Mark Mayo

The epidemiology of melioidosis in Australia and Papua New Guinea.

Melioidosis was first described in Australia in an outbreak in sheep in 1949 in north Queensland (22 degrees S). Human melioidosis was first described from Townsville (19 degrees S) in 1950. Melioidosis is hyperendemic in the Top End of the Northern Territory (NT) and as in parts of northeastern Thailand it is the commonest cause of fatal community-acquired septicemic pneumonia. In the 9 years since 1989 the prospective NT melioidosis study at Royal Darwin Hospital (12 degrees S) has documented 206 culture confirmed cases of melioidosis, with an average annual incidence of 16.5/100,000. Melioidosis is also seen in the north of Western Australia and north Queensland, including the Torres Strait Islands, but is uncommon in adjacent Papua New Guinea. Serological studies suggest that infection is rare in the Port Moresby region, but there is emerging evidence of melioidosis from Western Province. The NT study has documented inoculating events in 52 (25%) of cases, with an incubation period of 1-21 days (mean 9 days); 84% of cases had acute disease from presumed recent acquisition and 13% had chronic disease (sick, > 2 months). In 4% there was evidence of possible reactivation from a latent focus; 28 of 153 (18%) males had prostatic abscesses. The overall mortality was 21% (43 cases), with a mortality rate in septicemic cases (95) of 39% and in non-septicemic cases (103) of 4%. Pneumonia was the commonest presentation in both groups and, in addition, eight patients (two deaths) presented with melioidosis encephalomyelitis. Melioidosis clusters in temperate Australia are attributed to animals imported from the north. Molecular typing of Burkholderia pseudomallei isolates from temperate southwest Western Australia showed clonality over 25 years. In this outbreak and in studies from the NT, some soil isolates are molecularly identical to epidemiologically related animal and human isolates. Molecular typing has implicated the water supply in two clonal outbreaks in remote aboriginal communities in northern Australia. Further prospective collaborative studies are required to evaluate whether there are truly regional differences in clinical features of melioidosis and to better understand how B. pseudomallei is acquired from the environment.

Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion System of Burkholderia pseudomallei

ABSTRACT Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 × 103 genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 × 103 CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.

Animal melioidosis in Australia.

Melioidosis was first diagnosed in Australia in sheep in 1949. While it has been considered endemic in tropical Australia, there have been animal outbreaks in southwest Western Australia and southern Queensland. Infection occurs in many species, with both latency and a wide range of clinical manifestations. Some species may develop melioidosis only if immunocompromised. Sheep and goats are particularly susceptible, resulting in the requirement for pasteurisation of tropical commercial goats milk. Nine out of 43 (21%) goats had aortic lesions at autopsy and seven died from aortic aneurysm rupture. Transplacental transmission in goats has also been documented. Asymptomatic organ abscesses are common in pigs but bovine melioidosis is very rare. Camels moved north and an alpaca brought to Darwin have died from melioidosis. It also occurs in wildlife, including birds, crocodiles and kangaroos. Zoonotic transmission to humans is extremely unusual, but there are many similar epidemiological and clinical features of melioidosis in animals and humans. There have been three possible zoonotic cases in Australia. Molecular typing has found identical Burkholderia pseudomallei organisms from animals, humans and soil. The study of melioidosis in animals, especially the use of molecular genetic techniques for organism identification and typing, will continue to unravel aspects of the disease that remain unclear in humans.

Burkholderia pseudomallei virulence: definition, stability and association with clonality

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.

Within-Host Evolution of Burkholderia pseudomallei over a Twelve-Year Chronic Carriage Infection

ABSTRACT Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR, an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. IMPORTANCE Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens. Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.

Landscape Changes Influence the Occurrence of the Melioidosis Bacterium Burkholderia pseudomallei in Soil in Northern Australia

Background The soil-dwelling saprophyte bacterium Burkholderia pseudomallei is the cause of melioidosis, a severe disease of humans and animals in southeast Asia and northern Australia. Despite the detection of B. pseudomallei in various soil and water samples from endemic areas, the environmental habitat of B. pseudomallei remains unclear. Methodology/Principal Findings We performed a large survey in the Darwin area in tropical Australia and screened 809 soil samples for the presence of these bacteria. B. pseudomallei were detected by using a recently developed and validated protocol involving soil DNA extraction and real-time PCR targeting the B. pseudomallei–specific Type III Secretion System TTS1 gene cluster. Statistical analyses such as multivariable cluster logistic regression and principal component analysis were performed to assess the association of B. pseudomallei with environmental factors. The combination of factors describing the habitat of B. pseudomallei differed between undisturbed sites and environmentally manipulated areas. At undisturbed sites, the occurrence of B. pseudomallei was found to be significantly associated with areas rich in grasses, whereas at environmentally disturbed sites, B. pseudomallei was associated with the presence of livestock animals, lower soil pH and different combinations of soil texture and colour. Conclusions/Significance This study contributes to the elucidation of environmental factors influencing the occurrence of B. pseudomallei and raises concerns that B. pseudomallei may spread due to changes in land use.

