RESUMO
Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.
Assuntos
Antibacterianos , Bacitracina , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/química , Bacitracina/farmacologia , Bacitracina/química , Relação Estrutura-Atividade , Farmacorresistência Bacteriana/efeitos dos fármacos , Vancomicina/farmacologia , Vancomicina/química , Vancomicina/análogos & derivados , Desenho de Fármacos , Fosfatos de Poli-Isoprenil/metabolismo , Fosfatos de Poli-Isoprenil/química , Fosfatos de Poli-Isoprenil/farmacologiaRESUMO
An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C18 cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C18 column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R2 > 0.9980) across a 0.01-1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B1, B2, and B3), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2-20 µg/kg were achieved for bacitracin A, B1, B2, and B3. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.
Assuntos
Ração Animal , Bacitracina , Limite de Detecção , Espectrometria de Massas em Tandem , Bacitracina/análise , Espectrometria de Massas em Tandem/métodos , Ração Animal/análise , Cromatografia Líquida de Alta Pressão/métodos , Modelos Lineares , Reprodutibilidade dos Testes , Extração em Fase Sólida/métodos , Salicilatos/análise , Animais , Resíduos de Drogas/análiseRESUMO
Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.
Assuntos
Bacillus licheniformis , Bacitracina , Fezes , Animais , Bacitracina/farmacologia , Fezes/microbiologia , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Bacillus licheniformis/isolamento & purificação , SuínosRESUMO
BACKGROUND: In cariology studies, mitis-salivarius-bacitracin (MSB) agar has been commonly considered as the selective medium for Streptococcusmutans growth. The present study was the part of a funded project (a noninferiority randomized controlled trial) which compared the efficacy of a plant extract-based mouth rinse with that of a fluoride mouth rinse on the S.mutans counts of the children. AIM: This study aimed to identify the frequency of detection of S.mutans and nonstreptococcal bacterial species from the dental plaque of caries active children using a combined technique of anaerobic culture and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. SETTINGS AND METHODS: Caries-active children (8-12 years old) were enrolled from a pediatric dental outpatient department at a tertiary care hospital. From each participant, dental plaque samples were collected from carious surfaces under sterilized conditions and then subjected to anaerobic culture. After 48 h of incubation, the bacterial colonies were isolated by sub-culture and identified by the MALDI-TOF. RESULTS: In all, 13 different bacterial species were isolated from the MSB agar medium. Other than S.mutans species, colonies of bacterial species such as Veillonelladispar,Streptococcusanginosus, Veillonellaparvula, and Streptococcusgordonii were also frequently observed from the medium. CONCLUSIONS: The study concluded that several bacterial strains, both streptococcal and nonstreptococcal, could be isolated from the MSB agar medium; hence, this medium should no longer be considered selective medium for the culture of S.mutans in clinical and epidemiological studies.
Assuntos
Cárie Dentária , Placa Dentária , Criança , Humanos , Ágar , Bacitracina , Suscetibilidade à Cárie Dentária , Antissépticos BucaisRESUMO
It is common knowledge that prolonged and excessive use of antibiotics can lead to antimicrobial resistance. However, the characteristics and mechanism of resistant-bacteria induced by clinically recommended and prophylactic dose drugs remain largely unclear. This study aimed to observe the trends of drug resistance of the bacitracin-susceptible Staphylococcus aureus strain FS127 under exposure to bacitracin (BAC), which were induced in vitro and in chicken gut. Antimicrobial susceptibility testing was used to detect the susceptibility of S. aureus induced in vitro and in the chicken gut to gentamicin, chloramphenicol, tetracycline, doxycycline, penicillin and chloramphenicol. The research results showed that bacitracin could induce drug resistance in S. aureus both in vitro and in vivo. The bacitracin-resistance rate of S. aureus isolated from chicken gut was positively correlated with the dose and time of bacitracin administration. The findings revealed that bacitracin-resistant S. aureus induced in vivo had enhanced susceptibility to chloramphenicol but no such change in vitro. Meanwhile, RT-qPCR assay was used to detect the expression levels of vraD, braD, braR and bacA in typical strains with different bacitracin-resistance levels. It was found that BacA may play a key role in the bacitracin resistance of S. aureus. In conclusion, this work reveals the characteristics and mechanism of bacitracin-resistant S. aureus induced by bacitracin in vivo and in vitro respectively.
