Ching-Lin Shyu

Characterization of a novel thermophilic, cellulose-degrading bacterium Paenibacillus sp. strain B39.

Aims:  The aims of this study were to identify and characterize the novel thermophilic, cellulose‐degrading bacterium Paenibacillus sp. strain B39.

Species Diversity and Substrate Utilization Patterns of Thermophilic Bacterial Communities in Hot Aerobic Poultry and Cattle Manure Composts

This study investigated the species diversity and substrate utilization patterns of culturable thermophilic bacterial communities in hot aerobic poultry and cattle manure composts by coupling 16S rDNA analysis with Biolog data. Based on the phylogenetic relationships of 16S rDNA sequences, 34 thermophilic (grown at 60°C) bacteria isolated during aerobic composting of poultry manure and cattle manure were classified as Bacillus licheniformis, B. atrophaeus, Geobacillus stearothermophilus, G. thermodenitrificans, Brevibacillus thermoruber, Ureibacillus terrenus, U. thermosphaericus, and Paenibacillus cookii. In this study, B. atrophaeus, Br. thermoruber, and P. cookii were recorded for the first time in hot compost. Physiological profiles of these bacteria, obtained from the Biolog Gram-positive (GP) microplate system, were subjected to principal component analysis (PCA). All isolates were categorized into eight different PCA groups based on their substrate utilization patterns. The bacterial community from poultry manure compost comprised more divergent species (21 isolates, seven species) and utilized more diverse substrates (eight PCA groups) than that from cattle manure compost (13 isolates, five species, and four PCA groups). Many thermophilic bacteria isolated in this study could use a variety of carboxylic acids. Isolate B110 (from poultry manure compost), which is 97.6% similar to U. terrenus in its 16S rDNA sequence, possesses particularly high activity in utilizing a broad spectrum of substrates. This isolate may have potential applications in industry.

Differential heat stability of amphenicols characterized by structural degradation, mass spectrometry and antimicrobial activity

Heat stability of amphenicols and the relationship between structural degradation and antimicrobial activity after heating has not been well investigated. Florfenicol (FF), thiamphenicol (TAP), and chloramphenicol (CAP) were heated at 100 degrees C in water, salt water, soybean sauce and chicken meat for up to 2h. Degradation and antimicrobial activity of the compounds was evaluated using capillary electrophoresis (CE) with UV-DAD spectrometry, minimum inhibitory concentration (MIC) assay, and gas chromatography with electron impact ionization mass spectrometry (GC-EI-MS). Heat stability of amphenicols in matrices was ranked as water> or =salt water>soybean sauce>meat, suggesting that heat degradation of amphenicols was accelerated in soybean sauce and was not protected in meat. Heat stability by drug and matrices was ranked as FF>TAP=CAP in water, FF=TAP>CAP in salt water, TAP> or =FF=CAP in soybean sauce, and TAP> or =FF=CAP in meat, indicating differential heat stability of amphenicols among the 3 drugs and in different matrices. In accordance with the less than 20% degradation, the MIC against Escherichia coli and Staphylococcus aureus did not change after 2h heating in water. A 5-min heating of amphenicols in water by microwave oven generated comparable percentage degradation to boiling in water bath for 30 min to 1h. Both CE and GC-MS analysis showed that heating of FF produced TAP but not FF amine as one of its breakdown products. In conclusion, despite close similarity in structure; amphenicols exhibited differential behavior toward heating degradation in solutions and protein matrices. Although higher degradations of amphenicols were observed in soybean sauce and meat, heating treatment may generate product with antimicrobial activity (FF to TAP), therefore, heating of amphenicol residues in food cannot always be assumed safe.

