Synthesis, Characterization, Cytotoxicity Analysis and Evaluation of Novel Heterocyclic Derivatives of Benzamidine against Periodontal Disease Triggering Bacteria.

Periodontal disease (PD) is multifactorial oral disease that damages tooth-supporting tissue. PD treatment includes proper oral hygiene, deep cleaning, antibiotics therapy, and surgery. Despite the availability of basic treatments, some of these are rendered undesirable in PD treatment due to side effects and expense. Therefore, the aim of the present study is to develop novel molecules to combat the PD triggering pathogens. The study involved the synthesis of 4-((5-(substituted-phenyl)-1,3,4-oxadiazol-2-yl)methoxy)benzamidine (5a-e), by condensation of 2-(4-carbamimidoylphenoxy)acetohydrazide (3) with different aromatic acids; and synthesis of 4-((4-(substituted benzylideneamino)-4H-1,2,4-triazol-3-yl)methoxy)benzamidine (6a-b) by treatment of compound 3 with CS2 followed by hydrazination and a Schiff reaction with different aromatic aldehydes. Synthesized compounds were characterized based on the NMR, FTIR, and mass spectrometric data. To assess the effectiveness of the newly synthesized compound in PD, new compounds were subjected to antimicrobial evaluation against P. gingivalis and E. coli using the micro-broth dilution method. Synthesized compounds were also subjected to cytotoxicity evaluation against HEK-293 cells using an MTT assay. The present study revealed the successful synthesis of heterocyclic derivatives of benzamidine with significant inhibitory potential against P. gingivalis and E. coli. Synthesized compounds exhibited minimal to the absence of cytotoxicity. Significant antimicrobial potential and least/no cytotoxicity of new heterocyclic analogs of benzamidine against PD-triggering bacteria supports their potential application in PD treatment.

Synthesis, Characterization and Biological Evaluation of Novel Benzamidine Derivatives: Newer Antibiotics for Periodontitis Treatment.

Periodontal disease (PD) is complex polymicrobial disease which destroys tooth-supporting tissue. Although various synthetic inhibitors of periodontitis-triggering pathogens have been recognized, their undesirable side effects limit their application. Hence, the present study intended to perform the synthesis, characterization, antimicrobial evaluation, and cytotoxicity analysis of novel benzamidine analogues (NBA). This study involved the synthesis of novel imino bases of benzamidine (4a-c), by reacting different aromatic aldehydes with 2-(4-carbamimidoylphenoxy) acetohydrazide (3), which was synthesized by the hydrazination of ethyl 2-(4-carbamimidoylphenoxy) acetate (2), the derivative of 4-hydroxybenzene carboximidamide (1). This was followed by characterization using FTIR, 1H, 13C NMR and mass spectrometry. All synthesized compounds were further tested for antimicrobial potential against PD-triggering pathogens by the micro broth dilution method. The cytotoxicity analysis of the NBA against HEK 293 cells was conducted using an MTT assay. The present study resulted in a successful synthesis of NBA and elucidated their structures. The synthesized NBA exhibited significant antimicrobial activity values between 31.25 and 125 µg/mL against tested pathogens. All NBA exhibited weak cytotoxicity against HEK 293 cells at 7.81 µg, equally to chlorhexidine at 0.2%. The significant antimicrobial activity of NBA against PD-triggering pathogens supports their potential application in periodontitis treatment.

Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples.

Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

A rapid DNA biosensor for the molecular diagnosis of infectious disease.

Treating patients with infectious diseases relies heavily on rapid and proper diagnosis. Molecular detection such as PCR has become increasingly important and efforts have been made to simplify these detection methods. This study reports the development of a glass fibre-based lateral flow DNA biosensor that uses capture reagents coupled to carrier beads and detector reagent bioconjugated to gold nanoparticles, for the detection of foodborne pathogen, Vibrio cholerae. The DNA biosensor contains a test line which captures target PCR amplicons, an internal amplification control (IC) line which captures IC amplicons and a control line which acts as membrane control to validate the functionality of this device. The test line captures biotin labelled DNA, while the IC line captures digoxigenin labelled DNA. The detector reagent recognizes the fluorescein haptens of the amplified DNA and produces visual red lines. Scanning electron microscopy (SEM) studies performed indicated that the capture reagents remained relatively immobile within the matrix of the membrane even after binding of the detector reagent. The DNA biosensor recorded a limit of detection (LoD) of 5 ng of target DNA. A clinical evaluation was carried out with 174 strains of V. cholerae and non V. cholerae bacteria and the DNA biosensor recorded 100% for both sensitivity and specificity when compared to conventional agarose gel detection of DNA. Thus it is a viable alternative to agarose gel analysis and is easy-to-use, disposable and do not require any specialized equipment and use of carcinogenic chemicals.

