Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Sensitizing methicillin-resistant Staphylococcus aureus (MRSA) to cefuroxime: the synergic effect of bicarbonate and the wall teichoic acid inhibitor ticlopidine.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major challenge for clinicians due, in part, to their resistance to most ß-lactams, the first-line treatment for methicillin-susceptible S. aureus. A phenotype termed "NaHCO3-responsiveness" has been identified, wherein many clinical MRSA isolates are rendered susceptible to standard-of-care ß-lactams in the presence of physiologically relevant concentrations of NaHCO3, in vitro and ex vivo; moreover, such "NaHCO3-responsive" isolates can be effectively cleared by ß-lactams from target tissues in experimental infective endocarditis (IE). One mechanistic impact of NaHCO3 exposure on NaHCO3-responsive MRSA is to repress WTA synthesis. This NaHCO3 effect mimics the phenotype of tarO-deficient MRSA, including sensitization to the PBP2-targeting ß-lactam, cefuroxime (CFX). Herein, we further investigated the impacts of NaHCO3 exposure on CFX susceptibility in the presence and absence of a WTA synthesis inhibitor, ticlopidine (TCP), in a collection of clinical MRSA isolates from skin and soft tissue infections (SSTI) and bloodstream infections (BSI). NaHCO3 and/or TCP enhanced susceptibility to CFX in vitro, by both minimum inhibitor concentration (MIC) and time-kill assays, as well as in an ex vivo simulated endocarditis vegetations (SEV) model, in NaHCO3-responsive MRSA. Furthermore, in experimental IE (presumably in the presence of endogenous NaHCO3), pre-exposure to TCP prior to infection sensitized the NaHCO3-responsive MRSA strain (but not the non-responsive strain) to enhanced clearances by CFX in target tissues. These data support the notion that NaHCO3 is acting similarly to WTA synthesis inhibitors, and that such inhibitors have potential translational applications in the treatment of certain MRSA strains in conjunction with specific ß-lactam agents.

Participant Diversity in United States Randomized Controlled Trials of Antibacterials for Staphylococcus aureus Infections, 2000-2021.

BACKGROUND: Equitable representation of members from historically marginalized groups is important in clinical trials, which inform standards of care. The goal of this study was to characterize the demographics and proportional subgroup reporting and representation of participants enrolled in randomized controlled trials (RCTs) of antibacterials used to treat Staphylococcus aureus infections. METHODS: We examined randomized controlled registrational and strategy trials published from 2000-2021 to determine the sex, race, and ethnicity of participants. Participation to incidence ratios (PIRs) were calculated by dividing the percentage of study participants in each demographic group by the percentage of the disease population in each group. Underrepresentation was defined as a PIR <0.8. RESULTS: Of the 87 included studies, 82 (94.2%) reported participant sex; 69 (79.3%) reported participant race; and 20 (23.0%) included ethnicity data. Only 17 (19.5%) studies enrolled American Indian/Alaskan Native participants. Median PIRs indicated that Asian and Black participants were underrepresented in RCTs compared with the incidence of methicillin-resistant S. aureus (MRSA) infections in these subgroups. Underrepresentation of Black participants was associated with a larger study size, international sites, industry sponsorship, and Phase 2/3 trials compared with Phase 4 trials (P<0.05 for each). Black participants had over 4 times the odds of being underrepresented in Phase 2/3 trials compared with Phase 4 trials (OR 4.57; 95% CI 1.14-18.3). CONCLUSIONS: Standardized reporting methods for race and ethnicity and efforts to increase recruitment of marginalized groups would help ensure equity, rigor, and generalizability in RCTs of antibacterial agents and reduce health inequities.

In vitro activity of sulbactam-durlobactam against colistin-resistant and/or cefiderocol-non-susceptible, carbapenem-resistant Acinetobacter baumannii collected in U.S. hospitals.

The activity of a novel ß-lactamase inhibitor combination, sulbactam-durlobactam (SUL-DUR), was tested against 87 colistin-resistant and/or cefiderocol-non-susceptible carbapenem-resistant Acinetobacter baumannii clinical isolates collected from U.S. hospitals between 2017 and 2019. Among them, 89% and 97% were susceptible to SUL-DUR and imipenem plus SUL-DUR, with MIC50/MIC90 values of 2 µg/mL/8 µg/mL and 1 µg/mL/4 µg/mL, respectively. The presence of amino acid substitutions in penicillin-binding protein 3, including previously reported A515V or T526S, was associated with SUL-DUR non-susceptibility.

