Restriction Endonuclease Digestion Patterns Research Articles

Isolation, characterization, and effectiveness of bacteriophage Pɸ-Bw-Ab against XDR Acinetobacter baumannii isolated from nosocomial burn wound infection.

Objective(s):With emergence of drug resistance, novel approaches such as phage therapy for treatment of bacterial infections have received significant attention. The purpose of this study was to isolate and identify effective bacteriophages on extremely drug-resistant (XDR) bacteria isolated from burn wounds. Materials and Methods:Pathogenic bacteria were isolated from hospitalized patient wounds in specialized burn hospitals in Iran, and their identification was performed based on biochemical testing and sequencing of the gene encoding 16S rRNA. Bacteriophages were isolated from municipal sewage, Isfahan, Iran. The phage morphology was observed by TEM. After detection of the host range, adsorption rate, and one-step growth curve, the phage proteomics pattern and restriction enzyme digestion pattern were analyzed.Results:All isolates of bacteria were highly resistant to antibiotics. Among isolates, Acinetobacter baumannii strain IAU_FAL101 (GenBank accession number: MW845680), which was an XDR bacterium, showed significant sensitivity to phage Pɸ-Bw-Ab. TEM determined the phage belongs to Siphoviridae. They had double-stranded DNA. This phage showed the highest antibacterial effect at 15 °C and pH 7. Analysis of the restriction enzyme digestion pattern showed Pɸ-Bw-Ab phage was sensitive to most of the used enzymes and based on SDS-PAGE, protein profiles were revealed 43 to 90 kDa.Conclusion:Considering the potential ability of the isolated phage, it had an antibacterial impact on other used bacterial spp and also strong antibacterial effects on XDR A. baumannii. Also, it had long latency and low burst size. This phage can be a suitable candidate for phage therapy.

Cryptosporidium parvum in Children with Diarrhea in Zahedan, Iran

Background: Cryptosporidium is one of the most important causes of gastroenteritis in humans. Objectives: This study aimed to investigate the distribution of Cryptosporidium species in children with acute diarrheal disease in Zahedan, Iran. Methods: Stool specimens collected from 764 children aged < 10 years with diarrheal disease admitted to Aliasghar Pediatric Hospital and the Reference Laboratory in Zahedan were assessed for the presence of Cryptosporidium oocysts using the conventional microscopic examination and acid-fast staining. To characterize the oocysts at the molecular level, the 18S rRNA was nested-PCR amplified and sequenced. PCR products were also digested using Vsp1 restriction enzyme for genotype differentiation. Results: Of the 764 stool specimens, 7 (0.91%) were positive for Cryptosporidium oocysts using the microscopic examination. Restriction endonuclease digestion pattern successfully revealed the presence of Cryptosporidium parvum in two microscopic positive samples that were confirmed by DNA sequencing. Conclusions: Cryptosporidium parvum is responsible for cryptosporidiosis in children under 10 years of age in the study region. Although C. parvum may be an important pathogen associated with diarrhea, it is the cause of only a small proportion of diarrheal episodes.

Differentiation of European Corn Borer (Lepidoptera: Crambidae) and American Lotus Borer (Lepidoptera: Crambidae), Ostrinia penitalis, from North American Field Collections.

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is a perennial insect pest of cultivated maize that was inadvertently introduced into North America in the early 1900s, but population densities have decreased since the widespread adoption of transgenic hybrids that express Bacillus thuringiensis (Bt) toxins. The native American lotus borer, Ostrinia penitalis (Lepidoptera: Crambidae), is among the most ancestral species described in the genus Ostrinia, and has a geographic range that coincides with that of O. nubilalis across major maize growing regions of North America. Due to the recent decrease in O. nubilalis populations, O. penitalis has become more pronounced in light trap samples intended to monitor O. nubilalis. A molecular tool based on variation in restriction endonuclease digestion pattern of a polymerase chain reaction amplified fragment of the mitochondrial cytochrome c oxidase subunit I (coxI) gene was developed and validated to differentiate these two species. This method was applied to light trap samples over a 2-yr period and achieved accurate quantification of species, and shows that O. penitalis can be prevalent in O. nubilalis first flight sampling. These methods are useful for contemporary O. nubilalis field research in North America.

