REGULATION OF TOXIN SYNTHESIS BY CLOSTRIDIUM DIFFICILE

Clostridium difficile is the leading definable cause of nosocomial diarrhea worldwide due to its virulence, multi-drug resistance, spore-forming ability, and environmental persistence. The incidence of C. difficile infection (CDI) has been increasing exponentially in the last decade. Virulent strains of C. difficile produce either toxin A and/or toxin B, which are essential for the pathogenesis of this bacterium. Current methods for diagnosing CDI are mostly qualitative tests that detect the bacterium, the toxins, or the toxin genes. These methods do not differentiate virulent C. difficile strains that produce active toxins from non-virulent strains that do not produce toxins or produce inactive toxins. Based on the knowledge that C. difficile toxins A and B cleave a substrate that is stereochemically similar to the native substrate of the toxins, uridine diphosphoglucose, a quantitative, cost-efficient assay, the Cdifftox activity assay, was developed to measure C. difficile toxin activity. The concept behind the activity assay was modified to develop a novel, rapid, sensitive, and specific assay for C. difficile toxins in the form of a selective and differential agar plate culture medium, the Cdifftox Plate assay (CDPA). This assay combines in a single step the specific identification of C. difficile strains and the detection of active toxin(s). The CDPA was determined to be extremely accurate (99.8% effective) at detecting toxin-producing strains based on the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. This new assay advances and improves the culture methodology in that only C. difficile strains will grow on this medium and virulent strains producing active toxins can be differentiated from non-virulent strains. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains and provides a clinical isolate for antibiotic susceptibility testing and strain typing. The Cdifftox activity assay was used to screen for inhibitors of toxin activity. Physiological levels of the common human conjugated bile salt, taurocholate, was found to inhibit toxin A and B in vitro activities. When co-incubated ex vivo with purified toxin B, taurocholate protected Caco-2 colonic epithelial cells from the damaging effects of the toxin. Furthermore, using a caspase-3 detection assay, taurocholate reduced the extent of toxin B-induced Caco-2 cell apoptosis. These results suggest that bile salts can be effective in protecting the gut epithelium from C. difficile toxin damage, thus, the delivery of physiologic amounts of taurocholate to the colon, where it is normally in low concentration, could be useful in CDI treatment. These findings may help to explain why bile rich small intestine is spared damage in CDI, while the bile salt poor colon is vulnerable in CDI. Toxin synthesis in C. difficile occurs during the stationary phase, but little is known about the regulation of these toxins. It was hypothesized that C. difficile toxin synthesis is regulated by a quorum sensing mechanism. Two lines of evidence supported this hypothesis. First, a small (KDa), diffusible, heat-stable toxin-inducing activity accumulates in the medium of high-density C. difficile cells. This conditioned medium when incubated with low-density log-phase cells causes them to produce toxin early (2-4 hrs instead of 12-16 hrs) and at elevated levels when compared with cells grown in fresh medium. These data suggested that C. difficile cells extracellularly release an inducing molecule during growth that is able to activate toxin synthesis prematurely and demonstrates for the first time that toxin synthesis in C. difficile is regulated by quorum signaling. Second, this toxin-inducing activity was partially purified from high-density stationary-phase culture supernatant fluid by HPLC and confirmed to induce early toxin synthesis, even in C. difficile virulent strains that over-produce the toxins. Mass spectrometry analysis of the purified toxin-inducing fraction from HPLC revealed a cyclic compound with a mass of 655.8 Da. It is anticipated that identification of this toxin-inducing compound will advance our understanding of the mechanism involved in the quorum-dependent regulation of C. difficile toxin synthesis. This finding should lead to the development of even more sensitive tests to diagnose CDI and may lead to the discovery of promising novel therapeutic targets that could be harnessed for the treatment C. difficile infections

Secondary Plant Metabolites as Potent Drug Candidates against antimicrobial-Resistant Pathogens

Antibiotic resistance is a major public health threat of the twenty-first century and represents an important risk to the global economy. Healthcare-associated infections mainly caused by drug-resistant bacteria are wreaking havoc in patient care worldwide. The spread of such pathogens limits the utility of available drugs and complicates the treatment of bacterial diseases. As a result, there is an urgent need for new drugs with mechanisms of action capable of curbing resistance. Plants synthesize and utilize various metabolic compounds to deter pathogens and predators. Utilizing these plant-based metabolites is a promising option in identifying novel bioactive compounds that could be harnessed to develop new potent antimicrobial drugs to treat multidrug-resistant pathogens. The purpose of this review is to highlight medicinal plants as important sources of novel antimicrobial agents that could be developed to help combat antimicrobial resistance

