RESUMEN
Introduction: The emergence of disinfectant resistance has become a severe threat due to reduced effectiveness. This study was undertaken to determine how bacteria adapt to survive exposure to disinfectants in the busiest section of a tertiary care hospital in Varanasi, India. Methods: Four isolates (two Klebsiella pneumoniae, Kp1 and Kp2; two Pseudomonas aeruginosa, Pa1 and Pa2) were obtained from chlorhexidine (CHX)-based handwash during microbiological surveillance of "in-use disinfectants" in hospital. Six disinfectants [4% CHX, 2% glutaraldehyde, 7.5% hydrogen peroxide, 1% sodium hypochlorite and 0.1% benzalkonium chloride (BAC), and 70% ethyl alcohol] were tested against these four isolates to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Antibiotic profile, change in MIC on exposure to disinfectants and biofilm formation in the presence and absence of disinfectants was studied. Whole genome sequencing (WGS) was done to identify the resistance mechanisms. Result: The isolates showed the highest MBC/MIC ratio (4) against glutaraldehyde. Exposure to supra-inhibitory concentration of BAC for 21 days resulted in doubling of MIC/MBC. The majority (75%) of the isolates were multidrug resistant. All the isolates were strong biofilm producers. The reduction rate of biofilm formation decreased with an increase in the concentration of disinfectants (p = 0.05 for BAC). WGS revealed multiple AMR genes including bla DIM-1, disinfectant-resistant gene and efflux pump genes. Conclusion: The study emphasized the various adaptation strategies of these isolates for survival in disinfectant environment, thus posing a huge challenge for their control in the hospital environment.
Asunto(s)
Biopelículas , Desinfectantes , Klebsiella pneumoniae , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa , Desinfectantes/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , India , Humanos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Klebsiella pneumoniae/efectos de los fármacos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Hospitales , Secuenciación Completa del Genoma , Viabilidad Microbiana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Centros de Atención Terciaria , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Bacterias/clasificación , Bacterias/aislamiento & purificaciónRESUMEN
Clinical application of anticancer drugs is mostly limited due to their hydrophobic nature, which often results in lower bioavailability and lesser retention in systemic circulation. Despite extensive research on the development of targeted drug delivery systems for cancer treatment, delivery of hydrophobic therapeutic drugs to tumor cells remains a major challenge in the field. To address these concerns, we have precisely engineered a new hyperbranched polymer for the targeted delivery of hydrophobic drugs by using a malonic acid-based A2B monomer and 1,6-hexanediol. The choice of monomer systems in our design allows for the formation of higher molecular weight polymers with hydrophobic cavities for the efficient encapsulation of therapeutic drugs that exhibit poor water solubility. Using several experimental techniques such as NMR, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), Fourier transform-infrared (FT-IR), and gel permeation chromatography (GPC), the synthesized polymer was characterized, which indicated its dendritic structure, thermal stability, and amorphous nature, making it suitable as a drug delivery system. Following characterizations, theranostic nanoplatforms were formulated using a one-pot solvent diffusion method to coencapsulate hydrophobic drugs, BQU57 and doxorubicin. To achieve targeted delivery of loaded therapeutic drugs in A549 cancer cells, the surface of the polymeric nanoparticle was conjugated with folic acid. The therapeutic efficacy of the delivery system was determined by various cell-based in vitro experiments, including cytotoxicity, cell internalizations, reactive oxygen species (ROS), apoptosis, migration, and comet assays. Overall, findings from this study indicate that the synthesized dendritic polymer is a promising carrier for hydrophobic anticancer drugs with higher biocompatibility, stability, and therapeutic efficacy for applications in cancer therapy.
RESUMEN
Green fluorescent protein has long been a favorite protein for demonstrating protein purification in the biochemistry lab course. The protein's vivid green color helps demonstrate to students the concept(s) behind affinity or ion exchange chromatography. We designed a series of introduction to biochemistry labs utilizing a thermostable green protein (TGP-E) engineered to have unusually high thermostability. This protein allows students to proceed through purification and characterization without the need to keep protein samples on ice. The 5-week lab series begins with an introduction to molecular biology techniques during weeks 1 and 2, where site-directed mutagenesis is used introduce, a single nucleotide change that shifts the fluorescent spectra of TGP-E to either cyan (CTP-E) or yellow (YTP-E). Students identify successful mutagenesis reaction by the color of a small expression sample after induction with IPTG. Next, students purify either the TGP-E (control-typically one group volunteers), YTP-E, or CTP-E protein as a 1-week lab. During the following week's lab, students run SDS-PAGE to verify protein purity, bicinchoninic acid assay to quantify protein yield, and absorbance and fluorescence spectra to characterize their protein's fluorescent character. The final lab in the series investigates the thermostability of YTP-E and CTP-E compared with TGP-E using a fluorescence plate reader. This 5-week series of experiments provide students with experience in several key biochemistry techniques and allows the students to compare properties of mutations. At the end of the course, the students will write a research report and give a short presentation over their results.
