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1.
PLoS Biol ; 18(8): e3000805, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32810152

RESUMO

Antibiotics are losing efficacy due to the rapid evolution and spread of resistance. Treatments targeting bacterial virulence factors have been considered as alternatives because they target virulence instead of pathogen viability, and should therefore exert weaker selection for resistance than conventional antibiotics. However, antivirulence treatments rarely clear infections, which compromises their clinical applications. Here, we explore the potential of combining antivirulence drugs with antibiotics against the opportunistic human pathogen Pseudomonas aeruginosa. We combined two antivirulence compounds (gallium, a siderophore quencher, and furanone C-30, a quorum sensing [QS] inhibitor) together with four clinically relevant antibiotics (ciprofloxacin, colistin, meropenem, tobramycin) in 9×9 drug concentration matrices. We found that drug-interaction patterns were concentration dependent, with promising levels of synergies occurring at intermediate drug concentrations for certain drug pairs. We then tested whether antivirulence compounds are potent adjuvants, especially when treating antibiotic resistant (AtbR) clones. We found that the addition of antivirulence compounds to antibiotics could restore growth inhibition for most AtbR clones, and even abrogate or reverse selection for resistance in five drug combination cases. Molecular analyses suggest that selection against resistant clones occurs when resistance mechanisms involve restoration of protein synthesis, but not when efflux pumps are up-regulated. Altogether, our work provides a first systematic analysis of antivirulence-antibiotic combinatorial treatments and suggests that such combinations have the potential to be both effective in treating infections and in limiting the spread of antibiotic resistance.


Assuntos
Antibacterianos/farmacologia , Ciprofloxacino/farmacologia , Colistina/farmacologia , Furanos/farmacologia , Gálio/farmacologia , Meropeném/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Biossíntese de Proteínas/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/efeitos dos fármacos , Virulência
2.
PLoS Pathog ; 16(7): e1008591, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32645118

RESUMO

Reactive arthritis, an autoimmune disorder, occurs following gastrointestinal infection with invasive enteric pathogens, such as Salmonella enterica. Curli, an extracellular, bacterial amyloid with cross beta-sheet structure can trigger inflammatory responses by stimulating pattern recognition receptors. Here we show that S. Typhimurium produces curli amyloids in the cecum and colon of mice after natural oral infection, in both acute and chronic infection models. Production of curli was associated with an increase in anti-dsDNA autoantibodies and joint inflammation in infected mice. The negative impacts on the host appeared to be dependent on invasive systemic exposure of curli to immune cells. We hypothesize that in vivo synthesis of curli contributes to known complications of enteric infections and suggest that cross-seeding interactions can occur between pathogen-produced amyloids and amyloidogenic proteins of the host.


Assuntos
Artrite Infecciosa/imunologia , Proteínas de Bactérias/imunologia , Febre Tifoide/imunologia , Animais , Anticorpos Antinucleares/imunologia , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Infecciosa/metabolismo , Proteínas de Bactérias/biossíntese , Intestino Grosso/imunologia , Intestino Grosso/microbiologia , Camundongos , Febre Tifoide/metabolismo
3.
PLoS One ; 15(7): e0233945, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701964

RESUMO

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.


Assuntos
Brevibacterium/metabolismo , Queijo/microbiologia , Etanolamina/metabolismo , Microbiologia de Alimentos , Regulação Bacteriana da Expressão Gênica , Listeria monocytogenes/genética , Propilenoglicol/metabolismo , Psychrobacter/metabolismo , Transcriptoma , Aclimatação , Ágar , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Técnicas de Cocultura , Meios de Cultura , Transporte de Elétrons/genética , Fermentação/genética , Listeria monocytogenes/metabolismo , Listeria monocytogenes/patogenicidade , Plasmídeos , RNA Bacteriano/biossíntese , RNA Bacteriano/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Virulência/genética
4.
J Med Microbiol ; 69(6): 792-796, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32459618

