RESUMEN
BACKGROUND: Expert guidelines discourage use of antipseudomonal ß-lactams and fluoroquinolones in lower-risk patients with community-acquired complicated intra-abdominal infection (CA cIAI). Compliance with these recommendations across US hospitals is unclear. This study sought to determine treatment patterns and associated outcomes among adult hospitalized lower-risk patients with CA cIAI. METHODS: A study using data from the Premier Healthcare Database (10/2015-12/2017) was performed. Inclusion criteria: age ≥18 years; hospitalized; had a cIAI at admission; and received antibiotics within the first 4 hospital days. Patients were excluded if they were high risk, were transferred from another health care facility, had a recent hospital admission, or received dialysis within 30 days of admission. Empiric antibiotic treatment patterns and associated outcomes were quantified. RESULTS: Overall, 46â 722 (66%) patients with cIAIs met the lower-risk CA IAI study criteria. Among lower-risk CA IAI patients, the mean (SD) age was 53.4 (18.2) years, and 71% had a Charlson Comorbidity Index score of 0. The most common diagnosis was acute appendicitis with peritonitis (59.7%). Among lower-risk CA IAI patients, 54% received piperacillin/tazobactam, 20% received a fluoroquinolone (FQ), 11% received ceftriaxone, and 7% received ampicillin/sulbactam. Overall, the median hospital length of stay was 4 days and median costs were $12â 345 USD. Nearly 90% of patients were discharged home, and <1% died. Outcomes were similar across all empiric treatments received. CONCLUSIONS: Overuse of antipseudomonal ß-lactams and fluoroquinolones was commonplace among lower-risk CA IAI patients. These findings can serve as the basis for an antimicrobial stewardship initiative in hospitals aspiring to reduce the use of broad-spectrum antibiotics.
RESUMEN
The activity of the novel, fully synthetic fluorocycline antibiotic TP-6076 against carbapenem-resistant Acinetobacter baumannii (CRAB) isolates with defined carbapenem resistance mechanisms was compared against reference antimicrobials with known activity against Acinetobacter spp. The susceptibility of 323 non-duplicate CRAB isolates to TP-6076, amikacin, ampicillin/sulbactam (SAM), cefepime, colistin, doxycycline, eravacycline, imipenem, levofloxacin, meropenem, minocycline, rifampicin, sulbactam, tigecycline, tobramycin and trimethoprim/sulfamethoxazole (SXT) was determined by the broth microdilution method. TP-6076 showed greater activity than comparator antimicrobials of the tetracycline class, SAM, levofloxacin, amikacin, tobramycin, SXT and colistin. MIC50 and MIC90 values for TP-6076 were 0.06 mg/L and 0.25 mg/L, respectively. In comparison, doxycycline, eravacycline, minocycline and tigecycline MIC50/90 values were 32/≥64, 0.5/1, 4/8 and 1/2 mg/L, respectively. Compared with other compounds, TP-6076 was the most active antimicrobial against CRAB, including isolates that were resistant to other anti-Acinetobacter reference drugs including SAM, colistin, the aminoglycosides amikacin and tobramycin, and levofloxacin. TP-6076 is a promising new agent that may be a useful addition to the limited armamentarium of drugs targeting this problematic pathogen.
Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Carbapenémicos/farmacología , Infecciones por Acinetobacter/microbiología , Aminoglicósidos/farmacología , Ampicilina/farmacología , Colistina/farmacología , Farmacorresistencia Bacteriana Múltiple , Humanos , Minociclina/farmacología , Sulbactam/farmacología , Tetraciclinas/farmacología , Tigeciclina/farmacologíaRESUMEN
Eravacycline is a novel, fully synthetic fluorocycline antibiotic being developed for the treatment of serious infections, including those caused by resistant Gram-positive pathogens. Here, we evaluated the in vitro activities of eravacycline and comparator antimicrobial agents against a recent global collection of frequently encountered clinical isolates of Gram-positive bacteria. The CLSI broth microdilution method was used to determine in vitro MIC data for isolates of Enterococcus spp. (n = 2,807), Staphylococcus spp. (n = 4,331), and Streptococcus spp. (n = 3,373) isolated primarily from respiratory, intra-abdominal, urinary, and skin specimens by clinical laboratories in 37 countries on three continents from 2013 to 2017. Susceptibilities were interpreted using both CLSI and EUCAST breakpoints. There were no substantive differences (a >1-doubling-dilution increase or decrease) in eravacycline MIC90 values for different species/organism groups over time or by region. Eravacycline showed MIC50 and MIC90 results of 0.06 and 0.12 µg/ml, respectively, when tested against Staphylococcus aureus, regardless of methicillin susceptibility. The MIC90 values of eravacycline for Staphylococcus epidermidis and Staphylococcus haemolyticus were equal (0.5 µg/ml). The eravacycline MIC90s for Enterococcus faecalis and Enterococcus faecium were 0.06 µg/ml and were within 1 doubling dilution regardless of the vancomycin susceptibility profile. Eravacycline exhibited MIC90 results of ≤0.06 µg/ml when tested against Streptococcus pneumoniae and beta-hemolytic and viridans group streptococcal isolates. In this surveillance study, eravacycline demonstrated potent in vitro activity against frequently isolated clinical isolates of Gram-positive bacteria (Enterococcus, Staphylococcus, and Streptococcus spp.), including isolates collected over a 5-year period (2013 to 2017), underscoring its potential benefit in the treatment of infections caused by common Gram-positive pathogens.
Asunto(s)
Bacterias Grampositivas/efectos de los fármacos , Streptococcus/efectos de los fármacos , Tetraciclinas/farmacología , Enterococcus faecalis/efectos de los fármacos , Infecciones por Bacterias Grampositivas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Streptococcus pneumoniae/efectos de los fármacos , Vancomicina/farmacologíaRESUMEN
Eravacycline is a novel, fully synthetic fluorocycline antibiotic developed for the treatment of serious infections, including those caused by multidrug-resistant (MDR) pathogens. Here, we evaluated the in vitro activities of eravacycline and comparator antimicrobial agents against a global collection of frequently encountered clinical isolates of Gram-negative bacilli. The CLSI broth microdilution method was used to determine MIC data for isolates of Enterobacterales (n = 13,983), Acinetobacter baumannii (n = 2,097), Pseudomonas aeruginosa (n = 1,647), and Stenotrophomonas maltophilia (n = 1,210) isolated primarily from respiratory, intra-abdominal, and urinary specimens by clinical laboratories in 36 countries from 2013 to 2017. Susceptibilities were interpreted using both CLSI and EUCAST breakpoints. Multidrug-resistant (MDR) isolates were defined by resistance to agents from ≥3 different antimicrobial classes. The MIC90s ranged from 0.25 to 1 µg/ml for Enterobacteriaceae and were 1 µg/ml for A. baumannii and 2 µg/ml for S. maltophilia, Proteus mirabilis, and Serratia marcescens Eravacycline's potency was up to 4-fold greater than that of tigecycline against genera/species of Enterobacterales, A. baumannii, and S. maltophilia The MIC90s for five of six individual genera/species of Enterobacterales and A. baumannii were within 2-fold of the MIC90s for their respective subsets of MDR isolates, while the MDR subpopulation of Klebsiella spp. demonstrated 4-fold higher MIC90s. Eravacycline demonstrated potent in vitro activity against the majority of clinical isolates of Gram-negative bacilli, including MDR isolates, collected over a 5-year period. This study further underscores the potential benefit of eravacycline in the treatment of infections caused by MDR Gram-negative pathogens.
