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Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796–1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1–2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei. Author summary: Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to Southeast Asia and northern Australia. The evaluation of diversity within B. pseudomallei has largely been conducted utilizing clinical isolates despite almost all infections emerging from environmental exposure. In this study, we surveyed the genomic diversity of 169 isolates collected from a single soil sample in Ubon Ratchathani Province, Thailand. Seven different sequence types were identified, and substantial within-sequence-type gene diversity was observed. To test for differential virulence, 30 isolates were challenged in a mouse melioidosis model. A small number of isolates killed all mice, but most killed none, demonstrating the variable virulence potential of different B. pseudomallei isolates present in the single sample. A comparative genomics analysis identified multiple genes associated with virulence, including Hcp1, a component of the type VI secretion system and a known virulence factor. The results of this study have implications for the comprehensive environmental surveillance, environmental exposure, and differential virulence of B. pseudomallei. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro . These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used. [ABSTRACT FROM AUTHOR]

Background: Yersinia pestis is the causative agent of human plague and is endemic in various African, Asian and American countries. In Madagascar, the disease represents a significant public health problem with hundreds of human cases a year. Unfortunately, poor infrastructure makes outbreak investigations challenging. Methodology/Principal Findings: DNA was extracted directly from 93 clinical samples from patients with a clinical diagnosis of plague in Madagascar in 2007. The extracted DNAs were then genotyped using three molecular genotyping methods, including, single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. These methods provided increasing resolution, respectively. The results of these analyses revealed that, in 2007, ten molecular groups, two newly described here and eight previously identified, were responsible for causing human plague in geographically distinct areas of Madagascar. Conclusions/Significance: Plague in Madagascar is caused by numerous distinct types of Y. pestis. Genotyping method choice should be based upon the discriminatory power needed, expense, and available data for any desired comparisons. We conclude that genotyping should be a standard tool used in epidemiological investigations of plague outbreaks. Author Summary: Yersinia pestis is a highly pathogenic bacterium and the causative agent of human plague. It has caused three recognized pandemics and is a current human health problem in many countries of Africa, Asia and the Americas, including Madagascar. The pathogen cannot be eradicated from natural plague foci as it persists in various known and cryptic rodent reservoir species. Genotyping is a critical tool in understanding the molecular epidemiology and possible kinetics of plague. In the present study, we succeeded in extracting DNA and genotyping directly from human clinical samples from Madagascar. We applied three different methods, including single nucleotide polymorphism (SNP) typing, multi-locus variable-number tandem repeat (VNTR) analysis (MLVA), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) analysis. Relative to their discriminatory power, all three methods provided important genotype information useful for understanding the molecular epidemiology of the disease, revealing that multiple, distinct genotypes caused human plague in Madagascar within one year, 2007. [ABSTRACT FROM AUTHOR]

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited. [ABSTRACT FROM AUTHOR]

Background Rhipicephalus (Boophilus) microplus is a highly-invasive tick that transmits the cattle parasites (Babesia bovis and B. bigemina) that cause cattle fever. R. microplus and Babesia are endemic in Mexico and ticks persist in the United States inside a narrow tick eradication quarantine area (TEQA) along the Rio Grande. This containment area is threatened by unregulated movements of illegal cattle and wildlife like white-tailed deer (WTD; Odocoileus virginianus). Methods Using 11 microsatellite loci we genotyped 1,247 R. microplus from 63 Texas collections, including outbreak infestations from outside the TEQA. We used population genetic analyses to test hypotheses about ecological persistence, tick movement, and impacts of the eradication program in southern Texas. We tested acaricide resistance with larval packet tests (LPTs) on 47 collections. Results LPTs revealed acaricide resistance in 15/47 collections (32%); 11 were outside the TEQA and three were resistant to multiple acaricides. Some collections highly resistant to permethrin were found on cattle and WTD. Analysis of genetic differentiation over time at seven properties revealed local gene pools with very low levels of differentiation (FST 0.00- 0.05), indicating persistence over timespans of up to 29 months. However, in one neighborhood differentiation varied greatly over a 12-month period (FST 0.03-0.13), suggesting recurring immigration from distinct sources as another persistence mechanism. Ticks collected from cattle and WTD at the same location are not differentiated (FST = 0), implicating ticks from WTD as a source of ticks on cattle (and vice versa) and emphasizing the importance of WTD to tick control strategies. We identified four major genetic groups (K = 4) using Bayesian population assignment, suggesting multiple introductions to Texas. Conclusions Two dispersal mechanisms give rise to new tick infestations: 1) frequent short-distance dispersal from the TEQA; and 2) rare long-distance, human-mediated dispersal from populations outside our study area, probably Mexico. The threat of cattle fever tick transport into Texas is increased by acaricide resistance and the ability of R. microplus to utilize WTD as an alternate host. Population genetic analyses may provide a powerful tool for tracking invasions in other parts of the world where these ticks are established. [ABSTRACT FROM AUTHOR]

