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.

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.

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.

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.

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.

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.

Cost-effective interrogation of single nucleotide polymorphisms using the mismatch amplification mutation assay and capillary electrophoresis.

The ability to characterize SNPs is an important aspect of many clinical diagnostic, genetic and evolutionary studies. Here, we designed a multiplexed SNP genotyping method to survey a large number of phylogenetically informative SNPs within the genome of the bacterium Bacillus anthracis. This novel method, CE universal tail mismatch amplification mutation assay (CUMA), allows for PCR multiplexing and automatic scoring of SNP genotypes, thus providing a rapid, economical and higher throughput alternative to more expensive SNP genotyping techniques. CUMA delivered accurate B. anthracis SNP genotyping results and, when multiplexed, saved reagent costs by more than 80% compared with TaqMan real-time PCR. When real-time PCR technology and instrumentation is unavailable or the reagents are cost-prohibitive, CUMA is a powerful alternative for SNP genotyping.

A single introduction of Yersinia pestis to Brazil during the 3rd plague pandemic.

Yersinia pestis was introduced to Brazil during the third plague pandemic and currently exists in several recognized foci. There is currently limited available phylogeographic data regarding Y. pestis in Brazil. We generated whole genome sequences for 411 Y. pestis strains from six Brazilian foci to investigate the phylogeography of Y. pestis in Brazil; these strains were isolated from 1966 to 1997. All 411 strains were assigned to a single monophyletic clade within the 1.ORI population, indicating a single Y. pestis introduction was responsible for the successful establishment of endemic foci in Brazil. There was a moderate level of genomic diversity but little population structure among the 411 Brazilian Y. pestis strains, consistent with a radial expansion wherein Y. pestis spread rapidly from the coast to the interior of Brazil and became ecologically established. Overall, there were no strong spatial or temporal patterns among the Brazilian strains. However, strains from the same focus tended to be more closely related and strains isolated from foci closer to the coast tended to fall in more basal positions in the whole genome phylogeny than strains from more interior foci. Overall, the patterns observed in Brazil are similar to other locations affected during the 3rd plague pandemic such as in North America and Madagascar.

Evaluating the long-term persistence of Bacillus spores on common surfaces.

Bacillus spores resist inactivation, but the extent of their persistence on common surfaces is unclear. This work addresses knowledge gaps regarding biothreat agents in the environment to reduce uncertainty in risk assessment models. Studies were conducted to investigate the long-term inactivation of Bacillus anthracis and three commonly used surrogate organisms - B. cereus, B. atrophaeus and B. thuringiensis on three materials: laminate countertop, stainless steel and polystyrene Petri dishes. Viable spores were measured at 1, 30, 90, 196, 304 and 1038 days. Twelve different persistence models were fit to the data using maximum likelihood estimation and compared. The study found that (1) spore inactivation was not log-linear, as commonly modelled; (2) B. thuringiensis counts increased at 24 h on all materials, followed by a subsequent decline; (3) several experiments showed evidence of a 'U' shape, with spore counts apparently decreasing and then increasing between 1 and 304 days; (4) spores on polystyrene showed little inactivation; and (5) the maximum inactivation of 56% was observed for B. atrophaeus spores on steel at 196 days. Over the range of surfaces, time durations and conditions (humidity controlled vs. uncontrolled) examined, B. thuringiensis most closely matched the behaviour of B. anthracis.

Low cost, low tech SNP genotyping tools for resource-limited areas: Plague in Madagascar as a model.

BACKGROUND: Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs. METHODOLOGY/PRINCIPAL FINDINGS: To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort. CONCLUSIONS: The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for resource constraint laboratories. This is a practical formula that reduces resource-driven limitations to genetic research and promises to advance global collective knowledge of infectious diseases emanating from resource limited regions of the world.

Temporal phylogeography of Yersinia pestis in Madagascar: Insights into the long-term maintenance of plague.

BACKGROUND: Yersinia pestis appears to be maintained in multiple, geographically separate, and phylogenetically distinct subpopulations within the highlands of Madagascar. However, the dynamics of these locally differentiated subpopulations through time are mostly unknown. To address that gap and further inform our understanding of plague epidemiology, we investigated the phylogeography of Y. pestis in Madagascar over an 18 year period. METHODOLOGY/PRINCIPAL FINDINGS: We generated whole genome sequences for 31 strains and discovered new SNPs that we used in conjunction with previously identified SNPs and variable-number tandem repeats (VNTRs) to genotype 773 Malagasy Y. pestis samples from 1995 to 2012. We mapped the locations where samples were obtained on a fine geographic scale to examine phylogeographic patterns through time. We identified 18 geographically separate and phylogenetically distinct subpopulations that display spatial and temporal stability, persisting in the same locations over a period of almost two decades. We found that geographic areas with higher levels of topographical relief are associated with greater levels of phylogenetic diversity and that sampling frequency can vary considerably among subpopulations and from year to year. We also found evidence of various Y. pestis dispersal events, including over long distances, but no evidence that any dispersal events resulted in successful establishment of a transferred genotype in a new location during the examined time period. CONCLUSIONS/SIGNIFICANCE: Our analysis suggests that persistent endemic cycles of Y. pestis transmission within local areas are responsible for the long term maintenance of plague in Madagascar, rather than repeated episodes of wide scale epidemic spread. Landscape likely plays a role in maintaining Y. pestis subpopulations in Madagascar, with increased topographical relief associated with increased levels of localized differentiation. Local ecological factors likely affect the dynamics of individual subpopulations and the associated likelihood of observing human plague cases in a given year in a particular location.

