Insights into the molecular basis of reduced vancomycin susceptibility among three prominent Staphylococcus aureus clonal complexes.

Staphylococcus aureus is a leading cause of healthcare-associated infections globally. Vancomycin-resistant S. aureus (VRSA), those with high-level resistance [minimum inhibitory concentration (MIC) of 16-32 µg/mL vancomycin], are uncommon, whereas vancomycin-intermediate S. aureus (VISA; MIC of 4-8 µg/mL), are isolated more frequently and develop during long-term and/or repeated use of the antibiotic. VISA can be difficult to eradicate and infections may persist. Our knowledge of mechanisms that underlie the development of VISA is incomplete. We used a genomics approach to investigate the VISA phenotype in three prominent S. aureus lineages. All VISA clinical isolates tested had increased cell wall thickness compared with vancomycin-susceptible S. aureus strains. Growth rates of clonal complex (CC) 5, CC8, and CC45 clinical isolates were reduced in 2 µg/mL vancomycin compared to media alone. Culture in 2 and 4 µg/mL vancomycin sequentially for two weeks reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin in a majority of CC5, CC8, and CC45 isolates tested. We identified alleles reported previously to contribute to the VISA phenotype, but unexpectedly, these alleles were unique to each CC. A subtherapeutic concentration of vancomycin elicited changes in the VISA transcriptome-common and unique-among the three CCs tested. Multiple genes, including those encoding a glycerate kinase, an M50 family metallopeptidase, and an uncharacterized membrane protein, were upregulated among all three lineages and not reported previously as associated with VISA. Although there are lineage-specific changes in DNA sequence, our findings suggest changes in the VISA transcriptome constitute a general response to stress that confers reduced susceptibility to multiple antibiotics. IMPORTANCE: Our understanding of the mechanisms that underlie the development of vancomycin-intermediate Staphylococcus aureus (VISA) is incomplete. To provide a more comprehensive view of this process, we compared genome sequences of clonal complex (CC) 5, CC8, and CC45 VISA clinical isolates and measured changes in the transcriptomes of these isolates during culture with a subtherapeutic concentration of vancomycin. Notably, we identified differentially expressed genes that were lineage-specific or common to the lineages tested, including genes that have not been previously reported to contribute to a VISA phenotype. Changes in gene expression were accompanied by reduced growth rate, increased cell wall thickness, and reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin. Our results provide support to the idea that changes in gene expression contribute to the development of VISA among three CCs that are a prominent cause of human infections.

Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Drug resistant Mycobacterium tuberculosis strains have altered cell envelope hydrophobicity that influences infection outcomes in human macrophages.

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is considered one of the top infectious killers in the world. In recent decades, drug resistant (DR) strains of M.tb have emerged that make TB even more difficult to treat and pose a threat to public health. M.tb has a complex cell envelope that provides protection to the bacterium from chemotherapeutic agents. Although M.tb cell envelope lipids have been studied for decades, very little is known about how their levels change in relation to drug resistance. In this study, we examined changes in the cell envelope lipids [namely, phthiocerol dimycocerosates (PDIMs)], glycolipids [phosphatidyl-myo-inositol mannosides (PIMs)], and the PIM associated lipoglycans [lipomannan (LM); mannose-capped lipoarabinomannan (ManLAM)] of 11 M.tb strains that range from drug susceptible (DS) to multi-drug resistant (MDR) to pre-extensively drug resistant (pre-XDR). We show that there was an increase in the PDIMs:PIMs ratio as drug resistance increases, and provide evidence of PDIM species only present in the DR-M.tb strains studied. Overall, the LM and ManLAM cell envelope levels did not differ between DS- and DR-M.tb strains, but ManLAM surface exposure proportionally increased with drug resistance. Evaluation of host-pathogen interactions revealed that DR-M.tb strains have decreased association with human macrophages compared to DS strains. The pre-XDR M.tb strain with the largest PDIMs:PIMs ratio had decreased uptake, but increased intracellular growth rate at early time points post-infection when compared to the DS-M.tb strain H37Rv. These findings suggest that PDIMs may play an important role in drug resistance and that this observed increase in hydrophobic cell envelope lipids on the DR-M.tb strains studied may influence M.tb-host interactions.

In vitro activity of cefoxitin, imipenem, meropenem, and ceftaroline in combination with vaborbactam against Mycobacterium abscessus.

Mycobacterium abscessus (MAB) infections pose a growing public health threat. Here, we assessed the in vitro activity of the boronic acid-based ß-lactamase inhibitor, vaborbactam, with different ß-lactams against 100 clinical MAB isolates. Enhanced activity was observed with meropenem and ceftaroline with vaborbactam (1- and >4-fold MIC50/90 reduction). CRISPRi-mediated blaMAB gene knockdown showed a fourfold MIC reduction to ceftaroline but not the other ß-lactams. Our findings demonstrate vaborbactam's potential in combination therapy against MAB infections.

