Polyurethane/Nanosilver-Doped Halloysite Nanocomposites: Thermal, Mechanical Properties, and Antibacterial Properties.

In this study, the researchers successfully embellished the surface of halloysite (Ag/HNTs) with silver using halloysite, silver nitrate (AgNO3), and polyvinylpyrrolidone (PVP). The researchers then prepared polyurethane that contained pyridine ring by using 4,4'-diphenylmethane diisocyanate (MDI) and polytetramethylene glycol (PTMG) as the hard chain segment and the soft chain segment of polyurethane (PU), as well as 2,6-pyridinedimethanol (2,6-PDM) as the chain extension agent. This was followed by the preparation of Ag/HNTs/PUs nanocomposite thin films, achieved by mixing Ag/HNTs with different ratios into polyurethane that contains pyridine ring. First, the Ag/HNTs powders were analyzed using energy-dispersive X-ray spectroscopy, X-ray diffraction, and transmission electron microscopy. Subsequently, Fourier-transform infrared spectroscopy was used to examine the dispersibility of Ag/HNTs in PU, whereas the thermal stability and the viscoelasticity of Ag/HNTs/PU were examined using thermal gravimetric analysis, differential scanning calorimetry, and dynamic mechanical analysis. When the mechanical properties of Ag/HNTs/PU were tested using a universal strength tester, the results indicated a maximum increase of 109.5% in tensile strength. The researchers then examined the surface roughness and the hydrophobic ability of the Ag/HNTs/PU thin films by using atomic force microscopy and water contact angle. Lastly, antibacterial testing on Escherichia coli revealed that when the additive of Ag/HNTs reached 2.0 wt%, 99.3% of the E. coli were eliminated. These results indicated that the addition of Ag/HNTs into PU could enhance the thermal stability, mechanical properties, and antibacterial properties of PU, implying the potential of Ag/HNTs-02 as biomedicine material.

Designing primers and evaluation of the efficiency of propidium monoazide - Quantitative polymerase chain reaction for counting the viable cells of Lactobacillus gasseri and Lactobacillus salivarius.

The purpose of this study is to evaluate the efficiency of using propidium monoazide (PMA) real-time quantitative polymerase chain reaction (qPCR) to count the viable cells of Lactobacillus gasseri and Lactobacillus salivarius in probiotic products. Based on the internal transcription spacer and 23S rRNA genes, two primer sets specific for these two Lactobacillus species were designed. For a probiotic product, the total deMan Rogosa Sharpe plate count was 8.65±0.69 log CFU/g, while for qPCR, the cell counts of L. gasseri and L. salivarius were 8.39±0.14 log CFU/g and 8.57±0.24 log CFU/g, respectively. Under the same conditions, for its heat-killed product, qPCR counts for L. gasseri and L. salivarius were 6.70±0.16 log cells/g and 7.67±0.20 log cells/g, while PMA-qPCR counts were 5.33±0.18 log cells/g and 5.05±0.23 log cells/g, respectively. For cell dilutions with a viable cell count of 8.5 log CFU/mL for L. gasseri and L. salivarius, after heat killing, the PMA-qPCR count for both Lactobacillus species was near 5.5 log cells/mL. When the PMA-qPCR counts of these cell dilutions were compared before and after heat killing, although some DNA might be lost during the heat killing, significant qPCR signals from dead cells, i.e., about 4-5 log cells/mL, could not be reduced by PMA treatment. Increasing PMA concentrations from 100 µM to 200 µM or light exposure time from 5 minutes to 15 minutes had no or, if any, only minor effect on the reduction of qPCR signals from their dead cells. Thus, to differentiate viable lactic acid bacterial cells from dead cells using the PMA-qPCR method, the efficiency of PMA to reduce the qPCR signals from dead cells should be notable.

The correlation of the presence and expression levels of cry genes with the insecticidal activities against Plutella xylostella for Bacillus thuringiensis strains.

