Analysis of the Frequency, Antibiotic Susceptibility, and Related Genes among Foodborne Pathogenic Bacteria Isolated from Hospital Refrigerators in Tehran, Iran.

Background: Hospital refrigerators as essential food storage can be important source of food contamination. We aimed to investigate the frequency and antibiotic susceptibility of the pathogenic bacteria in three hospital refrigerators in Tehran. Methods: This study was performed on 254 samples, collected from 60 refrigerators of the various wards of three hospitals, A, B, and C, in Tehran, Iran from 2020 to 2021. Following isolation and identification of isolates, the antibiotic susceptibility pattern was determined. PCR-based assays were used to screen the presence of antibiotic resistance genes of resistant isolates. Results: From 254 collected samples, 236 samples (92.9%) were contaminated. Most strains were isolated from refrigerators with poorly cleaned, temperatures above 8 °C in non-critical wards. Most bacteria belonging to Enterobacteriaceae (68.8%), followed by Staphylococcus (11.9%), and Enterococcus (10.6%), while the frequency of non-Enterobacteriaceae isolates was 8.9%. The highest antibiotic resistant bacteria were in extended spectrum beta-lactamase (ESBL) 9.7%, vancomycin-resistant enterococci (VRE) 5.3%, methicillin-resistant S. epidermidis (MRSE) 0.4%, and methicillin-resistant S. aureus (MRSA) 0.4%, respectively. The bla OXA-48, bla CTX, and bcla TEM genes were found only in 10% of Enterobacteriaceae isolates. The bla OXA-51 gene was found in all non-Enterobacteriaceae isolates. The vanA and mecA genes were detected in antibiotic-resistant Enterococcus and Staphylococcus. Conclusion: Our findings suggests major concern about cross-contamination and the emergence of antibiotic-resistant isolates as a potential health threat with hospital refrigerators origin. More attention to hospital refrigerators cleaning is necessary to prevent foodborne diseases and nosocomial infections.

Designing and developing of high-resolution melting technique for separating different types of Toxoplasma gondii by analysis of B1 and ROP8 gene regions.

BACKGROUND: Determination of Toxoplasma gondii genotypes plays an important role in the health management and epidemiology of toxoplasmosis. We developed HRM analysis to differentiate genotypes of T. gondii using the B1 and ROP8 genes, through comparing the sensitivity and specificity of both genes and methods used for the detection of T. gondii. METHODS: A total of 96 DNA samples of muscle tissue of livestock and poultry brain tissue with three standard strains RH (type I), PRU (type II) and VEG (type III) were prepared and analyzed. Three methods of nested PCR, PCR-PCR and nested-qPCR-HRM were used. Specific new primers were designed and synthesized for developing HRM. Thirty positive samples obtained from nested-qPCR-HRM were sequenced (18 B1 and 12 ROP8). RESULTS: Overall, 87 infected samples were identified using both genes. Through the B1 gene, we could separate type I (Tm = 84.8 °C) from II/III types (Tm = 84.6 °C). Also, the ROP8 gene could separate type II (Tm = 84.5 °C) from I/III types (Tm = 84.12 °C). Highest sensitivity (100%) and specificity (78.72%) were observed by nested-qPCR-HRM assays of the B1 and ROP8 genes than by other methods, respectively. Thus, the B1 gene can be used to most accurately detect T. gondii, while the ROP8 gene was more appropriate for T. gondii genotyping. PCR-sequencing results were consistent with HRM results in most selected samples. CONCLUSION: HRM analysis is a powerful diagnostic tool for rapid detection and determination of main clonal lineages, and even unusual T. gondii genotypes.

Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran.

BACKGROUND: A high correlation is observed between specific clonal lineages and host types in toxoplasmosis. The main objectives of this study were comparing polymorphism and evolutionary analysis of the B1 and ROP8 genes, as well as the evaluation of phylogenic and Toxoplasma gondii isolates obtained from different hosts and regions. METHODS: Overall 96 brain/diaphragm tissue samples of livestock and poultry from three provinces of Iran (cows: 9 from Yazd, 9 from Qom; sheep: 19 from Yazd, 7 from Qom; goats: 7 from Yazd, 4 from Qom; one camel from Yazd and 37 chickens, 2 roosters and one duck from Golestan) were tested during 2018-19. A nested PCR and PCR-PCR methods were developed with the B1 and ROP8 genes. Evaluation of genetic proximity, genetic diversity and evolutionary analysis were done using MEGA-X and DnaSP5 software. Thirty samples of both genes were sequenced (18 B1 and 12 ROP8 genes), and submitted to the GenBank (MN275903-MN275932). RESULTS: Tajima's D index analyses showed that both genes were in the negative direction of evolution. The B1 gene was more sensitive than the ROP8 gene. The ROP8 gene showed better and more acceptable results in terms of the relationship between the host and the genotyping of the samples. CONCLUSION: The B1 gene was only an attractive target for rapid detection of T. gondii parasites, whereas the ROP8 gene due to a high level of polymorphism was able to isolate the three clonal lineages (type I, II and III), intertypes and even atypical strains from different isolates of T. gondii.

