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.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

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.

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.

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.

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.

Bacterial Two Component Systems: Overexpression and Purification: In Vitro and In Vivo Inhibitor Screens.

Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy, such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses upon cell wall or cell membrane damage. However, (i) activity assays using truncated kinase proteins, that is, the cytoplasmic domains containing the conserved histidine residue for phosphorylation, have been shown to produce artifacts, and (ii) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from Gram-positive bacteria that do not harbor more than two trans-membrane domains in an Escherichia coli host. This guide also presents in vitro and in vivo phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases, either using a traditional radioactively labeled ATP assay to quantify histidine kinase phosphorylation or Phos-tag acrylamide gel electrophoresis to examine histidine kinase phosphorylation through mobility shift in the polyacrylamide gel. In addition, we describe the use of Phos-tag combined with a western blot approach to visualize the phosphorylation of a response regulator in vivo.

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.

From cloning to mutant in 5 days: rapid allelic exchange in Staphylococcus aureus.

In the last 10 years, the barriers preventing the uptake of foreign DNA by clinical Staphylococcus aureus isolates have been identified and powerful mutagenesis techniques such as allelic exchange are now possible in most genotypes. However, these targeted approaches can still be cumbersome, and the construction of unmarked deletions/point mutations may take many weeks or months. Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. With this optimized approach, a site-specific mutation can be introduced into S. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. This streamlined protocol considerably reduces the time required to introduce a specific, unmarked mutation in S. aureus and should dramatically improve the scalability of gene-function studies.

Comprehensive Genomic Investigation of Adaptive Mutations Driving the Low-Level Oxacillin Resistance Phenotype in Staphylococcus aureus.

Antistaphylococcal penicillins such as oxacillin are the key antibiotics in the treatment of invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections; however, mec gene-independent resistance adaptation can cause treatment failure. Despite its clinical relevance, the basis of this phenomenon remains poorly understood. Here, we investigated the genomic adaptation to oxacillin at an unprecedented scale using a large collection of 503 clinical mec-negative isolates and 30 in vitro-adapted isolates from independent oxacillin exposures. By combining comparative genomics, evolutionary convergence, and genome-wide association analysis, we found 21 genetic loci associated with low-level oxacillin resistance, underscoring the polygenic nature of this phenotype. Evidence of adaptation was particularly strong for the c-di-AMP signal transduction pathways (gdpP and dacA) and in the clpXP chaperone-protease complex. The role of mutations in gdpP in conferring low-level oxacillin resistance was confirmed by allele-swapping experiments. We found that resistance to oxacillin emerges at high frequency in vitro (median, 2.9 × 10-6; interquartile range [IQR], 1.9 × 10-6 to 3.9 × 10-6), which is consistent with a recurrent minimum inhibitory concentration (MIC) increase across the global phylogeny of clinical isolates. Nevertheless, adaptation in clinical isolates appears sporadically, with no stably adapted lineages, suggesting a high fitness cost of resistance, confirmed by growth assessment of mutants in rich media. Our data provide a broader understanding of the emergence and dynamics of oxacillin resistance adaptation in S. aureus and a framework for future surveillance of this clinically important phenomenon.IMPORTANCE The majority of Staphylococcus aureus strains causing human disease are methicillin-susceptible (MSSA) and can be treated with antistaphylococcal penicillins (such as oxacillin). While acquisition of the mec gene represents the main resistance mechanism to oxacillin, S. aureus can acquire low-level resistance through adaptive mutations in other genes. In this study, we used genomic approaches to understand the basis of S. aureus adaption to oxacillin and its dynamic at the population level. By combining a genome analysis of clinical isolates from persistent MSSA infections, in vitro selection of oxacillin resistance, and genome-wide association analysis on a large collection of isolates, we identified 21 genes linked to secondary oxacillin resistance. Adaptive mutations in these genes were easy to select when S. aureus was exposed to oxacillin, but they also came at a substantial cost in terms of bacterial fitness, suggesting that this phenotype emerges preferentially in the setting of sustained antibiotic exposure.