Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Transmission of Antimicrobial Resistant Yersinia pestis During a Pneumonic Plague Outbreak.

BACKGROUND: Pneumonic plague (PP), caused by Yersinia pestis, is the most feared clinical form of plague due to its rapid lethality and potential to cause outbreaks. PP outbreaks are now rare due to antimicrobial therapy. METHODS: A PP outbreak in Madagascar involving transmission of a Y. pestis strain resistant to streptomycin, the current recommended first-line treatment in Madagascar, was retrospectively characterized using epidemiology, clinical diagnostics, molecular characterization, and animal studies. RESULTS: The outbreak occurred in February 2013 in the Faratsiho district of Madagascar and involved 22 cases, including 3 untreated fatalities. The 19 other cases participated in funeral practices for the fatal cases and fully recovered after combination antimicrobial therapy: intramuscular streptomycin followed by oral co-trimoxazole. The Y. pestis strain that circulated during this outbreak is resistant to streptomycin resulting from a spontaneous point mutation in the 30S ribosomal protein S12 (rpsL) gene. This same mutation causes streptomycin resistance in 2 unrelated Y. pestis strains, one isolated from a fatal PP case in a different region of Madagascar in 1987 and another isolated from a fatal PP case in China in 1996, documenting this mutation has occurred independently at least 3 times in Y. pestis. Laboratory experiments revealed this mutation has no detectable impact on fitness or virulence, and revertants to wild-type are rare in other species containing it, suggesting Y. pestis strains containing it could persist in the environment. CONCLUSIONS: Unique antimicrobial resistant (AMR) strains of Y. pestis continue to arise in Madagascar and can be transmitted during PP outbreaks.

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.

Phylogenetic Analysis of Francisella tularensis Group A.II Isolates from 5 Patients with Tularemia, Arizona, USA, 2015-2017.

We examined 5 tularemia cases in Arizona, USA, during 2015-2017. All were caused by Francisella tularensis group A.II. Genetically similar isolates were found across large spatial and temporal distances, suggesting that group A.II strains are dispersed across long distances by wind and exhibit low replication rates in the environment.

Pneumonic Plague Transmission, Moramanga, Madagascar, 2015.

During a pneumonic plague outbreak in Moramanga, Madagascar, we identified 4 confirmed, 1 presumptive, and 9 suspected plague case-patients. Human-to-human transmission among close contacts was high (reproductive number 1.44) and the case fatality rate was 71%. Phylogenetic analysis showed that the Yersinia pestis isolates belonged to group q3, different from the previous outbreak.

Clonality of erythromycin resistance in Francisella tularensis.

OBJECTIVES: We analysed diverse strains of Francisella tularensis subsp. holarctica to assess if its division into biovars I and II is associated with specific mutations previously linked to erythromycin resistance and to determine the distribution of this resistance trait across this subspecies. METHODS: Three-hundred and fourteen F. tularensis subsp. holarctica strains were tested for erythromycin susceptibility and whole-genome sequences for these strains were examined for SNPs in genes previously associated with erythromycin resistance. Each strain was assigned to a global phylogenetic framework using genome-wide canonical SNPs. The contribution of a specific SNP to erythromycin resistance was examined using allelic exchange. The geographical distribution of erythromycin-resistant F. tularensis strains was further investigated by literature search. RESULTS: There was a perfect correlation between biovar II strains (erythromycin resistance) and the phylogenetic group B.12. Only B.12 strains had an A → C SNP at position 2059 in the three copies of the rrl gene. Introducing 2059C into an rrl gene of an erythromycin-susceptible F. tularensis strain resulted in resistance. An additional 1144 erythromycin-resistant strains were identified from the scientific literature, all of them from Eurasia. CONCLUSIONS: Erythromycin resistance in F. tularensis is caused by an A2059C rrl gene mutation, which exhibits a strictly clonal inheritance pattern found only in phylogenetic group B.12. This group is an extremely successful clone, representing the most common type of F. tularensis throughout Eurasia.

Water as Source of Francisella tularensis Infection in Humans, Turkey.

