribution. For both genotypes the main pathological findings in hares associated with tularemia were severe (sub)acute necrotizing hepatitis and splenitis as well as necrotizing lesions and hemorrhages in several other organs. Pneumonia was significantly more common in the B.6 than in the B.12 cases. In conclusion, the two major basal clades present in different parts in Europe are both present in the Netherlands. In hares found dead, both genotypes were associated with severe acute disease affecting multiple organs. Hepatitis and splenitis were common pathological findings in hares infected with either genotype, but pneumonia occurred significantly more frequently in hares infected with the B.6 genotype compared to hares infected with the B.12 genotype.
Here we report the complete, accurate 1.89-Mb genome sequence of Francisella tularensis subsp. holarctica strain FSC200, isolated in 1998 in the Swedish municipality Ljusdal, which is in an area ...where tularemia is highly endemic. This genome is important because strain FSC200 has been extensively used for functional and genetic studies of Francisella and is well-characterized.
To develop effective and accurate typing of strains of Francisella tularensis, a potent human pathogen and a putative bioterrorist agent, we combined analysis of insertion-deletion (indel) markers ...with multiple-locus variable-number tandem repeat analysis (MLVA). From 5 representative F. tularensis genome sequences, 38 indel markers with canonical properties, i.e., capable of sorting strains into major genetic groups, were selected. To avoid markers with a propensity for homoplasy, we used only those indels with 2 allelic variants and devoid of substantial sequence repeats. MLVA included sequences with much diversity in copy number of tandem repeats. The combined procedure allowed subspecies division, delineation of clades A.I and A.II of subspecies tularensis, differentiation of Japanese strains from other strains of subspecies holarctica, and high-resolution strain typing. The procedure uses limited amounts of killed bacterial preparations and, because only 1 single analytic method is needed, is time- and cost-effective.
After 27 years with no detected cases, an outbreak of anthrax occurred in a beef cattle herd in the south of Sweden. The outbreak was unusual as it occurred in winter, in animals not exposed to ...meat-and-bone meal, in a non-endemic country. The affected herd consisted of 90 animals, including calves and young stock. The animals were kept in a barn on deep straw bedding and fed only roughage. Seven animals died during 10 days, with no typical previous clinical signs except fever. The carcasses were reportedly normal in appearance, particularly as regards rigor mortis, bleeding and coagulation of the blood. Subsequently, three more animals died and anthrax was suspected at necropsy and confirmed by culture and PCR on blood samples. The isolated strain was susceptible to tetracycline, ciprofloxacin and ampicillin. Subtyping by MLVA showed the strain to cluster with isolates in the A lineage of Bacillus anthracis. Environmental samples from the holding were all negative except for two soil samples taken from a spot where infected carcasses had been kept until they were picked up for transport. The most likely source of the infection was concluded to be contaminated roughage, although this could not be substantiated by laboratory analysis. The suspected feed was mixed with soil and dust and originated from fields where flooding occurred the previous year, followed by a dry summer with a very low water level in the river allowing for the harvesting on soil usually not exposed. In the early 1900s, animal carcasses are said to have been dumped in this river during anthrax outbreaks and it is most likely that some anthrax spores could remain in the area. The case indicates that untypical cases in non-endemic areas may be missed to a larger extent than previously thought. Field tests allowing a preliminary risk assessment of animal carcasses would be helpful for increased sensitivity of detection and prevention of further exposure to the causative agent.
•Land use influenced bacteria in lake inflows, enabling downstream consequences.•Standard fecal indicators are insufficient to detect downstream effects from land use.•Associated with inflows and ...land use, Legionella exemplifies downstream effects.•Reliable access to clean water needs consideration of indirect downstream effects.•This framework can assess new sources in case of drinking water disturbances.
Intensified land use can disturb water quality, potentially increasing the abundance of bacterial pathogens, threatening public access to clean water. This threat involves both direct contamination of faecal bacteria as well as indirect factors, such as disturbed water chemistry and microbiota, which can lead to contamination. While direct contamination has been well described, the impact of indirect factors is less explored, despite the potential of severe downstream consequences on water supply. To assess direct and indirect downstream effects of buildings, farms, pastures and fields on potential water sources, we studied five Swedish lakes and their inflows. We analysed a total of 160 samples in a gradient of anthropogenic activity spanning four time points, including faecal and water-quality indicators. Through species distribution modelling, Random Forest and network analysis using 16S rRNA amplicon sequencing data, our findings highlight that land use indirectly impacts lakes via inflows. Land use impacted approximately one third of inflow microbiota taxa, in turn impacting ∼20–50 % of lake taxa. Indirect effects via inflows were also suggested by causal links between e.g. water colour and lake bacterial taxa, where this influenced the abundance of several freshwater bacteria, such as Polynucleobacter and Limnohabitans. However, it was not possible to identify direct effects on the lakes based on analysis of physiochemical- or microbial parameters. To avoid potential downstream consequences on water supply, it is thus important to consider possible indirect effects from upstream land use and inflows, even when no direct effects can be observed on lakes. Legionella (a genus containing bacterial pathogens) illustrated potential consequences, since the genus was particularly abundant in inflows and was shown to increase by the presence of pastures, fields, and farms. The approach presented here could be used to assess the suitability of lakes as alternative raw water sources or help to mitigate contaminations in important water catchments. Continued broad investigations of stressors on the microbial network can identify indirect effects, avoid enrichment of pathogens, and help secure water accessibility.
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