Assuntos
Antibacterianos , Bacitracina , Galinhas , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas , Staphylococcus aureus , Bacitracina/farmacologia , Animais , Galinhas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Infecções Estafilocócicas/microbiologia , Cloranfenicol/farmacologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/efeitos dos fármacos , Proteínas de Bactérias/genéticaRESUMO
In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2â³abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2â³abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.
Assuntos
Bacillus licheniformis , Proteínas de Bactérias , Quitinases , Bacillus licheniformis/genética , Bacillus licheniformis/enzimologia , Bacillus thuringiensis/genética , Bacillus thuringiensis/enzimologia , Bacitracina , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/biossíntese , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Família Multigênica , Proteínas Recombinantes/biossíntese , TemperaturaRESUMO
Enterococcal infections frequently show high levels of antibiotic resistance, including to cell envelope-acting antibiotics like daptomycin (DAP). While we have a good understanding of the resistance mechanisms, less is known about the control of such resistance genes in enterococci. Previous work unveiled a bacitracin resistance network, comprised of the sensory ABC transporter SapAB, the two-component system (TCS) SapRS and the resistance ABC transporter RapAB. Interestingly, components of this system have recently been implicated in DAP resistance, a role usually regulated by the TCS LiaFSR. To better understand the regulation of DAP resistance and how this relates to mutations observed in DAP-resistant clinical isolates of enterococci, we here explored the interplay between these two regulatory pathways. Our results show that SapR regulates an additional resistance operon, dltXABCD, a known DAP resistance determinant, and show that LiaFSR regulates the expression of sapRS. This regulatory structure places SapRS-target genes under dual control, where expression is directly controlled by SapRS, which itself is up-regulated through LiaFSR. The network structure described here shows how Enterococcus faecalis coordinates its response to cell envelope attack and can explain why clinical DAP resistance often emerges via mutations in regulatory components.
Assuntos
Antibacterianos , Bacitracina , Proteínas de Bactérias , Daptomicina , Farmacorresistência Bacteriana , Enterococcus faecalis , Regulação Bacteriana da Expressão Gênica , Óperon , Daptomicina/farmacologia , Enterococcus faecalis/genética , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/metabolismo , Bacitracina/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Parede Celular/metabolismo , Parede Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
Multicomponent therapy combining antibiotics with enhancer molecules known as adjuvants is an emerging strategy to combat antimicrobial resistance. Niclosamide is a clinically relevant anthelmintic drug with potential to be repurposed for its inherent antibacterial activity against Gram-positive bacteria and its ability to potentiate the antibacterial activity of colistin against susceptible and resistant Gram-negative bacteria. Herein, sulfonamide analogs of niclosamide were prepared and found to enhance colistin activity against Gram-negative bacteria. The ability of niclosamide and the new sulfonamide analogs to synergize with bacitracin against vancomycin-resistant Enterococcus faecium was also discovered.
Assuntos
Antibacterianos , Bacitracina , Colistina , Testes de Sensibilidade Microbiana , Niclosamida , Sulfonamidas , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Colistina/farmacologia , Colistina/química , Relação Estrutura-Atividade , Niclosamida/farmacologia , Niclosamida/química , Niclosamida/síntese química , Sulfonamidas/farmacologia , Sulfonamidas/química , Sulfonamidas/síntese química , Bacitracina/farmacologia , Bacitracina/química , Bacitracina/síntese química , Estrutura Molecular , Bactérias Gram-Negativas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Enterococcus faecium/efeitos dos fármacos , Sinergismo Farmacológico , Bactérias Gram-Positivas/efeitos dos fármacosRESUMO
OBJECTIVE: Triple antibiotic paste (TAP) is known to have an essential role in the success of endodontic treatment by eliminating pathogens from the root canal system. Unfortunately, it causes discolouration and cytotoxicity at high concentrations. The objective of this research was to assess and compare the antimicrobial effectiveness of various concentrations (1 mg, 5 mg, 10 mg) of TAP, TAP hydrogel (TAPH), M-TAP, and M-TAP hydrogel (MTAPH) against Enterococcus faecalis. METHODS: The agar well diffusion method was used to assess the antibiotic sensitivity of the following intracanal medicaments: TAP (ciprofloxacin, metronidazole, and minocycline) mixed in a ratio of 1: 1: 1; TAPH, M-TAP (ciprofloxacin, metronidazole, and amoxicillin), M-TAPH and plain hydrogel. Each tested medicament was individually evaluated for its antimicrobial activity against Enterococcus faecalis. Structural and topographical characterisation were analysed using a Scanning Electron Microscope (SEM) and interpreted using ImageJ software. A microdilution broth test was performed to examine the minimum inhibitory concentration and minimum bactericidal concentration (MBC) of M-TAP and TAP. RESULTS: Except for the plain hydrogel, M-TAP and hydrogel and TAP and hydrogel showed significantly varied inhibitory zones at different concentrations. M-TAPH showed the highest mean zone of inhibition of 21.6, 33.33 and 38.0 mm at a concentration of 1, 5, and 10 mg/mL when compared to TAPH, which showed a mean zone of inhibition of 3.3 mm,12.3 mm, 21.3 mm at the respective concentrations. The MIC study shows that more than 75% of Enterococcus faecalis growth was inhibited by M-TAP at a concentration of 5 µg/mL, whereas TAP showed inhibition at a concentration of 35 µg/mL. MBC results indicate that almost 99.9% of the bacterial population was killed at a concentration of 100 µg/mL (10-1) for TAP and 10 µg/mL (10-2) for M-TAP. CONCLUSION: The antibacterial efficacy of M-TAP was significantly higher than TAP. Application of M-TAP at lower doses is advised to overcome the disadvantages seen with TAP.
