Comparative analysis of qPCR and metagenomics for detecting antimicrobial resistance in wastewater: a case study.

Objective: The World Health Organization (WHO) has declared antimicrobial resistance (AMR) as one of the top threats to global public health. While AMR surveillance of human clinical isolates is well-established in many countries, the increasing threat of AMR has intensified efforts to detect antibiotic resistance genes (ARGs) accurately and sensitively in environmental samples, wastewater, animals, and food. Using five ARGs and the 16S rRNA gene, we compared quantitative PCR (qPCR) and metagenomic sequencing (MGS), two commonly used methods to uncover the wastewater resistome.

Towards the identification of body fluids using RT-LAMP isothermal amplification coupled with CRISPR-Cas12a.

While often necessary in sexual assault cases, confirmatory identification of body fluids can be a lengthy and/or costly process. In particular, the detection of vaginal fluid and menstrual fluid in forensic casework is limited to endpoint reverse-transcription PCR to detect fluid-specific messenger RNA (mRNA) markers as there are no robust chemical or enzymatic techniques available for these fluids. Similarly, testing for rectal mucosa is not possible with standard methods, the presence of which would provide probative value in cases of alleged anal penetration, although mRNA-based markers have recently been described.

An automated method for the generation of bloodstain pattern metrics from images of blood spatter patterns.

An improved automated bloodstain pattern analysis method has been developed and validated, which utilises computer vision techniques to identify bloodstains on a plain background within a digital image. The method generates metrics relating to the individual stains as well as the overall pattern, including bloodstain pattern specific metrics such as the gamma angle, circularity, solidity, area of convergence, stain density and pattern linearity. This method provides an objective approach to the analysis of bloodstains and bloodstain patterns and can generate a wealth of quantitative data that is currently not obtainable using manual techniques or other image-based programs currently utilised in the discipline.

Jointly estimating epidemiological dynamics of Covid-19 from case and wastewater data in Aotearoa New Zealand.

Background: Timely and informed public health responses to infectious diseases such as COVID-19 necessitate reliable information about infection dynamics. The case ascertainment rate (CAR), the proportion of infections that are reported as cases, is typically much less than one and varies with testing practices and behaviours, making reported cases unreliable as the sole source of data. The concentration of viral RNA in wastewater samples provides an alternate measure of infection prevalence that is not affected by clinical testing, healthcare-seeking behaviour or access to care.

Soil conditions are a more important determinant of microbial community composition and functional potential than neighboring plant diversity.

Replanting is an important tool for ecological recovery. Management strategies, such as planting areas with monocultures or species mixtures, have implications for restoration success. We used 16S and ITS rRNA gene amplicon sequencing and shotgun metagenomics to assess how the diversity of neighboring tree species impacted soil bacterial and fungal communities, and their functional potential, within the root zone of mānuka () trees.

Genetic diversity of murine norovirus populations less susceptible to chlorine.

High genetic diversity in RNA viruses contributes to their rapid adaptation to environmental stresses, including disinfection. Insufficient disinfection can occur because of the emergence of viruses that are less susceptible to disinfection. However, understanding regarding the mechanisms underlying the alteration of viral susceptibility to disinfectants is limited.

Black soldier fly-based bioconversion of biosolids: Microbial community dynamics and fate of antibiotic resistance genes.

Biosolids as by-products of wastewater treatment can contain a large spectrum of pathogens and antibiotic resistance genes (ARGs). Insect-based bioconversion using black soldier fly larvae (BSFL) is an emerging technology that has shown to reduce significant amounts of biosolids quickly and produce larvae biomass containing low heavy metal concentrations. However, to the best of our knowledge, this is the first study investigating the transfer of pathogens and ARGs from biosolids into the process' end-products, BSFL and frass.

Using RNA-Stable isotope probing to investigate methane oxidation metabolites and active microbial communities in methane oxidation coupled to denitrification.

The active denitrifying communities performing methane oxidation coupled to denitrification (MOD) were investigated using samples from an aerobic reactor (∼20% O and 2% CH) and a microaerobic reactor (2% O, 2% CH) undertaking denitrification. The methane oxidation metabolites excreted in the reactors were acetate, methanol, formate and acetaldehyde. Using anaerobic batch experiments supplemented with exogenously supplied C-labelled metabolites, the active denitrifying bacteria were identified using 16S rRNA amplicon sequencing and RNA-stable isotope probing (RNA-SIP).

Extended-spectrum beta-lactamase, AmpC, and carbapenemase-producing Gram-negative wastewater isolates from Aotearoa New Zealand.

Extended-spectrum beta-lactamase, AmpC, and carbapenemase-producing bacteria were isolated from raw sewage, effluent, oxidation pond water, and sediment from a wastewater treatment plant in Aotearoa New Zealand. Here, we report the assemblies of 17 isolates belonging to the species , , , , , , , , , and .