The use of Bacillus thuringiensis (Bt) strains with high insecticidal activity is essential for the preparation of bioinsecticide. In this study, for 60 Bt strains isolated in Taiwan, their genotypes and the correlation of some cry genes as well as the expression levels of cry1 genes, with their insecticidal activities against Plutella xylostella, were investigated. Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) results revealed that the genotypes of these Bt strains are highly diversified. Also, a considerable number of the Bt strains isolated in Taiwan were found to have high insecticidal activities. Since strains that showed individual combined patterns of PFGE and RAPD exhibited distinct insecticidal activities against P. xylostella, thus, these genotypes may be useful for the identification of the new Bt strains and those which have been used in bioinsecticides. In addition, although the presence of cry2Aa1 may have a greater effect on the insecticidal activity of Bt strains in bioassay than other cry genes, only high expression level of cry1 genes plays a key role to determine the insecticidal activity of Bt strains. In conclusion, both RAPD and PFGE are effective in the differentiation of Bt strains. The presence of cry2Aa1 and, especially, the expression level of cry1 genes are useful for the prediction of the insecticidal activities of Bt strains against P. xylostella.

Effect of fermentation conditions on the enterotoxigenicity, cytotoxicity and pesticidal activity of Bacillus thuringiensis strains isolated in Taiwan.

A total of 75 Bacillus thuringiensis strains, among them 62 of Taiwan's microbiota, were screened for their enterotoxin genes, hemolysin BL activity and cytotoxicity. All the strains harbored enterotoxin genes and were cytotoxic to the cultivated Chinese hamster ovary (CHO) cells. The hemolysin BL and cytotoxicity titers of the B. thuringiensis culture in casitone yeast sucrose (CYS) broth were lower than those in brain heart infusion (BHI) broth, and when the B. thuringiensis strains were cultivated in CYS broth for 5 days, no cytotoxicity was detected. The spores and crystal toxins collected from 40 isolates showed high levels of insecticidal activity against Plutella xylostella. All strains exhibiting low cytotoxicity also had low pesticidal activity. Our study demonstrated that it is difficult to find B. thuringiensis strains that are both effective against insect targets and do not produce enterotoxins or cytotoxic effects in CHO cells. However, it is possible to avoid or reduce unwanted properties, but not the insecticidal activity, of some B. thuringiensis preparations by alteration of culture media and conditions.

Cloning and expression of an antifungal chitinase gene of a novel Bacillus subtilis isolate from Taiwan potato field.

A chitinase producing Bacillus subtilis CHU26 was isolated from Taiwan potato field. This strain exhibited a strong extra-cellular chitinase activity on the colloidal chitin containing agar plate, and showed a potential inhibit activity against phytopathogen, Rhizoctonia solani. The gene encoding chitinase (chi18) was cloned from the constructed B. subtilis CHU26 genomic DNA library. The chi18 consisted of an open reading frame of 1791 nucleotides and encodes 595 amino acids with a deduced molecular weight of 64kDa, next to a promoter region containing a 9 base pair direct repeat sequence (ATTGATGAA). The deduced amino acid sequence of the chitinase from Bacillus subtilis CHU26 exhibits 62% and 81% similarity to those from B. circulans WL-12 and B. licheniformis, respectively. Subcloned chi18 into vector pGEM3Z and pYEP352 to construct recombinant plasmid pGCHI18 and pYCHI18, respectively, chitinase activity could be observed on the colloidal chitin agar plate from recombinant plasmid containing Escherichia coli transformant. Cell-free culture broth of pYCHI18 containing E. coli transformant decreased R. solani pathogenic activity more than 90% in the antagonistic test on the radish seedlings (Raphanus sativus Linn.).

A pulsed field gel electrophoresis (PFGE) study that suggests a major world-wide clone of Salmonella enterica serovar Enteritidis.

Since human infections by Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) have been increasing world-wide over the past years and epidemiological studies have implicated the consumption of meat, poultry, eggs and egg products, elucidation of the predominant subtypes for this Salmonella spp. is important. In this study, 107 poultry and food isolates of Salmonella Enteritidis obtained from Germany were analyzed by pulsed field gel electrophoresis (PFGE), and the subtypes were compared with those of the 124 human isolates obtained in Taiwan. Results showed that for these 107 poultry and food isolates, when XbaI, SpeI and NotI were used for chromosomal DNA digestion followed by PFGE analysis, a total of 19, 20 and 19 PFGE patterns, respectively, were identified. Of them, 51 (47.7%), 52 (48.6%) and 42 (39.3%) strains belong to a single pattern of X3, S3 and N3, respectively, and 34 strains belong to a pattern combination of X3S3N3, which was the major subtype. When PFGE patterns of these 107 German isolates were compared with those of the 124 human isolates obtained in Taiwan, pattern combination of X3S3N3 was found as the most common pattern shared by isolates from both areas. PT4 is a major phage type for German and Taiwan isolates. Although most of the X3S3N3 strains are of this phage type, some strains of other PFGE patterns are also of this phage type. Since strains used in this study were unrelated, i.e., they were isolated from different origins in areas geographically far apart from each other, the PFGE study suggests a major world-wide clone of S. enterica serovar Enteritidis.

