Comparison of Antimicrobial Susceptibility of Campylobacter Strains Isolated from Food Samples and Patients with Diarrhea

Background:

Campylobacter infections may lead to serious conditions, including septicemia or other invasive forms of the disease, which require rapid and accurate laboratory diagnosis and subsequently appropriate antimicrobial therapy. The aim of this study was to compare the species distribution and antimicrobial susceptibility pattern of Campylobacter spp. strains isolated from patients and food samples.

Methods:

Biochemical identification was performed on 15 clinical and 30 food isolates of Campylobacter recovered onto Brucella agar containing 5% sheep blood. PCR was carried out to confirm the identity of Campylobacter spp. using primers for cadF, hipO, and asp genes of Campylobacter. To determine antibiotic sensitivity of isolates, Kirby-Bauer assay was carried out using 16 different antibiotic discs.

Results:

PCR assay and biochemical tests confirmed all 45 isolates as Campylobacter: 20 (44.44%) as C. jujeni, 10 (22.22%) as C. coli, and 15 (33.34%) as other Campylobacter strains. The maximum resistance was observed to cefotaxime and imipenem (each 86.49%) and the maximum sensitivity to erythromycin (48.65%).

Conclusion:

C. jujeni is dominant among isolates from clinical and food samples. In addition, tetracycline remains the first-line therapeutic agent against Campylobacter infections in Iran. Key Words: Campylobacter, Genetic diversity, Drug resistance     

Prevalence of Class 1 Integrons and Extended Spectrum Beta Lactamases among Multi-Drug Resistant Escherichia coli Isolates from North of Iran

Background:

Extended spectrum beta lactamases (ESBLs) are an important cause of transferable multidrug resistance (MDR) in gram-negative bacteria. The most described ESBL genes are generally found within integron-like structures as mobile genetic elements. The aim of this study was to identify the accompanying of class 1 integrons and ESBLs in the MDR E. coli isolates.

Methods:

Susceptibility to antimicrobial agents was determined for 33 E. coli strains by the disk diffusion method. Double-disk synergy test was applied for screening ESBL. To identify the strains carrying integrons, the conserved regions of integron-encoded integrase gene intI1 were amplified. For detection of gene cassettes, 5′CS and 3′CS primers were used.

Results:

All E. coli isolates were identified as multi-drug resistant. More than 50% of the isolates were resistant to tetracycline, cephalothin, cefuroxime, amoxicillin-clavulanic acid, and third generation cephalosporines. Nearly all of the isolates displayed sensitivity to piperacillin. There was a significant correlation between production of ESBL and resistance to all antibiotics except for ciprofloxacin and piperacillin (P < 0.01). Thirty two MDR strains (97%) included class 1 integron, and some isolates that included integrons were similar in the size of gene cassettes. The isolates were different in the resistance profiles; however, some others had similar resistance profiles. Of eight ESBL positive isolates, seven (87.5%) carried class 1 integrons.

Conclusion:

Class 1 integrons were frequent in MDR and also ESBL-producing E. coli isolates. High prevalence of class 1 integrons confirms that integron-mediated antimicrobial gene cassettes are important in E. coli resistance profile. Key Words: Antibiotic, Integrons, Escherichia coli     

Identification and Characterization of Metallo-β-Lactamases Producing Pseudomonas aeruginosa Clinical Isolates in University Hospital from Zanjan Province,Iran