Development of a dry reagent-based triplex PCR for the detection of toxigenic and non-toxigenic Vibrio cholerae.

Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q10 method and it was found to be stable for approximately 7 months at 24 °C. The limit of detection of the thermostabilized triplex PCR assay was 2×10(4) c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.

Construction and characterization of rtxA and rtxC mutants of auxotrophic O139 Vibrio cholerae.

Vibrio cholerae is a Gram-negative bacterium that causes diarrheal disease. V. cholerae O1 and O139 serogroups are toxigenic and are known to cause epidemic cholera. These serogroups produce cholera toxin and other accessory toxins such as accessory cholera enterotoxin, zonula occludens toxin, and multifunctional, autoprocessing repeat in toxin (MARTX). In the present study, we incorporated mutated rtxA and rtxC genes that encode MARTX toxin into the existing aminolevulinic acid (ALA) auxotrophic vaccine candidate VCUSM2 of V. cholerae O139 serogroup. The rtxC mutant was named VCUSM9 and the rtxC/rtxA mutant was named VCUSM10. VCUSM9 and VCUSM10 were able to colonize intestinal cells well, compared with the parent vaccine strain, and produced no fluid accumulation in a rabbit ileal loop model. Cell rounding and western blotting assays indicated that mutation of the rtxC gene alone (VCUSM9 strain) did not abolish MARTX toxicity. However mutation of both the rtxA and rtxC genes (VCUSM10) completely abolished MARTX toxicity. Thus we have produced a new, less reactogenic, auxotrophic rtxC/rtxA mutated vaccine candidate against O139 V. cholerae.

Comparison of histopathological features of Vibrio cholerae O1 El Tor and O139 Bengal infections in rabbit intestinal mucosa.

Vibrio cholerae is the causative agent of the infectious disease, cholera. The bacteria adhere to the mucosal membrane and release cholera toxin, leading to watery diarrhea. There are >100 serovars of V. cholerae, but the O1 and O139 serovars are the main causative agents of cholera. The present study aimed to compare the severity of intestinal mucosal infection caused by O1 El Tor and O139 V. cholerae in a rabbit ileal loop model. The results showed that although the fluid accumulation was similar in the loops inoculated with O1 and O139 V. cholerae, the presence of blood was detected only in the loops inoculated with the O139 serovar. Serosal hemorrhage was confirmed by histopathological examination and the loops inoculated with O139 showed massive destruction of villi and loss of intestinal glands. The submucosa and muscularis mucosa of the ileum showed the presence of edema with congested blood vessels, while severe hemorrhage was seen in the muscularis propria layer. The loops inoculated with O1 El Tor showed only minimal damage, with intact intestinal villi and glands. Diffuse colonies of the O139 serovar were seen to have infiltrated deep into the submucosal layer of the intestine. Although the infection caused by the O1 serovar was focal and invasive, it was more superficial than that due to O139, and involved only the villi. These observations were confirmed by immunostaining with O1 and O139 V. cholerae-specific monoclonal antibodies. The peroxidase reaction demonstrated involvement of tissues down to the submucosal layer in O139 V. cholerae infection, while in O1 El Tor infection, the reaction was confined mainly to the villi, and was greatly reduced in the submucosal region. This is the first reported study to clearly demonstrate the histopathological differences between infections caused by the O139 Bengal and O1 El Tor pathogenic serovars of V. cholerae.

Enzyme-linked amperometric electrochemical genosensor assay for the detection of PCR amplicons on a streptavidin-treated screen-printed carbon electrode.

A general purpose enzyme-based amperometric electrochemical genosensor assay was developed wherein polymerase chain reaction (PCR) amplicons labeled with both biotin and fluorescein were detected with peroxidase-conjugated antifluorescein antibody on a screen-printed carbon electrode (SPCE). As a proof of principle, the response selectivity of the genosensor was evaluated using PCR amplicons derived from lolB gene of Vibrio cholerae. Factors affecting immobilization, hybridization, and nonspecific binding were optimized to maximize sensitivity and reduce assay time. On the basis of the background amperometry signals obtained from nonspecific organisms and positive signals obtained from known V. cholerae, a threshold point of 4.20 microA signal was determined as positive. Under the optimum conditions, the limit of detection (LOD) of the assay was 10 CFU/mL of V. cholerae. The overall precision of this assay was good, with the coefficient of variation (CV) being 3.7% using SPCE and intermittent pulse amperometry (IPA) as an electrochemical technique. The assay is sensitive, safe, and cost-effective when compared to conventional agarose gel electrophoresis, real-time PCR, and other enzyme-linked assays for the detection of PCR amplicons. Furthermore, the use of a hand-held portable reader makes it suitable for use in the field.

A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species.

BACKGROUND: Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene. RESULTS: Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases. CONCLUSION: The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1.

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.