Asunto(s)
Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Tetraciclinas/farmacología , Acinetobacter baumannii/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Tigeciclina/farmacologíaRESUMEN
BACKGROUND: Carbapenems are a frequent firstline therapy in complicated intra-abdominal infections (cIAIs). We examined the microbiology, epidemiology, and outcomes among patients hospitalized in the United States with culture-positive cIAIs in the context of their exposure to empiric carbapenem treatment (ECT). METHODS: We performed a multicenter retrospective cohort study of Premier database of ~180 hospitals, 2013-2017. Using an International Classification of Diseases (ICD)-9/10-based algorithm, we identified all culture-positive adult patients hospitalized with cIAI and examined their microbiology, epidemiology, and outcomes. RESULTS: Among 4453 patients with cIAIs, 3771 (84.7%) had a gram-negative (GN) and 1782 (40.0%) a gram-positive organism; 1185 (26.6%) received ECT. Compared with those on non-ECT, patients on ECT were less frequently admitted from home (82.5% vs 86.0%) or emergently (76.0% vs 81.4%; P < .05 for each); E. coli were less frequent, whereas P. aeruginosa and Enterococcus spp. were more prevalent and resistance to third-generation cephalosporins (C3R; 10.1% vs 5.1%; P < .001) and carbapenems (CR; 3.6% vs 1.2%; P < .001) was more common. In adjusted analyses, ECT was associated with no rise in mortality, shorter postinfection length of stay (-0.59 days; 95% confidence interval [CI], -1.15 to -0.03), but higher postinfection costs ($3844; 95% CI, $1921 to $5767) and risk of Clostridioides difficile (odds ratio, 2.15; 95% CI, 1.02 to 4.50). CONCLUSIONS: Among patients hospitalized with cIAI, the majority were gram-negative. Despite a 10% prevalence of C3R, fully one-quarter of all empiric regimens contained a carbapenem. ECT was a marker for slightly lower postinfection length of stay, but higher costs and risk of hospital complications.
RESUMEN
Aim: Recently approved for use in complicated intra-abdominal infection, eravacycline is a novel fluorocycline with broad spectrum of activity against resistant Gram-negative pathogens. This manuscript is a pooled analysis of two Phase III trials. Clinical efficacy: Clinical cure rates were 86.8% for eravacycline versus 87.6% for ertapenem, and 90.8% for eravacycline versus 91.2% for meropenem in the Intent to Treat (micro-ITT) populations, and 87.0% for eravacycline versus 88.8% ertapenem, and 92.4 versus 91.6% for meropenem in the Modified Intent to Treat (MITT) populations. Safety: Eravacycline is well tolerated, with lower rates of nausea, vomiting and diarrhea than other tetracyclines. Conclusion: Eravacycline is an effective new option for use in complicated intra-abdominal infections, and in particular, for the treatment of extended-spectrum ß-lactamase- and carbapenem-resistant Enterobacteriaceae-expressing organisms.
Asunto(s)
Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana Múltiple , Enterobacteriaceae/efectos de los fármacos , Infecciones Intraabdominales/tratamiento farmacológico , Infecciones Intraabdominales/microbiología , Tetraciclinas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carbapenémicos/uso terapéutico , Interpretación Estadística de Datos , Enterobacteriaceae/enzimología , Ertapenem/uso terapéutico , Femenino , Humanos , Infecciones Intraabdominales/complicaciones , Masculino , Meropenem/uso terapéutico , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Estudios Prospectivos , Adulto Joven , beta-LactamasasRESUMEN
BACKGROUND: Trends in antimicrobial resistance help inform infection control efforts. We examined trends in resistance for Enterobacteriaceae and Acinetobacter spp. from 2013 to 2017 in hospitalized US patients. METHODS: We analyzed antimicrobial susceptibility of non-duplicate isolates in hospitalized patients (not limited to hospital-acquired infections) in the US BD Insights Research Database. Resistance profiles of interest were extended-spectrum beta-lactamase (ESBL)-producing, multidrug resistant (MDR), and carbapenem-nonsusceptible (Carb-NS) phenotypes of Enterobacteriaceae, and MDR and Carb-NS Acinetobacter spp. Time series models were used to evaluate the patterns of resistance trends in rate per 100 hospital admissions and proportion per isolates tested. RESULTS: More than 1 million Enterobacteriaceae isolates were obtained from 411 hospitals; 12.05% were ESBL, 1.21% Carb-NS, and 7.08% MDR. Urine was the most common source. For Acinetobacter spp. (n = 19,325), 37.48% were Carb-NS, 47.66% were MDR, and the most common source was skin/wound cultures. Trend analyses showed that the rates of ESBL and Carb-NS Enterobacteriaceae per 100 hospital admissions increased significantly between 2013 and 2017. Rates of MDR Enterobacteriaceae and Carb-NS and MDR Acinetobacter spp. decreased during this time period. Trends in proportions of resistant isolates generally mirrored trends in rates per 100 hospital admissions. MDR Enterobacteriaceae and Carb-NS and MDR Acinetobacter spp. were more common in winter than summer. CONCLUSIONS: In this large-scale study of patients in US hospitals, rates of ESBL and Carb-NS Enterobacteriaceae per 100 hospital admissions increased between 2013 and 2017. MDR Enterobacteriaceae and MDR and Carb-NS Acinetobacter spp. isolates decreased over this period. These data support continuing infection control and stewardship efforts and the development of new therapeutic options.