The article discusses study that examined potential mechanisms accounting for survival of a colony Gunnison's prairie dogs in the Aubrey Valley (AV) of northern Arizona. Prairie dogs are highly susceptible to Yersinia pestis, but a colony in AV was able to survive several regional epizootics of plague. The immune responses of the Gunnison's prairie dog to Y. pestis is investigated. The importance of innate immunity is highlighted.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha+] or orfX+). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. [ABSTRACT FROM AUTHOR]

Cefiderocol is a siderophore cephalosporin designed mainly for treatment of infections caused by β-lactam and multidrug-resistant Gram-negative bacteria. Burkholderia pseudomallei clinical isolates are usually highly cefiderocol susceptible, with in vitro resistance found in a few isolates. Resistance in clinical B. pseudomallei isolates from Australia is caused by a hitherto uncharacterized mechanism. We show that, like in other Gram-negatives, the PiuA outer membrane receptor plays a major role in cefiderocol nonsusceptibility in isolates from Malaysia., Competing Interests: The authors declare no conflict of interest.

Onchocerca lupi (Rodonaja, 1967) is an understudied, vector-borne, filarioid nematode that causes ocular onchocercosis in dogs, cats, coyotes, wolves, and is also capable of infecting humans. Onchocercosis in dogs has been reported with increasing incidence worldwide. However, despite the growing number of reports describing canine O. lupi cases as well as zoonotic infections globally, the disease prevalence in endemic areas and vector species of this parasite remains largely unknown. Here, our study aimed to identify the occurrence of O. lupi infected dogs in northern Arizona, New Mexico, and Utah, United States and identify the vector of this nematode. A total of 532 skin samples from randomly selected companion animals with known geographic locations within the Navajo Reservation were collected and molecularly surveyed by PCR for the presence of O. lupi DNA (September 2019-June 2022) using previously published nematode primers (COI) and DNA sequencing. O. lupi DNA was detected in 50 (9.4%) sampled animals throughout the reservation. Using positive animal samples to target geographic locations, pointed hematophagous insect trapping was performed to identify potential O. lupi vectors. Out of 1,922 insects screened, 38 individual insects and 19 insect pools tested positive for the presence of O. lupi, all of which belong to the Diptera family. This increased surveillance of definitive host and biological vector/intermediate host is the first large scale prevalence study of O. lupi in companion animals in an endemic area of the United States, and identified an overall prevalence of 9.4% in companion animals as well as multiple likely biological vector and putative vector species in the southwestern United States. Furthermore, the identification of these putative vectors in close proximity to human populations coupled with multiple, local zoonotic cases highlight the One Health importance of O. lupi ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Roe, Holiday, Upshaw-Bia, Benally, Williamson, Urbanz, Verocai, Ridenour, Nottingham, Ford, Lake, Kennedy, Hepp and Sahl.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deatherage Kaiser, Birdsell, Hutchison, Thelaus, Jenson, Andrianaivoarimanana, Byström, Myrtennäs, McDonough, Nottingham, Sahl, Schweizer, Rajerison, Forsman, Wunschel and Wagner.)

