Addressing a future pandemic: how can non-biological complex drugs prepare us for antimicrobial resistance threats?

Loss of effective antibiotics through antimicrobial resistance (AMR) is one of the greatest threats to human health. By 2050, the annual death rate resulting from AMR infections is predicted to have climbed from 1.27 million per annum in 2019, up to 10 million per annum. It is therefore imperative to preserve the effectiveness of both existing and future antibiotics, such that they continue to save lives. One way to conserve the use of existing antibiotics and build further contingency against resistant strains is to develop alternatives. Non-biological complex drugs (NBCDs) are an emerging class of therapeutics that show multi-mechanistic antimicrobial activity and hold great promise as next generation antimicrobial agents. We critically outline the focal advancements for each key material class, including antimicrobial polymer materials, carbon nanomaterials, and inorganic nanomaterials, and highlight the potential for the development of antimicrobial resistance against each class. Finally, we outline remaining challenges for their clinical translation, including the need for specific regulatory pathways to be established in order to allow for more efficient clinical approval and adoption of these new technologies.

A high-throughput amplicon sequencing approach for population-wide species diversity and composition survey.

Management of agricultural pests requires an understanding of pest species diversity, their interactions with beneficial insects and spatial-temporal patterns of pest abundance. Invasive and agriculturally important insect pests can build up very high populations, especially in cropping landscapes. Traditionally, sampling effort for species identification involves small sample sizes and is labour intensive. Here, we describe a multiprimer high throughput sequencing (HTS) metabarcoding method and associated analytical workflow for a rapid, intensive, high-volume survey of pest species compositions. We demonstrate our method using the taxonomically challenging Bemisia pest cryptic species complex as examples. The whiteflies Bemisia including the"tabaci" species are agriculturally important capable of vectoring diverse plant viruses that cause diseases and crop losses. Our multiprimer metabarcoding HTS amplicon approach simultaneously process high volumes of whitefly individuals, with efficiency to detect rare (i.e., 1%) test-species, while our improved whitefly primers for metabarcoding also detected beneficial hymenopteran parasitoid species from whitefly nymphs. Field-testing our redesigned Bemisia metabarcoding primer sets across the Tanzania, Uganda and Malawi cassava cultivation landscapes, we identified the sub-Saharan Africa 1 Bemisia putative species as the dominant pest species, with other cryptic Bemisia species being detected at various abundances. We also provide evidence that Bemisia species compositions can be affected by host crops and sampling techniques that target either nymphs or adults. Our multiprimer HTS metabarcoding method incorporated two overlapping amplicons of 472 bp and 518 bp that spanned the entire 657 bp 3' barcoding region for Bemisia, and is particularly suitable to molecular diagnostic surveys of this highly cryptic insect pest species complex that also typically exhibited high population densities in heavy crop infestation episodes. Our approach can be adopted to understand species biodiversity across landscapes, with broad implications for improving transboundary biosecurity preparedness, thus contributing to molecular ecological knowledge and the development of control strategies for high-density, cryptic, pest-species complexes.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains.

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia.

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the "Debug" Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Within-Season Changes in Land-Use Impact Pest Abundance in Smallholder African Cassava Production Systems.

Cassava (Manihot esculenta Crantz), an important commercial and food security crop in East and Central Africa, continues to be adversely affected by the whitefly Bemisia tabaci. In Uganda, changes in smallholder farming landscapes due to crop rotations can impact pest populations but how these changes affect pest outbreak risk is unknown. We investigated how seasonal changes in land-use have affected B. tabaci population dynamics and its parasitoids. We used a large-scale field experiment to standardize the focal field in terms of cassava age and cultivar, then measured how Bemisia populations responded to surrounding land-use change. Bemisia tabaci Sub-Saharan Africa 1 (SSA1) was identified using molecular diagnostics as the most prevalent species and the same species was also found on surrounding soybean, groundnut, and sesame crops. We found that an increase in the area of cassava in the 3-7-month age range in the landscape resulted in an increase in the abundance of the B. tabaci SSA1 on cassava. There was a negative relationship between the extent of non-crop vegetation in the landscape and parasitism of nymphs suggesting that these parasitoids do not rely on resources in the non-crop patches. The highest abundance of B. tabaci SSA1 nymphs in cassava fields occurred at times when landscapes had large areas of weeds, low to moderate areas of maize, and low areas of banana. Our results can guide the development of land-use strategies that smallholder farmers can employ to manage these pests.

