Molecular detection, quantification, and isolation of Streptococcus gallolyticus bacteria colonizing colorectal tumors: inflammation-driven potential of carcinogenesis via IL-1, COX-2, and IL-8

<p>Abstract</p> <p>Background</p> <p>Colorectal cancer (CRC) has long been associated with bacteremia and/or endocarditis by <it>Streptococcus gallolyticus </it>member bacteria (SGMB) but the direct colonization of SGMB along with its molecular carcinogenic role, if any, has not been investigated. We assessed the colonization of SGMB in CRC patients with history of bacteremia (CRC-w/bac) and without history of bacteremia (CRC-wo/bac) by isolating SGMB from feces, mucosal surfaces of colorectum, and colorectal tissues and detecting SGMB DNA, via PCR and in situ hybridization (ISH) assays targeting <it>SodA </it>gene in colorectal tissues. Moreover, mRNA of IL1, IL-8, COX-2, IFN-γ, c-Myc, and Bcl-2 in colorectal tissues of studied groups was assessed via ISH and RT-PCR.</p> <p>Results</p> <p>SGMB were found to be remarkably isolated in tumorous (TU) and non-tumorous (NTU) tissues of CRC-w/bac, 20.5% and 17.3%, and CRC-wo/bac, 12.8% and 11.5%, respectively while only 2% of control tissues revealed SGMB (P < 0.05); such contrast was not found in mucosal and fecal isolation of SGMB. The positive detection of SGMB DNA in TU and NTU of CRC-w/bac and CRC-wo/bac via PCR, 48.7%, 35.9%, 32.7%, and 23%, respectively, and ISH, 46.1%, 30.7%, 28.8%, and 17.3%, respectively, was higher than in control tissues, 4 and 2%, respectively (P < 0.05). SGMB count measured via quantitative PCR of SGMB DNA in terms of copy number (CN), in TU and NTU of CRC-w/bac and CRC-wo/bac, 2.96-4.72, 1.29-2.81, 2.16-2.92, and 0.67-2.07 log₁₀CN/g respectively, showed higher colonization in TU than in NTU and in CRC-w/bac than in CRC-wo/bac (P < 0.05). The PCR-based mRNA ratio and ISH-based percentage of positively stained cells of IL-1, 1.77 and 70.3%, COX-2, 1.63 and 44.8%, and IL-8, 1.73 and 70.3%, respectively, rather than IFN-γ, c-Myc, and Bcl-2, were higher in SGMB positive patients than in control or SGMB negative patients (P < 0.05).</p> <p>Conclusions</p> <p>The current study indicated that colorectal cancer is remarkably associated with SGMB; moreover, molecular detection of SGMB in CRC was superior to link SGMB with CRC tumors highlighting a possible direct and active role of SGMB in CRC development through most probably inflammation-based sequel of tumor development or propagation via, but not limited to, IL-1, COX-2, and IL-8.</p

The association of Streptococcus bovis/gallolyticus with colorectal tumors: The nature and the underlying mechanisms of its etiological role

Streptococcus bovis (S. bovis) bacteria are associated with colorectal cancer and adenoma. S. bovis is currently named S. gallolyticus. 25 to 80% of patients with S. bovis/gallolyticus bacteremia have concomitant colorectal tumors. Colonic neoplasia may arise years after the presentation of bacteremia or infectious endocarditis of S. bovis/gallolyticus. The presence of S. bovis/gallolyticus bacteremia and/or endocarditis is also related to the presence of villous or tubular-villous adenomas in the large intestine. In addition, serological relationship of S. gallolyticus with colorectal tumors and direct colonization of S. gallolyticus in tissues of colorectal tumors were found. However, this association is still under controversy and has long been underestimated. Moreover, the etiological versus non-etiological nature of this associationis not settled yet. Therefore, by covering the most of up to date studies, this review attempts to clarify the nature and the core of S. bovis/gallolyicus association with colorectal tumors and analyze the possible underlying mechanisms

