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1.
Microbiology (Reading) ; 170(2)2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38373028

RESUMO

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.


Assuntos
Proteínas de Transporte , Mycobacterium tuberculosis , ATPases do Tipo-P , Mycobacterium tuberculosis/genética , Levofloxacino , Norfloxacino , Antibacterianos/farmacologia , Oxacilina
2.
Ann Clin Microbiol Antimicrob ; 23(1): 7, 2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38245727

RESUMO

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.


Assuntos
Anti-Infecciosos , Staphylococcus aureus Resistente à Meticilina , Nisina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Oxacilina/farmacologia , Nisina/farmacologia , Nisina/uso terapêutico , Staphylococcus epidermidis , Staphylococcus aureus Resistente à Meticilina/genética , Peptídeos Antimicrobianos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Anti-Infecciosos/farmacologia , Staphylococcus , Biofilmes , Testes de Sensibilidade Microbiana
3.
Clin Chim Acta ; 554: 117704, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38185284

RESUMO

BACKGROUND: Systemically administered antibiotics are thought to penetrate the wounds more effectively during negative pressure wound therapy (NPWT).To test this hypothesis total and free antibiotic concentrations were quantified in serum and wound exudate. METHODS: UHPLC-MS/MS methods were developed and validated for the determination of ceftazidime, cefepime, cefotaxime, cefuroxime, cefazolin, meropenem, oxacillin, piperacillin with tazobactam, clindamycin, ciprofloxacin, sulfamethoxazole/trimethoprim (cotrimoxazole), gentamicin, vancomycin, and linezolid. The unbound antibiotic fraction was obtained by ultrafiltration using a Millipore Microcon-30kda Centrifugal Filter Unit. Analysis was performed on a 1.7-µm Acquity UPLC BEH C18 2.1 × 100-mm column with a gradient elution. RESULTS: The validation was performed for serum, exudates and free fractions. For all matrices, requirements were met regarding linearity, precision, accuracy, limit of quantitation, and matrix effect. The coefficient of variation was in the range of 1.2-13.6%.and the recovery 87.6-115.6%, respectively. Among the 29 applications of antibiotics thus far, including vancomycin, clindamycin, ciprofloxacin, oxacillin, cefepime, cefotaxime, cotrimoxazole, and gentamicin, total and free antibiotic concentrations in serum and exudate were correlated. CONCLUSION: This method can accurately quantify the total and free concentrations of 16 antibiotics. Comparison of concentration ratios between serum and exudates allows for monitoring individual antibiotics' penetration capacity in patients receiving NPWT.


Assuntos
Tratamento de Ferimentos com Pressão Negativa , Infecção dos Ferimentos , Humanos , Antibacterianos , Espectrometria de Massas em Tandem/métodos , Cefepima , Vancomicina , Combinação Trimetoprima e Sulfametoxazol , Clindamicina , Esternotomia , Cromatografia Líquida/métodos , Ciprofloxacina , Cefotaxima , Oxacilina , Gentamicinas , Exsudatos e Transudatos , Cromatografia Líquida de Alta Pressão/métodos
4.
PLoS Biol ; 22(1): e3002457, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38175839

RESUMO

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.


Assuntos
Daptomicina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus/genética , Vancomicina , Linezolida/uso terapêutico , Teicoplanina/uso terapêutico , Prevalência , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/tratamento farmacológico , Oxacilina/uso terapêutico , Mutação , Gentamicinas
5.
Artigo em Inglês | MEDLINE | ID: mdl-38160527

