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1.
J Microbiol Methods ; 226: 107047, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-39303991

RESUMEN

We report the creation of 17 Escherichia coli strains harboring the conjugative plasmid pLCasCureT with a CRISPR-Cas9 system to surgically "cure" the most common plasmids among Enterobacterales species. This approach can create isogenic pairs of strains to study host-plasmid interactions, correlate plasmid genotype and phenotype, and create plasmid-free cloning strains.


Asunto(s)
Sistemas CRISPR-Cas , Conjugación Genética , Escherichia coli , Plásmidos , Plásmidos/genética , Escherichia coli/genética , Vectores Genéticos/genética
2.
Antimicrob Agents Chemother ; 68(10): e0020824, 2024 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-39162528

RESUMEN

We characterized the molecular determinants of meropenem-vaborbactam (MV) non-susceptibility among non-metallo-ß-lactamase-producing KPC-Klebsiella pneumoniae (KPC-KP). Whole-genome sequencing was performed to identify mutations associated with MV non-susceptibility. Isolates with elevated MV MICs were found to have mutations encoding truncated or altered OmpK36 porins and increased blaKPC copy numbers. KPC-KP isolates with decreased susceptibility to MV were detected among a collection of isolates predating the availability of MV.


Asunto(s)
Antibacterianos , Proteínas Bacterianas , Ácidos Borónicos , Klebsiella pneumoniae , Meropenem , Pruebas de Sensibilidad Microbiana , Secuenciación Completa del Genoma , beta-Lactamasas , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Meropenem/farmacología , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Antibacterianos/farmacología , Ácidos Borónicos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Porinas/genética , Porinas/metabolismo , Humanos , Mutación , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/tratamiento farmacológico , Combinación de Medicamentos , Farmacorresistencia Bacteriana Múltiple/genética , Compuestos Heterocíclicos con 1 Anillo
3.
Microbiol Spectr ; 12(8): e0048624, 2024 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-38916317

RESUMEN

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.


Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus , Resistencia a la Vancomicina , Vancomicina , Vancomicina/farmacología , Antibacterianos/farmacología , Humanos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Infecciones Estafilocócicas/microbiología , Resistencia a la Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina/genética , Staphylococcus aureus Resistente a Vancomicina/efectos de los fármacos , Staphylococcus aureus Resistente a Vancomicina/metabolismo , Daptomicina/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
4.
Antimicrob Agents Chemother ; 68(5): e0017424, 2024 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-38557171

RESUMEN

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.


Asunto(s)
Antibacterianos , Ácidos Borónicos , Cefoxitina , Ceftarolina , Cefalosporinas , Imipenem , Meropenem , Pruebas de Sensibilidad Microbiana , Mycobacterium abscessus , Mycobacterium abscessus/efectos de los fármacos , Meropenem/farmacología , Ácidos Borónicos/farmacología , Antibacterianos/farmacología , Cefalosporinas/farmacología , Imipenem/farmacología , Cefoxitina/farmacología , Humanos , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Inhibidores de beta-Lactamasas/farmacología
5.
ACS Infect Dis ; 10(5): 1767-1779, 2024 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-38619138

RESUMEN

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.


Asunto(s)
Aminoaciltransferasas , Antituberculosos , Mycobacterium tuberculosis , Inhibidores de beta-Lactamasas , beta-Lactamasas , Aminoaciltransferasas/antagonistas & inhibidores , Antituberculosos/farmacología , Antituberculosos/química , Compuestos de Azabiciclo/farmacología , Compuestos de Azabiciclo/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Inhibidores de beta-Lactamasas/farmacología , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/metabolismo , beta-Lactamasas/química , Cinética , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología
6.
Antimicrob Agents Chemother ; 68(4): e0134623, 2024 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-38426743

RESUMEN

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.


Asunto(s)
Antibacterianos , Aztreonam , Ácidos Borónicos , Meropenem/farmacología , Aztreonam/farmacología , Antibacterianos/farmacología , Klebsiella pneumoniae/genética , beta-Lactamasas/genética , Combinación de Medicamentos , Pruebas de Sensibilidad Microbiana , Compuestos de Azabiciclo/farmacología
7.
iScience ; 27(2): 108875, 2024 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-38313058

RESUMEN

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.

8.
Clin Infect Dis ; 78(2): 248-258, 2024 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-37738153

RESUMEN

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.