Identification of Klebsiella oxytoca by VITEK-2 System in Baghdad Hospitals

Background: Klebsiella oxytoca is a Gram-negative rod-shaped bacterium that is becoming resistant to multiple drugs and is frequently endangering patients' lives. It is a member of the human microbiota. Objectives: To assess the value of identifying K. oxytoca using an automated diagnostic system (VITEK-2) as compared to traditional manual methods. Materials and Methods: A total of 136 clinical specimens were collected from patients in Baghdad hospitals during a period from July to November 2022. VITEK-2 system was used to recognize the isolated bacteria to the genus and species level. The biochemical indole test was used as a confirmatory test at the species level. Results: K. oxytoca was more common in urine samples 49 (36.0%) followed by blood samples 21 (15.4%). Of the total collected samples 77 (56.6%) were from inpatients and (43.3%) were from outpatients. The primary identification by cultural and microscopic examinations diagnosed all the isolates as Klebsiella. VITEK-2 system recognized them as K. pneumoniae. The indole test confirmed the species as K. oxytoca by the formation of the red ring. Conclusion: using a simple biochemical test like indole is crucial in clinical laboratories to investigate the accuracy of bacterial identification at the species level. Continuous evaluation for the identification results of the automated systems is needed and can be done by updating the system software for the new emerging pathogens in the hospitals.     Received June. 2022 Accepted Sept. 2023 Published Jan. 2024

Inhibition of Growth of Highly Resistant Bacterial and Fungal Pathogens by a Natural Product

The continuous escalation of resistant bacteria against a wide range of antibiotics necessitates discovering novel unconventional sources of antibiotics. B. oleracea L (red cabbage) is health-promoting food with proven anticancer and anti-inflammatory activities. However, it has not been researched adequately for its antimicrobial activity on potential resistant pathogens. The methanol crude extract of B. oleracea L. was investigated for a possible anti-microbial activity. The screening method was conducted using disc diffusion assay against 22 pathogenic bacteria and fungi. It was followed by evaluation of the minimum inhibitory concentration (MIC). Moreover, the antibacterial and the antifungal activities were confirmed using the minimum bactericidal concentration (MBC) and the minimum fungicidal concentration (MFC), respectively. Remarkable, antibacterial activity was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus, Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. Red cabbage is a rich source of phenolic compounds, anthocyanins being the most abundant class, which might explain its potent antimicrobial action. This extract is potentially novel for future antimicrobials, inexpensive, and readily available at a large scale for pharmaceutical companies for further investigation and processing

Investigation into the controversial association of Streptococcus gallolyticus with colorectal cancer and adenoma

Background: The seroprevalence of IgG antibodies of Streptococcus gallolyticus subspecies gallolyticus, CIP 105428, was evaluated to investigate the controversial association of S. gallolyticus with colorectal carcinoma and adenoma in attempt to investigate the nature of such association if any, by exploring the mRNA expression of NF-κB and IL-8. Moreover, the serological behavior of S. gallolyticus IgG antibodies was compared to that of an indicator bacterium of bowel, Bacteroides fragilis. Methods: ELISA was used to measure IgG antibodies of S. gallolyticus and B. fragilis in sera of 50 colorectal cancer, 14 colorectal adenoma patients, 30 age- and sex- matched apparently healthy volunteers (HV) and 30 age- and sex- matched colonoscopically-proven tumor-free control subjects. NF-κB and IL-8 mRNA expression was evaluated in tumorous and non-tumorous tissue sections of carcinoma and adenoma patients in comparison with that of control subjects by using in situ hybridization assay. Results: Colorectal cancer and adenoma patients were associated with higher levels of serum S. Gallolyticus IgG antibodies in comparison with HV and control subjects (P 0.05). ELISA cutoff value for the seropositivity of S. gallolyticus IgG was calculated from tumor-free control group. The expression of NF-κB mRNA was higher in tumorous than non-tumorous tissue sections of adenoma and carcinoma, higher in carcinoma/adenoma sections than in control subjects, higher in tumorous sections of carcinoma than in adenoma patients, and higher in S. gallolyticus IgG seropositive than in seronegative groups in both tumorous and non-tumorous sections (P < 0.05). IL-8 mRNA expression in tumorous sections of adenoma and carcinoma was higher than in non-tumorous sections, higher in carcinoma/adenoma than in control subjects, and higher in S. gallolyticus IgG seropositive than in seronegative groups in tumorous rather than non-tumorous sections (P < 0.05). Conclusion: S. gallolyticus most likely plays an essential role in the oncogenic progression of normal colorectal mucosa to adenoma and to CRC. This promoting/propagating role of S. gallolyticus might take place by utilizing certain inflammatory, anti-apoptotic, and angiogenic factors of transformation including NF-κB and IL-8.Ahmed S Abdulamir, Rand R Hafidh, Layla K Mahdi, Tarik Al-jeboori and Fatimah Abubake

Antimicrobial, immunomodulatory and tumor cells-selective cytotoxic activities of Brassica oleracea L. and Vigna radiata L

