RESUMO
BACKGROUND: Acinetobacter baumannii is an opportunistic pathogen that can cause a variety of nosocomial infections in humans. This study aimed to molecularly characterize extended-spectrum beta-lactamase (ESBL) producing and carbapenem-resistant Acinetobacter species isolated from surgical site infections (SSI). METHODS: A multicentre cross-sectional study was performed among SSI patients at four hospitals located in Northern, Southern, Southwest, and Central parts of Ethiopia. The isolates were identified by microbiological methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibility was determined using disk diffusion. The presence of phenotypic ESBL and carbapenemase production was detected by employing standard microbiological tests, including combined disk diffusion (CDT). ESBL and carbapenem resistance determinants genes were studied by polymerase chain reaction (PCR) and sequencing. RESULTS: A total of 8.7% Acinetobacter species were identified from 493 culture-positive isolates out of 752 SSI wounds. The species identified by MALDI-TOF MS were 88.4% A. baumannii, 4.7% Acinetobacter pittii, 4.7% Acinetobacter soli, and 2.3% Acinetobacter lactucae. Of all isolates 93% were positive for ESBL enzymes according to the CDT. Using whole genome sequencing 62.8% of the A. baumannii harbored one or more beta-lactamase genes, and 46.5% harbored one or more carbapenemase producing genes. The distribution of beta-lactamases among Acinetobacter species by hospitals was 53.8%, 64.3%, 75%, and 75% at JUSH, TASH, DTCSH, and HUCSH respectively. Among ESBL genes, blaCTX-M alleles were detected in 21.4% of isolates; of these 83.3% were blaCTX-M-15. The predominant carbapenemase gene of blaOXA type was detected in 24 carbapenem-resistant A. baumannii followed by blaNDM alleles carried in 12 A. baumannii with blaNDM-1 as the most common. CONCLUSIONS: The frequency of Acinetobacter species that produce metallobetalactamases (MBLs) and ESBLs that were found in this study is extremely scary and calls for strict infection prevention and control procedures in health facilities helps to set effective antibiotics stewardship.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Proteínas de Bactérias , Testes de Sensibilidade Microbiana , Infecção da Ferida Cirúrgica , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/metabolismo , Humanos , Acinetobacter baumannii/genética , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/isolamento & purificação , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/epidemiologia , Etiópia/epidemiologia , Estudos Transversais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Infecção da Ferida Cirúrgica/microbiologia , Infecção da Ferida Cirúrgica/epidemiologia , Adulto , Masculino , Pessoa de Meia-Idade , Feminino , Antibacterianos/farmacologia , Adulto Jovem , Adolescente , Idoso , Criança , Pré-Escolar , Carbapenêmicos/farmacologia , Idoso de 80 Anos ou mais , LactenteRESUMO
BACKGROUND: Cholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia. METHODS: Across-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value < 0.05 was considered as statically significant. RESULTS: The prevalence of V. cholerae in the fecal samples was 30.1%. Majority of the isolates were from Oromia National Regional State 43.2% (n = 54) followed by Amhara National Regional State 31.2% (n = 39) and Addis Ababa City Administration 25.6% (n = 32). Most of the V. cholerae isolates were O1 serogroups 90.4% (n = 113) and Ogawa serotypes 86.4% (n = 108). Majority of the isolates were susceptible to ciprofloxacin 100% (n = 125), tetracycline 72% (n = 90) and gentamycin 68% (n = 85). More than half of the isolates were resistant to trimethoprim-sulfamethoxazole 62.4% (n = 78) and ampicillin 56.8% (n = 71). In this study, participants unable to read and write were about four times more at risk for V. cholerae infection (AOR: 3.8, 95% CI: 1.07-13.33). In addition, consumption of river water were about three times more at risk for V. cholerae infection (AOR: 2.8, 95% CI: 1.08-7.08). CONCLUSION: our study revealed a high prevalence of V. cholerae from fecal samples. The predominant serogroups and serotypes were O1 and Ogawa, respectively. Fortunately, the isolates showed susceptible to most tested antibiotics. Drinking water from river were the identified associated risk factor for V. cholerae infection. Protecting the community from drinking of river water and provision of safe and treated water could reduce cholera outbreaks in the study areas.
