Development of a Multiplex Polymerase Chain Reaction-Based DNA Lateral Flow Assay as a Point-of-Care Diagnostic for Fast and Simultaneous Detection of MRSA and Vancomycin Resistance in Bacteremia

To reduce high mortality and morbidity rates, timely and proper treatment of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is required. A multiplex polymerase reaction (mPCR)-based DNA lateral flow assay (MBDLFA) was developed as a point-of-care diagnostic for simultaneous identification of S. aureus, methicillin resistance, and vancomycin resistance directly from blood or blood cultures. A mPCR was developed to detect nuc, mecA, and vanA/B; its sensitivity, specificity, and limit of detection (LOD) were determined. The developed reaction was further modified for use in MBDLFA and its sensitivity for detection of target genes from artificially inoculated blood samples was checked. The optimized mPCR successfully detected nuc, mecA, and vanA/B from genomic DNA of bacterial colonies with LODs of 107, 107, and 105 CFU/mL, respectively. The reaction was sensitive and specific. The optimized mPCR was used in MBDLFA that detected nuc, mecA, and vanA/B with LODs of 107, 108, and 104 CFU/mL, respectively, directly from artificially inoculated blood. The developed MBDLFA can be used as a rapid, cheap point-of-care diagnostic for detecting S. aureus, MRSA, and vancomycin resistance directly from blood and blood cultures in ~2 h with the naked eye. This will reduce morbidity, mortality, and treatment cost in S. aureus bacteremia

Identification of Potential Drug Targets in Helicobacter pylori Using In Silico Subtractive Proteomics Approaches and Their Possible Inhibition through Drug Repurposing

The class 1 carcinogen, Helicobacter pylori, is one of the World Health Organization’s high priority pathogens for antimicrobial development. We used three subtractive proteomics approaches using protein pools retrieved from: chokepoint reactions in the BIOCYC database, the Kyoto Encyclopedia of Genes and Genomes, and the database of essential genes (DEG), to find putative drug targets and their inhibition by drug repurposing. The subtractive channels included non-homology to human proteome, essentiality analysis, sub-cellular localization prediction, conservation, lack of similarity to gut flora, druggability, and broad-spectrum activity. The minimum inhibitory concentration (MIC) of three selected ligands was determined to confirm anti-helicobacter activity. Seventeen protein targets were retrieved. They are involved in motility, cell wall biosynthesis, processing of environmental and genetic information, and synthesis and metabolism of secondary metabolites, amino acids, vitamins, and cofactors. The DEG protein pool approach was superior, as it retrieved all drug targets identified by the other two approaches. Binding ligands (n = 42) were mostly small non-antibiotic compounds. Citric, dipicolinic, and pyrophosphoric acid inhibited H. pylori at an MIC of 1.5–2.5 mg/mL. In conclusion, we identified potential drug targets in H. pylori, and repurposed their binding ligands as possible anti-helicobacter agents, saving time and effort required for the development of new antimicrobial compounds

Prevalence of CagA and antimicrobial sensitivity of H. pylori isolates of patients with gastric cancer in Egypt

Abstract Background Helicobacter pylori (H. pylori) infection has been recognized as a significant threat for gastric cancer. However, studies that investigated the oncogenic factors and antimicrobial resistance of H. pylori in Egyptian isolates with gastric cancer are rare. The current study aimed to examine: (1) The pattern of antimicrobial resistance of H. pylori isolates of Egyptian gastric cancer patients, and (2) the prevalence of Cytotoxin-associated gene A (CagA). Methods Samples were collected from patients with gastric cancer. Isolation of H. pylori was performed using Columbia blood agar supplemented with 10% horse blood, and selective supplement of H. pylori for 3 to 5 days at 37 °C under microaerophilic condition. Isolates were identified by biochemical traits of H. pylori: oxidase, urease and catalase tests. Antimicrobial susceptibility of H. pylori isolates was examined against five antimicrobial agents using disc diffusion method. After that, extraction of DNA and Polymerase Chain Reaction (PCR) were performed to amplify the target genes. Results Twelve samples were collected from six males and six females Egyptian patients with cancer with an age range from 22 to 65 years. These cases are scarce and samples were collected over a period of almost eleven months. All isolates were confirmed as positive H. pylori through colony morphology and biochemical tests. The most effective antibiotic found was ciprofloxacin whereas all isolates showed resistance to metronidazole and erythromycin. The target CagA oncogene gene with expected product size was reported and seven (out of twelve) isolates (58%) were identified as CagA positive. Conclusion The current study is unique in two main aspects. First, it reported the pattern of antimicrobial susceptibility and prevalence of CagA gene in H. pylori from Egyptian patients. Second, it exclusively recruited isolates from gastric cancer patients which were confirmed by clinical and laparoscopic examination. The moderately high prevalence of CagA gene in Egyptian cancer patients calls for more vigilance against that oncogene

Data_Sheet_1_Aspartate α-decarboxylase a new therapeutic target in the fight against Helicobacter pylori infection.docx

Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: β-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague–Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing β-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.</p

A Novel Surface-Exposed Polypeptide Is Successfully Employed as a Target for Developing a Prototype One-Step Immunochromatographic Strip for Specific and Sensitive Direct Detection of Staphylococcus aureus Causing Neonatal Sepsis

Neonatal sepsis is a life-threatening condition and Staphylococcus aureus is one of its major causes. However, to date, no rapid and sensitive diagnostic tool has been developed for its direct detection. Bioinformatics analyses identified a surface-exposed 112-amino acid polypeptide of the cell wall protein NWMN_1649, a surface protein involved in cell aggregation and biofilm formation, as being a species-specific and highly conserved moiety. The polypeptide was cloned, purified, and used to immunize mice to raise specific immunoglobulins. The purified antibodies were conjugated to gold nano-particles and used to assemble an immunochromatographic strip (ICS). The developed prototype ICS detected as low as 5 &micro;g purified polypeptide and 102 CFU/mL S. aureus within 15 min. The strip showed superior ability to directly detect S. aureus in neonatal sepsis blood specimens without prior sample processing. Moreover, it showed no cross-reaction in specimens infected with two other major causes of neonatal sepsis; coagulase-negative staphylococci and Klebsiella pneumoniae. The selected NWMN_1649-derived polypeptide demonstrates success as a promising biomolecule upon which a prototype ICS has been developed. This ICS provides a rapid, direct, sensitive, and specific option for the detection of S. aureus causing neonatal sepsis. Such a tool is urgently needed especially in resources-limited countries

Table_1_Aspartate α-decarboxylase a new therapeutic target in the fight against Helicobacter pylori infection.DOCX

Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: β-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague–Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing β-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.</p

Table_2_Aspartate α-decarboxylase a new therapeutic target in the fight against Helicobacter pylori infection.DOCX

Effective eradication therapy for Helicobacter pylori is a worldwide demand. Aspartate α-decarboxylase (ADC) was reported as a drug target in H. pylori, in an in silico study, with malonic acid (MA) as its inhibitor. We evaluated eradicating H. pylori infection through ADC inhibition and the possibility of resistance development. MA binding to ADC was modeled via molecular docking. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of MA were determined against H. pylori ATCC 43504, and a clinical H. pylori isolate. To confirm selective ADC inhibition, we redetermined the MIC in the presence of products of the inhibited enzymatic pathway: β-alanine and pantothenate. HPLC was used to assay the enzymatic activity of H. pylori 6x-his tagged ADC in the presence of different MA concentrations. H. pylori strains were serially exposed to MA for 14 passages, and the MICs were determined. Cytotoxicity in different cell lines was tested. The efficiency of ADC inhibition in treating H. pylori infections was evaluated using a Sprague–Dawley (SD) rat infection model. MA spectrum of activity was determined in different pathogens. MA binds to H. pylori ADC active site with a good docking score. The MIC of MA against H. pylori ranged from 0.5 to 0.75 mg/mL with MBC of 1.5 mg/mL. Increasing β-alanine and pantothenate concentrations proportionally increased MA MIC. The 6x-his tagged ADC activity decreased by increasing MA concentration. No resistance to ADC inhibition was recorded after 14 passages; MA lacked cytotoxicity in all tested cell lines. ADC inhibition effectively eradicated H. pylori infection in SD rats. MA had MIC between 0.625 to 1.25 mg/mL against the tested bacterial pathogens. In conclusion, ADC is a promising target for effectively eradicating H. pylori infection that is not affected by resistance development, besides being of broad-spectrum presence in different pathogens. MA provides a lead molecule for the development of an anti-helicobacter ADC inhibitor. This provides hope for saving the lives of those at high risk of infection with the carcinogenic H. pylori.</p

Potential Antigenic Candidates for the Development of Peptide-Based Vaccines to Induce Immunization against Helicobacter pylori Infection in BALB/c Mice

Helicobacter pylori (H. pylori) has been identified as a group-1 definite carcinogen. As of yet, there is no available vaccine for this microorganism. Our study aimed to identify antigenic peptides in H. pylori using an in silico proteomic approach, and to evaluate their effectiveness as potential vaccine candidates. Four different peptide sequences were prioritized using the reverse vaccinology, namely, CagA1, CagA2, VacA, and SabA. Peptides emulsified with Freunde&rsquo;s adjuvant were used to immunize BALB/C mice. Subcutaneously immunized mice were challenged by oral administration of H. pylori. IgG, IgA, IL4, and IL17 were detected in mice sera. Histopathology of the dissected stomach of vaccinated and control mice were assessed using H&amp;E stain. IgG was significantly higher in mice vaccinated with SabA. IL-4 was significantly increased in CagA1, CagA2, VacA, and SabA vaccinated mice compared to the adjuvant group. Additionally, histopathological examination of gastric tissue showed a protective effect in the vaccinated groups compared to adjuvant and PBS groups. Our findings indicate a promising effect of the tested epitopes, particularly the SabA antigen, to induce an immune response against H. pylori