RESUMEN
Elucidating the underlying principles of amyloid protein self-assembly at nanobio interfaces is extremely challenging due to the diversity in physicochemical properties of nanomaterials and their physical interactions with biological systems. It is, therefore, important to develop nanoscale materials with dynamic features and heterogeneities. In this work, through engineering of hierarchical polyethylene glycol (PEG) structures on gold nanoparticle (GNP) surfaces, tailored nanomaterials with different surface properties and conformations (GNPs-PEG) are created for modulating the self-assembly of a widely studied protein, insulin, under amyloidogenic conditions. Important biophysical studies including thioflavin T (ThT) binding, circular dichroism (CD), surface plasmon resonance (SPR), and atomic force microscopy (AFM) showed that higher-molecular weight GNPs-PEG triggered the formation of amyloid fibrils by promoting adsorption of proteins at nanoparticle surfaces and favoring primary nucleation rate. Moreover, the modulation of fibrillation kinetics reduces the overall toxicity of insulin oligomers and fibrils. In addition, the interaction between the PEG polymer and amyloidogenic insulin examined using MD simulations revealed major changes in the secondary structural elements of the B chain of insulin. The experimental findings provide molecular-level descriptions of how the PEGylated nanoparticle surface modulates protein adsorption and drives the self-assembly of insulin. This facile approach provides a new avenue for systematically altering the binding affinities on nanoscale surfaces by tailoring their topologies for examining adsorption-induced fibrillogenesis phenomena of amyloid proteins. Together, this study suggests the role of nanobio interfaces during surface-induced heterogeneous nucleation as a primary target for designing therapeutic interventions for amyloid-related neurodegenerative disorders.
Asunto(s)
Amiloide , Oro , Insulina , Nanopartículas del Metal , Polietilenglicoles , Oro/química , Nanopartículas del Metal/química , Humanos , Insulina/metabolismo , Insulina/química , Polietilenglicoles/química , Amiloide/metabolismo , Amiloide/química , Microscopía de Fuerza Atómica , Propiedades de Superficie , Dicroismo Circular , Simulación de Dinámica Molecular , Resonancia por Plasmón de SuperficieRESUMEN
Colostrum has been shown to be suitable for oral and/or topical applications. Colostrum decreases the amount of discharge from wounds and also accelerates healing, leading to a decrease in the number of dressings. In this study, 40 patients with chronic non-healing wounds were divided into two groups, considering the inclusion and exclusion criteria. Group I included 15 patients with conventional dressings, and Group II included 25 patients with added topical colostrum dressings. All patients were assessed at the time of presentation and after 21 days. The results of the present study indicate that colostrum powder dressings may be used as an adjunct in the management of chronic non-healing wounds.
RESUMEN
PURPOSE: Antimicrobial resistance (AMR) in Pseudomonas aeruginosa has been ever-increasing. Among other reasons, colistin resistance might be attributed to limited routine testing by approved methods. Both broth microdilution (BMD) and colistin broth disc elution (CBDE) methods have been advocated, with limited data on the performance of these methods in the Indian settings. This prospective study was conducted to determine colistin resistance in P. aeruginosa, compare the BMD and CBDE methods with special reference to heteroresistance. MATERIALS AND METHODS: A total of 100 isolates of P. aeruginosa from admitted patients were included. Antimicrobial susceptibility testing was done against standard antibiotics by disc diffusion test. Minimum inhibitory concentration (MIC) against polymyxins was studied by BMD and CBDE (for colistin only). Heteroresistance to colistin was studied by population analysis profile (PAP). CBDE and BMD were compared by performance calculations. Discrepancy in results were analyzed based on heteroresistance. RESULTS: Majority of the P. aeruginosa isolates were from pus samples (62, 62 â%). Disc diffusion method revealed maximum susceptibility towards aztreonam (74, 74 â%) followed by meropenem (68, 68 â%) and piperacillin-tazobactam (65, 65 â%). Polymyxin B resistance was seen in 6 â% (6) while colistin resistance was seen in 9 â% (9) isolates by BMD. CBDE revealed 8 â% (8) resistance to colistin, having 97 â% essential agreement and 95 â% categorical agreement with BMD. Further, by PAP analysis, 9 isolates were resistant to colistin which included 9 resistant isolates by BMD. On discrepancy analysis, 1 isolate was found to be heteroresistant to colistin. No heteroresistance was seen in the isolates that were susceptible by all the methods. CONCLUSIONS: Heteroresistance to colistin in P. aeruginosa accounted for the discrepancy in results where CBDE method failed to detect heteroresistant isolate. As heteroresistance is a least studied phenotype, it's exact prevalence should be studied so that challenges in susceptibility testing could be addressed.