Assuntos
Antibacterianos , Cólera , Surtos de Doenças , Vibrio cholerae , Humanos , Cólera/epidemiologia , Cólera/microbiologia , Vibrio cholerae/isolamento & purificação , Vibrio cholerae/efeitos dos fármacos , Etiópia/epidemiologia , Prevalência , Estudos Transversais , Adulto , Feminino , Adolescente , Masculino , Antibacterianos/farmacologia , Adulto Jovem , Fezes/microbiologia , Pessoa de Meia-Idade , Testes de Sensibilidade Microbiana , Criança , Pré-EscolarRESUMO
The phytochemical investigation of the leaves of Inula confertiflora, a medicinal plant endemic to Ethiopia, led to the isolation of 15 terpenoids; 1ß-hydroxy-α-costic acid (1), 3α-hydroxycostic acid (2), isotelekin (3), asperilin (4), carabrone (5), carpesioline (6), graveolide (7), inuviscolide (8), 8-epi-inuviscolide (9), 1ß,4ß-dihydroxy-5α(H)-guaia-10(14),11(13)-dien-8α,12-olide (10), isoinuviscolide (11), 4ß,10ß-dihydroxy-5α(H)-1,11(13)-guaidien-8α,12-olide (12), 4ß,10ß-dihydroxy-1ß(H)-5α(H)-guai-11(13)-en-8α,12-olide (13), 4ß,10α-dihydroxy-1ß(H)-5α(H)-guai-11(13)-en-8α,12-olide (14), 4ß,10α-dihydroxy-1α(H)-5α(H)-guai-11(13)-en-8α,12-olide (15). Herein, structural elucidation and full NMR data for compound 1 are presented for the first time. The structures were elucidated using NMR, HRESIMS, and by comparison with literature data. The relative configurations were defined by NOESY correlations and single-crystal X-ray crystallography. Herein, crystallography data of 6 and 7 were reported for the first time. The antibacterial efficacy of some of the isolated compounds was evaluated against two commonly dispersed environmental strains of Escherichia coli and Staphylococcus aureus. Compounds 1, 3, 6, 7, and 8 exhibited moderate antibacterial activities against the tested organisms. The chemotaxonomic significance of compounds is discussed.
Assuntos
Antibacterianos , Inula , Lactonas , Testes de Sensibilidade Microbiana , Sesquiterpenos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/isolamento & purificação , Lactonas/química , Lactonas/farmacologia , Lactonas/isolamento & purificação , Inula/química , Sesquiterpenos/química , Sesquiterpenos/farmacologia , Sesquiterpenos/isolamento & purificação , Folhas de Planta/química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Estrutura Molecular , Conformação MolecularRESUMO
BACKGROUND: Globally, surgical site infections (SSI) are the most commonly reported healthcare-associated infections. METHODS: A multicentre study was conducted among patients who underwent surgical procedures at four hospitals located in Northern (Debre Tabor), Southern (Hawassa), Southwest (Jimma), and Central (Tikur Anbessa) parts of Ethiopia. A total of 752 patients clinically studied for surgical site infection were enrolled. The number of patients from Debre Tabor, Hawassa, Jimma, and Tikur Anbessa, hospitals was 172, 184, 193, and 203, respectively. At each study site, SSI discharge culture was performed from all patients, and positive cultures were characterized by colony characteristics, Gram stain, and conventional biochemical tests. Each bacterial species was confirmed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF). An antimicrobial susceptibility test (AST) was done on Mueller-Hinton agar using the disk diffusion method. Logistic regression analysis was used to assess associations of dependent and independent variables. A p-value < 0.05 was considered statistically significant. Data were analysed using STATA 16 software. RESULTS: Among 752 wound discharge cultures performed, 65.5% yielded growth. Among these, 57.9% and 42.1% were Gram-negative and Gram-positive isolates, respectively. In this study, a total of 494 bacteria were isolated; Staphylococcus aureus (31%), Escherichia coli (20.7%), and Klebsiella pneumoniae (9.8%) were the most common. Rare isolates (0.8% each) included Raoultella ornithinolytica, Stenotrophomonas maltophilia, Alcalignes faecalis, Pantoea ecurina, Bacillus flexus, and Paenibacillus tylopili. Enterobacteriaceae showed high levels of