RESUMEN
The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.
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Cromosomas Bacterianos , Elementos Transponibles de ADN , Nisina , Streptococcus pneumoniae , Nisina/farmacología , Nisina/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/efectos de los fármacos , Cromosomas Bacterianos/genética , Elementos Transponibles de ADN/genética , Regulación Bacteriana de la Expresión Génica , Enterococcus/genética , Enterococcus/efectos de los fármacos , Vectores Genéticos/genética , Conjugación Genética , Streptococcus/genética , Streptococcus/efectos de los fármacos , Streptococcus/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Streptococcus pneumoniae, a medically important opportunistic bacterial pathogen of the upper respiratory tract, is a major public health concern, causing a wide range of pneumococcal illnesses, both invasive and noninvasive. It is associated with significant global morbidity and mortality, including pneumonia, meningitis, sepsis, and acute otitis media. The major purpose of this study was to determine the molecular epidemiology of Streptococcus pneumoniae strains that cause invasive and noninvasive infections in Ethiopia. A prospective study was undertaken in two regional hospitals between January 2018 and December 2019. Whole-genome sequencing was used to analyze all isolates. Serotypes and multilocus sequence types (MLST) were derived from genomic data. The E-test was used for antimicrobial susceptibility testing. Patient samples obtained 54 Streptococcus pneumoniae isolates, 33 from invasive and 21 from noninvasive specimens. Our findings identified 32 serotypes expressed by 25 Global Pneumococcal Sequence Clusters (GPSCs) and 42 sequence types (STs), including 21 new STs. The most common sequence types among the invasive isolates were ST3500, ST5368, ST11162, ST15425, ST15555, ST15559, and ST15561 (2/33, 6% each). These sequence types were linked to serotypes 8, 7 C, 15B/C, 16 F, 10 A, 15B, and 6 A, respectively. Among the noninvasive isolates, only ST15432, associated with serotype 23 A, had numerous isolates (4/21, 19%). Serotype 14 was revealed as the most resistant strain to penicillin G, whereas isolates from serotypes 3, 8, 7 C, and 10 A were resistant to erythromycin. Notably, all serotype 6 A isolates were resistant to both erythromycin and penicillin G. Our findings revealed an abnormally significant number of novel STs, as well as extremely diversified serotypes and sequence types, implying that Ethiopia may serve as a breeding ground for novel STs. Recombination can produce novel STs that cause capsular switching. This has the potential to influence how immunization campaigns affect the burden of invasive pneumococcal illness. The findings highlight the importance of continuous genetic surveillance of the pneumococcal population as a vital step toward enhancing future vaccine design.
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Antibacterianos , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Tipificación de Secuencias Multilocus , Infecciones Neumocócicas , Serogrupo , Streptococcus pneumoniae , Secuenciación Completa del Genoma , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/clasificación , Humanos , Etiopía/epidemiología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/epidemiología , Masculino , Niño , Femenino , Preescolar , Adolescente , Adulto , Persona de Mediana Edad , Estudios Prospectivos , Lactante , Adulto Joven , Antibacterianos/farmacología , AncianoRESUMEN
In the final phases of bacterial cell wall synthesis, penicillin-binding proteins (PBPs) catalyze the cross-linking of peptidoglycan. For many decades, effective and non-toxic ß-lactam antibiotics have been successfully used as mimetics of the d-Ala-d-Ala moiety of the natural substrate and employed as irreversible inhibitors of PBPs. In the years following their discovery, the emergence of resistant bacteria led to a decline in their clinical efficacy. Using Staudinger cycloaddition, we synthesized a focused library of novel monocyclic ß-lactams in which different substituents were introduced at the C4 position of the ß-lactam ring, at the C3 amino position, and at the N1 lactam nitrogen. In biochemical assays, the compounds were evaluated for their inhibitory effect on the model enzyme PBP1b from Streptococcus pneumoniae. Upon investigation of the antibacterial activity of the newly prepared compounds against ESKAPE pathogens, some compounds showed moderate inhibition. We also examined their reactivity and selectivity in a biochemical assay with other enzymes that have a catalytic serine in the active site, such as human cholinesterases, where they also showed no inhibitory activity, highlighting their specificity for bacterial targets. These compounds form the basis for further work on new monocyclic ß-lactams with improved antibacterial activity.
