Case Report: Gestational Melioidosis through Perinatal Transmission.

Burkholderia pseudomallei is an emerging pathogen in the Americas. Cases of mother-to-child transmission of B. pseudomallei are rare and probably occur by placental or perinatal infection. We report the first case of native gestational and neonatal melioidosis in the Western hemisphere. The isolated strains in the mother and newborn were confirmed by whole-genome sequencing and identified as a novel sequence type ST1748. The comparison of both genomes revealed a nucleotide similarity of 100%. Melioidosis should be considered within the differential diagnosis of febrile illness or pneumonia in pregnant women and newborns from endemic areas of the Americas.

Comparative genomics confirms a rare melioidosis human-to-human transmission event and reveals incorrect phylogenomic reconstruction due to polyclonality.

Human-to-human transmission of the melioidosis bacterium, Burkholderia pseudomallei, is exceedingly rare, with only a handful of suspected cases documented to date. Here, we used whole-genome sequencing (WGS) to characterize one such unusual B. pseudomallei transmission event, which occurred between a breastfeeding mother with mastitis and her child. Two strains corresponding to multilocus sequence types (STs)-259 and -261 were identified in the mother's sputum from both the primary culture sweep and in purified colonies, confirming an unusual polyclonal infection in this patient. In contrast, primary culture sweeps of the mother's breast milk and the child's cerebrospinal fluid and blood samples contained only ST-259, indicating monoclonal transmission to the child. Analysis of purified ST-259 isolates showed no genetic variation between mother and baby isolates, providing the strongest possible evidence of B. pseudomallei human-to-human transmission, probably via breastfeeding. Next, phylogenomic analysis of all isolates, including the mother's mixed ST-259/ST-261 sputum sample, was performed to investigate the effects of mixtures on phylogenetic inference. Inclusion of this mixture caused a dramatic reduction in the number of informative SNPs, resulting in branch collapse of ST-259 and ST-261 isolates, and several instances of incorrect topology in a global B. pseudomallei phylogeny, resulting in phylogenetic incongruence. Although phylogenomics can provide clues about the presence of mixtures within WGS datasets, our results demonstrate that this methodology can lead to phylogenetic misinterpretation if mixed genomes are not correctly identified and omitted. Using current bioinformatic tools, we demonstrate a robust method for bacterial mixture identification and strain parsing that avoids these pitfalls.

Melioidosis fatalities in captive slender-tailed meerkats (Suricata suricatta): combining epidemiology, pathology and whole-genome sequencing supports variable mechanisms of transmission with one health implications.

BACKGROUND: Melioidosis is a tropical infectious disease which is being increasingly recognised throughout the globe. Infection occurs in humans and animals, typically through direct exposure to soil or water containing the environmental bacterium Burkholderia pseudomallei. Case clusters of melioidosis have been described in humans following severe weather events and in exotic animals imported into melioidosis endemic zones. Direct transmission of B. pseudomallei between animals and/or humans has been documented but is considered extremely rare. Between March 2015 and October 2016 eight fatal cases of melioidosis were reported in slender-tailed meerkats (Suricata suricatta) on display at a Wildlife Park in Northern Australia. To further investigate the melioidosis case cluster we sampled the meerkat enclosure and adjacent park areas and performed whole-genome sequencing (WGS) on all culture-positive B. pseudomallei environmental and clinical isolates. RESULTS: WGS confirmed that the fatalities were caused by two different B. pseudomallei sequence types (STs) but that seven of the meerkat isolates were highly similar on the whole-genome level. Used concurrently with detailed pathology data, our results demonstrate that the seven cases originated from a single original source, but routes of infection varied amongst meerkats belonging to the clonal outbreak cluster. Moreover, in some instances direct transmission may have transpired through wounds inflicted while fighting. CONCLUSIONS: Collectively, this study supports the use of high-resolution WGS to enhance epidemiological investigations into transmission modalities and pathogenesis of melioidosis, especially in the instance of a possible clonal outbreak scenario in exotic zoological collections. Such findings from an animal outbreak have important One Health implications.

Isolation of Burkholderia pseudomallei from a Pet Green Iguana, Belgium.

We isolated Burkholderia pseudomallei, the causative agent of melioidosis, from liver granulomas of a pet green iguana (Iguana iguana) in Belgium. This case highlights a risk for imported green iguanas acting as a reservoir for introduction of this high-threat, zoonotic pathogen into nonendemic regions.