Assuntos
Anti-Infecciosos , Hidrazonas , Metronidazol , Tiofenos , Metronidazol/farmacologia , Enterococcus faecalis , Hidrogéis/farmacologia , Antibacterianos/farmacologia , Ciprofloxacina , Bacitracina , Polimixina B , FramicetinaRESUMO
Bacitracin is an antimicrobial used in the feed or water of poultry in the U.S. for the prevention, treatment, and control of clostridial diseases such as necrotic enteritis. Concern has been raised that bacitracin can select for antimicrobial-resistant bacteria that can be transmitted to humans and subsequently cause disease that is more difficult to treat because of the resistance. The objective of the present study was to perform a quantitative risk assessment (QRA) to estimate the potential risk in the U.S. of human infection with antimicrobial-resistant Enterococcus faecalis and E. faecium derived from chicken and turkey products as a result of bacitracin usage in U.S. poultry. The modeling approach estimated the annual number of healthcare-associated enterococcal infections in the U.S. that would be resistant to antimicrobial therapy and that would be derived from poultry sources because of bacitracin use in poultry. Parameter estimates were developed to be "maximum risk" to overestimate the risk to humans. While approximately 60% of E. faecalis and E. faecium derived from poultry were predicted to possess bacitracin resistance based on the presence of the bcrABDR gene locus, very few human-derived isolates possessed this trait. Furthermore, no vancomycin or linezolid-resistant strains of E. faecalis or E. faecium were detected in poultry sources between the years 2002 and 2019. The model estimated the number of antimicrobial-resistant E. faecalis and E. faecium cases per year that might resist therapy due to bacitracin use in poultry as 0.86 and 0.14, respectively, which translates to an annual risk estimate for E. faecalis of less than 1 in 350 million and for E. faecium of less than 1 in 2 billion for members of the U.S. population. Even with the use of risk-maximizing assumptions, the results indicate that there is a high probability that the use of bacitracin according to label instructions in U.S. poultry presents a negligible risk to human health.
Assuntos
Antibacterianos , Bacitracina , Farmacorresistência Bacteriana , Bacitracina/farmacologia , Animais , Humanos , Antibacterianos/farmacologia , Medição de Risco , Enterococcus faecalis/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Aves Domésticas , Galinhas , Enterococcus faecium/efeitos dos fármacosRESUMO
The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.