Background: Infectious by Pseudomonas aeruginosa has spread worldwide and metallo-beta-lactamases (MBL) are being reported with increasing frequency. The aim of this study was to investigate the antibiotic susceptibility and distribution of bla_VIM and bla_IMP genes in P. aeruginosa isolates from Zanjan Province of Iran. Methods: A total of 70 P. aeruginosa isolates were identified from patients admitted at intensive care units. The antimicrobial susceptibility was tested by disk diffusion (Kirby-Bauer) method and for production of MBL using double-disk synergy test (DDST). After DNA extraction, the presence of bla_VIM and bla_IMP genes and class 1 integron were detected by PCR. RESULTS: Most of the isolates were resistant to meropenem, cefotaxime and imipenem (IPM). Also, 44/70 (62.85%) IPM resistant isolates were confirmed by DDST. Of the 44 clinical isolates, 41 (93%) isolates showed MIC≥4 µg/ml for IPM. Based on the DDST results, 36 (87.8%) were confirmed to be MBL producers. PCR amplification showed that 23/41 (56%) carried bla_VIM and 10/41 (24.3%) possessed bla_IMP gene. Also, 31/44 (70.5%) isolates contained class 1 integron gene. Conclusion: Our results highlight that the genes for Verona integron-encoded metallo-β-lactamase, IPM β-lactamases and class 1 integrons were predominantly present among the IPM-resistant P. aeruginosa tested in our province and also the frequency of bla_VIM type is higher than bla_IMP. This is the first report of P. aeruginosa strains producing bla_IMP with high frequency from Zanjan province of Iran. Key Words: Pseudomonas aeruginosa, Beta-lactamases, PCR     

Antibiotic Resistance Pattern of Different Escherichia coli Phylogenetic Groups Isolated from Human Urinary Tract Infection and Avian Colibacillosis

Background : The emergence and propagation of different phylogenetic groups of antimicrobial-resistant E. coli have become a worldwide health concern in human and veterinary medicine. Therefore, the evaluation of the phylogenetic distribution of antibiotic-resistant E. coli is important for therapeutic and economic purposes. The aims of this study were to determine phylogenetic groups and patterns of antibiotic resistance of E. coli strains isolated from human urinary tract infection and avian colibacillosis. Methods: A total of 50 E. coli isolates (25 from human urinary tract infection and 25 from avian colibacillosis) were characterized by culture and assigned as different phylogenetic groups (A, B1, B2, and D) by triplex PCR assay. Kirby-Bauer disk diffusion method was used to assess the susceptibility of all isolates to ten antibiotics. Results: Results showed that the majority of the human and poultry isolates belonged to phylogenetic groups A and B2 and phylogenetic group B1 of the avian pathogenic strain isolates were the most drug-resistant isolates. Most of the isolates were resistant to at least five antibiotics, and multiple drug resistance was observed in 98% of E. coli isolates. A high degree of resistance was seen against penicillin and erythromycin. Conclusion: According to the results of this study, multidrug-resistance among isolates and high relation between phylogenetic groups and resistance in both human and poultry isolates were observed. Key Words: Escherichia coli, Avian colibacillosis, Phylogenetic grouping     

Screening of three sweet potato (Ipomoea batatas L.) cultivars for resistance to different virulence groups of root-knot nematodes (Meloidogyne spp.) under controlled conditions

The susceptibility of three sweet potato cultivars (Ipomoea batatas L.) C4, TIS 3290 and TIS 9162 was evaluated against 156 isolates of Meloidogyne spp. with the aim to include resistant/tolerant sweet potato cultivars in a crop rotation scheme for the management of root-knot nematodes. The nematode isolates corresponded to races 1, 2 and 3 of Meloidogyne arenaria (n = 7), races 1, 2, 3 and 4 of M. incognita (n = 131) and Meloidogyne javanica (n = 18). Also, the isolates of M. incognita were differentiated in virulence groups: Pepper (n = 35), Pepper-Mi (n = 25), Tomato (n = 41) and Tomato-Mi (n = 30), depending on their ability to parasitize resistant pepper and tomato cultivars. The tested isolates of M. javanica parasitized C4 and TIS 3290, but not TIS 9162, whereas M. arenaria parasitized C4 and TIS 9162, but not TIS 3290, and M. incognita was able to parasitize the three sweet potato cultivars tested. C4 was the most susceptible cultivar to all nematode species tested, especially M. incognita, TIS 3290 was the most resistant and TIS 9162 was in between (7.2, 62.9 and 26.9% of resistant plants, respectively). Susceptibility of the sweet potato cultivars showed slight variations depending on the race or virulence group of M. incognita. The results suggest that sweet potato cultivars TIS 3290 and TIS 9162 may be used as rotation crops in fields where root-knot nematodes are present, their selection depending on the Meloidogyne isolates present. The use of resistant sweet potato cultivars would be preferably combined with other management practices to avoid virulence selection in nematode isolates.     