RESUMO

Cloxacillin and oxacillin are group M penicillins. The therapeutic monitoring of plasma concentrations of these antibiotics and those of their hydroxymethylated metabolites is of great clinical interest, especially in the choice of an adequate dosage allowing an effective treatment while limiting the occurrence of undesirable effects and the development of bacterial resistance. In this context, we conducted this work aiming at developing and validating a method allowing the determination of cloxacillin and oxacillin as well as the identification of their active metabolites in different biological matrices (CSF and plasma) using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. To do this, we carried out several optimisation tests. Subsequently, we validated our method according to the latest bioanalytical validation recommendations of the European Medicines Agency. The validation results showed that our method is specific and sensitive. We obtained good linearity in the range 0.5 to 100 µg/mL with correlation coefficients above 0.995. The lower limit of quantification was 0.5 µg/mL for each analyte. The method was found to be accurate with repeatability and reproducibility coefficients of variation below 15 %. Our method is also accurate with bias values below 15 %. Recovery values ranged from 87 % to 95 %. Finally, we were able to apply our method to the therapeutic monitoring of the analysed molecules and to identify their active metabolites. Our results suggest that LC-MS shows superiority in the therapeutic monitoring of these antibiotics due to the superiority of specificity shown by this method. This assay method can be routinely used for the daily plasma assays of patients treated with these antibiotics in the context of therapeutic monitoring.


Assuntos
Cloxacilina , Oxacilina , Humanos , Cloxacilina/análise , Monitoramento de Medicamentos/métodos , Reprodutibilidade dos Testes , Antibacterianos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos
6.
Nat Commun ; 14(1): 7065, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37923729

RESUMO

Staphylococcus pseudintermedius is historically understood as a prevalent commensal and pathogen of dogs, though modern clinical diagnostics reveal an expanded host-range that includes humans. It remains unclear whether differentiation across S. pseudintermedius populations is driven primarily by niche-type or host-species. We sequenced 501 diagnostic and commensal isolates from a hospital, veterinary diagnostic laboratory, and within households in the American Midwest, and performed a comparative genomics investigation contrasting human diagnostic, animal diagnostic, human colonizing, pet colonizing, and household-surface S. pseudintermedius isolates. Though indistinguishable by core and accessory gene architecture, diagnostic isolates harbor more encoded and phenotypic resistance, whereas colonizing and surface isolates harbor similar CRISPR defense systems likely reflective of common household phage exposures. Furthermore, household isolates that persist through anti-staphylococcal decolonization report elevated rates of base-changing mutations in - and parallel evolution of - defense genes, as well as reductions in oxacillin and trimethoprim-sulfamethoxazole susceptibility. Together we report parallel niche-specific bolstering of S. pseudintermedius defense mechanisms through gene acquisition or mutation.


Assuntos
Doenças do Cão , Infecções Estafilocócicas , Humanos , Animais , Cães , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/veterinária , Staphylococcus/genética , Oxacilina , Mecanismos de Defesa , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana
7.
Vet Med Sci ; 9(6): 2885-2892, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37792167

RESUMO

BACKGROUND: Literature is scarce on the occurrence of bovine mastitis and antimicrobial resistance among dairy animals kept by pastoralists in the Kenya. OBJECTIVES: A cross-sectional study was carried out to investigate the prevalence and risk factors of subclinical mastitis (SCM) and evaluate the antibiotic sensitivity of bacteria isolated from dairy cattle kept by farmers in Kajiado Central sub-county, Kenya. METHODS: A total of 202 lactating cows from 40 farms were sampled. Milk from the cows was screened for SCM using the California mastitis test, and the bacteria present in the milk samples were determined using standard bacteriological methods. The sensitivity of the isolated coagulase-negative staphylococci (CNS) and Staphylococcus aureus against antibiotics was tested using the Kirby-Bauer disk diffusion method. RESULTS: The prevalence of SCM at quarter- and cow-level was 31.7% and 53%, respectively. The prevalence of SCM was significantly higher (p < 0.05) in exotic breeds of cattle and those kept under an extensive system of production. A total of 19 bacterial species were isolated with the majority being CNS (40.1%), S. aureus (15.8%) and Micrococcus spp. (10.4%). S. aureus isolates showed varied resistance to the tested antibiotics with the highest resistance being against ceftazidime (75%), amoxycillin (50%) and streptomycin (46.9%). Several S. aureus isolates were resistant to oxacillin (34.4%) and cefoxitin (12.5%). CNSs were more resistant against ceftazidime (79.1%), amoxycillin (34.6%) and oxacillin (32.1%). Majority (92%-100%) of the Staphylococcus spp. were highly sensitive to ciprofloxacin a fluoroquinolone and augmentin. CONCLUSIONS: The high prevalence of SCM and bacteria resistant to antibiotics shows a need for animal health professionals and farmers to develop strategies for the management of mastitis and antibiotic resistance in dairy cows in the study area.


