New Insights into the Molecular Mechanism of Clostridium perfringens Spore Germination

C. perfringens is a spore-forming, gram-positive, anaerobic pathogenic bacterium capable of causing a wide variety of diseases in both humans and animals. However, the two most common illnesses in humans are C. perfringens type A food poisoning (FP) and non-food-borne (NFB) gastrointestinal (GI) illnesses. Interestingly, these two major diseases are caused only by C. perfringens Type A isolates that are able to produce the C. perfringens enterotoxin (CPE). Importantly, the CPE-encoding gene (cpe) can be found in different places in FP and NFB isolates. C. perfringens FP isolates carry the cpe gene on the chromosome, while NFB isolates causing GI illnesses (i.e., sporadic diarrhea and antibiotic-associated diarrhea) carry a plasmid-borne copy of the cpe. C. perfringens spores are highly resistant and can survive in the environment for decades. When spores are in a favorable environment, they can initiate germination and return to active growth to cause disease. Spore germination is an early step and essential stage in the progression of C. perfringens infection in humans and animals. Recent findings have identified the germinants of spores of C. perfringens FP and NFB isolates. A variety of germinants can initiate the process, including nutrients, amino acids, cationic surfactants, and enzymes termed germinant. Further understanding of the germination of Clostridium species is needed, since Bacillus species spore germination has been studied well. In the first study, we demonstrated the individual role of each of these germinant receptors (GRs) in C. perfringens spore germination. To reach the goal, we constructed mutant strains carrying single mutations in gerKA or gerKC, and double mutant gerKB gerAA, and characterized the germination phenotypes of their spores in the presence of nutrient and non-nutrient germinants. Through Western blot analyses, the precise location of GerKC protein in spores was also determined. This study offers the following findings: (i) Spores of gerKC mutant did not germinate with KCl, L-asparagine, a mixture of asparagine and KCl, and NaPi at pH 6.0. (ii) The gerKC spores germinated poorly compared to wild-type and other GRs mutant spores with the non-nutrient germinants dodecylamine and a 1:1 chelate of Ca²⁺ and dipicolinic acid. (iii) The germination defect in gerKC spores was restored by complementing the gerKC mutant with wild-type gerK operon, indicating that GerKC is essential for germination of C. perfringens spores. (iv) GerKC is essential for the release of DPA from the spore's core during germination with KCl and dodecylamin. (v) GerKC is also essential for spore’s viability; Finally, (vi) GerKC localizes in the spore’s inner membrane. A second study of this work investigated the precise location for the Csp proteases CspB and cortex lytic enzymes SleC in spores of C. perfringens FP strain SM101. It was shown that CspB and pro-SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. In addition, quantitative Western blot analyses demonstrated that there are approximately 2,000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore. The third study was to identify and characterize the germinants and receptors involved in C. perfringens NFB strain F4969. Results from these studies indicate that NFB strain F4969 germinates with AK in a cooperative manner, while FP strain SM101 mixture of L-Asparagine and KCl (AK) components are capable of triggering spore germination through independent pathways. Spores of gerKA-KC, gerKA, gerKC, gerAA, and gerKB knock-out mutants indicate that (i) germination of FP and NFB spores differs significantly in rich media and several defined germinants; (ii) L-asn and KCl induced germination in NFP F4969 spores cooperatively, while either germinant alone triggers germination of FP spores; (iii) GerKC and GerAA proteins are required for normal germination of NFB spores with AK and L-cys;(iv) The colony-forming ability of gerKC or gerKA-KC spores was significantly lower than that of wild-type spores; although the ability of gerAA spores to outgrow was significantly affected, they gave rise to similar titers as wild-type spores. Together, this dissertation study will help further understanding of the mechanism of spore germination of C. perfringens and the insights into the roles of spore germination for both FP and NFB CPE-producing C. perfringens isolates

Patient Safety Culture and Nursing Shortage among Nurses in Al-Medina Hospitals

This article explores the current relationship of patient safety culture to the nursing shortage in general hospitals in the city of Al-Medina, Saudi Arabia. A mixed methods article was conducted on 335 respondents drawn from 10 government hospitals in the region. We utilized an AHRQ Questionnaire for data collection, and we analyzed the data with statistical approaches such as the mean, percentages, and standard deviation. This analysis established significant relationship between patient safety cultures and nursing shortages. Furthermore, we found a weak and positive relationship between some of patient SC’s components and nursing shortages in the hospitals studied; these components included communications (r=0.144, p<0.05), the frequency of event reports (r=0.151, p<0.05), and hospital work area (r=0.329, p<0.05). Most of the nurses who participated in the current article had low incident report rates, with only three to five event reports in one year. However, among the dimensions of patient safety culture, it was found in this article that the nurses had a high level of communication (3.91±0.54/High). The overall grading of patient safety culture was moderate (3.27±0.93) in Al-Medina City. The findings of this investigation will help the Ministry of Health (MOH) decide how to improve patient safety. Moreover, to increase the condition of nursing care provided to the patients and to boost patient safety culture, the healthcare sector must consider the factors and other causes. It is also crucial that nurse leaders learn to maintain and secure patient safety and that nurses learn to frequently report incidents, including any actual errors. Keywords: Patient Safety, Culture, Nursing Shortage DOI: 10.7176/JHMN/100-07 Publication date:May 31st 202

Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa

Indexación: Web of Science.Clostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CBI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.http://journal.frontiersin.org/article/10.3389/fcimb.2016.00099/ful

Utilizing Andrographis paniculata leaves and roots by effective usage of the bioactive andrographolide and its nanodelivery: investigation of antikindling and antioxidant activities through in silico and in vivo studies

To valorise the bioactive constituents abundant in leaves and other parts of medicinal plants with the objective to minimize the plant-based wastes, this study was undertaken. The main bioactive constituent of Andrographis paniculata, an Asian medicinal plant, is andrographolide (AG, a diterpenoid), which has shown promising results in the treatment of neurodegenerative illnesses. Continuous electrical activity in the brain is a hallmark of the abnormal neurological conditions such as epilepsy (EY). This can lead to neurological sequelae. In this study, we used GSE28674 as a microarray expression profiling dataset to identify DEGs associated with andrographolide and those with fold changes >1 and p-value <0.05 GEO2R. We obtained eight DEG datasets (two up and six down). There was marked enrichment under various Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms for these DEGs (DUSP10, FN1, AR, PRKCE, CA12, RBP4, GABRG2, and GABRA2). Synaptic vesicles and plasma membranes were the predominant sites of DEG expression. AG acts as an antiepileptic agent by upregulating GABA levels. The low bioavailability of AG is a significant limitation of its application. To control these limitations, andrographolide nanoparticles (AGNPs) were prepared and their neuroprotective effect against pentylenetetrazol (PTZ)-induced kindling epilepsy was investigated using network pharmacology (NP) and docking studies to evaluate the antiepileptic multi-target mechanisms of AG. Andrographolide is associated with eight targets in the treatment of epilepsy. Nicotine addiction, GABAergic synapse, and morphine addiction were mainly related to epilepsy, according to KEGG pathway enrichment analysis (p < 0.05). A docking study showed that andrographolide interacted with the key targets. AG regulates epilepsy and exerts its therapeutic effects by stimulating GABA production. Rats received 80 mg/kg body weight of AG and AGNP, phenytoin and PTZ (30 mg/kg i.p. injection on alternate days), brain MDA, SOD, GSH, GABAand histological changes of hippocampus and cortex were observed. PTZ injected rats showed significantly (***p < 0.001) increased kindling behavior, increased MDA, decreased GSH, SOD, GABA activities, compared with normal rats, while treatment AGNPs significantly reduced kindling score and reversed oxidative damage. Finally, we conclude that the leaves and roots of A. Paniculata can be effectively utilized for its major bioactive constituent, andrographolide as a potent anti-epileptic agent. Furthermore, the findings of novel nanotherapeutic approach claim that nano-andrographolide can be successfully in the management of kindling seizures and neurodegenerative disorders

ZnO Nano-swirlings for Azo Dye AR183 photocatalytic degradation and antimycotic activity

The sol-gel technique was used to fabricate ZnO Nano-swirlings (ZNsw) at a predetermined agitation rate (of \u3e\u3e 1900 rpm), with around 21.94 gm of zinc acetate dihydrate and 0.2 g cetyltrimethylammoniumbromide (CTAB) and a cationic surfactant (drop-wise). The impact of the predetermined agitation condition on the molecular size and morphology of ZNsw is examined, and the outcomes are dissected by useful characterization tools and techniques viz. XRD, SEM embedded with EDS, TEM, FT-IR and UV–visible. The SEM and TEM results suggest that the product formed into a big cluster of adequate ZNsw, containing a significant quantity of folded long thread-lengths. Each group indicated a fair amount of the volume of these lengths. The photocatalytic process of ZNsw was carried out as a result of the irradiation time due to the deterioration of Azo Dye AR183, resulting in approximately 79 percent dye discoloration following an 80-min UV light irradiation in the presence of ZNsw. Additionally, the synthesized ZNsw was tested for antagonistic activity, and the growth hindrance of two plant pathogenic fungal strains found. Per cent inhibition in growth of Rhizoctonia solani and Alternaria alternata were observed in response to ZNsw

Systematic Review and Meta-Analysis on the Frequency of Antibiotic-Resistant Clostridium Species in Saudi Arabia