Asunto(s)
Infecciones por Acinetobacter/epidemiología , Acinetobacter/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Infecciones por Enterobacteriaceae/epidemiología , Enterobacteriaceae/efectos de los fármacos , Acinetobacter/genética , Acinetobacter/aislamiento & purificación , Infecciones por Acinetobacter/microbiología , Antibacterianos/farmacología , Carbapenémicos/farmacología , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Infecciones por Enterobacteriaceae/microbiología , Hospitales/estadística & datos numéricos , Humanos , Pruebas de Sensibilidad Microbiana , Estudios Retrospectivos , Estados Unidos/epidemiología , beta-Lactamasas/metabolismoRESUMEN
OBJECTIVES: To determine antimicrobial nonsusceptibility rates for Enterobacteriaceae and Acinetobacter spp. in US hospitals. METHODS: We analyzed antimicrobial susceptibilities of non-duplicate Enterobacteriaceae and Acinetobacter spp. isolates reported in 2017 from 375 US hospitals in the BD Insights Research Database. Logistic and Poisson regression modeling methods were used to estimate proportions of resistant isolates and rates per 1000 hospital admissions. National projections were generated based on raking (weighting) methods. RESULTS: The nationwide proportions of resistant isolates in inpatients were an estimated 12.6%, 6.6%, and 1.2% for Enterobacteriaceae with extended-spectrum beta-lactamase (ESBL), multidrug resistant (MDR), and carbapenem-nonsusceptible (Carb-NS) phenotypes, respectively, and 42.4% and 34.5% for Acinetobacter spp. with MDR and Carb-NS phenotypes. Resistance varied by geographic region and hospital size/type. Estimated nationwide rates per 1000 hospital admissions ranged from a high of 7.1 for ESBL Enterobacteriaceae to a low of 0.3 for Carb-NS Acinetobacter spp. The estimated number of isolates occurring in US inpatients each year was 290,220 ESBL, 173,984 MDR, and 30,194 Carb-NS for Enterobacteriaceae and 12,274 MDR and 9,991 Carb-NS for Acinetobacter spp. CONCLUSIONS: National prevalence estimates suggest high levels of antimicrobial resistance and a substantial number of patients with resistant Enterobacteriaceae and Acinetobacter spp. in US hospitals.
Asunto(s)
Acinetobacter/efectos de los fármacos , Farmacorresistencia Bacteriana , Enterobacteriaceae/efectos de los fármacos , Acinetobacter/aislamiento & purificación , Antibacterianos/farmacología , Carbapenémicos/farmacología , Enterobacteriaceae/aislamiento & purificación , Hospitalización , Humanos , Estados UnidosRESUMEN
Eravacycline (7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline or TP-434) is a novel, fully synthetic broad-spectrum fluorocycline with potent activity against Gram-positive bacteria, anaerobes, and multidrug-resistant Enterobacteriaceae We characterized the plasma pharmacokinetics of eravacycline and conducted a comprehensive analysis of the eravacycline tissue distribution in rabbits after multiple-day dosing. For single-dose pharmacokinetic analysis, eravacycline was administered to New Zealand White (NZW) rabbits at 1, 2, 4, 8, and 10 mg/kg of body weight intravenously (i.v.) once a day (QD) (n = 20). For multidose pharmacokinetic analysis, eravacycline was administered at 0.5, 1, 2, and 4 mg/kg i.v. QD (n = 20) for 6 days. Eravacycline concentrations in plasma and tissues were analyzed by a liquid chromatography-tandem mass spectrometry assay. Mean areas under the concentration-time curves (AUCs) following a single eravacycline dose ranged from 5.39 µg · h/ml to 183.53 µg · h/ml. Within the multidose study, mean AUCs ranged from 2.53 µg · h/ml to 29.89 µg · h/ml. AUCs correlated linearly within the dosage range (r = 0.97; P = 0.0001). In the cardiopulmonary system, the concentrations were the highest in the lung, followed by the heart > pulmonary alveolar macrophages > bronchoalveolar lavage fluid; for the intra-abdominal system, the concentrations were the highest in bile, followed by the liver > gallbladder > spleen > pancreas; for the renal system, the concentrations were the highest in urine, followed by those in the renal cortex > renal medulla; for the musculoskeletal tissues, the concentrations were the highest in muscle psoas, followed by those in the bone marrow > adipose tissue; for the central nervous system, the concentrations were the highest in cerebrum, followed by those in the aqueous humor > cerebrospinal fluid > choroid > vitreous. The prostate and seminal vesicles demonstrated relatively high mean concentrations. The plasma pharmacokinetic profile of 0.5 to 4 mg/kg in NZW rabbits yields an exposure comparable to that in humans (1 or 2 mg/kg every 12 h) and demonstrates target tissue concentrations in most sites.