In vitro activity of meropenem-vaborbactam plus aztreonam against metallo-ß-lactamase-producing Klebsiella pneumoniae.

We evaluated the in vitro activity of meropenem-vaborbactam plus aztreonam (MEV-ATM) against 140 metallo-ß-lactamase (MBL)-producing Klebsiella pneumoniae isolates. Among them, 25 isolates (17.9%) displayed minimum inhibitory concentrations (MIC) ≥ 8 µg/mL, while 112 (80.0%) had MIC ≤ 2 µg/mL. Genomic analysis and subsequent gene cloning experiments revealed OmpK36 134-135GD-insertion and increased carbapenemase gene (blaNDM-1 and blaOXA-48-like) copy numbers are the main factors responsible for MEV-ATM non-susceptibility. Notably, MEV-ATM is actively against aztreonam-avibactam-resistant mutants due to CMY-16 mutations.

Next generation antibiotic combinations to combat pan-drug resistant Klebsiella pneumoniae.

Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-ß-lactamases. Unexpectedly, the ß-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug ß-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated ß-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel ß-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.

Differential mucosal tropism and dissemination of classical and hypervirulent Klebsiella pneumoniae infection.

Klebsiella pneumoniae (Kp) infection is an important healthcare concern. The ST258 classical (c)Kp strain is dominant in hospital-acquired infections in North America and Europe, while ST23 hypervirulent (hv)Kp prevails in community-acquired infections in Asia. This study aimed to develop symptomatic mucosal infection models in mice that mirror natural infections in humans to gain a deeper understanding of Kp mucosal pathogenesis. We showed that cKp replicates in the nasal cavity instead of the lungs, and this early infection event is crucial for the establishment of chronic colonization in the cecum and colon. In contrast, hvKp replicates directly in the lungs to lethal bacterial load, and early infection of esophagus supported downstream transient colonization in the ileum and cecum. Here, we have developed an in vivo model that illuminates how differences in Kp tropism are responsible for virulence and disease phenotype in cKp and hvKp, providing the basis for further mechanistic study.

Emergence of OXA-48-producing hypervirulent Klebsiella pneumoniae strains in Taiwan.

The OXA-48-producing hypervirulent Klebsiella pneumoniae (hvKP) strains were rarely reported. In this study, we characterized three carbapenem-resistant hvKP strains (KP2185, NCRE61, and KP2683-1) isolated from renal abscess, scrotal abscess, and blood samples in a Taiwan hospital. The three strains belonged to two different clones: ST23 K1 (KP2683-1) and ST11 KL64 (KP2185 and NCRE61). KP2683-1 exhibited the highest virulence in an in vivo model. Whole-genome sequencing analysis showed that KP2185 and NCRE61 acquired IncFIB type plasmids containing a set of virulence genes (iroBCDN, iucABCD, rmpA, rmpA2, and iutA), while KP2683-1 acquired an IncL type plasmid harboring blaOXA-48.

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.

CRISPR-Cas9-mediated IncF plasmid curing in extraintestinal pathogenic Escherichia coli.

IMPORTANCE: Understanding the role of IncF plasmids in the success of drug-resistant bacteria has far-reaching implications for tackling antibiotic resistance. The study's use of a novel CRISPR-Cas9-mediated plasmid-curing system provides a precision tool for dissecting the specific impact of IncF plasmids on ExPEC clones, especially high-risk, multidrug-resistant strains like ST131, ST1193, and ST410. The study offers a crucial stepping stone for future research into understanding how these plasmids influence more complex aspects of bacterial behavior, such as cell invasion and in vivo fitness.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence.

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Interaction of multidrug-resistant hypervirulent Klebsiella pneumoniae with components of human innate host defense.

IMPORTANCE: Klebsiella pneumoniae strains with a combination of multidrug resistance and hypervirulence genotypes (MDR hvKp) have emerged as a cause of human infections. The ability of these microbes to avoid killing by the innate immune system remains to be tested fully. To that end, we compared the ability of a global collection of hvKp and MDR hvKp clinical isolates to survive in human blood and resist phagocytic killing by human neutrophils. The two MDR hvKp clinical isolates tested (ST11 and ST147) were killed in human blood and by human neutrophils in vitro, whereas phagocytic killing of hvKp clinical isolates (ST23 and ST86) required specific antisera. Although the data were varied and often isolate specific, they are an important first step toward gaining an enhanced understanding of host defense against MDR hvKp.

Molecular and Clinical Epidemiology of SARS-CoV-2 Infection among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey.

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha (n = 714), Delta (n = 1877), and Omicron (n = 1802). Omicron isolates were further sub-typed as BA.1 (n = 899), BA.2 (n = 853), or BA.4/BA.5 (n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

The impact of COVID on bacterial sepsis.