Assuntos
Antibacterianos , Bacitracina , Antibacterianos/química , Testes de Sensibilidade Microbiana , BactériasRESUMO
The poultry industry is evolving towards antibiotic-free production to meet market demands and decelerate the increasing spread of the antimicrobial resistance. The growing need for antibiotic free products has challenged producers to decrease or completely stop using antimicrobials as feed supplements in broiler diet to improve feed efficiency, growth rate, and intestinal health. Natural feed additives (e.g., probiotics and phytobiotics) are promising alternatives to substitute antimicrobial growth promoters. The goal of our study was to characterize the effects of a Probiotic and an Essential Oils blend on broilers' performance and perform a time-series analysis to describe their excreta microbiome. A total of 320 Cobb 500 (1-day-old) chicks were raised for 21 d in 32 randomly allocated cages. Treatments consisted of 4 experimental diets: a basal diet, and a basal diet mixed with an Antibiotic (bacitracin methylene disalicylate), an essential oils blend (oregano oil, rosemary, and red pepper), or a Probiotic (Bacillus subtilis). Body weight (on 1, 10, and 21d), and feed intake (10d and 21d) were recorded and feed conversion ratio was calculated. Droppings were collected daily (1-21d) to characterize broilers' excreta microbiota by targeted sequencing of the bacterial 16S rRNA gene. The Probiotic significantly improved feed conversion ratio for starter phase 1 to 10d (P = 0.03), grower phase 10 to 21d (P = 0.05), and total period 1 to 21d (P = 0.01) compared to the Antibiotic. Feed supplements did not affect alpha diversity but did impact microbial beta diversity (P < 0.01). Age also impacted microbiome turnover as differences in alpha and beta diversity were detected. Furthermore, when compared to the basal diet, the probiotic and antibiotic significantly impacted relative abundance of Bifidobacterium (log2 fold change -1.44, P = 0.03), Intestinimonas (log2 fold change 0.560, P < 0.01) and Ligilactobacillus (log2 fold change -1.600, P < 0.01). Overall, Probiotic supplementation but not essential oils supplementation positively impacted broilers' growth performance by directly causing directional shifts in broilers' excreta microbiota structure.
Assuntos
Ração Animal , Antibacterianos , Galinhas , Dieta , Suplementos Nutricionais , Óleos Voláteis , Probióticos , Salicilatos , Animais , Galinhas/crescimento & desenvolvimento , Galinhas/microbiologia , Ração Animal/análise , Probióticos/administração & dosagem , Probióticos/farmacologia , Dieta/veterinária , Suplementos Nutricionais/análise , Óleos Voláteis/administração & dosagem , Óleos Voláteis/farmacologia , Antibacterianos/farmacologia , Antibacterianos/administração & dosagem , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Bacitracina/farmacologia , Bacitracina/administração & dosagem , Distribuição Aleatória , Bacillus subtilis/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Masculino , Óleos de Plantas/farmacologia , Óleos de Plantas/administração & dosagemRESUMO
BACKGROUND: The use of irrigation with bacitracin-containing solution is common among surgeons, as it was widely thought to have antibacterial properties and prevent postoperative infection. Current literature, however, suggests that antibiotic-containing irrigation confers little added benefit. On January 31, 2020, the Food and Drug Administration instituted a ban on bacitracin-containing irrigation for operative use. This study aimed to determine whether bacitracin has a beneficial effect on postoperative infection rates by analyzing infection rates before and after the Food and Drug Administration ban on bacitracin irrigation. METHODS: A single-institution retrospective chart review was conducted. Eligible patients underwent implant-based breast reconstruction after mastectomy from October 1, 2016, to July 31, 2022. Procedure date, reconstruction type, patient comorbidities, use of bacitracin irrigation, postoperative infection, and secondary outcomes were collected. Univariate and multivariable logistic regression analyses were performed. RESULTS: A total of 188 female patients were included in the study. Bacitracin use did not protect against infection in univariate or multivariable analysis. Age greater than 50 years was associated with an increased risk of postoperative infection ( P = 0.0366). The presence of comorbidities, smoker status, neoadjuvant therapy treatment before surgery, implant placement, and laterality were all not significantly associated with postoperative infection development. CONCLUSIONS: The results of this study demonstrate a lack of association between bacitracin use and postoperative infection. Additional research into the optimal antibiotic for perioperative irrigation is needed, as bacitracin is not encouraged for use.
Assuntos
Implantes de Mama , Neoplasias da Mama , Mamoplastia , Humanos , Feminino , Pessoa de Meia-Idade , Bacitracina/uso terapêutico , Estudos Retrospectivos , Neoplasias da Mama/complicações , Mastectomia/efeitos adversos , Antibacterianos/uso terapêutico , Mamoplastia/métodos , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Infecção da Ferida Cirúrgica/etiologia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/prevenção & controle , Implantes de Mama/efeitos adversosRESUMO
Haemophilus influenzae is an important pathogen able to cause various forms of respiratory and invasive disease. To provide high sensitivity for detection, culture media must inhibit growth of residential flora from the respiratory tract. This study aimed to identify and compare the diagnostic and economic advantages of using bacitracin containing selective agar (SEL) or oleandomycin disk supplemented chocolate agar (CHOC). Growth and semi-quantitative abundance of H. influenzae and growth suppression of residential flora was prospectively assessed in a 28-week period. H. influenzae was identified in 164 (5 %) of all included samples: CHOC and SEL, CHOC only, and SEL only were positive in 95, 24, and 45 cases. Diagnostic superiority of SEL was primarily attributable to the results of throat swabs. However, on average, 200 had to be spent for the detection of each additional isolate that was recovered only because of additional incubation on SEL.
