Rifaximin prophylaxis causes resistance to the last-resort antibiotic daptomycin.

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.

Optimization of Reverse Transcription Loop-Mediated Isothermal Amplification for In Situ Detection of SARS-CoV-2 in a Micro-Air-Filtration Device Format.

The Coronavirus disease 2019 (COVID-19) pandemic has supercharged innovation in the field of molecular diagnostics and led to the exploration of systems that permit the autonomous identification of airborne infectious agents. Airborne virus detection is an emerging approach for determining exposure risk, although current methods limit intervention timeliness. Here, we explore reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for one-pot detection of Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) (SCV2) run on membrane filters suitable for micro-air-filtration of airborne viruses. We use a design of experiments statistical framework to establish the optimal additive composition for running RT-LAMP on membrane filters. Using SCV2 liquid spike-in experiments and fluorescence detection, we show that single-pot RT-LAMP on glass fiber filters reliably detected 0.10 50% tissue culture infectious dose (TCID50) SCV2 per reaction (3600 E-gene copies) and is an order of magnitude more sensitive than conventional RT-LAMP.

Staphylococcus aureus adapts to exploit collagen-derived proline during chronic infection.

Staphylococcus aureus is a pulmonary pathogen associated with substantial human morbidity and mortality. As vaccines targeting virulence determinants have failed to be protective in humans, other factors are likely involved in pathogenesis. Here we analysed transcriptomic responses of human clinical isolates of S. aureus from initial and chronic infections. We observed upregulated collagenase and proline transporter gene expression in chronic infection isolates. Metabolomics of bronchiolar lavage fluid and fibroblast infection, growth assays and analysis of bacterial mutant strains showed that airway fibroblasts produce collagen during S. aureus infection. Host-adapted bacteria upregulate collagenase, which degrades collagen and releases proline. S. aureus then imports proline, which fuels oxidative metabolism via the tricarboxylic acid cycle. Proline metabolism provides host-adapted S. aureus with a metabolic benefit enabling out-competition of non-adapted strains. These data suggest that clinical settings characterized by airway repair processes and fibrosis provide a milieu that promotes S. aureus adaptation and supports infection.

Marine sponge microbe provides insights into evolution and virulence of the tubercle bacillus.

Reconstructing the evolutionary origins of Mycobacterium tuberculosis, the causative agent of human tuberculosis, has helped identify bacterial factors that have led to the tubercle bacillus becoming such a formidable human pathogen. Here we report the discovery and detailed characterization of an exceedingly slow growing mycobacterium that is closely related to M. tuberculosis for which we have proposed the species name Mycobacterium spongiae sp. nov., (strain ID: FSD4b-SM). The bacterium was isolated from a marine sponge, taken from the waters of the Great Barrier Reef in Queensland, Australia. Comparative genomics revealed that, after the opportunistic human pathogen Mycobacterium decipiens, M. spongiae is the most closely related species to the M. tuberculosis complex reported to date, with 80% shared average nucleotide identity and extensive conservation of key M. tuberculosis virulence factors, including intact ESX secretion systems and associated effectors. Proteomic and lipidomic analyses showed that these conserved systems are functional in FSD4b-SM, but that it also produces cell wall lipids not previously reported in mycobacteria. We investigated the virulence potential of FSD4b-SM in mice and found that, while the bacteria persist in lungs for 56 days after intranasal infection, no overt pathology was detected. The similarities with M. tuberculosis, together with its lack of virulence, motivated us to investigate the potential of FSD4b-SM as a vaccine strain and as a genetic donor of the ESX-1 genetic locus to improve BCG immunogenicity. However, neither of these approaches resulted in superior protection against M. tuberculosis challenge compared to BCG vaccination alone. The discovery of M. spongiae adds to our understanding of the emergence of the M. tuberculosis complex and it will be another useful resource to refine our understanding of the factors that shaped the evolution and pathogenesis of M. tuberculosis.

Polyclonal but not monoclonal circulating memory CD4+ T cells attenuate the severity of Staphylococcus aureus bacteremia.

Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.

Emergence and clonal expansion of a qacA-harbouring sequence type 45 lineage of methicillin-resistant Staphylococcus aureus.

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus.

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.

Stringent Response-Mediated Control of GTP Homeostasis Is Required for Long-Term Viability of Staphylococcus aureus.