Asunto(s)
Infecciones por Acinetobacter , Acinetobacter baumannii , Humanos , Acinetobacter baumannii/genética , Carbapenémicos/farmacología , Carbapenémicos/uso terapéutico , Estudios Prospectivos , Pruebas de Sensibilidad Microbiana , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/epidemiología , Infecciones por Acinetobacter/microbiología , beta-Lactamasas/genética , Proteínas Bacterianas/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico
9.
Eur J Clin Microbiol Infect Dis ; 43(2): 389-393, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38062176

RESUMEN

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.


Asunto(s)
Infecciones por Klebsiella , beta-Lactamasas , Humanos , beta-Lactamasas/genética , Klebsiella pneumoniae , Taiwán/epidemiología , Absceso , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/microbiología , Plásmidos/genética , Antibacterianos/farmacología
10.
Microbiol Spectr ; 12(1): e0369223, 2024 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-38018989

RESUMEN

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.


Asunto(s)
Infecciones por Escherichia coli , Escherichia coli Patógena Extraintestinal , Humanos , Infecciones por Escherichia coli/microbiología , Sistemas CRISPR-Cas , Plásmidos/genética , Antibacterianos
11.
Nat Commun ; 14(1): 7962, 2023 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-38042959

RESUMEN

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.


Asunto(s)
Klebsiella pneumoniae , Factores de Virulencia , Humanos , Animales , Ratones , Virulencia/genética , Factores de Virulencia/genética , Antibacterianos/farmacología , Plásmidos , Sideróforos
12.
mBio ; 14(5): e0194923, 2023 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-37671860

RESUMEN

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.


Asunto(s)
Infecciones por Klebsiella , Klebsiella pneumoniae , Humanos , Virulencia/genética , Neutrófilos , Genotipo , Antibacterianos
13.
Eur J Clin Microbiol Infect Dis ; 42(10): 1173-1181, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37597051

RESUMEN

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.


Asunto(s)
Bacteriemia , COVID-19 , Sepsis , Infecciones Estafilocócicas , Humanos , Persona de Mediana Edad , Staphylococcus aureus , COVID-19/complicaciones , Sepsis/complicaciones , Sepsis/epidemiología , Bacteriemia/complicaciones , Bacteriemia/epidemiología , Pacientes Internos , Escherichia coli
14.
Viruses ; 15(8)2023 08 05.
Artículo en Inglés | MEDLINE | ID: mdl-37632041

RESUMEN

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.


Asunto(s)
COVID-19 , Humanos , COVID-19/epidemiología , COVID-19/prevención & control , SARS-CoV-2/genética , New Jersey/epidemiología , Vacunas contra la COVID-19 , Pandemias , Estudios Retrospectivos , Glicoproteína de la Espiga del Coronavirus , Infección Irruptiva
15.
Sci Adv ; 9(23): eadg4205, 2023 06 09.
Artículo en Inglés | MEDLINE | ID: mdl-37294761

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas , Colistina , Colistina/farmacología , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacología , Antibacterianos/metabolismo , Bacterias/metabolismo , Hierro , Homeostasis
16.
Front Cell Infect Microbiol ; 13: 1081070, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36761897

RESUMEN

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.


Asunto(s)
Epidemias , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Estados Unidos , Humanos , Filogenia , Infecciones Estafilocócicas/epidemiología , Genoma Bacteriano , Evolución Molecular
17.
Lancet Microbe ; 4(3): e159-e170, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36774938

RESUMEN

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.


Asunto(s)
Antibacterianos , Infecciones por Pseudomonas , Estados Unidos , Humanos , Antibacterianos/uso terapéutico , Pseudomonas aeruginosa/genética , Estudios Prospectivos , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/epidemiología , Carbapenémicos/uso terapéutico
18.
EBioMedicine ; 88: 104445, 2023 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-36696817

RESUMEN

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].


Asunto(s)
Proteínas Bacterianas , Klebsiella pneumoniae , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Klebsiella pneumoniae/genética , Sistemas CRISPR-Cas , China , Plásmidos/genética , Antibacterianos , beta-Lactamasas/genética
19.
Clin Infect Dis ; 76(2): 229-237, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36173830