RESUMO

Introduction. Klebsiella rods, belonging to the family Enterobacteriaceae, are generally opportunistic pathogens commonly associated with nosocomial infections, especially in intensive care units. Interestingly, strains of this genus also show multi-drug resistance. In recent years, multiple studies have indicated that the prevalence of carbapenem resistance has increased rapidly among Klebsiella representatives.Aim. The aim of this study was to assess the usefulness of selected phenotypic and genotypic methods for the detection of the most important carbapenemases in Klebsiella strains.Methodology. The study involved 51 Klebsiella strains. The ability to produce carbapenemases was determined by phenotypic methods (double disc synergy test, test with four discs and three inhibitors, CarbaNP test, culture on chromogenic medium, panels of automatic method - Phoenix, CIM test and modified Hodge test). The potential for carbapenemase synthesis was also evaluated using real-time PCR, detecting bla VIM/IMP, bla KPC, bla NDM and bla OXA-48 genes.Results. Using the phenotypic methods, positive results were obtained for all of the analysed strains. Using PCR, carbapenemase synthesis potential was confirmed on the molecular level; the bla VIM gene was detected in 23 strains, the bla NDM gene in 26 strains and the bla OXA-48 gene in two strains.Conclusion. There was complete agreement between the carbapenemases detected by the genetic method and the results obtained with phenotypic methods.


Assuntos
Proteínas de Bactérias/análise , Klebsiella/enzimologia , beta-Lactamases/análise , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Genótipo , Fenótipo , beta-Lactamases/biossíntese , beta-Lactamases/genética
5.
PLoS One ; 15(5): e0233301, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469926

RESUMO

Bacterial spot is a destructive disease of tomato in Florida that prior to the early 1990s was caused by Xanthomonas euvesicatoria. X. perforans was first identified in Florida in 1991 and by 2006 was the only xanthomonad associated with bacterial spot disease in tomato. The ability of an X. perforans strain to outcompete X. euvesicatoria both in vitro and in vivo was at least in part associated with the production of three bacteriocins designated Bcn-A, Bcn-B, and Bcn-C. The objective of this study was to characterize the genetic determinants of these bacteriocins. Bcn-A activity was confined to one locus consisting of five ORFs of which three (ORFA, ORF2 and ORF4) were required for bacteriocin activity. The fifth ORF is predicted to encode an immunity protein to Bcn-A based on in vitro and in vivo assays. The first ORF encodes Bcn-A, a 1,398 amino acid protein, which bioinformatic analysis predicts to be a member of the RHS family of toxins. Based on results of homology modeling, we hypothesize that the amino terminus of Bcn-A interacts with a protein in the outer membrane of X. euvesicatoria. The carboxy terminus of the protein may interact with an as yet unknown protein(s) and puncture the X. euvesicatoria membrane, thereby delivering the accessory proteins into the target and causing cell death. Bcn-A appears to be activated upon secretion based on cell fractionation assays. The other two loci were each shown to be single ORFs encoding Bcn-B and Bcn-C. Both gene products possess homology toward known proteases. Proteinase activity for both Bcn-B and Bcn-C was confirmed using a milk agar assay. Bcn-B is predicted to be an ArgC-like serine protease, which was confirmed by PMSF inhibition of proteolytic activity, whereas Bcn-C has greater than 50% amino acid sequence identity to two zinc metalloproteases.


Assuntos
Proteínas de Bactérias/genética , Bacteriocinas/genética , Loci Gênicos , Lycopersicon esculentum/microbiologia , Doenças das Plantas/microbiologia , Xanthomonas/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Bacteriocinas/biossíntese , Homologia de Sequência , Xanthomonas/classificação , Xanthomonas/genética , Xanthomonas/metabolismo
6.
Arch Microbiol ; 202(6): 1507-1515, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32222778

RESUMO

Pyocyanin produced by Pseudomonas aeruginosa is a key virulence factor that often causes heavy damages to airway and lung in patients. Conversion of phenazine-1-carboxylic acid to pyocyanin involves an extrametabolic pathway that contains two enzymes encoded, respectively, by phzM and phzS. In this study, with construction of the rpoS-deficient mutant, we first found that although phenazine production increased, pyocyanin produced in the mutant YTΔrpoS was fourfold much higher than that in the wild-type strain YT. To investigate this issue, we constructed phzM-lacZ fusion on a vector and on the chromosome. By quantifying ß-galactosidase activities, we confirmed that expression of the phzM was up-regulated when the rpoS gene was inactivated. However, no changes occurred in the expression of phzS and phzH when the rpoS was knocked out. Taken together, overproduction of the SAM-dependent methyltransferase (PhzM) might contribute to the increased pyocyanin in the absence of RpoS in P. aeruginosa.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Metiltransferases/biossíntese , Oxigenases de Função Mista/biossíntese , Pseudomonas aeruginosa/metabolismo , Piocianina/biossíntese , Fator sigma/genética , Humanos , Metiltransferases/genética , Oxigenases de Função Mista/genética , Fenazinas/metabolismo , Pseudomonas aeruginosa/genética , Fatores de Virulência/metabolismo
7.
Arch Microbiol ; 202(6): 1517-1527, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32222779

