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Background: Plague, caused by the bacterium Yersinia pestis, remains an important disease in Madagascar, where the oriental rat flea, Xenopsylla cheopis, is a primary vector. To control fleas, synthetic pyrethroids (SPs) have been used for >20 years, resulting in resistance in many X. cheopis populations. The most common mechanisms of SP resistance are target site mutations in the voltage-gated sodium channel (VGSC) gene. Methodology/Principal findings: We obtained 25 collections of X. cheopis from 22 locations across Madagascar and performed phenotypic tests to determine resistance to deltamethrin, permethrin, and/or dichlorodiphenyltrichloroethane (DDT). Most populations were resistant to all these insecticides. We sequenced a 535 bp segment of the VGSC gene and identified two different mutations encoding distinct substitutions at amino acid position 1014, which is associated with knockdown resistance (kdr) to SPs in insects. Kdr mutation L1014F occurred in all 25 collections; a rarer mutation, L1014H, was found in 12 collections. There was a significant positive relationship between the frequency of kdr alleles and the proportion of individuals surviving exposure to deltamethrin. Phylogenetic comparisons of 12 VGSC alleles in Madagascar suggested resistant alleles arose from susceptible lineages at least three times. Because genotype can reasonably predict resistance phenotype, we developed a TaqMan PCR assay for the rapid detection of kdr resistance alleles. Conclusions/Significance: Our study provides new insights into VGSC mutations in Malagasy populations of X. cheopis and is the first to report a positive correlation between VGSC genotypes and SP resistance phenotypes in fleas. Widespread occurrence of these two SP resistance mutations in X. cheopis populations in Madagascar reduces the viability of these insecticides for flea control. However, the TaqMan assay described here facilitates rapid detection of kdr mutations to inform when use of these insecticides is still warranted to reduce transmission of plague. Author summary: In Madagascar, the Oriental rat flea Xenopsylla cheopis is the primary vector of Yersinia pestis, which causes plague. This flea has been reported to be resistant to insecticides used for vector control. The objective of this study was to examine the target site mutations in the voltage-gated sodium channel (VGSC) gene in X. cheopis populations sampled throughout the island and previously tested for synthetic pyrethroid (SPs) susceptibility. Two widespread mutations associated with knock-down resistance (kdr) to SPs were identified, and a significant relationship between the frequency of kdr alleles and the SP resistance phenotype was revealed. The results raise concerns about the overall efficacy of SPs for controlling Yersinia pestis transmission by X. cheopis in Madagascar. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis, an opportunistic pathogen found in the environment, is a bacterium closely related to B. pseudomallei, the cause of melioidosis. Human B. thailandensis infections are uncommon. We isolated B. thailandensis from water in Texas and Puerto Rico and soil in Mississippi in the United States, demonstrating a potential public health risk. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology. Methodology/Principal findings: We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup. Conclusions/Significance: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics. Author summary: Leptospirosis is a common zoonotic disease worldwide, but more prevalent in the tropics. Cases are more common following severe weather events, possibly due to flooding, which may more readily distribute soil and/or water contaminated with Leptospira spp., the disease agents. Human cases increased following the 2017 hurricanes that ravaged Puerto Rico (Maria and Irma), prompting environmental sampling of soil and water to assess the presence, abundance, and possible persistence of pathogenic leptospires in these environments. The goal was to better understand these potential reservoirs of human and animal disease. Diverse and novel groups of pathogenic Leptospira were abundant and widespread in soil and water in Puerto Rico and sometimes were consistently detected in these environments for >1 year. However, most groups we identified have not previously been described from humans and/or other animals, so the disease potential of these novel organisms is unknown. The results of this study reveal a tremendous amount of previously uncharacterized Leptospira diversity in soil and water in Puerto Rico. The description and characterization of these novel types improves our understanding of the genus Leptospira, and will aid in the development of improved diagnostics and preventative tools to advance public health outcomes. [ABSTRACT FROM AUTHOR]

The distribution of Burkholderia pseudomallei in the Caribbean is poorly understood. We isolated B. pseudomallei from US Virgin Islands soil. The soil isolate was genetically similar to other isolates from the Caribbean, suggesting that B. pseudomallei might have been introduced to the islands multiple times through severe weather events. [ABSTRACT FROM AUTHOR]