Illumina MiSeq sequencing analysis of fungal diversity in stored dates.

BACKGROUND: Date palm has been a major fruit tree in the Middle East over thousands of years, especially in the Arabian Peninsula. Dates are consumed fresh (Rutab) or after partial drying and storage (Tamar) during off-season. The aim of the study was to provide in-depth analysis of fungal communities associated with the skin (outer part) and mesocarp (inner fleshy part) of stored dates (Tamar) of two cultivars (Khenizi and Burny) through the use of Illumina MiSeq sequencing. RESULTS: The study revealed the dominance of Ascomycota (94%) in both cultivars, followed by Chytridiomycota (4%) and Zygomycota (2%). Among the classes recovered, Eurotiomycetes, Dothideomycetes, Saccharomycetes and Sordariomycetes were the most dominant. A total of 54 fungal species were detected, with species belonging to Penicillium, Alternaria, Cladosporium and Aspergillus comprising more than 60% of the fungal reads. Some potentially mycotoxin-producing fungi were detected in stored dates, including Aspergillus flavus, A. versicolor and Penicillium citrinum, but their relative abundance was very limited (<0.5%). PerMANOVA analysis revealed the presence of insignificant differences in fungal communities between date parts or date cultivars, indicating that fungal species associated with the skin may also be detected in the mesocarp. It also indicates the possible contamination of dates from different cultivars with similar fungal species, even though if they are obtained from different areas. CONCLUSION: The analysis shows the presence of different fungal species in dates. This appears to be the first study to report 25 new fungal species in Oman and 28 new fungal species from date fruits. The study discusses the sources of fungi on dates and the presence of potentially mycotoxin producing fungi on date skin and mesocarp.

Efficiency of Electronic Nose in Detecting the Microbial Spoilage of Fresh Sardines (Sardinella longiceps).

The assessment of microbial spoilage in fresh fish is a major concern for the fish industry. This study aimed to evaluate the efficiency and reliability of an electronic nose (E-nose) to detect microbial spoilage of fresh sardines (Sardinella longiceps) by comparing its measurements with Total Bacterial Count (TBC), Hydrogen Sulfide (H2S) producing bacterial count and Trimethylamine Oxide (TMAO) reducing bacterial count after variable storage conditions. The samples were stored at 0 °C (0, 2, 4, 6, and 8 days) and 25 °C (0, 3, 6, and 9 h), while day 0 was used as a control. The E-nose measurements were analyzed by Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA) and Artificial Neural Network (ANN). Microbial counts increased significantly and simultaneously with the changes in E-nose measurements during storage. The LDA and ANN showed a good classification of E-nose data for different storage times at two storage temperatures (0 °C and 25 °C) compared to PCA. It is expected as PCA is based on linear relationships between the factors, while ANN is based on non-linear relationships. Correlation coefficients between E-nose and TBC, TMAO-reducing bacterial and H2S-producing bacterial counts at 0 °C were 0.919, 0.960 and 0.915, respectively, whereas at 25 °C, the correlation coefficients were 0.859, 0.945 and 0.849, respectively. These positive correlations qualify the E-nose as an efficient and reliable device for detecting microbial spoilage of fish during storage.

Effect of frozen storage on the characteristics of a developed and commercial fish sausages.

The effect of frozen storage on the physiochemical, chemical and microbial characteristics of two types of fish sausages was studied. Fish sausages developed (DFS) with a spice-sugar formulation and commercial fish sausages (CFS) were stored at -20 °C for 3 months. Fresh DFS contained 12.22% lipids and had a 3.53 cfu/g total bacteria count (TBC) whereas, CFS contained 5.5% lipids and had a 4.81 cfu/g TBC. During storage, TBC decreased significantly (p < 0.05) in DFS whereas it did not change (p > 0.05) in CFS. A peroxide value (PV) was not detectable until week four and eight of storage in CFS and DFS, respectively. The salt-soluble proteins (SSP) level was stable in DFS but in CFS it declined significantly (p < 0.05). Colour values did not change significantly (p > 0.05) in both sausage types. This study showed that the effect of storage at -20 °C on fish sausages characteristics varied between formulations and depended on the ingredients of fish sausages.