Prevalence and molecular typing of methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin gene.

Panton-Valentine leukocidin (pvl) toxin is an important virulence factor of Staphylococcus aureus. The main genes are coa and spa for distinguishing and typing of S. aureus isolates. The aim of this study was to investigate antibiotic resistance, presence of mecA and pvl genes, as well as epidemiological typing of these isolates according to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in clinical sample isolated from Rasht city, Iran. A total of 250 clinical samples have been isolated from different hospitals. First, isolates of S. aureus were identified through microbiological methods and their antibiotic sensitivity was determined by disk diffusion agar based on a standard method of Clinical and Laboratory Standards Institute. DNA was extracted by boiling and presence of pvl and mecA genes was investigated by PCR using specific primers. To type these isolates, amplification of fragments of coa and spa genes was done and restriction enzyme digestion pattern was determined by PCR-RFLP method. Among the 250 samples, 50 isolates belonged to S. aureus and results of antibiotic sensitivity showed that 68% (34 samples) of isolates were methicillin resistant. Frequency of mecA and pvl genes among S. aureus isolates were 60% (30 samples) and 20% (10 samples). The PCR of coa gene showed three patterns whereas that of spa gene showed two patterns for enzyme digestion. Result of PCR-RFLP using HaeIII enzymes for coa gene and Bsp1431 for spa gene showed three patterns for enzyme digestion. Recent studies indicated increase in the resistance of S. aureus to different antibiotics, which is a serious problem in the treatment of infections resulting from S. aureus in this region. The result of PCR of pvl showed high frequency of this gene in this region, and coa and spa typing by PCR-RFLP was a useful tool for typing of S. aureus isolates.

Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

Pseudomonas syringae pv. actinidiae causes bacterial canker disease in kiwifruit. Owing to the prohibition of agricultural antibiotic use in major kiwifruit-cultivating countries, alternative methods need to be developed to manage this disease. Bacteriophages are viruses that specifically infect target bacteria and have recently been reconsidered as potential biological control agents for bacterial pathogens owing to their specificity in terms of host range. In this study, we isolated bacteriophages against P. syringae pv. actinidiae from soils collected from kiwifruit orchards in Korea and selected seven bacteriophages for further characterization based on restriction enzyme digestion patterns of genomic DNA. Among the studied bacteriophages, two belong to the Myoviridae family and three belong to the Podoviridae family, based on morphology observed by transmission electron microscopy. The host range of the selected bacteriophages was confirmed using 18 strains of P. syringae pv. actinidiae, including the Psa2 and Psa3 groups, and some were also effective against other P. syringae pathovars. Lytic activity of the selected bacteriophages was sustained in vitro until 80 h, and their activity remained stable up to 50°C, at pH 11, and under UV-B light. These results indicate that the isolated bacteriophages are specific to P. syringae species and are resistant to various environmental factors, implying their potential use in control of bacterial canker disease in kiwifruits.

Genome Wide Analysis for Searching Novel Markers to Rapidly Identify Clostridium Strains.

Microbial classification is based largely on the 16S rRNA (rrs) gene sequence, which is conserved throughout the prokaryotic domain. The Ribosomal Database Project (RDP) has become a reference point for almost all practical purposes. The use of this gene is limited by the fact that it can be used to identify only to the extent to what has been known and is available in the RDP. In order to identify an organism whose rrs is not present in the RDP database, we need to generate novel markers to place the unknown on the evolutionary map. Here, sequenced genomes of 27 Clostridium strains belonging to 9 species have been used to identify two sets of genes: (1) common to most of the species, and (2) unique to a species. Combinations of genes (recN, dnaJ, secA, mutS, and/or grpE) and their unique restriction endonuclease digestion (AluI, BfaI and/or Tru9I) patterns have been established to rapidly identify Clostridium species. This strategy for identifying novel markers can be extended to all other organisms and diagnostic applications.

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y.enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y.pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y.pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. There was a clear association between the presence of the host bacteria and specific phages in the stools. The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.

A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10–11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg−1 fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae.

Isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against Salmonella enterica serovars

The use of bacteriophages is considered as a viable alternative to chemical antimicrobials against foodborne pathogens. The objective of this study was to develop a collection of lytic bacteriophages which will be able to infect different pathogenic Salmonella enterica serovars. Phages were isolated from animal feces and sewage samples, purified, characterized morphologically and by DNA fingerprinting, and host ranges were determined. Spot test and efficiency of plaquing (EOP) data indicated that two phages, SEA1 and SEA2 had the broadest host range against Salmonella among all isolated phages. SEA2 was highly efficient to infect S. Typhimurium DT104 (0.5–1 EOP value). Only phage SSA1 was able to infect S. Montevideo. Transmission electron microscopy (TEM) revealed the phages in the collection were mostly (4 out of 6) Siphoviridae, while SEA1 and SEA2 were Myoviridae T4-like phages. SEA1 and SEA2 had the largest genome sizes in the collection, 190 and 170 kb, respectively. Pulsed field gel electrophoresis (PFGE) analysis demonstrated distinct digestion profiles with EcoRI for phages SSA1, STD3, STE3 and STF1. However, SEA1 and SEA2 shared a similar restriction enzyme (RE) digestion pattern with same morphotype, but distinct profiles in lysing Salmonella strains. These anti-Salmonella phages were highly host specific with few exceptions of lytic phages that were able to infect a wide variety of Salmonella. These phages have potential for use in applications controlling Salmonella on different matrices.

Protective effects of the Aeromonas phages pAh1-C and pAh6-C against mass mortality of the cyprinid loach (Misgurnus anguillicaudatus) caused by Aeromonas hydrophila

To investigate methods to control the mass mortality of cyprinid loaches (Misgurnus anguillicaudatus) caused by multiple-antibiotic-resistant Aeromonas hydrophila on a private fish farm in Korea, we isolated bacteriophages (phages), designated pAh1-C and pAh6-C, from the Han River in Seoul. The two isolated phages were morphologically classified as Myoviridae and showed similar infection patterns for A. hydrophila isolates. The two phages possessed approximately 55kb (pAh1-C) and 58kb (pAh6-C) of double-stranded genomic DNA, and their gDNAs showed different restriction endonuclease digestion patterns. Both phages showed efficient bacteriolytic activity against fish-pathogenic A. hydrophila from loaches. The latent periods of the phages were estimated to be approximately 30min (pAh1-C) and 20min (pAh6-C), while the burst sizes were 60PFU/cell (pAh1-C) and 10PFU/cell (pAh6-C). The phages proved to be efficient in the inhibition of bacterial growth, as demonstrated by their in vitro bactericidal effects. Additionally, a single administration of either phage to cyprinid loaches resulted in noticeable protective effects, with increased survival rates against A. hydrophila infection. These results suggest that the phages pAh1-C and pAh6-C constitute potential therapeutic agents for the treatment of A. hydrophila infection in fish.

Development of Genomic Tools for the Identification of Certain Pseudomonas up to Species Level

Pseudomonas is a highly versatile bacterium at the species level with great ecological significance. These genetically and metabolically diverse species have undergone repeated taxonomic revisions. We propose a strategy to identify Pseudomonas up to species level, based on the unique features of their 16S rDNA (rrs) gene sequence, such as the frame work of sequences, sequence motifs and restriction endonuclease (RE) digestion patterns. A species specific phylogenetic framework composed of 31 different rrs sequences, allowed us to segregate 1,367 out of 2,985 rrs sequences of this genus, which have been classified at present only up to genus (Pseudomonas) level, as follows: P. aeruginosa (219 sequences), P. fluorescens (463 sequences), P. putida (347 sequences), P. stutzeri (197 sequences), and P. syringae (141 sequences). These segregations were validated by unique 30-50 nucleotide long motifs and RE digestion patterns in their rrs. A single gene thus provides multiple makers for identification and surveillance of Pseudomonas.