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Antibacterianos , Proteínas de Unión a las Penicilinas , Streptococcus pneumoniae , beta-Lactamas , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Proteínas de Unión a las Penicilinas/metabolismo , Streptococcus pneumoniae/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , beta-Lactamas/farmacología , beta-Lactamas/síntesis química , beta-Lactamas/química , Relación Estructura-Actividad , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Streptococcus pneumoniae carriage studies are crucial to monitor changes induced by use of pneumococcal conjugate vaccines and inform vaccination policies. In this cross-sectional study, we examined changes within the pneumococcal population following introduction of PCV13 in 2015 in the National Immunization Program (NIP), in Portugal. In 2018-2020 (NIP-PCV13), we obtained 1450 nasopharyngeal samples from children ≤6 years attending day-care. We assessed serotypes, antimicrobial resistance, and genotypes (MLST and GPSC) and compared findings with earlier periods: 2009-2010 (pre-PCV13), 2011-2012 (early-PCV13), and 2015-2016 (late-PCV13). Pneumococcal carriage prevalence remained stable at 60.2 %. Carriage of PCV13 serotypes was 10.7 %, markedly reduced compared to pre-PCV13 period (47.6 %). The most prevalent PCV13 serotypes were 19F, 3, and 19A all showing a significant decreasing trend compared to the pre-PCV13 period (from 7.1 % to 4.7 %, 10.1 % to 1.8 %, and 14.1 % to 1.8 %, respectively), a notable observation given the described limited effectiveness of PCV13 against serotype 3. Non-vaccinated children and children aged 4-6 years were more likely to carry PCV13 serotypes (2.5-fold, 95 %CI [1.1-5.6], and 2.9-fold, 95 %CI [1.3-6.8], respectively). The most prevalent non-PCV13 serotypes were 15B/C, 11A, 23B, 23A, and NT, collectively accounting for 51.9 % of all isolates. In total, 30.5 % of all pneumococci were potentially covered by PCV20. Resistance to penicillin (low-level) and macrolides increased significantly, from 9.3 % and 13.4 %, respectively, in the late-PCV13 period, to approximately 20 % each, mostly due to lineages expressing non-PCV13 serotypes, nearing pre-PCV13 levels. An expansion of lineages traditionally associated with PCV13 serotypes, like CC156-GPSC6 (serotype 14) and CC193-GPSC11 (serotype 19F), but now predominantly expressing non-PCV13 serotypes (11A, 15B/C, and 24F for GPSC6; and 15A and 21 for GPSC11) was noted. These findings indicate that the pneumococcal population is adapting to the pressures conferred by PCV13 and antimicrobial use and indicate the need to maintain close surveillance.
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Portador Sano , Genotipo , Programas de Inmunización , Nasofaringe , Infecciones Neumocócicas , Vacunas Neumococicas , Serogrupo , Streptococcus pneumoniae , Humanos , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Vacunas Neumococicas/inmunología , Vacunas Neumococicas/administración & dosificación , Estudios Transversales , Portugal/epidemiología , Preescolar , Infecciones Neumocócicas/prevención & control , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/microbiología , Femenino , Masculino , Portador Sano/epidemiología , Portador Sano/microbiología , Lactante , Nasofaringe/microbiología , Niño , Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Prevalencia , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/administración & dosificación , Pruebas de Sensibilidad MicrobianaRESUMEN
Ketolides (3-keto) such as TE-802 and acylides (3-O-acyl) like TEA0929 are ineffective against constitutively resistant pathogens harboring erythromycin ribosomal methylation (erm) genes. Following our previous work on alkylides (3-O-alkyl), we explored the structure-activity relationships of hybrids combining (R/S) 3-descladinosyl erythromycin with 6/7-quinolone motifs, featuring extended ether-linked spacers, with a focus on their efficacy against pathogens bearing constitutive erm gene resistance. Optimized compounds 17a and 31f not only reinstated efficacy against inducibly resistant pathogens but also demonstrated significantly augmented activities against constitutively resistant strains of Streptococcus pneumoniae and Streptococcus pyogenes, which are typically refractory to existing C-3 modified macrolides. Notably, hybrid 31f (coded ZN-51) represented a pioneering class of agents distinguished by its dual modes of action, with ribosomes as the primary target and topoisomerases as the secondary target. As a novel chemotype of macrolide-quinolone hybrids, alkylide 31f is a valuable addition to our armamentarium against macrolide-resistant bacteria.
