Five
isolates (susceptible and resistant) recovered from the urine of a kidney transplant patient treated with voriconazole (VRC) 200 mg twice daily for 20 days were studied. Eight unrelated clinical ...isolates of
were exposed
to VRC 0.001 μg/ml for 30 days. Development of VRC transient resistance occurred
, and induction of permanent resistance occurred
Mostly,
and
genes were overexpressed, and a homozygous T418C mutation in the
gene was found.
Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate ...a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay.
One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined.
Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed.
FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.
Background Roux-en-Y gastric bypass (RYGB) surgery is one of the most efficient procedures for the treatment of obesity, also improving metabolic and inflammatory status, in patients with mild ...obesity. The underlying mechanisms have not been fully understood, but gut microbiota is hypothesized to play a key role. Our aim was to evaluate the association between gut microbiota changes and anthropometric, metabolic and inflammatory profiles after metabolic surgery compared with medical therapy, in type 2 diabetic (T2DM) adults with mild obesity (BMI 30-35 kg/m.sup.2). Methods DM.sup.2 was an open-label, randomised controlled clinical trial (RCT: ISRCTN53984585) with 2 arms: (i) surgical, and (ii) medical. The main outcome was gut microbiota changes after: metabolic surgery (Roux-en-Y gastric bypass--RYGB) versus standard medical therapy. Secondary outcomes included anthropometric, metabolic and inflammatory profiles. Clinical visits, blood workup, and stool samples were collected at baseline and months (M)1, 3, 6, 12. Gut microbiota was profiled using 16S rRNA targeted sequencing. Results Twenty patients were included: 10 in surgical and 10 in medical arm. Anthropometric and metabolic comparative analysis favoured RYGB over medical arm. At M12, the percentage of weight loss was 25.5 vs. 4.9% (p < 0.001) and HbA1c was 6.2 vs. 7.7% (p < 0.001) respectively. We observed a continuous increase of genus richness after RYGB up until M12. In the medical arm, genus richness ended-up being significantly lower at M12. Composition analysis indicated significant changes of the overall microbial ecosystem (permanova p = 0.004, R.sup.2 = 0.17) during the follow-up period after RYGB. There was a strong association between improvement of anthropometric/metabolic/inflammatory biomarkers and increase in microbial richness and Proteobacterial lineages. Conclusions This was the first RCT studying composite clinical, analytic, and microbiome changes in T2DM patients with class 1 obesity after RYGB versus standard medical therapy. The remarkable phenotypic improvement after surgery occurred concomitantly with changes in the gut microbiome, but at a lower level. Trial registration: ISRCTN53984585 Keywords: Diabetes mellitus, Insulin resistance, Microbiome, Roux-en-Y gastric bypass, Weight loss
Candida krusei is an important agent of opportunistic infections that often displays resistance to several antifungals. We describe here the in vivo acquisition of resistance to voriconazole (VRC) by ...C. krusei isolates recovered from a leukemia patient during a long period of VRC therapy. In order to mimic the in vivo development of VRC resistance, a susceptible C. krusei isolate was exposed daily to 1 μg/ml of VRC in vitro. Interestingly, after 5 days of exposure to VRC, a MIC of 4 μg/ml was achieved; this value remained constant after 25 additional days of treatment with VRC and also after 30 consecutive days of incubation in VRC-free medium. Our objective was to determine the associated molecular resistance mechanisms, such as expression of efflux pump genes and ERG11 gene mutations, among the resistant strains. Synergistic effects between the efflux blocker tacrolimus (FK506) and VRC were found in all of the resistant strains. Moreover, ABC1 gene expression increased over time in both the in vivo- and in vitro-induced resistant strains, in contrast to the ABC2 and ERG11 genes, whose expression was invariably lower and constant. ERG11 gene sequencing showed two different types of mutations, i.e., heterozygosity at T1389T/C, corresponding to synonymous mutations, in C. krusei strains and a missense mutation at position T418C, resulting in a change from Tyr to His, among resistant C. krusei clinical isolates. This study highlights the relevance of ATP-dependent efflux pump (namely, Abc1p) activity in VRC resistance and describes new mutations in the ERG11 gene among resistant C. krusei clinical isolates.