Clostridium is a genus comprising Gram-positive, rod-shaped, spore-forming, anaerobic bacteria that cause a variety of diseases. However, there is a shortage of information regarding antibiotic resistance in the genus in Saudi Arabia. This comprehensive analysis of research results published up until December 2021 intends to highlight the incidence of antibiotic resistance in Clostridium species in Saudi Arabia. PubMed, Google Scholar, Web of Science, SDL, and ScienceDirect databases were searched using specific keywords, and ten publications on antibiotic resistance in Clostridium species in Saudi Arabia were identified. We found that the rates of resistance of Clostridium difficile to antibiotics were as follows: 42% for ciprofloxacin, 83% for gentamicin, 28% for clindamycin, 25% for penicillin, 100% for levofloxacin, 24% for tetracycline, 77% for nalidixic acid, 50% for erythromycin, 72% for ampicillin, and 28% for moxifloxacin; whereas those of C. perfringens were: 21% for metronidazole, 83% for ceftiofur, 39% for clindamycin, 59% for penicillin, 62% for erythromycin, 47% for oxytetracycline, and 47% for lincomycin. The current findings suggest that ceftiofur, erythromycin, lincomycin, and oxytetracycline should not be used in C. perfringens infection treatments in humans or animals in Saudi Arabia

Clostridium difficile Infections: A Global Overview of Drug Sensitivity and Resistance Mechanisms

Clostridium difficile (C. difficile) is the most prevalent causative pathogen of healthcare-associated diarrhea. Notably, over the past 10 years, the number of Clostridium difficile outbreaks has increased with the rate of morbidity and mortality. The occurrence and spread of C. difficile strains that are resistant to multiple antimicrobial drugs complicate prevention as well as potential treatment options. Most C. difficile isolates are still susceptible to metronidazole and vancomycin. Incidences of C. difficile resistance to other antimicrobial drugs have also been reported. Most of the antibiotics correlated with C. difficile infection (CDI), such as ampicillin, amoxicillin, cephalosporins, clindamycin, and fluoroquinolones, continue to be associated with the highest risk for CDI. Still, the detailed mechanism of resistance to metronidazole or vancomycin is not clear. Alternation in the target sites of the antibiotics is the main mechanism of erythromycin, fluoroquinolone, and rifamycin resistance in C. difficile. In this review, different antimicrobial agents are discussed and C. difficile resistance patterns and their mechanism of survival are summarized

Divalent Cation Signaling in <i>Clostridium perfringens</i> Spore Germination

Spore germination plays an essential role in the pathogenesis of Clostridium perfringens-associated food poisoning. Germination is initiated when bacterial spores sense various stimuli, including chemicals and enzymes. A previous study showed that dipicolinic acid (DPA) chelated with calcium (Ca-DPA) significantly stimulated spore germination in C. perfringens. However, whether Ca2+ or DPA alone can induce germination is unknown. Therefore, we aimed to evaluate the possible roles of Ca2+ and other divalent cations present in the spore core, such as Mn2+ and Mg2+, in C. perfringens spore germination. Our study demonstrated that (i) Ca-DPA, but not DPA alone, induced C. perfringens spore germination, suggesting that Ca2+ might play a signaling role; (ii) all tested calcium salts induced spore germination, indicating that Ca2+ is critical for germination; (iii) the spore-specific divalent cations Mn2+ and Mg2+, but not Zn2+, induced spore germination, suggesting that spore core-specific divalent cations are involved in C. perfringens spore germination; and (iv) endogenous Ca2+ and Mg2+ are not required for induction of C. perfringens spore germination, whereas exogenous and partly endogenous Mn2+ are required. Collectively, our results suggest that exogenous spore core-specific divalent cation signals are more important than endogenous signals for the induction of spore germination

Prevalence of Multidrug-Resistant Bacteria in Healthcare-Associated Bloodstream Infections at Hospitals in Riyadh, Saudi Arabia

Bloodstream infection (BSI) prevalence in hospitalized patients has increased owing to the spread of antibiotic-resistant pathogens; moreover, antimicrobial resistance in bacteria is a global problem. Here, BSIs are investigated in several patients at a hospital in Saudi Arabia, and the resistance of bacterial isolates to widely used drugs is determined. Throughout 2020, bacteria isolated from patients were identified and subjected to antibiotic susceptibility testing. In total, 1125 bacterial isolates were obtained from 1039 patients; among them, gram-positive bacteria were significantly more abundant than gram-negative bacteria. The most prevalent bacteria were Staphylococcus epidermidis and Klebsiella pneumoniae. Notably, gram-negative bacteria were mainly isolated from adult patients, and 20.63% of the gram-positive isolates were from pediatric patients, which was significantly higher than the corresponding percentages in elders and adults. The gram-positive isolates were mainly resistant to cephalothin, oxacillin, amoxicillin-clavulanate, and erythromycin and susceptible to penicillin, gentamicin, ciprofloxacin, and vancomycin. Additionally, the gram-negative isolates were mainly resistant to ampicillin, cephalothin, and amoxicillin-clavulanate and susceptible to amikacin, ertapenem, aztreonam, colistin, and trimethoprim-sulfamethoxazole. Consequently, the high prevalence of infective multidrug-resistant bacteria may account as a significant health issue; it is considered a hazard in Riyadh hospitals and must be prevented at all costs