Antimicrobial Susceptibility of Fresh Produce-Associated Enterobacteriaceae and Enterococci in Oman.

Fresh produce bacteria may have phenotypic and/or genotypic antimicrobial resistance traits that may lead to various consequences on the environment and human health. This study evaluated the susceptibility of fresh produce bacteria (banana, cabbage, capsicum, carrots, cucumber, dates, lettuce, mango, papaya, pomegranate, radish, tomato and watermelon) to chlorhexidine and the antibiotic resistance of enterococci. Eighty-eight Enterobacteriaceae bacteria and 31 enterococci were screened for their susceptibility to chlorhexidine using the broth microdilution method. Susceptibility of enterococci to various antibiotics was determined using agar dilution, colorimetric, and Kirby-Bauer disc diffusion methods. Enterococci were more susceptible to chlorhexidine than Enterobacteriaceae indicated by chlorhexidine minimum inhibitory concentration (MIC) of 1 to 8 µg/mL for the former and 1 to 64 µg/mL for the latter. The IntI 1, qacEΔ1, qacE and qacG genes were distributed weakly in three, two, two, and three Enterobacteriaceae isolates, respectively. Enterococci had resistance to chloramphenicol (3%), tetracycline (19%), erythromycin (68%), ciprofloxacin (55%), and vancomycin (10%) while 19% of them were multi-drug resistant. In conclusion, this research detected a low to moderate level of antibiotic resistance in enterococci. Some Enterobacteriaceae bacteria had reduced chlorhexidine MICs that were not 10x less than the recommended concentration (100-200 µg/mL) in food production areas which might challenge the success of the disinfection processes or have clinical implications if the involved bacteria are pathogens. The prevalence of antimicrobial-resistant bacteria in fresh produce should be monitored in the future.

Antibiotic Resistance of Enterobacteriaceae Isolated from Fresh Fruits and Vegetables and Characterization of their AmpC ß-Lactamases.

Enterobacteria may gain antibiotic resistance and be potent pathogens wherever they are present, including in fresh fruits and vegetables. This study tested the antibiotic resistance of enterobacteria isolated from 13 types of local and imported fresh fruits and vegetables (n = 105), using the standard Kirby-Bauer disk diffusion method. Phenotypic and genotypic characterizations of AmpC ß-lactamases were determined in cefoxitin-resistant isolates. Ten percent of the enterobacteria tested (n = 88) were pansusceptible, 74% were resistant to at least one antibiotic, and 16% were multidrug resistant. Enterobacteria isolates showed the highest antibiotic resistance against ampicillin (66%), cephalothin (57%), amoxicillin-clavulanic acid (33%), cefoxitin (31%), tetracycline (9%), nalidixic acid (7%), trimethoprim (6%), and kanamycin (5%). Three isolates showed intermediate resistance to the clinically important antibiotic imipenem. Escherichia coli isolated from lettuce exhibited multidrug resistance against five antibiotics. Fifteen isolates were confirmed to have AmpC ß-lactamase, using the inhibitor-based test and the antagonism test; the latter test confirmed that the enzyme was an inducible type. Four types of ampC ß-lactamase genes (CIT, EBC, FOX, and MOX) were detected in eight isolates: four Enterobacter cloacae isolates and one isolate each of Citrobacter freundii, Enterobacter asburiae, Enterobacter hormaechei, and Enterobacter ludwigii. It was concluded that fresh fruits and vegetables might play a role as a source or vehicle for transferring antibiotic-resistant bacteria that might spread to other countries through exportation. The clinically significant AmpC ß-lactamase was rarely documented in the literature on bacteria isolated from fruits and vegetables, and to our knowledge, this is the first report on the detection of an inducible type in such commodities.

The Protective Effect of Curcumin against Nitrosamine-Induced Gastric Oxidative Stress in Rats.

