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.

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Multitarget Quantitative PCR Improves Detection and Predicts Cultivability of the Pathogen Burkholderia pseudomallei

Applied and Environmental Microbiology ◽

10.1128/aem.03212-16 ◽

2017 ◽

Vol 83 (8) ◽

Cited By ~ 8

Author(s):

Andre Göhler ◽

Trinh Thanh Trung ◽

Verena Hopf ◽

Christian Kohler ◽

Jörg Hartleib ◽

...

Keyword(s):

Quantitative Pcr ◽

Burkholderia Pseudomallei ◽

Detection Rate ◽

Soil Samples ◽

Single Type ◽

Comparative Genomic ◽

Environmental Distribution ◽

Content Type ◽

The World ◽

Culture Positive

ABSTRACT Burkholderia pseudomallei is present in the environment in many parts of the world and causes the often-fatal disease melioidosis. The sensitive detection and quantification of B. pseudomallei in the environment are a prerequisite for assessing the risk of infection. We recently reported the direct detection of B. pseudomallei in soil samples using a quantitative PCR (qPCR) targeting a single type three secretion system 1 (TTSS1) gene. Here, we extend the qPCR-based analysis of B. pseudomallei in soil by validating novel qPCR gene targets selected from a comparative genomic analysis. Two hundred soil samples from two rice paddies in northeast Thailand were evaluated, of which 47% (94/200) were B. pseudomallei culture positive. The TTSS1 qPCR and two novel qPCR assays that targeted open reading frames (ORFs) BPSS0087 and BPSS0745 exhibited detection rates of 76.5% (153/200), 34.5% (69/200), and 74.5% (150/200), respectively. The combination of TTSS1 and BPSS0745 qPCR increased the detection rate to 90% (180/200). Combining the results of the three qPCR assays and the BPSS1187 nested PCR previously published, all 200 samples were positive by at least one PCR assay. Samples positive by either TTSS1 (n = 153) or BPSS0745 (n = 150) qPCR were more likely to be direct-culture positive, with odds ratios of 4.0 (95% confidence interval [CI], 1.7 to 9.5; P < 0.001) and 9.0 (95% CI, 3.1 to 26.4; P < 0.001), respectively. High B. pseudomallei genome equivalents correlated with high CFU counts by culture. In conclusion, multitarget qPCR improved the B. pseudomallei detection rate in soil samples and predicted culture positivity. This approach has the potential for use as a sensitive environmental screening method for B. pseudomallei. IMPORTANCE The worldwide environmental distribution of the soil bacterium Burkholderia pseudomallei remains to be determined. So far, most environmental studies have relied on culture-based approaches to detect this pathogen. Since current culture methods are laborious, are time consuming, and have limited sensitivity, culture-independent and more sensitive methods are needed. In this study, we show that a B. pseudomallei-specific qPCR approach can detect significantly higher numbers of B. pseudomallei-positive soil samples from areas where it is endemic compared with that from culture. The use of multiple independent B. pseudomallei-specific qPCR targets further increased the detection rate of B. pseudomallei compared with that from single targets. Samples with a high molecular B. pseudomallei load were more likely to be culture positive. We conclude that our quantitative multitarget approach might be useful in defining areas where there is a risk of B. pseudomallei infections in different parts of the world.

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Highly Sensitive Direct Detection and Quantification of Burkholderia pseudomallei Bacteria in Environmental Soil Samples by Using Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.00735-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6486-6494 ◽

Cited By ~ 31

Author(s):

Trinh Thanh Trung ◽

Adrian Hetzer ◽

André Göhler ◽

Eylin Topfstedt ◽

Vanaporn Wuthiekanun ◽

...

Keyword(s):

Nested Pcr ◽

Burkholderia Pseudomallei ◽

Geometric Mean ◽

Soil Samples ◽

Specific Sequence ◽

Soil Dna ◽

Content Type ◽

Highly Sensitive ◽

Two Samples ◽

Pcr Targeting

ABSTRACTThe soil bacterium and potential biothreat agentBurkholderia pseudomalleicauses the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection ofB. pseudomalleirepresents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification ofB. pseudomalleifrom soil. We established a protocol forB. pseudomalleisoil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that wereB. pseudomalleipositive by direct culture wereB. pseudomalleiqPCR positive, with a median of 1.84 × 104genome equivalents (range, 3.65 × 102to 7.85 × 105) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detectedB. pseudomalleiin seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independentB. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance ofB. pseudomallei.

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Expanding the Burkholderia pseudomallei complex with the addition of two novel species: Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov.