RESUMO

Biofilm formation is a harmful phenomenon in many areas, such as in industry and clinically, but offers advantages in the field of biocatalysis for the generation of robust biocatalytic platforms. In this work, we optimised growth conditions for the production of Escherichia coli biofilms by three strains (PHL644, a K-12 derivative with enhanced expression of the adhesin curli; the commercially-used strain BL21; and the probiotic Nissle 1917) on a variety of surfaces (plastics, stainless steel and PTFE). E. coli PHL644 and PTFE were chosen as optimal strain and substratum, respectively, and conditions (including medium, temperature, and glucose concentration) for biofilm growth were determined. Finally, the impact of these growth conditions on expression of the curli genes was determined using flow cytometry for planktonic and sedimented cells. We reveal new insights into the formation of biofilms and expression of curli in E. coli K-12 in response to environmental conditions.


Assuntos
Adesinas Bacterianas/biossíntese , Proteínas de Bactérias/biossíntese , Biofilmes/crescimento & desenvolvimento , Exposição Ambiental , Escherichia coli/metabolismo , Adesinas Bacterianas/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Plásticos/química , Politetrafluoretileno/química , Aço Inoxidável/química , Propriedades de Superfície
8.
Microb Cell Fact ; 19(1): 45, 2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32093734

RESUMO

BACKGROUND: Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT: Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION: We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.


Assuntos
Bacillus licheniformis/metabolismo , Proteínas de Bactérias/biossíntese , Endopeptidases/biossíntese , Bacillus licheniformis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Engenharia Genética , Microbiologia Industrial , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Plasmídeos/genética
9.
Science ; 367(6476): 464-468, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31974256

RESUMO

Expression of proteins inside cells is noisy, causing variability in protein concentration among identical cells. A central problem in cellular control is how cells cope with this inherent noise. Compartmentalization of proteins through phase separation has been suggested as a potential mechanism to reduce noise, but systematic studies to support this idea have been missing. In this study, we used a physical model that links noise in protein concentration to theory of phase separation to show that liquid droplets can effectively reduce noise. We provide experimental support for noise reduction by phase separation using engineered proteins that form liquid-like compartments in mammalian cells. Thus, phase separation can play an important role in biological signal processing and control.


Assuntos
Células/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Transição de Fase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
10.
Nat Commun ; 11(1): 466, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980604

RESUMO

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/enzimologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Surtos de Doenças , Enterobacter/enzimologia , Enterobacter/genética , Infecções por Enterobacteriaceae/epidemiologia , beta-Lactamases/biossíntese , beta-Lactamases/genética , Queimaduras/microbiologia , Enterobacteriáceas Resistentes a Carbapenêmicos/patogenicidade , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Enterobacter/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Genoma Bacteriano , Humanos , Queensland/epidemiologia , Fatores R/genética , Engenharia Sanitária , Centros de Atenção Terciária , Sequenciamento Completo do Genoma/métodos , Resistência beta-Lactâmica/genética
11.
Int J Antimicrob Agents ; 55(3): 105903, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31954832

RESUMO

This study characterizes four KPC-2-producing Klebsiella pneumoniae isolates from neonates belonging to a single sequence type 147 (ST147) in relation to carbapenem resistance and explores probable mechanisms of differential colistin resistance among the clonal cluster. Whole genome sequencing (WGS) revealed that the isolates were nearly 100% identical and harbored resistance genes (blaKPC-2,OXA-9,CTX-M-15,SHV-11,OXA-1,TEM-1B, oqxA, oqxB, qnrB1, fosA, arr-2, sul1, aacA4, aac(6')Ib-cr, aac(6')Ib), and several virulence genes. blaKPC-2 was the only carbapenem-resistant gene found, bracketed between ISKpn7 and ISKpn6 of Tn4401b on a non-conjugative IncFII plasmid. Remarkably, one of the clonal isolates was resistant to colistin, the mechanistic basis of which was not apparent from comparative genomics. The transmissible colistin resistance gene, mcr, was absent. Efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) rendered a 32-fold decrease in the minimum inhibitory concentration (MIC) of colistin in the resistant isolate only. acrB, tolC, ramA, and soxS genes of the AcrAB-TolC pump system overexpressed exclusively in the colistin-resistant isolate, although the corresponding homologs of the AcrAB-TolC pump, regulators and promoters were mutually identical. No change was observed in the expression of other efflux genes (kpnE/F and kpnG/H) or two-component system (TCS) genes (phoP/phoQ, pmrA/pmrB). Colistin resistance in one of the clonal KPC-2-producing isolates is postulated to be due to overexpression of the AcrAB-TolC pump. This study is probably the first to report clinical clonal K. pneumoniae isolates with differences in colistin susceptibility. The presence of carbapenem-resistant isolates with differential behavior in the expression of a genomically identical pump system indicates the nuances of the resistance mechanisms and the difficulty of treatment thereof.