Staphylococcus aureus is an opportunistic bacterial pathogen that often results in difficult-to-treat infections. One mechanism used by S. aureus to enhance survival during infection is the stringent response. This is a stress survival pathway that utilizes the nucleotides (p)ppGpp to reallocate bacterial resources, shutting down growth until conditions improve. Small colony variants (SCVs) of S. aureus are frequently associated with chronic infections, and this phenotype has previously been linked to a hyperactive stringent response. Here, we examine the role of (p)ppGpp in the long-term survival of S. aureus under nutrient-restricted conditions. When starved, a (p)ppGpp-null S. aureus mutant strain ((p)ppGpp0) initially had decreased viability. However, after 3 days we observed the presence and dominance of a population of small colonies. Similar to SCVs, these small colony isolates (p0-SCIs) had reduced growth but remained hemolytic and sensitive to gentamicin, phenotypes that have been tied to SCVs previously. Genomic analysis of the p0-SCIs revealed mutations arising within gmk, encoding an enzyme in the GTP synthesis pathway. We show that a (p)ppGpp0 strain has elevated levels of GTP, and that the mutations in the p0-SCIs all lower Gmk enzyme activity and consequently cellular GTP levels. We further show that in the absence of (p)ppGpp, cell viability can be rescued using the GuaA inhibitor decoyinine, which artificially lowers the intracellular GTP concentration. Our study highlights the role of (p)ppGpp in GTP homeostasis and underscores the importance of nucleotide signaling for long-term survival of S. aureus in nutrient-limiting conditions, such as those encountered during infections. IMPORTANCE Staphylococcus aureus is a human pathogen that upon invasion of a host encounters stresses, such as nutritional restriction. The bacteria respond by switching on a signaling cascade controlled by the nucleotides (p)ppGpp. These nucleotides function to shut down bacterial growth until conditions improve. Therefore, (p)ppGpp are important for bacterial survival and have been implicated in promoting chronic infections. Here, we investigate the importance of (p)ppGpp for long-term survival of bacteria in nutrient-limiting conditions similar to those in a human host. We discovered that in the absence of (p)ppGpp, bacterial viability decreases due to dysregulation of GTP homeostasis. However, the (p)ppGpp-null bacteria were able to compensate by introducing mutations in the GTP synthesis pathway that led to a reduction in GTP build-up and a rescue of viability. This study therefore highlights the importance of (p)ppGpp for the regulation of GTP levels and for long-term survival of S. aureus in restricted environments.

Staphylococcus aureus stimulates neutrophil itaconate production that suppresses the oxidative burst.

Neutrophils are critical in the host defense against Staphylococcus aureus, a major human pathogen. However, even in the setting of a robust neutrophil response, S. aureus can evade immune clearance. Here, we demonstrate that S. aureus impairs neutrophil function by triggering the production of the anti-inflammatory metabolite itaconate. The enzyme that synthesizes itaconate, Irg1, is selectively expressed in neutrophils during S. aureus pneumonia. Itaconate inhibits neutrophil glycolysis and oxidative burst, which impairs survival and bacterial killing. In a murine pneumonia model, neutrophil Irg1 expression protects the lung from excessive inflammation but compromises bacterial clearance. S. aureus is thus able to evade the innate immune response by targeting neutrophil metabolism and inducing the production of the anti-inflammatory metabolite itaconate.

Improved Genome Sequence of Australian Methicillin-Resistant Staphylococcus aureus Strain JKD6159.

Staphylococcus aureus strain JKD6159 represents a prominent community-acquired methicillin-resistant S. aureus (MRSA) clone in Australia. Here, we report an improved assembly of the original S. aureus JKD6159 genome sequence. By using deep sequencing with multiple technologies combined with carefully curated assembly and polishing, we believe the assembly to contain zero errors.

Serine-threonine phosphoregulation by PknB and Stp contributes to quiescence and antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp, which encodes the only annotated functional serine-threonine phosphatase in S. aureus, increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Staphylococcus aureus host interactions and adaptation.

Invasive Staphylococcus aureus infections are common, causing high mortality, compounded by the propensity of the bacterium to develop drug resistance. S. aureus is an excellent case study of the potential for a bacterium to be commensal, colonizing, latent or disease-causing; these states defined by the interplay between S. aureus and host. This interplay is multidimensional and evolving, exemplified by the spread of S. aureus between humans and other animal reservoirs and the lack of success in vaccine development. In this Review, we examine recent advances in understanding the S. aureus-host interactions that lead to infections. We revisit the primary role of neutrophils in controlling infection, summarizing the discovery of new immune evasion molecules and the discovery of new functions ascribed to well-known virulence factors. We explore the intriguing intersection of bacterial and host metabolism, where crosstalk in both directions can influence immune responses and infection outcomes. This Review also assesses the surprising genomic plasticity of S. aureus, its dualism as a multi-mammalian species commensal and opportunistic pathogen and our developing understanding of the roles of other bacteria in shaping S. aureus colonization.