RESUMEN

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is the most prevalent carbapenem-resistant Enterobacterales in the United States. We evaluated CRKp clustering in patients in US hospitals. METHODS: From April 2016 to August 2017, 350 patients with clonal group 258 CRKp were enrolled in the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae, a prospective, multicenter, cohort study. A maximum likelihood tree was constructed using RAxML. Static clusters shared ≤21 single-nucleotide polymorphisms (SNP) and a most recent common ancestor. Dynamic clusters incorporated SNP distance, culture timing, and rates of SNP accumulation and transmission using the R program TransCluster. RESULTS: Most patients were admitted from home (n = 150, 43%) or long-term care facilities (n = 115, 33%). Urine (n = 149, 43%) was the most common isolation site. Overall, 55 static and 47 dynamics clusters were identified involving 210 of 350 (60%) and 194 of 350 (55%) patients, respectively. Approximately half of static clusters were identical to dynamic clusters. Static clusters consisted of 33 (60%) intrasystem and 22 (40%) intersystem clusters. Dynamic clusters consisted of 32 (68%) intrasystem and 15 (32%) intersystem clusters and had fewer SNP differences than static clusters (8 vs 9; P = .045; 95% confidence interval [CI]: -4 to 0). Dynamic intersystem clusters contained more patients than dynamic intrasystem clusters (median [interquartile range], 4 [2, 7] vs 2 [2, 2]; P = .007; 95% CI: -3 to 0). CONCLUSIONS: Widespread intrasystem and intersystem transmission of CRKp was identified in hospitalized US patients. Use of different methods for assessing genetic similarity resulted in only minor differences in interpretation.


Asunto(s)
Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Klebsiella , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Klebsiella pneumoniae/genética , Estudios de Cohortes , Estudios Prospectivos , Infecciones por Klebsiella/epidemiología , Infecciones por Klebsiella/tratamiento farmacológico , Carbapenémicos/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Hospitales , Farmacorresistencia Bacteriana
20.
mBio ; 13(6): e0290622, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36445082

RESUMEN

It is unknown whether bacterial bloodstream infections (BSIs) are commonly caused by single organisms or mixed microbial populations. We hypothesized that contemporaneous carbapenem-resistant Klebsiella pneumoniae (CRKP) strains from blood cultures of individual patients are genetically and phenotypically distinct. We determined short-read whole-genome sequences of 10 sequence type 258 (ST258) CRKP strains from blood cultures in each of 6 patients (Illumina HiSeq). Strains clustered by patient by core genome and pan-genome phylogeny. In 5 patients, there was within-host strain diversity by gene mutations, presence/absence of antibiotic resistance or virulence genes, and/or plasmid content. Accessory gene phylogeny revealed strain diversity in all 6 patients. Strains from 3 patients underwent long-read sequencing for genome completion (Oxford Nanopore) and phenotypic testing. Genetically distinct strains within individuals exhibited significant differences in carbapenem and other antibiotic responses, capsular polysaccharide (CPS) production, mucoviscosity, and/or serum killing. In 2 patients, strains differed significantly in virulence during mouse BSIs. Genetic or phenotypic diversity was not observed among strains recovered from blood culture bottles seeded with index strains from the 3 patients and incubated in vitro at 37°C. In conclusion, we identified genotypic and phenotypic variant ST258 CRKP strains from blood cultures of individual patients with BSIs, which were not detected by the clinical laboratory or in seeded blood cultures. The data suggest a new paradigm of CRKP population diversity during BSIs, at least in some patients. If validated for BSIs caused by other bacteria, within-host microbial diversity may have implications for medical, microbiology, and infection prevention practices and for understanding antibiotic resistance and pathogenesis. IMPORTANCE The long-standing paradigm for pathogenesis of bacteremia is that, in most cases, a single organism passes through a bottleneck and establishes itself in the bloodstream (single-organism hypothesis). In keeping with this paradigm, standard practice in processing positive microbiologic cultures is to test single bacterial strains from morphologically distinct colonies. This study is the first genome-wide analysis of within-host diversity of Klebsiella pneumoniae strains recovered from individual patients with bloodstream infections (BSIs). Our finding that positive blood cultures comprised genetically and phenotypically heterogeneous carbapenem-resistant K. pneumoniae strains challenges the single-organism hypothesis and suggests that at least some BSIs are caused by mixed bacterial populations that are unrecognized by the clinical laboratory. The data support a model of pathogenesis in which pressures in vivo select for strain variants with particular antibiotic resistance or virulence attributes and raise questions about laboratory protocols and treatment decisions directed against single strains.


Asunto(s)
Bacteriemia , Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Klebsiella , Sepsis , Animales , Ratones , Klebsiella pneumoniae/genética , Cultivo de Sangre , Antibacterianos/uso terapéutico , Carbapenémicos , Enterobacteriaceae Resistentes a los Carbapenémicos/genética , Bacteriemia/microbiología , Sepsis/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Pruebas de Sensibilidad Microbiana , beta-Lactamasas
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