Occurrence of methicillin‐resistant Staphylococci in surgically treated dogs and the environment in a Swedish animal hospital

To investigate whether hospitalised dogs treated surgically may become culture positive for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. Surgically treated dogs (n=45) were sampled for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus on admission, before and after surgery and at the time of removal of surgical stitches. The hospital environment (n=57), including healthy dogs in the veterinary hospital environment (n=34), were sampled for methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. Genetic variations among methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus isolates were identified through detection of restriction fragment polymorphisms. No dogs developed a wound infection due to methicillin-resistant Staphylococcus pseudintermedius or methicillin-resistant Staphylococcus aureus. However, there was a significant increase in the number of dogs carrying methicillin-resistant Staphylococcus pseudintermedius after hospitalisation compared to admission (P<0·001). No methicillin-resistant Staphylococcus aureus was isolated from dogs, but was present in the environment. Methicillin-resistant Staphylococcus pseudintermedius isolates were recovered from environmental surfaces and hospitalised animals, but not from healthy dogs. Methicillin-resistant Staphylococcus pseudintermedius isolates representing nine different restriction endonuclease digestion patterns were found, with two of these occurring in both the environment and on dogs. Dogs may contract methicillin-resistant Staphylococcus pseudintermedius in association with surgery and hospitalisation. Resistant bacteria may be transmitted between dogs, staff and the environment. Dogs colonised with methicillin-resistant Staphylococcus pseudintermedius may be a source for hospital- and community-acquired infections.

Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods

Bacillus cereus is a foodborne bacterial pathogen that causes diarrhea and vomiting. In this study, the usefulness of bacteriophages to eradicate B. cereus from fermented foods was investigated. A total of 13 phages were isolated from Korean fermented food products, and 2 (BCP1-1 and BCP8-2) were further characterized. Transmission electron microscopy (TEM), restriction enzyme digestion pattern analysis, and SDS-PAGE of the structural proteins suggest that both phages belong to the family Myoviridae, containing approximately 150 kbp-long genomes. The host ranges of both phages were limited to B. cereus group species (12/13), as they were not able to lyse other Gram-positive or negative strains including Bacillus subtilis. Purified phages were used to inhibit B. cereus growth in a model fermented food system, cheonggukjang, a fast-fermented soybean paste product. BCP1-1 and BCP8-2 were able to effectively eradicate B. cereus from the food only if divalent cations (Ca2+, Mg2+, or Mn2+) were added to the medium. Further studies reveal that divalent cations are essential for phage adsorption, while a monovalent cation (Na+) is required for the post-adsorption phase of phage infection. Taken together, our findings imply that a phage could be an ideal anti-bacterial agent for use in fermented food products that require the presence of beneficial microflora and, during phage application, optimization of phage reaction conditions is critical for the successful utilization of phage biocontrol.

Factor VIII Point Mutations with Higher Risk for Inhibitor Development in Mild/Moderate Hemophilia A: A Rapid, Specific Test Panel.

Abstract 2264The conversion of mild or moderate Factor VIII-deficient patients into those with a severe bleeding disorder can be an alarming consequence of inhibitor development, which is not typically anticipated in these patients. Cross-reactivity of the inhibitor antibodies with the patient’s own circulating FVIII can reduce their baseline FVIII activity from that of 5–10% to 0–1%, with severe bleeding diathesis. Immune tolerance can be effective, as with more severely deficient cases. But patients must undergo daily desensitization and use costly bypassing agents. While cross-reactivity of the inhibitor antibodies with the patient’s circulating FVIII remains poorly understood, there is growing evidence that mild/moderate hemophilia A patients with specific FVIII point mutations are at greater risk than others for developing this life-threatening complication. Current hypotheses suggest that certain mutations result in greater conformational changes in the patient’s circulating FVIII, so that the infused FVIII becomes immunogenic.Based on review of previously published reports and the Haemophilia A Mutation, Structure, Test, and Resource Site (HAMSTeRS) database, we have selected nine point mutations, associated with multiple cases of mild/moderate hemophilia A, where at least 10% of those patients reported have developed inhibitors. Six of the nine are in the FVIII C-domain (Arg2150His, Trp2229Cys, Tyr2105Cys, Asn2286Lys, Pro2300Leu, and Arg2307Gln) and the other three are in the A-domain (Arg593Cys, Arg1997Trp, and Glu1999Gly).As an alternative to complete DNA sequencing or heteroduplex analysis, we have developed a more straightforward panel to rapidly test for these point mutations directly. Specific restriction endonucleases (New England BioLabs) were identified with overlapping recognition sites for each of the nine point mutations. For seven of the mutations, the restriction site is lost if a mutation occurs. For the other two, the restriction site is gained when the mutation is present. We evaluated this panel by digesting PCR amplicons from synthetic, control DNA templates (IDT), representing the wild-type and mutated FVIII exons encompassing each of the nine mutations. Differences between the resulting restriction enzyme digestion patterns were successfully detected with a BioAnalyzer DNA 1000 Kit (Agilent). A pilot study has further tested genomic DNA from eleven pediatric hemophilia A patients, all of whom were negative for each of the nine mutations. The results from testing of a larger bank of pediatric hemophilia A genomic DNA will also be presented.A more rapid, specific molecular screen that could be performed at the initial diagnosis of mild/moderate FVIII deficiency would allow providers to more readily identify those patients at risk for developing a FVIII inhibitor. This form of “personalized medicine” could help to prevent the potentially tragic outcome of inhibitor development, which can last from months to many years, in those patients that unexpectedly convert into a severe bleeding phenotype. Disclosures:No relevant conflicts of interest to declare.