Assuntos
Antibacterianos/farmacologia , Colistina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , beta-Lactamases/biossíntese , Proteínas de Bactérias/biossíntese , Proteínas de Transporte/biossíntese , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Humanos , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Infecções por Klebsiella/tratamento farmacológico , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/metabolismo
12.
Microb Cell Fact ; 19(1): 11, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964372

RESUMO

BACKGROUND: In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS: High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS: We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.


Assuntos
Proteínas de Bactérias/biossíntese , Técnicas Biossensoriais , Corynebacterium glutamicum/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas de Bactérias/metabolismo , Biotecnologia/métodos , Engenharia Genética , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/metabolismo , Via Secretória , Proteínas de Transporte Vesicular/metabolismo
13.
PLoS One ; 15(1): e0220095, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910206

RESUMO

There are numerous reports on poly-ß-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.


Assuntos
Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Hidroxibutiratos/química , Plásticos/química , Poliésteres/química , Poluentes do Solo/química , Stenotrophomonas/enzimologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Biodegradação Ambiental , Hidrolases de Éster Carboxílico/biossíntese , Hidrolases de Éster Carboxílico/isolamento & purificação , Cromatografia de Afinidade , Meios de Cultura/química , Humanos , Hidrólise , Peso Molecular , Solo/química , Resíduos Sólidos , Stenotrophomonas/química
14.
Int J Infect Dis ; 93: 118-120, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31978576

RESUMO

KPC-producing K. pneumoniae is an endemic challenge. Seven KPC-producing Enterobacterales showed unusual carbapenems susceptibility profile. These strains were resistance at least one carbapenem and the ertapenem MIC was lower than imipenem and/or meropenem MICs using Vitek 2™ system (bioMerieux). When E-test™ and disk diffusion methods were performed the carbapenems showed susceptible results.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Carbapenêmicos/farmacologia , Gammaproteobacteria/efeitos dos fármacos , beta-Lactamases/biossíntese , Farmacorresistência Bacteriana , Ertapenem/farmacologia , Imipenem/farmacologia , Klebsiella pneumoniae/enzimologia , Meropeném/farmacologia , Testes de Sensibilidade Microbiana
15.
Diagn Microbiol Infect Dis ; 96(3): 114968, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31924425

RESUMO

Klebsiella pneumoniae strain is an important opportunistic pathogen that causes severe nosocomial infections. In the present study a molecular characterization of carbapenem resistant K. pneumoniae, isolated from blood samples of hospitalized patients of Verona University Hospital, was performed. The simultaneous presence of SHV-1/CTX-M-15/KPC-3 and SHV-1/CTX-M-15/OXA-48 serin-ß-lactamases was ascertained in the 89% and 11% of K. pneumoniae ST512 and K. pneumoniae ST14, respectively. Molecular characterization of bla genes showed that blaKPC-3 was found in Tn4401a transposon with the tnpR, tnpA, ISKpn6, and ISKpn7 mobile elements whereas blaCTX-M-15 was detected downstream ISEcp1 genetic element. A class 1 integron with a gene cassette of 780 bp corresponding to aadA2 gene was identified in 33 K. pneumoniae ST512 isolates.


Assuntos
Proteínas de Bactérias/biossíntese , Infecções por Klebsiella/sangue , Klebsiella pneumoniae/genética , Centros de Atenção Terciária/estatística & dados numéricos , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla , Variação Genética , Hospitais Universitários/estatística & dados numéricos , Humanos , Unidades de Terapia Intensiva/estatística & dados numéricos , Itália , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Quartos de Pacientes/estatística & dados numéricos , beta-Lactamases/genética
16.
Immunology ; 159(2): 231-241, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31713233

RESUMO

Regulatory T (Treg) cell-specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP-Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP-Cre mice with Insrfl/fl mice. Using a conventional two-band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR-based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1 -generation mice showed evidence of ectopic recombination and that ~10% of the F2 -generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell-specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP-Cre allele, particularly when studying genes that are systemically expressed.