Clinical relevance of topical antibiotic use in co-selecting for multidrug-resistant Staphylococcus aureus: Insights from in vitro and ex vivo models.

Topical antibiotic preparations, such as fusidic acid (FA) or mupirocin, are used in the prevention and treatment of superficial skin infections caused by staphylococci. Previous genomic epidemiology work has suggested an association between the widespread use of topical antibiotics and the emergence of methicillin resistant Staphylococcus aureus in some settings. In this study, we provide experimental proof of co-selection for multidrug resistance in S. aureus following exposure to FA or mupirocin. Through targeted mutagenesis and phenotypic analyses, we confirmed that fusC carriage confers resistance to FA, and mupA carriage confers high-level resistance to mupirocin in multiple S. aureus genetic backgrounds. In vitro experiments demonstrated that carriage of fusC and mupA confer a competitive advantage in the presence of sub-inhibitory concentrations of FA and mupirocin, respectively. Further, we used a porcine skin colonisation model to show that clinically relevant concentrations of topical antibiotics can co-select for presence of unrelated antimicrobial resistance determinants, such as mecA, blaZ, and qacA, in fusC or mupA harbouring S. aureus These findings provide valuable insights on the role of acquired FA or mupirocin resistance in co-selecting for broader antibiotic resistance in S. aureus, prompting greater need for judicious use of topical antibiotics.

Antimicrobial tolerance and its role in the development of resistance: Lessons from enterococci.

Bacteria have developed resistance against every antimicrobial in clinical use at an alarming rate. There is a critical need for more effective use of antimicrobials to both extend their shelf life and prevent resistance from arising. Significantly, antimicrobial tolerance, i.e., the ability to survive but not proliferate during antimicrobial exposure, has been shown to precede the development of bona fide antimicrobial resistance (AMR), sparking a renewed and rapidly increasing interest in this field. As a consequence, problematic infections for the first time are now being investigated for antimicrobial tolerance, with increasing reports demonstrating in-host evolution of antimicrobial tolerance. Tolerance has been identified in a wide array of bacterial species to all bactericidal antimicrobials. Of particular interest are enterococci, which contain the opportunistic bacterial pathogens Enterococcus faecalis and Enterococcus faecium. Enterococci are one of the leading causes of hospital-acquired infection and possess intrinsic tolerance to a number of antimicrobial classes. Persistence of these infections in the clinic is of growing concern, particularly for the immunocompromised. Here, we review current known mechanisms of antimicrobial tolerance, and include an in-depth analysis of those identified in enterococci with implications for both the development and prevention of AMR.

Barriers to genetic manipulation of Enterococci: Current Approaches and Future Directions.

Enterococcus faecalis and Enterococcus faecium are Gram-positive commensal gut bacteria that can also cause fatal infections. To study clinically relevant multi-drug resistant E. faecalis and E. faecium strains, methods are needed to overcome physical (thick cell wall) and enzymatic barriers that limit the transfer of foreign DNA and thus prevent facile genetic manipulation. Enzymatic barriers to DNA uptake identified in E. faecalis and E. faecium include type I, II and IV restriction modification systems and CRISPR-Cas. This review examines E. faecalis and E. faecium DNA defence systems and the methods with potential to overcome these barriers. DNA defence system bypass will allow the application of innovative genetic techniques to expedite molecular-level understanding of these important, but somewhat neglected, pathogens.

RNase III-CLASH of multi-drug resistant Staphylococcus aureus reveals a regulatory mRNA 3'UTR required for intermediate vancomycin resistance.

Treatment of methicillin-resistant Staphylococcus aureus infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA-RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA-RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA-mRNA interactions are recovered and we find that an mRNA encoding a long 3' untranslated region (UTR) (termed vigR 3'UTR) functions as a regulatory 'hub' within the RNA-RNA interaction network. We demonstrate that the vigR 3'UTR promotes expression of folD and the cell wall lytic transglycosylase isaA through direct mRNA-mRNA base-pairing. Deletion of the vigR 3'UTR re-sensitised VISA to glycopeptide treatment and both isaA and vigR 3'UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that S. aureus uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.