Outbreak of equine endometritis caused by a genotypically identical strain of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that has been recognized as a cause of endometritis in mares. Pulsed field gel electrophoresis was used to characterize and compare isolates of P. aeruginosa from an outbreak of endometritis and unrelated isolates collected at the same time as the outbreak. The restriction endonuclease digestion patterns and antimicrobial resistance profiles of all outbreak isolates were identical. Therefore, a single strain of P. aeruginosa was responsible for the cases of endometritis. The unrelated isolates could be distinguished from the outbreak strain using the techniques outlined in the present study. The results establish that this pathogen was not venereally transmitted between all the horses from which it was isolated, but rather must have been disseminated, at least initially, from a contaminated water source. Once the water used to clean the mares and stallions was replaced, there were no further reports of endometritis caused by this organism on the affected stud. Furthermore, the fertility of the stallions was not affected, in spite of persistent carriage for 1 to 2 months. The current study has shown that the use of pulsed field gel electrophoresis has considerable value in epidemiological investigations of equine urogenital tract infections with P. aeruginosa.

Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901

Bacillus cereus is widely distributed in the environment and is responsible for foodborne illnesses, causing diarrhea and vomiting. In this study, the prevalence of B. cereus bacteriophages in Korean fermented foods was studied using 6 reference strains, and JBP901 was further characterized. In a total of 47 samples, 19 (40.4%) contained more than one kind of B. cereus phage. Fourteen phages were purified and determined for host lysis profile. Among them, JBP901 was specific to B. cereus group strains (forming plaques in 13 of 14 isolates) and not able to lyse other tested Gram-positive and Gram-negative strains including Bacillus subtilis. Transmission electron microscopy analysis suggested that JBP901 is a member of the family Myoviridae. Restriction enzyme digestion pattern analysis and one-step growth study indicated a genome size of approximately 150 kbp and a burst size of 100. Furthermore, bacterial lysis studies revealed that JBP901 could be used to control the growth of B. cereus in liquid culture and in food. This is the first report to examine the prevalence of phages in fermented foods in Korea, and suggests that fermented foods are useful sources of B. cereus phage isolation; moreover, B. cereus phages could provide an additional tool to tackle problems associated with B. cereus contamination.

Establishment of a humankeratinocyte cell line (HaCaT) stably expressing human papillomavirus type 16 E7 (HPV16E7)protein

：Objective To establish ahuman keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein. Methods HPV16E7gene was amplified from CaSki cells using PCR and inserted into the eukaryotic expressionplasmid pcDNA3.1. Then, the recombinant expression plasmid pcDNA3.1-HPV16E7 wastransfected into HaCaT cells followed by G418 selection and identification by RT-PCR andWestern blot. Results The recombinant eukaryotic expression plasmid pcDNA3.1-HPV16E7 wassuccessfully identified by restriction enzyme digestion pattern and sequence analysis.Agarose gel electrophoresis of RT-PCR products detected the 297-bp fragment of HPV16E7cDNA, and Western blot confirmed the stable expression of HPV16E7 protein. Conclusion Ahuman keratinocyte cell line (HaCaT) stably expressing HPV16E7 protein is successfullyestablished.