resistance to most of the tested antibiotics but lower levels of ertapenem (32.9%), amikacin (24.3%), imipenem (20.3%), and meropenem (17.6%) resistance. Multidrug-resistant (MDR) frequency of Enterobacteriaceae at Debre Tabor, Hawassa, Jimma, and Tikur Anbessa hospitals was 84.5%, 96.5%, 97.3%, and 94%, respectively. Ages ≥ 61 years (AOR = 2.83, 95% CI: 1.02-7.99; P 0.046), prolonged duration of hospital stay (AOR = 4.15, 95% CI: 2.87-6.01; P 0.000), history of previous antibiotics use (AOR = 2.83, 95% CI: 1.06-2.80; P 0.028), history of smoking (AOR = 2.35, 95% CI: 1.44-3.83; P 0.001), emergency surgery (AOR = 2.65, 95% CI: 1.92-3.66; P 0.000), and duration of operation (AOR = 0.27, 95% CI: 0.181-0.392; P 0.000) were significant risk factors. CONCLUSION: The most prevalent isolates from Gram-positive and Gram-negative bacteria across all hospitals were S. aureus and E. coli, respectively. Many newly emerging Gram-negative and Gram-positive bacteria were identified. Variation between hospitals was found for both SSI etiology type and MDR frequencies. Hence, to prevent the emergence and spread of MDR bacteria, standard bacteriological tests and their AST are indispensable for effective antimicrobial stewardship.
Assuntos
Antibacterianos , Infecção da Ferida Cirúrgica , Humanos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/tratamento farmacológico , Estudos Transversais , Staphylococcus aureus , Escherichia coli , Etiópia/epidemiologia , Estudos Prospectivos , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Bactérias , Farmacorresistência Bacteriana MúltiplaRESUMO
DNA processing chain A (DprA) is a DNA-binding protein that is ubiquitous in bacteria and expressed in some archaea. DprA is active in many bacterial species that are competent for transformation of DNA, but its role in Neisseriameningitidis (Nm) is not well characterized. An Nm mutant lacking DprA was constructed, and the phenotypes of the wild-type and ΔdprA mutant were compared. The salient feature of the phenotype of dprA null cells is the total lack of competence for genetic transformation shown by all of the donor DNA substrates tested in this study. Here, Nm wild-type and dprA null cells appeared to be equally resistant to genotoxic stress. The gene encoding DprANm was cloned and overexpressed, and the biological activities of DprANm were further investigated. DprANm binds ssDNA more strongly than dsDNA, but lacks DNA uptake sequence-specific DNA binding. DprANm dimerization and interaction with the C-terminal part of the single-stranded binding protein SSBNmwere demonstrated. dprA is co-expressed with smg, a downstream gene of unknown function, and the gene encoding topoisomerase 1, topA.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Neisseria meningitidis/metabolismo , Transformação Bacteriana , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Neisseria meningitidis/química , Neisseria meningitidis/genética , Alinhamento de SequênciaRESUMO
BACKGROUND: DNA processing chain A (DprA) is a DNA binding protein which is ubiquitous in bacteria, and is required for DNA transformation to various extents among bacterial species. However, the interaction of DprA with competence and recombination proteins is poorly understood. Therefore, the proteomes of whole Neisseria meningitidis (Nm) wildtype and dprA mutant cells were compared. Such a comparative proteomic analysis increases our understanding of the interactions of DprA with other Nm components and may elucidate its potential role beyond DNA processing in transformation. RESULTS: Using label-free quantitative proteomics, a total of 1057 unique Nm proteins were identified, out of which 100 were quantified as differentially abundant (P ≤ 0.05 and fold change ≥ |2|) in the dprA null mutant. Proteins involved in homologous recombination (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT1, PilT2, PilM, PilO, PilQ, PilF and PilE), cell division, including core energy metabolism, and response to oxidative stress were downregulated in the Nm dprA null