Asunto(s)
Colistina , Infecciones por Pseudomonas , Humanos , Colistina/farmacología , Pseudomonas aeruginosa , Estudios Prospectivos , Antibacterianos/farmacología , Polimixina B/farmacología , Pruebas de Sensibilidad MicrobianaRESUMEN
Background & objectives: During the course of a retrospective survey on healthcare associated infections (HAIs) due to carbapenem-resistant organisms, an unusual prevalence of HAIs due to carbapenem-resistant Providencia stuartii (CRPS) was found. Hence this study aimed to conduct the occurrence of P. stuartii associated HAIs with special reference to the drug resistance profiling of these isolates. Methods: Of the eight total HAI cases (7.5% of total HAIs and 33.3% of HAIs due to Enterobacterales) of CRPS infections included in this study, three were reported from ventilator-associated pneumonia (VAP), three were surgical site infections (SSIs), one was a catheter-associated urinary tract infection (CAUTI) and one was a bloodstream infection. All the eight CRPS isolates were tested for extended-spectrum ß-lactamases production, AmpC hyperproduction as well as carbapenem resistance. Typing of the isolates was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Results: All the eight isolates of CRPS were found to be AmpC hyperproducers, carbapenemase producers, and harboured chromosomally located blaNDM in seven isolates and blaIMP genes in one. All the cases with CRPS infections had prior history of colistin therapy along with prolonged hospital stay (>20 days). The cases were located in five different wards/intensive care unit (ICU) within the hospital in one year. However, strain typing by REP-PCR revealed 100 per cent similarity and clonal relatedness in all the seven isolates carrying blaNDM genes. Interestingly, routine hospital surveillance revealed a high carriage of P. stuartii in the axilla of patients admitted to the ICU. Interpretation & conclusions: The study findings suggest CRPS as an important cause of HAIs. This organism often goes unnoticed due to the burden of carbapenem resistance in other Enterobacterales and non-fermenters.
Asunto(s)
Proteínas Bacterianas , Infección Hospitalaria , Humanos , Estudios Retrospectivos , Pruebas de Sensibilidad Microbiana , Proteínas Bacterianas/genética , beta-Lactamasas/genética , Carbapenémicos/uso terapéutico , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/epidemiología , Hospitales , Antibacterianos/efectos adversosRESUMEN
Low macrophage viability in chronic diabetic foot ulcers (DFUs) may lead to inadequate interleukin (IL) expression and the persistence of infection. This study evaluates the association between macrophage function, IL-2 expression, and wound microflora in chronic DFUs. Diabetic patients with DFUs (group 1, n = 40) and without DFUs (group 2, n = 40) were compared for macrophage function in serum by viability testing. Immunological response was measured by serum IL-1ß, IL-2ß, and IL-10 levels. The aerobic and anaerobic microflora of the DFUs were assessed by culture and molecular methods. Demographic, clinical, and biochemical factors were statistically analyzed by χ2 test and Student t test. Multiple correspondence analysis (MCA) was used to detect the pattern of association between glycosylated hemoglobin (hemoglobin A1c), serum IL-2 levels, and macrophage viability. Of the total DFU cases, 22 (55%) showed the presence of polymicrobial microflora. Low macrophage viability with predominant Gram-negative flora was seen in 10 (25%) cases in group 1. Serum IL-2 levels were significantly lower (P = .004) in patients in group 1 along with elevated levels of hemoglobin A1c (P = .038). MCA showed an association between low viability of macrophages and lower IL-2 levels and elevated hemoglobin A1c levels with lower serum IL-2 levels. As compared to group 2, the low viability of macrophages was significantly associated (P = .007) with lower IL-2 levels in group 1. Elevated hemoglobin A1c levels are strongly associated with lower IL-2 levels and low macrophage viability. This might be a contributing factor to the persistence of infections in chronic DFUs.
