Jean Y. H. Lee

An exquisitely sensitive quantitative PCR (qPCR) assay targeting the high-copy number insertion sequence, IS 2404 , is the gold standard diagnostic test for Mycobacterium ulcerans , the agent of the neglected tropical skin disease Buruli ulcer. Here, we designed and tested an alternative M. ulcerans diagnostic test, a fluorescent probe-based, loop-mediated isothermal amplification (P-LAMP) assay that also targets IS 2404 . Benchmarked against IS 2404 qPCR, P-LAMP was equally specific and nearly as sensitive (analytical sensitivity of four vs two M. ulcerans genome copies). Clinical and environmental specimen validation against IS 2404 qPCR showed P-LAMP had 100% sensitivity and specificity. P-LAMP was twice as fast as qPCR with an average time-to-positive at the limit-of-detection of 19 minutes. P-LAMP targeting IS 2404 is a versatile assay that addresses the performance issues of previously described IS 2404 LAMP formats. This study tackles a key research priority for Buruli ulcer and represents another avenue for the development of rapid and accessible molecular diagnostics for this neglected tropical disease. IMPORTANCE: Buruli ulcer is a neglected tropical disease caused by infection with Mycobacterium ulcerans . Correct diagnosis is essential before appropriate treatment for Buruli ulcer can be started. Development of a portable, easy-to-use diagnostic test for M. ulcerans has been identified by the World Health Organization as a research priority. Buruli ulcer most commonly occurs in remote, rural areas; therefore, an ideal test is one that can be used at (or near) the point of care (community health centres) without the need for specialized laboratories. Here, we describe a molecular test using loop-mediated isothermal amplification (LAMP) to detect DNA specific to M. ulcerans and show that this new test has equivalent performance to the gold standard M. ulcerans PCR test currently used worldwide. Our new test is rapid (30 minutes to run), simple to perform, and could be further developed into a robust, portable format to provide accessible and affordable M. ulcerans diagnostics anywhere.

Antimicrobial resistance (AMR) is a critical global public health threat, with bacterial pathogens of primary concern. Pathogen genomics has revolutionized the study of bacterial pathogens and provided deep insights into the mechanisms and dissemination of AMR, with the precision of whole-genome sequencing informing better control strategies. However, generating actionable data from genomic surveillance and diagnostic efforts requires integration at the public health and clinical interface that goes beyond academic efforts to identify resistance mechanisms, undertake post hoc analyses of outbreaks, and share data after research publications. In addition to timely genomics data, consideration also needs to be given to epidemiological sampling frames, analysis, and reporting mechanisms that meet International Organization for Standardization (ISO) standards and generation of reports that are interpretable and actionable for public health and clinical “end-users.” Importantly, ensuring all countries have equitable access to data and technology is critical, through timely data sharing following the FAIR principles (findable, accessible, interoperable, and re-usable). In this review, we describe (i) advances in genomic approaches for AMR research and surveillance to understand emergence, evolution, and transmission of AMR and the key requirements to enable this work and (ii) discuss emerging and future applications of genomics at the clinical and public health interface, including barriers to implementation. Harnessing advances in genomics-enhanced AMR research and embedding robust and reproducible workflows within clinical and public health practice promises to maximize the impact of pathogen genomics for AMR globally in the coming decade.

Multidrug-resistant bacterial pathogens like vancomycin-resistant Enterococcus faecium (VREfm) are a critical threat to human health¹. Daptomycin is a last-resort antibiotic for VREfm infections with a novel mode of action², but for which resistance has been widely reported but is unexplained. Here we show that rifaximin, an unrelated antibiotic used prophylactically to prevent hepatic encephalopathy in patients with liver disease³, causes cross-resistance to daptomycin in VREfm. Amino acid changes arising within the bacterial RNA polymerase in response to rifaximin exposure cause upregulation of a previously uncharacterized operon (prdRAB) that leads to cell membrane remodelling and cross-resistance to daptomycin through reduced binding of the antibiotic. VREfm with these mutations are spread globally, making this a major mechanism of resistance. Rifaximin has been considered ‘low risk’ for the development of antibiotic resistance. Our study shows that this assumption is flawed and that widespread rifaximin use, particularly in patients with liver cirrhosis, may be compromising the clinical use of daptomycin, a major last-resort intervention for multidrug-resistant pathogens. These findings demonstrate how unanticipated antibiotic cross-resistance can undermine global strategies designed to preserve the clinical use of critical antibiotics.