Assuntos
Doenças dos Bovinos , Mastite Bovina , Bovinos , Animais , Feminino , Staphylococcus aureus , Leite/microbiologia , Lactação , Ceftazidima , Prevalência , Mastite Bovina/epidemiologia , Mastite Bovina/microbiologia , Quênia/epidemiologia , Estudos Transversais , Staphylococcus , Antibacterianos/farmacologia , Bactérias , Oxacilina , Fatores de Risco , Amoxicilina
8.
J Microorg Control ; 28(3): 101-107, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37866891

RESUMO

Considering the lack of detailed research on the antibacterial mechanism of polyoxometalates, we examined the synergistic effect of novel bulky mixed Ti/W hetero-polyoxometalates (K9.5H2.5 ï¼»α-Ge2Ti4W20O78]・ 29H2O; αTi4, K9H5 ï¼»α-Ge2Ti6W18O77]・16H2O; αTi6, K23H5ï¼»α-Ge4Ti12W36O154]・39H2O; αTi12, K9H5 ï¼»ß-Ge2Ti6W18O77]・ 45H2O; ßTi6) with the antibiotic oxacillin against vancomycin intermediate-resistant Staphylococcus aureus (VISA) using fractional inhibitory concentration (FIC) index and growth curve in this study. All polyoxometalates used in this study showed remarkable synergistic effects with oxacillin. Its synergistic antibacterial mechanism was examined using reverse transcription PCR (RT-PCR) and penicillin binding protein-2' (PBP2') latex agglutination test. The results suggested that these polyoxometalates did not inhibit mecA gene transcription but resulted in PBP2' protein malfunction. From these results, we concluded that the substances producing resistance in VISA were affected by polyoxometalates depending on their molecular size, facilitating a synergistic antibacterial effect with oxacillin.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Oxacilina , Oxacilina/farmacologia , Vancomicina/farmacologia , Sinergismo Farmacológico , Titânio/farmacologia , Antibacterianos/farmacologia , Staphylococcus aureus
9.
Eur J Med Chem ; 261: 115823, 2023 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-37839345

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe public health challenges throughout the world, and the multi-drug resistance (MDR) of MRSA to antibiotics necessitates the development of more effective antibiotics. Natural 2,4-diacetylphloroglucinol (DAPG), produced by Pseudomonas, displays moderate inhibitory activity against MRSA. A series of DAPG derivatives was synthesized and evaluated for their antibacterial activities, and some showed excellent activities (MRSA MIC = 0.5-2 µg/mL). Among these derivatives, 7g demonstrated strong antibacterial activity without resistance development over two months. Mechanistic studies suggest that 7g asserted its activity by targeting bacterial cell membranes. In addition, 7g exhibited significant synergistic antibacterial effects with oxacillin both in vitro and in vivo, with a tendency to eradicate MRSA biofilms. 7g is a promising lead for the treatment of MRSA.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Sinergismo Farmacológico , Antibacterianos/farmacologia , Oxacilina/farmacologia , Testes de Sensibilidade Microbiana
10.
Antimicrob Agents Chemother ; 67(10): e0071623, 2023 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-37655923