Assuntos
Proteínas de Bactérias/genética , Fatores de Transcrição Forkhead/genética , Integrases/genética , Proteínas Luminescentes/genética , Receptor de Insulina/genética , Recombinação Genética , Linfócitos T Reguladores/imunologia , Animais , Proteínas de Bactérias/biossíntese , Linhagem da Célula , Cruzamentos Genéticos , Genes Reporter , Genótipo , Integrases/metabolismo , Proteínas Luminescentes/biossíntese , Camundongos Knockout , Camundongos Transgênicos , Fenótipo , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Receptor de Insulina/deficiência , Linfócitos T Reguladores/metabolismo
17.
Proc Natl Acad Sci U S A ; 117(1): 371-380, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871149

RESUMO

Microbial natural products represent a rich resource of evolved chemistry that forms the basis for the majority of pharmacotherapeutics. Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a particularly interesting class of natural products noted for their unique mode of biosynthesis and biological activities. Analyses of sequenced microbial genomes have revealed an enormous number of biosynthetic loci encoding RiPPs but whose products remain cryptic. In parallel, analyses of bacterial metabolomes typically assign chemical structures to only a minority of detected metabolites. Aligning these 2 disparate sources of data could provide a comprehensive strategy for natural product discovery. Here we present DeepRiPP, an integrated genomic and metabolomic platform that employs machine learning to automate the selective discovery and isolation of novel RiPPs. DeepRiPP includes 3 modules. The first, NLPPrecursor, identifies RiPPs independent of genomic context and neighboring biosynthetic genes. The second module, BARLEY, prioritizes loci that encode novel compounds, while the third, CLAMS, automates the isolation of their corresponding products from complex bacterial extracts. DeepRiPP pinpoints target metabolites using large-scale comparative metabolomics analysis across a database of 10,498 extracts generated from 463 strains. We apply the DeepRiPP platform to expand the landscape of novel RiPPs encoded within sequenced genomes and to discover 3 novel RiPPs, whose structures are exactly as predicted by our platform. By building on advances in machine learning technologies, DeepRiPP integrates genomic and metabolomic data to guide the isolation of novel RiPPs in an automated manner.


Assuntos
Proteínas de Bactérias/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Descoberta de Drogas/métodos , Peptídeos/isolamento & purificação , Software , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Produtos Biológicos/metabolismo , Genômica/métodos , Aprendizado de Máquina , Metabolômica/métodos , Biossíntese Peptídica/genética , Peptídeos/genética , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo
18.
Biomed Res Int ; 2019: 9308593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31828148

RESUMO

This study aims to achieve high-level soluble expression and characterization of a thermostable industrially important enzyme, i.e., beta-glucosidase (BglA; EC: 3.2.1.21), from Clostridium thermocellum (C. thermocellum) by cloning in an Escherichia coli (E. coli) expression system. BglA was expressed as a partially soluble component of total cellular protein (TCP) having a molecular weight of ∼53 kDa with 50% of it as soluble fraction. Purification in two steps, namely, heat inactivation and Ni-chromatography, yielded approximately 30% and 15% of BglA, respectively. The purified (∼98%) BglA enzyme showed promising activity against the salicin substrate having a K m of 19.83 mM and a V max of 0.12 µmol/min. The enzyme had an optimal temperature and pH of 50°C and 7.0, respectively, while retaining its catalytic activity up till 60°C and at pH 7. The optimized maximum expression level was attained in M9NG medium with lactose as an inducer. Circular dichroism revealed presence of alpha helix (43.50%) and small percentage of beta sheets (10.60%). Factors like high-end cellulolytic activity, fair thermal stability, stability against low pH, and ease of purification make BglA from C. thermocellum a potential candidate in industrial applications.


Assuntos
Proteínas de Bactérias , Clonagem Molecular , Clostridium thermocellum , Escherichia coli , Temperatura Alta , beta-Glucosidase , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Clostridium thermocellum/enzimologia , Clostridium thermocellum/genética , Estabilidade Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , beta-Glucosidase/biossíntese , beta-Glucosidase/química , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação
19.
Clin Lab ; 65(12)2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31850708

