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.

Inter-species gene flow drives ongoing evolution of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of human infection with invasive disease incidence and clinical manifestations comparable to the closely related species, Streptococcus pyogenes. Through systematic genomic analyses of 501 disseminated SDSE strains, we demonstrate extensive overlap between the genomes of SDSE and S. pyogenes. More than 75% of core genes are shared between the two species with one third demonstrating evidence of cross-species recombination. Twenty-five percent of mobile genetic element (MGE) clusters and 16 of 55 SDSE MGE insertion regions were shared across species. Assessing potential cross-protection from leading S. pyogenes vaccine candidates on SDSE, 12/34 preclinical vaccine antigen genes were shown to be present in >99% of isolates of both species. Relevant to possible vaccine evasion, six vaccine candidate genes demonstrated evidence of inter-species recombination. These findings demonstrate previously unappreciated levels of genomic overlap between these closely related pathogens with implications for streptococcal pathobiology, disease surveillance and prevention.

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.

A statistical genomics framework to trace bacterial genomic predictors of clinical outcomes in Staphylococcus aureus bacteremia.

Outcomes of severe bacterial infections are determined by the interplay between host, pathogen, and treatments. While human genomics has provided insights into host factors impacting Staphylococcus aureus infections, comparatively little is known about S. aureus genotypes and disease severity. Building on the hypothesis that bacterial pathoadaptation is a key outcome driver, we developed a genome-wide association study (GWAS) framework to identify adaptive mutations associated with treatment failure and mortality in S. aureus bacteremia (1,358 episodes). Our research highlights the potential of vancomycin-selected mutations and vancomycin minimum inhibitory concentration (MIC) as key explanatory variables to predict infection severity. The contribution of bacterial variation was much lower for clinical outcomes (heritability <5%); however, GWASs allowed us to identify additional, MIC-independent candidate pathogenesis loci. Using supervised machine learning, we were able to quantify the predictive potential of these adaptive signatures. Our statistical genomics framework provides a powerful means to capture adaptive mutations impacting severe bacterial infections.

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence.

Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

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 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.

Genomic investigation of the emergence of vanD vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus (VRE) is an increasingly identified cause of human disease, with most infections resulting from the vanA and vanB genotypes; less is known about other clinically relevant genotypes. Here we report a genomic exploration of a vanD VRE faecium (VREfm), which arose de novo during a single infectious episode. The genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm were subjected to long-read sequencing and closed, with whole-genome alignments, cross-mapping and orthologue clustering used to identify genomic variation. Three key differences were identified. (i) The VREfm chromosome gained a 142.6 kb integrative conjugative element (ICE) harbouring the vanD locus. (ii) The native ligase (ddl) was disrupted by an ISEfm1 insertion. (iii) A large 1.74 Mb chromosomal inversion of unknown consequence occurred. Alignment and phylogenetic-based comparisons of the VREfm with a global collection of vanD-harbouring genomes identified strong similarities in the 120-160 kb genomic region surrounding vanD, suggestive of a common mobile element and integration site, irrespective of the diverse taxonomic, geographical and host origins of the isolates. This isolate diversity revealed that this putative ICE (and its source) is globally disseminated and is capable of being acquired by different genera. Although the incidence of vanD VREfm is low, understanding its emergence and potential for spread is crucial for the ongoing efforts to reduce antimicrobial resistance.

Significant variability exists in the cytotoxicity of global methicillin-resistant Staphylococcus aureus lineages.

Staphylococcus aureus is a major human pathogen where the emergence of antibiotic resistant lineages, such as methicillin-resistant S. aureus (MRSA), is a major health concern. While some MRSA lineages are restricted to the healthcare setting, the epidemiology of MRSA is changing globally, with the rise of specific lineages causing disease in healthy people in the community. In the past two decades, community-associated MRSA (CA-MRSA) has emerged as a clinically important and virulent pathogen associated with serious skin and soft-tissue infections (SSTI). These infections are primarily cytotoxin driven, leading to the suggestion that hypervirulent lineages/multi-locus sequence types (STs) exist. To examine this, we compared the cytotoxicity of 475 MRSA isolates representing five major MRSA STs (ST22, ST93, ST8, ST239 and ST36) by employing a monocyte-macrophage THP-1 cell line as a surrogate for measuring gross cytotoxicity. We demonstrate that while certain MRSA STs contain highly toxic isolates, there is such variability within lineages to suggest that this aspect of virulence should not be inferred from the genotype of any given isolate. Furthermore, by interrogating the accessory gene regulator (Agr) sequences in this collection we identified several Agr mutations that were associated with reduced cytotoxicity. Interestingly, the majority of isolates that were attenuated in cytotoxin production contained no mutations in the agr locus, indicating a role of other undefined genes in S. aureus toxin regulation.