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Antibacterianos , Macrólidos , Pruebas de Sensibilidad Microbiana , Quinolonas , Streptococcus pneumoniae , Relación Estructura-Actividad , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Quinolonas/química , Quinolonas/farmacología , Quinolonas/síntesis química , Macrólidos/química , Macrólidos/farmacología , Streptococcus pneumoniae/efectos de los fármacos , Estructura Molecular , Diseño de Fármacos , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/enzimología , Relación Dosis-Respuesta a Droga , Éteres/química , Éteres/farmacología , Éteres/síntesis químicaRESUMEN
BACKGROUND: In early 2021, the 10-valent Pneumococcal conjugate vaccine (PCV10) was replaced with 13-valent (PCV13) by the federal directorate of immunization (FDI), Pakistan. We assessed the impact of a higher valent vaccine, PCV13, on the serotype distribution of nasopharyngeal carriage in rural Pakistan. METHODS: Children <2 years were randomly selected from two rural union councils of Matiari, Sindh in Pakistan between September-October,2022. Clinical, sociodemographic and vaccination histories were recorded. Nasopharyngeal swabs were collected and processed at Infectious Disease Research Laboratory, Aga Khan University, Karachi. Whole genome sequencing was performed on the culture positive isolates. RESULTS: Of the 200 children enrolled, pneumococcus was detected in 140(70 %) isolates. Majority of age-eligible children (60.1 %,110/183) received 3 PCV13 doses. PCV10 carriage declined from 13.2 %(78/590) in 2017/18 to 7.2 % (10/140) in 2022, additional PCV13 serotypes (3, 6A/6C and 19A) decreased from 18.5 %(109/590) to 11.4 %(16/140) while non-PCV13 serotypes increased from 68.3 %(403/590) to 81.4 %(114/140). There were 88.5 %(n = 124), 80.7 %(n = 113), 55.0 %(n = 77), and 46.0 %(n = 65) isolates predicted to be resistant to cotrimoxazole, penicillin(meningitis cut-off), tetracycline, and erythromycin respectively. CONCLUSION: Replacing PCV10 with PCV13 rapidly decreased prevalence of PCV13 carriage among vaccinated children in Matiari, Pakistan. Vaccine-driven selection pressure may have been responsible for the increase of non-PCV13 serotypes.
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Portador Sano , Nasofaringe , Infecciones Neumocócicas , Vacunas Neumococicas , Serogrupo , Streptococcus pneumoniae , Humanos , Pakistán/epidemiología , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/efectos de los fármacos , Vacunas Neumococicas/inmunología , Vacunas Neumococicas/administración & dosificación , Infecciones Neumocócicas/prevención & control , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/microbiología , Lactante , Portador Sano/epidemiología , Portador Sano/microbiología , Masculino , Femenino , Nasofaringe/microbiología , Antibacterianos/farmacología , Preescolar , Secuenciación Completa del Genoma , Población Rural/estadística & datos numéricos , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/administración & dosificaciónRESUMEN
Antimicrobial resistance (AMR) is a public health threat worldwide. Next-generation sequencing (NGS) has opened unprecedented opportunities to accelerate AMR mechanism discovery and diagnostics. Here, we present an integrative approach to investigate trimethoprim (TMP) resistance in the key pathogen Streptococcus pneumoniae. We explored a collection of 662 S. pneumoniae genomes by conducting a genome-wide association study (GWAS), followed by functional validation using resistance reconstruction experiments, combined with machine learning (ML) approaches to predict TMP minimum inhibitory concentration (MIC). Our study showed that multiple additive mutations in the folA and sulA loci are responsible for TMP non-susceptibility in S. pneumoniae and can be used as key features to build ML models for digital MIC prediction, reaching an average accuracy within ±1 twofold dilution factor of 86.3%. Our roadmap of in silico analysis-wet-lab validation-diagnostic tool building could be adapted to explore AMR in other combinations of bacteria-antibiotic. IMPORTANCE: In the age of next-generation sequencing (NGS), while data-driven methods such as genome-wide association study (GWAS) and machine learning (ML) excel at finding patterns, functional validation can be challenging due to the high numbers of candidate variants. We designed an integrative approach combining a GWAS on S. pneumoniae clinical isolates, followed by whole-genome transformation coupled with NGS to functionally characterize a large set of GWAS candidates. Our study validated several phenotypic folA mutations beyond the standard Ile100Leu mutation, and showed that the overexpression of the sulA locus produces trimethoprim (TMP) resistance in Streptococcus pneumoniae. These validated loci, when used to build ML models, were found to be the best inputs for predicting TMP minimal inhibitory concentrations. Integrative approaches can bridge the genotype-phenotype gap by biological insights that can be incorporated in ML models for accurate prediction of drug susceptibility.