RESUMEN
Infections by Entamoeba histolytica (E. histolytica) lead to considerable morbidity and mortality worldwide and treatment is reliant on a single class of drugs, nitroimidazoles. Treatment failures and intermittent reports of relapse from different parts of world indicate towards development of clinical drug resistance. In the present study, susceptibility testing of clinical isolates of E. histolytica was carried against metronidazole and tinidazole. Additionally, anti-amoebic property of active compounds of Andrographis paniculata was also evaluated. Prevalence of metronidazole resistance gene (nim) in patients attending hospital was also done to get comprehensive insight of present situation of drug resistance in E. histolytica. Mean inhibitory concentration 50 (IC50) value of E. histolytica isolates against metronidazole and tinidazole was 20.01 and 16.1 µM respectively. Andrographolide showed minimum mean IC50 value (3.06 µM). Significant percentage inhibition of E. histolytica isolates by andrographolide was seen as compared to metronidazole (p = 0.0495). None of E. histolytica isolates showed presence of nim gene. However, in stool samples from hospital attending population, prevalence of nimE gene was found to be 76.6% (69/90) and 62.2% (56/90) in diarrheal and non-diarrheal samples respectively. Inhibitory concentration of commonly used nitroimidazoles against clinical isolates of E. histolytica are on rise. Percentage inhibition of E. histolytica isolates by andrographolide was significantly higher than control drug metronidazole.
Asunto(s)
Entamoeba histolytica , Absceso Hepático Amebiano , Nitroimidazoles , Humanos , Nitroimidazoles/farmacología , Nitroimidazoles/uso terapéutico , Entamoeba histolytica/genética , Absceso Hepático Amebiano/diagnóstico , Absceso Hepático Amebiano/tratamiento farmacológico , Metronidazol/farmacología , Metronidazol/uso terapéutico , Tinidazol/uso terapéutico , Reposicionamiento de MedicamentosRESUMEN
PURPOSE: Neonatal sepsis has been a global concern considering the mortality and morbidity. This study was undertaken to determine the effects of implementation of interventions namely healthcare associated infection (HAI) surveillance and hand hygiene (HH) monitoring in the neonatal intensive care unit (NICU). MATERIALS AND METHODS: The cohort study was conducted in the NICU of a tertiary care hospital over a period of June-September 2021 (pre-intervention) to October-March 2022 (post-intervention). HAI surveillance of primary bloodstream infections (BSI) and HH monitoring was initiated as interventions post outbreak due to non-albicans Candida (NAC). The primary outcome of the interventions was to record any improvement in HH rates or any change in HAI rates in the 6 months intervention period. Characteristics of the pre- and post-intervention period were compared by Fisher exact test. RESULTS: There was significant reduction in BSI cases in the post-intervention period (p â< â0.05). Mortality and BSI due to NAC were significantly more in the pre-intervention period even though low birth weight neonates (<2500 âg) were significantly more in the post-intervention period (p â< â0.05). The HAI rate for primary BSI in the NICU was 10.82 per 1000 patient days. The overall adherence rate to HH was 10.68% (complete) and 73.35% (partial). HAI rates were seen to change reciprocally with changes in HH rates. CONCLUSIONS: HAI rates of primary BSI in the NICU could be regulated by the effective implementation of HAI surveillance, HH monitoring, feedback meetings with the NICU staff and other simple interventional measures even in resource-limited setups.