Genomic Epidemiology and Global Population Structure of Exfoliative Toxin A-Producing Staphylococcus aureus Strains Associated With Staphylococcal Scalded Skin Syndrome.

Staphylococci producing exfoliative toxins are the causative agents of staphylococcal scalded skin syndrome (SSSS). Exfoliative toxin A (ETA) is encoded by eta, which is harbored on a temperate bacteriophage ΦETA. A recent increase in the incidence of SSSS in North America has been observed; yet it is largely unknown whether this is the result of host range expansion of ΦETA or migration and emergence of established lineages. Here, we detail an outbreak investigation of SSSS in a neonatal intensive care unit, for which we applied whole-genome sequencing (WGS) and phylogenetic analysis of Staphylococcus aureus isolates collected from cases and screening of healthcare workers. We identified the causative strain as a methicillin-susceptible S. aureus (MSSA) sequence type 582 (ST582) possessing ΦETA. To then elucidate the global distribution of ΦETA among staphylococci, we used a recently developed tool to query extant bacterial WGS data for biosamples containing eta, which yielded 436 genomes collected between 1994 and 2019 from 32 countries. Applying population genomic analysis, we resolved the global distribution of S. aureus with lysogenized ΦETA and assessed antibiotic resistance determinants as well as the diversity of ΦETA. The population is highly structured with eight dominant sequence clusters (SCs) that generally aligned with S. aureus ST clonal complexes. The most prevalent STs included ST109 (24.3%), ST15 (13.1%), ST121 (10.1%), and ST582 (7.1%). Among strains with available data, there was an even distribution of isolates from carriage and disease. Only the SC containing ST121 had significantly more isolates collected from disease (69%, n = 46) than carriage (31%, n = 21). Further, we identified 10.6% (46/436) of strains as methicillin-resistant S. aureus (MRSA) based on the presence of mecA and the SCCmec element. Assessment of ΦETA diversity based on nucleotide identity revealed 27 phylogroups, and prophage gene content further resolved 62 clusters. ΦETA was relatively stable within lineages, yet prophage variation is geographically structured. This suggests that the reported increase in incidence is associated with migration and expansion of existing lineages, not the movement of ΦETA to new genomic backgrounds. This revised global view reveals that ΦETA is diverse and is widely distributed on multiple genomic backgrounds whose distribution varies geographically.

Evolutionary dynamics of multidrug resistant Salmonella enterica serovar 4,[5],12:i:- in Australia.

Salmonella enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-) is a monophasic variant of Salmonella Typhimurium that has emerged as a global cause of multidrug resistant salmonellosis. We used Bayesian phylodynamics, genomic epidemiology, and phenotypic characterization to describe the emergence and evolution of Salmonella 4,[5],12:i:- in Australia. We show that the interruption of the genetic region surrounding the phase II flagellin, FljB, causing a monophasic phenotype, represents a stepwise evolutionary event through the accumulation of mobile resistance elements with minimal impairment to bacterial fitness. We identify three lineages with different population dynamics and discrete antimicrobial resistance profiles emerged, likely reflecting differential antimicrobial selection pressures. Two lineages are associated with travel to South-East Asia and the third lineage is endemic to Australia. Moreover antimicrobial-resistant Salmonella 4,[5],12:i- lineages efficiently infected and survived in host phagocytes and epithelial cells without eliciting significant cellular cytotoxicity, suggesting a suppression of host immune response that may facilitate the persistence of Salmonella 4,[5],12:i:-.

Cell Membrane Adaptations Mediate ß-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro.

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected ß-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such ß-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin ('daptomycin-resensitized'). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected ß-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

Re-evaluation of a Neonatal Mouse Model of Infection With Enterotoxigenic Escherichia coli.

Enterotoxigenic E. coli (ETEC) is a common cause of diarrhea in children in low- and middle-income countries, and in travelers to these countries. ETEC is also an important cause of morbidity and premature mortality in piglets, calves, goat kids and lambs. The major virulence determinants of ETEC are enterotoxins and colonization factors, which enable the pathogen to colonize the small intestine and deliver enterotoxins, such as the heat-stable enterotoxins, STp and STh, to epithelial cells. Because most ETEC strains are host-specific, there are few convenient animal models to investigate the pathogenesis of ETEC infections or to evaluate specific anti-ETEC interventions, such as drugs and vaccines. An exception is ETEC strains bearing F41 pili, which mediate intestinal colonization of various young animals, including neonatal mice, to cause disease and in some cases death. In this study, we used the archetypal F41-producing bovine ETEC strain, B41 (O101:NM; K99, F41, STp) to validate and further explore the contribution of F41 and STp to bacterial virulence. By using targeted gene deletion and trans-complementation studies, augmented by whole genome sequencing, and in vitro and animal studies of virulence, we established that F41 mediates colonization of the mouse intestine and is essential for bacterial virulence. In addition, we showed for the first time that STp is as important as F41 for virulence. Together, these findings validate the use of neonatal mice to study the pathogenesis of F41-bearing ETEC and to investigate possible specific anti-ETEC interventions including vaccines that target heat-stable enterotoxins.

