Hesham Elhariry

Biofilm Formation by Aeromonas hydrophila on Green-Leafy Vegetables: Cabbage and Lettuce

Aeromonas hydrophila is the most well known of the six species of Aeromonas, which has been linked to two groups of human diseases: septicemia and gastroenteritis. Reference strain ATCC 7966 and biofilm strains TUB19, TUB20, and TUB21 were investigated for their ability to form biofilm in vitro (after 48 h on polystyrene surface) and on the surface of two green-leafy vegetables, cabbage and lettuce (after 1, 2, 4, and 24 h). Attachment strength (S(R)) of these strains to the vegetable surface was also measured in the same time intervals. The ATCC 7966 and TUB19 had high ability to form biofilm in vitro compared with TUB20 and TUB21 in full strength tryptone soy broth or under starvation conditions in diluted tryptone soy broth (1:20, v/v). Cell surface hydrophobicity of the biofilm strains was lower than that of the reference strain. The biofilm of all tested strains on polystyrene surfaces differed from that on the vegetable surfaces. All strains studied rapidly attached to both green leafy vegetables (after 1 h). S(R) and cell populations (loosely and strongly attached cells) significantly (p < 0.05) increased with contact time; however, no significant (p > 0.05) differences in cell populations were recorded after 4 and 24 h. The highest S(R) and cell population (log CFU cm⁻²) were recorded by TUB19. In conclusion, the use of A. hydrophila strains isolated from environmental biofilm samples may be more useful for understanding biofilm formation on green-leafy vegetables than the reference or laboratory strains. The attachment of A. hydrophila was significantly affected by the surfaces of green-leafy vegetables. Further studies are required to improve our understanding of the interaction between human microbial pathogens and surfaces of raw vegetables.

Genotypic identification of Penicillium expansum and the role of processing on patulin presence in juice.

This work aimed at isolation and identification of patulin producing fungi and to follow the presence of patulin during apple juice processing. Among 34 Penicillium isolates, eight isolates (five from healthy appeared apples and 12 from rot spotted apples) were considered as patulin producers using thin-layer chromatography. These isolates were classically identified as a Penicillium expansum. PCR utilizing primers based on the polygalacturonase gene of P. expansum was applied for detecting this mold. The PCR amplified a 404-bp DNA product from all tested P. expansum isolates, but not in other common food spoilage Penicillium species. RAPD technique using P1 or M13 primers was applied to determine the similarity of the P. expansum isolates. RAPD results revealed that the tested strains showed high percentage of similarity and no correlation was observed between cluster analysis and the sources of isolation. Patulin could not be detected in healthy appeared apples and their extracted juice during different stages of juice process. In apple juice made from the healthy parts of apples decayed by P. expansum contained patulin which may present health hazard. The obtained results assured that patulin is known to be stable in apple juice even after pasteurization. In conclusion, the removal of the rotten part from the fruit is not sufficient to eliminate the mycotoxin patulin from apple juice. Although, the enzyme treatment (pectinase and amylase) and pasteurization (95 °C for 7 min) significantly (p < 0.05) reduced patulin level, its level is still higher than the level of <50 μg/kg considered by Codex alimentarius when the apple juice processed from the healthy parts of rot spotted fruits.

Molecular Screening of Streptomyces Isolates for Antifungal Activity and Family 19 Chitinase Enzymes

Thirty soil-isolates of Streptomyces were analyzed to determine their antagonism against plant-pathogenic fungi including Fusarium oxysporum, Pythium aristosporum, Colletotrichum gossypii, and Rhizoctonia solani. Seven isolates showed antifungal activity against one or more strain of the tested fungi. Based on the 16S rDNA sequence analysis, these isolates were identified as Streptomyces tendae (YH3), S. griseus (YH8), S. variabilis (YH21), S. endus (YH24), S. violaceusniger (YH27A), S. endus (YH27B), and S. griseus (YH27C). The identity percentages ranged from 98 to 100%. Although some isolates belonged to the same species, there were many differences in their cultural and morphological characteristics. Six isolates out of seven showed chitinase activity according to a chitinolytic activity test and on colloidal chitin agar plates. Based on the conserved regions among the family 19 chitinase genes of Streptomyces sp. two primers were used for detection of the chitinase (chiC) gene in the six isolates. A DNA fragment of 1.4 kb was observed only for the isolates YH8, YH27A, and YH27C. In conclusion, six Streptomyces strains with potential chitinolytic activity were identified from the local environment in Taif City, Saudi Arabia. Of these isolates, three belong to family 19 chitinases. To our knowledge, this is the first reported presence of a chiC gene in S. violaceusniger YH27A.