Asunto(s)
Infección Hospitalaria , Higiene de las Manos , Sepsis , Recién Nacido , Humanos , Unidades de Cuidado Intensivo Neonatal , Estudios de Cohortes , Infección Hospitalaria/epidemiología , Infección Hospitalaria/prevención & control , Sepsis/epidemiología , Sepsis/prevención & controlRESUMEN
Objective Coagulase-negative staphylococci (CoNS) are being implicated as one of the leading causes of bloodstream infection (BSI). To study the spectrum, prevalence, and antimicrobial susceptibility of CoNS causing BSI in neonates. Materials and Methods A cross-sectional study was done in level III neonatal intensive care unit (NICU). Blood samples in automated culture bottles were processed as per the standard technique. Previously validated methods were followed for the characterization of CoNS and for AST of standard antibiotics by Kirby Bauer disk diffusion and vancomycin by agar dilution. The prevalence of causative organisms and susceptibility of CoNS were statistically analyzed. Categorical variables were compared by chi-square or Fisher's exact probability tests. Result In total, 1,365 blood samples (1,365 neonates) were studied, of which 383 (28.05%) were positive and 982 (71.94%) were negative. Gram-positive organisms (GPC) predominated ( n = 238; 62.14%) ( p < 0.001) with 41.77% (160/383) S. aureus and 13.83% (53/383) CoNS. CoNS included S. epidermidis (19, 38%), S . haemolyticus (7, 14%), S. hominis (6, 12%), S. simulans (6,12%), S. capitis (5,10%), S. cohnii (4, 8%), S. warneri (1, 2%), and S. xylosus (1, 2%). The susceptibility to netilmicin, linezolid, and vancomycin was 100% ( p ≤ 0.001), and 54% ( n = 27) had vancomycin MIC of 0.125 µg/mL but methicillin-resistant CoNS (MRCoNS) was 70%. Methicillin-susceptible (MS) CoNS had lower MIC of vancomycin ( p < 0.05) than MRCoNS. Conclusion The spectrum of pathogens causing BSI in neonates is changing with predominance of GPC and among CoNS, S. epidermidis . Considerable proportion of MRCoNS with the emergence of MIC creep for vancomycin requires immediate attention.
RESUMEN
The bubbling community of microorganisms, consisting of diverse colonies encased in a self-produced protective matrix and playing an essential role in the persistence of infection and antimicrobial resistance, is often referred to as a biofilm. Although apparently indolent, the biofilm involves not only inanimate surfaces but also living tissue, making it truly ubiquitous. The mechanism of biofilm formation, its growth, and the development of resistance are ever-intriguing subjects and are yet to be completely deciphered. Although an abundance of studies in recent years has focused on the various ways to create potential anti-biofilm and antimicrobial therapeutics, a dearth of a clear standard of clinical practice remains, and therefore, there is essentially a need for translating laboratory research to novel bedside anti-biofilm strategies that can provide a better clinical outcome. Of significance, biofilm is responsible for faulty wound healing and wound chronicity. The experimental studies report the prevalence of biofilm in chronic wounds anywhere between 20 and 100%, which makes it a topic of significant concern in wound healing. The ongoing scientific endeavor to comprehensively understand the mechanism of biofilm interaction with wounds and generate standardized anti-biofilm measures which are reproducible in the clinical setting is the challenge of the hour. In this context of "more needs to be done", we aim to explore various effective and clinically meaningful methods currently available for biofilm management and how these tools can be translated into safe clinical practice.
Asunto(s)
Antiinfecciosos , Infección de Heridas , Humanos , Desbridamiento/métodos , Infección de Heridas/terapia , Cicatrización de Heridas , BiopelículasRESUMEN
Mycobacterium spp. intimidated mankind since time immemorial. The triumph over this organism was anticipated with the introduction of potent antimicrobials in the mid-20th century. However, the emergence of drug resistance in mycobacteria, Mycobacterium tuberculosis, in particular, caused great concern for the treatment. With the enemy growing stronger, there is an immediate need to equip the therapeutic arsenal with novel and potent chemotherapeutic agents. The task seems intricating as our understanding of the dynamic nature of the mycobacteria requires intense experimentation and research. Targeting the mycobacterial cell envelope appears promising, but its versatility allows it to escape the lethal effect of the molecules acting on it. The unique ability of hiding (inactivity during latency) also assists the bacterium to survive in a drug-rich environment. The drug delivery systems also require upgradation to allow better bioavailability and tolerance in patients. Although the resistance to the novel drugs is inevitable, our commitment to the research in this area will ensure the discovery of effective weapons against this formidable opponent.