Clinical Outcomes and Bacterial Characteristics of Carbapenem-resistant Acinetobacter baumannii Among Patients From Different Global Regions.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAb) is 1 of the most problematic antimicrobial-resistant bacteria. We sought to elucidate the international epidemiology and clinical impact of CRAb. METHODS: In a prospective observational cohort study, 842 hospitalized patients with a clinical CRAb culture were enrolled at 46 hospitals in five global regions between 2017 and 2019. The primary outcome was all-cause mortality at 30 days from the index culture. The strains underwent whole-genome analysis. RESULTS: Of 842 cases, 536 (64%) represented infection. By 30 days, 128 (24%) of the infected patients died, ranging from 1 (6%) of 18 in Australia-Singapore to 54 (25%) of 216 in the United States and 24 (49%) of 49 in South-Central America, whereas 42 (14%) of non-infected patients died. Bacteremia was associated with a higher risk of death compared with other types of infection (40 [42%] of 96 vs 88 [20%] of 440). In a multivariable logistic regression analysis, bloodstream infection and higher age-adjusted Charlson comorbidity index were independently associated with 30-day mortality. Clonal group 2 (CG2) strains predominated except in South-Central America, ranging from 216 (59%) of 369 in the United States to 282 (97%) of 291 in China. Acquired carbapenemase genes were carried by 769 (91%) of the 842 isolates. CG2 strains were significantly associated with higher levels of meropenem resistance, yet non-CG2 cases were over-represented among the deaths compared with CG2 cases. CONCLUSIONS: CRAb infection types and clinical outcomes differed significantly across regions. Although CG2 strains remained predominant, non-CG2 strains were associated with higher mortality. Clinical Trials Registration. NCT03646227.

Moving Beyond Mortality: Development and Application of a Desirability of Outcome Ranking (DOOR) Endpoint for Hospital-Acquired Bacterial Pneumonia and Ventilator-Associated Bacterial Pneumonia.

BACKGROUND: Hospital-acquired and ventilator-associated bacterial pneumonia (HABP/VABP) are frequently caused by multidrug-resistant organisms. Patient-centered endpoints in clinical trials are needed to develop new antibiotics for HABP/VABP. Desirability of outcome ranking (DOOR) is a paradigm for the design, analysis, and interpretation of clinical trials based on a patient-centered, benefit-risk evaluation. METHODS: A multidisciplinary committee created an infectious diseases DOOR endpoint customized for HABP/VABP, incorporating infectious complications, serious adverse events, and mortality. We applied this to 2 previously completed, large randomized controlled trials for HABP/VABP. ZEPHyR compared vancomycin to linezolid and VITAL compared linezolid to tedizolid. For each trial, we evaluated the DOOR distribution and probability, including DOOR component and partial credit analyses. We also applied DOOR in subgroup analyses. RESULTS: In both trials, the HABP/VABP DOOR demonstrated similar overall clinical outcomes between treatment groups. In ZEPHyR, the probability that a participant treated with linezolid would have a more desirable outcome than a participant treated with vancomycin was 50.2% (95% confidence interval [CI], 45.1%--55.3%). In VITAL, the probability that a participant treated with tedizolid would have a more desirable outcome than a participant treated with linezolid was 48.7% (95% CI, 44.8%-52.6%). The DOOR component analysis revealed that participants treated with tedizolid had a less desirable outcome than those treated with linezolid when considering clinical response alone. However, participants with decreased renal function had improved overall outcomes with tedizolid. CONCLUSIONS: The HABP/VABP DOOR provided more granular information about clinical outcomes than is typically presented in clinical trials. HABP/VABP trials would benefit from prospectively using DOOR.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by Methicillin-Resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to Next Generation ß-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance is classically mediated by PBP2a, a Penicillin-Binding Protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as Methicillin-Resistant Lacking mec (MRLM) are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of ß-lactam resistant S. aureus infections.

Contemporary Management of Staphylococcus aureus Bacteremia-Controversies in Clinical Practice.

