The interaction of 2,2,2-trifluoroethanol (TFE) with concanavalin A has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry (ITC), circular ...dichroism (CD), and fluorescence spectroscopy at pH 2.5 and 5.2. All of the calorimetric transitions at both the pH values were found to be irreversible. In the presence of 4 mol kg-1 TFE at pH 2.5, concanavalin A is observed to be in a partially folded state with significant loss of native tertiary structure. The loss of specific side chain interactions in the transition from native to the TFE-induced partially folded state is demonstrated by the loss of cooperative thermal transition and reduction of the CD bands in the aromatic region. Acrylamide quenching, 8-anilinonaphthalene sulfonate (ANS) binding, and energy transfer also suggest that in the presence of 4 mol kg-1 TFE at pH 2.5 concanavalin A is in a molten globule state. ITC has been used for the first time to characterize the energetics of ANS binding to the molten globule state. ITC results indicate that the binding of ANS to the molten globule state and acid-induced state at pH 2.5 displays heterogeneity with two classes of non-interacting binding sites. The results provide insights into the role of hydrophobic and electrostatic interactions in the binding of ANS to concanavalin A. The results also demonstrate that ITC can be used to characterize the partially folded states of the protein both qualitatively and quantitatively.
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.
•Cholera toxin (CT) must enter the host cytosol to elicit a cytopathic effect.•Only 4% of cell-associated CT reaches the cytosol after 2 h of intoxication.•More than 700 molecules of cytosolic toxin are required for a cellular response.•Intoxication is blocked when CT cannot reach its threshold quantity in the cytosol.•Limiting or reducing the amount of cytosolic CT represents a new therapeutic strategy.
Fluoroquinolone resistance in both Gram-positive and Gram-negative bacteria has increased with the widespread use of fluoroquinolones. Fluoroquinolone resistance in Gram-negative bacilli has been ...widely studied, though staphylococci and enterococci are also notably resistant. Enterococci being the second most common cause of healthcare-associated urinary tract infections (UTIs) fluoroquinolones are often the drug of choice. This study was undertaken to assess the risk factors associated with fluoroquinolone-resistant enterococcal UTI in a tertiary level health facility in north India.
A total of 365 patients with UTI caused by enterococci were studied over a period of two years. Patients with ciprofloxacin-resistant and susceptible UTI were considered as cases and controls, respectively. Resistance profile of the isolates against common antibiotics was studied by minimum inhibitory concentration (MIC) determination. Mechanisms for fluoroquinolone resistance was studied by efflux pump inhibitor activity and multiplex PCR targeting the qnr genes.
A total of 204 (55.89%) cases and 161 (44.1%) controls were identified. The fluoroquinolone-resistant isolates were significantly resistant to ampicillin, high strength aminoglycosides and vancomycin. The majority (78%) of the resistant isolates showed efflux pump activity. Treatment in indoor locations, presence of urinary catheters and pregnancy along with recent exposure to antibiotics especially fluoroquinolones, third generation cephalosporins and piperacillin-tazobactam were identified as independent risk factors.
Our results showed that fluoroquinolone resistance in enterococcal UTI was largely associated with indoor usage of antibiotics and use of indwelling devices. Knowledge of risk factors is important to curb this emergence of resistance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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 β-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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cholera toxin (CT) is an AB
protein toxin that activates the stimulatory alpha subunit of the heterotrimeric G protein (Gsα) through ADP-ribosylation. Activation of Gsα produces a cytopathic effect ...by stimulating adenylate cyclase and the production of cAMP. To reach its cytosolic Gsα target, CT binds to the plasma membrane of a host cell and travels by vesicle carriers to the endoplasmic reticulum (ER). The catalytic CTA1 subunit then exploits the quality control mechanism of ER-associated degradation to move from the ER to the cytosol. ER-associated degradation is functionally linked to another quality control system, the unfolded protein response (UPR). However, the role of the UPR in cholera intoxication is unclear. We report here that CT triggers the UPR after 4 h of toxin exposure. A functional toxin was required to induce the UPR, but, surprisingly, activation of the adenylate cyclase signaling pathway was not sufficient to trigger the process. Toxin-induced activation of the UPR coincided with increased toxin accumulation in the cytosol. Chemical activation of the heterotrimeric G protein or the UPR also enhanced the onset of CTA1 delivery to the cytosol, thus producing a toxin-sensitive phenotype. These results indicate there is a cAMP-independent response to CT that activates the UPR and thereby enhances the efficiency of intoxication.
Abstract
A healthy young adult male presented with complaints of frequent (>3/day) formed stools and passage of excessive mucous in stool for 3 months. He did not complain of nocturnal motions, ...recent diarrhea, blood in stool, straining, weight loss, or pain abdomen. Stool test was normal. He was counseled and treated as a case of irritable bowel syndrome. Due to inadequate relief with empirical therapy, colonoscopy was performed in a subsequent visit. Club-shaped small, round organisms with moving proboscis were seen in the cecum. Organism was later identified as a trematode
Gastrodiscoides hominis
, a rare foodborne trematode. The patient was treated with praziquantel, without complete relief. Trematode infection might not be the cause of symptoms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
Background:
Alarming rise of vancomycin-resistant enterococci (VRE) is a global cause of concern. Several factors have been held responsible for such rise, of which antibiotic usage is a ...prominent one.
Objectives:
This study was undertaken to determine the intestinal VRE colonization rate amongst hospitalized patients in relation to use of various antibiotics in the Intensive Care Unit (ICU) of a tertiary care university hospital, India.
Materials and Methods:
Stool samples were collected weekly from all the patients in the adult ICU for a period of 6 months and processed for isolation and phenotypic and genotypic characterization of VRE isolates. Patient and treatment details were noted and cases (those with VRE in stool) and controls (those without VRE in stool) were compared statistically. Further, a multivariate analysis was done to identify those antibiotics as independent risk factors for VRE colonization.
Results:
VRE colonization was found in 34.56% (28/81) of the patients studied, with the majority 75% (21/28) carrying the
vanA
gene. The cases had significantly more (
P
< 0.05) duration of hospital stay and antibiotic exposure. Intake of metronidazole, vancomycin, and piperacillin-tazobactam were identified as significant risk factors both in univariate and multivariate analysis.
Conclusion:
A potential reservoir of VRE was thus revealed even in low VRE prevalence setting. Based on this high colonization status, restriction of empirical antibiotic use, reviewing of the ongoing antibiotic policy, and active VRE surveillance as an integral part of infection control strategy were suggested.
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