Public health response to an imported case of canine melioidosis.

Melioidosis in humans presents variably as fulminant sepsis, pneumonia, skin infection and solid organ abscesses. It is caused by Burkholderia pseudomallei, which in the United States is classified as a select agent, with "potential to pose a severe threat to both human and animal health, to plant health or to animal and plant products" (Federal Select Agent Program, http://www.selectagents.gov/, accessed 22 September 2016). Burkholderia pseudomallei is found in soil and surface water in the tropics, especially South-East Asia and northern Australia, where melioidosis is endemic. Human cases are rare in the United States and are usually associated with travel to endemic areas. Burkholderia pseudomallei can also infect animals. We describe a multijurisdictional public health response to a case of subclinical urinary B. pseudomallei infection in a dog that had been adopted into upstate New York from a shelter in Thailand. Investigation disclosed three human contacts with single, low-risk exposures to the dog's urine at his residence, and 16 human contacts with possible exposure to his urine or culture isolates at a veterinary hospital. Contacts were offered various combinations of symptom/fever monitoring, baseline and repeat B. pseudomallei serologic testing, and antibiotic post-exposure prophylaxis, depending on the nature of their exposure and their personal medical histories. The dog's owner accepted recommendations from public health authorities and veterinary clinicians for humane euthanasia. A number of animal rescue organizations actively facilitate adoptions into the United States of shelter dogs from South-East Asia. This may result in importation of B. pseudomallei into almost any community, with implications for human and animal health.

Global and regional dissemination and evolution of Burkholderia pseudomallei.

The environmental bacterium Burkholderia pseudomallei causes an estimated 165,000 cases of human melioidosis per year worldwide and is also classified as a biothreat agent. We used whole genome sequences of 469 B. pseudomallei isolates from 30 countries collected over 79 years to explore its geographic transmission. Our data point to Australia as an early reservoir, with transmission to Southeast Asia followed by onward transmission to South Asia and East Asia. Repeated reintroductions were observed within the Malay Peninsula and between countries bordered by the Mekong River. Our data support an African origin of the Central and South American isolates with introduction of B. pseudomallei into the Americas between 1650 and 1850, providing a temporal link with the slave trade. We also identified geographically distinct genes/variants in Australasian or Southeast Asian isolates alone, with virulence-associated genes being among those over-represented. This provides a potential explanation for clinical manifestations of melioidosis that are geographically restricted.

Unprecedented Melioidosis Cases in Northern Australia Caused by an Asian Burkholderia pseudomallei Strain Identified by Using Large-Scale Comparative Genomics.

Melioidosis is a disease of humans and animals that is caused by the saprophytic bacterium Burkholderia pseudomallei. Once thought to be confined to certain locations, the known presence of B. pseudomallei is expanding as more regions of endemicity are uncovered. There is no vaccine for melioidosis, and even with antibiotic administration, the mortality rate is as high as 40% in some regions that are endemic for the infection. Despite high levels of recombination, phylogenetic reconstruction of B. pseudomallei populations using whole-genome sequencing (WGS) has revealed surprisingly robust biogeographic separation between isolates from Australia and Asia. To date, there have been no confirmed autochthonous melioidosis cases in Australia caused by an Asian isolate; likewise, no autochthonous cases in Asia have been identified as Australian in origin. Here, we used comparative genomic analysis of 455 B. pseudomallei genomes to confirm the unprecedented presence of an Asian clone, sequence type 562 (ST-562), in Darwin, northern Australia. First observed in Darwin in 2005, the incidence of melioidosis cases attributable to ST-562 infection has steadily risen, and it is now a common strain in Darwin. Intriguingly, the Australian ST-562 appears to be geographically restricted to a single locale and is genetically less diverse than other common STs from this region, indicating a recent introduction of this clone into northern Australia. Detailed genomic and epidemiological investigations of new clinical and environmental B. pseudomallei isolates in the Darwin region and ST-562 isolates from Asia will be critical for understanding the origin, distribution, and dissemination of this emerging clone in northern Australia.

Melioidosis in New Caledonia: a dominant strain in a transmission hotspot.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a bacterium endemic in Southeast Asia and northern Australia. In New Caledonia, sporadic cases were first described in 2005; since then, more cases have been identified. To improve our understanding of melioidosis epidemiology in New Caledonia, we compared the local cases and B. pseudomallei isolates with those from endemic areas. Nineteen melioidosis cases have been diagnosed in New Caledonia since 1999, mostly severe and with frequent bacteraemia, leading to three (16%) fatalities. All but one occurred in the North Province. Besides sporadic cases caused by non-clonal strains, we also identified a hotspot of transmission related to a clonal group of B. pseudomallei that is phylogenetically related to Australian strains.