mutant. The mass spectrometry data are available via ProteomeXchange with identifier PXD006121. Immunoblotting and co-immunoprecipitation were employed to validate the association of DprA with PilG. The analysis revealed reduced amounts of PilG in the dprA null mutant and reduced amounts of DprA in the Nm pilG null mutant. Moreover, a number of pilus biogenesis proteins were shown to interact with DprA and /or PilG. CONCLUSIONS: DprA interacts with proteins essential for Nm DNA recombination in transformation, pilus biogenesis, and other functions associated with the inner membrane. Inverse downregulation of Nm DprA and PilG expression in the corresponding mutants indicates a link between DNA processing and pilus biogenesis.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Fímbrias/genética , Proteínas de Membrana/genética , Neisseria meningitidis/genética , Proteômica/métodos , Proteínas Recombinantes/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Metabolismo Energético , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Imunoprecipitação , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Mutação , Neisseria meningitidis/metabolismo , Estresse Oxidativo , Proteínas Recombinantes/metabolismo , Recombinação Genética , Transformação BacterianaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of severe surgical site infections (SSI). The molecular epidemiology of MRSA is poorly documented in Ethiopia. This study is designed to determine the prevalence of MRSA and associated factors among patients diagnosed with SSI. A multicenter study was conducted at four hospitals in Ethiopia. A wound culture was performed among 752 SSI patients. This study isolated S. aureus and identified MRSA using standard bacteriology, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), and cefoxitin disk diffusion test. The genes mecA, femA, vanA, and vanB were detected through PCR tests. S. aureus was identified in 21.6% of participants, with 24.5% of these being methicillin-resistant Staphylococci and 0.6% showing vancomycin resistance. Using MALDI-TOF MS for the 40 methicillin-resistant Staphylococci, we confirmed that 31 (77.5%) were S. aureus, 6 (15%) were Mammaliicoccus sciuri, and the other 3 (2.5%) were Staphylococcus warneri, Staphylococcus epidermidis, and Staphylococcus haemolyticus. The gene mecA was detected from 27.5% (11/40) of Staphylococci through PCR. Only 36.4% (4/11) were detected in S. aureus, and no vanA or vanB genes were identified. Out of 11 mecA-gene-positive Staphylococci, 8 (72.7%) were detected in Debre Tabor Comprehensive Specialized Hospital. Methicillin-resistant staphylococcal infections were associated with the following risk factors: age ≥ 61 years, prolonged duration of hospital stay, and history of previous antibiotic use, p-values < 0.05. Hospitals should strengthen infection prevention and control strategies and start antimicrobial stewardship programs.
RESUMO
Novel coronavirus disease (COVID-19) is spreading rapidly and creating a huge economic, social and public health challenge worldwide. Although currently an effective vaccine is ready, its distribution is limited, and hence the only currently available lever to reduce transmission is to identify and isolate individuals who are contagious. Thus, testing for SARS CoV-2 has a paramount importance. However, testing in many African countries including Ethiopia has multidimensional growing challenges. Here, we tried to identify, categorize and summarize the challenges of COVID-19 testing in Africa from Ethiopian experience.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , África , Etiópia , HumanosRESUMO
Three complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Ethiopian patients were compared with deposited global genomes. Two genomes belonged to genetic group 20A/B.1/GH, and the other belonged to genetic group 20A/B.1.480/GH. Enhancing genomic capacity is important to investigate the transmission and to monitor the evolution and mutational patterns of SARS-CoV-2 in this country.