In Australia, native possums are a major wildlife reservoir for Mycobacterium ulcerans , the causative agent of the neglected tropical skin disease Buruli ulcer (BU). Large-scale possum excreta surveys that use PCR to detect M. ulcerans in 100–1,000 s of excreta specimens are an important tool that can inform geospatial modeling and predict locations of future human BU risk. However, the significant expense of commercial kits used to extract DNA from specimens is a major barrier to routine implementation. Here, we developed a low-cost method for DNA extraction from possum excreta, possum tissue, and pure mycobacterial cultures, using a guanidinium isothiocyanate lysis solution and paramagnetic beads. In a 96-well plate format for high-throughput processing, the paramagnetic bead DNA extraction method was threefold less sensitive but only 1/6 the cost of a commonly used commercial kit. Applied to tissue swabs, the method was fourfold more sensitive and 1/5 the cost of a commercial kit. When used for preparing DNA from pure mycobacterial cultures, the method yielded purified genomic DNA with quality metrics comparable to more lengthy techniques. Our paramagnetic bead method is an economical means to undertake large-scale M. ulcerans environmental surveillance that will directly inform efforts to halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCE: Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans . Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

In Australia, native possums are an animal reservoir for Mycobacterium ulcerans , the causative agent of Buruli ulcer, a neglected tropical skin disease that can progress to extensive ulceration with deformity and disability. Surveillance of possum excreta for shedding of M. ulcerans can be used to inform geospatial modelling to predict locations at which humans are at increased risk of disease acquisition. This discovery provides opportunities to disrupt transmission pathways. However, the significant expense of commercial kits used for DNA extraction from environmental samples in large scale surveillance studies can hinder implementation of public health measures. To address this barrier, we developed a low-cost method for extraction of nucleic acids from possum excreta, possum tissue swabs, and mycobacterial cultures, using a guanidinium thiocyanate lysis solution and paramagnetic beads for DNA clean-up. In 96-well plate format for high-throughput processing, the SPRI-bead DNA extraction method for possum excreta was 3-fold less sensitive but only 1/6 the cost of a widely used commercial kit. While a SPRI-bead microtube-based extraction method for tissue swabs sampled from possums was 4-fold more sensitive and 1/5 the cost of the corresponding commercial kit. Furthermore, when used for preparing DNA from pure mycobacterial cultures, the SPRI-bead method produced genomic DNA with quality metrics comparable to more laborious techniques. The methods described here provide an economical means to continue large-scale M. ulcerans environmental surveillance that should facilitate efforts to halt the spread of Buruli ulcer in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCE: Buruli ulcer is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In this region of the world Buruli ulcer is a zoonosis, Australian native possums are a major wildlife reservoir of the causative agent, Mycobacterium ulcerans , and mosquitoes the vector to humans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria.

Buruli ulcer is a chronic ulcerative disease of skin and subcutaneous tissues caused by infection with Mycobacterium ulcerans. Although Australian possums are known to be susceptible to Buruli ulcer, many aspects of the disease in possums, including welfare impacts, remain largely unreported. Severe clinical Buruli ulcer was identified in four common ringtail possums (Pseudocheirus peregrinus) from Melbourne, Victoria. All four possums were euthanised due to welfare concerns and poor prognosis, based on the presence of deep ulcerative lesions to paws, some of which exposed carpal or tarsal bones. These lesions were considered likely to cause partial or complete loss of limb function, which would severely affect the welfare and survival of these wild arboreal animals. Histologically, there was severe ulcerative necrotising pyogranulomatous dermatitis, panniculitis, and myositis, with intralesional acid-fast bacteria. M. ulcerans was detected by real-time PCR in all swabs and biological tissues collected from all four cases. Buruli ulcer may be an important and under-recognised cause of poor possum welfare in endemic areas. In addition to severe cutaneous lesions, infection extends to underlying bones and joints, and the systemic distribution of M. ulcerans throughout all major tissues and internal organs, as observed in these four cases, may further impact the health and fitness of infected possums. Further research is needed to investigate the epidemiology, pathogenesis and impacts of Buruli ulcer in possums, and to inform the design of interventions that may protect the health and welfare of possums and other affected animal species.