The presence of major world-wide clones for phage type 4 and 8 Salmonella enterica serovar Enteritidis and the evaluation of their virulence levels by invasiveness assays in vitro and in vivo.

Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.

Characterisation of antimicrobial resistance patterns and class 1 integrons among Escherichia coli and Salmonella enterica serovar Choleraesuis strains isolated from humans and swine in Taiwan.

Escherichia coli isolates from humans (n=110) and swine (n=61) and Salmonella enterica serovar Choleraesuis isolates (n=95) from swine in southern Taiwan were characterised for antimicrobial resistance patterns and class 1 integrons. All E. coli isolates and S. Choleraesuis isolates were multidrug resistant and demonstrated high resistance to beta-lactams, aminoglycosides, tetracycline, sulfonamides, spectinomycin, chloramphenicol and nalidixic acid. By polymerase chain reaction and DNA sequencing, 104 (61%) E. coli isolates and 31 (33%) S. Choleraesuis isolates were found to carry class 1 integrons. The gene cassette array dfrA12-orfF-aadA2 was the most prevalent (24%) among the human and swine E. coli isolates, whilst the gene cassette array dfrA12-orfF-aadA2-sul1 was the most prevalent (24%) among S. Choleraesuis strains. For E. coil isolates, all class 1 integrons were located on conjugated plasmids. Meanwhile, human and swine E. coli isolates carrying identical gene cassettes were genetically unrelated. Our results revealed that multidrug resistance and class 1 integrons were widely present in E. coli and S. Choleraesuis isolates obtained in Taiwan and that class 1 integrons might play an important role in contributing to the horizontal transfer of antimicrobial resistance.

Development of PCR primers for the detection of Salmonella enterica serovar Choleraesuis based on the fliC gene.

Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Because the conventional method for detection of this Salmonella serovar may take 3 to 5 days, a PCR method for detection was evaluated. By comparing the sequence of the phase 1 flagellin (fliC) gene of Salmonella Choleraesuis with that of other Salmonella serovars and of other bacteria species available in GenBank, two PCR primers (flinC-F and flinC-R) were designed. Using these primers, all 97 Salmonella Choleraesuis strains assayed generated the expected PCR product, with a molecular mass of 963 bp. Except for S. enterica Paratyphi C, Salmonella isolates other than Salmonella Choleraesuis and non-Salmonella isolates, including strains of Enterobacteriaceae, all generated negative PCR results. Salmonella Paratyphi C could be differentiated from Salmonella Choleraesuis through the use of primers designed from the viaB gene. When Salmonella Choleraesuis isolates from swine stool, pork, liver, feed, and human whole blood samples were assayed with a preenrichment step, as low as 1 CFU/g or ml of the original sample could be detected.

Enterotoxigenicity and cytotoxicity of Bacillus thuringiensis strains and development of a process for Cry1Ac production.

Bacillus thuringiensis is indistinguishable from Bacillus cereus except for the production of insecticidal crystal proteins (ICPs). B. thuringiensis strains may show enterotoxin profiles and toxin levels similar to those of B. cereus strains isolated from food-poisoning cases. It is important for the food industry and farmers to consider that with the application of B. thuringiensis strains to crops, their spores may be introduced into the human food chain. In this study, 59 B. thuringiensis strains were assayed for their hemolysin BL (HBL) using a BCET-RPLA kit and their cytotoxicity to Chinese hamster ovary (CHO) cells. The enterotoxin titer was as high as that of B. cereus diarrheal-type strain ATCC 49064. In an attempt to obtain a food safety strain for bioinsecticide use, in this study, a 3.5-kb cry1Ac DNA fragment was amplified with PCR from the total DNA of B. thuringiensis subsp. kurstaki CCRC 11502 and cloned into the Bacillus expression vector pHY300PLK. The alpha-amylase promoter, amyE, was then introduced into the promoter region and, afterward, the recombinant plasmid pHYe1Ac35 was introduced into a non-enterotoxigenic and non-cytotoxic B. thuringiensis subsp. kurstaki Tt14 strain. The transformant, without any detectable enterotoxigenicity or cytotoxicity, produced Cry1Ac toxin properly, and its insecticidal activity against Trichoplusia ni larvae was found to be satisfactory.