Staphylococcus aureus bacteremia (SAB) carries a high risk for excess morbidity and mortality. Despite its prevalence, significant practice variation continues to permeate clinical management of this syndrome. Since the publication of the 2011 Infectious Diseases Society of America (IDSA) guidelines on management of methicillin-resistant Staphylococcus aureus infections, the field of SAB has evolved with the emergence of newer diagnostic strategies and therapeutic options. In this review, we seek to provide a comprehensive overview of the evaluation and management of SAB, with special focus on areas where the highest level of evidence is lacking to inform best practices.

Spatial clusters of extended-spectrum beta-lactamase-producing Escherichia coli causing community-onset bacteriuria due to repeat infections: cluster analysis from a large urban medical center, San Francisco, 2014-2020.

BACKGROUND: Urinary tract infections caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (ESBL-E. coli) may occur as outbreaks due to common-source exposures. Yet, it is currently unknown if they cluster geographically as would be expected as part of an outbreak. METHODS: We collected electronic health record data on all patients living in San Francisco with culture-documented community-onset E. coli bacteriuria in a safety-net public healthcare system from January 2014 to March 2020 (diagnosed < 48 h after hospital admission or in outpatient clinical settings without a hospitalization in the past 90 days). We assessed the presence of spatial clusters of (1) ESBL-E. coli bacteriuria episodes, and (2) individuals with any ESBL-E. coli bacteriuria episode, with Global and Local Moran's I. We evaluated differences in prevalence of bacteriuria recurrence by ESBL-production by Poisson regression. RESULTS: Out of 4,304 unique individuals, we identified spatial clusters of ESBL-E. coli bacteriuria episodes (n = 461) compared to non-ESBL-E. coli bacteriuria episodes (n = 5477; Global Moran's p < 0.001). Spatial clusters of individuals with any bacteriuria caused by ESBL-E. coli were not identified (p = 0.43). Bacteriuria recurrence was more likely to occur with ESBL-E. coli (odds ratio [OR] 2.78, 95% confidence interval [95% CI] 2.10, 3.66, p < 0.001), particularly after an initial ESBL-E. coli bacteriuria episode (OR 2.27, 95% CI 1.82, 2.83, p < 0.001). CONCLUSION: We found spatial clusters of ESBL-E. coli bacteriuria episodes. However, this was partly explained by clustering within individuals more than between individuals, as having an ESBL-E. coli bacteriuria was associated with recurrence with ESBL-E. coli. These findings may help better tailor clinical treatment of patients with recurrent urinary tract infections after an initial episode caused by ESBL-E. coli.

Under the Hood: The Scientific Leadership, Clinical Operations, Statistical and Data Management, and Laboratory Centers of the Antibacterial Resistance Leadership Group.

Developing and implementing the scientific agenda of the Antibacterial Resistance Leadership Group (ARLG) by soliciting input and proposals, transforming concepts into clinical trials, conducting those trials, and translating trial data analyses into actionable information for infectious disease clinical practice is the collective role of the Scientific Leadership Center, Clinical Operations Center, Statistical and Data Management Center, and Laboratory Center of the ARLG. These activities include shepherding concept proposal applications through peer review; identifying, qualifying, training, and overseeing clinical trials sites; recommending, developing, performing, and evaluating laboratory assays in support of clinical trials; and designing and performing data collection and statistical analyses. This article describes key components involved in realizing the ARLG scientific agenda through the activities of the ARLG centers.

The Future Ain't What It Used to Be Out With the Old In With the Better: Antibacterial Resistance Leadership Group Innovations.

Clinical research networks conduct important studies that would not otherwise be performed by other entities. In the case of the Antibacterial Resistance Leadership Group (ARLG), such studies include diagnostic studies using master protocols, controlled phage intervention trials, and studies that evaluate treatment strategies or dynamic interventions, such as sequences of empiric and definitive therapies. However, the value of a clinical research network lies not only in the results from these important studies but in the creation of new approaches derived from collaborative thinking, carefully examining and defining the most important research questions for clinical practice, recognizing and addressing common but suboptimal approaches, and anticipating that the standard approaches of today may be insufficient for tomorrow. This results in the development and implementation of new methodologies and tools for the design, conduct, analyses, and reporting of research studies. These new methodologies directly impact the studies conducted within the network and have a broad and long-lasting impact on the field, enhancing the scientific value and efficiency of generations of research studies. This article describes innovations from the ARLG in diagnostic studies, observational studies, and clinical trials evaluating interventions for the prevention and treatment of antibiotic-resistant bacterial infections.