RESUMO
BACKGROUND: Antimicrobial resistance is one of the major public health challenges in Ethiopia. However, there is no comprehensive summary of existing AMR data in the country. AIM: To determine the prevalence of antimicrobial resistance and its clinical implications in Ethiopia. METHODS: A systematic literature search was performed on the PubMed/Medline database. Original studies on antimicrobial resistance conducted in Ethiopia between 1st January 2009 and 31st July 2019 were included. The outcome measure was the number of isolates resistant to antimicrobial agents in terms of specific pathogens, and disease condition. Data was calculated as total number of resistant isolates relative to the total number of isolates per specific pathogen and medication. RESULTS: A total of 48,021 study participants enrolled from 131 original studies were included resulting in 15,845 isolates tested for antimicrobial resistance. The most common clinical sample sources were urine (28%), ear, nose, and throat discharge collectively (27%), and blood (21%). All the studies were cross-sectional and 83% were conducted in hospital settings. Among Gram-positive bacteria, the reported level of resistance to vancomycin ranged from 8% (Enterococcus species) to 20% (S. aureus). E. coli, K. pneumoniae and P. aeruginosa were the most common Gram-negative pathogens resistant to key antimicrobial agents described in the national standard treatment guideline and were associated with diverse clinical conditions: urinary tract infections, diarrhea, surgical site infections, pneumonia, ocular infections, and middle ear infections. CONCLUSION: Overall, there is a high prevalence of antimicrobial resistance in Ethiopia. Empirical treatment of bacterial infections needs to be guided by up-to-date national guidelines considering local antimicrobial susceptibility patterns. Equipping diagnostic laboratories with culture and drug susceptibility testing facilities, and establishing a strong antimicrobial stewardship program should be high priorities.
Assuntos
Antibacterianos/farmacologia , Infecções Bacterianas/epidemiologia , Farmacorresistência Bacteriana Múltipla , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/epidemiologia , Bactérias Gram-Positivas/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/epidemiologia , Infecções Bacterianas/tratamento farmacológico , Etiópia/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Testes de Sensibilidade Microbiana , PrevalênciaRESUMO
BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has revealed the global public health importance of robust diagnostic testing. To overcome the challenge of nucleic acid (NA) extraction and testing kit availability, an efficient method is urgently needed. OBJECTIVES: To establish an efficient, time and resource-saving and cost-effective methods, and to propose an ad hoc pooling approach for mass screening of SARS-CoV-2. METHODS: We evaluated pooling approach on both direct clinical and NA samples. The standard reverse transcriptase polymerase chain reaction (RT-PCR) test of the SARS CoV-2 was employed targeting the nucleocapsid (N) and open reading frame (ORF1ab) genomic region of the virus. The experimental pools were created using SARS CoV-2 positive clinical samples and extracted RNA spiked with up to 9 negative samples. For the direct clinical samples viral NA was extracted from each pool to a final extraction volume of 200µL, and subsequently both samples tested using the SARS CoV-2 RT-PCR assay. RESULTS: We found that a single positive sample can be amplified and detected in pools of up to 7 samples depending on the cycle threshold (Ct) value of the original sample, corresponding to high, and low SARS CoV-2 viral copies per reaction. However, to minimize false negativity of the assay with pooling strategies and with unknown false negativity rate of the assay under validation, we recommend pooling of 4/5 in 1 using the standard protocols of the assay, reagents and equipment. The predictive algorithm indicated a pooling ratio of 5 in 1 was expected to retain accuracy of the test irrespective of the Ct value samples spiked, and result in a 137% increase in testing efficiency. CONCLUSIONS: The approaches showed its concept in easily customized and resource-saving manner and would allow expanding of current screening capacities and enable the expansion of detection in the community. We recommend clinical sample pooling of 4 or 5 in 1. However, we don't advise pooling of clinical samples when disease prevalence is greater than 7%; particularly when sample size is large.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Algoritmos , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/economia , Humanos , Programas de Rastreamento/economia , Programas de Rastreamento/métodos , RNA Viral/genética , SARS-CoV-2/genética , Sensibilidade e Especificidade , Manejo de Espécimes/economia , Manejo de Espécimes/métodosRESUMO