Self-Medication with Antibiotics for Protection against COVID-19: The Role of Psychological Distress, Knowledge of, and Experiences with Antibiotics.

: Self-medication with antibiotics is a major contributing factor to antimicrobial resistance. Prior research examining factors associated with antibiotic self-medication has focused on an individual's knowledge about antibiotics, antibiotic usage practices, accessibility to antibiotic medication, and demographic characteristics. The role of psychological distress associated with perceived health risks in explaining antibiotic self-medication is less understood. This study was designed to address this knowledge gap in the context of the COVID-19 pandemic in Australia. An online survey of 2217 participants was conducted at the height of the initial outbreak and revealed that 19.5% of participants took antibiotics to protect themselves from COVID-19. Multivariate logistic analysis examined the predictors of taking antibiotics for protection against COVID-19. An integrative framework developed from the results illustrates potential pathways and facilitating factors that may contribute to prophylactic self-medication with antibiotics. Specifically, COVID-19 pandemic-induced psychological distress was significantly positively related to self-medication. Preventive use of antibiotics was also facilitated by a lack of understanding about antibiotics, inappropriate antibiotics usage practices, the nature of the patient-doctor relationship, and demographic characteristics. The findings highlight that to combat antimicrobial resistance due to self-medication, interventions need to focus on interrupting entrenched behavioural responses and addressing emotional responses to perceived health risks.

Improving climate suitability for Bemisia tabaci in East Africa is correlated with increased prevalence of whiteflies and cassava diseases.

Projected climate changes are thought to promote emerging infectious diseases, though to date, evidence linking climate changes and such diseases in plants has not been available. Cassava is perhaps the most important crop in Africa for smallholder farmers. Since the late 1990's there have been reports from East and Central Africa of pandemics of begomoviruses in cassava linked to high abundances of whitefly species within the Bemisia tabaci complex. We used CLIMEX, a process-oriented climatic niche model, to explore if this pandemic was linked to recent historical climatic changes. The climatic niche model was corroborated with independent observed field abundance of B. tabaci in Uganda over a 13-year time-series, and with the probability of occurrence of B. tabaci over 2 years across the African study area. Throughout a 39-year climate time-series spanning the period during which the pandemics emerged, the modelled climatic conditions for B. tabaci improved significantly in the areas where the pandemics had been reported and were constant or decreased elsewhere. This is the first reported case where observed historical climate changes have been attributed to the increase in abundance of an insect pest, contributing to a crop disease pandemic.

Publisher Correction: Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Insight into the microbial world of Bemisia tabaci cryptic species complex and its relationships with its host.

The 37 currently recognized Bemisia tabaci cryptic species are economically important species and contain both primary and secondary endosymbionts, but their diversity has never been mapped systematically across the group. To achieve this, PacBio sequencing of full-length bacterial 16S rRNA gene amplicons was carried out on 21 globally collected species in the B. tabaci complex, and two samples from B. afer were used here as outgroups. The microbial diversity was first explored across the major lineages of the whole group and 15 new putative bacterial sequences were observed. Extensive comparison of our results with previous endosymbiont diversity surveys which used PCR or multiplex 454 pyrosequencing platforms showed that the bacterial diversity was underestimated. To validate these new putative bacteria, one of them (Halomonas) was first confirmed to be present in MED B. tabaci using Hiseq2500 and FISH technologies. These results confirmed PacBio is a reliable and informative venue to reveal the bacterial diversity of insects. In addition, many new secondary endosymbiotic strains of Rickettsia and Arsenophonus were found, increasing the known diversity in these groups. For the previously described primary endosymbionts, one Portiera Operational Taxonomic Units (OTU) was shared by all B. tabaci species. The congruence of the B. tabaci-host and Portiera phylogenetic trees provides strong support for the hypothesis that primary endosymbionts co-speciated with their hosts. Likewise, a comparison of bacterial alpha diversities, Principal Coordinate Analysis, indistinct endosymbiotic communities harbored by different species and the co-divergence analyses suggest a lack of association between overall microbial diversity with cryptic species, further indicate that the secondary endosymbiont-mediated speciation is unlikely to have occurred in the B. tabaci species group.