Staphylococcus aureus specific lung resident memory CD4+ Th1 cells attenuate the severity of influenza virus induced secondary bacterial pneumonia.

Staphylococcus aureus is a major cause of severe pulmonary infections. The evolution of multi-drug resistant strains limits antibiotic treatment options. To date, all candidate vaccines tested have failed, highlighting the need for an increased understanding of the immunological requirements for effective S. aureus immunity. Using an S. aureus strain engineered to express a trackable CD4+ T cell epitope and a murine model of S. aureus pneumonia, we show strategies that lodge Th1 polarised bacterium specific CD4+ tissue resident memory T cells (Trm) in the lung can significantly attenuate the severity of S. aureus pneumonia. This contrasts natural infection of mice that fails to lodge CD4+ Trm cells along the respiratory tract or provide protection against re-infection, despite initially generating Th17 bacterium specific CD4+ T cell responses. Interestingly, lack of CD4+ Trm formation after natural infection in mice appears to be reflected in humans, where the frequency of S. aureus reactive CD4+ Trm cells in lung tissue is also low. Our findings reveal the protective capacity of S. aureus specific respiratory tract CD4+ Th1 polarised Trm cells and highlight the potential for targeting these cells in vaccines that aim to prevent the development of S. aureus pneumonia.

Low-Cost, Open-Source Device for High-Performance Fluorescence Detection of Isothermal Nucleic Acid Amplification Reactions.

The ability to detect SARS-CoV-2 is critical to implementing evidence-based strategies to address the COVID-19 global pandemic. Expanding SARS-CoV-2 diagnostic ability beyond well-equipped laboratories widens the opportunity for surveillance and control efforts. However, such advances are predicated on the availability of rapid, scalable, accessible, yet high-performance diagnostic platforms. Methods to detect viral RNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP) show promise as rapid and field-deployable tests; however, the per-unit costs of the required diagnostic hardware can be a barrier for scaled deployment. Here, we describe a diagnostic hardware configuration for LAMP technology, named the FABL-8, that can be built for approximately US$380 per machine and provide results in under 30 min. Benchmarking showed that FABL-8 has a similar performance to a high-end commercial instrument for detecting fluorescence-based LAMP reactions. Performance testing of the instrument with RNA extracted from a SARS-CoV-2 virus dilution series revealed an analytical detection sensitivity of 50 virus copies per microliter-a detection threshold suitable to detect patient viral load in the first few days following symptom onset. In addition to the detection of SARS-CoV-2, we show that the system can be used to detect the presence of two bacterial pathogens, demonstrating the versatility of the platform for the detection of other pathogens. This cost-effective and scalable hardware alternative allows democratization of the instrumentation required for high-performance molecular diagnostics, such that it could be available to laboratories anywhere-supporting infectious diseases surveillance and research activities in resource-limited settings.

Accessible Platform for High-Throughput COVID-19 Molecular Diagnostics and Genome Sequencing Using a Repurposed 3D Printer for RNA Extraction.

The COVID-19 pandemic has exposed the dependence of diagnostic laboratories on a handful of large corporations with market monopolies on the worldwide supply of reagents, consumables, and hardware for molecular diagnostics. Global shortages of key consumables for RT-qPCR detection of SARS-CoV-2 RNA have impaired the ability to run essential, routine diagnostic services. Here, we describe a workflow for rapid detection of SARS-CoV-2 RNA in upper respiratory samples including nasal swabs and saliva, utilizing low-cost equipment and readily accessible reagents. Using repurposed Creality3D Ender-3 three-dimensional (3D) printers, we built a semiautomated paramagnetic bead RNA extraction platform. The hardware for the system was built for $300 USD, and the material cost per reaction was $1 USD. Named the Ender VX500, instrument performance when paired with RT-qPCR for SARS-CoV-2 detection in nasal and saliva specimens was two virus copies per microliter. There was a high-performance agreement (assessed using 458 COVID-19 nasal swab specimens) with the Aptima SARS-CoV-2 assay run on the Hologic Panther, a commercial automated RNA extraction and detection platform. Inter- and intrainstrument precision was excellent (coefficients of variation (CoV) of 1.10 and 0.66-1.32%, respectively) across four instruments. The platform is scalable with throughput ranging from 23 specimens on a single instrument run by one user in 50 min to 364 specimens on four instruments run by four users in 190 min. Step-by-step instructions and protocols for building and running the Ender VX500 have been made available without restriction.