Occurrence and characterization of fungi and oomycetes transmitted via potting mixtures and organic manures

A study was conducted to investigate the most common fungal and oomycete pathogens introduced into farms in Oman via potting mixtures and organic manures. A total of 37 commercial types of potting mixtures (2 local and 35 imported from overseas), 4 commercial types of organic manures and 11 non-commercial types of organic manures were included in the study. Identification of the isolated species was based on morphological characteristics, except for the most common species which were further identified using sequences of the internal transcribed spacer region of the ribosomal DNA (ITS rDNA). Fusarium spp. (14%), Pythium aphanidermatum (3%), Alternaria spp. (5%), Helminthosporium spp. (5%) and Cladosporium spp. (3%) were recovered at different frequencies from samples of potting mixtures. Fusarium solani (40%) and Fusarium equiseti (47%) were recovered at high frequencies from samples of organic manures. Isolations from organic manures also yielded Pythium periplocum (7%), Rhizoctonia solani (7%), Fusarium lichenicola (7%), Helminthosporium spp. (27%) and Alternaria spp. (27%). Trichoderma spp., Penicillium spp., Aspergillus spp. and Rhizopus spp. were found to be common in samples of potting mixtures and organic manures. Investigating sensitivity to hymexazol among 9 isolates of F. equiseti and 13 isolates of F. solani revealed variations among different isolates. The EC₅₀ values ranged from 1 to over 1200 (avg. 192 μg ml⁻¹) for F. equiseti isolates and from 135 to 789 (avg. 324 μg ml⁻¹) for F. solani isolates, indicating presence of resistance to this important fungicide among some Fusarium isolates. This appears to be the first report of contamination with R. solani, P. periplocum, F. solani, F. equiseti and F. lichenicola of organic manures. This study appears to report for the first time F. lichenicola in Oman and appears to be the first report of occurrence of resistance to hymexazol among F. equiseti and F. solani isolates.     

Multi-gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with Eucalyptus stem cankers

Colletogloeopsis zuluensis, previously known as Coniothyrium zuluense, causes a serious stem canker disease on Eucalyptus spp. grown as non-natives in many tropical and sub-tropical countries. This stem canker disease was first reported from South Africa and it has subsequently been found on various species and hybrids of Eucalyptus in other African countries as well as in countries of South America and South-East Asia. In previous studies, phylogenetic analyses based on DNA sequence data of the ITS region suggested that all material of C. zuluensis was monophyletic. However, the occurrence of the fungus in a greater number of countries, and analyses of DNA sequences with additional isolates has challenged the notion that a single species is involved with Coniothyrium canker. The aim of this study was to consider the phylogenetic relationships amongst C. zuluensis isolates from all available locations and to support these analyses with phenotypic and morphological comparisons. Individual and combined phylogenies were constructed using DNA sequences from the ITS region, exons 3 through 6 of the β-tubulin gene, the intron of the translation elongation factor 1-α gene, and a partial sequence of the mitochondrial ATPase 6 gene. Both phylogenetic data and morphological characteristics showed clearly that isolates of C. zuluensis represent at least two taxa. One of these is C. zuluensis as it was originally described from South Africa, and we provide an epitype for it. The second species occurs in Argentina and Uruguay, and is newly described as C. gauchensis. Both fungi are serious pathogens resulting in identical symptoms. Recognising them as different species has important quarantine consequences.Taxonomic novelty: Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf. sp. nov.     

Functional Food Quality of Curcuma caesia, Curcuma zedoaria and Curcuma aeruginosa Endemic to Northeastern India