African cassava whitefly, Bemisia tabaci, cassava colonization preferences and control implications.

Cassava is a staple food for people across sub-Saharan Africa. Over the last 20 years, there has been an increased frequency of outbreaks and crop damage in this region caused by the cassava-adapted Bemisia tabaci putative species. Little is known about when and why B. tabaci adults move and colonize new cassava crops, especially in farming systems that contain a mixture of cultivar types and plant ages. Here, we assessed experimentally whether the age and variety of cassava affected the density of B. tabaci. We also tested whether the age and variety of the source cassava field affected the variety preference of B. tabaci when they colonized new cassava plants. We placed uninfested potted "sentinel" plants of three cassava varieties (Nam 130, Nase 14, and Njule Red) in source fields containing one of two varieties (Nam 130 or Nase 14) and one of three age classes (young, medium, or old). After two weeks, the numbers of nymphs on the sentinel plants were used as a measure of colonization. Molecular identification revealed that the B. tabaci species was sub-Saharan Africa 1 (SSA1). We found a positive correlation between the density of nymphs on sentinel plants and the density of adults in the source field. The density of nymphs on the sentinels was not significantly related to the age of the source field. Bemisia tabaci adults did not preferentially colonize the sentinel plant of the same variety as the source field. There was a significant interactive effect, however, between the source and sentinel variety that may indicate variability in colonization. We conclude that managing cassava source fields to reduce B. tabaci abundance will be more effective than manipulating nearby varieties. We also suggest that planting a "whitefly sink" variety is unlikely to reduce B. tabaci SSA1 populations unless fields are managed to reduce B. tabaci densities using other integrative approaches.

Spectral separability and mapping potential of cassava leaf damage symptoms caused by whiteflies (Bemisia tabaci).

BACKGROUND: This study examines whether leaf spectra can be used to measure damage to cassava plants from whitefly (Bemisia tabaci), and the potential to translate measurements from leaf to landscape scale in eastern Africa. Symptoms of the cassava brown streak disease (CBSD) and cassava mosaic disease (CMD) viruses, and sooty mould (SM) blackening of lower leaves from whiteflies feeding on the upper leaves, were measured at the leaf scale with a high-resolution spectroradiometer and a single photon avalanche diode (SPAD) meter, which retrieves relative chlorophyll concentration. Spectral measurements were compared to the five-level visual scores used to assess the severity of each of the three damaging agents in the field, and also to leaf chemistry data. RESULTS: Leaves exhibiting severe CBSD and CMD were spectrally indistinguishable from leaves without any symptoms. Severe SM was spectrally distinctive but is likely to be difficult to map because of its occurrence in the lower crown. SPAD measurements were highly correlated with most foliar chemistry measurements and field scores of disease severity. Regression models between simulated Sentinel 2 bands, field scores and SPAD measurements were strongest using wavelengths with high importance weightings in random forest models. CONCLUSION: SPAD measurements are highly correlated to many foliar chemistry parameters, and should be considered for use in mapping disease severity over larger areas. Remaining challenges for mapping relate to the subtle expression of symptoms, the spatial distribution of disease severity within fields, and the small size and complex structure of the cassava fields themselves. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Recommendations for Laboratory Containment and Management of Gene Drive Systems in Arthropods.

Versatile molecular tools for creating driving transgenes and other invasive genetic factors present regulatory, ethical, and environmental challenges that should be addressed to ensure their safe use. In this article, we discuss driving transgenes and invasive genetic factors that can potentially spread after their introduction into a small proportion of individuals in a population. The potential of invasive genetic factors to increase their number in natural populations presents challenges that require additional safety measures not provided by previous recommendations regarding accidental release of arthropods. In addition to providing physical containment, invasive genetic factors require greater attention to strain management, including their distribution and identity confirmation. In this study, we focus on insects containing such factors with recommendations for investigators who are creating them, institutional biosafety committees charged with ensuring safety, funding agencies providing support, those managing insectaries handling these materials who are responsible for containment, and other persons who will be receiving insects-transgenic or not-from these facilities. We give specific examples of efforts to modify mosquitoes for mosquito-borne disease control, but similar considerations are relevant to other arthropods that are important to human health, the environment, and agriculture.