Pediatric Diarrhea Patients Living in Urban Areas Have a Higher Incidence of Clostridioides difficile Infection

Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea and an unappreciated contributor to child mortality in low- and middle-income countries where the diagnosis may be difficult. There is little information about the prevalence of CDI among infants, children, and adolescents in Africa. Using a cross-sectional design, seventy-six samples were collected from pediatric patients presenting with diarrhea, including infants (≤ 2 years old), children (2-12 years), and adolescents (13 ≤17 years) from three hospitals between January and December 2019. Demographic data, medical history, and prior antibiotic use were recorded. Toxigenic culture and PCR were used to detect and validate the presence of C. difficile in the samples. Data obtained were analyzed using descriptive and inferential statistics. A total of 29 (38.7%), 39 (52.0%) and 7 (9.3%) samples were from infants, children, and adolescents, respectively. The average age of the patients was 4.4 years. Of these samples, 31 (41%) were positive for C. difficile by culture and were verified by PCR amplification of C. difficile-specific genes (tcdA and tcdB). The most positive cases were children (53.3%) and infants (40.0%) with the majority of them residing in urban areas. Forty-nine (66.2%) of the patients had no known antibiotics exposure, whereas 29.0% and 29.7% reported the use of over-the-counter antibiotics at 14 and 90 days, prior to the hospital visit, respectively. CDI is relatively common among children with diarrhea in Northern Nigeria. Therefore, for effective management and treatment, more attention should be given to testing for C. difficile as one of the causative agents of diarrhea

Emergence of Dual and Multicarbapenemase Coproducing organisms in the United States

Carbapenemase-producing organisms commonly carry a single carbapenemase gene conferring resistant to carbapenems and other β-lactam antibiotics. Here, we report rare cases of multidrug-resistan

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme

Multistate Outbreaks of Foodborne Illness in the United States associated With Fresh Produce From 2010 to 2017

In the United States, the consumption of fresh fruits and vegetables has increased during recent years as consumers seek to make healthier lifestyle choices. However, the number of outbreaks associated with fresh produce that involve cases in more than one state (multistate) has increased concomitantly. As the distance along the farm-to-fork continuum has lengthened over time, there are also more opportunities for fresh produce contamination with bacterial pathogens before it reaches the consumer. This review provides an overview of the three bacterial pathogens (i.e., pathogeni

Dietary Habits, Meal Timing, and Meal Frequency in Kuwaiti adults: analysis of the Kuwait National Nutrition Surveillance Data

Dietary habits, including meal frequency, meal timing, and skipping meals, have been extensively studied due to their association with the development of noncommunicable diseases (NCDs). This study describes dietary habits, meal timing, frequency, skipping meals, and late-night eating in Kuwaiti adults. Kuwait National Nutrition Surveillance System data were utilized to reach the objectives of this study. The findings reveal that approximately 54% of the adults in Kuwait eat after 10 p.m., 29% skip breakfast, and 9.8% skip dinner. Furthermore, adults in Kuwait consume 4.4 meals per day on average. Women skip breakfast more often and have more extended night fasting than men

Food Safety attitudes, Behaviors, and Hygiene Measures among Predominantly Low-income Parents in Houston, Texas

ABSTRACT: Foodborne infections in the United States affect racial-ethnic minority and low-income populations at higher rates than the general population. to identify the prevalence of food safety behaviors and demographic characteristics associated with food handling practices among a susceptible, high-risk population, a cross-sectional survey was administered to 106 parents with children enrolled at two elementary schools serving predominantly low-income families in Houston, Texas. Relationships between demographic characteristics and food safety behavioral outcomes were examined using cross-tabulations and Fisher\u27s exact test. Most respondents were female (93.4%), Hispanic, Latino, or Mexican American (94.9%), and had no previous food handling employment experience (75.0%). The primary source of food safety information reported was the Internet (32.7%), and nearly half of parents (42.7%) reported that they did not consider contamination of food with germs a serious food safety problem. Hand washing before food preparation was more common (98.0%) than before touching the refrigerator handle (66.3%), after electronic device use (55.6%), or after handling raw animal proteins (77.6%). The prevalence of fresh fruit (98.0%) and vegetable (97.9%) washing and appropriate contaminated cutting board handling (89.0%) was high among parents. Self-reported gaps in food handling behaviors identified included lack of food thermometer ownership (80.4%), use of reusable cleaning tools (71.0%), inappropriate defrosting methods (67.4%), and washing of raw poultry (86.3%), seafood (84.9%), and meat (74.7%). Hand washing after electronic device use and defrosting methods were observed to vary significantly according to demographic characteristics. Food safety education with messages targeted to specific demographic groups may be necessary to reduce the risk of foodborne disease among low-income parents and young children