Curcuma spp. (Zingiberaceae) is one of the significant ingredients in food and traditional medicines. The current study was to investigate health-benefits of the rhizomes of endemic Curcuma caesia, Curcuma zedoaria and Curcuma aeruginosa using in vitro antioxidant, antiinflammatory and human tumor cell proliferation inhibitory activities. Among these, C. caesia (black turmeric) showed the best overall biological activities based on [3-(4, 5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) and lipid peroxidation (LPO), cyclooxygenase (COX-1 and -2) enzymes, and tumor cell growth inhibitory assays. The hexane and methanolic extracts of C. caesia (CCH and CCM) showed LPO inhibition by 31 and 43 %, and COX-2 enzyme by 29 and 38 %, respectively, at 100 μg/ml. Eleven terpenoids were isolated and identified. The MTT antioxidant assay revealed that the extracts of three Curcuma spp. at 250 μg/ml and isolates at 5 μg/ml demonstrated activity comparable to positive controls vitamin C and t-butyl hydroquinone (TBHQ) at 25 μg/ml. The extracts inhibited LPO by 40 % at 250 μg/ml whereas pure isolates 1–11 by about 20 %. The extracts and isolates inhibited COX-1 and -2 enzymes between the ranges of 3–56 and 5–30 %, respectively. The in vitro biological activity exhibited by the extracts and isolates of C. caesia rhizome further supported its use in traditional medicine.     

Distribution of enterococcal species and detection of vancomycin resistance genes by multiplex PCR in Tehran sewage

BACKGROUND: Enterococci are important because of their role as the leading cause of nosocomial infections which have a significant role in the dissemination and persistence of antimicrobial resistance genes. METHODS: In this study, we determined the distribution of enterococcal species in the sewage treatment plants in Iran. Furthermore, we improved a rapid and specific PCR method using primers (sodA and ddl genes) for identification of enterococci spp. RESULTS AND CONCLUSION: A total number of 712 enterococci spp. were isolated and the results showed that 56%, 24%, 12%, 4%, 2%, 1% and 1% isolates were E. faecium, E. hirae, E. faecalis, E. gallinarum, E. casseliflavus, E. mundtii and other enterococcal spp., respectively. The use of species-specific PCR was in agreement with the biochemical tests. Furthermore, multiplex PCR was developed to study the presence of vancomycin resistant genes in E. faecium or E. faecalis. The multiplex PCR appeared to be a useful, rapid and specific method for detecting and discriminating genotypes for vancomycin-resistant Enterococcus.     

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.     

Virulence Types of Magnaporthe oryzae to Hybrid Rice in Sichuan,China

A total of 638 isolates of rice blast(Magnaporthe oryzae) were isolated in 2002-2009 from different rice varieties in different regions of Sichuan,China and inoculated onto seven rice varieties(Lijiangxintuanheigu,IR24,Minghui 63,Duohui 1,Chenghui 448,Neihui 99-14 and RHR-1) to differentiate the virulence types of the fungus and trace the changes.The virulence to the seven varieties was respectively scored at 1,2,4,8,16,32 and 64.The total scores of individual M.grisea isolates which were the sum of scores infecting differential varieties could,in turn,be used for the nomenclature of the virulence types due to their accordance to the special virulence patterns.The 638 tested isolates were then differentiated into 56 different virulence types.Type 15 virulent to Lijiangxintuanheigu,IR24 and Minghui 63,and Type 127 virulent to all of the seven varieties were the most dominant virulence types respectively with the occurrence frequencies of 15.99% and 15.83%.Type 19 and other seven virulence types were not monitored during 2002-2009.Type 15 was the predominant virulence type in 2002,2003,2004 and 2007,whereas Type 127 had been the most dominant virulence type after 2005 except for the year 2007 when the province underwent severe drought.Five hundred and seven out of the 638 tested isolates were virulent to Minghui 63,and 89.58% of the 384 isolates virulent to either Duohui 1,Chenghui 448 or Neihui 99-14 were virulent to Minghui 63,which indicated the impact of the extensive plantation of hybrid rice Minghui 63 as the restorer line on the virulence evolution of M.oryzae in Sichuan.The virulence pattern of the dominant virulence types suggested that the acquiring of virulence to all the major resistant restorer lines was the main routes of the evolution in virulence of M.oryzae to hybrid rice in Sichuan.The virulence frequencies of the 638 tested isolates to IR24,Minghui 63,Duohui 1,Chenghui 448,Neihui 99-14 and RHR-1 were respectively 74.6%,79.5%,73.8%,37.0%,39.0% and 40.4%.The analysis for the sources of the different virulence type isolates indicated the pathogen on the newly released resistant varieties were stronger than conventional rice varieties which had become susceptible in the field since 1980s.     