Standardized molecular diagnostic tool for the identification of cryptic species within the Bemisia tabaci complex.

BACKGROUND: The whitefly Bemisia tabaci complex harbours over 40 cryptic species that have been placed in 11 phylogenetically distinct clades based on the molecular characterization of partial mitochondrial DNA COI (mtCOI) gene region. Four cryptic species are currently within the invasive clade, i.e. MED, MEAM1, MEAM2 and IO. Correct identification of these species is a critical step towards implementing reliable measures for plant biosecurity and border protection; however, no standardized B. tabaci-specific primers are currently available which has caused inconsistencies in the species identification processes. RESULTS: We report three sets of polymerase chain reaction (PCR) primers developed to amplify the mtCOI region which can be used for genotyping MED, MEAM1 and IO species, and tested these primers on 91 MED, 35 MEAM1 and five IO individuals. PCR and sequencing of amplicons identified a total of 21, six and one haplotypes in MED, MEAM1 and IO respectively, of which six haplotypes were new to the B. tabaci database. CONCLUSION: These primer pairs enabled standardization and robust molecular species identification via mtCOI screening of the targeted invasive cryptic species and will improve quarantine decisions. Use of this diagnostic tool could be extended to other species within the complex. © 2017 Society of Chemical Industry.

Updated mtCOI reference dataset for the Bemisia tabaci species complex.

Members of the whitefly Bemisia tabaci species complex cause millions of dollars of damage globally and are considered one of the world's most invasive species. They are capable of causing extensive damage to major vegetable, grain legume and fiber crops. All member of the species complex are morphologically identical therefore, data from the partial mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequence has been used to identify the various species. The current reference dataset that is widely used is found on the CSIRO data portal. However, the reference set stored on the CSIRO data does not include newly added sequences (2013-2017), therefore an updated reference dataset is needed.  All mtCOI data for the Bemisia tabaci species complex were downloaded on 22 May 2017 from GenBank and after quality checking, a dataset of 1,071 unique sequences and 696 base pairs was generated (https://doi.org/10.6084/m9.figshare.5437420.v1).

The Trouble with MEAM2: Implications of Pseudogenes on Species Delimitation in the Globally Invasive Bemisia tabaci (Hemiptera: Aleyrodidae) Cryptic Species Complex.

Molecular species identification using suboptimal PCR primers can over-estimate species diversity due to coamplification of nuclear mitochondrial (NUMT) DNA/pseudogenes. For the agriculturally important whitefly Bemisia tabaci cryptic pest species complex, species identification depends primarily on characterization of the mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene. The lack of robust PCR primers for the mtDNA COI gene can undermine correct species identification which in turn compromises management strategies. This problem is identified in the B. tabaci Africa/Middle East/Asia Minor clade which comprises the globally invasive Mediterranean (MED) and Middle East Asia Minor I (MEAM1) species, Middle East Asia Minor 2 (MEAM2), and the Indian Ocean (IO) species. Initially identified from the Indian Ocean island of Réunion, MEAM2 has since been reported from Japan, Peru, Turkey and Iraq. We identified MEAM2 individuals from a Peruvian population via Sanger sequencing of the mtDNA COI gene. In attempting to characterize the MEAM2 mitogenome, we instead characterized mitogenomes of MEAM1. We also report on the mitogenomes of MED, AUS, and IO thereby increasing genomic resources for members of this complex. Gene synteny (i.e., same gene composition and orientation) was observed with published B. tabaci cryptic species mitogenomes. Pseudogene fragments matching MEAM2 partial mtDNA COI gene exhibited low frequency single nucleotide polymorphisms that matched low copy number DNA fragments (<3%) of MEAM1 genomes, whereas presence of internal stop codons, loss of expected stop codons and poor primer annealing sites, all suggested MEAM2 as a pseudogene artifact and so not a real species.

Global threat to agriculture from invasive species.