RESUMO

Acquisition of PBP2a (encoded by the mec gene) is the key resistance mechanism to ß-lactams in Staphylococcus aureus. The mec gene can be easily detected by PCR assays; however, these tools will miss mec-independent oxacillin resistance. This phenotype is mediated by mutations in cell wall metabolism genes that can be acquired during persistent infections under prolonged antibiotic exposure. The complex case presented by Hess et al. (Antimicrob Agents Chemother 67:e00437-23, 2023, https://doi.org/10.1128/aac.00437-23) highlights the diagnostic and therapeutic challenges in the management of mec-independent oxacillin resistance.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Oxacilina/farmacologia , Oxacilina/uso terapêutico , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo
11.
J Med Microbiol ; 72(9)2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37707372

RESUMO

Introduction. Antibiotic resistance is a major threat to public health, particularly with methicillin-resistant Staphylococcus aureus (MRSA) being a leading cause of antimicrobial resistance. To combat this problem, drug repurposing offers a promising solution for the discovery of new antibacterial agents.Hypothesis. Menadione exhibits antibacterial activity against methicillin-sensitive and methicillin-resistant S. aureus strains, both alone and in combination with oxacillin. Its primary mechanism of action involves inducing oxidative stress.Methodology. Sensitivity assays were performed using broth microdilution. The interaction between menadione, oxacillin, and antioxidants was assessed using checkerboard technique. Mechanism of action was evaluated using flow cytometry, fluorescence microscopy, and in silico analysis.Aim. The aim of this study was to evaluate the in vitro antibacterial potential of menadione against planktonic and biofilm forms of methicillin-sensitive and resistant S. aureus strains. It also examined its role as a modulator of oxacillin activity and investigated the mechanism of action involved in its activity.Results. Menadione showed antibacterial activity against planktonic cells at concentrations ranging from 2 to 32 µg ml-1, with bacteriostatic action. When combined with oxacillin, it exhibited an additive and synergistic effect against the tested strains. Menadione also demonstrated antibiofilm activity at subinhibitory concentrations and effectively combated biofilms with reduced sensitivity to oxacillin alone. Its mechanism of action involves the production of reactive oxygen species (ROS) and DNA damage. It also showed interactions with important targets, such as DNA gyrase and dehydroesqualene synthase. The presence of ascorbic acid reversed its effects.Conclusion. Menadione exhibited antibacterial and antibiofilm activity against MRSA strains, suggesting its potential as an adjunct in the treatment of S. aureus infections. The main mechanism of action involves the production of ROS, which subsequently leads to DNA damage. Additionally, the activity of menadione can be complemented by its interaction with important virulence targets.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Oxacilina , Oxacilina/farmacologia , Vitamina K 3/farmacologia , Meticilina , Staphylococcus aureus , Espécies Reativas de Oxigênio , Antibacterianos/farmacologia , Biofilmes
12.
BMC Microbiol ; 23(1): 267, 2023 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-37742008

RESUMO

BACKGROUND: Peritonitis is the most important complication of peritoneal dialysis (PD) and coagulase-negative staphylococci (CNS) are a frequent cause of dialysis-related infections. The association between SCCmec typing with psm-mec positivity in staphylococci and PD-related infections has not been identified. We aim to investigate the molecular epidemiology of CNS isolated from PD-peritonitis in a single Chinese center, focusing on the genetic determinants conferring methicillin resistance. METHODS: We collected 10 genetically unrelated CNS isolates from 10 patients with CNS PD-related peritonitis. The patients were divided into two groups based on the results of MIC to oxacillin: the methicillin-resistant CNS (MRCNS) and methicillin-sensitive CNS (MSCNS) groups. The biofilm formation group (BFG) and the non-biofilm formation group (NBFG) were used as the control groups. Phenotypic and molecular methods were used to analyze SCCmec types I, II and III, associated genes and biofilm formation and the existence of psm-mec. The demographic data and clinical indicators were collected. RESULTS: Ten CNS PD-related peritonitis patients were enrolled for this study. There were 6 MRCNS and 4 MRCNS isolates. SCCmec types were fully determined in 10 isolates. Seven staphylococci (70%) carried SCCmec, of which 4 isolates carried single SCCmec type I (40%) and 3 isolates had multiple SCCmec elements (I + III). Of the 6 MRCNS isolates, 3 carried SCCmec type I (50%) and 2 isolates carried SCCmec type I + III (33.3%). A high diversity of ccr types, mec complexes and ccr-mec complex combinations was identified among the 10 CNS isolates. The psm-mec gene was detected in 2/10 (20%) CNS isolates. There was no mutation in the psm-mec gene. CONCLUSIONS: The majority of isolates were hospital-associated isolates. Furthermore, 2 psm-mec positive isolates were MRCNS in the NBFG. The PD patients frequent exposure to hospital would be the main risk factor. The presence of the psm-mec signal in the spectra of the MRCNS tested here demonstrates the presence of certain SCCmec cassettes that convey methicillin resistance.