Among the 16 two-component systems in the opportunistic human pathogen Staphylococcus aureus , only WalKR is essential. Like the orthologous systems in other Bacillota, S. aureus WalKR controls autolysins involved in peptidoglycan remodeling and is therefore intimately involved in cell division. However, despite the importance of WalKR in S. aureus , the basis for its essentiality is not understood and the regulon is poorly defined. Here, we defined a consensus WalR DNA-binding motif and the direct WalKR regulon by using functional genomics, including chromatin immunoprecipitation sequencing, with a panel of isogenic walKR mutants that had a spectrum of altered activities. Consistent with prior findings, the direct regulon includes multiple autolysin genes. However, this work also revealed that WalR directly regulates at least five essential genes involved in lipoteichoic acid synthesis ( ltaS ): translation ( rplK ), DNA compaction ( hup ), initiation of DNA replication ( dnaA , hup ) and purine nucleotide metabolism ( prs ). Thus, WalKR in S. aureus serves as a polyfunctional regulator that contributes to fundamental control over critical cell processes by coordinately linking cell wall homeostasis with purine biosynthesis, protein biosynthesis, and DNA replication. Our findings further address the essentiality of this locus and highlight the importance of WalKR as a bona fide target for novel anti-staphylococcal therapeutics. IMPORTANCE: The opportunistic human pathogen Staphylococcus aureus uses an array of protein sensing systems called two-component systems (TCS) to sense environmental signals and adapt its physiology in response by regulating different genes. This sensory network is key to S. aureus versatility and success as a pathogen. Here, we reveal for the first time the full extent of the regulatory network of WalKR, the only staphylococcal TCS that is indispensable for survival under laboratory conditions. We found that WalKR is a master regulator of cell growth, coordinating the expression of genes from multiple, fundamental S. aureus cellular processes, including those involved in maintaining cell wall metabolism, protein biosynthesis, nucleotide metabolism, and the initiation of DNA replication.

Global spread of multidrug-resistant, hospital-adapted Staphylococcus epidermidis lineages underscores the need for new therapeutic strategies. Here we show that many S. epidermidis isolates belonging to these lineages display cryptic susceptibility to penicillin/β-lactamase inhibitor combinations under in vitro conditions, despite carrying the methicillin resistance gene mecA. Using a mouse thigh model of S. epidermidis infection, we demonstrate that single-dose treatment with amoxicillin/clavulanic acid significantly reduces methicillin-resistant S. epidermidis loads without leading to detectable resistance development. On the other hand, we also show that methicillin-resistant S. epidermidis is capable of developing increased resistance to amoxicillin/clavulanic acid during long-term in vitro exposure to these drugs. These findings suggest that penicillin/β-lactamase inhibitor combinations could be a promising therapeutic candidate for treatment of a high proportion of methicillin-resistant S. epidermidis infections, although the in vivo risk of resistance development needs to be further addressed before they can be incorporated into clinical trials.

This paper aims to explore the interplay between smart city projects and international development cooperation initiatives while identifying potential challenges associated with their ongoing processes. Numerous studies have investigated the potential economic development opportunities through smart city initiatives at the local level. This paper contributes to existing literature by examining the convergence of smart city and international development projects, providing a comprehensive perspective on these projects in terms of governance, motivation, financing, and inclusivity. This research includes a critical assessment of these projects, with the hope that both communities can actively engage in dialogue and cooperation to share their shortcomings and successful experiences, ultimately achieving a ‘smart’ balance in their development landscape.

Multidrug-resistant bacterial pathogens like vancomycin-resistant Enterococcus faecium (VREfm) are a critical threat to human health. Daptomycin is a last-resort antibiotic for VREfm infections with a novel mode-of-action, but for which resistance has surprisingly been widely reported but unexplained. Here we show that rifaximin, an unrelated antibiotic used prophylactically to prevent hepatic encephalopathy in liver disease patients, causes cross-resistance to daptomycin in VREfm. Amino acid changes arising within the bacterial RNA polymerase in response to rifaximin exposure cause upregulation of a previously uncharacterised operon ( prdRAB ) that leads to cell membrane remodelling and cross-resistance to daptomycin through reduced binding of the antibiotic. Alarmingly, VREfm with these mutations are spread globally, making this a major mechanism of resistance. Rifaximin has been considered ‘low-risk’ for antibiotic resistance development. Our study shows this assumption is flawed and widespread rifaximin use, particularly in patients with liver cirrhosis, may be compromising the clinical use of daptomycin, a major last-resort intervention for multidrug-resistant pathogens. These findings demonstrate how unanticipated antibiotic cross-resistance can undermine global strategies designed to preserve the clinical use of critical antibiotics.