Identification of (Z)-2,3-Diphenylacrylonitrileas Anti-Cancer Molecule in Persian Gulf Sea Cucumber Holothuria parva.

Hepatocellular carcinoma (HCC), also named cancerous hepatoma, is the most common type of malignant neoplasia of the liver. In this research, we screened the Persian Gulf sea cucumber Holothuria parva (H. parva) methanolic sub-fractions for the possible existence of selective toxicity on liver mitochondria isolated from an animal model of HCC. Next, we purified the most active fraction. Thus the structure of the active molecule was identified. HCC was induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) protocol. Rat liver mitochondria for evaluation of the selective cytotoxic effects of sub-fractions of H. parva were isolated and then mitochondrial parameters were determined. Our results showed that C1 sub-fraction of methanolic extract of H. parva considerably increased reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), swelling in mitochondria and cytochrome c release only on HCC liver mitochondria. Furthermore, the methanolic extract of H. parva was investigated furthermore and the active fraction was extracted. In this fraction, (Z)-2,3-diphenylacrylonitrile molecule, which is also known as α-cyanostilbene, was identified by mass analysis. This molecule increased ROS generation, collapse of MMP, swelling in mitochondria and finally cytochrome c release only on HCC liver mitochondria. The derivatives of (Z)-2,3-diphenylacrylonitrile in other natural products were also reported as an anti-cancer agent. These results suggest the eligibility of the (Z)-2,3-diphenylacrylonitrile as a complementary therapeutic agent for patients with HCC.

Selective toxicity of persian gulf sea cucumber holothuria parva on human chronic lymphocytic leukemia b lymphocytes by direct mitochondrial targeting.

Natural products isolated from marine environment are well known for their pharmacodynamic potential in diversity of disease treatments such as cancer or inflammatory conditions. Sea cucumbers are one of the marine animals of the phylum Echinoderm. Many studies have shown that the sea cucumber contains antioxidants and anti-cancer compounds. Chronic lymphocytic leukemia (CLL) is a disease characterized by the relentless accumulation of CD5+ B lymphocytes. CLL is the most common leukemia in adults, about 25-30% of all leukemias. In this study B lymphocytes and their mitochondria (cancerous and non-cancerous) were obtained from peripheral blood of human subjects and B lymphocyte cytotoxicity assay, and caspase 3 activation along with mitochondrial upstream events of apoptosis signaling including reactive oxygen species (ROS) production, collapse of mitochondrial membrane potential (MMP) and mitochondrial swelling were determined following the addition of Holothuria parva extract to both cancerous and non-cancerous B lymphocytes and their mitochondria. Our in vitro finding showed that mitochondrial ROS formation, MMP collapse, and mitochondrial swelling and cytochrome c release were significantly (P < 0.05) increased after addition of different concentrations of H. parva only in cancerous BUT NOT normal non-cancerous mitochondria. Consistently, different concentrations of H. parva significantly (P < 0.05) increased cytotoxicity and caspase 3 activation only in cancerous BUT NOT normal non-cancerous B lymphocytes. These results showed that H. parva methanolic extract has a selective mitochondria mediated apoptotic effect on chronic lymphocytic leukemia B lymphocytes hence may be promising in the future anticancer drug development for treatment of CLL. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1158-1169, 2017.

Selective Toxicity of Persian Gulf Sea Cucumber (Holothuria parva) and Sponge (Haliclona oculata) Methanolic Extracts on Liver Mitochondria Isolated from an Animal Model of Hepatocellular Carcinoma.