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Antibacterianos , Estudio de Asociación del Genoma Completo , Aprendizaje Automático , Pruebas de Sensibilidad Microbiana , Streptococcus pneumoniae , Resistencia al Trimetoprim , Trimetoprim , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/efectos de los fármacos , Trimetoprim/farmacología , Antibacterianos/farmacología , Humanos , Resistencia al Trimetoprim/genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Infecciones Neumocócicas/microbiología , MutaciónRESUMEN
Introduction: the present study aimed at isolating and characterizing actinomycete from unexplored Windhoek rocky crest mountainous soil and extracting bioactive metabolites as possible therapeutics against common life-threatening Streptococcus pneumonia (S. pneumonia) and Stachybotrys chartarum (S. chartarum). Methods: chemotaxonomy and biochemical methods were used to identify the isolates. The solvent extraction method was used to extract bioactive compounds. Agar overlay and disc diffusion methods were used to determine the antimicrobial activity of isolates and extracted bioactive metabolites against S. pneumonia and S. chartarum. The antioxidant activity of the extracted bioactive metabolites was determined using 2.2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method with ascorbic acid as a positive control. Comparison between groups was done using a Two-way ANOVA, followed by Bonferroni post-test. Results: three distinct isolates from 3 soil samples were identified on starch casein agar and distinguished using biochemical tests. All three isolates showed strong inhibitory activity against S. pneumonia with average growth inhibition zones between 18.0±1.00 and 27±0.00 mm p< 0.005. All isolates showed potent inhibitory activity against S. chartarum with the average inhibition zones ranging between 42.0±1.00 and 48±0.00 mm, p< 0.005. The chloroform extracts showed potent DPPH activity of up to 73± 1.41%. Conclusion: growth conditions and extraction solvents can influence the antimicrobial and antioxidant properties of bioactive metabolites.
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Actinobacteria , Antibacterianos , Antioxidantes , Microbiología del Suelo , Streptococcus pneumoniae , Antioxidantes/farmacología , Antioxidantes/aislamiento & purificación , Humanos , Actinobacteria/aislamiento & purificación , Actinobacteria/metabolismo , Antibacterianos/farmacología , Antibacterianos/aislamiento & purificación , Namibia , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
BACKGROUND: Gram-positive bacteria residing in the nasopharynx can lead to severe illnesses in children, such as otitis media, pneumonia, and meningitis. Despite the potential threat, there is a lack of comprehensive data regarding the carriage rates of these bacteria among children in outpatient departments in the study area. OBJECTIVE: This study aimed to assess the nasopharyngeal carriage, antimicrobial resistance patterns, and associated factors of Gram-positive bacteria among children attending the outpatient department at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. METHODS: A hospital-based cross-sectional study was conducted from May 1, 2023, to August 30, 2023. A total of 424 nasopharyngeal swab samples were collected using sterile nasopharyngeal swabs, inoculated on Blood Agar and Mannitol Salt Agar plates, and identified through colony morphology, Gram stain, and biochemical tests. Antimicrobial susceptibility of the identified bacterial isolates was determined employing both the Kirby-Bauer and modified Kirby-Bauer methods. D-tests were conducted using clindamycin and erythromycin discs to detect inducible clindamycin resistance, while cefoxitin disc tests were utilized to ascertain methicillin resistance. Data entry was executed using Epi-Data version 4.6, and subsequent analysis was performed utilizing SPSS version 25. Bivariable and multivariable logistic regression analyses were employed to identify associated factors. An adjusted odds ratio at a 95% confidence interval with a P-value of < 0.05 was considered statistically significant. RESULTS: The overall nasopharyngeal carriage rate of Gram-positive bacteria was 296/424 (69.8%, 95% CI: 65.3-74.0). Staphylococcus aureus was the most prevalent 122/424 (28.8%), followed by Streptococcus pneumoniae 92/424 (21.7%). Methicillin resistance was observed in 19/122 (15.6%) of S. aureus and 3/60 (5%) of coagulase-negative staphylococcus (CoNS) species. Inducible clindamycin resistance was 10/122 (8.2%) in S. aureus and 4/53 (7.5%) in coagulase-negative staphylococcus species. Multidrug resistance was found in 146/296 (49.3%, 95% CI: 43.6-55.0) of the isolates. Associated factors with a bacterial