Burkholderia stagnalis sp nov and Burkholderia territorii sp nov., two novel Burkholderia cepacia complex species from environmental and human sources

Nine Burkholderia cepacia complex (Bcc) bacteria were isolated during environmental surveys for the ecological niche of Burkholderia pseudomallei, the aetiological agent of melioidosis, in the Northern Territory of Australia. They represented two multi-locus sequence analysis-based clusters, referred to as Bcc B and Bcc L. Three additional environmental and clinical Bcc B isolates were identified upon deposition of the sequences in the PubMLST database. Analysis of the concatenated nucleotide sequence divergence levels within both groups (1.4 and 1.9%, respectively) and towards established Bcc species (4.0 and 3.9%, respectively) demonstrated that the two taxa represented novel Bcc species. All 12 isolates were further characterized using 16S rRNA and recA gene sequence analysis, RAPD analysis, DNA base content determination, fatty acid methyl ester analysis and biochemical profiling. Analysis of recA gene sequences revealed a remarkable diversity within each of these taxa, but, together, the results supported the affiliation of the two taxa to the Bcc. Bcc B strains can be differentiated from most other Bcc members by the assimilation of maltose. Bcc L strains can be differentiated from other Bcc members by the absence of assimilation of N-acetylglucosamine. The names Burkholderia stagnalis sp. nov. with type strain LMG 28156(T) ( = CCUG 65686(T)) and Burkholderia territorii sp. nov. with type strain LMG 28158(T) ( = CCUG 65687(T)) are proposed for Bcc B and Bcc L bacteria, respectively.

Sensitive and Specific Molecular Detection of Burkholderia pseudomallei, the Causative Agent of Melioidosis, in the Soil of Tropical Northern Australia

ABSTRACT Burkholderia pseudomallei, the cause of the severe disease melioidosis in humans and animals, is a gram-negative saprophyte living in soil and water of areas of endemicity such as tropical northern Australia and Southeast Asia. Infection occurs mainly by contact with wet contaminated soil. The environmental distribution of B. pseudomallei in northern Australia is still unclear. We developed and evaluated a direct soil B. pseudomallei DNA detection method based on the recently published real-time PCR targeting the B. pseudomallei type III secretion system. The method was evaluated by inoculating different soil types with B. pseudomallei dilution series and by comparing B. pseudomallei detection rate with culture-based detection rate for 104 randomly collected soil samples from the Darwin rural area in northern Australia. We found that direct soil B. pseudomallei DNA detection not only was substantially faster than culture but also proved to be more sensitive with no evident false-positive results. This assay provides a new tool to detect B. pseudomallei in soil samples in a fast and highly sensitive and specific manner and is applicable for large-scale B. pseudomallei environmental screening studies or in outbreak situations. Furthermore, analysis of the 104 collected soil samples revealed a significant association between B. pseudomallei-positive sites and the presence of animals at these locations and also with moist, reddish brown-to-reddish gray soils.

Tandem repeat regions within the Burkholderia pseudomallei genome and their application for high resolution genotyping

BackgroundThe facultative, intracellular bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a serious infectious disease of humans and animals. We identified and categorized tandem repeat arrays and their distribution throughout the genome of B. pseudomallei strain K96243 in order to develop a genetic typing method for B. pseudomallei. We then screened 104 of the potentially polymorphic loci across a diverse panel of 31 isolates including B. pseudomallei, B. mallei and B. thailandensis in order to identify loci with varying degrees of polymorphism. A subset of these tandem repeat arrays were subsequently developed into a multiple-locus VNTR analysis to examine 66 B. pseudomallei and 21 B. mallei isolates from around the world, as well as 95 lineages from a serial transfer experiment encompassing ~18,000 generations.ResultsB. pseudomallei contains a preponderance of tandem repeat loci throughout its genome, many of which are duplicated elsewhere in the genome. The majority of these loci are composed of repeat motif lengths of 6 to 9 bp with 4 to 10 repeat units and are predominately located in intergenic regions of the genome. Across geographically diverse B. pseudomallei and B.mallei isolates, the 32 VNTR loci displayed between 7 and 28 alleles, with Neis diversity values ranging from 0.47 and 0.94. Mutation rates for these loci are comparable (>10-5 per locus per generation) to that of the most diverse tandemly repeated regions found in other less diverse bacteria.ConclusionThe frequency, location and duplicate nature of tandemly repeated regions within the B. pseudomallei genome indicate that these tandem repeat regions may play a role in generating and maintaining adaptive genomic variation. Multiple-locus VNTR analysis revealed extensive diversity within the global isolate set containing B. pseudomallei and B. mallei, and it detected genotypic differences within clonal lineages of both species that were identical using previous typing methods. Given the health threat to humans and livestock and the potential for B. pseudomallei to be released intentionally, MLVA could prove to be an important tool for fine-scale epidemiological or forensic tracking of this increasingly important environmental pathogen.

Development of a Prototype Lateral Flow Immunoassay (LFI) for the Rapid Diagnosis of Melioidosis

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the “gold standard” for the diagnosis of melioidosis; results can take 3–7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.