The world today faces a great challenge in the emergence of drug-resistant microbes and dangerous cancers against most of the commonly known antimicrobial and anticancer drugs, respectively. Screening for effective, new, and cheap remedies from natural resources has become the goal of many scientists all around the world. The plants’ methanol extracts used in this study were red cabbage (RC) and mung bean sprout (MBS) which both showed remarkable antiviral activities against herpes simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The mode of action of RC extract was shown to be prophylactic against RSV (the concentration that inhibit 50% of virus cytopathic effect (IC50) = 13.9 mg/ml and selectivity index (SI) = 17.67). Against HSV-1, RC extract showed a remarkable viricidal activity (IC50 = 11.51 mg/ml and SI = 34.52) with less prophylactic activity (IC50 = 32.97 mg/ml and SI = 11.71). MBS extract had viricidal effect (IC50 = 15.62 mg/ml and SI = 14.18), (IC50 = 7.62 mg/ml and SI = 18.23) and to a lesser extent prophylactic (IC50 = 17.23 mg/ml and SI = 12.82), (IC50 = 12.72 mg/ml and SI = 10.9) against RSV and HSV-1 viruses, respectively. One of the good explaination on the effective prophylactic activities of MBS and RC extracts was their excuisite ability to induce production of high levels of IFNβ, TNFα, IL-1, and IL-6 cytokines (300-900% higher levels when compared to untreated cells; P<0.0001) in virally infected cells. These antiviral cytokines proved to play critical and synergistic role in the antiviral resistance of human cells. For the cytotoxic effects of the tested extracts, RC extract showed selective cytotoxic effect against human cervix adenocarcinoma cells (HeLa), (SI = 10.88) while it showed less selectivity against human hepatocellular carcinoma cells (HepG2), (SI = 8.93). On the contrary, MBS extract showed a selective cytotoxic effect against both HeLa and HepG2 cells with SI values of 12.44 and 11.94, respectively. In an attempt to disclose part of the underlying mechansims of cytotoxic effect of both extracts using concentrations lower than IC50, RC induced the production of the anticancer cytokine TNFα (up to 600% increase;P<0.0001) whereas MBS induced the production of both anticancer cytokines TNFα (up to 700% increase; P<0.0001) and IFNβ (up to 300% increase; P<0.001). The increase in the anticancer cytokine levels was dose-dependent. In addition, the IC50 of RC extract induced cell cycle arrest in G0/G1 phase in cancer cells increasing the percentage of cells from 66.87 to 76.96% and from 57.83 to 70.41% in the treated HeLa and HepG2 cells, respectively. On the other hand, the IC50 of MBS extract induced cell cycle arrest in G0/G1 phase only in HeLa cells (62.87 to 80.48%). Both extracts induced remarkably apoptosis in the treated human cancer cells in a doseand time-dependent manner. The induction of apoptosis in cancerous cells 12-16h after exposure to RC and MBS extracts was significant via caspase-dependent pathways including the extrinsic pathway by upregulating caspase 8 (about 8-32 folds) and the intrinsic pathway by upregulating both caspase 9 (about 32-256 folds) and Bax genes (about 8-32 folds). Furthermore, both extracts upregulated highly caspase 7 (about 32-512 folds) which could induce apoptosis via caspaseindependent pathway. More to the point, Cdk-inhibitor proteins were upregulated (p21: about 8-512 folds, p27: about 4-16 folds, and p53: about 16-32 folds) 12-16h after exposure to both extracts and their upregulation might be the main mechanism used by both extracts to exert G1 cell cycle arrest and ultimately the final fate of cells, apoptosis. For the immunomodulatory effect of tested extracts, RC extract showed potent anti-inflammatory activity by inhibiting the production of the proinflammatory cytokine, IFNγ (about 80% decrease; P<0.01) in peripheral blood mononuclear cells (PBMC). On the other hand, MBS extract was effective immunomodulator agent as TNFα and IFNβ cytokines are also potent immunomodulators. In addition, MBS extract showed an immunopolarizing effect by inducing IFNγ (about 300% increase; P<0.01) and inhibiting IL-4 production (about 75% decrease; P<0.01) in PBMC. The immunopolarization effect of MBS extract was dose-dependent. For the antibacterial and antifungal potential of the tested extracts, antibacterial and antifungal activities against multiple drug resistant (MDR), non-MDR bacteria, and fungi were shown to be significant for both extracts. The antibacterial and antifungal activities were ose-dependent. Remarkably, antibacterial activity of RC extract was evident particularly against highly infectious microorganisms such as Methicillin-resistant Staphylococcus aureus (MRSA),Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae,Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. MBS extract revealed potential antibacterial and antifungal activities against MRSA, Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumonaie,Staphylococcus aureus, and Salmonella Typhimurium as well as against human fungal pathogens, Trichophyton rubrum and Trichoderma harzianum. The findings of the electron microscopy together with phenotype microarray (PM) supported these results for the antibacterial activity. PM screening was a valuable tool in the search for compounds that can inhibit bacterial growth by affecting certain metabolic pathways like peptidoglycan synthesis pathway. This pathway appeared to be targeted by both RC and MBS extracts at metabolic points differ from that of other available drugs. These points could be targets for discovering new therapeutic agents for the currently studied microbes. The considerable results of this study could be explained by the powerful antioxidant activity of RC and MBS which is most likely due to the phenolic and antioxidant compounds present in these plants which act individually or synergistically to bring to light the importance of these plants as candidates for new therapeutic agents

