Assessment of antibiotic resistance in Klebsiella pneumoniae exposed to sequential in vitro antibiotic treatments.

BACKGROUND: Bacteria treated with different classes of antibiotics exhibit changes in susceptibility to successive antibiotic treatments. This study was designed to evaluate the influence of sequential antibiotic treatments on the development of antibiotic resistance in Klebsiella pneumoniae associated with ß-lactamase and efflux pump activities. METHODS: The antibiotic susceptibility, ß-lactamase activity, and efflux activity were determined in K. pneumoniae grown at 37 °C by adding initial (0 h) and second antibiotics (8 or 12 h). Treatments include control (CON; no first and second antibiotic addition), no initial antibiotic addition followed by 1 MIC ciprofloxacin addition (CON-CIP), no initial antibiotic addition followed by 1 MIC meropenem addition (CON-MER), initial 1/4 MIC ciprofloxacin addition followed by no antibiotic addition (1/4CIP-CON), initial 1/4 MIC ciprofloxacin addition followed by 1 MIC ciprofloxacin addition (1/4CIP-CIP), and initial 1/4 MIC ciprofloxacin addition followed by 1 MIC meropenem addition (1/4CIP-MER). RESULTS: Compared to the CON, the initial addition of 1/4 MIC ciprofloxacin inhibited the growth of K. pneumoniae throughout the incubation period. The ciprofloxacin treatments (CON-CIP and 1/4CIP-CIP) showed significant reduction in the number of K. pneumoniae cells compared to meropenem (CON-MER and 1/4CIP-MER). The 1/4CIP-CIP achieved a further 1 log reduction of K. pneumoniae, when compared to the 1/4CIP-CON and 1/CIP-MER. The increase in sensitivity of K. pneumoniae to cefotaxime, kanamycin, levofloxacin, nalidixic acid was observed for CON-CIP. Noticeable cross-resistance pattern was observed at the 1/4CIP-CIP, showing the increased resistance of K. pneumoniae to chloramphenicol, ciprofloxacin, kanamycin, levofloxacin, nalidixic acid norfloxacin, sulphamethoxazole/trimethoprim, and tetracycline. The levels of ß-lactamase activities were estimated to be 8.4 µmol/min/ml for CON, 7.7 µmol/min/ml for 1/4CIP-CON and as low as 2.9 µmol/min/ml for CON-CIP. Compared to the absence of phenylalanine-arginine-ß-naphthylamide (PAßN), the fluorescence intensity of EtBr was increased in K. pneumoniae cells treated at the CON, CON-CIP, and CON-MER in the presence of PAßN. However, the efflux pump activity remained in K. pneumoniae cells treated at the 1/CIP, 1/CIP-CIP, and 1/CIP-MER in the presence of PAßN. CONCLUSION: The results suggest that the pre-exposed antibiotic history, treatment order, and concentrations influenced the development of multiple antibiotic resistant associated with ß-lactamase and efflux pump activities. This study highlights the importance of antibiotic treatment conditions, which would be taken into consideration when new antibiotic strategy is designed to prevent antibiotic resistance.

Characterization of Bacillus subtilis bacteriophage BasuTN3 isolated from Thua Nao, a thai fermented soybean food product.

The infection of bacteriophage is of great concern in food industry as this can result in complete fermentation failure. In this study, a virulent bacteriophage, named BasuTN3, was isolated from Thua Nao, a Thai fermented soybean. The stability of BasuTN3 was evaluated under various ranges of temperature, pH, chloroform, UV, and disinfectants. The results showed that the isolated BasuTN3 appeared to be specific to its bacterial host, which was identified as Bacillus subtilis strain TN3 based on the 16 S rRNA gene sequence analysis. Under TEM, the BasuTN3 belonged to the family Myoviridae. The isolated BasuTN3 could withstand wide temperature ranges (4-45 °C) and pH conditions (5-11). The BasuTN3 was susceptible to chloroform, UV, and commonly used disinfectants. The results obtained expand the knowledge of the Bacillus bacteriophage diversity in the fermented soybean products and provide useful information for the bacteriophage and its bacterial starter cultures.