Applied and Environmental Microbiology ◽

10.1128/aem.01583-21 ◽

2021 ◽

Author(s):

Carina M. Hall ◽

Anthony L. Baker ◽

Jason W. Sahl ◽

Mark Mayo ◽

Holger C. Scholz ◽

...

Keyword(s):

Type Strain ◽

Burkholderia Pseudomallei ◽

Novel Species ◽

Phage Type ◽

Distinct Species ◽

Soil Samples ◽

Northern Australia ◽

Coding Regions ◽

Scientific Disciplines ◽

Distinct Cluster

Distinct Burkholderia strains were isolated from soil samples collected in tropical northern Australia (Northern Territory and the Torres Strait Islands, Queensland). Phylogenetic analysis of 16S rRNA and whole genome sequences revealed these strains were distinct from previously described Burkholderia species and assigned them to two novel clades within the B. pseudomallei complex (Bpc). Because average nucleotide identity and digital DNA-DNA hybridization calculations are consistent with these clades representing distinct species, we propose the names Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov. Strains assigned to B. mayonis sp. nov. include type strain BDU6 T (=TSD-80; LMG 29941; ASM152374v2) and BDU8. Strains assigned to B. savannae sp. nov. include type strain MSMB266 T (=TSD-82; LMG 29940; ASM152444v2), MSMB852, BDU18, and BDU19. Comparative genomics revealed unique coding regions for both putative species, including clusters of orthologous genes associated with phage. Type strains of both B. mayonis sp. nov. and B. savannae sp. nov. yielded biochemical profiles distinct from each other and other species in the Bpc, and profiles also varied among strains within B. mayonis sp. nov. and B. savannae sp. nov. Matrix-assisted laser desorption ionization–time of flight analysis revealed a B. savannae sp. nov. cluster separate from other species, whereas B. mayonis sp. nov. strains did not form a distinct cluster. Neither B. mayonis sp. nov. nor B. savannae sp. nov. caused mortality in mice when delivered via the subcutaneous route. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in eight species currently in the Bpc. IMPORTANCE Burkholderia species can be important sources of novel natural products and new species are of interest to diverse scientific disciplines. Although many Burkholderia species are saprophytic, Burkholderia pseudomallei is the causative agent of the disease melioidosis. Understanding the genomics and virulence of the closest relatives to B. pseudomallei ( i.e., the other species within the Bpc) is important for identifying robust diagnostic targets specific to B. pseudomallei and understanding evolution of virulence in B. pseudomallei . Two proposed novel species, B. mayonis sp. nov. and B. savannae sp. nov., were isolated from soil samples collected from multiple locations in northern Australia. The two proposed species belong to the Bpc but are phylogenetically distinct from all other members of this complex. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species within this significant complex of bacteria that are available for future studies.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03013-15 ◽

2015 ◽

Vol 82 (3) ◽

pp. 954-963 ◽

Cited By ~ 31

Author(s):

Erin P. Price ◽

Derek S. Sarovich ◽

Emma J. Smith ◽

Barbara MacHunter ◽

Glenda Harrington ◽

...

Keyword(s):

Burkholderia Pseudomallei ◽

Large Scale ◽

Phylogenetic Reconstruction ◽

Genomic Analysis ◽

Comparative Genomic ◽

Antibiotic Administration ◽

Northern Australia ◽

Content Type ◽

Clone Sequence ◽

Asian Isolate

ABSTRACTMelioidosis is a disease of humans and animals that is caused by the saprophytic bacteriumBurkholderia pseudomallei. Once thought to be confined to certain locations, the known presence ofB. pseudomalleiis 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 ofB. pseudomalleipopulations 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 455B. pseudomalleigenomes 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 environmentalB. pseudomalleiisolates 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.

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Salmonella in Captive Reptiles and Their Environment—Can We Tame the Dragon?

Microorganisms ◽

10.3390/microorganisms9051012 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1012

Author(s):

Magdalena Zając ◽

Magdalena Skarżyńska ◽

Anna Lalak ◽

Renata Kwit ◽

Aleksandra Śmiałowska-Węglińska ◽

...