Assuntos
Diálise Peritoneal , Peritonite , Infecções Estafilocócicas , Humanos , Staphylococcus/genética , Coagulase/genética , Oxacilina , Diálise Peritoneal/efeitos adversos , Infecções Estafilocócicas/epidemiologia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia
13.
Clin Lab ; 69(9)2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37702668

RESUMO

BACKGROUND: We aimed to analyze the infection characteristics of multidrug-resistant organisms (MDROs) and their resistance to antibiotics in patients with diabetic foot and provide guidance for the use of antibiotics in clinical practice. METHODS: The clinical data of 737 patients with diabetic foot who were hospitalized at our institution from February 2020 to January 2023 were retrospectively analyzed. Purulent secretions were collected from the patient's ulcers and bacterial culture, identification, and drug susceptibility tests were performed. The multidrug resistance (MDR) rate of different bacteria, composition ratio of MDROs, drug resistance characteristics of the main MDROs, distribution characteristics of multidrug-resistant gram-positive cocci and gram-negative bacilli in patients with different Wagner Grades, MDR in patients with different Wagner Grades, bacterial infection rate, and other indicators were analyzed. RESULTS: Pathogenic bacteria from wound secretions of 505 patients were cultured, and 509 pathogenic bacteria were obtained. Among the pathogenic bacteria, 225 strains were gram-positive cocci, of which 172 (76.44%) were MDROs, and 284 were gram-negative bacilli, of which 232 (81.69%) were MDROs. Among the 404 multidrug-resistant strains, gram-positive cocci and gram-negative bacilli accounted for 42.57% and 57.43%, respectively. The top five dominant MDROs were Staphylococcus aureus (18.56%), coagulase-negative Staphylococcus (10.89%), Escherichia coli (10.15%), Proteus mirabilis (8.17%), Proteus vulgaris (6.19%), and Pseudomonas aeruginosa (6.19%). Staphylococcus aureus and coagulase-negative Staphylococcus were more resistant to penicillin, oxacillin, erythromycin, azithromycin, and clarithromycin, with resistance rates of 50.0 - 95.0%. The resistance rates of E. coli to ampicillin, cefazolin, cefuroxime, ceftriaxone, and cefepime were > 75%. With an increase in Wagner Grade, the proportion of gram-negative bacilli among the pathogenic bacteria of MDROs increased significantly (p < 0.05), as did the infection rate of MDROs in patients with diabetic foot (χ2 = 14.045, p < 0.05). CONCLUSIONS: MDROs in patients with diabetic foot are mainly gram-negative bacilli, followed by gram-positive cocci. The drug resistance of various MDROs varies greatly. With the increase in Wagner Grade and MDR of diabetic foot patients, the infection rate of drug-resistant bacteria has increased significantly. Therefore, clinicians should use drugs rationally according to drug sensitivity results.