Curcumin has a wide spectrum of biological, pharmaceutical, and antioxidant effects in cancer experimental models. Nitrosamine is commonly used as an experimental oxidizing agent which induces gastric oxidative stress and gastric carcinogenesis in rats. We examined the antioxidant potential effect of curcumin against nitrosamine-induced gastric oxidative stress in rats. Forty Sprague-Dawley rats were randomly divided into 4 groups (10 rats/group). The control group was fed a standard diet and received a single dose of normal saline, the nitrosamine-treated group was fed a standard diet and received an intraperitoneal injection of nitrosamine at a single dose of 100 mg/kg body weight (b.w.). The other two groups received a daily dose of curcumin (200 mg/kg b.w.) via intra-gastric intubation in the presence or absence of nitrosamine injection. After 16 weeks, all rats were sacrificed, and the gastric tissues were dissected for histopathological examination and for biochemical measurements of oxidative stress indices. Our results showed that nitrosamine causes oxidative stress in gastric tissues as evidenced by glutathione depletion, increased level of lipid peroxides, nitric oxide release, impairment of total antioxidant capacity, DNA oxidative damage, and inhibition of antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase). Histopathological findings revealed abnormal gastric architecture in association with nitrosamine injection compared to the non-treated control group. Curcumin significantly suppressed the gastric oxidative damage associated with nitrosamine treatment and mitigated its histopathological effect. These results suggest that curcumin, as an antioxidant, has a therapeutic effect against oxidative stress-mediated gastric diseases.

Hiding in Fresh Fruits and Vegetables: Opportunistic Pathogens May Cross Geographical Barriers.

Different microbial groups of the microbiome of fresh produce can have diverse effects on human health. This study was aimed at identifying some microbial communities of fresh produce by analyzing 105 samples of imported fresh fruits and vegetables originated from different countries in the world including local samples (Oman) for aerobic plate count and the counts of Enterobacteriaceae, Enterococcus, and Staphylococcus aureus. The isolated bacteria were identified by molecular (PCR) and biochemical methods (VITEK 2). Enterobacteriaceae occurred in 60% of fruits and 91% of vegetables. Enterococcus was isolated from 20% of fruits and 42% of vegetables. E. coli and S. aureus were isolated from 22% and 7% of vegetables, respectively. Ninety-seven bacteria comprising 21 species were similarly identified by VITEK 2 and PCR to species level. E. coli, Klebsiella pneumoniae, Enterococcus casseliflavus, and Enterobacter cloacae were the most abundant species; many are known as opportunistic pathogens which may raise concern to improve the microbial quality of fresh produce. Phylogenetic trees showed no relationship between clustering of the isolates based on the 16S rRNA gene and the original countries of fresh produce. Intercountry passage of opportunistic pathogens in fresh produce cannot be ruled out, which requires better management.

Speciation of Gram-positive bacteria in fresh and ambient-stored sub-tropical marine fish.

This study identified Gram-positive bacteria in three sub-tropical marine fish species; Pseudocaranx dentex (silver trevally), Pagrus auratus (snapper) and Mugil cephalus (sea mullet). It further elucidated the role played by fish habitat, fish body part and ambient storage on the composition of the Gram-positive bacteria. A total of 266 isolates of Gram-positive bacteria were identified by conventional biochemical methods, VITEK, PCR using genus- and species-specific primers and/or 16S rRNA gene sequencing. The isolates were found to fall into 13 genera and 30 species. In fresh fish, Staphylococcus epidermidis and Micrococcus luteus were the most frequent isolates. After ambient storage, S. epidermidis, S. xylosus and Bacillus megaterium were no longer present whereas S. warneri, B. sphaericus, Brevibacillus borstelensis, Enterococcus faecium and Streptococcus uberis increased in frequency. Micrococcus luteus and S. warneri were the most prevalent isolates from P. dentex, while E. faecium and Strep. uberis were the most frequent isolates from P. auratus and M. cephalus. With respect to different parts of the fish body, E. faecium, Strep. uberis and B. sphaericus were the most frequent isolates from the muscles, E. faecium, Strep. uberis from the gills and M. luteus from the gut. This study showed a diversity of Gram-positive bacteria in sub-tropical marine fish; however, their abundance was affected by fish habitat, fish body part and ambient storage.