Assuntos
Bacitracina , Chocolate , Humanos , Ágar , Bacitracina/farmacologia , Haemophilus influenzae , Oleandomicina , Meios de CulturaRESUMO
Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. σM responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall-inhibiting drugs, such as bacitracin and cefuroxime, induce the σM-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics, we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which σM-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes involved in cell wall homeostasis.IMPORTANCEPeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the σM regulon, which is critical for intrinsic antibiotic resistance. The σM-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Furthermore, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the undecaprenyl-pyrophosphate lipid carrier that sustains peptidoglycan synthesis.
Assuntos
Bacillus subtilis , Bacitracina , Bacitracina/farmacologia , Bacitracina/metabolismo , Bacillus subtilis/genética , Peptidoglicano/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismo , Óperon , Hidrolases/metabolismo , Lipídeos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Mast cells (MCs) are primary effector cells involved in immediate allergic reactions. Mas-related G protein-coupled receptor-X2 (MrgX2), which is highly expressed on MCs, is involved in receptor-mediated drug-induced pseudo-anaphylaxis. Many small-molecule drugs and peptides activate MrgX2, resulting in MC activation and allergic reactions. Although small-molecule drugs can be identified using existing MrgX2 ligand-screening systems, there is still a lack of effective means to screen peptide ligands. In this study, to screen for peptide drugs, the MrgX2 high-affinity endogenous peptide ligand substance P (SP) was used as a recognition group to design a fluorescent peptide probe. Spectroscopic properties and fluorescence imaging of the probe were assessed. The probe was then used to screen for MrgX2 agonists among peptide antibiotics. In addition, the effects of peptide antibiotics on MrgX2 activation were investigated in vivo and in vitro. The environment-sensitive property of the probe was revealed by the dramatic increase in fluorescence intensity after binding to the hydrophobic ligand-binding domain of MrgX2. Based on these characteristics, it can be used for in situ selective visualization of MrgX2 in live cells. The probe was used to screen ten types of peptide antibiotics, and we found that caspofungin and bacitracin could compete with the probe and are hence potential ligands of MrgX2. Pharmacological experiments confirmed this hypothesis; caspofungin and bacitracin activated MCs via MrgX2 in vitro and induced local anaphylaxis in mice. Our research can be expected to provide new ideas for screening MrgX2 peptide ligands and reveal the mechanisms of adverse reactions caused by peptide drugs, thereby laying the foundation for improving their clinical safety.
Assuntos
Anafilaxia , Hipersensibilidade a Drogas , Camundongos , Animais , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/metabolismo , Ligantes , Bacitracina/metabolismo , Bacitracina/farmacologia , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Caspofungina/metabolismo , Caspofungina/farmacologia , Peptídeos/farmacologia , Antibacterianos/farmacologia , Mastócitos/metabolismo , Degranulação Celular/fisiologiaRESUMO
Bacitracin is a broad-spectrum antibiotics mainly produced by Bacillus, and is used as veterinary medicine in the fields of livestock and poultry breeding. Insufficient supply of precursor amino acids might be an important factor that hinders high-level microbial production of bacitracin. We investigated the effect of strengthening L-cysteine supply on bacitracin production by an industrial bacitracin producer, Bacillus licheniformis DW2. Overexpression of cysK encoding L-cysteine synthase led to a 9.17% increase of the bacitracin titer. Moreover, overexpression of cysE encoding L-serine acetyltransferase and cysP encoding thiosulfate/sulfate intracellular transporter increased the bacitracin titers by 7.23% and 8.52%, respectively. Moreover, overexpression of a putative cystine importer TcyP led to a 29.19% increase of intracellular L-cysteine, and bacitracin titer was increased by 7.79%. Subsequently, the strong promoter PbacA was used to replace the promoters of genes cysP, cysE and tcyP in strain DW2::ysK, respectively. The resulted strain CYS4 (DW2::cysK-PbacA-(cysP)-PbacA(cysE)- PbacA(tcyP) produced 910.02 U/mL bacitracin, which was 21.10% higher than that of the original strain DW2 (747.71 U/mL). Together with the experiments in 3 L fermenters, this research demonstrated that enhancing intracellular L-cysteine supply is an effective strategy to increase bacitracin production of B. licheniformis.