Analysis of the biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum

The aim of this study was to investigate biosynthesis genes and chemical components of the capsule of Avibacterium paragallinarum. The sequence of a 10-kb region containing the capsule biosynthetic locus of Av. paragallinarum was determined. Two reference strains, i.e., 221 (serovar A) and H18 (serovar C), together with four Taiwanese field strains (all serovar C) were sequenced. The results showed that there are two genotypes (I and II) of the capsule biosynthetic locus in Av. paragallinarum, and the capsule genotype is independent of the serovar. The capsule biosynthetic loci of genotypes I and II consisted of six and five genes, respectively. The genotype I genes encoded proteins that are most similar to proteins from Pasteurella multocida capsule types A and F while the genotype II genes encoded proteins most similar to proteins from P. multocida capsule type D and Escherichia coli K5. The results suggested that genotype I strains contain hyaluronan or chondroitin in the capsule wall while genotype II contain heparosan. Enzymatic digestion of the capsule materials extracted from Av. paragallinarum showed that genotype I strains contained chondroitin while genotype II strains contained heparosan in the capsule. This is the first report on the existence of different genotypes of capsule biosynthesis genes in Av. paragallinarum and the presence of chondroitin and heparosan as chemical components of the capsule of Av. paragallinarum.

Antibacterial and Synergistic Activity of Pentacyclic Triterpenoids Isolated from Alstonia scholaris.

(1) Background: Alstonia scholaris (Apocynaceae) is an important medicinal plant that has been historically used in “Dai” ethnopharmacy to treat infectious diseases in China. Although various pharmacological activities have been reported, the antimicrobial constitutes of A. scholaris have not yet been identified. The objective of this study is to evaluate the antibacterial constitutes from the leaf extract of A. scholaris and to assess the synergistic effects of isolated compounds with antibiotics against bacterial pathogens.; (2) Methods: The chemical constitutes isolated from the leaf extract of A. scholaris were structurally identified by NMR. The antibacterial and synergistic effect of compounds was assessed by calculating the minimal inhibitory concentration (MIC), checkerboard dilution test, and time-kill assay.; (3) Results: Six pentacyclic triterpenoids were structurally identified as (1) lupeol, (2) betulin, (3) 3-hydroxy-11-ursen-28,13-olide, (4) betulinic acid, (5) oleanolic acid and (6) ursolic acid. Both oleanolic and ursolic acid showed antibacterial activity but were limited to Gram-positive bacteria. Ursolic acid showed a synergistic effect with ampicillin and tetracycline against both Bacillus cereus and S. aureus.; (4) Conclusion: These findings reflect that pentacyclic triterpenoids are the antibacterial chemicals in A. scholaris. The ability of ursolic acid to enhance the activity of antibiotics can constitute a valuable group of therapeutic agents in the future.

Occurrence and molecular characterization of reptilian Campylobacter fetus strains isolated in Taiwan.

The number of people who raise reptiles as pets has increased, but information about zoonotic Campylobacter carried by reptiles is limited. A survey of zoonotic Campylobacter species isolated from reptiles was undertaken to understand the possibility of this zoonotic bacterial pathogen causing human infection. A total of 179 fresh reptile fecal samples were collected from human-raised, pet shop and wild reptiles to survey the Campylobacter species. Basic biochemical reactions and 16S rRNA gene sequencing were used to identify the Campylobacter species. In the 179 fecal samples, 6.7% (12/179) were Campylobacter positive; all positive samples were identified as Campylobacter fetus. For the different reptile species, the prevalence of C. fetus in turtles was 9.7% (10/103), 1.7% (1/56) in lizards and 5.0% (1/20) in snakes. Based on published C. fetus subspecies-specific sequences, 9 of the C. fetus bacterial isolates were identified as C. fetus subsp. fetus by multiplex PCR. In addition, multilocus sequence typing (MLST) was used to analyze the Campylobacter epidemiology and population genetics. Most of the C. fetus strains isolated from the reptiles were genetically distinct from classical mammalian C. fetus. Only the new type of ST-43, isolated from Chelonoidis carbonaria (turtle), was closely related to mammalian strains. Strain Campy-pet-3 possesses a urease activity in this study is the first to be described in C. fetus and this strain is the only one of lizard origin. This study provides the first information of Campylobacter species distribution in reptilian feces and supports the possibility of zoonotic Campylobacter infectious diseases caused by reptiles.

