Extending the new era of genomic testing into pregnancy management: A proposed model for Australian prenatal services.

BACKGROUND: Trio exome sequencing can be used to investigate congenital abnormalities identified on pregnancy ultrasound, but its use in an Australian context has not been assessed. AIMS: Assess clinical outcomes and changes in management after expedited genomic testing in the prenatal period to guide the development of a model for widespread implementation. MATERIALS AND METHODS: Forty-three prospective referrals for whole exome sequencing, including 40 trios (parents and pregnancy), two singletons and one duo were assessed in a tertiary hospital setting with access to a state-wide pathology laboratory. Diagnostic yield, turn-around time (TAT), gestational age at reporting, pregnancy outcome, change in management and future pregnancy status were assessed for each family. RESULTS: A clinically significant genomic diagnosis was made in 15/43 pregnancies (35%), with an average TAT of 12 days. Gestational age at time of report ranged from 16 + 5 to 31 + 6 weeks (median 21 + 3 weeks). Molecular diagnoses included neuromuscular and skeletal disorders, RASopathies and a range of other rare Mendelian disorders. The majority of families actively used the results in pregnancy decision making as well as in management of future pregnancies. CONCLUSIONS: Rapid second trimester prenatal genomic testing can be successfully delivered to investigate structural abnormalities in pregnancy, providing crucial guidance for current and future pregnancy management. The time-sensitive nature of this testing requires close laboratory and clinical collaboration to ensure appropriate referral and result communication. We found the establishment of a prenatal coordinator role and dedicated reporting team to be important facilitators. We propose this as a model for genomic testing in other prenatal services.

Hb Mizuho Case Report; Early Genomic Testing Facilitates a Life Changing Diagnosis.

Unstable variant hemoglobinopathies are an uncommon cause of hemolysis in the pediatric patient and may cause a delay in diagnosis if there is not a high index of suspicion. Hemoglobin (Hb) Mizuho is a rare unstable hemoglobinopathy caused by a pathogenic variant of the HBB gene with a severe phenotype. Here we report on the first known case of Hb Mizuho in Australia, presenting with features of acute and chronic hemolysis. The morphological features on blood film review, in conjunction with biochemical findings and other clinical features, did not immediately suggest an alternative diagnosis and a Next Generation Sequencing gene analysis approach was taken to investigate genes associated with red blood cell disorders and atypical uremic syndrome. The HBB Mizuho variant was detected and established the diagnosis. This report highlights the challenge of diagnosing Hb Mizuho on conventional testing and the need for early genomic testing to clarify a diagnosis.

Re-use of remediated soils for the bioremediation of waste oil sludge.

We investigated the possibility of re-using remediated soils for new bioremediation projects by spiking these soils with waste oil sludge in laboratory based microcosms. The level of Total Petroleum Hydrocarbon (TPH) reduction was high (>80%) in naturally attenuated microcosms and was not significantly improved by biostimulation, bioaugmentation and the combined treatment of bioaugmentation and biostimulation by week 12. This indicated that the observed TPH reduction might have been related to the soil's inherent hydrocarbon-degrading potential. Microbial community analysis (16S rDNA and ITS-based Denaturing Gradient Gel Electrophoresis fingerprints) confirmed the dominance of hydrocarbon degrading genera such as Alcanivorax and Scedosporium. Cluster and Shannon diversity analysis revealed similar but stable bacterial and fungal communities in naturally attenuated and amended microcosms indicating that rapid reduction in TPH may not always be accompanied by changes in soil microbial communities. This study has therefore shown that soils previously used for bioremediation can have an improved hydrocarbon degrading potential which was successfully re-harnessed for new projects. This ability to re-harness this potential is attractive because it substantially reduces operational costs as no additional bioremediation treatments are needed. It can also extend a landfill's lifespan as soils can be re-used again before landfill disposal.

The application of a carrier-based bioremediation strategy for marine oil spills.