Phylogeny of Chaetothyriaceae in northern Thailand including three new species.

In a recent study unusual taxa of epiphyllous ascomycota belonging to Chaetothyriaceae (Eurotiomycetes) were collected in northern Thailand. This family is poorly understood due to morphological confusion and lack of phylogenetic studies. This paper deals with three new species, Ceramothyrium thailandicum, Chaetothyrium brischofiacola and Phaeosaccardinula ficus, which are fully described and illustrated. A DNA sequence analyses of LSU and ITS rDNA genes shows that the new species cluster in the Chaetothyriaceae. This paper adds six sequences for Chaetothyriaceae to GenBank, providing much needed data for the family.

Two new Kirschsteiniothelia species with Dendryphiopsis anamorphs cluster in Kirschsteiniotheliaceae fam. nov.

Two new Kirschsteiniothelia species are proposed in this study; both were collected on decaying wood from Chiang Mai and Chiang Rai provinces in northern Thailand. The taxa were isolated and the morphological characters are described and illustrated. ITS, LSU and SSU combined sequence analysis showed taxa of Kirschsteiniothelia separating into three lineages: (i) K. elaterascus grouped within Morosphaeriaceae (Pleosporales); (ii) K. maritima clustered with Mytilinidion spp. as a sister group in the Mytilinidiaceae clade; and (iii) the two new Kirschsteiniothelia species, which produce Dendryphiopsis anamorphs in culture, clustered with K. aethiops (the generic type) and the anamorph D. atra. The new family Kirschsteiniotheliaceae is introduced to accommodate taxa grouping with K. aethiops. K. elaterascus is transferred to Morosphaeria (Morosphaeriaceae) and a new genus Halokirschteiniothelia is introduced to accommodate K. maritima (Mytilinidiaceae).

Capnodiaceae.

In this paper we revisit the Capnodiaceae with notes on selected genera. Type specimens of the ascomycetous genera Aithaloderma, Anopeltis, Callebaea, Capnodaria, Echinothecium, Phragmocapnias and Scorias were re-examined, described and illustrated. Leptoxyphium is anamorphic Capnodiaceae and Polychaeton is a legitimate and earlier name for Capnodium, but in order to maintain nomenclatural stability we propose that the teleomorphic name should be conisdered for the approved lists of names currently in preparation for fungi. Notes are provided on the ascomycetous genus Scoriadopsis. However, we were unable to locate the type of this genus during the time frame of this study. The ascomycetous genera Aithaloderma, Ceramoclasteropsis, Hyaloscolecostroma and Trichomerium are excluded from Capnodiaceae on the basis of having ascostromata and trans-septate hyaline ascospores and should be accommodated in Chaetothyriaceae. Callebaea is excluded as the ascomata are thyriothecia and the genus is placed in Micropeltidaceae. Echinothecium is excluded as synonym of Sphaerellothecium and is transferred to Mycosphaerellaceae. The type specimen of Capnophaeum is lost and this should be considered as a doubtful genus. The coelomycetous Microxiphium is polyphyletic, while the status of Fumiglobus, Polychaetella and Tripospermum is unclear. Fourteen new collections of sooty moulds made in Thailand were isolated and sequenced. The nuclear large and small rDNA was partially sequenced and compared in a phylogeny used to build a more complete understanding of the relationships of genera in Capnodiaceae. Four new species are described and illustrated, while Phragmocapnias and Scorias are epitypified with fresh collections.

Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product.

Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

Recalibration of mapping quality scores in Illumina short-read alignments improves SNP detection results in low-coverage sequencing data.

