Selective pks+ Escherichia coli strains induce cell cycle arrest and apoptosis in colon cancer cell line.

pks+ Escherichia coli (E. coli) triggers genomic instability in normal colon cells which leads to colorectal cancer (CRC) tumorigenesis. Previously, we reported a significant presentation of pks+ E. coli strains in CRC patients' biopsies as compared to healthy cohorts. In this work, using an in vitro infection model, we further explored the ability of these strains in modulating cell cycle arrest and activation of apoptotic mediators in both primary colon epithelial cells (PCE) and CRC cells (HCT-116). Sixteen strains, of which eight tumours and the matching non-malignant tissues, respectively, from eight pks+ E. coli CRC patients were subjected to BrDU staining and cell cycle analysis via flow cytometry, while a subset of these strains underwent analysis of apoptotic mediators including caspase proteins, cellular reactive oxygen species (cROS) and mitochondrial membrane potential (MMP) via spectrophotometry as well as proinflammatory cytokines via flow cytometry. Data revealed that all strains exerted S-phase cell cycle blockade in both cells and G2/M phase in PCE cells only. Moreover, more significant upregulation of Caspase 9, cROS, proinflammatory cytokines and prominent downregulation of MMP were detected in HCT-116 cells indicating the potential role of pks related bacterial toxin as anticancer agent as compared to PCE cells which undergo cellular senescence leading to cell death without apparent upregulation of apoptotic mediators. These findings suggest the existence of discrepancies underlying the mechanism of action of pks+ E. coli on both cancer and normal cell lines. This work propounds the rationale to further understand the mechanism underlying pks+ E. coli-mediated CRC tumorigenesis and cancer killing.

Lactobacillus probiotics restore vaginal and gut microbiota of pregnant women with vaginal candidiasis.

The development of probiotics has now included the areas along the gut-vaginal axis. We thus aimed to investigate the effects of lactobacilli probiotic to modulate and restore vaginal and gut microbiota of pregnant women with vaginal candidiasis (VC). A randomised, double-blind and placebo-controlled study was performed in 78 pregnant women with VC. Patients were randomised to either the probiotic (SynForU-HerCare) or placebo which were administered at baseline and continued for 8-weeks (two capsules/day of 9.5 log cfu/capsule). Microbiota profiles were assessed at time points of weeks-0, 4 and 8 for high vaginal swab and faecal samples. Shannon diversity index showed that after 8-weeks amid VC, a shift in microbial community compositional changes occurred in the high vaginal region at both genus (P=0.025) and species (P=0.044) levels, where the administration of probiotic prevented such a shift. These changes were mainly attributed to a decreased in abundance of Lactobacillus (P=0.042) accompanied by increased abundance of Prevotella (P=0.002) and Atopobium (P=0.002) in the placebo group while the probiotic group remained unchanged over time. The administration of probiotics also prevented a reduced abundance of faecal phylum Firmicutes after 8-weeks as seen in the placebo group (P<0.0001), which also showed reduction at subsequent taxonomic levels of class, family, genera and species. VC has not only altered the microbiota of vagina regions but also gut microbiota profiles, causing lessening of gut microbiota that are crucial for gut nutrient availability, protection and immunity. The administration of lactobacilli probiotics has prevented such a shift, leading to better modulated gut and vaginal microenvironment amid VC. The study was registered at ClinicalTrials.gov: identifier number NCT03940612.

Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species.

The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.

A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species.

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 105 CFU/ml, while PCR displayed a limit of 5.9 x 107 CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 104 CFU/g, whereas PCR was 3.6 x 105 CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species

The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.

Simultaneous detection of methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by multiplex PCR.

A PCR-based assay that can simultaneously detect and differentiate five different types of nosocomial bacterial pathogens was developed. Six pairs of selected primers targeting femA (132 bp) and mecA (310 bp) of methicillin-resistant Staphylococcus aureus, gltA (722 bp) of Acinetobacter baumannii, phoA (903 bp) of Escherichia coli, mdh (364 bp) of Klebsiella pneumoniae and oprL (504 bp) of Pseudomonas aeruginosa were used in this study. The conditions were optimized for the multiplex PCR to ensure specific amplification of the selected targets. Sensitivity and specificity tests were also carried out using a blind test approach on 50 bacterial cultures and resulted in 100% for both positive and negative predictive values.

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes.

AIMS: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. METHODS AND RESULTS: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 x 10(4) CFU ml(-1) (c. 200 CFU per PCR) to 2 x 10(3) CFU ml(-1) (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. CONCLUSIONS: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. SIGNIFICANCE AND IMPACT OF THE STUDY: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.