Asunto(s)
Antiinfecciosos , Mycobacterium tuberculosis , Humanos , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Pruebas de Sensibilidad Microbiana , Antiinfecciosos/farmacologíaRESUMEN
PURPOSE: Recent reports have noted an emergence of unusual organisms in microflora of pilonidal sinus (PNS); this study was undertaken to identify the primary microbial flora associated with infected primary PNS over a period of 1 year. DESIGN: A prospective multiple case series. SUBJECTS AND SETTING: A case series of 20 patients with primary PNS in a tertiary care center in Varanasi, India, was studied. The study was conducted at the Department of Microbiology and General Surgery, Institute of Medical Sciences, Varanasi, over a period of 1 year (September 2016 to July 2017). METHODS: Purulent exudate (pus) samples were collected from 20 patients with primary PNS from the discharging sinuses by aseptic methods. Samples were assessed for aerobic and anaerobic flora by conventional culture and molecular methods. Antimicrobial susceptibility testing was done for bacterial isolates. Bacterial diversity was compared with the demographic and clinical profile of the sinuses by multiple correspondence analysis. RESULTS: Of the total cases, 11 (55%) had purulent discharge, among which all showed polymicrobial flora. The ratio of aerobic to anaerobic organisms was 1:2 (16/32). Escherichia coli (E. coli, 4, 36.36%) and Enterococcus faecalis (E. faecalis, 4, 36.36%) were commonly isolated. Bifidobacterium was the most frequent anaerobe. Detailed molecular analysis revealed the presence of Kocuria flava as an unusual pathogen. On statistical analysis, factors like male gender, increased body mass index, absence of hair in sinus, presence of features of hirsutism, and absence of Fusobacteria were closely associated with one another in these PNS cases. CONCLUSIONS: The case series revealed the predominance of anaerobes in primarily infected PNS cases. Bifidobacterium spp and unusual pathogens like K. flava were among the emerging pathogens in infected PNS. Use of better molecular diagnostic facilities in addition to the conventional methods might enhance the verified diversity of microorganisms in such cases.
Asunto(s)
Seno Pilonidal , Humanos , Masculino , Escherichia coli , Estudios Prospectivos , IndiaRESUMEN
Frequent outbreaks of food-borne pathogens, particularly E. coli O157:H7, continue to impact human health and the agricultural economy tremendously. The required cell count for this pathogenic strain of E. coli O157:H7 is relatively low and hence it is vital to detect at low colony forming unit (CFU) counts. Available detection methods, though sensitive, fall short in terms of timeliness and often require extensive sample processing. To overcome these limitations, we propose a novel magneto-plasmonic nanosensor (MPnS) by integrating surface plasmon resonance (SPR) properties with spin-spin magnetic relaxation (T2 MR) technology. We engineered MPnS by encapsulating several gold nanoparticles (GNPs) within the polymer-coating of iron oxide nanoparticles (IONPs). First, the polyacrylic acid (PAA)-coated IONPs were synthesized using a solvent precipitation method, then gold chloride solution was used to synthesize GNPs and encapsulate them within the PAA-coatings of IONPs in one step. A magnetic separation technique was used to purify the MPnS and the presence of GNPs within IONPs was characterized using transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and other spectroscopic methods. The synthesized MPnS exhibits MR relaxation properties while possessing amplified optical properties than conventional GNPs. This allows for rapid and ultrasensitive detection of E. coli O157:H7 by SPR, T2 MR, and colorimetric readout. Experiments conducted in simple buffer and in milk as a complex media demonstrated that our MPnS-based assay could detect as low as 10 CFUs of this pathogenic strain of E. coli O157:H7 in minutes with no cross-reactivity. Overall, the formulated MPnS is robust and holds great potential for the ultrasensitive detection of E. coli O157:H7 in a simple and timely fashion. Moreover, this platform is highly customizable and can be used for the detection of other foodborne pathogens.
