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1.
Int J Nanomedicine ; 15: 199-213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021174

RESUMO

Purpose: Resistance to antibiotics is a major problem of public health. One of the alternative therapies is silver - more and more popular because of nanotechnology development and new possibilities of usage. As a component of colloid, powder, cream, bandages, etc., nanosilver is often recommended to treat the multidrug-resistant pathogens and we can observe its overuse also outside of the clinic where different physicochemical forms of silver nanoformulations (e.g. size, shape, compounds, surface area) are introduced. In this research, we described the consequences of long-term bacteria exposure to silver nanoformulations with different physicochemical properties, including changes in genome and changes of bacterial sensitivity to silver nanoformulations and/or antibiotics. Moreover, the prevalence of exogenous resistance to silver among multidrug-resistant bacteria was determined. Materials and Methods: Gram-negative and Gram-positive bacteria strains are described as sensitive and multidrug-resistant strains. The sensitivity of the tested bacterial strains to antibiotics was carried out with disc diffusion methods. The sensitivity of bacteria to silver nanoformulations and development of bacterial resistance to silver nanoformulations has been verified via determination of the minimal inhibitory concentrations. The presence of sil genes was verified via PCR reaction and DNA electrophoresis. The genomic and phenotypic changes have been verified via genome sequencing and bioinformatics analysis. Results: Bacteria after long-term exposure to silver nanoformulations may change their sensitivity to silver forms and/or antibiotics, depending on the physicochemical properties of silver nanoformulations, resulting from phenotypic or genetic changes in the bacterial cell. Finally, adaptants and mutants may become more sensitive or resistant to some antibiotics than wild types. Conclusion: Application of silver nanoformulations in the case of multiple resistance or multidrug-resistant bacterial infection can enhance or decrease their resistance to antibiotics. The usage of nanosilver in a clinic and outside of the clinic should be determined and should be under strong control. Moreover, each silver nanomaterial should be considered as a separate agent with a potential different mode of antibacterial action.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nanoestruturas/química , Prata/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Testes de Sensibilidade Microbiana , Prata/química
2.
BMC Infect Dis ; 20(1): 99, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32007106

RESUMO

BACKGROUND: The identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema. METHODS: A prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions. RESULTS: Overall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [31] followed by gram-positive cocci [10] and gram-negative rods [4]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008). CONCLUSION: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.


Assuntos
Empiema Pleural/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Derrame Pleural/microbiologia , Idoso , Idoso de 80 Anos ou mais , Bactérias Anaeróbias/genética , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/microbiologia , Estudos de Coortes , Empiema Pleural/tratamento farmacológico , Exsudatos e Transudatos/microbiologia , Feminino , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Derrame Pleural/tratamento farmacológico , Derrame Pleural/mortalidade , Estudos Prospectivos
3.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194489, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935527

RESUMO

Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain essential functions and modulate accessory functions in response to environmental cues. Over the years, it has become clear that bacterial regulation of gene expression is still far from fully understood. This review focuses on antisense RNAs (asRNAs), a class of RNA regulators defined by their location in cis and their perfect complementarity with their targets, as opposed to small RNAs (sRNAs) which act in trans with only short regions of complementarity. For a long time, only few functional asRNAs in bacteria were known and were almost exclusively found on mobile genetic elements (MGEs), thus, their importance among the other regulators was underestimated. However, the extensive application of transcriptomic approaches has revealed the ubiquity of asRNAs in bacteria. This review aims to present the landscape of studied asRNAs in bacteria by comparing 67 characterized asRNAs from both Gram-positive and Gram-negative bacteria. First we describe the inherent ambiguity in the existence of asRNAs in bacteria, second, we highlight their diversity and their involvement in all aspects of bacterial life. Finally we compare their location and potential mode of action toward their target between Gram-negative and Gram-positive bacteria and present tendencies and exceptions that could lead to a better understanding of asRNA functions.


Assuntos
Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , RNA Antissenso/fisiologia , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Sequências Repetitivas Dispersas , RNA Antissenso/metabolismo
4.
Ecotoxicol Environ Saf ; 187: 109852, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31670243

RESUMO

Microplastics have become emerging pollutants and served as potential vectors for harmful bacteria, while rare information on the emergency and propagation of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) on the surface of microplastics is available. This study investigated the enrichment of ARB, especially multi-antibiotic resistant bacteria (MARB), on the surface of microplastics in mariculture system. Polyethylene terephthalate accounted for the highest proportion (75%) in the collected microplastics. The counts of cultivable ARB in microplastic samples were 6.40 × 106-2.48 × 108 cfu/g, which were 100-5000 times higher than those in water samples. The ratios of cultivable ARB to total cultivable bacteria from microplastic samples were higher than those from water samples. High-throughput sequencing showed that the diversity and abundance of cultivable ARB in the microplastic samples was high with the predominant bacterial genera of Vibrio, Muricauda and Ruegeria. Total 160 MARB isolates were obtained and most of isolates were obtained from the microplastic samples. MARB isolates resisting or intermediating to four and three antibiotics accounted for much higher proportions in the microplastic samples, and the higher percentage of antibiotic resistance was to penicillin, sulfafurazole, erythromycin and tetracycline. The dominant multiple antibiotic resistance profile was TET-SFX-ERY-PEN, which accounted for 25.4% in microplastic samples and 23.9% in water samples. In typical MARB isolates, the positive detection rate of ARGs was up to 80.0% in microplastic samples while that was 65.3% in water samples. Five types of class 1 integrons (intI1) associated gene cassette arrays and seven types of gene cassettes were detected in microplastic samples, which were more than those in water samples. These results revealed that microplastics were hazardous pollutants for the enrichment of ARB, especially superbugs, and the spread of antibiotic resistance.


Assuntos
Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Microplásticos/toxicidade , Rhodobacteraceae/crescimento & desenvolvimento , Poluentes Químicos da Água/toxicidade , Antibacterianos/farmacologia , Bactérias Gram-Negativas/genética , Integrons/genética , Microplásticos/química , Rhodobacteraceae/genética , Propriedades de Superfície , Poluentes Químicos da Água/química
5.
BMC Genomics ; 20(1): 731, 2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31606035

RESUMO

BACKGROUND: TetR-family transcriptional regulators (TFTRs) are DNA binding factors that regulate gene expression in bacteria. Well-studied TFTRs, such as AcrR, which regulates efflux pump expression, are usually encoded alongside target operons. Recently, it has emerged that there are many TFTRs which act as global multi-target regulators. Our classical view of TFTRs as simple, single-target regulators therefore needs to be reconsidered. As some TFTRs regulate essential processes (e.g. metabolism) or processes which are important determinants of resistance and virulence (e.g. biofilm formation and efflux gene expression) and as TFTRs are present throughout pathogenic bacteria, they may be good drug discovery targets for tackling antimicrobial resistant infections. However, the prevalence and conservation of individual TFTR genes in Gram-negative species, has to our knowledge, not yet been studied. RESULTS: Here, a wide-scale search for TFTRs in available proteomes of clinically relevant pathogens Salmonella and Escherichia species was performed and these regulators further characterised. The majority of identified TFTRs are involved in efflux regulation in both Escherichia and Salmonella. The percentage variance in TFTR genes of these genera was found to be higher in those regulating genes involved in efflux, bleach survival or biofilm formation than those regulating more constrained processes. Some TFTRs were found to be present in all strains and species of these two genera, whereas others (i.e. TetR) are only present in some strains and some (i.e. RamR) are genera-specific. Two further pathogens on the WHO priority pathogen list (K. pneumoniae and P. aeruginosa) were then searched for the presence of the TFTRs conserved in Escherichia and Salmonella. CONCLUSIONS: Through bioinformatics and literature analyses, we present that TFTRs are a varied and heterogeneous family of proteins required for the regulation of numerous important processes, with consequences to antimicrobial resistance and virulence, and that the roles and responses of these proteins are frequently underestimated.


Assuntos
Biologia Computacional/métodos , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/classificação , Fatores de Transcrição/genética , Proteínas de Bactérias/genética , Sequência Conservada , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/genética , Klebsiella pneumoniae/genética , Funções Verossimilhança , Família Multigênica , Filogenia , Salmonella typhimurium/genética
6.
PLoS Negl Trop Dis ; 13(9): e0007729, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568511

RESUMO

INTRODUCTION: The prevalence of bacteremia caused by Gram negative non-fermentative (GNNF) bacteria has been increasing globally over the past decade. Many studies have investigated their epidemiology but focus on the common GNNF including Pseudomonas aeruginosa and Acinetobacter baumannii. Knowledge of the uncommon GNNF bacteremias is very limited. This study explores invasive bloodstream infection GNNF isolates that were initially unidentified after testing with standard microbiological techniques. All isolations were made during laboratory-based surveillance activities in two rural provinces of Thailand between 2006 and 2014. METHODS: A subset of GNNF clinical isolates (204/947), not identified by standard manual biochemical methodologies were run on the BD Phoenix automated identification and susceptibility testing system. If an organism was not identified (12/204) DNA was extracted for whole genome sequencing (WGS) on a MiSeq platform and data analysis performed using 3 web-based platforms: Taxonomer, CGE KmerFinder and One Codex. RESULTS: The BD Phoenix automated identification system recognized 92% (187/204) of the GNNF isolates, and because of their taxonomic complexity and high phenotypic similarity 37% (69/187) were only identified to the genus level. Five isolates grew too slowly for identification. Antimicrobial sensitivity (AST) data was not obtained for 93/187 (50%) identified isolates either because of their slow growth or their taxa were not in the AST database associated with the instrument. WGS identified the 12 remaining unknowns, four to genus level only. CONCLUSION: The GNNF bacteria are of increasing concern in the clinical setting, and our inability to identify these organisms and determine their AST profiles will impede treatment. Databases for automated identification systems and sequencing annotation need to be improved so that opportunistic organisms are better covered.


Assuntos
Bacteriemia/microbiologia , Técnicas de Tipagem Bacteriana/métodos , Bactérias Gram-Negativas/isolamento & purificação , DNA Bacteriano/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Tailândia , Sequenciamento Completo do Genoma/métodos
7.
Biomed Res Int ; 2019: 2165316, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534954

RESUMO

The aim of this study was to determine the species distribution of Staphylococcus, Gram negative bacteria (GNB) and the occurrence of Methicillin Resistant Staphylococci (MRS) and Extended-Spectrum ß-lactamase- (ESBL-) producing GNB. Bacterial culture of 300 clinical mastitis milk samples from 30 different farms across different regions of Tunisia during four seasons was realized. The obtained results showed the presence of high frequency of the tested samples with a positive growth for bacteria (64%). In addition a high recovery rate of Staphylococci and/or GNB in these clinical mastitis milk samples (87%) was detected. In addition, a high percentage of GNB (68.2%) compared to Staphylococcus species (32%) was noted. Moreover, a significant variation of the number of these bacteria according to the farm location, the seasons, and cows age was detected. The highest percentage was observed in the North of Tunisia during the winter and the spring seasons in adult cows with a dominance of GNB growth. Coagulase negative Staphylococci (CNS) (n=11) and GNB (n=16) species were identified. Escherichia coli (E. coli) was the most frequently found bacterium followed by Klebsiella pneumoniae. The dominant Staphylococcus isolates was S. xylosus followed by S. aureus the major pathogen isolated. Methicillin resistance was confirmed by the presence of the mecA gene in 3 S. aureus and 14 CNS isolates; all of these isolates were lacking the mecC gene. Various species of GNB, resistant to cefotaxime, were detected (n=15). ESBLs were detected on selective medium in 10 E. coli and 4 K. pneumoniae. All ESBL producers strains carry the blaCTX-M. The presence of different resistant mastitis pathogens in dairy farms may complicate therapeutic options and contaminated animals could become zoonotic agent reservoir for human.


Assuntos
Proteínas de Bactérias/genética , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas , Mastite Bovina , Infecções Estafilocócicas , Staphylococcus , beta-Lactamases/genética , Animais , Bovinos , Feminino , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/enzimologia , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/genética , Mastite Bovina/enzimologia , Mastite Bovina/epidemiologia , Mastite Bovina/genética , Mastite Bovina/microbiologia , Infecções Estafilocócicas/enzimologia , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/genética , Staphylococcus/enzimologia , Staphylococcus/genética , Tunísia/epidemiologia
8.
J Ayub Med Coll Abbottabad ; 31(3): 299-307, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31535495

RESUMO

Background: Metallo-beta-lactamases (MBL) catalyze the hydrolysis of beta-lactam antibiotics including carbapenems. A novel MBL subtype, New Delhi MBL (NDM), poses a serious public health problem. The aims of this study were to determine the frequency of NDM producers among the Carbapenem-resistant gram-negative bacilli (GNB) in hospitalized patients and carrying out the molecular analysis of the NDM genes as reliable data on this is not available in Pakistan. Methods: We carried out a cross-sectional study on prospectively collected clinical samples from 113 patients hospitalised at Shaikh Zayed Hospital Lahore, Pakistan. All the samples that were carbapenem-resistant on routine sensitivity testing were selected for this study. Various microbiological and genotypic analyses of the samples were performed. Results: The mean age of the patients was 47.8±20.8 years. About a quarter (25.7%) of the samples was from the urology ward and 43% were urine samples. Around two-third of the samples (n=74, 65.5%) tested positive for Non-Enterobacteriaceae GNB. Pseudomonas spp was the most common isolate among the Non-Enterobacteriaceae and E-coli amongst the Enterobacteriaceae. NDM gene was detected in 22 patients (19.5%). We did not find any association of the NDM gene with the demographic and clinical characteristics.. Conclusion: NDMpositive GNB are present in our hospitalized patients, which is worrisome as these bacteria can disseminate globally and lead to an extensive and uncontrollable spread of pandemic clones for which efficient antibiotic therapy is currently not available. Systemic surveillance network and infection control strategies should be established to curtail dissemination of NDM-producing GNB in Pakistan.


Assuntos
Carbapenêmicos/farmacologia , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas/microbiologia , Resistência beta-Lactâmica/genética , beta-Lactamases , Adulto , Idoso , Antibacterianos/farmacologia , Estudos Transversais , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Tipagem Molecular , Paquistão , Centros de Atenção Terciária
9.
Clin Lab ; 65(8)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31414749

RESUMO

BACKGROUND: The use of carbapenem antibiotics in the treatment of serious Gram-negative bacterial infections is under threat due to the emergence of the blaIMP gene amongst the bacterial pathogens. METHODS: The present descriptive cross-sectional study aimed to determine the occurrence of the blaIMP gene and minimum inhibitory concentrations (MICs) of the bacterial pathogens. The carbapenem-resistant isolates were screened out for the detection of MBLs by using the modified Hodge test and disk potentiation method. MBL producing strains were tested for the presence of the blaIMP gene by using PCR technique. The MICs of the blaIMP gene positive bacterial isolates were detected on the Vitek 2 system (bioMerieux). RESULTS: The primary source of MBLs and blaIPM gene carrying bacterial pathogens was blood (38.5%). We isolated 104 bacterial isolates in the initial screening of carbapenem resistance. Metallo-beta-lactamases (MBLs) were detected in 76 (73%) of the isolates which predominantly included 27 (26%) Klebsiella pneumoniae, 20 (19.2%) Acinetobacter baumannii, 16 (15.4%) Pseudomonas aeruginosa, and 11 (10.6%) E. coli while the other Gram-nega-tive MBL producing bacteria were few in number. The blaIPM gene was detected in 1 (1.3%) case of Acinetobacter baumannii and 1 (1.3%) case of Stenotrophomonas maltophilia. These strains were found to be multi-drug resistant with high MICs (≥ 8 to ≥ 256 µg/mL) against the majority of the drugs. CONCLUSIONS: The emergence of the blaIPM gene is a matter of serious concern as it left us with limited treatment options of minocycline, tigecycline, and levofloxacin. The horizontal transfer of blaIPM gene in other Gram-negative isolates can lead the epidemics of multidrug resistance.


Assuntos
Proteínas de Bactérias/genética , Carbapenêmicos/farmacologia , Genes Bacterianos/genética , Bactérias Gram-Negativas/efeitos dos fármacos , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Proteínas de Bactérias/metabolismo , Estudos Transversais , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , beta-Lactamases/metabolismo
10.
Eur J Clin Microbiol Infect Dis ; 38(11): 2029-2036, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31385145

RESUMO

Carbapenemase-producing microorganisms are increasingly isolated and often associated with treatment failures and outbreaks. The need for reliable and timely detection and/or confirmation of carbapenemase production is paramount; therefore, a real-time PCR assay targeting IMP, NDM, VIM, KPC and OXA-48-like carbapenemases was designed and validated. All available allele variants of the above carbapenemases were downloaded from the Beta-Lactamase DataBase ( http://bldb.eu/ ), aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized for the QuantStudio 6-Flex (Applied Biosystems) using the PowerUp SYBR Green Master Mix (Life Technologies) and validated using a panel of 204 characterised strains carrying a wide range of beta-lactamases, sometimes in combination. Melt-curve analysis was used to confirm positive results. The in silico approach allowed primers to be designed in conserved regions of the KPC and NDM alignments, while three primer sets for IMP and two for VIM were necessary to ensure amplification of the different variants. One primer set was designed for OXA-48-like; however, it is unlikely to detect all variants. Expected results were obtained for all 204 tested strains, with 100% sensitivity and specificity. Melt-curve analysis showed consistent Tm results for KPC, NDM, and OXA-48-like; differences were instead noted for IMP and VIM as likely consequence of higher variability in the PCR target regions. Inhibition was not observed. The assay is rapid, easy to perform and implement. It enables unequivocal detection of IMP, NDM, VIM, KPC and OXA-48-like carbapenemases even when more than one type is present simultaneously.


Assuntos
Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , beta-Lactamases/genética , Testes Diagnósticos de Rotina , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/diagnóstico , Infecções por Enterobacteriaceae/microbiologia , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Sensibilidade e Especificidade
11.
J Med Microbiol ; 68(10): 1431-1437, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385779

RESUMO

Purpose. Rapid and accurate detection of carbapenem resistance is a critical requirement for the selection of appropriate therapy and initiation of infection control measures. Although several tests are available, their use is limited by one or more factors. Phenotypic tests are lengthy, have variable sensitivity and specificity and do not generally identify the carbapenemase. Molecular assays overcome many of these issues but cost can be a barrier to adoption, particularly in low-resource settings. To address the need for affordable, molecular tools, we have assessed the performance characteristics of loop-mediated isothermal amplification (LAMP)-based assays for the major carbapenemase genes, blaNDM, blaKPC, blaOXA-48, blaOXA-23 blaVIM and blaIMP.Methodology. The assays were validated using 1849 Gram-negative Indian clinical isolates obtained from seven hospitals and diagnostic centres.Results. The assays had diagnostic sensitivities of 98.14 %, 98.92 %, 100 %, 98.48 %, and diagnostic specificities of 98.94 %, 99.61 %, 97.42 %, 99.38 % for blaNDM, blaOXA-48, blaOXA-23 and blaVIM, respectively. Due to a low number of isolates positive for blaKPC and blaIMP, the performance characteristics of assays for these two genes could not be suitably evaluated.Conclusion. The performance characteristics suggest suitability for diagnostic and surveillance purposes.


Assuntos
Proteínas de Bactérias/genética , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , beta-Lactamases/genética , Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/classificação , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/diagnóstico , Humanos , beta-Lactamases/metabolismo
12.
BMC Res Notes ; 12(1): 464, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362783

RESUMO

OBJECTIVE: The aim of this study was to determine the predominant bacterial species causing bacteremia among febrile cancer patients, and their antibacterial resistance profiles at the Uganda Cancer Institute. RESULTS: We enrolled in-patients with a documented fever (≥ 37.5 °C). Bacteria from positive blood cultures were identified using standard methods biochemically. Antibacterial susceptibility testing was performed with the Kirby-Bauer disc diffusion method. From a total of 170 febrile episodes, positive blood cultures were obtained from 24 (14.1%). A positive culture was more likely to be obtained from a patient with neutropenia (P = 0.017). Of 22 (66.7%) Gram-negative bacteria isolated, half were E. coli (n = 11). Gram-negative compared to Gram-positive bacteria were most likely to be isolated from patients with a hematologic malignancy (P = 0.02) or patients with neutropenia (P = 0.006). Of the isolated Enterobacteriaceae 85% (n = 20) were resistant to three or more classes of antibiotic and 41% (n = 7) had extended spectrum beta-lactamases. Of the 11 Gram-positive bacteria isolated, the S. aureus isolate was methicillin resistant but susceptible to vancomycin. Multidrug resistant Gram-negative bacteria are the main cause of bacteremia in febrile cancer patients at the Uganda Cancer Institute. There is need for ongoing microbial surveillance, infection prevention and control, and antibiotic stewardship programs.


Assuntos
Bacteriemia/microbiologia , Febre/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Positivas/microbiologia , Neoplasias/microbiologia , Neutropenia/microbiologia , Adulto , Antibacterianos/uso terapêutico , Bacteriemia/complicações , Bacteriemia/tratamento farmacológico , Bacteriemia/patologia , Hemocultura , Criança , Estudos Transversais , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Febre/complicações , Febre/tratamento farmacológico , Febre/patologia , Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , Infecções por Bactérias Gram-Negativas/complicações , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/patologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/complicações , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/patologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Neoplasias/complicações , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neutropenia/complicações , Neutropenia/tratamento farmacológico , Neutropenia/patologia , Uganda , beta-Lactamases/genética , beta-Lactamases/metabolismo
13.
BMC Infect Dis ; 19(1): 609, 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296179

RESUMO

BACKGROUND: Bloodstream infections (BSI) are associated with high morbidity and mortality. This scenario worsens with the emergence of drug-resistant pathogens, resulting in infections which are difficult to treat or even untreatable with conventional antimicrobials. The aim of this study is to describe the epidemiological aspects of BSI caused by multiresistant gram-negative bacilli (MDR-GNB). METHODS: We conducted a laboratory-based surveillance for gram-negative bacteremia over a 1-year period. The bacterial isolates were identified by MALDI-TOF/MS and the antimicrobial susceptibility testing was performed by VITEK®2. Resistance genes were identified through PCR assays. RESULTS: Of the 143 patients, 28.7% had infections caused by MDR-GNB. The risk factors for MDR bacteremia were male sex, age ≥ 60, previous antimicrobial use, liver disease and bacteremia caused by K. pneumoniae. K. pneumoniae was the most frequently observed causative agent and had the highest resistance level. Regarding the resistance determinants, SHV, TEM, OXA-1-like and CTX-M-gp1 were predominant enzymatic variants, whereas CTX-M-gp9, CTX-M-gp2, KPC, VIM, GES, OXA-48-like, NDM and OXA-23-like were considered emerging enzymes. CONCLUSIONS: Here we demonstrate that clinically relevant antibiotic resistance genes are prevalent in this setting. We hope our findings support the development of intervention measures by policy makers and healthcare professionals to face antibiotic resistance.


Assuntos
Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Adolescente , Adulto , Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Brasil/epidemiologia , Criança , Pré-Escolar , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Feminino , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Infecções por Klebsiella/microbiologia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Prevalência
14.
J Dermatol ; 46(9): 787-790, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31290561

RESUMO

The skin microbiome plays important roles in the pathogenesis and development of acne. We aimed to investigate the facial skin microbiome of acne and microbiome differences related to different grades of acne. Skin swabs from nine healthy controls and 67 acne patients were collected, and the skin microbiomes were analyzed using 16S rRNA gene sequencing. Compared with healthy controls, acne patients harbored significantly altered skin microbiomes. The skin microbiomes of patients with grade 1-3 acne were similar, but patients with grade 4 acne showed a significantly different skin microbiome compared with grade 1-3 acne, including increased alpha diversity and increased proportions of four Gram-negative bacteria (Faecalibacterium, Klebsiella, Odoribacter and Bacteroides). In conclusion, acne patients harbored an altered skin microbiome, and more significant dysbiosis was found in patients with grade 4 acne (severe acne). Our findings may provide evidence for the pathogenic mechanisms of acne and microbial-based strategies to avoid and treat acne, especially grade 4 acne.


Assuntos
Acne Vulgar/diagnóstico , Disbiose/diagnóstico , Microbiota/genética , Índice de Gravidade de Doença , Pele/microbiologia , Acne Vulgar/microbiologia , DNA Bacteriano/isolamento & purificação , Disbiose/microbiologia , Feminino , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Masculino , RNA Ribossômico 16S/genética , Adulto Jovem
15.
Forensic Sci Int Genet ; 42: 103-112, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31302459

RESUMO

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40-80 °C in wet conditions; microbial decomposition: 1-5 days in humid soil; and ultraviolet (UV) irradiation: 0.01-1 J/cm2). We compared saliva markers' detectability from the degraded samples-oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1-5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose-dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2-5 days) and UV (0.1-1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.


Assuntos
DNA Bacteriano/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Saliva/microbiologia , Adulto , Biomarcadores/metabolismo , Impressões Digitais de DNA , DNA Mitocondrial/genética , Exposição Ambiental/efeitos adversos , Feminino , Temperatura Alta/efeitos adversos , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Saliva/metabolismo , Raios Ultravioleta/efeitos adversos , Adulto Jovem , alfa-Amilases/metabolismo
16.
RNA ; 25(9): 1091-1097, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31186369

RESUMO

We previously reported a large collection of structured noncoding RNAs (ncRNAs) that includes many riboswitch candidates identified through comparative sequence analysis of bacterial intergenic regions. One of these candidates, initially named the "folE motif," adopts a simple architecture commonly found upstream of folE genes. FolE enzymes catalyze the first enzyme in the de novo folate biosynthesis pathway. Herein, we demonstrate that folE motif RNAs selectively bind the enzyme cofactor tetrahydrofolate (THF) and several of its close derivatives. These aptamers, commonly found in Gram-negative bacteria, are distinct from aptamers of the previous validated THF riboswitch class found in Gram-positive bacteria. Our findings indicate that folE motif RNAs are aptamer domains for a second THF riboswitch class, named THF-II. The biochemical validation of THF-II riboswitches further highlights the ability of bacteria to utilize diverse RNA structures to sense universal enzyme cofactors that are predicted to be of ancient origin.


Assuntos
Bactérias Gram-Negativas/genética , RNA não Traduzido/metabolismo , Tetra-Hidrofolatos/metabolismo , Regulação Bacteriana da Expressão Gênica , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA não Traduzido/química , Riboswitch
17.
Artigo em Inglês | MEDLINE | ID: mdl-31157176

RESUMO

Background: Rapid screening of patients for colonization with carbapenemase-producing organisms (CPO), coupled with implementation of infection prevention strategies, has the potential to contain the spread of CPO. Methods: We first evaluated the performance of Xpert Carba-R assay (in comparison with other phenotypic methods) for carbapenemase detection using clinical isolates, and then used it to determine the intestinal CPO colonization in hospitalized patients. We then assessed the effectiveness of patient isolation in controlling the spread of CPO in a medical intensive care unit. Results: The Xpert Carba-R assay required the least processing time to reveal results and showed a 94.5% sensitivity and specificity in carbapenemase detection, except for IMP-8 (n = 4). During a 6-month study period, 134 patients in one ward were studied for CPO colonization and infection. Fifteen patients (11.2%) were colonized by CPO as detected by Xpert Carba-R assay, including three NDM, three IMP, and nine KPC possessing strains. The overall colonization and CPO infection rates were both 11.2% each. Isolation of patients with CPO led to a reduction in both colonization (from 28.6 to 5.6%) and infection rates (from 35.7 to 2.8%) during the study period (p < 0.05). Conclusion: Active surveillance of CPO utilizing the Xpert Carba-R assay supplemented with immediate patient isolation, proved to be an effective strategy to limit the spread of CPO in a health care setting.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas/métodos , Isolamento de Pacientes , Conduta Expectante/métodos , beta-Lactamases/isolamento & purificação , beta-Lactamases/metabolismo , Proteínas de Bactérias/genética , Assistência à Saúde , Testes Diagnósticos de Rotina , Feminino , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e Especificidade , beta-Lactamases/genética
18.
Future Microbiol ; 14: 691-704, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31148474

RESUMO

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.


Assuntos
Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética , beta-Lactamases/biossíntese , beta-Lactamases/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Criança , DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Feminino , Perfilação da Expressão Gênica , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/isolamento & purificação , Infecções por Bactérias Gram-Negativas/epidemiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Masculino , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Paquistão/epidemiologia , Plasmídeos , Análise de Sequência de DNA
19.
mBio ; 10(3)2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31213552

RESUMO

Although distinct lipid phosphatases are thought to be required for processing lipid A (component of the outer leaflet of the outer membrane), glycerophospholipid (component of the inner membrane and the inner leaflet of the outer membrane), and undecaprenyl pyrophosphate (C55-PP; precursors of peptidoglycan and O antigens of lipopolysaccharide) in Gram-negative bacteria, we report that the lipid A 1-phosphatases, LpxEs, functionally connect multiple layers of cell envelope biogenesis in Gram-negative bacteria. We found that Aquifex aeolicus LpxE structurally resembles YodM in Bacillus subtilis, a phosphatase for phosphatidylglycerol phosphate (PGP) with a weak in vitro activity on C55-PP, and rescues Escherichia coli deficient in PGP and C55-PP phosphatase activities; deletion of lpxE in Francisella novicida reduces the MIC value of bacitracin, indicating a significant contribution of LpxE to the native bacterial C55-PP phosphatase activity. Suppression of plasmid-borne lpxE in F. novicida deficient in chromosomally encoded C55-PP phosphatase activities results in cell enlargement, loss of O-antigen repeats of lipopolysaccharide, and ultimately cell death. These discoveries implicate LpxE as the first example of a multifunctional regulatory enzyme that orchestrates lipid A modification, O-antigen production, and peptidoglycan biogenesis to remodel multiple layers of the Gram-negative bacterial envelope.IMPORTANCE Dephosphorylation of the lipid A 1-phosphate by LpxE in Gram-negative bacteria plays important roles in antibiotic resistance, bacterial virulence, and modulation of the host immune system. Our results demonstrate that in addition to removing the 1-phosphate from lipid A, LpxEs also dephosphorylate undecaprenyl pyrophosphate, an important metabolite for the synthesis of the essential envelope components, peptidoglycan and O-antigen. Therefore, LpxEs participate in multiple layers of biogenesis of the Gram-negative bacterial envelope and increase antibiotic resistance. This discovery marks an important step toward understanding the regulation and biogenesis of the Gram-negative bacterial envelope.


Assuntos
Proteínas de Bactérias/metabolismo , Bactérias Gram-Negativas/enzimologia , Lipídeo A/metabolismo , Proteínas de Membrana/metabolismo , Biogênese de Organelas , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Bactérias Gram-Negativas/genética , Lipídeo A/genética , Proteínas de Membrana/genética , Antígenos O/genética , Antígenos O/metabolismo , Peptidoglicano/genética , Peptidoglicano/metabolismo , Monoéster Fosfórico Hidrolases/genética , Fosfatos de Poli-Isoprenil/metabolismo , Homologia de Sequência de Aminoácidos
20.
Biomed Res Int ; 2019: 8219748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214618

RESUMO

Background: The prevalence of a variety of carbapenemases in Gram-negative bacteria (GNB) has posed a global threat on clinical control and management. Monitoring and controlling the carbapenemase-producing GNB became imperative tasks for many healthcare centers. The aim of this study was to develop a high-throughput, specific, sensitive, and rapid DNA microarray-based method for the diagnosis, phenotypic confirmation, and molecular epidemiological study of carbapenemase genes. Methods: We targeted a panel of eight carbapenemase genes, including bla KPC, bla NDM-1, bla OXA-23, bla OXA-48, bla OXA-51, bla IMP, bla VIM, and bla DIM for detection. Ultrasensitive chemiluminescence (CL) detection method was developed and used to simultaneously detect eight carbapenemase genes, and plasmids were established as positive or limit of detection (LOD) reference materials. Antibiotic susceptibility was determined by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines in order to screen clinical isolates resistant to carbapenem antibiotics as well as Sanger sequencing which was used to confirm the reliability of the results presented by DNA microarray. Results: Eight carbapenemase genes could be detected with high sensitivity and specificity. The absolute LOD of this strategy to detect serially diluted plasmids of eight carbapenemase genes was 102- 103copies/µL. Then, 416 specimens collected from hospital were detected and the results showed 96.6% concordance between the phenotypic and microarray tests. Compared with Sanger sequencing, a specificity and sensitivity of 100% were recorded for bla NDM-1, bla IMP, bla VIM, and bla DIM genes. The specificity for bla KPC, bla OXA-23, bla OXA-48, and bla OXA-51 genes was 100% and the sensitivity was 98.5%, 97.6%, 95.7%, and 97.9%, respectively. The overall consistency rate between the sequencing and microarray is 97.8%. Conclusions: The proposed ultrasensitive CL imaging DNA hybridization has high specificity, sensitivity, and reproducibility and could detect and differentiate clinical specimens that carried various carbapenemase genes, suggesting that the method can conveniently be customized for high-throughput detection of the carbapenemase-producing GNB and can be easily adapted for various clinical applications.


Assuntos
Proteínas de Bactérias/genética , DNA Bacteriano/genética , Bactérias Gram-Negativas , Análise de Sequência com Séries de Oligonucleotídeos , beta-Lactamases/genética , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Negativas/genética
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