RESUMO
The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.
Assuntos
Antibacterianos , RNA Polimerases Dirigidas por DNA , Staphylococcus aureus Resistente à Meticilina , Testes de Sensibilidade Microbiana , Oxacilina , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Oxacilina/farmacologia , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Antibacterianos/farmacologia , beta-Lactamas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mutação de Sentido Incorreto , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Parede Celular/genética , Humanos , Mutação , MetabolômicaRESUMO
In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.
Assuntos
Sistemas CRISPR-Cas , Capsídeo , Staphylococcus aureus Resistente à Meticilina , Mutação , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Capsídeo/metabolismo , Antibacterianos/farmacologia , Bacteriófagos/genéticaRESUMO
Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC, the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary.IMPORTANCEThe combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.
Assuntos
Antibacterianos , Infecções por Escherichia coli , Escherichia coli O157 , Fosfomicina , Antibacterianos/farmacologia , Escherichia coli O157/virologia , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Fosfomicina/farmacologia , Farmacorresistência Bacteriana , Bacteriófagos/genética , Bacteriófagos/fisiologia , Bacteriófagos/efeitos dos fármacos , Terapia por Fagos/métodos , Colífagos/genética , Colífagos/efeitos dos fármacos , Colífagos/fisiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.
Assuntos
Escherichia coli O157 , Escherichia coli O157/virologia , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Humanos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/diagnóstico , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Colífagos/genética , Colífagos/isolamento & purificação , Sensibilidade e Especificidade , Genoma ViralRESUMO
The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents. Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise; however, a thorough comparison and evaluation of their bactericidal efficacy are lacking. This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes, with the goal of harnessing them for antibacterial therapy. First, we examined the bactericidal activity of spanins, endolysins, and holins derived from 2 Escherichia coli model phages, T1 and T7. Among these, T1-spanin exhibited the highest bactericidal activity against E. coli. Subsequently, we expressed T1-spanin within bacterial cells and assessed its bactericidal activity. T1-spanin showed potent bactericidal activity against all clinical isolates tested, including bacterial strains of 111 E. coli, 2 Acinetobacter spp., 3 Klebsiella spp., and 3 Pseudomonas aeruginosa. In contrast, T1 phage-derived endolysin showed bactericidal activity against E. coli and P. aeruginosa, yet its efficacy against other bacteria was inferior to that of T1-spanin. Finally, we developed a phage-based technology to introduce the T1-spanin gene into target bacteria. The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria. The potent bactericidal activity exhibited by spanins, combined with the novel phage synthetic technology, holds promise for the development of innovative antimicrobial agents.
RESUMO
Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 µM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.
Assuntos
Monkeypox virus , Ácido Micofenólico , Guanosina/farmacologia , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Ácido Micofenólico/farmacologia , Trifluridina , Monkeypox virus/efeitos dos fármacosRESUMO
BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.
Assuntos
Mefloquina , Monkeypox virus , Humanos , Atovaquona/farmacologia , Atovaquona/uso terapêutico , Mefloquina/farmacologia , Mefloquina/uso terapêutico , Monkeypox virus/efeitos dos fármacosRESUMO
Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.
Assuntos
Terapia por Fagos , Infecções Estafilocócicas , Camundongos , Animais , Terapia por Fagos/métodos , Fagos de Staphylococcus/ultraestrutura , Staphylococcus aureus , Staphylococcus , Infecções Estafilocócicas/terapia , Myoviridae/ultraestrutura , Imunoglobulina GRESUMO
In the concept of central dogma (RNA is transcribed from DNA to produce proteins), RNA was thought to be merely an intermediary for genetic information to synthesize proteins from DNA. Since the discovery of RNA interference in 2000, research on RNA has progressed remarkably, especially in mammals. On the other hand, the role of RNA in bacterial infections was largely unknown. At that time, we started research on RNA and bacterial infection and revealed that miR-210, a small RNA in the gastric epithelial cells, is involved in gastric diseases caused by Helicobacter pylori in-fection. Furthermore, we have successfully developed sequence-specific antimicrobials by loading CRISPR-Cas13, an RNA-targeting CRISPR-Cas, on bacteriophage. The constructed antimicrobials were effective against at least Escherichia coli and Staphylococcus aureus. In this paper, we would like to introduce the importance of RNA in bacteriology.
Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Humanos , RNA/genética , Sistemas CRISPR-Cas , Escherichia coli/genética , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/genéticaRESUMO
In the ongoing COVID-19 pandemic, rapid and sensitive diagnosis of viral infection is a critical deterrent to the spread of SARS-CoV-2. To this end, we developed an automated amplification-free digital RNA detection platform using CRISPR-Cas13a and microchamber device (opn-SATORI), which automatically completes a detection process from sample mixing to RNA quantification in clinical specimens within ~9 min. Using the optimal Cas13a enzyme and magnetic beads technology, opn-SATORI detected SARS-CoV-2 genomic RNA with a LoD of < 6.5 aM (3.9 copies µL-1), comparable to RT-qPCR. Additionally, opn-SATORI discriminated between SARS-CoV-2 variants of concern, including alpha, delta, and omicron, with 98% accuracy. Thus, opn-SATORI can serve as a rapid and convenient diagnostic platform for identifying several types of viral infections.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Pandemias , RNA Viral/genética , SARS-CoV-2/genéticaRESUMO
Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.