Genetic variation at the MHC DRB1 locus is similar across Gunnison's prairie dog (Cynomys gunnisoni) colonies regardless of plague history.

Yersinia pestis was introduced to North America around 1900 and leads to nearly 100% mortality in prairie dog (Cynomys spp.) colonies during epizootic events, which suggests this pathogen may exert a strong selective force. We characterized genetic diversity at an MHC class II locus (DRB1) in Gunnison's prairie dog (C. gunnisoni) and quantified population genetic structure at the DRB1 versus 12 microsatellite loci in three large Arizona colonies. Two colonies, Seligman (SE) and Espee Ranch (ES), have experienced multiple plague-related die-offs in recent years, whereas plague has never been documented at Aubrey Valley (AV). We found fairly low allelic diversity at the DRB1 locus, with one allele (DRB1*01) at high frequency (0.67-0.87) in all colonies. Two other DRB1 alleles appear to be trans-species polymorphisms shared with the black-tailed prairie dog (C. ludovicianus), indicating that these alleles have been maintained across evolutionary time frames. Estimates of genetic differentiation were generally lower at the MHC locus (F ST = 0.033) than at microsatellite markers (F ST = 0.098). The reduced differentiation at DRB1 may indicate that selection has been important for shaping variation at MHC loci, regardless of the presence or absence of plague in recent decades. However, genetic drift has probably also influenced the DRB1 locus because its level of differentiation was not different from that of microsatellites in an F ST outlier analysis. We then compared specific MHC alleles to plague survivorship in 60 C. gunnisoni that had been experimentally infected with Y. pestis. We found that survival was greater in individuals that carried at least one copy of the most common allele (DRB1*01) compared to those that did not (60% vs. 20%). Although the sample sizes of these two groups were unbalanced, this result suggests the possibility that this MHC class II locus, or a nearby linked gene, could play a role in plague survival.

Whole Genome Analysis of Injectional Anthrax Identifies Two Disease Clusters Spanning More Than 13 Years.

BACKGROUND: Anthrax is a rare disease in humans but elicits great public fear because of its past use as an agent of bioterrorism. Injectional anthrax has been occurring sporadically for more than ten years in heroin consumers across multiple European countries and this outbreak has been difficult to trace back to a source. METHODS: We took a molecular epidemiological approach in understanding this disease outbreak, including whole genome sequencing of Bacillus anthracis isolates from the anthrax victims. We also screened two large strain repositories for closely related strains to provide context to the outbreak. FINDINGS: Analyzing 60 Bacillus anthracis isolates associated with injectional anthrax cases and closely related reference strains, we identified 1071 Single Nucleotide Polymorphisms (SNPs). The synapomorphic SNPs (350) were used to reconstruct phylogenetic relationships, infer likely epidemiological sources and explore the dynamics of evolving pathogen populations. Injectional anthrax genomes separated into two tight clusters: one group was exclusively associated with the 2009-10 outbreak and located primarily in Scotland, whereas the second comprised more recent (2012-13) cases but also a single Norwegian case from 2000. INTERPRETATION: Genome-based differentiation of injectional anthrax isolates argues for at least two separate disease events spanning > 12 years. The genomic similarity of the two clusters makes it likely that they are caused by separate contamination events originating from the same geographic region and perhaps the same site of drug manufacturing or processing. Pathogen diversity within single patients challenges assumptions concerning population dynamics of infecting B. anthracis and host defensive barriers for injectional anthrax. FUNDING: This work was supported by the United States Department of Homeland Security grant no. HSHQDC-10-C-00,139 and via a binational cooperative agreement between the United States Government and the Government of Germany. This work was supported by funds from the German Ministry of Defense (Sonderforschungsprojekt 25Z1-S-431,214). Support for sequencing was also obtained from Illumina, Inc. These sources had no role in the data generation or interpretation, and had not role in the manuscript preparation. PANEL 1 RESEARCH IN CONTEXT SYSTEMATIC REVIEW: We searched PubMed for any article published before Jun. 17, 2015, with the terms "Bacillus anthracis" and "heroin", or "injectional anthrax". Other than our previously published work (Price et al., 2012), we found no other relevant studies on elucidating the global phylogenetic relationships of B. anthracis strains associated with injectional anthrax caused by recreational heroin consumption of spore-contaminated drug. There were, however, publically available genome sequences of two strains involved (Price et al., 2012, Grunow et al., 2013) and the draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user (Ruckert et al., 2012) with only limited information on the genotyping of closely related strains (Price et al., 2012, Grunow et al., 2013). LAY PERSON INTERPRETATION: Injectional anthrax has been plaguing heroin drug users across Europe for more than 10 years. In order to better understand this outbreak, we assessed genomic relationships of all available injectional anthrax strains from four countries spanning a > 12 year period. Very few differences were identified using genome-based analysis, but these differentiated the isolates into two distinct clusters. This strongly supports a hypothesis of at least two separate anthrax spore contamination events perhaps during the drug production processes. Identification of two events would not have been possible from standard epidemiological analysis. These comprehensive data will be invaluable for classifying future injectional anthrax isolates and for future geographic attribution.