carriage were large family size (AOR = 3.061, 95% CI: 1.595-5.874, P = 0.001), having siblings under five years old (AOR = 1.991, 95% CI: 1.196-3.313, P = 0.008), indoor cooking (AOR = 2.195, 95% CI: 1.275-3.778, P = 0.005), an illiterate mother (AOR = 3.639, 95% CI: 1.691-7.829, P = 0.001), and hospital visits (AOR = 2.690, 95% CI: 1.405-5.151, P = 0.003). CONCLUSION: The study found a high nasopharyngeal carriage of Gram-positive bacteria in outpatient children, including notable levels of methicillin-resistant S. aureus and multi-drug-resistant isolates. Clindamycin, rifampin, and erythromycin were the most effective antimicrobials for the tested isolates. Factors contributing to bacterial carriage include visits to healthcare facilities, larger family sizes, having younger siblings, maternal illiteracy, and indoor cooking. This emphasizes the need for methicillin-resistant S. aureus surveillance in pediatric outpatient settings and community health education, especially for children's guardians. Additionally, improving household ventilation by separating kitchens from sleeping areas and regular screening of younger siblings in healthcare environments were recommended to reduce bacterial transmission within family members. The study also called for studies with advanced procedures like minimum inhibitory concentration testing and molecular characterization to better comprehend the resistance patterns and genes in circulating bacteria.
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Antibacterianos , Pruebas de Sensibilidad Microbiana , Nasofaringe , Humanos , Etiopía/epidemiología , Femenino , Masculino , Nasofaringe/microbiología , Preescolar , Niño , Estudios Transversales , Lactante , Antibacterianos/farmacología , Bacterias Grampositivas/efectos de los fármacos , Bacterias Grampositivas/aislamiento & purificación , Pacientes Ambulatorios/estadística & datos numéricos , Portador Sano/microbiología , Portador Sano/epidemiología , Infecciones por Bacterias Grampositivas/microbiología , Infecciones por Bacterias Grampositivas/epidemiología , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Farmacorresistencia Bacteriana , Adolescente , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
This study aimed to investigate the bacterial characteristics of pneumococcal isolates obtained from a tertiary care hospital in Japan. We analyzed the antimicrobial susceptibility, possession of macrolide resistance genes, pneumococcal serogroup/serotype, and sequence type (ST) of pneumococcal isolates from patients aged 15 years or older between 2011 and 2020 at Nagasaki University Hospital. Of the 73 isolates analyzed, 86.3% showed resistance to macrolides, and 28.8%, 46.6%, and 11.0% harbored mefA, ermB, and both, respectively. Of the isolates possessing ermB, 97.6% showed high levels of macrolide resistance [minimal inhibitory concentration (MIC) range, > 16 µg/mL]. Solithromycin (MIC range, 0.03-0.25 µg/mL), regardless of the presence of macrolide resistance genes, and lascufloxacin (MIC range, 0.06-0.5 µg/mL) showed potent in vitro activity against pneumococci. Serotype 19A was the most prevalent (six isolates), followed by serotypes 10A, 15A, and 15B/C (five isolates each). Four serotypes (11A, 19A, 22F, and 23B) and five STs (36, 99, 433, 558, and 3111) were significantly correlated with the presence of macrolide resistance genes. All four isolates with serotype 11A/ST99 and three isolates with serotype 19A/ST3111 harbored both mefA and ermB. No macrolide resistance genes were detected in either of the two isolates with serotype 22F/ST433, while all ten isolates with serogroup 15 (serotypes 15A and 15B/C, five isolates each) possessed ermB alone. Our study revealed the bacterial characteristics of the pneumococcal isolates obtained from our hospital. In vitro activity of solithromycin and lascufloxacin against these isolates was confirmed.
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Antibacterianos , Farmacorresistencia Bacteriana , Macrólidos , Pruebas de Sensibilidad Microbiana , Infecciones Neumocócicas , Serogrupo , Streptococcus pneumoniae , Centros de Atención Terciaria , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/aislamiento & purificación , Streptococcus pneumoniae/clasificación , Humanos , Infecciones Neumocócicas/microbiología , Japón , Antibacterianos/farmacología , Macrólidos/farmacología , Farmacorresistencia Bacteriana/genética , Adulto Joven , Adolescente , Fenotipo , Anciano , Persona de Mediana Edad , Adulto , Proteínas Bacterianas/genética , Femenino , Masculino , Metiltransferasas/genética , Anciano de 80 o más Años , Pueblos del Este de Asia , Proteínas de la MembranaRESUMEN
Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.