RESUMO

BACKGROUND: KPC-producing Klebsiella pneumoniae (KPC-Kp) has become a serious threat to patients worldwide, as the treatment options are limited. The combination of fosfomycin with other antibiotics has been reported but with inconsistent results. Thus, we performed synergy testing of fosfomycin combined with tigecycline by E-test, which was easy to perform and to determine results, compared with microdilution checkerboard, which is considered to be the "gold standard", to evaluate the agreement between the two methods. METHODS: Thirty non-repetitive KPC-Kp isolates from different patients were included in this study. Bacterial identification and routine antibiotic susceptibility testing were performed by a VITEK 2 Compact automated system. The KPC producing isolates were identified by modified Carbapenem Inhibitory Method (mCIM) and PCR amplification of carbapenemase genes. Synergy testing of fosfomycin combined with tigecycline was performed by E-test (E-test stripes were placed at 90° angle), with microdilution chequerboard performed in parallel. Fractional inhibitory concentration index (FICI) was calculated. Statistical analyses were performed by SPSS 18.0 software. RESULTS: All 30 KPC-Kp were mCIM test positive and KPC-2 producing. The susceptibility rates of fosfomycin and tigecycline were 36.7% (11/30) and 63.3% (19/30), respectively. Both checkerboard and E-test results showed that most MICs of fosfomycin and tigecycline decreased in the combination group. FICI showed 13.3% - 16.7% isolates were synergistic, 30.0% - 36.7% were additive, and 50.0% - 53.3% were indifferent. No antagonism was found. There was no significant difference between the two groups (p > 0.05), and the overall agreement (with FICI difference ≤ 0.25 in each isolate) between the two methods was 76.7% (23/30). CONCLUSIONS: The synergy testing results determined by E-test correlated well with microdilution checkerboard. Thus E-test synergy testing has the potential to be used in routine clinical laboratory.


Assuntos
Proteínas de Bactérias/biossíntese , Fosfomicina/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Tigeciclina/farmacologia , beta-Lactamases/biossíntese , Antibacterianos/farmacologia , Sinergismo Farmacológico , Humanos , Klebsiella pneumoniae/metabolismo , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/estatística & dados numéricos , Reprodutibilidade dos Testes
20.
Infez Med ; 27(4): 357-364, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31846984

RESUMO

Antimicrobial resistance (AMR) is a global health security threat requiring actions across government sectors and society. Many factors are involved in this phenomenon, being overuse of antibiotics, incorrect antibiotic prophylaxis, and use of antibiotics for zootechnic reasons the main causes of the increasing rate of multi-drug resistant (MDR) bacteria. The impact of resistance to antimicrobials is an important threat due also to the emergence of MDR Gram-negative bacteria resistant to carbapenems, and the lack of the research for new active molecules. The production of extended spectrum beta-lactamase enzymes has been the first threatening mechanism for Gram-negative resistance to antibiotics, which prompted the development of new classes of antibiotics such as carbapenems. Unfortunately, resistance to carbapenems developed because of multiple mechanisms including efflux pumps, porin mutations and enzyme production, being the latter particularly relevant in terms of diffusion due to the genes located within plasmids that drive their horizontal diffusion. In this scenario, antimicrobial stewardship programs (ASP) are a mandatory resource in fighting the resistance spread. The reduction of total amount of antibiotics administration in the hospital setting and guiding prescribers in the correct administration of antibiotics for the smallest period possible, at the correct dosage, can be defined as the first goals of an ASP. Anyway, in an efficacious ASP, apart from antibiotic administration, efforts must been made in ensuring the lowest probability of spreading of MDR by efficacious measures of isolation of carriers, and by offering tools for a rapid diagnosis of viral infections avoiding the administration of unnecessary antibiotics. A continuous audit of the ASP programs and a correct assessment of the allergy to drugs such as penicillin have to complete the program. Currently, only a few options are available for patients with an infection sustained by Gram-negative MDR bacteria. All the options actually available are based on the administration of colystin, an old drug whose real efficacy is reduced due to its relevant toxicity, or on the administration of recently proposed drugs such as ceftolozane-tazobactam, ceftazidime-avibactam and meropenem-vaborbactam. All these new drugs do not have a novel mechanism of action and have limited spectrum in term of activity against MDR bacteria. In conclusion, antimicrobial resistance is a global emergence and AMP is the most powerful tool actually available. Few limited options are available to treat infections due to Carbapenem Resistant Enterobacteria. Antimicrobial molecules with true novel mechanism of action are needed to win the fight against antimicrobial resistance.


Assuntos
Gestão de Antimicrobianos/normas , Proteínas de Bactérias/biossíntese , Enterobacteriáceas Resistentes a Carbapenêmicos , Farmacorresistência Bacteriana , Infecções por Enterobacteriaceae/tratamento farmacológico , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , beta-Lactamases/biossíntese , Humanos
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