Detection of matrix metalloproteinase (MMP)-2 and MMP-9 in canine seminal plasma

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality.

Structural and biological characterization of mastoparans in the venom of Vespa species in Taiwan

Mastoparans, a family of small peptides, are isolated from the wasp venom. In this study, six mastoparans were identified in the venom of six Vespa species in Taiwan. The precursors of these mastoparans are composed of N-terminal signal sequence, prosequence, mature mastoparan, and appendix glycine at C-terminus. These mature mastoparans all have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bond. Therefore, these peptides could be predicted to adopt an amphipathic α-helical secondary structure. In fact, the CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 8 mM SDS or 40% 2,2,2-trifluoroethanol (TFE). All mastoparans exhibit mast cell degranulation activity, antimicrobial activity against both Gram-positive and -negative bacteria tested, various degree of hemolytic activity on chicken, human, and sheep erythrocytes as well as membrane permeabilization on Escherichia coli BL21. Our results also show that the hemolytic activity of mastoparans is correlated to mean hydrophobicity and mean hydrophobic moment.

In vitro activity of mastoparan-AF alone and in combination with clinically used antibiotics against multiple-antibiotic-resistant Escherichia coli isolates from animals

The in vitro activity of mastoparan-AF, an amphipathic antimicrobial peptide isolated from the hornet venom of Vespa affinis, alone and in combination with various clinically used antibiotics, was investigated against 21 Escherichia coli isolates/strains. Most E. coli isolates tested were detected containing multiple-antimicrobial resistance genes. Antimicrobial activity of mastoparan-AF was measured by MIC, MBC, time-kill kinetic assay and chequerboard titration method. Mastoparan-AF exhibited potent antimicrobial activity against most multiple-antibiotic-resistant E. coli isolates at the concentrations ranging from 4 to 16 μg/ml. Combination studies showed that mastoparan-AF acts synergistically with certain antibiotics, i.e., cephalothin or gentamicin, against some multiple-antibiotic-resistant E. coli isolates. In conclusion, mastoparan-AF alone or in combination with other antibiotics could be promising as alternatives for combating multiple-antibiotic-resistant E. coli in future clinical applications.

Liquid chromatography incorporating ultraviolet and electrochemical analyses for dual detection of zeranol and zearalenone metabolites in mouldy grains.

BACKGROUND Zeranol (Z) is a semi-synthetic mycotoxin that is used in some countries as a growth-promoting agent in livestock. In view of the known oestrogenic actions by Z and certain Z analogues, significant concerns exist with regard to the presence of Z residues in human foods and the potential for untoward effects, including carcinogenicity within the reproductive system. In order to confirm that foods are free from harmful Z residues, regulators need a quick and reliable analytical method that can be used for routine confirmation of Z-positive samples identified by enzyme-linked immunosorbent assay (ELISA) screening. In this study the authors have developed and validated a simple and rapid high-performance liquid chromatography method incorporating ultraviolet (UV) absorbance (wavelength 274 nm) and electrochemical (EC) dual-mode detection for simultaneous determination of Z-related mycotoxins produced from mouldy grain matrices, including rice, soybean and corn flakes. RESULTS Recoveries for all analytes were around 80% and the limits of detection ranged from 10 to 25 ng mL(-1) for UV and from 50 to 90 ng mL(-1) for EC detection with good accuracy and reproducibility. Differential profiles and occurrence rates of Z, β-zearalenol, β-zearalanol and α-zearalenol in naturally moulded grain matrices were observed, indicating different metabolite patterns and possibly grain-specific effects of mycotoxin exposure for humans and animals. The strength of this dual detection method lies in its selectivity characterised by a carbon screen-printed electrode such that aflatoxin interference is precluded. CONCLUSION The combined dual detection technique affords quick and reliable semi-confirmative and quantitative information on multiple types of Z analogues in mouldy grains without the necessity of using expensive mass spectrometry. The method is considered a superior supplement to ELISA, which only screens total Z immunoreactivity.