Assuntos
Diabetes Mellitus , Pé Diabético , Infecções Estafilocócicas , Humanos , Farmacorresistência Bacteriana Múltipla , Pé Diabético/tratamento farmacológico , Coagulase , Escherichia coli , Estudos Retrospectivos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Oxacilina , Staphylococcus
14.
Microb Pathog ; 184: 106342, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37704062

RESUMO

Antimicrobial resistance is a global issue that limits therapeutic options for infections. S. aureus being a member of the ESKAPE group is capable of "escaping" the biocidal action of antimicrobial agents. There are phenotypic and genotypic methods used for the identification of antibiotic resistant genes harboring S. aureus but these methods do not always show concordant results. To address these discrepancies, a total of 335 equine nasal swab samples from four districts of Punjab were collected using a convenient sampling technique. These samples were first subjected to common microbial techniques to identify S. aureus. The disc diffusion assay was performed for the phenotypic identification of antibiotic resistant S. aureus by using discs of oxacillin, penicillin, vancomycin, gentamycin, and tetracycline. After this, PCR was performed by targeting mecA, blaZ, vanB, aaca-aphd, and tetK genes for genotypic identification of respective antibiotic-resistant S. aureus. Phenotypic discrepancies (number of antibiotic resistant isolates found from disc diffusion who appeared to be negative for the resistant gene), and genotypic discrepancies (number of antibiotic sensitive isolates found from disc diffusion who appeared to be positive for the resistant gene) were calculated. The discrepancy ratio for mecA, blaZ, vanB, aaca-aphd, and tetK genes were 3.09, 1.96, 2.67, 1.93, and 1.67 respectively. These discrepant results indicate that the absence or presence of only one gene is not a true marker of resistant or sensitive isolates. There are multiple resistance determinants and resistance mechanisms. This study also highlighted the phenomenon of silencing of antibiotic resistance determinants.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Cavalos , Antibacterianos/farmacologia , Staphylococcus aureus/genética , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Oxacilina , Infecções Estafilocócicas/veterinária
15.
PLoS Pathog ; 19(7): e1011536, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37486930

RESUMO

Central metabolic pathways control virulence and antibiotic resistance, and constitute potential targets for antibacterial drugs. In Staphylococcus aureus the role of the pentose phosphate pathway (PPP) remains largely unexplored. Mutation of the 6-phosphogluconolactonase gene pgl, which encodes the only non-essential enzyme in the oxidative phase of the PPP, significantly increased MRSA resistance to ß-lactam antibiotics, particularly in chemically defined media with physiologically-relevant concentrations of glucose, and reduced oxacillin (OX)-induced lysis. Expression of the methicillin-resistance penicillin binding protein 2a and peptidoglycan architecture were unaffected. Carbon tracing and metabolomics revealed extensive metabolic reprogramming in the pgl mutant including increased flux to glycolysis, the TCA cycle, and several cell envelope precursors, which was consistent with increased ß-lactam resistance. Morphologically, pgl mutant cells were smaller than wild-type with a thicker cell wall and ruffled surface when grown in OX. The pgl mutation reduced resistance to Congo Red, sulfamethoxazole and oxidative stress, and increased resistance to targocil, fosfomycin and vancomycin. Levels of lipoteichoic acids (LTAs) were significantly reduced in pgl, which may limit cell lysis, while the surface charge of pgl cells was significantly more positive. A vraG mutation in pgl reversed the increased OX resistance phenotype, and partially restored wild-type surface charge, but not LTA levels. Mutations in vraF or graRS from the VraFG/GraRS complex that regulates DltABCD-mediated d-alanylation of teichoic acids (which in turn controls ß-lactam resistance and surface charge), also restored wild-type OX susceptibility. Collectively these data show that reduced levels of LTAs and OX-induced lysis combined with a VraFG/GraRS-dependent increase in cell surface positive charge are accompanied by significantly increased OX resistance in an MRSA pgl mutant.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Via de Pentose Fosfato/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Oxacilina/farmacologia , Parede Celular/metabolismo , Monobactamas/metabolismo , Resistência beta-Lactâmica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Testes de Sensibilidade Microbiana
16.
Eur J Clin Microbiol Infect Dis ; 42(9): 1125-1133, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37515660