Pharmacological evaluation of new generation OXIZID synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the largest classes of new psychoactive substances, and are increasingly associated with severe adverse effects and death compared to the phytocannabinoid Δ-tetrahydrocannabinol (THC). In the attempt to circumvent the rapid emergence of novel SCRAs, several nations have implemented 'generic' legislations, or 'class-wide' bans based on common structural scaffolds. However, this has only encouraged the incorporation of new chemical entities, including distinct core and linker structures, for which there is a dearth of pharmacological data.

Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples.

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV).

Draft genome sequences of spp. isolates from New Zealand environmental sources.

is often used as a fecal indicator bacterium for water quality monitoring. We report the draft genome sequences of 500 isolates including newly described species, namely , and , obtained from diverse environmental sources to assist with improved public health risk assessments.

Testing methods to estimate population size for wastewater treatment plants using census data: Implications for wastewater-based epidemiology.

In wastewater-based epidemiology (WBE), wastewater loads are commonly reported as a per capita value. Census population counts are often used to obtain a population size to normalise wastewater loads. However, the methods used to calculate the population size of wastewater treatment plants (WWTPs) from census data are rarely reported in the WBE literature.

High-resolution genomic analysis to investigate the impact of the invasive brushtail possum (Trichosurus vulpecula) and other wildlife on microbial water quality assessments.

Escherichia coli are routine indicators of fecal contamination in water quality assessments. Contrary to livestock and human activities, brushtail possums (Trichosurus vulpecula), common invasive marsupials in Aotearoa/New Zealand, have not been thoroughly studied as a source of fecal contamination in freshwater. To investigate their potential role, Escherichia spp.

Persistence of Infectious Human Norovirus in Estuarine Water.

Norovirus is the predominant cause of viral acute gastroenteritis globally. While person-to-person is the most reported transmission route, norovirus is also associated with waterborne and foodborne illness, including from the consumption of contaminated bivalve molluscan shellfish. The main cause of shellfish contamination is via the bioaccumulation of norovirus from growing waters impacted by human wastewater.

Partial validation of multiplexed real-time quantitative PCR assays for forensic body fluid identification.

Confirmatory body fluid identification using messenger RNA (mRNA) is a well-established technique to address issues encountered with conventional testing - such as poor sensitivity, specificity, and a lack of available tests for all body fluids of interest. For over a decade, endpoint reverse-transcription polymerase chain reaction (RT-PCR) assays have been used in forensic casework for such purposes. However, in comparison with real-time quantitative RT-PCR (RT-qPCR), endpoint RT-PCR has lower sensitivity, precision, and linear dynamic range.

Microaerobic methane-driven denitrification in a biotrickle bed - Investigating the active microbial biofilm community composition using RNA-stable isotope probing.

A microaerobic (2% O v/v) biotrickle bed reactor supplied continuously with 2% methane to drive nitrate removal (MAME-D) was investigated using 16S rDNA and rRNA amplicon sequencing in combination with RNA-stable isotope probing (RNA-SIP) to identify the active microorganisms. Methane removal rates varied from 500 to 1000 mmol mh and nitrate removal rates from 25 to 58 mmol mh over 55 days of operation. Biofilm samples from the column were incubated in serum bottles supplemented with CH.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000).

Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch.

The Potential of Myrtaceae Species for the Phytomanagement of Treated Municipal Wastewater.

The use of native plants in land application systems for treated municipal wastewater (TMW) can contribute to ecological restoration. However, research on the potential of native species to manage the nutrients and contaminants contained in TMW is scarce. At a 10-hectare field site irrigated with TMW at >4000 mm yr, we investigated the distribution of nutrients and trace elements in the soil-plant system, comparing the New Zealand native Myrtaceae species and with pasture.

Diagnosis of protozoa diarrhoea in Campylobacter patients increases markedly with molecular techniques.

Cryptosporidium and Giardia are major causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques.

One-step endpoint RT-PCR assays for confirmatory body fluid identification.

Messenger RNA (mRNA) expression analysis is increasingly used in casework, in the form of multiplex two-step reverse transcriptase PCR (RT-PCR) assays such as CellTyper 2 (CT2), developed by the Institute of Environmental Science and Research (ESR). This paper presents the development of a one-step endpoint RT-PCR workflow to improve the efficiency and precision of confirmatory body fluid identification. A comparative study of commercial one-step RT-PCR kits was undertaken, with the highest performing kit (RNA to C) retained for further development.

A clinical review of mpox for the Aotearoa New Zealand clinician.

The World Health Organization declared mpox (formerly monkeypox) a Public Health Emergency of International Concern in July 2022. Aotearoa New Zealand has reported cases of mpox since July, with reports of locally acquired cases since October 2022. The 2022 global mpox outbreak highlights many features of the illness not previously described, including at-risk populations, mode of transmission, atypical clinical features, and complications.