RESUMO
The bacteriophage (or phage for short) has been used as an antibacterial agent for over a century but was abandoned in most countries after the discovery and broad use of antibiotics. The worldwide emergence and high prevalence of antimicrobial-resistant (AMR) bacteria have led to a revival of interest in the long-forgotten antibacterial therapy with phages (phage therapy) as an alternative approach to combatting AMR bacteria. The rapid progress recently made in molecular biology and genetic engineering has accelerated the generation of phage-related products with superior therapeutic potentials against bacterial infection. Nowadays, phage-based technology has been developed for many purposes, including those beyond the framework of antibacterial treatment, such as to suppress viruses by phages, gene therapy, vaccine development, etc. Here, we highlighted the current progress in phage engineering technology and its application in modern medicine.
RESUMO
Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.
Assuntos
Adaptação Fisiológica/genética , Infecções por Helicobacter/patologia , Helicobacter pylori/fisiologia , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Neoplasias Gástricas/microbiologia , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Carcinogênese , Modelos Animais de Doenças , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano/genética , Gerbillinae , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Helicobacter pylori/patogenicidade , Interações entre Hospedeiro e Microrganismos , Humanos , Masculino , Mutação , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , RNA-Seq , Neoplasias Gástricas/patologiaRESUMO
A high prevalence of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) may call for monitoring in geriatric long-term care facilities (g-LTCFs). We surveyed the distribution of ESBL-causative gene types and antimicrobial susceptibility in ESBL-PE strains from residents in g-LTCFs, and investigated the association between ESBL-causative gene types and antimicrobial susceptibility. First, we analyzed the types of ESBL-causative genes obtained from 141 ESBL-PE strains collected from the feces of residents in four Japanese g-LTCFs. Next, we determined the minimum inhibitory concentration values for alternative antimicrobial agents against ESBL-PE, including ß-lactams and non-ß-lactams. Escherichia coli accounted for 96% of the total ESBL-PE strains. Most strains (94%) contained blaCTX-M group genes. The genes most commonly underlying resistance were of the blaCTX-M-9 and blaCTX-M-1 groups. Little difference was found in the distribution of ESBL-causative genes among the facilities; however, antimicrobial susceptibility differed widely among the facilities. No specific difference was found between antimicrobial susceptibility and the number of ESBL-causative genes. Our data showed that ESBL-PEs were susceptible to some antimicrobial agents, but the susceptibility largely differed among facilities. These findings suggest that each g-LTCF may require specific treatment strategies based on their own antibiogram. Investigations into drug resistance should be performed in g-LTCFs as well as acute medical facilities.
RESUMO
Cancer, especially the solid tumor sub-set, poses considerable challenges to modern medicine owing to the unique physiological characteristics and substantial variations in each tumor's microenvironmental niche fingerprints. Though there are many treatment methods available to treat solid tumors, still a considerable loss of life happens, due to the limitation of treatment options and the outcomes of ineffective treatments. Cancer cells evolve with chemo- or radiation-treatment strategies and later show adaptive behavior, leading to failed treatment. These challenges demand tailored and individually apt personalized treatment methods. Bacteriophages (or phages) and phage-based theragnostic vectors are gaining attention in the field of modern cancer medicine, beyond their bactericidal ability. With the invention of the latest techniques to fine-tune phages, such as in the field of genetic engineering, synthetic assembly methods, phage display, and chemical modifications, noteworthy progress in phage vector research for safe cancer application has been realized, including use in pre-clinical studies. Herein, we discuss the distinct fingerprints of solid tumor physiology and the potential for bacteriophage vectors to exploit specific tumor features for improvised tumor theragnostic applications.
Assuntos
Bacteriófagos/fisiologia , Neoplasias/diagnóstico , Neoplasias/terapia , Terapia por Fagos , Animais , Vacinas Anticâncer/imunologia , Humanos , Microambiente TumoralRESUMO
Staphylococcus aureus strains that are susceptible to the ß-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire ß-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0-256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of ß-lactam resistance in OS-MRSA.
Assuntos
Proteínas de Bactérias/genética , Mutação/genética , Oxacilina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Testes de Sensibilidade Microbiana/métodos , Filogenia , Infecções Estafilocócicas/tratamento farmacológico , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Daptomicina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Mutação/genética , Infecções Estafilocócicas/tratamento farmacológico , Vancomicina/farmacologia , Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/genética , Seguimentos , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Genótipo , Humanos , Testes de Sensibilidade Microbiana/métodos , Fenótipo , Infecções Estafilocócicas/microbiologiaRESUMO
The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.
Assuntos
Anti-Infecciosos/farmacologia , Sistemas CRISPR-Cas/genética , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.
RESUMO
Helicobacter pylori ATCC 43504 is a type strain isolated from a gastric cancer patient in Australia and is commonly used for pathogenicity studies. In this study, we report the complete genome sequence of a strain that can infect gerbils. The data provide a basis for future H. pylori research.