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Antibacterianos , Pared Celular , Farmacorresistencia Bacteriana , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Streptococcus pneumoniae , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Lipopéptidos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Péptidos Cíclicos/farmacología , HumanosAsunto(s)
Antibacterianos , Cefalosporinas , Streptococcus pneumoniae , Animales , Cefalosporinas/farmacocinética , Cefalosporinas/farmacología , Cefalosporinas/uso terapéutico , Cefalosporinas/administración & dosificación , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Antibacterianos/administración & dosificación , Ratones , Streptococcus pneumoniae/efectos de los fármacos , Modelos Animales de Enfermedad , Pulmón/metabolismo , Pulmón/efectos de los fármacos , Pulmón/microbiología , Humanos , Neumonía Neumocócica/tratamiento farmacológico , Neumonía Neumocócica/microbiologíaRESUMEN
Streptococcus pneumoniae infection is a major public health concern with high morbidity and mortality rates. This study aimed to evaluate the serotype distribution, antimicrobial resistance changes, clonal composition, and virulence factors of S. pneumoniae isolates causing pneumococcal disease in northeast China from 2000 to 2021. A total of 1,454 S. pneumoniae isolates were included, with 568 invasive strains and 886 non-invasive strains. The patients from whom the S. pneumoniae were isolated ranged in age from 26 days to 95 years, with those ≤ 5 years old comprising the largest group (67.19%). 19 F, 19 A, 23 F, 14, and 6B were the most common serotypes, of which 19 A and 19 F were the main serotypes of invasive and non-invasive S. pneumoniae, respectively. CC271 was the most common multilocus sequence type. Serotype 14 had the lowest expression of cbpA, rrgA, and psrP genes, but expression levels of 19 A and 19 F genes were similar. All isolates were sensitive to ertapenem, moxifloxacin, linezolid, and vancomycin but highly resistant to macrolides, tetracyclines, and cotrimoxazole. Simultaneous resistance to erythromycin, clindamycin, tetracyclines, and trimethoprim/sulfamethoxazole was common pattern among multidrug-resistant isolates. Non-invasive S. pneumoniae had higher resistance to ß-lactam antibiotics than invasive strains. 19 A and 19 F were the main strains of penicillin-resistant S. pneumoniae. The resistance rate of ß-lactam antibiotics decreased from 2017 to 2021 compared to previous periods. Including PCV13 in the national immunization program can reduce the morbidity and mortality rates of pneumococcal disease effectively.
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Antibacterianos , Tipificación de Secuencias Multilocus , Infecciones Neumocócicas , Serogrupo , Streptococcus pneumoniae , Factores de Virulencia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/patogenicidad , Streptococcus pneumoniae/aislamiento & purificación , Humanos , China/epidemiología , Factores de Virulencia/genética , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/epidemiología , Preescolar , Lactante , Persona de Mediana Edad , Adolescente , Antibacterianos/farmacología , Adulto , Niño , Anciano , Adulto Joven , Anciano de 80 o más Años , Recién Nacido , Pruebas de Sensibilidad Microbiana , Femenino , Masculino , Farmacorresistencia Bacteriana , Farmacorresistencia Bacteriana Múltiple/genéticaRESUMEN
We studied toxicity of recombinant Streptococcus pneumoniae pneumolysin protein in experiments on mice and its cytopathogenic effect on cultures of Vero green monkey kidney cells and human lung carcinoma A549 cells in vitro. In vivo and in vitro experiments proved the absence of compromised toxicity and direct cytopathogenic action of the recombinant protein.
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Proteínas Bacterianas , Proteínas Recombinantes , Streptococcus pneumoniae , Estreptolisinas , Estreptolisinas/toxicidad , Estreptolisinas/genética , Animales , Proteínas Bacterianas/toxicidad , Proteínas Bacterianas/genética , Chlorocebus aethiops , Ratones , Células Vero , Streptococcus pneumoniae/efectos de los fármacos , Humanos , Proteínas Recombinantes/toxicidad , Proteínas Recombinantes/genética , Células A549RESUMEN
Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.