Use of Whole-Genome Sequencing to Link Burkholderia pseudomallei from Air Sampling to Mediastinal Melioidosis, Australia.

The frequency with which melioidosis results from inhalation rather than percutaneous inoculation or ingestion is unknown. We recovered Burkholderia pseudomallei from air samples at the residence of a patient with presumptive inhalational melioidosis and used whole-genome sequencing to link the environmental bacteria to B. pseudomallei recovered from the patient.

Airborne Transmission of Melioidosis to Humans from Environmental Aerosols Contaminated with B. pseudomallei.

Melioidosis results from an infection with the soil-borne pathogen Burkholderia pseudomallei, and cases of melioidosis usually cluster after rains or a typhoon. In an endemic area of Taiwan, B. pseudomallei is primarily geographically distributed in cropped fields in the northwest of this area, whereas melioidosis cases are distributed in a densely populated district in the southeast. We hypothesized that contaminated cropped fields generated aerosols contaminated with B. pseudomallei, which were carried by a northwesterly wind to the densely populated southeastern district. We collected soil and aerosol samples from a 72 km2 area of land, including the melioidosis-clustered area and its surroundings. Aerosols that contained B. pseudomallei-specific TTSS (type III secretion system) ORF2 DNA were well distributed in the endemic area but were rare in the surrounding areas during the rainy season. The concentration of this specific DNA in aerosols was positively correlated with the incidence of melioidosis and the appearance of a northwesterly wind. Moreover, the isolation rate in the superficial layers of the contaminated cropped field in the northwest was correlated with PCR positivity for aerosols collected from the southeast over a 2-year period. According to pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses, PFGE Type Ia (ST58) was the predominant pattern linking the molecular association among soil, aerosol and human isolates. Thus, the airborne transmission of melioidosis moves from the contaminated soil to aerosols and/or to humans in this endemic area.

Tracing melioidosis back to the source: using whole-genome sequencing to investigate an outbreak originating from a contaminated domestic water supply.

Melioidosis, a disease of public health importance in Southeast Asia and northern Australia, is caused by the Gram-negative soil bacillus Burkholderia pseudomallei. Melioidosis is typically acquired through environmental exposure, and case clusters are rare, even in regions where the disease is endemic. B. pseudomallei is classed as a tier 1 select agent by the Centers for Disease Control and Prevention; from a biodefense perspective, source attribution is vital in an outbreak scenario to rule out a deliberate release. Two cases of melioidosis within a 3-month period at a residence in rural northern Australia prompted an investigation to determine the source of exposure. B. pseudomallei isolates from the property's groundwater supply matched the multilocus sequence type of the clinical isolates. Whole-genome sequencing confirmed the water supply as the probable source of infection in both cases, with the clinical isolates differing from the likely infecting environmental strain by just one single nucleotide polymorphism (SNP) each. For the first time, we report a phylogenetic analysis of genomewide insertion/deletion (indel) data, an approach conventionally viewed as problematic due to high mutation rates and homoplasy. Our whole-genome indel analysis was concordant with the SNP phylogeny, and these two combined data sets provided greater resolution and a better fit with our epidemiological chronology of events. Collectively, this investigation represents a highly accurate account of source attribution in a melioidosis outbreak and gives further insight into a frequently overlooked reservoir of B. pseudomallei. Our methods and findings have important implications for outbreak source tracing of this bacterium and other highly recombinogenic pathogens.

[Melioidosis, first four cases in Iceland]. / Melioidosis á Íslandi, fyrstu fjögur tilfelli.

We report the first four cases of Melioidosis treated in Iceland and review the literature. Melioidosis is caused by the saprophytic Gram negative bacteria Burkholderia pseudomallei. Most disease occurs in residents of Southeast-Asia and North-Australia. The most common presentation of Melioidosis is pneumonia but as these cases demonstrate the infection has protean manifestations and B. pseudomallei can infect nearly every organ. It is important to notify the laboratory of the possibilty of Melioidosis as the bacteria can be difficult to identify and poses an infection risk to laboratory staff. Also, B. pseudomallei is resistant to many of the antibiotics normally used to treat pneumonia and due to its intracellular persistance requires prolonged therapy.