PURPOSE: To identify the predictors of morbidity and mortality in matched COVID-19 positive and negative patients who were septic with Gram positive or Gram negative infections. METHODS: We conducted a retrospective review, from March to October 2020, of matched septic patients at five Hackensack Meridian Health hospitals who had bacteremia with Staphylococcus aureus, Klebsiella pneumoniae or Escherichia coli with and without COVID-19. We extracted patient demographics, comorbidities and clinical outcomes data using ICD-10 codes. Bacterial isolates were compared by whole genome sequencing analysis. Multivariate logistic regression was used to analyze independent predictors of morbidity and mortality. RESULTS: A total of 208 patients were grouped by positive bloodstream infection (BSI) with COVID-19 (n = 104) and without COVID-19 (n = 104). Most patients were over age 50 (90% vs. 89%) and Caucasian (78% vs. 86%). Inpatient mortality was higher in patients with COVID-19 for both GP (35% vs. 8%, p < 0.05) and GN (28% vs. 10%, p < 0.05) BSIs. Patients with Gram positive (GP) BSIs had a significant increase in mortality risk (OR 4.5, CI 1.4-14.5, p < 0.05) in contrast to those with Gram negative (GN) infections (OR 0.4, CI 0.4-4.0, p = 0.4). CONCLUSION: Concurrent COVID-19 infection is associated with a significant increase in morbidity and mortality in patients with GP and GN BSIs. Patients with S. aureus BSIs with COVID-19 are more likely to develop shock and respiratory failure and have higher rates and odds of mortality than those without COVID-19. These findings provide an essential insight into the care of these patients, especially those co-infected with Staphylococcus aureus.

Clonal diversity of Staphylococcus aureus isolates in clinical specimens from selected health facilities in Ethiopia.

Staphylococcus aureus is among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology of S. aureus in hospital settings, with limited molecular genotyping results. Molecular characterization of S. aureus is essential for identification of strains, and contributes to the control and prevention of S. aureus infection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. The spa typing analysis showed diversity of S. aureus with 56 distinct spa types. Spa type t355 was most prevalent (56/170, 32.9%), while eleven new spa types were detected including t20038, t20039, and t20042. The identified spa types were clustered into 15 spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknown spa types were further subjected to MLST analysis. The majority of isolates belonged to spa-CC 152 (62/170, 36.4%), followed by spa-CC 121 (19/170, 11.2%), and spa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) were spa-CC 239 with staphylococcal cassette chromosome (SCC)mec III. These findings highlight the diversity of S. aureus strains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization of S. aureus for antimicrobial resistance detection and infection prevention purposes.

Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy.

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

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.

Pre-epidemic evolution of the MRSA USA300 clade and a molecular key for classification.

Introduction: USA300 has remained the dominant community and healthcare associated methicillin-resistant Staphylococcus aureus (MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American epidemic (SAE) clade and North American epidemic (NAE) clade. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Methods: Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome, to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Results: Phylogenetic analysis with these genomes confirms that the NAE and SAE USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We provide evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South and North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

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.

Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae.

BACKGROUND: Mobile plasmids play a key role in spurring the global dissemination of multidrug-resistant (MDR) K. pneumoniae, while plasmid curing has been recognized as a promising strategy to combat antimicrobial resistance. Here we exploited a K. pneumoniae native CRISPR system to cure the high-risk IncFII plasmids. METHODS: We examined matched protospacers in 725 completely sequenced IncFII plasmids from K. pneumoniae genomes. Then, we re-engineered a native CRISPR-Cas3 system and deliver the CRISPR-Cas3 system via conjugation. Plasmid killing efficiency and G. mellonella infection model were applied to evaluate the CRISPR-Cas3 immunity in vitro and in vivo. FINDINGS: Genomic analysis revealed that most IncFII plasmids could be targeted by the native CRISPR-Cas3 system with multiple matched protospacers, and the targeting regions were highly conserved across different IncFII plasmids. This conjugative endogenous CRISPR-Cas3 system demonstrated high plasmid curing efficiency in vitro (8-log decrease) and in vivo (∼100% curing) in a Galleria mellonella infection model, as well as provided immunization against the invasion of IncFII plasmids once the system entering a susceptible bacterial host. INTERPRETATION: Overall, our work demonstrated the applicability of using native CRISPR-mediated plasmid curing to re-sensitize drug-resistant K. pneumoniae to multiple antibiotics. This work provided strong support for the idea of utilizing native CRISPR-Cas systems to tackle AMR in K. pneumoniae. FUNDING: This work was supported by research grants National Natural Science Foundation of China [grant numbers 81871692, 82172315, 82102439, and 82202564], the Shanghai Science and Technology Commission [grant number 19JC1413002], and Shanghai Sailing Program [grant number 22YF1437500].