Scaling up of diagnostic capacity is needed to mitigate the global pandemic of SARS-CoV2. However, there are challenges including shortage of sample collection swabs and transport medium. Saliva has been recommended as a simple, low-cost, non-invasive option. However, data from different populations and settings are limited. Here, we showed that saliva could be a good alternative sample to diagnose COVID-19 patients. Pair of NPS-saliva samples was collected from 152 symptomatic; confirmed COVID-19 patients, and compared their positivity rate, viral load, and duration of viral shedding. From 152 patients, 80 (52.63%) tested positive and 72 (47.37%) were negative for SARSA-CoV2 in NPS sample. In saliva, 129 (92.14%) were tested positive and 11 (7.86%) were negative on the day of admission to hospital. The overall percent agreement of RT-PCR result of Saliva to NPS was 70% (196/280). A comparison of viral load from 72 NPS-saliva pair samples on day of admission shows saliva contains significantly higher viral load (P < 0.001). In conclusion, saliva has higher yield in detecting SARS-CoV2, and COVID-19 patients show higher viral load and prolonged period of viral shedding in saliva. Therefore, we recommend saliva as a better alternative sample to NPS to diagnose COVID-19 patients.
Assuntos
COVID-19/diagnóstico , Nasofaringe/virologia , SARS-CoV-2/genética , Saliva/virologia , Manejo de Espécimes/métodos , Teste de Ácido Nucleico para COVID-19 , Hospitalização , Humanos , Pandemias , RNA Viral , Carga Viral , Eliminação de Partículas ViraisRESUMO
Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5' â 3' directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5' â 3' directionality and prefers DNA substrates containing a 5'-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Neisseria meningitidis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Helicases/química , Regulação da Expressão Gênica no Desenvolvimento , Instabilidade Genômica , Mitomicina/efeitos adversos , Modelos Moleculares , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Filogenia , Estrutura Terciária de ProteínaRESUMO
Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant.
Assuntos
Clonagem Molecular/métodos , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Bacteriano/metabolismo , Neisseria meningitidis/crescimento & desenvolvimento , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Domínio Catalítico , Sequência Conservada , DNA Helicases/química , Reparo do DNA , Replicação do DNA , Modelos Moleculares , Neisseria meningitidis/enzimologia , Neisseria meningitidis/genética , Polimorfismo de Nucleotídeo Único , Recombinação Genética , Transformação BacterianaRESUMO
Expression of type IV pili (Tfp), filamentous appendages emanating from the bacterial surface, is indispensable for efficient neisserial transformation. Tfp pass through the secretin pore consisting of the membrane protein PilQ. PilG is a polytopic membrane protein, conserved in Gram-positive and Gram-negative bacteria, that is required for the biogenesis of neisserial Tfp. PilG null mutants are devoid of pili and non-competent for transformation. Here, recombinant full-length, truncated and mutated variants of meningococcal PilG were overexpressed, purified and characterized. We report that meningococcal PilG directly binds DNA in vitro, detected by both an electromobility shift analysis and a solid phase overlay assay. PilG DNA binding activity was independent of the presence of the consensus DNA uptake sequence. PilG-mediated DNA binding affinity was mapped to the N-terminus and was inactivated by mutation of residues 43 to 45. Notably, reduced meningococcal transformation of DNA in vivo was observed when PilG residues 43 to 45 were substituted by alanine in situ, defining a biologically significant DNA binding domain. N-terminal PilG also interacted with the N-terminal region of PilQ, which previously was shown to bind DNA. Collectively, these data suggest that PilG and PilQ in concert bind DNA during Tfp-mediated transformation.