Physicochemical factors affecting the growth of Burkholderia pseudomallei in soil microcosm.

Burkholderia pseudomallei causes melioidosis, the third most common cause of death from infectious diseases in northeast Thailand. Four physicochemical factors were set so that their values covered the range of the northeast, which is an endemic area. The soil pH was set at pH 4-10, soil salinity was 0.0-5.0% NaCl, total iron was 50-150 mg/kg soil, and carbon to nitrogen ratio (C/N) was 10:1 to 40:1. The experiments were carried out at 37°C, and soil moisture was maintained for 7 days. The number of viable bacterial cells was counted daily. Soil pH, salinity, Fe, and C/N ratio affected the bacterial growth. The bacterial colony was significantly (P < 0.05) reduced at soil pH > 8, soil salinity > 1% NaCl, and C/N ratio > 40:1. However, the growth of B. pseudomallei was enhanced by increasing the concentrations of iron significantly (P < 0.05). We propose using these findings to control B. pseudomallei in situ.

Case cluster shifting and contaminant source as determinants of melioidosis in Taiwan.

OBJECTIVES: To assess the geographical distribution of melioidosis contamination sources and the association between the location of melioidosis cases and positive sampling sites for Burkholderia pseudomallei in Taiwan. METHODS: Data on the location of melioidosis cases from 2002 to 2011 were combined with the geographical distribution of B. pseudomallei as indicated by the detection of specific flagella gene products measured from 2005 to 2011. Temporal and spatial analyses were used to determine the incidence, cluster shifts and associations between the two datasets. RESULTS: Melioidosis cases clustered in two 'hot-spot' areas with incidence rates that were significantly higher than in neighbouring towns. The incidence rates in the northern area gradually decreased, while the rates in the southern area increased and were temporally associated with the appearance of B. pseudomallei-specific flagella genes in water samples. CONCLUSIONS: Melioidosis hot-spot areas were present in Taiwan. Water contaminated with B. pseudomallei serves as a potential transmission vehicle and is correlated with an increase in melioidosis cases; this correlation was stronger than that for B. pseudomallei-contaminated soil.

Burkholderia pseudomallei in unchlorinated domestic bore water, Tropical Northern Australia.

To determine whether unchlorinated bore water in northern Australia contained Burkholderia pseudomallei organisms, we sampled 55 bores; 18 (33%) were culture positive. Multilocus sequence typing identified 15 sequence types. The B. pseudomallei sequence type from 1 water sample matched a clinical isolate from a resident with melioidosis on the same property.

Melioidosis acquired by traveler to Nigeria.

We describe melioidosis associated with travel to Nigeria in a woman with diabetes, a major predisposing factor for this infection. With the prevalence of diabetes projected to increase dramatically in many developing countries, the global reach of melioidosis may expand.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade.

Burkholderia pseudomallei and Burkholderia thailandensis are related pathogens that invade a variety of cell types, replicate in the cytoplasm, and spread to nearby cells. We have investigated temporal and spatial requirements for virulence determinants in the intracellular life cycle, using genetic dissection and photothermal nanoblade delivery, which allows efficient placement of bacterium-sized cargo into the cytoplasm of mammalian cells. The conserved Bsa type III secretion system (T3SS(Bsa)) is dispensable for invasion, but is essential for escape from primary endosomes. By nanoblade delivery of B. thailandensis we demonstrate that all subsequent events in intercellular spread occur independently of T3SS(Bsa) activity. Although intracellular movement was essential for cell-cell spread by B. pseudomallei and B. thailandensis, neither BimA-mediated actin polymerization nor the formation of membrane protrusions containing bacteria was required for B. thailandensis. Surprisingly, the cryptic (fla2) flagellar system encoded on chromosome 2 of B. thailandensis supported rapid intracellular motility and efficient cell-cell spread. Plaque formation by both pathogens was dependent on the activity of a type VI secretion system (T6SS-1) that functions downstream from T3SS(Bsa)-mediated endosome escape. A remarkable feature of Burkholderia is their ability to induce the formation of multinucleate giant cells (MNGCs) in multiple cell types. By infection and nanoblade delivery, we observed complete correspondence between mutant phenotypes in assays for cell fusion and plaque formation, and time-course studies showed that plaque formation represents MNGC death. Our data suggest that the primary means for intercellular spread involves cell fusion, as opposed to pseudopod engulfment and bacterial escape from double-membrane vacuoles.