Invasive species present significant threats to global agriculture, although how the magnitude and distribution of the threats vary between countries and regions remains unclear. Here, we present an analysis of almost 1,300 known invasive insect pests and pathogens, calculating the total potential cost of these species invading each of 124 countries of the world, as well as determining which countries present the greatest threat to the rest of the world given their trading partners and incumbent pool of invasive species. We find that countries vary in terms of potential threat from invasive species and also their role as potential sources, with apparently similar countries sometimes varying markedly depending on specifics of agricultural commodities and trade patterns. Overall, the biggest agricultural producers (China and the United States) could experience the greatest absolute cost from further species invasions. However, developing countries, in particular, Sub-Saharan African countries, appear most vulnerable in relative terms. Furthermore, China and the United States represent the greatest potential sources of invasive species for the rest of the world. The analysis reveals considerable scope for ongoing redistribution of known invasive pests and highlights the need for international cooperation to slow their spread.

Risk Associated with the Release of Wolbachia-Infected Aedes aegypti Mosquitoes into the Environment in an Effort to Control Dengue.

BACKGROUND: In an effort to eliminate dengue, a successful technology was developed with the stable introduction of the obligate intracellular bacteria Wolbachia pipientis into the mosquito Aedes aegypti to reduce its ability to transmit dengue fever due to life shortening and inhibition of viral replication effects. An analysis of risk was required before considering release of the modified mosquito into the environment. METHODS: Expert knowledge and a risk assessment framework were used to identify risk associated with the release of the modified mosquito. Individual and group expert elicitation was performed to identify potential hazards. A Bayesian network (BN) was developed to capture the relationship between hazards and the likelihood of events occurring. Risk was calculated from the expert likelihood estimates populating the BN and the consequence estimates elicited from experts. RESULTS: The risk model for "Don't Achieve Release" provided an estimated 46% likelihood that the release would not occur by a nominated time but generated an overall risk rating of very low. The ability to obtain compliance had the greatest influence on the likelihood of release occurring. The risk model for "Cause More Harm" provided a 12.5% likelihood that more harm would result from the release, but the overall risk was considered negligible. The efficacy of mosquito management had the most influence, with the perception that the threat of dengue fever had been eliminated, resulting in less household mosquito control, and was scored as the highest ranked individual hazard (albeit low risk). CONCLUSIONS: The risk analysis was designed to incorporate the interacting complexity of hazards that may affect the release of the technology into the environment. The risk analysis was a small, but important, implementation phase in the success of this innovative research introducing a new technology to combat dengue transmission in the environment.

Phosphine resistance in Australian Cryptolestes species (Coleoptera: Laemophloeidae): perspectives from mitochondrial DNA cytochrome oxidase I analysis.

BACKGROUND: The flat grain beetle (FGB) species Cryptolestes ferrugineus, C. pusillus, C. pusilloides and C. turcicus are major stored-product pests worldwide, of which the first three are present in Australia. C. ferrugineus is also a species with high phosphine resistance status in various countries. Morphological identification of Cryptolestes species is difficult and represents an additional barrier to effective management of phosphine resistance in FGBs. RESULT: Mitochondrial DNA cytochrome oxidase I (mtDNA COI) gene characterisation enabled differentiation of the four major FGB pest species through direct sequence comparison, and enabled the development of a PCR-RFLP method for rapid species differentiation. We detected two mtDNA haplotypes (Cunk-01, 02) present at low frequencies with an average nucleotide divergence rate of 0.079 ± 0.011 (SE) from C. pusillus. This nucleotide divergence rate is similar to that between C. ferrugineus and C. pusilloides (0.088 ± 0.012). Male and female genitalia morphologies of the Cunk-02 individuals indicated they were consistent with C. pusillus, yet DNA sequence analyses suggested species-level divergence. The mtDNA COI gene of phosphine-bioassayed, lab-reared F1 generation survivors supported the presence of strong phosphine resistance in C. ferrugineus, but unexpectedly also in C. pusilloides and C. pusillus F1 survivors. CONCLUSION: We demonstrated the utility of molecular DNA techniques for differentiating closely related insect species, and its usefulness in assisting the management of pest insect species. The likely presence of a cryptic C. pusillus species in Australia and the possible development of strong phosphine resistance in Australian FGB pest species require further investigation. © 2014 Society of Chemical Industry.