Complete Genome Sequence of Exfoliative Toxin-Producing Staphylococcus aureus Strain MSSA_SSSS_01, Obtained from a Case of Staphylococcal Scalded-Skin Syndrome.

Here, we announce the complete genome sequence of an exfoliative toxin-producing strain of Staphylococcus aureus sequence type 582 (ST582), isolated from a case of staphylococcal scalded-skin syndrome. The genome consists of a single circularized unitig with a total length of 2,792,190 bp carrying 2,699 genes. The genome is the basis for future epidemiological and genomic 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.

Complete microbial genomes for public health in Australia and the Southwest Pacific.

Complete genomes of microbial pathogens are essential for the phylogenomic analyses that increasingly underpin core public health laboratory activities. Here, we announce a BioProject (PRJNA556438) dedicated to sharing complete genomes chosen to represent a range of pathogenic bacteria with regional importance to Australia and the Southwest Pacific; enriching the catalogue of globally available complete genomes for public health while providing valuable strains to regional public health microbiology laboratories. In this first step, we present 26 complete high-quality bacterial genomes. Additionally, we describe here a framework for reconstructing complete microbial genomes and highlight some of the challenges and considerations for accurate and reproducible genome reconstruction.

Population genomics and antimicrobial resistance in Corynebacterium diphtheriae.

BACKGROUND: Corynebacterium diphtheriae, the agent of diphtheria, is a genetically diverse bacterial species. Although antimicrobial resistance has emerged against several drugs including first-line penicillin, the genomic determinants and population dynamics of resistance are largely unknown for this neglected human pathogen. METHODS: Here, we analyzed the associations of antimicrobial susceptibility phenotypes, diphtheria toxin production, and genomic features in C. diphtheriae. We used 247 strains collected over several decades in multiple world regions, including the 163 clinical isolates collected prospectively from 2008 to 2017 in France mainland and overseas territories. RESULTS: Phylogenetic analysis revealed multiple deep-branching sublineages, grouped into a Mitis lineage strongly associated with diphtheria toxin production and a largely toxin gene-negative Gravis lineage with few toxin-producing isolates including the 1990s ex-Soviet Union outbreak strain. The distribution of susceptibility phenotypes allowed proposing ecological cutoffs for most of the 19 agents tested, thereby defining acquired antimicrobial resistance. Penicillin resistance was found in 17.2% of prospective isolates. Seventeen (10.4%) prospective isolates were multidrug-resistant (≥ 3 antimicrobial categories), including four isolates resistant to penicillin and macrolides. Homologous recombination was frequent (r/m = 5), and horizontal gene transfer contributed to the emergence of antimicrobial resistance in multiple sublineages. Genome-wide association mapping uncovered genetic factors of resistance, including an accessory penicillin-binding protein (PBP2m) located in diverse genomic contexts. Gene pbp2m is widespread in other Corynebacterium species, and its expression in C. glutamicum demonstrated its effect against several beta-lactams. A novel 73-kb C. diphtheriae multiresistance plasmid was discovered. CONCLUSIONS: This work uncovers the dynamics of antimicrobial resistance in C. diphtheriae in the context of phylogenetic structure, biovar, and diphtheria toxin production and provides a blueprint to analyze re-emerging diphtheria.

Prolonged Exposure to ß-Lactam Antibiotics Reestablishes Susceptibility of Daptomycin-Nonsusceptible Staphylococcus aureus to Daptomycin.

Daptomycin-nonsusceptible (DAP-NS) Staphylococcus aureus often exhibits gain-in-function mutations in the mprF gene (involved in positive surface charge maintenance). Standard ß-lactams, although relatively inactive against methicillin-resistant S. aureus (MRSA), may prevent the emergence of mprF mutations and DAP-NS. We determined if ß-lactams might also impact DAP-NS isolates already possessing an mprF mutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory ß-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates ("seesaw" effect). Prolonged (28-d) ß-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexisting mprF polymorphisms, accumulation of additional mprF mutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both mprF-dependent phenotypes). ß-lactams that either promiscuously or specifically target PBP-1 have significant DAP "resensitizing" effects against DAP-NS S. aureus strains. This may relate to the acquisition of multiple mprF single nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.