RESUMO

The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin. A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS) were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA (11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmec IVc and ST59-t1950-SCCmec V lineages. OS-MRSA were characterized by mecA promoter mutations at - 33 (C→T) or - 7 (G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried mutations in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level resistance and treatment failures.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Oxacilina/farmacologia , Staphylococcus aureus/genética , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , beta-Lactamas/farmacologia , Testes de Sensibilidade Microbiana , Proteínas de Ligação às Penicilinas/genética , Proteínas de Bactérias/genética , Infecções Estafilocócicas/microbiologia , Meticilina , Genômica
17.
Biochimie ; 214(Pt B): 165-175, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37437685

RESUMO

Lectins presents the ability to interact with glycans and trigger varied responses, including the inhibition of the development of various pathogens. Structural studies of these proteins are essential to better understand their functions. In marine sponges, so far only a few lectins have their primary structures completely determined. Thus, the objective of this work was to structurally characterize and evaluate antibacterial potential, in association with different antibiotics, of the lectin isolated from the marine sponge Aplysina lactuta (ALL). ALL is a homotetramer of 60 kDa formed by four 15 kDa-subunits. The lectin showed affinity only for the glycoproteins fetuin, asialofetuin, mucin type III, and bovine submaxillary mucin type I. The complete amino acid sequences of two isoforms of ALL, named ALL-a and ALL-b, were determined by a combination of Edman degradation and overlapped peptides sequenced by tandem mass spectrometry. ALL-a and ALL-b have 144 amino acids with molecular masses of 15,736 Da and 15,985 Da, respectively. Both structures contain conserved residues typical of the galectin family. ALL is a protein with antibacterial potential, when in association with ampicillin and oxacillin the lectin potentiates its antibiotic effect, included Methicillin-resistant Staphylococcus strains. Thus, ALL shows to be a molecule with potential for the development of new antibacterial drugs.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Poríferos , Animais , Bovinos , Antibacterianos/farmacologia , Antibacterianos/química , Galectinas , Oxacilina
18.
Microbiol Spectr ; 11(4): e0022023, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37409947

RESUMO

Staphylococcus aureus can produce ß-lactamases capable of hydrolyzing penicillins and first-generation cephalosporins. The propensity of type A and type C ß-lactamase-producing S. aureus (TAPSA and TCPSA) to hydrolyze cefazolin at a high inoculum is termed the cefazolin inoculum effect (CIE). Strains with a CIE have a theoretical risk of causing treatment failure and are unable to be detected routinely by most laboratories. We developed a high-performing yet straightforward ß-lactamase disc test that identifies and differentiates both TAPSA and TCPSA and is suitable for routine diagnostic laboratory workflows. Clinical isolates of S. aureus resistant to penicillin were identified, and their blaZ genes were sequenced. MICs were determined at low and high inocula (5 × 105 CFU/mL and 5 × 107 CFU/mL), and isolates demonstrating a CIE were characterized. A semimechanistic model was established to describe differential hydrolysis patterns, and candidate models were iteratively assessed using area-under-the-curve analysis from competitor receiver operating characteristic (ROC) curves. Biomarker thresholds were derived from Youdon index-derived optimal cutoff values. Genetic analysis of 99 isolates identified 26 TAPSA isolates and 45 TCPSA isolates. The model best differentiating TAPSA from non-TAPSA utilized cefazolin-to-cephalothin ratio analysis (sensitivity, 96.2%; specificity, 98.6%). The model best differentiating TCPSA from non-TCPSA incorporated cefazolin, cephalothin, and oxacillin (sensitivity, 88.6%; specificity, 96.6%). TAPSA and TCPSA can be differentiated using three antibiotic discs on a single agar plate. The test has potential value in typing the ß-lactamase type from isolates from patients that are candidates for or have failed cefazolin therapy. IMPORTANCE The key significance of this article is that it details a straightforward method of performing a disc test that can differentiate Staphylococcus aureus isolates that are likely to be associated with a cefazolin inoculum effect and theoretical risk of cefazolin treatment failure from isolates that are less likely to be associated with a cefazolin inoculum effect.