The urgent need for rapid antimicrobial susceptibility is broadly apparent from government reports to the lay press. Accordingly, we developed a flow-cytometry assay (FCM) for evaluating ...ceftolozane-tazobactam (C/T) susceptibility directly on blood cultures (BC) requiring < 2 h from flag positivity to report. The protocol was optimized with C/T-susceptible and C/T-resistant gram-negative bacilli inoculated in BC aerobic bottles (Becton-Dickinson, USA), and afterward optimized for different C/T concentrations (1/4, 2/4, 4/4, and 8/4 mg/L) for 1 h incubation (37 °C), followed by FCM and software analysis. Fluorescent membrane permeability and membrane potential dyes were comparatively used to detect early cell lesions using the CytoFLEX cytometer (Beckman-Coulter, USA). Repeatability, reproducibility, and stability of the assay up to 48 h after BC positivity were determined. Internal validation was performed in spiked BC bottles with 130
Enterobacterales
and 32
Pseudomonas aeruginosa
isolates from Porto University (Portugal), including 13 ATCC isolates. Additionally, 64 gram-negative bacilli recovered from positive BC at Ramon y Cajal Hospital (Madrid, Spain) were tested. Categorical agreement (CA) and analytical errors were calculated comparing FCM with broth microdilution results. Only the membrane potential dyes clearly distinguished CT-susceptible and CT-resistant isolates. Excellent repeatability, reproducibility, and inter-method concordance was observed. Overall, CA was 99.1% using EUCAST criteria with 2 major errors and 98.7% with CLSI criteria with 2 major and 1 minor errors. A new, accurate, and ultra-rapid FCM (< 2 h) for testing C/T susceptibility gave accurate results and would expand current FCM antimicrobial susceptibility assay.
A rapid flow cytometric antimicrobial susceptibility test for bacteria isolated from companion animals – the FAST
vet
assay, developed by FASTinov
®
, was evaluated. Bacterial strains isolated from ...different biological samples of companion animals with infectious diseases in progress were obtained from several veterinary clinical laboratories across the country. A total of 115 strains, comprising 65 Gram-negative and 50 Gram positive isolates, were incubated with 13 antimicrobial drugs (ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefpodoxime, imipenem, enrofloxacin, gentamicin, amikacin for Gram-negative; penicillin, cefoxitin, enrofloxacin, vancomycin and ampicillin for Gram-positive) at breakpoint concentrations following CLSI protocol (
CLSI Vet 01, 2018
) for 1 h and analyzed by flow cytometry. The overall categorical agreement was 95.6% in case of Gram-negative and of 96.7% in Gram-positive isolates when compared to microdilution. FAST
vet
kits contribute to reduce the turnaround time (2 vs. 24 h) with early determination of the antimicrobial susceptibility profile. The correct and rapid choice of the target antibiotic therapy, will have a positive impact on animal care, contributing for preventing antimicrobial resistance. In conclusion, FASTinov
®
vet kits showed an excellent performance, both for Gram-negative and Gram-positive isolates encouraging us to enlarge the sample size and planning multicentric studies.
The composition of the essential oil of Thymus pulegioides and its antifungal activity on Candida, Aspergillus and dermatophyte fungal strains were studied. Essential oil from the aerial parts of the ...plant was obtained by hydrodistillation and analysed by GC and GC-MS. The oil showed high contents of carvacrol and thymol. The MIC and minimal lethal concentration were used to evaluate the antifungal activity against Candida (seven clinical isolates and four ATCC type strains), Aspergillus five clinical isolates, and two Colección Española de Cultivos Tipo (CECT) and two ATCC type strains and five clinical dermatophyte strains. Antifungal activity was evaluated for the essential oil and for its main components. To clarify its mechanism of action on yeasts and filamentous fungi, flow-cytometric studies of cytoplasmic membrane integrity were performed, and the effect on the amount of ergosterol was investigated. Results showed that T. pulegioides essential oil exhibited a significant activity against clinically relevant fungi, mainly due to lesion formation in the cytoplasmic membrane and a considerable reduction of the ergosterol content. The present study indicates that T. pulegioides essential oil has considerable antifungal activity, deserving further investigation for clinical applications.
Candida albicans is the most prevalent cause of fungemia worldwide. Its ability to develop resistance in patients receiving azole antifungal therapy is well documented. In a murine model of systemic ...infection, we show that ibuprofen potentiates fluconazole antifungal activity against a fluconazole-resistant strain, drastically reducing the fungal burden and morbidity. The therapeutic combination of fluconazole with ibuprofen may constitute a new approach for the management of antifungal therapeutics to reverse the resistance conferred by efflux pump overexpression.
Obesity and type 2 diabetes are metabolic diseases that have reached epidemic proportions worldwide. Although their etiology is complex, both result from interplay between behaviour, environment and ...genetic factors. Within ambient determinants, human overall gut bacteria have been identified as a crucial mediator of obesity and its consequences. Gut microbiota plays a crucial role in gastro-intestinal mucosa permeability and regulates the fermentation and absorption of dietary polyssacharides, which may explain its importance in the regulation of fat accumulation and the resultant development of obesity-related diseases. The main objective of this review is to address the pathogenic association between gut microbiota and obesity and to explore related innovative therapeutic targets. New insights into the role of the small bowel and gut microbiota in diabetes and obesity may make possible the development of integrated strategies to prevent and treat these metabolic disorders.
PDF