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 μM dosage of stenoparib-below the approximated 25.5 μM half-maximally effective concentration (EC 50 )-combined with 0.5 μM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19. IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection., (Copyright © 2021 Stone et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

The Burkholderia pseudomallei isolate MSHR1435 is a fully virulent environmental sequence type 131 (ST131) isolate that is epidemiologically associated with a 17.5-year chronic melioidosis infection. The completed genome will serve as a reference for studies of environmental ecology, virulence, and chronic B. pseudomallei infections., (Copyright © 2018 Sahl et al.)

The lack of published data on effective disinfectants and contact times for use on the fungal pathogens Coccidioides immitis and C. posadasii prompted the authors to investigate the fungicidal activity of three commonly used laboratory disinfectants on arthroconidia harvested from C. immitis strain 2009. They tested the ability of 10% bleach, 70% ethanol, and Vesphene ® IIse to inactivate 10 7 arthroconidia in an aqueous suspension within 1, 2, 5, 10, or 20 minutes of contact time. Both 10% bleach and 70% ethanol provided a 7-log 10 reduction in arthroconidia in less than 1 minute, with no growth observed at any of the tested time points. Vesphene ® IIse was less effective, providing a 6-log 10 reduction in arthroconidia after 5 minutes, but was unable to completely inactivate all of the arthroconidia, even after 20 minutes of contact time.

Sequence analyses and subtyping of Bacillus anthracis strains from Georgia reveal a single distinct lineage (Aust94) that is ecologically established. Phylogeographic analysis and comparisons to a global collection reveals a clade that is mostly restricted to Georgia. Within this clade, many groups are found around the country, however at least one subclade is only found in the eastern part. This pattern suggests that dispersal into and out of Georgia has been rare and despite historical dispersion within the country, for at least for one lineage, current spread is limited.

Unlabelled: A cluster of human plague cases occurred in the seaport city of Mahajanga, Madagascar, from 1991 to 1999 following 62 years with no evidence of plague, which offered insights into plague pathogen dynamics in an urban environment. We analyzed a set of 44 Mahajanga isolates from this 9-year outbreak, as well as an additional 218 Malagasy isolates from the highland foci. We sequenced the genomes of four Mahajanga strains, performed whole-genome sequence single-nucleotide polymorphism (SNP) discovery on those strains, screened the discovered SNPs, and performed a high-resolution 43-locus multilocus variable-number tandem-repeat analysis of the isolate panel. Twenty-two new SNPs were identified and defined a new phylogenetic lineage among the Malagasy isolates. Phylogeographic analysis suggests that the Mahajanga lineage likely originated in the Ambositra district in the highlands, spread throughout the northern central highlands, and was then introduced into and became transiently established in Mahajanga. Although multiple transfers between the central highlands and Mahajanga occurred, there was a locally differentiating and dominant subpopulation that was primarily responsible for the 1991-to-1999 Mahajanga outbreaks. Phylotemporal analysis of this Mahajanga subpopulation revealed a cycling pattern of diversity generation and loss that occurred during and after each outbreak. This pattern is consistent with severe interseasonal genetic bottlenecks along with large seasonal population expansions. The ultimate extinction of plague pathogens in Mahajanga suggests that, in this environment, the plague pathogen niche is tenuous at best. However, the temporary large pathogen population expansion provides the means for plague pathogens to disperse and become ecologically established in more suitable nonurban environments., Importance: Maritime spread of plague led to the global dissemination of this disease and affected the course of human history. Multiple historical plague waves resulted in massive human mortalities in three classical plague pandemics: Justinian (6th and 7th centuries), Middle Ages (14th to 17th centuries), and third (mid-1800s to the present). Key to these events was the pathogen's entry into new lands by "plague ships" via seaport cities. Although initial disease outbreaks in ports were common, they were almost never sustained for long and plague pathogens survived only if they could become established in ecologically suitable habitats. Although plague pathogens' ability to invade port cities has been essential for intercontinental spread, these regions have not proven to be a suitable long-term niche. The disease dynamics in port cities such as Mahajanga are thus critical to plague pathogen amplification and dispersal into new suitable ecological niches for the observed global long-term maintenance of plague pathogens.