BACKGROUND: Low-coverage sequencing is a cost-effective way to obtain reads spanning an entire genome. However, read depth at each locus is low, making sequencing error difficult to separate from actual variation. Prior to variant calling, sequencer reads are aligned to a reference genome, with alignments stored in Sequence Alignment/Map (SAM) files. Each alignment has a mapping quality (MAPQ) score indicating the probability a read is incorrectly aligned. This study investigated the recalibration of probability estimates used to compute MAPQ scores for improving variant calling performance in single-sample, low-coverage settings. MATERIALS AND METHODS: Simulated tomato, hot pepper and rice genomes were implanted with known variants. From these, simulated paired-end reads were generated at low coverage and aligned to the original reference genomes. Features extracted from the SAM formatted alignment files for tomato were used to train machine learning models to detect incorrectly aligned reads and output estimates of the probability of misalignment for each read in all three data sets. MAPQ scores were then re-computed from these estimates. Next, the SAM files were updated with new MAPQ scores. Finally, Variant calling was performed on the original and recalibrated alignments and the results compared. RESULTS: Incorrectly aligned reads comprised only 0.16% of the reads in the training set. This severe class imbalance required special consideration for model training. The F1 score for detecting misaligned reads ranged from 0.76 to 0.82. The best performing model was used to compute new MAPQ scores. Single Nucleotide Polymorphism (SNP) detection was improved after mapping score recalibration. In rice, recall for called SNPs increased by 5.2%, while for tomato and pepper it increased by 3.1% and 1.5%, respectively. For all three data sets the precision of SNP calls ranged from 0.91 to 0.95, and was largely unchanged both before and after mapping score recalibration. CONCLUSION: Recalibrating MAPQ scores delivers modest improvements in single-sample variant calling results. Some variant callers operate on multiple samples simultaneously. They exploit every sample's reads to compensate for the low read-depth of individual samples. This improves polymorphism detection and genotype inference. It may be that small improvements in single-sample settings translate to larger gains in a multi-sample experiment. A study to investigate this is ongoing.

Incidence of viruses infecting pepper in Thailand.

This study was conducted to determine the incidence, diversity and distribution of viruses infecting pepper (Capsicum spp.) in the central, northern and northeastern parts of Thailand. During a survey in 2016 - 2019, a total of 2,149 leaf samples from symptomatic and asymptomatic peppers were collected randomly from farmer's fields, and preliminary tested by an enzyme-linked immunosorbent assay (ELISA) using 7 antibodies specific for cucumber mosaic virus (CMV), chilli veinal mottle virus (ChiVMV), tomato necrotic ringspot virus (TNRV), tobacco mosaic virus (TMV), potato virus Y (PVY), tomato spotted wilt virus (TSWV), and begomoviruses. Our data revealed that the incidence of the viruses infecting pepper in Thailand was high, accounting for nearly 70% (1,482 infected samples). The highest viral incidence was found in the central part (96%), followed by the north (74.4%) and the northeastern (52.8%), respectively. Begomoviruses, CMV, ChiVMV, and TNRV were detected in the samples at varying rates, whereas PVY, TMV, and TSWV were not detected. Of these, the most frequently found virus was Begomoviruses accounting for nearly 33%, with the highest rate (ca. 82%) in the central Provinces of Thailand. In addition, of the 1,482 infected samples, mixed infections among the four viruses were also found in 616 samples (ca. 42%), and CMV + ChiVMV (approximately 11%) was the most common mixed infection. This is the first report describing an occurrence of viruses in pepper of Thailand, and the results obtained have revealed that viruses infecting pepper are widespread, which may pose a threat to pepper production in Thailand.

Bacteriophage control of Salmonella Typhimurium in milk.

This study was designed to evaluate the effect of bacteriophage P22 on the inhibition of growth of Salmonella Typhimurium. The P22 belongs to Podoviridae family consisting of a hexagonal head and short tail. The inhibitory effect of phage in milk was noticeable at the early storage period, showing more than 3 log reduction at 4 h and day 3. The pH values of milk treated with P22 were significantly decreased from 6.7 to 6.3 after 24 h incubation at 37 °C, while no significant changes in pH values were observed for the control and bacteriophage treatment throughout the storage at 4 °C for 12 days. The slight color changes were observed in the control and bacteriophage treatment throughout the storage at 4 °C for 12 days and 37 °C for 24 h. These results provide useful information for enhancing microbiological safety and quality of milk and designing effective bacteriophage-based control in food system.