Assuntos
Aminoácidos , Bacillus licheniformis/genética , Bacitracina , Cisteína , Engenharia MetabólicaRESUMO
The objective of this study was to evaluate the effects of probiotics and synbiotics on the performance and Enterobacteriaceae count of broiler chickens. A total of 640 one-day-old male broiler chicks were distributed in a completely randomized design with four treatments and eight replicates with 20 birds each. The treatments were: ration with performance enhancer (zinc bacitracin; positive control); ration without performance enhancer and probiotic/synbiotic (negative control); ration with probiotics; and ration with synbiotics. At 35 days, five birds from each treatment were euthanized and intestinal contents were harvested for determining the Enterobacteriaceae count. The performance data and average colony-forming units (CFUs) transformed as log CFU/g were subjected to analysis of variance and Tukey's test. The effects of probiotics and synbiotics were observed in the initial phase, with supplemented birds exhibiting comparable weight gain to those supplemented with bacitracin. No effect of the treatment on broiler performance was observed after 42 days. The enterobacterial count was comparable among all experimental treatments. Supplementation with probiotics and synbiotics did not compromise the performance of broilers and did not alter the Enterobacteriaceae count.(AU)
Objetivou-se avaliar o efeito do probiótico e do simbiótico sobre o desempenho e a contagem de Enterobacteriaceae em frangos. Foram utilizados 640 pintos de corte, machos, de um dia de idade, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos, oito repetições com 20 aves cada. Os tratamentos foram: ração com melhorador de desempenho (bacitracina de zinco) (controle positivo); ração sem melhorador de desempenho e sem probiótico/simbiótico (controle negativo); ração com probiótico e ração com simbiótico. Aos 35 dias, cinco aves por tratamento foram eutanasiadas para retirada de conteúdo intestinal e determinação de Enterobacteriaceae. As médias das unidades formadoras de colônias, transformadas em log/UFC/g, e de desempenho foram submetidas à análise de variância e comparadas pelo teste Tukey. Foi observado efeito do probiótico e do simbiótico na fase inicial, sendo que aves apresentaram os mesmos resultados de ganho de peso e de peso corporal que o grupo de aves alimentado com bacitracina. Aos 42 dias, não houve efeito dos tratamentos sobre o desempenho. Aves que não receberam nenhum aditivo não apresentaram maior contagem de enterobactérias, sendo semelhantes aos demais tratamentos. A adição do probiótico e do simbiótico não compromete o desempenho dos frangos e não altera a contagem de Enterobacteriaceae.(AU)
Assuntos
Animais , Bacitracina/administração & dosagem , Galinhas/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Enterobacteriaceae , Escherichia coli , MicrobiotaRESUMO
Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.
Assuntos
Bacillus licheniformis , Metabolismo , Bacitracina , Metabolismo Energético , Genética , Microbiologia Industrial , MétodosRESUMO
Abstract: Background: Topical antimicrobial drugs are indicated for limited superficial pyodermitis treatment, although they are largely used as self-prescribed medication for a variety of inflammatory dermatoses, including atopic dermatitis. Monitoring bacterial susceptibility to these drugs is difficult, given the paucity of laboratory standardization. Objective: To evaluate the prevalence of Staphylococcus aureus topical antimicrobial drug resistance in atopic dermatitis patients. Methods: We conducted a cross-sectional study of children and adults diagnosed with atopic dermatitis and S. aureus colonization. We used miscellaneous literature reported breakpoints to define S. aureus resistance to mupirocin, fusidic acid, gentamicin, neomycin and bacitracin. Results: A total of 91 patients were included and 100 S. aureus isolates were analyzed. All strains were methicillin-susceptible S. aureus. We found a low prevalence of mupirocin and fusidic acid resistance (1.1% and 5.9%, respectively), but high levels of neomycin and bacitracin resistance (42.6% and 100%, respectively). Fusidic acid resistance was associated with more severe atopic dermatitis, demonstrated by higher EASI scores (median 17.8 vs 5.7, p=.009). Our results also corroborate the literature on the absence of cross-resistance between the aminoglycosides neomycin and gentamicin. Conclusions: Our data, in a southern Brazilian sample of AD patients, revealed a low prevalence of mupirocin and fusidic acid resistance of S. aureus atopic eczema colonizer strains. However, for neomycin and bacitracin, which are commonly used topical antimicrobial drugs in Brazil, high levels of resistance were identified. Further restrictions on the use of these antimicrobials seem necessary to keep resistance as low as possible.