Asunto(s)
Antibacterianos/farmacología , Antibacterianos/farmacocinética , Enterobacteriaceae/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Tetraciclinas/farmacología , Tetraciclinas/farmacocinética , Distribución Tisular/fisiología , Animales , Área Bajo la Curva , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Femenino , Masculino , Pruebas de Sensibilidad Microbiana/métodos , ConejosRESUMEN
Neisseria gonorrhoeae has two porins, PIA and PIB, whose genes (porA and porB, respectively) are alleles of a single por locus. We recently demonstrated that penB mutations at positions 120 and 121 in PIB, which are presumed to reside in loop 3 that forms the pore constriction zone, confer intermediate-level resistance to penicillin and tetracycline (M. Olesky, M. Hobbs, and R. A. Nicholas, Antimicrob. Agents Chemother. 46:2811-2820, 2002). In the present study, we investigated the electrophysiological properties as well as solute and antibiotic permeation rates of recombinant PIB proteins containing penB mutations (G120K, G120D/A121D, G120P/A121P, and G120R/A121H). In planar lipid bilayers, the predominant conducting state of each porin variant was 30 to 40% of the wild type, even though the anion selectivity and maximum channel conductance of each PIB variant was similar to that of the wild type. Liposome-swelling experiments revealed no significant differences in the permeation of sugars or beta-lactam antibiotics through the wild type or PIB variants. Although these results are seemingly contradictory with the ability of these variants to increase antibiotic resistance, they are consistent with MIC data showing that these porin mutations confer resistance only in strains containing an mtrR mutation, which increases expression of the MtrC-MtrD-MtrE efflux pump. Moreover, both the mtrR and penB mutations were required to decrease in vivo permeation rates below those observed in the parental strain containing either mtrR or porin mutations alone. Thus, these data demonstrate a novel mechanism of porin-mediated resistance in which mutations in PIB have no affect on antibiotic permeation alone but instead act synergistically with the MtrC-MtrD-MtrE efflux pump in the development of antibiotic resistance in gonococci.
Asunto(s)
Antibacterianos/metabolismo , Farmacorresistencia Bacteriana , Mutación/genética , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/metabolismo , Porinas/genética , Porinas/metabolismo , Antibacterianos/farmacología , Metabolismo de los Hidratos de Carbono , Regulación Bacteriana de la Expresión Génica , Neisseria gonorrhoeae/genéticaRESUMEN
Herpes simplex virus 1 (HSV-1) ICP27 and ICP8 proteins have both been implicated in the transcription of late genes and regulation of viral gene expression. We showed previously that ICP27 and ICP8 associate with the RNAP II holoenzyme (Zhou and Knipe, J. Virol. 76, 5893-5904). Here, we demonstrate that ICP27 and ICP8 coprecipitate from lysates of HSV-1-infected HEp2 cells and from lysates of insect cells expressing ICP27 and ICP8, the latter being in the absence of other HSV-1 proteins. By expressing and purifying hexahistidine-tagged ICP8 (His-ICP8) and maltose binding protein (MBP)-tagged ICP27 (MBP-27) proteins and performing in vitro immunoprecipitation and pull-down assays, we also demonstrate that ICP27 and ICP8 coprecipitate in the absence of other viral or cellular proteins. Taken together, these data provide evidence that ICP27 and ICP8 interact directly in vitro and in infected cells. We hypothesize that the ICP27-ICP8 interaction plays a role in the stimulation of late gene transcription.