Characterization of endophytic bacteria associated with rose plant (Rosa damascena trigintipeta) during flowering stage and their plant growth promoting traits

Little is known about the bacterial communities associated with the rose plants inhabiting dry desert ecosystems. The aim of this study was to isolate and characterize endophytic bacteria from different organs of rose plant. Endophytic bacteria were observed in healthy roots, stems, leaves, and flowers of rose plant, with a significantly higher density in roots, followed by stems, leaves, and petals. A total of 38 bacterial endophytes were isolated and are closely related phylogenetically to Acetobacter, Acinetobacter, Methylococcus, Bacillus, Micrococcus, Planococcus by 16S rRNA sequence analysis. Six endophytic bacteria were found to produce IAA, solubilize Ca3(PO4)2 and produce siderophore. The six endophytic bacteria all had the capacity to produce hydrolytic enzyme such as cellulase, xylanase, pectinase, amylase, protease, lipase, and chitinase, but difference existed among these isolates.

Mycobiota and Mycotoxins (Aflatoxins and Ochratoxin) Associated with Some Saudi Date Palm Fruits

This study aimed to determine the mycological profile of the retail date fruits distributed in different markets at Taif, Saudi Arabia. The presence of aflatoxins and ochratoxin A was also measured. Twenty-two fungal species belonging to 12 genera were isolated from 50 different date samples. Aspergillus flavus, A. niger, Penicillium chrysogenum, and Rhizopus stolonifer were the most prevalent species among isolated fungi. Eighty isolates of A. flavus and 36 of A. niger were detected for their aflatoxins and ochratoxin production potentials using thin layer chromatography. Toxicity test using Artimia larvae indicated that seven out of 18 A. flavus isolates had aflatoxins potentials, while nine out of 36 isolates of A. niger were ochratoxigenic. The quadruplex polymerase chain reaction using specific primers demonstrated the presence of four genes: nor A, ver 1, omt A, and avf A in seven A. flavus toxigenic isolates. Nine A. niger toxigenic isolates showed positive results for the presence of the PKS gene. In conclusion, the present study highlighted the potential hazards of mycotoxins on human health from consuming raw dates. Rapid molecular detection methods described here might help the food authorities to assure the safety of raw dates distributed in local markets.

Molecular characterization of black Aspergillus species from onion and their potential for ochratoxin A and fumonisin B2 production.

Onion bulbs can become contaminated with various molds during the storage period, the most important causal agents being black aspergilli (Aspergillus section Nigri). Taxonomic studies have revealed that this group of Aspergillus contains many species that cannot be reliably identified using standard morphological methods. Therefore, it is necessary to define the fungus causing this problem in the onion exactly, especially since some species assigned to section Nigri are well known as ochratoxin and/or fumonisin producers. Sixty fungal isolates belonging to 10 fungal genera were isolated from 40 onion samples originated from the Taif region in Saudi Arabia. Black aspergilli were detected in 37 onion samples. Using primer pairs (awaspec and Cmd6) designed based on partial calmodulin gene sequence data, 37 isolates were identified as A. welwitschiae. The ochratoxin A and fumonisin B2 contents of the onion samples were examined. No ochratoxins were detected in the collected samples, while fumonisin B2 was detected in 37.5% of the onion samples. Eighteen of 37 isolates of Aspergillus welwitschiae were recognized as potential producers for fumonisin B2. Multiplex polymerase chain reactions designed to detect biosynthetic genes of fumonisins confirmed these results.

Molecular Identification and Biofilm-Forming Ability of Culturable Aquatic Bacteria In Microbial Biofilms Formed in Drinking Water Distribution Networks