The Antibacterial Resistance Leadership Group: Scientific Advancements and Future Directions.

In this overview, we describe important contributions from the Antibacterial Resistance Leadership Group (ARLG) to patient care, clinical trials design, and mentorship while outlining future priorities. The ARLG research agenda is focused on 3 key areas: gram-positive infections, gram-negative infections, and diagnostics. The ARLG has developed an innovative approach to clinical trials design, the desirability of outcome ranking (DOOR), which uses an ordinal measure of global outcome to assess both benefits and harms. DOOR was initially applied to observational studies to determine optimal dosing of vancomycin for methicillin-resistant Staphylcococcus aureus bacteremia and the efficacy of ceftazidime-avibactam versus colistin for the treatment of carbapenem-resistant Enterobacterales infection. DOOR is being successfully applied to the analysis of interventional trials and, in collaboration with the US Food and Drug Administration (FDA), for use in registrational trials. In the area of diagnostics, the ARLG developed Master Protocol for Evaluating Multiple Infection Diagnostics (MASTERMIND), an innovative design that allows simultaneous testing of multiple diagnostic platforms in a single study. This approach will be used to compare molecular assays for the identification of fluoroquinolone-resistant Neisseria gonorrhoeae (MASTER GC) and to compare rapid diagnostic tests for bloodstream infections. The ARLG has initiated a first-in-kind randomized, double-blind, placebo-controlled trial in participants with cystic fibrosis who are chronically colonized with Pseudomonas aeruginosa to assess the pharmacokinetics and antimicrobial activity of bacteriophage therapy. Finally, an engaged and highly trained workforce is critical for continued and future success against antimicrobial drug resistance. Thus, the ARLG has developed a robust mentoring program targeted to each stage of research training to attract and retain investigators in the field of antimicrobial resistance research.

Daily fosfomycin versus levofloxacin for complicated urinary tract infections.

IMPORTANCE: Concerns over resistance and safety have been identified in the current treatment regimen for complicated urinary tract infections. Fosfomycin is a drug that is routinely used for the treatment of uncomplicated cystitis. This study shows that fosfomycin could be an oral alternative as step-down therapy for the treatment of complicated urinary tract infections, with a clinical cure rate comparable to levofloxacin but a lower microbiological success rate 3 weeks from start of antibiotics.

International Epidemiology of Carbapenemase-Producing Escherichia coli.

BACKGROUND: Carbapenemase-producing (CP) Escherichia coli (CP-Ec) are a global public health threat. We aimed to describe the clinical and molecular epidemiology and outcomes of patients from several countries with CP-Ec isolates obtained from a prospective cohort. METHODS: Patients with CP-Ec were enrolled from 26 hospitals in 6 countries. Clinical data were collected, and isolates underwent whole-genome sequencing. Clinical and molecular features and outcomes associated with isolates with or without metallo-ß-lactamases (MBLs) were compared. The primary outcome was desirability of outcome ranking (DOOR) at 30 days after the index culture. RESULTS: Of the 114 CP-Ec isolates in Consortium on resistance against carbapenems in Klebsiella and other Enterobacterales-2 (CRACKLE-2), 49 harbored an MBL, most commonly blaNDM-5 (38/49, 78%). Strong regional variations were noted with MBL-Ec predominantly found among patients in China (23/49). Clinically, MBL-Ec were more often from urine sources (49% vs 29%), less often met criteria for infection (39% vs 58%, P = .04), and had lower acuity of illness when compared with non-MBL-Ec. Among patients with infection, the probability of a better DOOR outcome for a randomly selected patient with MBL-Ec as compared with non-MBL-Ec was 62% (95% CI: 48.2-74.3%). Among infected patients, non-MBL-Ec had increased 30-day (26% vs 0%; P = .02) and 90-day (39% vs 0%; P = .001) mortality compared with MBL-Ec. CONCLUSIONS: Emergence of CP-Ec was observed with important geographic variations. Bacterial characteristics, clinical presentations, and outcomes differed between MBL-Ec and non-MBL-Ec. Mortality was higher among non-MBL isolates, which were more frequently isolated from blood, but these findings may be confounded by regional variations.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-ß-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care ß-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to ß-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-ß-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most ß-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to ß-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-ß-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using ß-lactams.