A comprehensive anticancer molecular study for genistein the promising anticancer drug

Objective Genistein a potent isoflavonoid isolated from dietary soybean with a wide range of biological and therapeutic activities, particularly, in cancer prevention. The molecular mechanism that explains this powerful chemopreventive activity is still not fully understood. The study designed to give a clear and complete picture about this mechanism using a flow cytometry analysis and a quantitative real-time PCR to investigate 16 gene families for 1023 genes. Methods Human cervical cancer cells were treated with genistein IC 50 for 48 h. Cell-cycle arrest and apoptosis induction were investigated using flow cytometry analysis. The high-throughput quantitative PCR array was used to explore the up- and down-regulated genes affected by the treatment. Results The quantitative real-time PCR analysis demonstrated the up- and down-regulation to 11 cancer-related gene families due to genistein treatment. Most of the up- and down-regulated genes have a role in cell proliferation, DNA replication and cell cycle progress. These findings were in harmony with the flow cytometry analysis in which a significant increase was manifested in the apoptotic cells (P < 0.05) of the treated cell cultures (18.34%) when compared with untreated cell cultures (5.7%). Conclusion The results highly pointed on the importance of genistein as promising anticancer drug. A comprehensive map regarding the anticancer molecular mechanism of this natural compound was given and many new therapeutic targets to control cancer development were uncovered. Keywords genistein, anticancer, cervical cancer, flavonoid

Novel molecular, cytotoxical, and immunological study on promising and selective anticancer activity of Mung bean sprouts

<p>Abstract</p> <p>Background</p> <p>The anticancer and immunomodulatory activity of mung bean sprouts (MBS) and the underlying mechanisms against human cervical and hepatocarcinoma cancer cells were explored.</p> <p>Methods</p> <p>MBS cytotoxicity and MBS-induced anticancer cytokines, TNF-α and IFN-β from cancer cells, and immunological cytokines, IL-4, IFN-γ, and IL-10 from peripheral mononuclear cells (PMNC) were assessed by MTS and ELISA assays. Apoptotic cells were investigated by flow cytometry. The expression level of apoptotic genes (Bax, BCL-2, Capsases 7–9) and cell cycle regulatory genes (cyclin D, E, and A) and tumor suppressor proteins (p27, p21, and p53) was assessed by real-time qPCR in the cancer cells treated with extract IC50.</p> <p>Results</p> <p>The cytotoxicity on normal human cells was significantly different from HeLa and HepG2 cells, 163.97 ± 5.73, 13.3 ± 0.89, and 14.04 ± 1.5 mg/ml, respectively. The selectivity index (SI) was 12.44 ± 0.83 for HeLa and 11.94 ± 1.2 for HepG2 cells. Increased levels of TNF-α and IFN-β were observed in the treated HeLa and HepG2 culture supernatants when compared with untreated cells. MBS extract was shown to be an immunopolarizing agent by inducing IFNγ and inhibiting IL-4 production by PBMC; this leads to triggering of CMI and cellular cytotoxicity. The extract induced apoptosis, in a dose and time dependent manner, in treated HeLa and HepG2, but not in untreated, cells (P < 0.05). The treatment significantly induced cell cycle arrest in G0/G1 in HeLa cells. The percentage of cells in G0/G1 phase of the treated HeLa cells increased from 62.87 ± 2.1%, in untreated cells, to 80.48 ± 2.97%. Interestingly, MBS IC50 induced the expression of apoptosis and tumor suppressor related genes in both HeLa and HepG2 cells. MBS extract succeeded in inducing cdk-inhibitors, p21, p53, and p27 in HeLa cells while it induced only p53 in HepG2 cells (P < 0.05). This is a clue for the cell type- specific interaction of the studied extract. These proteins inhibit the cyclin-cdk complexes apart from the presence of some other components that might stimulate some cyclins such as cyclin E, A, and D.</p> <p>Conclusion</p> <p>MBS extract was shown to be a potent anticancer agent granting new prospects of anticancer therapy using natural products.</p