The application of recycled marine materials to develop sustainable remediation technologies in marine environment was assessed. The remediation strategy consisted of a shell carrier mounted bacterial consortium composed of hydrocarbonoclastic strains enriched with nutrients (Bioaug SC). Pilot scale studies (5000 l) were used to examine the ability of Bioaug-SC to degrade weathered crude oil (10 g l(-1); initially 315,000±44,000 mg l(-1)) and assess the impacts of the introduction and biodegradation of oil. Total petroleum hydrocarbon mass was effectively reduced by 53.3 (±5.75)% to 147,000 (±21,000) mg l(-1) within 27 weeks. 16S rDNA bacterial community profiling using Denaturant Gradient Gel Electrophoresis revealed that cyanobacteria and Proteobacteria dominated the microbial community. Aquatic toxicity assessment was conducted by ecotoxicity assays using brine shrimp hatchability, Microtox and Phaeodactylum tricornutum. This study revealed the importance of combining ecotoxicity assays with oil chemistry analysis to ensure safe remediation methods are developed.

Confined aquifers as viral reservoirs.

Knowledge about viral diversity and abundance in deep groundwater reserves is limited. We found that the viral community inhabiting a deep confined aquifer in South Australia was more similar to reclaimed water communities than to the viral communities in the overlying unconfined aquifer community. This similarity was driven by high relative occurrence of the single-stranded DNA viral groups Circoviridae, Geminiviridae and Microviridae, which include many known plant and animal pathogens. These groups were present in a 1500-year-old water situated 80 m below the surface, which suggests the potential for long-term survival and spread of potentially pathogenic viruses in deep, confined groundwater. Obtaining a broader understanding of potentially pathogenic viral communities within aquifers is particularly important given the ability of viruses to spread within groundwater ecosystems.

Carrier mounted bacterial consortium facilitates oil remediation in the marine environment.

Marine oil pollution can result in the persistent presence of weathered oil. Currently, removal of weathered oil is reliant on chemical dispersants and physical removal, causing further disruption. In contrast few studies have examined the potential of an environmentally sustainable method using a hydrocarbon degrading microbial community attached to a carrier. Here, we used a tank mesocosm system (50 l) to follow the degradation of weathered oil (10 g l(-1)) using a bacterial consortium mobilised onto different carrier materials (alginate or shell grit). GCMS analysis demonstrated that the extent of hydrocarbon degradation was dependent upon the carrier material. Augmentation of shell grit with nutrients and exogenous hydrocarbon degraders resulted in 75±14% removal of >C32 hydrocarbons after 12 weeks compared to 20±14% for the alginate carrier. This study demonstrated the effectiveness of a biostimulated and bioaugmented carrier material to degrade marine weathered oil.

A complementary approach to identifying and assessing the remediation potential of hydrocarbonoclastic bacteria.

The isolation and assessment of hydrocarbonoclastic bacteria often represents a key strategy in the bioremediation of hydrocarbon-contaminated sites. However the isolation and assessment of such bacteria is often a lengthy and expensive procedure. The aim of this study was to identify potential isolates for use in the remediation of hydrocarbon contaminated sites using a combination of selective isolation plating, the Biolog system and subsequent multivariate analyses. The use of weathered oil as the main C source restricted the number of isolates growing to 5×10(2)CFUg soil(-1). Isolates (n=96) were then assessed individually using Biolog MT2 plates with seven different hydrocarbons (dodecane, tridecane, hexadecane, octadecane, eicosane, naphthalene and phenanthrene). The results indicated that all isolates were able to grow on at least one hydrocarbon from the seven chosen. This confirmed that the isolation media developed was selective in isolating hydrocarbonoclastic bacteria only. Cluster analysis of Biolog data separated the isolates into two discrete clusters with cluster 2 identifying hydrocarbonoclastic bacteria that are effective in degrading a variety of contaminants. Further study on the isolates from cluster 2 was carried out based on their phylogenetic analysis. Phylogenetic analysis of 28 bacterial isolates from cluster 2 based on the 1500bp sequences from 16S rDNA genes using MRBAYES confirmed all isolates as being hydrocarbonoclastic, providing supportive evidence that isolates from cluster 2 have a potential use in bioremediation. This approach could improve both the speed and efficiency of the commercial bioremediation process.