Global epidemiology and clinical outcomes of carbapenem-resistant Pseudomonas aeruginosa and associated carbapenemases (POP): a prospective cohort study.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a global threat, but the distribution and clinical significance of carbapenemases are unclear. The aim of this study was to define characteristics and outcomes of CRPA infections and the global frequency and clinical impact of carbapenemases harboured by CRPA. METHODS: We conducted an observational, prospective cohort study of CRPA isolated from bloodstream, respiratory, urine, or wound cultures of patients at 44 hospitals (10 countries) between Dec 1, 2018, and Nov 30, 2019. Clinical data were abstracted from health records and CRPA isolates were whole-genome sequenced. The primary outcome was 30-day mortality from the day the index culture was collected. We compared outcomes of patients with CRPA infections by infection type and across geographic regions and performed an inverse probability weighted analysis to assess the association between carbapenemase production and 30-day mortality. FINDINGS: We enrolled 972 patients (USA n=527, China n=171, south and central America n=127, Middle East n=91, Australia and Singapore n=56), of whom 581 (60%) had CRPA infections. 30-day mortality differed by infection type (bloodstream 21 [30%] of 69, respiratory 69 [19%] of 358, wound nine [14%] of 66, urine six [7%] of 88; p=0·0012) and geographical region (Middle East 15 [29%] of 52, south and central America 20 [27%] of 73, USA 60 [19%] of 308, Australia and Singapore three [11%] of 28, China seven [6%] of 120; p=0·0002). Prevalence of carbapenemase genes among CRPA isolates also varied by region (south and central America 88 [69%] of 127, Australia and Singapore 32 [57%] of 56, China 54 [32%] of 171, Middle East 27 [30%] of 91, USA ten [2%] of 527; p<0·0001). KPC-2 (n=103 [49%]) and VIM-2 (n=75 [36%]) were the most common carbapenemases in 211 carbapenemase-producing isolates. After excluding USA patients, because few US isolates had carbapenemases, patients with carbapenemase-producing CRPA infections had higher 30-day mortality than those with non-carbapenemase-producing CRPA infections in both unadjusted (26 [22%] of 120 vs 19 [12%] of 153; difference 9%, 95% CI 3-16) and adjusted (difference 7%, 95% CI 1-14) analyses. INTERPRETATION: The emergence of different carbapenemases among CRPA isolates in different geographical regions and the increased mortality associated with carbapenemase-producing CRPA infections highlight the therapeutic challenges posed by these organisms. FUNDING: National Institutes of Health.

Structural basis of broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Broad-spectrum ß-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden1,2. In clinical strains, resistance is largely controlled by BlaR13, a receptor that senses ß-lactams through the acylation of its sensor domain, inducing transmembrane signalling and activation of the cytoplasmic-facing metalloprotease domain4. The metalloprotease domain has a role in BlaI derepression, inducing blaZ (ß-lactamase PC1) and mecA (ß-lactam-resistant cell-wall transpeptidase PBP2a) expression3-7. Here, overcoming hurdles in isolation, we show that BlaR1 cleaves BlaI directly, as necessary for inactivation, with no requirement for additional components as suggested previously8. Cryo-electron microscopy structures of BlaR1-the wild type and an autocleavage-deficient F284A mutant, with or without ß-lactam-reveal a domain-swapped dimer that we suggest is critical to the stabilization of the signalling loops within. BlaR1 undergoes spontaneous autocleavage in cis between Ser283 and Phe284 and we describe the catalytic mechanism and specificity underlying the self and BlaI cleavage. The structures suggest that allosteric signalling emanates from ß-lactam-induced exclusion of the prominent extracellular loop bound competitively in the sensor-domain active site, driving subsequent dynamic motions, including a shift in the sensor towards the membrane and accompanying changes in the zinc metalloprotease domain. We propose that this enhances the expulsion of autocleaved products from the active site, shifting the equilibrium to a state that is permissive of efficient BlaI cleavage. Collectively, this study provides a structure of a two-component signalling receptor that mediates action-in this case, antibiotic resistance-through the direct cleavage of a repressor.