Francisella tularensis DNA extractions and isolates from the environment and humans were genetically characterized to elucidate environmental sources that cause human tularemia in Turkey. Extensive genetic diversity consistent with genotypes from human outbreaks was identified in environmental samples and confirmed water as a source of human tularemia in Turkey.

High Prevalence of Intermediate Leptospira spp. DNA in Febrile Humans from Urban and Rural Ecuador.

Leptospira spp., which comprise 3 clusters (pathogenic, saprophytic, and intermediate) that vary in pathogenicity, infect >1 million persons worldwide each year. The disease burden of the intermediate leptospires is unclear. To increase knowledge of this cluster, we used new molecular approaches to characterize Leptospira spp. in 464 samples from febrile patients in rural, semiurban, and urban communities in Ecuador; in 20 samples from nonfebrile persons in the rural community; and in 206 samples from animals in the semiurban community. We observed a higher percentage of leptospiral DNA-positive samples from febrile persons in rural (64%) versus urban (21%) and semiurban (25%) communities; no leptospires were detected in nonfebrile persons. The percentage of intermediate cluster strains in humans (96%) was higher than that of pathogenic cluster strains (4%); strains in animal samples belonged to intermediate (49%) and pathogenic (51%) clusters. Intermediate cluster strains may be causing a substantial amount of fever in coastal Ecuador.

Yersinia pestis DNA from skeletal remains from the 6(th) century AD reveals insights into Justinianic Plague.

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19(th) and 20(th) centuries, during which plague was spread around the world, and the second pandemic of the 14(th)-17(th) centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6(th)-8(th) centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.

An outbreak of respiratory tularemia caused by diverse clones of Francisella tularensis.

BACKGROUND: The bacterium Francisella tularensis is recognized for its virulence, infectivity, genetic homogeneity, and potential as a bioterrorism agent. Outbreaks of respiratory tularemia, caused by inhalation of this bacterium, are poorly understood. Such outbreaks are exceedingly rare, and F. tularensis is seldom recovered from clinical specimens. METHODS: A localized outbreak of tularemia in Sweden was investigated. Sixty-seven humans contracted laboratory-verified respiratory tularemia. F. tularensis subspecies holarctica was isolated from the blood or pleural fluid of 10 individuals from July to September 2010. Using whole-genome sequencing and analysis of single-nucleotide polymorphisms (SNPs), outbreak isolates were compared with 110 archived global isolates. RESULTS: There were 757 SNPs among the genomes of the 10 outbreak isolates and the 25 most closely related archival isolates (all from Sweden/Finland). Whole genomes of outbreak isolates were >99.9% similar at the nucleotide level and clustered into 3 distinct genetic clades. Unexpectedly, high-sequence similarity grouped some outbreak and archival isolates that originated from patients from different geographic regions and up to 10 years apart. Outbreak and archival genomes frequently differed by only 1-3 of 1 585 229 examined nucleotides. CONCLUSIONS: The outbreak was caused by diverse clones of F. tularensis that occurred concomitantly, were widespread, and apparently persisted in the environment. Multiple independent acquisitions of F. tularensis from the environment over a short time period suggest that natural outbreaks of respiratory tularemia are triggered by environmental cues. The findings additionally caution against interpreting genome sequence identity for this pathogen as proof of a direct epidemiological link.

Diversity of Francisella tularensis subsp. holarctica lineages, China.

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.

Francisella tularensis subsp. tularensis group A.I, United States.

We used whole-genome analysis and subsequent characterization of geographically diverse strains using new genetic signatures to identify distinct subgroups within Francisella tularensis subsp. tularensis group A.I: A.I.3, A.I.8, and A.I.12. These subgroups exhibit complex phylogeographic patterns within North America. The widest distribution was observed for A.I.12, which suggests an adaptive advantage.

High prevalence and two dominant host-specific genotypes of Coxiella burnetii in U.S. milk.