A Protocol for Extraction of High-quality RNA and DNA from Peanut Plant Tissues

Peanuts are an increasingly important global food source. However, until recently the lack of effective protocols for the extraction of nucleic acids has made molecular studies of peanut development and maturation difficult. Here, we describe a method to isolate high-quality RNA and DNA from peanut tissue and have successfully applied this method to peanut plant roots, stems, leaves, flowers, and seeds. Spectrophotometric analysis showed that the average yields of total RNA from 100mg of peanut materials ranged from 24.52 to 74.6μg, and those of genomic DNA from the same tissues ranged from 23.47 to 57.68μg. Using this protocol, we obtained OD260/280 values between 1.9 and 2.0 and isolated RNA which could be reverse transcribed in a manner suitable for RT-qPCR and expression analysis. In addition, genomic DNA isolated using this method produced reliable restriction enzyme digestion patterns and could be used for Southern blot hybridization.

Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium.

BackgroundBacterial taxonomy and phylogeny based on rrs (16S rDNA) sequencing is being vigorously pursued. In fact, it has been stated that novel biological findings are driven by comparison and integration of massive data sets. In spite of a large reservoir of rrs sequencing data of 1,237,963 entries, this analysis invariably needs supplementation with other genes. The need is to divide the genetic variability within a taxa or genus at their rrs phylogenetic boundaries and to discover those fundamental features, which will enable the bacteria to naturally fall within them. Within the large bacterial community, Clostridium represents a large genus of around 110 species of significant biotechnological and medical importance. Certain Clostridium strains produce some of the deadliest toxins, which cause heavy economic losses. We have targeted this genus because of its high genetic diversity, which does not allow accurate typing with the available molecular methods.ResultsSeven hundred sixty five rrs sequences (> 1200 nucleotides, nts) belonging to 110 Clostridium species were analyzed. On the basis of 404 rrs sequences belonging to 15 Clostridium species, we have developed species specific: (i) phylogenetic framework, (ii) signatures (30 nts) and (iii) in silico restriction enzyme (14 Type II REs) digestion patterns. These tools allowed: (i) species level identification of 95 Clostridium sp. which are presently classified up to genus level, (ii) identification of 84 novel Clostridium spp. and (iii) potential reduction in the number of Clostridium species represented by small populations.ConclusionsThis integrated approach is quite sensitive and can be easily extended as a molecular tool for diagnostic and taxonomic identification of any microbe of importance to food industries and health services. Since rapid and correct identification allows quicker diagnosis and consequently treatment as well, it is likely to lead to reduction in economic losses and mortality rates.

Enumeration and Strain Characterization of FecalEscherichia coliAssociated with Feeding Triticale Dried Distillers Grain with Solubles in Beef Cattle Diets

Triticale dried distillers grain with solubles (TDDGS), a major by-product of the bioethanol industry, has potential for utilization in animal feed. This study investigated the changes in generic fecal Escherichia coli strains associated with inclusion of TDDGS in cattle diets. Within this study, a longitudinal experiment (112 days) examined the effect of step-up increasing TDDGS inclusion from control to a final diet containing 30% TDDGS among cattle (n = 4), and a short-term experiment (28 days) compared animals (n = 16) fed control, 20%, 25%, or 30% TDDGS diets. We found that incorporation of either 20%, 25%, or 30% TDDGS did not have any effect on the amount of total E. coli shedding over either the longitudinal (p = 0.06) or the short-term (p = 0. 87) study. In both the experiments, 67% of the total E. coli isolates were found to be resistant to one or more of the 17 antimicrobials tested. Among the resistant isolates, cephalothin was the most prevalent resistance (44% isolates). Over the duration of the study, tet(C) was a commonly detected resistance gene in tetracycline-resistant E. coli. Significant diversity was observed among isolates with 33 and 31 pulsed-field gel electrophoresis patterns clustering into 11 and 10 restriction endonuclease digestion pattern clusters for the longitudinal and short-term studies, respectively. Neither the duration of feeding nor increasing the proportion of TDDGS within the diet affected the diversity of E. coli resistance phenotypes or the clonal relatedness of the observed strains. Individual animals retained similar or closely related strains. Based on this study, inclusion of TDDGS as a protein and fiber source in cattle diets is not associated with increased maintenance, shedding, or proliferation of resistant strains of generic E. coli, which is an important reservoir of antimicrobial resistance among cattle.