Asunto(s)
Técnicas Biosensibles , Escherichia coli O157 , Nanopartículas del Metal , Humanos , Animales , Microbiología de Alimentos , Oro/química , Nanopartículas del Metal/química , Leche , Técnicas Biosensibles/métodosRESUMEN
After binding to the surface of a target cell, cholera toxin (CT) moves to the endoplasmic reticulum (ER) by retrograde transport. In the ER, the catalytic CTA1 subunit dissociates from the rest of the toxin and is transferred to the cytosol where it is degraded by a ubiquitin-independent proteasomal mechanism. However, CTA1 persists long enough to induce excessive cAMP production through the activation of Gsα. It is generally believed that only one or a few molecules of cytosolic CTA1 are necessary to elicit a cytopathic effect, yet no study has directly correlated the levels of cytosolic toxin to the extent of intoxication. Here, we used the technology of surface plasmon resonance to quantify the cytosolic pool of CTA1. Our data demonstrate that only 4% of surface-bound CTA1 is found in the cytosol after 2 h of intoxication. This represented around 2600 molecules of cytosolic toxin per cell, and it was sufficient to produce a robust cAMP response. However, we did not detect elevated cAMP levels in cells containing less than 700 molecules of cytosolic toxin. Thus, a threshold quantity of cytosolic CTA1 is required to elicit a cytopathic effect. When translocation to the cytosol was blocked soon after toxin exposure, the pool of CTA1 already in the cytosol was degraded and was not replenished. The cytosolic pool of CTA1 thus remained below its functional threshold, preventing the initiation of a cAMP response. These observations challenge the paradigm that extremely low levels of cytosolic toxin are sufficient for toxicity, and they provide experimental support for the development of post-intoxication therapeutic strategies.
Asunto(s)
Toxina del Cólera , Retículo Endoplásmico , Cricetinae , Animales , Toxina del Cólera/farmacología , Toxina del Cólera/química , Toxina del Cólera/metabolismo , Citosol/metabolismo , Transporte de Proteínas , Células CHO , Unión Proteica , Retículo Endoplásmico/metabolismoRESUMEN
The endemicity of Acinetobacter baumannii in intensive care units (ICUs) is a serious concern. We studied the reservoirs of A. baumannii in the ICU and their effects on colonization pressure and transmission. A prospective surveillance (6 months) was conducted. Screening culture (rectal and axillary) swabs were collected within 48 hours admission and in 120 hours. Surveillance cultures from patients' surroundings, health care workers (HCWs), and hospital sewage were collected. A. baumannii was identified by phenotypic and genotypic methods. Carbapenem resistance and insertion sequence element were detected. Typing was done by repetitive extragenic palindromic-polymerase chain reaction and multilocus sequence typing. Colonization pressure was calculated and compared with environment colonizers. Of the 87 patients, 21.83% (19) were colonized with A. baumannii, 73.68% (14/19) were imported, and 26.31% (5/19) acquired carriers. Axilla was the commonest site. From the environment (15), bed rails 33.33% (5/15) and suction tubes 26.66% (4/15) were the common sites. HCWs showed 7.5% (3/40) carriage. Carbapenem resistance with blaOXA-51, blaOXA-23, and ISAba1 were 91.89% (34/37). Strong correlation between colonization pressures and environmental colonizers was seen (r2 = 0.719, p = 0.032). Carbapenem and polymyxin B were (p ≤ 0.05) significant exposures. Sequence type 623 was the predominant cluster with isolates from carriers, HCWs, and environment. Colonization pressure of carbapenem-resistant A. baumannii depends on their presence in the hospital. Hands of HCWs were an important vehicle for transmission. Infection control measure should consider reducing the environmental reservoir.
Asunto(s)
Acinetobacter baumannii , Humanos , Proteínas Bacterianas , Antibacterianos/farmacología , beta-Lactamasas , Estudios Prospectivos , Pruebas de Sensibilidad Microbiana , Carbapenémicos , Hospitales , Unidades de Cuidados IntensivosRESUMEN
BACKGROUND: The emergence of multidrug-resistant tuberculosis (MDR-TB) has complicated the situation due to the decline in potency of second-line anti-tubercular drugs. This limits the treatment option for extensively drug-resistant tuberculosis (XDR-TB). The aim of this study was to determine and compare the minimum inhibitory concentration (MIC) by agar dilution and resazurin microtiter assay (REMA) along with the detection of mutations against linezolid and clofazimine in confirmed XDR-TB clinical isolates. RESULTS: A total of 169 isolates were found positive for Mycobacterium tuberculosis complex (MTBC). The MIC was determined by agar dilution and REMA methods. The isolates which showed non-susceptibility were further subjected to mutation detection by targeting rplC gene (linezolid) and Rv0678 gene (clofazimine). The MIC for linezolid ranged from 0.125 µg/ml to > 2 µg/ml and for clofazimine from 0.25 µg/ml to > 4 µg/ml. The MIC50 and MIC90 for linezolid were 0.5 µg/ml and 1 µg/ml respectively while for clofazimine both were 1 µg/ml. The essential and categorical agreement for linezolid was 97.63% and 95.26% and for clofazimine, both were 100%. The sequencing result of the rplC gene revealed a point mutation at position 460 bp, where thymine (T) was substituted for cytosine (C) while seven mutations were noted between 46 to 220 bp in Rv0678 gene. CONCLUSION: REMA method has been found to be more suitable in comparison to the agar dilution method due to lesser turnaround time. Mutations in rplC and Rv0678 genes were reasons for drug resistance against linezolid and clofazimine respectively.