BACKGROUND: Coxiella burnetii causes Q fever in humans and Coxiellosis in animals; symptoms range from general malaise to fever, pneumonia, endocarditis and death. Livestock are a significant source of human infection as they shed C. burnetii cells in birth tissues, milk, urine and feces. Although prevalence of C. burnetii is high, few Q fever cases are reported in the U.S. and we have a limited understanding of their connectedness due to difficulties in genotyping. Here, we develop canonical SNP genotyping assays to evaluate spatial and temporal relationships among C. burnetii environmental samples and compare them across studies. Given the genotypic diversity of historical collections, we hypothesized that the current enzootic of Coxiellosis is caused by multiple circulating genotypes. We collected A) 23 milk samples from a single bovine herd, B) 134 commercial bovine and caprine milk samples from across the U.S., and C) 400 bovine and caprine samples from six milk processing plants over three years. RESULTS: We detected C. burnetii DNA in 96% of samples with no variance over time. We genotyped 88.5% of positive samples; bovine milk contained only a single genotype (ST20) and caprine milk was dominated by a second type (mostly ST8). CONCLUSIONS: The high prevalence and lack of genotypic diversity is consistent with a model of rapid spread and persistence. The segregation of genotypes between host species is indicative of species-specific adaptations or dissemination barriers and may offer insights into the relative lack of human cases and characterizing genotypes.

The activity of human aquaporin 1 as a cGMP-gated cation channel is regulated by tyrosine phosphorylation in the carboxyl-terminal domain.

In addition to a constitutive water channel activity, several studies suggest Aquaporin-1 (AQP1) functions as a nonselective monovalent cation channel activated by intracellular cGMP, although variability in responsiveness between preparations has led to controversy in the field. Data here support the hypothesis that responsiveness of the AQP1 ionic conductance to cGMP is governed by tyrosine phosphorylation. Wild-type and mutant human AQP1 channels expressed in Xenopus laevis oocytes were characterized by two-electrode voltage clamp and optical osmotic swelling analyses. Quadruple mutation by site-directed mutagenesis of barrier hydrophobic residues (Val50, Leu54, Leu170, Leu174) to alanines in the central pore induced inward rectification of the ionic current and shifted reversal potential by approximately +10 mV, indicating increased permeability of tetraethylammonium ion. Introduction of cysteine at lysine 51 in the central pore (K51C) in a cysteine-less template created new sensitivity to block of the conductance by mercuric ion. Mutations of candidate consensus sites and pharmacological manipulation of serine and threonine phosphorylation did not alter cGMP-dependent responses; however, mutation of tyrosine Y253C or pharmacological dephosphorylation prevented ion channel activation. Modification of Y253C by covalent addition of a negatively charged group [2-sulfonatoethyl methanethiosulfonate sodium salt (MTSES)] rescued the cGMP-activated conductance response, an effect reversed by dithiothreitol. Results support the proposal that phosphorylation of tyrosine Tyr253 in the carboxyl terminal domain, confirmed by Western blot, acts as a master switch regulating responsiveness of AQP1 ion channels to cGMP, and the tetrameric central pore is the ion permeation pathway. These findings advance resolution of a standing controversy and expand our understanding of AQP1 as a multifunctional regulated channel.

Phylogeography of Francisella tularensis subsp. holarctica, Europe.

Francisella tularensis subsp. holarctica isolates from Austria, Germany, Hungary, Italy, and Romania were placed into an existing phylogeographic framework. Isolates from Italy were assigned to phylogenetic group B.FTNF002-00; the other isolates, to group B.13. Most F. tularensis subsp. holarctica isolates from Europe belong to these 2 geographically segregated groups.

Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples.

Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.

Proteomic Signatures of Antimicrobial Resistance in Yersinia pestis and Francisella tularensis.

Antimicrobial resistance (AMR) is a well-recognized, widespread, and growing issue of concern. With increasing incidence of AMR, the ability to respond quickly to infection with or exposure to an AMR pathogen is critical. Approaches that could accurately and more quickly identify whether a pathogen is AMR also are needed to more rapidly respond to existing and emerging biological threats. We examined proteins associated with paired AMR and antimicrobial susceptible (AMS) strains of Yersinia pestis and Francisella tularensis, causative agents of the diseases plague and tularemia, respectively, to identify whether potential existed to use proteins as signatures of AMR. We found that protein expression was significantly impacted by AMR status. Antimicrobial resistance-conferring proteins were expressed even in the absence of antibiotics in growth media, and the abundance of 10-20% of cellular proteins beyond those that directly confer AMR also were significantly changed in both Y. pestis and F. tularensis. Most strikingly, the abundance of proteins involved in specific metabolic pathways and biological functions was altered in all AMR strains examined, independent of species, resistance mechanism, and affected cellular antimicrobial target. We have identified features that distinguish between AMR and AMS strains, including a subset of features shared across species with different resistance mechanisms, which suggest shared biological signatures of resistance. These features could form the basis of novel approaches to identify AMR phenotypes in unknown strains.