BACKGROUND: Natural products isolated from marine environments are well known for their pharmacodynamic potential in diverse disease treatments, such as for cancer or inflammatory conditions. Sea cucumbers are marine animals of the phylum Echinoderm and the class Holothuroidea, with leathery skin and gelatinous bodies. Sponges are important components of Persian Gulf animal communities, and the marine sponges of the genus Haliclona have been known to display broad-spectrum biological activity. Many studies have shown that sea cucumbers and sponges contain antioxidants and anti-cancer compounds. OBJECTIVES: This study was designed to determine the selective toxicity of Persian Gulf sea cucumber (Holothuria parva) and sponge (Haliclona oculata) methanolic extracts on liver mitochondria isolated from an animal model of hepatocellular carcinoma, as part of a national project that hopes to identify novel potential anticancer candidates among Iranian Persian Gulf flora and fauna. MATERIALS AND METHODS: To induce hepatocarcinogenesis, rats were given diethylnitrosamine (DEN) injections (200 mg/kg i.p. by a single dose), and then the cancer was promoted with 2-acetylaminofluorene (2-AAF) (0.02 w/w) for two weeks. Histopathological evaluations were performed, and levels of liver injury markers and a specific liver cancer marker (alpha-fetoprotein), were determined for confirmation of hepatocellular carcinoma induction. Finally, mitochondria were isolated from cancerous and non-cancerous hepatocytes. RESULTS: Our results showed that H. parva methanolic extracts (250, 500, and 1000 µg/mL) and H. oculata methanolic extracts (200, 400, and 800 µg/mL) increased reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), mitochondrial swelling, and cytochrome c release in the mitochondria obtained from cancerous hepatocytes, but not in mitochondria obtained from non-cancerous liver hepatocytes. These extracts also induced caspase-3 activation, which is known as a final mediator of apoptosis, in the hepatocytes obtained only from cancerous, not non-cancerous, rat livers. CONCLUSIONS: Our results suggest that H. parva and H. oculata may be promising therapeutic candidates for the treatment of HCC, following further confirmatory in vivo experiments and clinical trials.

Oxidative mechanisms of fish hepatocyte toxicity by the harmful dinoflagellate Cochlodinium polykrikoides.

Harmful Algal Blooms caused by the marine ichthyotoxic dinoflagellate Cochlodinium polykrikoides are responsible for mass mortalities of wild and farmed fish worldwide. In this research, we investigated the cytotoxic mechanisms of aqueous extract of C. polykrikoides on isolated Rainbow trout (Oncorhynchus mykiss) liver hepatocytes. Algal extract exposure with isolated trout hepatocytes caused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, lysosomal membrane rupture, collapse of mitochondrial membrane potential, ATP depletion and increase in ADP/ATP ratio, cytochrome C release into the hepatocyte cytosol, and activation of caspases cascade. Anti-oxidants, free radical scavengers, mitochondrial permeability transition (MPT) pore sealing agents, microsomal oxidases inhibitors, ATP generators and lysosomotropic agents protected fish hepatocytes against C. polykrikoides. Fish hepatocyte toxicity was also associated with mitochondrial and lysosomal membrane injury. These events caused cytochrome C release from the mitochondrial intra-membrane space into cytosol. The cytochrome C release could trigger activation of caspase-3 and apoptosis.

Ichthyotoxic Cochlodinium polykrikoides Induces Mitochondrial Mediated Oxidative Stress and Apoptosis in Rat Liver Hepatocytes.

In this research, we investigated the cytotoxic mechanisms of Cochlodinium polykrikoidescell lysate on isolated rat liver hepatocytes.This micro algae is responsible for a severe and widespread harmful algal bloom in the Persian Gulf and Gulf of Oman (2008-2009). Isolated hepatocytes were obtained by collagenase perfusion of Sprague-Dawley rat liver.According to our results, incubation of algal lysate with isolated rat hepatocytescaused hepatocyte membrane lysis, reactive oxygen species (ROS) formation, glutathione depletion, collapse of mitochondrial membrane potential,ATP depletion and increase in ADP/ATP ratio, cytochrome c release in to the hepatocyte cytosol,activation of caspase-3 (final mediator of apoptosis) and appearance of apoptosis phenotype. On the other hand, pre-treatment of antioxidants (α-tocopherol succinate and BHT), radical scavengers (mannitol and DMSO), mitochondrial permeability transition (MPT) pore sealing agents (cyclosporine A, carnitine and trifluoperazine), NADPH P450 reductase inhibitor (Diphenyliodonium chloride), CYP2E1 inhibitors (Phenylimidazole and 4-Methylpyrazole) and ATP generators (L-glutamine, Fructose and Xylitol)inhibitedcaspase-3 activation and cell death in algal lysate treated hepatocytes.Our data also confirmed that algal lysate activates apoptosis signaling via oxidative stress and mitochondrial pathway. Thus, ROS formation caused by the lysate exposure could directly be involved in mitochondrial MPT pore opening and activation of caspase-3 leading to C.polykrikoides lysateinduced apoptosis on rat hepatocytes. These findings contribute to a better understanding of C.polykrikoides-toxic effects on mammalian liver cells.

Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. In the following investigation, a novel chitinase with antifungal activity was characterized from a native Serratia marcescens B4A. Partially purified enzyme had an apparent molecular mass of 54 kDa. It indicated an optimum activity in pH 5 at 45ºC. Enzyme was stable in 55ºC for 20 min and at a pH range of 3-9 for 90 min at 25ºC. When the temperature was raised to 60ºC, it might affect the structure of enzymes lead to reduction of chitinase activity. Moreover, the Km and Vmax values for chitin were 8.3 mg/ml and 2.4 mmol/min, respectively. Additionally, the effect of some cations and chemical compounds were found to stimulate the chitinase activity. In addition, Iodoacetamide and Idoacetic acid did not inhibit enzyme activity, indicating that cysteine residues are not part of the catalytic site of chitinase. Finally, chitinase activity was further monitored by scanning electronic microscopy data in which progressive changes in chitin porosity appeared upon treatment with chitinase. This enzyme exhibited antifungal activity against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola, revealing a potential application for the industry with potentially exploitable significance. Fungal chitin shows some special features, in particular with respect to chemical structure. Difference in chitinolytic ability must result from the subsite structure in the enzyme binding cleft. This implies that why the enzyme didn't have significant antifungal activity against other Fungi.

Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A.

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. In the following investigation, a novel chitinase with antifungal activity was characterized from a native Serratia marcescens B4A. Partially purified enzyme had an apparent molecular mass of 54 kDa. It indicated an optimum activity in pH 5 at 45°C. Enzyme was stable in 55°C for 20 min and at a pH range of 3-9 for 90 min at 25°C. When the temperature was raised to 60°C, it might affect the structure of enzymes lead to reduction of chitinase activity. Moreover, the Km and Vmax values for chitin were 8.3 mg/ml and 2.4 mmol/min, respectively. Additionally, the effect of some cations and chemical compounds were found to stimulate the chitinase activity. In addition, Iodoacetamide and Idoacetic acid did not inhibit enzyme activity, indicating that cysteine residues are not part of the catalytic site of chitinase. Finally, chitinase activity was further monitored by scanning electronic microscopy data in which progressive changes in chitin porosity appeared upon treatment with chitinase. This enzyme exhibited antifungal activity against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola, revealing a potential application for the industry with potentially exploitable significance. Fungal chitin shows some special features, in particular with respect to chemical structure. Difference in chitinolytic ability must result from the subsite structure in the enzyme binding cleft. This implies that why the enzyme didn't have significant antifungal activity against other Fungi.

Radioactivity concentrations in sediments on the coast of the Iranian province of Khuzestan in the Northern Persian Gulf.

Gamma-ray spectrometric analyses were performed on sediment samples from the coast of Khuzestan province (south west of Iran, neighbor to Iraq and Kuwait) to study the concentration of natural as well as man-made radioactive sources. The coast of Khuzestan, which extends for approximately 400 km is mainly soft areas of mud flats within different ecosystems including river mouth, estuaries, creeps, and small bays. Suspended material from the Iranian rivers including Arvand (Karun), Bahmanshir, Jarrahi, and Zohreh has settled to form these extensive soft areas. Eighty three samples were taken at different points along the coast in undisturbed areas at intervals of about 5 km since Fall 2005 to Winter 2006. Collection was carried out during low-tide, where it was possible to collect sediments from the wet region that was covered by sea water during the high tide. At each of the sample sites, a sampling area of about 1 m(2) was considered. All samples were of a muddy nature, and were left to dry in open air before drying in the oven at 105 degrees C for 2-3 days to remove all water content. The average activity concentration of the radionuclides (226)Ra (30 Bq/Kg), (232)Th (11 Bq/kg), (238)U (18 Bq/kg), and (137)Cs (2.6 Bq/kg) along the shore of Khuzestan reaches are much less than the values commonly assigned as the world average. Nevertheless in case of (40)K which is a long lived naturally occurring radionuclide, the result (481 Bq/kg) was higher than the world average which could be due to a large Kuwaiti oil spill and also fallout and deposition of tremendous amount of fly ashes which resulted from ignited Kuwaiti oil fields during the 2nd Persian Gulf war (1990-91). For man-made (137)Cs and naturally occurring (232)Th, the western and eastern parts of Khuzestan shore showed higher concentrations than the middle part (Khooriat or creeps). For the long lived naturally occurring radionuclide (40)K and Gulf war (238)U (anti armor shells), there were no significant differences (P < 0.05) among the three regions.