Asunto(s)
Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/fisiología , Proteínas Virales/fisiología , Animales , Línea Celular , Proteínas de Unión al ADN , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Humano 1/química , Humanos , Proteínas Inmediatas-Precoces/análisis , Inmunoprecipitación , Proteínas Virales/análisisRESUMEN
The herpes simplex virus (HSV) immediate early ICP27 protein plays an essential role in stimulating viral early and late gene expression. ICP27 appears to be multifunctional in that it has been reported to stimulate viral late gene transcription, polyadenylation site usage, and RNA export. We report here on proteomic studies involving immunoprecipitation of ICP27 and mass spectrometric identification of co-precipitated proteins. These studies show an association of ICP27 with the cellular translation initiation factors poly A binding protein (PABP), eukaryotic initiation factor 3 (eIF3), and eukaryotic initiation factor 4G (eIF4G) in infected cells. Immunoprecipitation-western blot studies confirmed these associations. Finally, purified MBP-tagged ICP27 (MBP-27) can interact with eIF3 subunits p47 and p116 in vitro. These results suggest that ICP27 may also play a role in stimulating translation of certain viral and host mRNAs and/or in inhibiting host mRNA translation.
Asunto(s)
Factores Eucarióticos de Iniciación/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Mapeo de Interacción de Proteínas , Simplexvirus/fisiología , Western Blotting , Línea Celular , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Humanos , Inmunoprecipitación , Espectrometría de Masas , Proteínas de Unión a Poli(A)/metabolismo , Subunidades de Proteína/metabolismoRESUMEN
A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Neisseria gonorrhoeae/metabolismo , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , beta-Lactamas/metabolismo , Acilación , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , División Celular/genética , Supervivencia Celular/genética , Clonación Molecular , Farmacorresistencia Microbiana , Endopeptidasas/química , Endopeptidasas/metabolismo , Estabilidad de Enzimas , Regulación Bacteriana de la Expresión Génica , Hexosiltransferasas/antagonistas & inhibidores , Hexosiltransferasas/genética , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/antagonistas & inhibidores , Muramoilpentapéptido Carboxipeptidasa/genética , Neisseria gonorrhoeae/enzimología , Neisseria gonorrhoeae/crecimiento & desarrollo , Neisseria gonorrhoeae/ultraestructura , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/antagonistas & inhibidores , Peptidil Transferasas/genética , Unión Proteica , Especificidad por Sustrato , beta-Lactamas/químicaRESUMEN
PenB is the third resistance determinant in the stepwise acquisition of multiple resistance genes in chromosomally mediated resistant Neisseria gonorrhoeae (CMRNG). Alterations in por(IB), one of two alleles at the por locus that encodes the outer membrane protein porin IB (PIB), were recently reported to be responsible for the increased resistance to penicillin and tetracycline conferred by penB, but the specific mutations conferring antibiotic resistance were not identified experimentally. To determine which amino acids in PIB confer increased resistance, we transformed a recipient strain with chimeras of the por(IB) genes from strains FA1090 and FA140 (penB2). These studies revealed that two amino acid changes, G120D and A121D, were both necessary and sufficient to confer increased resistance to penicillin and tetracycline. Site-saturation and site-directed mutagenesis of Gly-120 and Ala-121 revealed that both a single mutation, G120K, and the double mutations G120R A121H and G120P A121P also conferred antibiotic resistance to the recipient strain. The identical mutations in PIA increased penicillin and tetracycline resistance either moderately or not at all. Analysis of por(IB) genes present in the GenBank database from 51 clinical isolates demonstrated that lysine and aspartate mutations at positions 120 and/or 121 also occur in nature. These studies demonstrate that charged amino acids at positions 120 and 121 in PIB are highly preferential for conferring resistance to penicillin and tetracycline in N. gonorrhoeae.
Asunto(s)
Mutación/genética , Neisseria gonorrhoeae/genética , Resistencia a las Penicilinas/genética , Porinas/genética , Resistencia a la Tetraciclina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/efectos de los fármacos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Western Blotting , ADN Bacteriano/efectos de los fármacos , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/crecimiento & desarrollo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transformación BacterianaRESUMEN
Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were > or = 1 microg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of beta-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 microg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.