Co-Occurrence of Francisella, Spotted Fever Group Rickettsia, and Midichloria in Avian-Associated Hyalomma rufipes.

The migratory behavior of wild birds contributes to the geographical spread of ticks and their microorganisms. In this study, we aimed to investigate the dispersal and co-occurrence of Francisella and spotted fever group Rickettsia (SFGR) in ticks infesting birds migrating northward in the African-Western Palaearctic region (AWPR). Birds were trapped with mist nests across the Mediterranean basin during the 2014 and 2015 spring migration. In total, 575 ticks were collected from 244 birds. We screened the ticks for the species Francisella tularensis, the genus Francisella, and SFGR by microfluidic real-time PCR. Confirmatory analyses and metagenomic sequencing were performed on tick samples that putatively tested positive for F. tularensis during initial screenings. Hyalomma rufipes was the most common tick species and had a high prevalence of Francisella, including co-occurrence of Francisella and SFGR. Metagenomic analysis of total DNA extracted from two H. rufipes confirmed the presence of Francisella, Rickettsia, and Midichloria. Average nucleotide identity and phylogenetic inference indicated the highest identity of the metagenome-assembled genomes to a Francisella-like endosymbiont (FLE), Rickettsia aeschlimannii, and Midichloria mitochondrii. The results of this study suggest that (i) FLE- and SFGR-containing ticks are dispersed by northbound migratory birds in the AWPR, (ii) H. rufipes likely is not involved in transmission of F. tularensis in the AWPR, and (iii) a dual endosymbiosis of FLEs and Midichloria may support some of the nutritional requirements of H. rufipes.

Phylogeography of Francisella tularensis subspecies holarctica from the country of Georgia.

BACKGROUND: Francisella tularensis, the causative agent of tularemia, displays subspecies-specific differences in virulence, geographic distribution, and genetic diversity. F. tularensis subsp. holarctica is widely distributed throughout the Northern Hemisphere. In Europe, F. tularensis subsp. holarctica isolates have largely been assigned to two phylogenetic groups that have specific geographic distributions. Most isolates from Western Europe are assigned to the B.Br.FTNF002-00 group, whereas most isolates from Eastern Europe are assigned to numerous lineages within the B.Br.013 group. The eastern geographic extent of the B.Br.013 group is currently unknown due to a lack of phylogenetic knowledge about populations at the European/Asian juncture and in Asia. In this study, we address this knowledge gap by describing the phylogenetic structure of F. tularensis subsp. holarctica isolates from the country of Georgia, and by placing these isolates into a global phylogeographic context. RESULTS: We identified a new genetic lineage of F. tularensis subsp. holarctica from Georgia that belongs to the B.Br.013 group. This new lineage is genetically and geographically distinct from lineages previously described from the B.Br.013 group from Central-Eastern Europe. Importantly, this new lineage is basal within the B.Br.013 group, indicating the Georgian lineage diverged before the diversification of the other known B.Br.013 lineages. Although two isolates from the Georgian lineage were collected nearby in the Ukrainian region of Crimea, all other global isolates assigned to this lineage were collected in Georgia. This restricted geographic distribution, as well as the high levels of genetic diversity within the lineage, is consistent with a relatively older origin and localized differentiation. CONCLUSIONS: We identified a new lineage of F. tularensis subsp. holarctica from Georgia that appears to have an older origin than any other diversified lineages previously described from the B.Br.013 group. This finding suggests that additional phylogenetic studies of F. tularensis subsp. holarctica populations in Eastern Europe and Asia have the potential to yield important new insights into the evolutionary history and phylogeography of this broadly dispersed F. tularensis subspecies.