Asunto(s)
Tuberculosis Extensivamente Resistente a Drogas , Mycobacterium tuberculosis , Tuberculosis Resistente a Múltiples Medicamentos , Agar , Antituberculosos/farmacología , Clofazimina/farmacología , Clofazimina/uso terapéutico , Citosina/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genética , Tuberculosis Extensivamente Resistente a Drogas/tratamiento farmacológico , Tuberculosis Extensivamente Resistente a Drogas/microbiología , Humanos , Linezolid/farmacología , Linezolid/uso terapéutico , Pruebas de Sensibilidad Microbiana , Mutación , Timina/uso terapéutico , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Increasing foodborne illnesses have led to global health and economic burdens. E. coli O157:H7 is one of the most common disease-provoking pathogens and known to be lethal Shiga toxin-producing E. coli (STEC) strains. With a low infection dose in addition to person-to-person transmission, STEC infections are easily spread. As a result, specific and rapid testing methods to identify foodborne pathogens are urgently needed. Nanozymes have emerged as enzyme-mimetic nanoparticles, demonstrating intrinsic catalytic activity that could allow for rapid, specific, and accurate pathogen identification in the agrifood industry. In this study, we developed a sensitive nanoplatform based on the traditional ELISA assay with the synergistic properties of gold and iron oxide nanozymes, replacing the conventional enzyme horseradish peroxidase (HRP). We designed an easily interchangeable sandwich ELISA composed of a novel, multifunctional magneto-plasmonic nanosensor (MPnS) with target antibodies (MPnS-Ab). Our experiments demonstrate a 100-fold increase in catalytic activity in comparison to HRP with observable color changes within 15 min. Results further indicate that the MPnS-Ab is highly specific for E. coli O157:H7. Additionally, effective translatability of catalytic activity of the MPnS technology in the lateral flow assay (LFA) platform is also demonstrated for E. coli O157:H7 detection. As nanozymes display more stability, tunable activity, and multi-functionality than natural enzymes, our platform could provide customizable, low-cost assay that combines high specificity with rapid detection for a variety of pathogens in a point-of-care setup.
Asunto(s)
Escherichia coli O157 , Enfermedades Transmitidas por los Alimentos , Oro , Peroxidasa de Rábano Silvestre , Humanos , Toxina ShigaRESUMEN
The rapid emergence of drug resistance in Acinetobacter baumannii has put forward the use of colistin as a last-resort treatment for infections with A. baumannii. Empirical colistin use without prior susceptibility testing has been one of the factors that has been promoting drug resistance in low-resource settings. In this regard, while the advocated broth microdilution (BMD) method for colistin susceptibility testing is often considered cumbersome, the preferable colistin broth disk elution (CBDE) method has not yet been approved for A. baumannii. To prevent the underreporting of colistin susceptibility, we tested the CBDE method for A. baumannii and compared the results with those of BMD. A total of 125 A. baumannii, including 100 susceptible and 25 resistant isolates were tested via the CBDE method and compared with the standard BMD method. The essential agreement, categorical agreement, sensitivity, and specificity for CBDE were 97.6% (n = 122), 98.4% (n = 123), 100%, and 98.40%, respectively. The percentage of major error found was 1.6% (n = 2), and no very major error was found. CBDE in A. baumannii could be considered in low-resource settings. IMPORTANCE The relatively cumbersome broth microdilution (BMD) method for routine colistin susceptibility testing has not been adopted, especially in low-resource settings, often leading to the underreporting of colistin susceptibility and the promotion of the empirical use of colistin. In this regard, the much-preferred colistin broth disk elution (CBDE) method has not yet been approved for A. baumannii. We evaluated colistin susceptibility via the CBDE method, compared the results with those of the BMD method in 125 A. baumannii isolates with various profiles, and inferred that the CBDE method using 50 µL inoculum could be helpful, at least in resource-limited setups, versus not reporting susceptibility testing for colistin.