Reorganized Genomic Taxonomy of Francisellaceae Enables Design of Robust Environmental PCR Assays for Detection of Francisella tularensis.

In recent years, an increasing diversity of species has been recognized within the family Francisellaceae. Unfortunately, novel isolates are sometimes misnamed in initial publications or multiple sources propose different nomenclature for genetically highly similar isolates. Thus, unstructured and occasionally incorrect information can lead to confusion in the scientific community. Historically, detection of Francisella tularensis in environmental samples has been challenging due to the considerable and unknown genetic diversity within the family, which can result in false positive results. We have assembled a comprehensive collection of genome sequences representing most known Francisellaceae species/strains and restructured them according to a taxonomy that is based on phylogenetic structure. From this structured dataset, we identified a small number of genomic regions unique to F. tularensis that are putatively suitable for specific detection of this pathogen in environmental samples. We designed and validated specific PCR assays based on these genetic regions that can be used for the detection of F. tularensis in environmental samples, such as water and air filters.

Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments.

Francisella tularensis, the causative agent of the zoonotic disease tularemia, can cause seasonal outbreaks of acute febrile illness in humans with disease peaks in late summer to autumn. Interestingly, its mechanisms for environmental persistence between outbreaks are poorly understood. One hypothesis is that F. tularensis forms biofilms in aquatic environments. We utilized two fully virulent wild-type strains: FSC200 (Francisella tularensis subsp. holarctica) and Schu S4 (Francisella tularensis subsp. tularensis) and three control strains, the attenuated live vaccine strain (LVS; F. tularensis subsp. holarctica), a Schu S4 ΔwbtI mutant that is documented to form biofilms, and the low-virulence strain U112 of the closely related species Francisella novicida Strains were incubated in saline solution (0.9% NaCl) microcosms for 24 weeks at both 4°C and 20°C, whereupon viability and biofilm formation were measured. These temperatures were selected to approximate winter and summer temperatures of fresh water in Scandinavia, respectively. U112 and Schu S4 ΔwbtI formed biofilms, but F. tularensis strains FSC200 and Schu S4 and the LVS did not. All strains exhibited prolonged viability at 4°C compared to 20°C. U112 and FSC200 displayed remarkable long-term persistence at 4°C, with only 1- and 2-fold log reductions, respectively, of viable cells after 24 weeks. Schu S4 exhibited lower survival, yielding no viable cells by week 20. At 24 weeks, cells from FSC200, but not from Schu S4, were still fully virulent in mice. Taken together, these results demonstrate biofilm-independent, long-term survival of pathogenic F. tularensis subsp. holarctica in conditions that mimic overwinter survival in aquatic environments.IMPORTANCE Tularemia, a disease caused by the environmental bacterium Francisella tularensis, is characterized by acute febrile illness. F. tularensis is highly infectious: as few as 10 organisms can cause human disease. Tularemia is not known to be spread from person to person. Rather, all human infections are independently acquired from the environment via the bite of blood-feeding arthropods, ingestion of infected food or water, or inhalation of aerosolized bacteria. Despite the environmental origins of human disease events, the ecological factors governing the long-term persistence of F. tularensis in nature between seasonal human outbreaks are poorly understood. The significance of our research is in identifying conditions that promote long-term survival of fully virulent F. tularensis outside a mammalian host or insect vector. These conditions are similar to those found in natural aquatic environments in winter and provide important new insights on how F. tularensis may persist long-term in the environment.

Complete Genome Sequence of Francisella tularensis subsp. holarctica Strain A271_1 (FDC408), Isolated from a Eurasian Beaver (Castor fiber).

Here, we report the complete genome sequence of Francisella tularensis subsp. holarctica strain A271_1, isolated from a Eurasian beaver (Castor fiber) in 2012 in the Berlin/Brandenburg region, Germany.

Accounting for Bacterial Overlap Between Raw Water Communities and Contaminating Sources Improves the Accuracy of Signature-Based Microbial Source Tracking.

Microbial source tracking (MST) analysis is essential to identifying and mitigating the fecal pollution of water resources. The signature-based MST method uses a library of sequences to identify contaminants based on operational taxonomic units (OTUs) that are unique to a certain source. However, no clear guidelines for how to incorporate OTU overlap or natural variation in the raw water bacterial community into MST analyses exist. We investigated how the inclusion of bacterial overlap between sources in the library affects source prediction accuracy. To achieve this, large-scale sampling - including feces from seven species, raw sewage, and raw water samples from water treatment plants - was followed by 16S rRNA amplicon sequencing. The MST library was defined using three settings: (i) no raw water communities represented; (ii) raw water communities selected through clustering analysis; and (iii) local water communities collected across consecutive years. The results suggest that incorporating either the local background or representative bacterial composition improves MST analyses, as the results were positively correlated to measured levels of fecal indicator bacteria and the accuracy at which OTUs were assigned to the correct contamination source increased fourfold. Using the proportion of OTUs with high source origin probability, underpinning a contaminating signal, is a solid foundation in a framework for further deciphering and comparing contaminating signals derived in signature-based MST approaches. In conclusion, incorporating background bacterial composition of water in MST can improve mitigation efforts for minimizing the spread of pathogenic and antibiotic resistant bacteria into essential freshwater resources.