Association of tcdA+/tcdB+ Clostridium difficile Genotype with Emergence of Multidrug-Resistant Strains Conferring Metronidazole Resistant Phenotype

Background:

Reduced susceptibility of Clostridium difficile to antibiotics is problematic in clinical settings. There is new evidence indicating the cotransfer of toxin-encoding genes and conjugative transposons encoding resistance to antibiotics among different C. difficile strains. To analyze this association, in the current study, we evaluated the frequency of toxigenic C. difficile among the strains with different multidrug-resistant (MDR) profiles in Iran.

Methods:

Antimicrobial susceptibility patterns and minimal inhibitory concentrations (MIC) of the isolates were determined against metronidazole, imipenem, ceftazidime, amikacin, and ciprofloxacin by agar dilution method. The association of the resistance profiles and toxigenicity of the strains were studied by PCR targeting tcdA and tcdB genes.

Results:

Among 86 characterized strains, the highest and lowest resistance rates were related to ciprofloxacin (97%) and metronidazole (5%), respectively. The frequency of resistance to other antibiotics was as follow: imipenem (48%), ceftazidime (76%), and amikacin (76.5%). Among the resistant strains, double drug resistance and MDR phenotypes were detected in the frequencies of 10.4% and 66.2%, respectively. All of the metronidazole-resistant strains belonged to tcdA ⁺/tcdB ⁺ genotype with triple or quintuple drug resistance phenotypes. MIC₅₀ and MIC₉₀ for this antibiotic was equally ≤ 8 μg/ml.

Conclusion:

These results proposed the association of tcdA ⁺/tcdB ⁺ genotype of C. difficile and the emergence of resistance strains to broad-spectrum antibiotics and metronidazole. Key Words: Multidrug resistance, Clostridium difficile, Metronidazole     

河北省玉米大斑病菌生理小种组成研究

采用鉴别寄主鉴定技术,对2009～ 2010年采自河北省的120份玉米大斑病菌菌株进行生理小种组成及毒性分析.结果表明,共鉴定出15个生理小种致病类型,即0、1、3、N、12、13、1N、23、2N、3N、123、12N、13N、23N和123N,其中,0号和1号生理小种为优势小种,分别占总分离数的24.2％和20.8％;其次为1N和N号生理小种,分别占总分离数的10.0％和9.2％.春玉米区大斑病菌生理小种的结构较复杂,出现多个生理小种类型,夏玉米区大斑病菌生理小种的结构相对较简单.将各菌株进行毒性分析发现,河北省玉米大斑病菌对Htl和HtN基因的毒性较高,毒性频率分别为57.5％和37.5％;对Ht2和Ht3基因毒性较低.     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

Pathogenic and genetic diversity of Didymella rabiei affecting chickpea in Syria

Simple sequence repeats and mating type markers were used to estimate the genetic diversity of 133 Didymella rabiei isolates collected from nine provinces of Syria. Moreover, phenotyping was done on 56 isolates randomly selected from the different genetic groups using five chickpea genotypes. The genetic diversity of D. rabiei population was high with inter-population variability accounting for 83% of the total variation, whereas the genetic diversity among populations was very low (17%). Principal component analysis grouped the isolates from Aleppo, Idlib, Hama, Homs and Hassakeh provinces together, while Daraa and Tartous were in different groups. Isolates from Lattakia and Suweida provinces formed very distinct clusters compared to the others. The 56 isolates were grouped into four pathotypes, namely, pathotype-1 (12 isolates), pathotype-2 (13 isolates), pathotype-3 (5 isolates) and pathotype-4 (26 isolates) with varying degrees of virulence on the chickpea genotypes. Our findings showed a clear genetic shift toward more virulence over time and space in the populations of D. rabiei in Syria. These results stress the need for chickpea breeding materials to be tested for resistance to the more virulent pathotypes. Also, concerted action should be taken to ensure the shipment of healthy seeds of international chickpea nurseries to avoid D. rabiei genotypes or pathotypes flow from Syria to other countries.     