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Antibacterianos , Proteínas Bacterianas , Percepción de Quorum , Streptococcus pneumoniae , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiología , Antibacterianos/farmacología , Percepción de Quorum/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Transformación BacterianaRESUMEN
Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location1,2. The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient.
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Aptitud Genética , Mapeo Geográfico , Streptococcus pneumoniae , Humanos , Aptitud Genética/efectos de los fármacos , Aptitud Genética/genética , Genoma Bacteriano/genética , Resistencia a las Penicilinas/efectos de los fármacos , Resistencia a las Penicilinas/genética , Penicilinas/farmacología , Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/transmisión , Vacunas Neumococicas/inmunología , Serogrupo , Sudáfrica/epidemiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/aislamiento & purificación , Vacunas Conjugadas/inmunología , Vacuna Neumocócica Conjugada Heptavalente/inmunología , LocomociónRESUMEN
In the face of increasing antimicrobial tolerance and resistance there is a global obligation to optimise oral antimicrobial dosing strategies including narrow spectrum penicillins, such as penicillin-V. We conducted a randomised, crossover study in healthy volunteers to characterise the influence of probenecid on penicillin-V pharmacokinetics and estimate the pharmacodynamics against Streptococcus pneumoniae. Twenty participants took six doses of penicillin-V (250 mg, 500 mg or 750 mg four times daily) with and without probenecid. Total and free concentrations of penicillin-V and probenecid were measured at two timepoints. A pharmacokinetic model was developed, and the probability of target attainment (PTA) calculated. The mean difference (95% CI) between penicillin-V alone and in combination with probenecid for serum total and free penicillin-V concentrations was significantly different at both timepoints (total: 45 min 4.32 (3.20-5.32) mg/L p < 0.001, 180 min 2.2 (1.58-3.25) mg/L p < 0.001; free: 45 min 1.15 (0.88-1.42) mg/L p < 0.001, 180 min 0.5 (0.35-0.76) mg/L p < 0.001). There was no difference between the timepoints in probenecid concentrations. PTA analysis shows probenecid allows a fourfold increase in MIC cover. Addition of probenecid was safe and well tolerated. The data support further research into improved dosing structures for complex outpatient therapy and might also be used to address penicillin supply shortages.
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Antibacterianos , Estudios Cruzados , Penicilina V , Probenecid , Humanos , Probenecid/farmacocinética , Probenecid/farmacología , Probenecid/administración & dosificación , Masculino , Adulto , Femenino , Antibacterianos/farmacocinética , Antibacterianos/administración & dosificación , Penicilina V/farmacocinética , Penicilina V/administración & dosificación , Streptococcus pneumoniae/efectos de los fármacos , Adulto Joven , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Voluntarios Sanos , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/microbiologíaRESUMEN
BACKGROUND: Bacterial etiologies of acute otitis media (AOM) have shifted from the introduction of pneumococcal conjugate vaccines (PCVs), antibiotic selection and competition among species. We characterized Streptococcus pneumoniae ( Spn ), Haemophilus influenzae ( Hflu ) and Moraxella catarrhalis ( Mcat ) in the nasopharynx during well-child healthy visits and at the onset of AOM, and in middle ear fluid (MEF) of children with AOM to assess anticipated effects of higher-valency PCVs (PCV15 and PCV20). METHODS: From September 2021 to September 2023, we conducted a prospective longitudinal cohort study of PCV13 immunized children 6-36 months old. MEF was collected via tympanocentesis. Serotyping and antibiotic susceptibility testing were performed on Spn , Hflu and Mcat isolates. RESULTS: We obtained 825 nasopharyngeal and 216 MEF samples from 301 children. The order of frequency of nasopharyngeal colonization was Mcat , Spn and Hflu ; Hflu was the predominant otopathogen in MEF. Among Spn isolates, non-PCV15, non-PCV20 serotypes predominated in the nasopharynx and in MEF; the most frequent serotype was 35B. Among MEF samples, 30% of Spn isolates were amoxicillin nonsusceptible; 23% of Hflu isolates and 100% of Mcat isolates were ß-lactamase-producing. CONCLUSION: The majority of Spn isolates among young children were non-PCV15, non-PCV20 serotypes, especially serotype 35B; therefore, the impact of higher-valency PCVs in reducing pneumococcal colonization or AOM is expected to be limited. Hflu continues to be the most frequent AOM pathogen. Antibiotic susceptibility data suggest a high dose of amoxicillin/clavulanate or alternative drugs that are effective against contemporary mix of otopathogens could be considered for optimal empiric selection to provide the best efficacy.