Drinking water distribution networks are known to harbor microbial biofilms. The aim of the present work is to (i) identify the culturable bacteria presented in the drinking-water distribution network, (ii) investigate the ability of isolated bacteria to form biofilm under some environmental stress conditions and some eliminating or removing treatments. To achieve it, 57 strains were isolated from biofilm (43 isolates) and water samples (14 isolates) collected from five stations in drinking-water distribution network in Taif city, Kingdom of Saudi Arabia (KSA). Partial sequences of 16S rRNA gene in the 57 isolates ensured the presence of only 22 different strains in biofilm samples. Among these strains, only 14 strains were also detected in water samples. Gram-negative Aeromonas hydrophila was the most occurred bacterium in the microbial biofilm obtained from the purified-water storage tanks followed by Gram-negative Pseudomonas sp. Gram-positive Bacillus subtilis was the most occurred bacterium in the microbial biofilm collected from the ends of the distribution pipes. Among the 22 isolated strains, 13 strains were strong biofilm producers at 30 and 37°C. The effects of environmental stresses including nutrient starvation (diluted TSB, 20:1), heating (100°C for 10 min), UV-treatment (240 nm for 10 min) and dynamic incubation (150 rpm min−1) on the formation of biofilm were also investigated. These conditions affected the biofilm formation ability of the isolated strains at different levels. Nutrient starvation enhanced biofilm formation by most of the isolates. Among some biofilm deforming treatments, SDS and trypsin had considerable effects on preventing biofilm formation by most of the isolated strains. In conclusion, the results of the present work indicated that not all biofilm strains released from biofilm to the drinking water. Also, not all biofilm strains were able to form biofilm. Most of isolated bacteria had ability to form biofilm at suboptimum temperature of growth. These results may provide basic information on formation of microbial biofilms and overcome the problem of deteriorating of water quality in the drinking-water distribution networks.

Molecular Barcoding of Microscopic Fungi with Emphasis on the Mucoralean Genera Mucor and Rhizopus

A broad range of fungi were isolated from different geographic regions and substrates and identified according to traditional and modern methods. A total of 120 different isolates were assigned to the phyla, Basidiomycota with 8 isolates, Ascomycota with 75 isolates, and “Zygomycota” with 37 isolates. Although morphological characters were able to differentiate the isolates to their phyla and in most cases to the correct genera, a combination of several methods is always recommended because characterization and identification of unknown fungal isolates is highly error-prone if relying on single methods. Sequence-based identification turned out to be reliable for most Ascomycetes and Zygomycetes. But with the ongoing questionable trend to rely on sequences as first source information for species separation, the most serious problems are the annotation problems in public reference databases, the inconsistency of described taxa, and the available reference data.

Endophytic fungi associated with high-altitude Juniperus trees and their antimicrobial activities

Fungal endophytes were isolated from twigs of Juniperus procera (Cupressaceae) collected from Taif region (Saudi Arabia). Twenty-six different taxa were recovered. The overall foliar colonization rate was 36%. A total of 144 isolates were obtained and identified into six distinct operational taxonomic units based on the sequencing of the internal transcribed spacer regions of the rRNA gene. The most prevalent fungi were Aspergillus fumigatus, Penicillium oxalicum, Preussia sp., Peyronellaea eucalyptica, Peyronellaea sancta and Alternaria tenuissima. A total of 144 isolates were tested for antibacterial and antifungal activities against Staphylococcus aureus, Klebsiella pneumoniae, Candida albicans and Fusarium solani in which 52 isolates showed antimicrobial activity against at least one of the tested microbes. Aspergillus fumigatus (7 isolates), Hypocrea lutea (4), Penicillium oxalicum (10) and Preussia sp. (5) presented the strongest antimicrobial activity. This study confirmed the variation of different isolates of the same species in terms of antibacterial activity. Also, it indicated that the endophytic fungi of Juniperus procera plants should be another potential source of bioactive antimicrobial agents.

Development of Co-Culture Sourdough Systems for Improving Bread Quality and Delaying Staling

Six different blends of lactic acid bacterial cultures (Lactobacillus plantarum or Pediococcus cerevisiae) combined with Saccharomyces cerevisiae were used as co-culture for preparing sourdoughs. These prefermented sourdoughs were applied for producing pan breads. Acidification power, leavening ability, and microbial counts were conducted at different sour fermentation intervals. The highest acidification power in sourdoughs were recorded when 2% wd/wd (dry weight of cells/dry weight of flour) of either L. plantarum or P. cerevisiae was used in combination with 2% wd/wd S. cerevisiae. The leavening ability of sourdoughs was significantly (p < 0.05) enhanced by increasing the addition percentage of the studied LAB. According to staling rate, physical, and sensory characteristics, the best two treatments were obtained when the co-culture of 2% wd/wd S. cerevisiae with 4% wd/wd L. plantarum or 4% wd/wd P. cerevisiae were used. These two treatments were successfully applied in production of the flat bread. According to staling rate, physical and sensory characteristics, sourdough prefermented with 2% wd/wd S. cerevisiae combined with 4% wd/wd L. plantarum was the best treatment for preparing of flat bread. Overall the co-culture of L. plantarum and with S. cerevisiae might be a useful tool for preparing sourdough starter that was most effective for improving sensory and physical properties of both pan and flat bread. In addition it can help extend the shelf life by delaying bread staling and inhibiting mold spoilage.