Investigating the effectiveness of economically sustainable carrier material complexes for marine oil remediation.

The application of bioremediation to marine oil spills is limited due to dilution of either nutrients or hydrocarbonoclastic organisms. This study investigated the effectiveness of three unique natural carrier materials (mussel shells, coir peat and mussel shell/agar complex) which allowed nutrients, hydrocarbonoclastic organisms and oil to be in contact, facilitating remediation. TPH analysis after 30 d showed that mussel shells exhibited the greatest capacity to degrade oil with a 55% reduction (123.3 mg l(-1) from 276 mg l(-1)) followed by mussel shell/agar complex (49%) and coir peat (36%). Both the mussel shells and mussel shell/agar complex carriers were significantly different to the control (P=0.008 and P=0.002, respectively). DGGE based cluster analysis of the seawater microbial community showed groupings based on time rather than carriers. This study demonstrated that inexpensive, accessible waste materials used as carriers of hydrocarbonoclastic bacteria led to significant degradation of hydrocarbon contaminants in seawater.

The importance of weathered crude oil as a source of hydrocarbonoclastic microorganisms in contaminated seawater.

This study investigated the hydrocarbonoclastic microbial community present on weathered crude oil and their ability to degrade weathered oil in seawater obtained from the Gulf St. Vincent (SA, Australia). Examination of the native seawater communities capable of utilizing hydrocarbon as the sole carbon source identified a maximum recovery of just 6.6 × 10(1) CFU/ml, with these values dramatically increased in the weathered oil, reaching 4.1 × 10(4) CFU/ml. The weathered oil (dominated by >C30 fractions; 750,000 +/- 150,000 mg/l) was subject to an 8 week laboratory-based degradation microcosm study. By day 56, the natural inoculums degraded the soluble hydrocarbons (initial concentrations 3,400 +/- 700 mg/l and 1,700 +/- 340 mg/l for the control and seawater, respectively) to below detectable levels, and biodegradation of the residual oil reached 62% (254,000 +/- 40,000 mg/l) and 66% (285,000 +/- 45,000 mg/l) in the control and seawater sources, respectively. In addition, the residual oil gas chromatogram profiles changed with the presence of short and intermediate hydrocarbon chains. 16S rDNA DGGE sequence analysis revealed species affiliated with the genera Roseobacter, Alteromonas, Yeosuana aromativorans, and Pseudomonas, renowned oil-degrading organisms previously thought to be associated with the environment where the oil contaminated rather than also being present in the contaminating oil. This study highlights the importance of microbiological techniques for isolation and characterisation, coupled with molecular techniques for identification, in understanding the role and function of native oil communities.

Identification of Vibrio natriegens uvrA and uvrB genes and analysis of gene regulation using transcriptional reporter plasmids.

Nucleotide excision repair (NER) rectifies a variety of chemically and structurally distinct DNA lesions. The current model of NER is based upon the enteric bacterium Escherichia coli and there is scarce information about how other bacterial species respond to, and correct, DNA damage. Here we report the isolation and functional analysis of the uvrA and uvrB genes from Vibrio natriegens, a naturally occurring marine bacterium. Genetic studies were completed to assess the repair capabilities of V. natriegens uvrA and uvrB in E. coli uvrA and uvrB mutants. In addition to the genetic studies, transcriptional fusions between the luciferase gene and the 5' regulatory regions of uvrA and uvrB gene of V. natriegens and E. coli were constructed. Luminescent measurements from E. coli transformed with these constructs showed that whilst the response to UV irradiation of either E. coli or V. natriegens uvrA regulatory sequences was similar, both the rate and induction of luminescence detected from the uvrB regulatory regions differed.