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Moraxella catarrhalis , Nasofaringe , Otitis Media , Vacunas Neumococicas , Streptococcus pneumoniae , Humanos , Vacunas Neumococicas/administración & dosificación , Otitis Media/microbiología , Otitis Media/prevención & control , Otitis Media/epidemiología , Lactante , Preescolar , Nasofaringe/microbiología , Estudios Prospectivos , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/aislamiento & purificación , Estudios Longitudinales , Femenino , Moraxella catarrhalis/efectos de los fármacos , Moraxella catarrhalis/aislamiento & purificación , Masculino , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/aislamiento & purificación , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Vacunas Conjugadas/administración & dosificación , Infecciones Neumocócicas/prevención & control , Infecciones Neumocócicas/microbiología , Infecciones Neumocócicas/epidemiología , Pruebas de Sensibilidad Microbiana , Enfermedad AgudaRESUMEN
Energy-coupling factor transporters (ECFTs) are membrane-bound ATP-binding cassette (ABC) transporters in prokaryotes that are found in pathogens against which novel antibiotics are urgently needed. To date, just 54 inhibitors of three molecular-structural classes with mostly weak inhibitory activity are known. Target repurposing is a strategy that transfers knowledge gained from a well-studied protein family to under-studied targets of phylogenetic relation. Forty-eight human ABC transporters are known that may harbor structural motifs similar to ECFTs to which particularly multitarget compounds may bind. We assessed 31 multitarget compounds which together target the entire druggable human ABC transporter proteome against ECFTs, of which nine showed inhibitory activity (hit rate 29.0%) and four demonstrated moderate to strong inhibition of an ECFT (IC50 values between 4.28 and 50.2 µM) as well as antibacterial activity against ECFT-expressing Streptococcus pneumoniae. Here, ivermectin was the most potent candidate (MIC95: 22.8 µM), and analysis of five ivermectin derivatives revealed moxidectin as one of the most potent ECFT-targeting antibacterial agents (IC50: 2.23 µM; MIC95: 2.91 µM). Distinct molecular-structural features of avermectins and derivatives as well as the differential biological response of the hit compounds in general provided first indications with respect to the structure-activity relationships and mode of action, respectively.
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Antibacterianos , Reposicionamiento de Medicamentos , Ivermectina , Pruebas de Sensibilidad Microbiana , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Humanos , Ivermectina/farmacología , Ivermectina/análogos & derivados , Ivermectina/química , Ivermectina/síntesis química , Relación Estructura-Actividad , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Streptococcus pneumoniae/efectos de los fármacos , Estructura Molecular , Relación Dosis-Respuesta a DrogaRESUMEN
Background: The use of antimicrobials to treat food animals may result in antimicrobial residues in foodstuffs of animal origin. The European Medicines Association (EMA) and World Health Organization (WHO) define safe antimicrobial concentrations in food based on acceptable daily intakes (ADIs). It is unknown if ADI doses of antimicrobials in food could influence the antimicrobial susceptibility of human-associated bacteria. Objectives: This aim of this study was to evaluate if the consumption of ADI doses of erythromycin could select for erythromycin resistance in a Galleria mellonella model of Streptococcus pneumoniae infection. Methods: A chronic model of S. pneumoniae infection in G. mellonella larvae was used for the experiment. Inoculation of larvae with S. pneumoniae was followed by injections of erythromycin ADI doses (0.0875 and 0.012 µg/ml according to EMA and WHO, respectively). Isolation of S. pneumoniae colonies was then performed on selective agar plates. Minimum inhibitory concentrations (MICs) of resistant colonies were measured, and whole genome sequencing (WGS) was performed followed by variant calling to determine the genetic modifications. Results: Exposure to single doses of both EMA and WHO ADI doses of erythromycin resulted in the emergence of erythromycin resistance in S. pneumoniae. Emergent resistance to erythromycin was associated with a mutation in rplA, which codes for the L1 ribosomal protein and has been linked to macrolide resistance in previous studies. Conclusion: In our in vivo model, even single doses of erythromycin that are classified as acceptable by the WHO and EMA induced significant increases in erythromycin MICs in S. pneumoniae. These results suggest the need to include the induction of antimicrobial resistance (AMR) as a significant criterion for determining ADIs.