Keyword(s):

Public Health ◽

Antimicrobial Resistance ◽

Detection Rate ◽

Large Scale ◽

Isolation And Identification ◽

Epidemiological Analysis ◽

High Occurrence ◽

Salmonella Serovars ◽

Floor Surfaces

Reptiles are considered a reservoir of a variety of Salmonella (S.) serovars. Nevertheless, due to a lack of large-scale research, the importance of Reptilia as a Salmonella vector still remains not completely recognized. A total of 731 samples collected from reptiles and their environment were tested. The aim of the study was to assess the prevalence of Salmonella in exotic reptiles kept in Poland and to confirm Salmonella contamination of the environment after reptile exhibitions. The study included Salmonella isolation and identification, followed by epidemiological analysis of the antimicrobial resistance of the isolates. Implementation of a pathway additional to the standard Salmonella isolation protocol led to a 21% increase in the Salmonella serovars detection rate. The study showed a high occurrence of Salmonella, being the highest at 92.2% in snakes, followed by lizards (83.7%) and turtles (60.0%). The pathogen was also found in 81.2% of swabs taken from table and floor surfaces after reptile exhibitions and in two out of three egg samples. A total of 918 Salmonella strains belonging to 207 serovars and serological variants were obtained. We have noted the serovars considered important with respect to public health, i.e., S. Enteritidis, S. Typhimurium, and S. Kentucky. The study proves that exotic reptiles in Poland are a relevant reservoir of Salmonella.

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Collateral impact of COVID-19: why should children continue to suffer?

European Journal of Pediatrics ◽

10.1007/s00431-021-03963-x ◽

2021 ◽

Cited By ~ 1

Author(s):

Prasad Nagakumar ◽

Ceri-Louise Chadwick ◽

Andrew Bush ◽

Atul Gupta

Keyword(s):

Young People ◽

Large Scale ◽

Hospital Admissions ◽

Severe Disease ◽

Children And Young People ◽

Collateral Effects ◽

Rsv Infection ◽

Syncytial Virus ◽

Set Up ◽

The Uk

AbstractThe COVID-19 pandemic caused by SARS-COV-2 virus fortunately resulted in few children suffering from severe disease. However, the collateral effects on the COVID-19 pandemic appear to have had significant detrimental effects on children affected and young people. There are also some positive impacts in the form of reduced prevalence of viral bronchiolitis. The new strain of SARS-COV-2 identified recently in the UK appears to have increased transmissibility to children. However, there are no large vaccine trials set up in children to evaluate safety and efficacy. In this short communication, we review the collateral effects of COVID-19 pandemic in children and young people. We highlight the need for urgent strategies to mitigate the risks to children due to the COVID-19 pandemic. What is Known:• Children and young people account for <2% of all COVID-19 hospital admissions• The collateral impact of COVID-19 pandemic on children and young people is devastating• Significant reduction in influenza and respiratory syncytial virus (RSV) infection in the southern hemisphere What is New:• The public health measures to reduce COVID-19 infection may have also resulted in near elimination of influenza and RSV infections across the globe• A COVID-19 vaccine has been licensed for adults. However, large scale vaccine studies are yet to be initiated although there is emerging evidence of the new SARS-COV-2 strain spreading more rapidly though young people.• Children and young people continue to bear the collateral effects of COVID-19 pandemic

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The utility of self-rated health in population surveys: the role of bodyweight

Population Health Metrics ◽

10.1186/s12963-021-00255-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Robert Bozick

Keyword(s):

Treatment Group ◽

Large Scale ◽

Well Being ◽

Control Group ◽

Self Rated Health ◽

Risk Of Mortality ◽

Highly Sensitive ◽

The Usa ◽

Group Members ◽

Nationally Representative

Abstract Background Self-rated health (SRH) is one of the most commonly used summary measures of overall health and well-being available to population scientists due to its ease of administration in large-scale surveys and to its efficacy in predicting mortality. This paper assesses the extent to which SRH is affected by its placement before or after questions about bodyweight on a survey, and whether differences in placement on the questionnaire affects SRH’s predictive validity. Methods I assessed the validity of SRH in predicting the risk of mortality by comparing outcomes of sample members who were asked to rate their health before reporting on their bodyweight (the control group) and sample members who were asked to rate their health after reporting on their bodyweight (the treatment group). Both the control and treatment group were randomly assigned via an experiment administered as a module in a nationally representative sample of adults in the USA in 2019 (N = 2523). Results The odds of reporting a more favorable appraisal of health are 30% lower for sample members who were in the treatment group when compared with the control group. Additionally, the SRH of treatment group members is significantly associated with their risk of mortality, while the SRH of control group members is not. Conclusion The findings from this study suggest that for researchers to maximize the utility of SRH, closer attention needs to be paid to the context of the survey within which it asked. SRH is highly sensitive to the questions that precede it, and this sensitivity may in turn mischaracterize the true health of the population that the survey is intending to measure.

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