Assuntos
Cefazolina , Infecções Estafilocócicas , Humanos , Cefazolina/uso terapêutico , Staphylococcus aureus/genética , beta-Lactamases/genética , Cefalotina/uso terapêutico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/tratamento farmacológico , Oxacilina , Testes de Sensibilidade Microbiana
19.
Microbiol Spectr ; 11(4): e0528222, 2023 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-37395643

RESUMO

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinical threat with high morbidity and mortality. Here, we describe a new simple, rapid identification method for MRSA using oxacillin sodium salt, a cell wall synthesis inhibitor, combined with Gram staining and machine vision (MV) analysis. Gram staining classifies bacteria as positive (purple) or negative (pink) according to the cell wall structure and chemical composition. In the presence of oxacillin, the integrity of the cell wall for methicillin-susceptible S. aureus (MSSA) was destroyed immediately and appeared Gram negative. In contrast, MRSA was relatively stable and appeared Gram positive. This color change can be detected by MV. The feasibility of this method was demonstrated in 150 images of the staining results for 50 clinical S. aureus strains. Based on effective feature extraction and machine learning, the accuracies of the linear linear discriminant analysis (LDA) model and nonlinear artificial neural network (ANN) model for MRSA identification were 96.7% and 97.3%, respectively. Combined with MV analysis, this simple strategy improved the detection efficiency and significantly shortened the time needed to detect antibiotic resistance. The whole process can be completed within 1 h. Unlike the traditional antibiotic susceptibility test, overnight incubation is avoided. This new strategy could be used for other bacteria and represents a new rapid method for detection of clinical antibiotic resistance. IMPORTANCE Oxacillin sodium salt destroys the integrity of the cell wall of MSSA immediately, appearing Gram negative, whereas MRSA is relatively stable and still appears Gram positive. This color change can be detected by microscopic examination and MV analysis. This new strategy has significantly reduced the time to detect resistance. The results show that using oxacillin sodium salt combined with Gram staining and MV analysis is a new, simple and rapid method for identification of MRSA.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Testes de Sensibilidade Microbiana , Oxacilina/farmacologia , Meticilina/farmacologia , Coloração e Rotulagem , Antibacterianos/farmacologia , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia
20.
Bioorg Chem ; 138: 106644, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37302315

RESUMO

Based on the readily available 3-organyl-5-(chloromethyl)isoxazoles, a number of previously unknown water-soluble conjugates of isoxazoles with thiourea, amino acids, some secondary and tertiary amines, and thioglycolic acid were synthesized. The bacteriostatic activity of aforementioned compounds has been studied against Enterococcus durans B-603, Bacillus subtilis B-407, Rhodococcus qingshengii Ac-2784D, and Escherichia coli B-1238 microorganisms (provided by All-Russian Collection of Microorganisms, VKM). The influence of the nature of the substituents in positions 3 and 5 of the isoxazole ring on the antimicrobial activity of the obtained compounds has been determined. It is found that the highest bacteriostatic effect is observed for compounds containing 4-methoxyphenyl or 5-nitrofuran-2-yl substituents in position 3 of the isoxazole ring as well as methylene group in position 5 bearing residues of l-proline or N-Ac-l-cysteine (5a-d, MIC 0.06-2.5 µg/ml). The leading compounds showed low cytotoxicity on normal human skin fibroblast cells (NAF1nor) and low acute toxicity on mice in comparison with the well-known isoxazole-containing antibiotic oxacillin.


Assuntos
Anti-Infecciosos , Nitrofuranos , Camundongos , Humanos , Animais , Isoxazóis/farmacologia , Isoxazóis/química , Antibacterianos/farmacologia , Antibacterianos/química , Oxacilina , Testes de Sensibilidade Microbiana
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