Complete Genome Sequence of Francisella guangzhouensis Strain 08HL01032T, Isolated from Air-Conditioning Systems in China.

We present the complete genome sequence of Francisella guangzhouensis strain 08HL01032(T), which consists of one chromosome (1,658,482 bp) and one plasmid (3,045 bp) with G+C contents of 32.0% and 28.7%, respectively.

Transmission of tularemia from a water source by transstadial maintenance in a mosquito vector.

Mosquitoes are thought to function as mechanical vectors of Francisella tularensis subspecies holarctica (F. t. holarctica) causing tularemia in humans. We investigated the clinical relevance of transstadially maintained F. t. holarctica in mosquitoes. Aedes egypti larvae exposed to a fully virulent F. t. holarctica strain for 24 hours, were allowed to develop into adults when they were individually homogenized. Approximately 24% of the homogenates tested positive for F. t. DNA in PCR. Mice injected with the mosquito homogenates acquired tularemia within 5 days. This novel finding demonstrates the possibility of transmission of bacteria by adult mosquitoes having acquired the pathogen from their aquatic larval habitats.

Brucellosis outbreak in a Swedish kennel in 2013: determination of genetic markers for source tracing.

Brucellosis is a highly infectious zoonotic disease but rare in Sweden. Nonetheless, an outbreak of canine brucellosis caused by an infected dog imported to Sweden was verified in 2013. In total 25 dogs were tested at least duplicated by the following approaches: real-time PCR for the detection of Brucella canis, a Brucella genus-specific real-time PCR, selective cultivation, and microscopic examination. The whole genome of B. canis strain SVA13 was analysed regarding genetic markers for epidemiological examination. The genome of an intact prophage of Roseobacter was detected in B. canis strain SVA13 with whole genome sequence prophage analysis (WGS-PA). It was shown that the prophage gene content in the American, African and European isolates differs remarkably from the Asian strains. The prophage sequences in Brucella may therefore serve of use as genetic markers in epidemiological investigations. Phage DNA fragments were also detected in clustered, regularly interspaced short palindromic repeats (CRISPR) in the genome of strain SVA13. In addition to the recommendations for genetic markers in Brucella outbreak tracing, our paper reports a validated two-step stand-alone real-time PCR for the detection of B. canis and its first successful use in an outbreak investigation.

Complete Genome Sequence of Francisella endociliophora Strain FSC1006, Isolated from a Laboratory Culture of the Marine Ciliate Euplotes raikovi.

A strain of Francisella endociliophora was isolated from a laboratory culture of the marine ciliate Euplotes raikovi. Here, we report the complete genome sequence of the bacterial strain FSC1006 (Francisella Strain Collection, Swedish Defence Research Agency, Umeå, Sweden).

Draft Genome Sequences of Brucella suis Biovar 4 Strain NCTC 10385, Brucella ceti Strain NCTC 12891T, Brucella inopinata Strain CAMP 6436T, and Brucella neotomae Strain ATCC 23459T.

With the aim of developing quantitative PCR methods for the detection and differentiation of Brucella species, the genomes of Brucella ceti, Brucella inopinata, Brucella netotomae, and Brucella suis biovar 4 were sequenced and analyzed.

Whole-Genome Sequence of Brucella canis Strain SVA13, Isolated from an Infected Dog.

An outbreak of canine brucellosis in Sweden was confirmed by the National Veterinary Institute (SVA) in August 2013. The whole genome of the causative agent was sequenced, assembled, and analyzed.

Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden.

In Sweden, human cases of tularemia caused by Francisella tularensis holarctica are assumed to be transmitted by mosquitoes, but how mosquito vectors acquire and transmit the bacterium is not clear. To determine how transmission of this bacterium occurs, mosquito larvae were collected in an area where tularemia is endemic, brought to the laboratory, and reared to adults in their original pond water. Screening of adult mosquitoes by real-time PCR demonstrated F. tularensis lpnA sequences in 14 of the 48 mosquito pools tested; lpnA sequences were demonstrated in 6 of 9 identified mosquito species. Further analysis confirmed the presence of F. tularensis holarctica-specific 30-bp deletion region sequences (FtM19inDel) in water from breeding containers and in 3 mosquito species (Aedes sticticus, Ae. vexans, and Ae. punctor) known to take blood from humans. Our results suggest that the mosquitoes that transmit F. tularensis holarctica during tularemia outbreaks acquire the bacterium already as larvae.

Anthrax outbreak in a Swedish beef cattle herd--1st case in 27 years: Case report.

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.

cDNA-arrays and real-time quantitative PCR techniques in the investigation of chronic Achilles tendinosis.

The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1+/-4.3 (years+/-SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at -80 degrees C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled (32P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrix-metalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue compared with control tissue. Using real-time PCR, 4/5 and 3/5 patients showed up-regulation of MMP-2 and FNRB mRNA, respectively. For decorin, VEGF, and MAPKp38, real-time PCR revealed a great variability among patients. Interestingly, the mRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue.