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1.
JBI Evid Synth ; 2021 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-34698707

RESUMO

OBJECTIVE: This review will systematically examine and synthesize the evidence evaluating the effectiveness and safety of interventions that enable non-allergy specialist health care workers to assess allergy risk in patients with reported penicillin allergies and subsequently remove erroneous allergy records. INTRODUCTION: The potential benefits of removing erroneous penicillin allergy labels (de-labeling) are wide-ranging. Penicillin allergy assessment and de-labeling is an antibiotic stewardship priority. Delivery of such assessment and de-labeling by non-allergy specialists has been reported in several studies, but the effectiveness and safety have not been formally synthesized. This is a necessary step in the upscaling of penicillin allergy assessment services. INCLUSION CRITERIA: This review will consider quantitative studies using appropriate designs. The studies will include adults and pediatric patients who have undergone penicillin allergy assessment and de-labeling by non-allergy specialists in any health care setting. METHODS: A range of databases will be searched to identify studies published in English, with no date limit. Unpublished studies and gray literature will also be searched. Title and abstract screening, and assessment of selected full texts against the inclusion criteria will be conducted by at least two independent reviewers. Identified studies will be assessed for methodological quality using standardized critical appraisal instruments. Data will be extracted and categorized using the EPOC taxonomy, and the effectiveness and safety of the intervention will be determined. Where possible, data will be pooled to facilitate meta-analysis. Data from heterogeneous studies will be reported narratively. The GRADE approach for grading the certainty of evidence will be followed. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020219044.

3.
Theranostics ; 11(10): 4910-4928, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754035

RESUMO

Antimicrobial resistance has been a global health challenge that threatens our ability to control and treat life-threatening bacterial infections. Despite ongoing efforts to identify new drugs or alternatives to antibiotics, no new classes of antibiotic or their alternatives have been clinically approved in the last three decades. A combination of antibiotics and non-antibiotic compounds that could inhibit bacterial resistance determinants or enhance antibiotic activity offers a sustainable and effective strategy to confront multidrug-resistant bacteria. In this review, we provide a brief overview of the co-evolution of antibiotic discovery and the development of bacterial resistance. We summarize drug-drug interactions and uncover the art of repurposing non-antibiotic drugs as potential antibiotic adjuvants, including discussing classification and mechanisms of action, as well as reporting novel screening platforms. A pathogen-by-pathogen approach is then proposed to highlight the critical value of drug repurposing and its therapeutic potential. Finally, general advantages, challenges and development trends of drug combination strategy are discussed.


Assuntos
Adjuvantes Farmacêuticos/uso terapêutico , Antibacterianos/uso terapêutico , Infecções Bacterianas/tratamento farmacológico , Reposicionamento de Medicamentos , Farmacorresistência Bacteriana Múltipla/fisiologia , Sinergismo Farmacológico , Interações Medicamentosas , Farmacorresistência Bacteriana , Quimioterapia Combinada , Humanos
4.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530493

RESUMO

The impact of the Gram-negative bacterium Escherichia coli (E. coli) on the microbiomic and pathogenic phenomena occurring in humans and other warm-blooded animals is relatively well-recognized. At the same time, there are scant data concerning the role of E. coli strains in the health and disease of cold-blooded animals. It is presently known that reptiles are common asymptomatic carriers of another human pathogen, Salmonella, which, when transferred to humans, may cause a disease referred to as reptile-associated salmonellosis (RAS). We therefore hypothesized that reptiles may also be carriers of specific E. coli strains (reptilian Escherichia coli, RepEC) which may differ in their genetic composition from the human uropathogenic strain (UPEC) and avian pathogenic E. coli (APEC). Therefore, we isolated RepECs (n = 24) from reptile feces and compared isolated strains' pathogenic potentials and phylogenic relations with the aforementioned UPEC (n = 24) and APEC (n = 24) strains. To this end, we conducted an array of molecular analyses, including determination of the phylogenetic groups of E. coli, virulence genotyping, Pulsed-Field Gel Electrophoresis-Restriction Analysis (RA-PFGE) and genetic population structure analysis using Multi-Locus Sequence Typing (MLST). The majority of the tested RepEC strains belonged to nonpathogenic phylogroups, with an important exception of one strain, which belonged to the pathogenic group B2, typical of extraintestinal pathogenic E. coli. This strain was part of the globally disseminated ST131 lineage. Unlike RepEC strains and in line with previous studies, a high percentage of UPEC strains belonged to the phylogroup B2, and the percentage distribution of phylogroups among the tested APEC strains was relatively homogenous, with most coming from the following nonpathogenic groups: C, A and B1. The RA-PFGE displayed a high genetic diversity among all the tested E. coli groups. In the case of RepEC strains, the frequency of occurrence of virulence genes (VGs) was lower than in the UPEC and APEC strains. The presented study is one of the first attempting to compare the phylogenetic structures of E. coli populations isolated from three groups of vertebrates: reptiles, birds and mammals (humans).


Assuntos
Doenças dos Animais/microbiologia , Infecções por Escherichia coli/veterinária , Filogenia , Répteis/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/genética , Animais , Proteínas de Escherichia coli/genética , Especificidade de Hospedeiro , Humanos , Tipagem de Sequências Multilocus , Doenças das Aves Domésticas/microbiologia , Virulência/genética , Fatores de Virulência/genética
5.
Pathog Dis ; 79(2)2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33503238

RESUMO

Galleria mellonella is a recognised model to study antimicrobial efficacy; however, standardisation across the scientific field and investigations of methodological components are needed. Here, we investigate the impact of weight on mortality following infection with Methicillin-resistant Staphylococcus aureus (MRSA). Larvae were separated into six weight groups (180-300 mg at 20 mg intervals) and infected with a range of doses of MRSA to determine the 50% lethal dose (LD50), and the 'lipid weight' of larvae post-infection was quantified. A model of LD50 values correlated with weight was developed. The LD50 values, as estimated by our model, were further tested in vivo to prove our model. We establish a weight-dependent LD50 in larvae against MRSA and demonstrate that G. mellonella is a stable model within 180-260 mg. We present multiple linear models correlating weight with: LD50, lipid weight, and larval length. We demonstrate that the lipid weight is reduced as a result of MRSA infection, identifying a potentially new measure in which to understand the immune response. Finally, we demonstrate that larval length can be a reasonable proxy for weight. Refining the methodologies in which to handle and design experiments involving G. mellonella, we can improve the reliability of this powerful model.

6.
iScience ; 23(8): 101423, 2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32795916

RESUMO

Bacteriocins are a distinct family of antimicrobial proteins postulated to porate bacterial membranes. However, direct experimental evidence of pore formation by these proteins is lacking. Here we report a multi-mode poration mechanism induced by four-helix bacteriocins, epidermicin NI01 and aureocin A53. Using a combination of crystallography, spectroscopy, bioassays, and nanoscale imaging, we established that individual two-helix segments of epidermicin retain antibacterial activity but each of these segments adopts a particular poration mode. In the intact protein these segments act synergistically to balance out antibacterial and hemolytic activities. The study sets a precedent of multi-mode membrane disruption advancing the current understanding of structure-activity relationships in pore-forming proteins.

8.
J Med Microbiol ; 69(4): 605-616, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32125268

RESUMO

Introduction. Against the backdrop of increasing resistance to conventional antibiotics, bacteriocins represent an attractive alternative, given their potent activity, novel modes of action and perceived lack of issues with resistance.Aim. In this study, the nature of the antibacterial activity of a clinical isolate of Streptococcus gallolyticus was investigated.Methods. Optimization of the production of an inhibitor from strain AB39 was performed using different broth media and supplements. Purification was carried out using size exclusion, ion exchange and HPLC. Gel diffusion agar overlay, MS/MS, de novo peptide sequencing and genome mining were used in a proteogenomics approach to facilitate identification of the genetic basis for production of the inhibitor.Results. Strain AB39 was identified as representing Streptococcus gallolyticus subsp. pasteurianus and the successful production and purification of the AB39 peptide, named nisin P, with a mass of 3133.78 Da, was achieved using BHI broth with 10 % serum. Nisin P showed antibacterial activity towards clinical isolates of drug-resistant bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and penicillin-resistant Streptococcus pneumoniae. In addition, the peptide exhibited significant stability towards high temperature, wide pH and certain proteolytic enzymes and displayed very low toxicity towards sheep red blood cells and Vero cells.Conclusion. To the best of our knowledge, this study represents the first production, purification and characterization of nisin P. Further study of nisin P may reveal its potential for treating or preventing infections caused by antibiotic-resistant Gram-positive bacteria, or those evading vaccination regimens.


Assuntos
Nisina/isolamento & purificação , Nisina/farmacologia , Streptococcus gallolyticus/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia , Cromatografia Líquida de Alta Pressão , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Estrutura Molecular , Nisina/química , Nisina/metabolismo , Ovinos , Streptococcus gallolyticus/química , Streptococcus gallolyticus/classificação , Streptococcus gallolyticus/genética , Espectrometria de Massas em Tandem
9.
Microbiol Resour Announc ; 8(18)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048392

RESUMO

We report the complete genome sequence of a colistin-resistant strain of uropathogenic Escherichia coli, isolated in January 2013 at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. The isolate (named SA186) was sequence type 131 (ST131) and belonged to serotype O25b-H4 and clade B (fimH22).

10.
J Med Microbiol ; 68(2): 188-196, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30561295

RESUMO

PURPOSE: Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence potential and are also highly virulent in insect infection models. However, strains of this lineage are reported in relatively low numbers in many studies. ST127 strains are also usually widely susceptible to antibiotics and, consequently, their true prevalence may be under-recognized as they will be eradicated during empirical therapy. A genuine concern is the possibility that members of this highly virulent lineage will acquire resistance, leading to a more serious threat. The aim of this study was to design and validate a PCR assay specific to ST127. METHODOLOGY: Genomic sequences obtained from various UPEC isolates from the leading clones were used in comparative genomic analyses to allow identification of highly discriminatory sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the upaG (autotransporter adhesin) gene were identified as meeting our criteria and were used to develop a multiplex PCR assay. A total of 143 UPEC isolates representing 99 different MLST clones from three locations (North West and South West England and Riyadh, Saudi Arabia) were used to validate the PCR assay. RESULTS: The multiplex PCR readily identified all 29 E. coli ST127 isolates but, equally importantly, produced no false positives with representatives of any of the other 98 STs tested. CONCLUSION: We report the design and validation of a specific multiplex PCR for the rapid and reliable identification of ST127, which can be used for enhanced surveillance for this high-risk clone.


Assuntos
Escherichia coli Extraintestinal Patogênica/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/patogenicidade , Flagelina/genética , Genômica , Humanos , Anotação de Sequência Molecular , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Sensibilidade e Especificidade , Fatores de Tempo , Infecções Urinárias/microbiologia , Urina/microbiologia , Virulência , Sequenciamento Completo do Genoma
11.
BMC Microbiol ; 17(1): 23, 2017 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-28109256

RESUMO

BACKGROUND: The composition of the skin microbiome is predicted to play a role in the development of conditions such as atopic eczema and psoriasis. 16S rRNA gene sequencing allows the investigation of bacterial microbiota. A significant challenge in this field is development of cost effective high throughput methodologies for the robust interrogation of the skin microbiota, where biomass is low. Here we describe validation of methodologies for 16S rRNA (ribosomal ribonucleic acid) gene sequencing from the skin microbiome, using the Illumina MiSeq platform, the selection of primer to amplify regions for sequencing and we compare results with the current standard protocols.. METHODS: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of healthy volunteers. This was amplified using primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3); and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 and sequenced. Both data sets were denoised, cleaned of chimeras and analysed using QIIME. RESULTS: There was no significant difference in the diversity indices at the phylum and the genus level observed between the platforms. The capture of diversity using the low density mock community samples demonstrated that the primer pair spanning the V3-V4 hypervariable region had better capture when compared to the primer pair for the V1-V3 region and was robust to spiking with human DNA. The pilot data generated using the V3-V4 region from the skin of healthy volunteers was consistent with these results, even at the genus level (Staphylococcus, Propionibacterium, Corynebacterium, Paracoccus, Micrococcus, Enhydrobacter and Deinococcus identified at similar abundances on both platforms). CONCLUSIONS: The results suggest that the bacterial community diversity captured using the V3-V4 16S rRNA hypervariable region from sequencing using the MiSeq platform is comparable to the Roche454 GS Junior platform. These findings provide evidence that the optimised method can be used in human clinical samples of low bacterial biomass such as the investigation of the skin microbiota.


Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Técnicas de Tipagem Bacteriana/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Microbiota/genética , Pele/microbiologia , Adulto , Bactérias/classificação , Sequência de Bases , Biomassa , Biologia Computacional/métodos , Contaminação por DNA , Primers do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Genes Bacterianos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos/microbiologia , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Análise de Sequência de DNA/métodos
12.
J Antimicrob Chemother ; 72(3): 778-781, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27999015

RESUMO

Objectives: To investigate the efficacy of a potent novel antimicrobial protein of mass 6 kDa, epidermicin NI01, for eradicating the nasal burden of MRSA in a cotton rat ( Sigmodon hispidus ) model. Methods: MRSA strain ATCC 43300 was used to establish a robust colonization of cotton rat nares. This model was used to evaluate the efficacy of topical 0.04% and 0.2% epidermicin NI01, administered twice daily for 3 days consecutively, and topical 0.8% epidermicin NI01 administered once, for reducing nasal MRSA burden. Control groups remained untreated or were administered vehicle only (0.5% hydroxypropylmethylcellulose) or 2% mupirocin twice daily for 3 days. The experiment was terminated at day 5 and MRSA quantitative counts were determined. Tissues recovered from animals treated with 0.2% epidermicin twice daily for 3 days were examined for histological changes. Results: Mupirocin treatment resulted in a reduction in burden of log 10 (log R) of 2.59 cfu/nares compared with vehicle ( P < 0.0001). Epidermicin NI01 administered once at 0.8% showed excellent efficacy, resulting in a log R of 2.10 cfu/nares ( P = 0.0004), which was equivalent to mupirocin. Epidermicin NI01 administered at 0.2% or 0.04% twice daily for 3 days did not have a significant impact on the tissue burden recovered from the nares. Mild to marked histological abnormalities were noted, but these were determined to be reversible. Conclusion: A single dose of topical epidermicin NI01 was as effective as mupirocin administered twice daily for 3 days in eradication of MRSA from the nares of cotton rats. This justifies further development of epidermicin for this indication.


Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Bacteriocinas/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Nariz/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Administração Tópica , Animais , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Carga Bacteriana/efeitos dos fármacos , Bacteriocinas/uso terapêutico , Relação Dose-Resposta a Droga , Mupirocina/uso terapêutico , Nariz/efeitos dos fármacos , Ratos , Sigmodontinae , Infecções Estafilocócicas/microbiologia
13.
J Water Health ; 14(5): 727-737, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27740540

RESUMO

The domestic environment can be a source of pathogenic bacteria. We show here that domestic shower hoses may harbour potentially pathogenic bacteria and fungi. Well-developed biofilms were physically removed from the internal surface of shower hoses collected in four locations in England and Scotland. Amplicon pyrosequencing of 16S and 18S rRNA targets revealed the presence of common aquatic and environmental bacteria, including members of the Actinobacteria, Alphaproteobacteria, Bacteroidetes and non-tuberculous Mycobacteria. These bacteria are associated with infections in immunocompromised hosts and are widely reported in shower systems and as causes of water-acquired infection. More importantly, this study represents the first detailed analysis of fungal populations in shower systems and revealed the presence of sequences related to Exophiala mesophila, Fusarium fujikuroi and Malassezia restricta. These organisms can be associated with the environment and healthy skin, but also with infection in compromised and immuno-competent hosts and occurrence of dandruff. Domestic showering may result in exposure to aerosols of bacteria and fungi that are potentially pathogenic and toxigenic. It may be prudent to limit development of these biofilms by the use of disinfectants, or regular replacement of hoses, where immuno-compromised persons are present.


Assuntos
Fenômenos Fisiológicos Bacterianos , Biofilmes/crescimento & desenvolvimento , Fungos/fisiologia , Microbiologia da Água , Bactérias/classificação , Bactérias/isolamento & purificação , Inglaterra , Fungos/classificação , Fungos/isolamento & purificação , Infecções Oportunistas/microbiologia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Escócia , Abastecimento de Água
14.
Mol Microbiol ; 101(6): 1069-87, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27309594

RESUMO

Uropathogenic Escherichia coli (UPEC) of sequence type 131 (ST131) are a pandemic multidrug resistant clone associated with urinary tract and bloodstream infections. Type 1 fimbriae, a major UPEC virulence factor, are essential for ST131 bladder colonization. The globally dominant sub-lineage of ST131 strains, clade C/H30-R, possess an ISEc55 insertion in the fimB gene that controls phase-variable type 1 fimbriae expression via the invertible fimS promoter. We report that inactivation of fimB in these strains causes altered regulation of type 1 fimbriae expression. Using a novel read-mapping approach based on Illumina sequencing, we demonstrate that 'off' to 'on' fimS inversion is reduced in these strains and controlled by recombinases encoded by the fimE and fimX genes. Unlike typical UPEC strains, the nucleoid-associated H-NS protein does not strongly repress fimE transcription in clade C ST131 strains. Using a genetic screen to identify novel regulators of fimE and fimX in the clade C ST131 strain EC958, we defined a new role for the guaB gene in the regulation of type 1 fimbriae and in colonisation of the mouse bladder. Our results provide a comprehensive analysis of type 1 fimbriae regulation in ST131, and highlight important differences in its control compared to non-ST131 UPEC.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Integrases/genética , Integrases/metabolismo , Receptores Imunológicos/metabolismo , Fatores de Virulência/metabolismo , Animais , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Escherichia coli Uropatogênica/metabolismo , Fatores de Virulência/genética
15.
Nucleic Acids Res ; 44(3): e21, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26405200

RESUMO

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.


Assuntos
Biossíntese de Proteínas , Transcrição Genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regiões Promotoras Genéticas , Riboswitch , Proteínas Virais/genética
16.
mBio ; 6(6): e01602-15, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26578678

RESUMO

UNLABELLED: Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located. IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.


Assuntos
Metilação de DNA , DNA Bacteriano/metabolismo , Genótipo , Metiltransferases/metabolismo , Escherichia coli Uropatogênica/enzimologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Saúde Global , Humanos , Metiltransferases/genética , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/isolamento & purificação
17.
Lancet ; 385 Suppl 1: S27, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26312849

RESUMO

BACKGROUND: Skin microbiota are likely to be important in the development of conditions such as psoriatic arthritis. Profiling the bacterial community in the psosriatic plaques will contribute to our understanding of the role of the skin microbiome in these conditions. The aim of this work was to determine the optimum study design for work on the skin microbiome with use of the MiSeq platform. The objectives were to compare data generated from two platforms for two primer pairs in a low density mock bacterial community. METHODS: DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of four healthy volunteers. The DNA was amplified with primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3), and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 platforms and sequenced. Both datasets were de-noised, cleaned of chimeras, and analysed by use of QIIME software (version 1.8.0). FINDINGS: No significant difference in the diversity indices at the phylum and the genus level between the platforms was seen. Comparison of the diversity indices for the mock community data for the two primer pairs demonstrated that the V3-V4 hypervariable region had significantly better capture of bacterial diversity than did the V1-V3 region. Amplification with the same primer pairs showed strong concordance within each platform (98·9-99·8%), with negligible effect of spiked human DNA contamination. Comparison at the family level classification between samples processed on the MiSeq and Roche454 platforms using the V3-V4 hypervariable region also showed a high level of concordance (87%), although less so for the V1-V3 primers (10%). The pilot data from healthy volunteers were similar. INTERPRETATION: Results obtained from the V3-V4 16S rRNA hypervariable region, sequencing on the MiSeq and Roche454 platforms, were concordant between replicates, and between each other. These findings suggest that the MiSeq platform, and these primers, is a comparable method for determining skin microbiota to the widely used Roche454 methodology. FUNDING: NIHR Manchester Musculoskeletal Biomedical Research Unit.

18.
J Antimicrob Chemother ; 70(10): 2757-62, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26183183

RESUMO

OBJECTIVES: We investigated the molecular epidemiology of uropathogenic Escherichia coli (UPEC) from a tertiary care hospital in Riyadh, Saudi Arabia, revealing, for the first time, the population structure of UPEC in the region. METHODS: A total of 202 UPEC isolates were recovered from hospital and community patients with urinary tract infection in December 2012 and January 2013. Strains were characterized by MLST, antibiotic susceptibility determination and virulence gene detection. RESULTS: The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%), ST10 (6.4%), ST127 (5.9%), ST95 (5.4%), ST12 (3.5%), ST998 (3.5%) and ST405 (3%). ST131 and ST405 isolates were significantly associated with high levels of antibiotic resistance (60% of ST131 carried CTX-M-14 or CTX-M-15 and 66.7% of ST405 isolates carried CTX-M-15). ST131, CTX-M-15-positive isolates were predominantly of the fimH30/clade C group, resistant to fluoroquinolones; members of this sub-group were more likely to carry a high number of genes encoding selected virulence determinants. The relatively high proportion of ST38 was notable and four of these isolates harboured aggR. CONCLUSIONS: Our findings highlight the presence of MDR, CTX-M-positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion of isolates with CTX-M is a particular concern. We suggest that ST38 UPEC warrant further study.


Assuntos
Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli Uropatogênica/classificação , Escherichia coli Uropatogênica/efeitos dos fármacos , Antibacterianos/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Prevalência , Arábia Saudita/epidemiologia , Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Virulência/genética
19.
PLoS One ; 10(4): e0122369, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25875675

RESUMO

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the blaCTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Infecções por Escherichia coli/genética , Escherichia coli/genética , Plasmídeos/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Genoma Bacteriano/efeitos dos fármacos , Humanos , Mutagênese , Replicon/genética , Análise de Sequência de DNA , beta-Lactamases/genética
20.
J Antimicrob Chemother ; 70(7): 1969-72, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25786480

RESUMO

OBJECTIVES: Escherichia coli ST131 is a globally disseminated MDR clone originally identified due to its association with the blaCTX-M-15 gene encoding an ESBL. It is thus assumed that blaCTX-M-15 is the major determinant for resistance to ß-lactam antibiotics in this clone. The complete sequence of EC958, a reference strain for E. coli ST131, revealed that it contains a chromosomally located blaCMY-23 gene with an upstream ISEcp1 element as well as several additional plasmid-encoded ß-lactamase genes. Here, we examined the genetic context of the blaCMY-23 element in EC958 and other E. coli ST131 strains and investigated the contribution of blaCMY-23 to EC958 resistance to a range of ß-lactam antibiotics. METHODS: The genetic context of blaCMY-23 and its associated mobile elements was determined by PCR and sequencing. Antibiotic susceptibility testing was performed using Etests. The activity of the blaCMY-23 promoter was assessed using lacZ reporter assays. Mutations were generated using λ-Red-recombination. RESULTS: The genetic structure of the ISEcp1-IS5-blaCMY-23 mobile element was determined and localized within the betU gene on the chromosome of EC958 and five other E. coli ST131 strains. The transcription of blaCMY-23, driven by a previously defined promoter within ISEcp1, was significantly higher than other ß-lactamase genes and could be induced by cefotaxime. Deletion of the blaCMY-23 gene resulted in enhanced susceptibility to cefoxitin, cefotaxime and ceftazidime. CONCLUSIONS: This is the first known report to demonstrate the chromosomal location of blaCMY-23 in E. coli ST131. In EC958, CMY-23 plays a major role in resistance to third-generation cephalosporins and cephamycins.


Assuntos
Resistência às Cefalosporinas , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , beta-Lactamases/metabolismo , Fusão Gênica Artificial , Cromossomos Bacterianos , DNA Bacteriano/química , DNA Bacteriano/genética , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Genes Reporter , Sequências Repetitivas Dispersas , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Análise de Sequência de DNA , beta-Galactosidase/análise , beta-Galactosidase/genética , beta-Lactamases/genética
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