西南地区玉米大斑病菌生理小种鉴定

2008～2010年对采自西南地区的玉米大斑病标样经过单孢分离、纯化,共获得146个菌株。通过Ht单基因鉴别寄主进行生理小种鉴定,共鉴定出9个生理小种,分别为0、1、2、3、12、1N、2N、3N、N。0号小种占鉴定菌株的62.33%,为优势小种,其次为1号小种,占鉴定菌株15.07%。毒性频率分析表明,所有供试菌株对Ht1抗性基因的毒性频率最高,为20.99%;对Ht3抗性基因的毒性频率最低,为1.36%;对Ht2和HtN抗性基因的毒性频率分别为13.01%和9.57%。目前,西南地区玉米大斑病生理小种分化明显,并且产生了新的生理小种,对玉米生产存在潜在威胁。     

Planting date and development of spring-seeded irrigated canola, brown mustard and camelina

With increased emphasis on bio-diesel fuels, the influence of spring planting on development of brown mustard (Brassica juncea cv. Arid), canola (B. napus cv. Hyola 401) and camelina (Camelina sativa cv. Boa) has become important. Field trials were conducted at Scottsbluff, NE, in 2005 and 2006 at planting dates of 24 February, 24 March, 7 April, 21 April and 5 May, and 3 March, 3 April, 10 April, 27 April, 11 May, and 2 Jun, respectively. Emergence time was shorter with later planting. Flowering date was later with later planting but occurred within a range of degree days (P-days). Fruiting was affected by date and P-days, but seed maturity was not affected by planting date and was unrelated to P-days. Fleabeetle (Phyllotreta spp.) damage was very high in brown mustard and canola. Bird, primarily house finch (Carppodacus mexicanus), feeding was a major problem with brown mustard planted before mid April and in canola, only with the first planting. Camelina was not affected by either. Planting in April gave the best yields, and canola could yield over 2200 kg ha⁻¹. Oil content of the Brassica was highest when planted from late March and later. For camelina, planting date had no effect. In brown mustard and canola, 60-65% of oil was C18:1, in camelina, about 15%. Later planting increased C18:1 content for the three crops. The second fatty acid was C18:2 with 20% in brown mustard, 18% in canola and 20% in camelina. Later planting increased C18:2 in camelina only. The major fatty acid in camelina was C18:3 at 32-37%; earlier planting increased the content of C18:3. In Camelina, C20:1 comprised about 12% of the oil and was highest with April planting. Canola and camelina seeded in April could be grown for oil successfully in western Nebraska.     

Box-Wilson Design for Optimization of in vitro Levan Production and Levan Application as Antioxidant and Antibacterial Agents

Background:Levan or fructan, a polysaccharide of fructose, is widely used in various commercial industries. Levan could be produced by many organisms, including plants and bacteria. The cloning of the gene from Bacillus licheniformis, which expressed levansucrase in Escherichia coli host, was carried out successfully. In the present study, we performed the in vitro production of levan and analyzed its potential application as antibacterial and antioxidant agents. Methods: In vitro levan production catalyzed by heterologous-expressed levansucrase Lsbl-bk1 and Lsbl-bk2 was optimized with BW design. The antibacterial activity of the produced levan was carried out using agar well diffusion method, while its antioxidant activity was tested by free radical scavenging assays. Results:The optimum conditions for levan production were observed at 36 °C and pH 7 in 12% (w/v) sucrose for levansucrase Lsbl-bk1, while the optimum catalysis of levansucrase Lsbl-bk2 was obtained at 32 ^oC and pH 8 in the same sucrose concentration. The in vitro synthesized levan showed an antibacterial activity within a concentration range of 10-20% (w/v) against Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa. The same levan was also able to inhibit the DPPH radical scavenging activity with the antioxidant strength of 75% compared to ascorbic acid inhibition. Conclusion:Our study, therefore, shows that the optimized heterologous expression of levansucrases encoded by Lsbl-bk1 and Lsbl-bk2 could open the way for industrial levan production as an antibacterial and antioxidant agent. Key Words: Antioxidants, Fructans, In vitro technique, Levan