Significance of bacteriophages in fermented soybeans: A review.

Bacteriophages are ubiquitous and have been reported to have been found in many food products. Their presence is important as they have the ability to interact with their bacterial host in food matrices. Fermented soybean products, one of the most widely consumed ethnic foods among Asian people, are prepared naturally and include Japanese Natto, Indian Kinema, Korean Chongkukjang and Thai Thua Nao. This review highlights bacteriophages which have been isolated from fermented soybean products and also includes an overview of their diversity, occurrence as well as their significance.

Racemization of l-lactic acid in pH-swing open fermentation of kitchen refuse by selective proliferation of Lactobacillus plantarum.

We have shown that stable lactic acid fermentation of model kitchen refuse occurs with intermittent pH adjustment under nonsterilized conditions. Nonetheless, the optical activity of the accumulated lactic acid was low, which is disadvantageous for the production of high-quality poly-l-lactic acid. Here, we attempt to increase optical purity by introducing l-lactic acid-producing strains under nonsterilized conditions and demonstrate that the inoculation of Lactobacillus rhamnosus or Lactococcus lactis, both of which are l-lactic acid producers, is partially effective in the early fermentation stage, but does not improve the final optical purity of the accumulated lactic acid. We confirmed by fluorescence in situ hybridization using group-specific and species-specific 16S rDNA probes that this is due to the selective proliferation of naturally existing L. plantarum. L. plantarum KY-1, which is isolated from model kitchen refuse, showing lactic acid racemase activity, as well as d-lactate dehydrogenase activity, in its membrane fraction. We conclude that racemase activity associated with L. plantarum is the main cause of decreased optical purity in the accumulated lactic acid.

The Diaporthe sojae species complex: Phylogenetic re-assessment of pathogens associated with soybean, cucurbits and other field crops.

Phytopathogenic species of Diaporthe are associated with a number of soybean diseases including seed decay, pod and stem blight and stem canker and lead to considerable crop production losses worldwide. Accurate morphological identification of the species that cause these diseases has been difficult. In this study, we determined the phylogenetic relationships and species boundaries of Diaporthe longicolla, Diaporthe phaseolorum, Diaporthe sojae and closely related taxa. Species boundaries for this complex were determined based on combined phylogenetic analysis of five gene regions: partial sequences of calmodulin (CAL), beta-tubulin (TUB), histone-3 (HIS), translation elongation factor 1-α (EF1-α), and the nuclear ribosomal internal transcribed spacers (ITS). Phylogenetic analyses revealed that this large complex of taxa is comprised of soybean pathogens as well as species associated with herbaceous field crops and weeds. Diaporthe arctii, Diaporthe batatas, D. phaseolorum and D. sojae are epitypified. The seed decay pathogen D. longicolla was determined to be distinct from D. sojae. D. phaseolorum, originally associated with stem and leaf blight of Lima bean, was not found to be associated with soybean. A new species, Diaporthe ueckerae on Cucumis melo, is introduced with description and illustrations.

Morphological and molecular characterisation of Diaporthe species associated with grapevine trunk disease in China.

Trunk diseases in grapevine (Vitis spp.) are major problems in the wine and table-grape industries reducing the productivity, quality and longevity of vineyards. Species of Diaporthe are important fungal pathogens of grapevine trunk disease worldwide. A survey of 14 grape vineyards located in different provinces of China was yielded Diaporthe isolates associated with symptomatic grapevine wood. These isolates were identified based on morphology and a combined data matrix of rDNA ITS, partial sequences of translation elongation factor 1-α (EF 1-α), ß-tubulin (TUB) and calmodulin (CAL) gene regions. Four species of Diaporthe were identified, which included Diaporthe eres, Diaporthe hongkongensis, Diaporthe phaseolorum and Diaporthe sojae. All isolates of Diaporthe caused disease on detached grape shoots in pathogenicity experiments but differed in virulence. The incidence in local vineyards and the pathogenicity results indicate that D. eres is an important pathogen of grapevine in Chinese vineyards, where it may significantly limit grape production. This is the first detailed report of Diaporthe species associated with grapevine trunk diseases in China with morphology, pathogenicity and molecular data.

Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes.

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.

Optimal conditions of mycelia growth of Laetiporus sulphureus sensu lato.

Laetiporus sulphureus is an edible wood-rotting basidiomycete, growing on decaying logs, stumps, and trunks of many deciduous and coniferous tree species. This fungus produces relatively large striking yellowish or orange-coloured bracket-like fruitbodies. L. sulphureus is widely consumed as a nutritional food because of its fragrance and texture. In this study, two L. sulphureus strains, MFLUCC 12-0546 and MFLUCC 12-0547, isolated from Chiang Rai, Thailand, were investigated for optimal conditions of mycelia growth. Potato dextrose agar and malt extract agar were observed as the favourable medium for mycelia growth. The optimum pH and temperature for the mushroom mycelia were 6-8 and 25-30°C, respectively.

Changes in biochemical and nutritional qualities of aerobic and vacuum-packaged Thua Nao during shelf-life storage.

Thai traditionally fermented soybean (Thua Nao) has been suggested as a good source of available amino acids and aglycone isoflavones. The objective of this research was to investigate the changes of biochemical and nutritional qualities in aerobic- and vacuum-packed thua Nao during the storage at 4 and 40 degrees C for 60 days. Three Thua Nao samples including Bacillus subtilis TN51-fermented Thua Nao (TNB51), spontaneously fermented Thua Nao (TNMX) and commercial product (MH) were used in this study. It was found that the storage of packed Thua Nao at 4 degrees C could prolong the product shelf-life up to 40 days. The moisture contents, pH values and colour L a* b* of these products were not different in both aerobic and vacuum-packaged products and remained stable throughout the experiment. The Thiobarbituric Acid (TBA) values of all storage Thua Nao were increased during storage; this is in particular for the aerobic packages of the TNMX and TNB51 products, indicating high oxidation of lipids. There was a slight decrease in DPPH radical scavenging effect (18%) and phenolic compounds (6%) of the vacuum-packaged product when stored at 4 degrees C. In contrast, the great losses in total phenolic content (44%), inhibitory activity of DPPH radicals (83%) and total antioxidant (41%) were observed when the products were stored aerobically at 40 degrees C. A reduction in total free amino acids was also found with the highest decrease of Arg in both aerobic- (69%) and vacuum-packages (68%).

Agaricus subrufescens: A review.

Medicinal mushrooms have currently become a hot issue due to their various therapeutic properties. Of these, Agaricus subrufescens, also known as the "almond mushroom", has long been valued by many societies (i.e., Brazil, China, France, and USA). Since its discovery in 1893, this mushroom has been cultivated throughout the world, especially in Brazil where several strains of A. subrufescens have been developed and used as health food and alternative medicine. This article presents up-to-date information on this mushroom including its taxonomy and health promoting benefits. Medicinal properties of A. subrufescens are emphasized in several studies which are reviewed here. In addition, safety issues concerning the use of this fungus will be discussed.

Cloning and sequence analysis of the cellobiohydrolase I genes from some basidiomycetes.

Genes encoding the cellobiohydrolase enzyme (CBHI), designated as cbhI, were isolated from the basidiomycetes Auricularia fuscosuccinea, Pleurotus giganteus, P. eryngii, P. ostreatus, and P. sajor-caju. Initially, the fungal genomic DNA was extracted using a modified cetyltrimethyl ammonium bromide (CTAB) protocol and used as a DNA template. The cbhI genes were then amplified and cloned using the pGEM-T Easy Vector Systems. The sizes of these PCR amplicons were between 700~800 bp. The DNA sequences obtained were similar showing high identity to the cbhI gene family. These cbhI genes were partial consisting of three coding regions and two introns. The deduced amino acid sequences exhibited significant similarity to those of fungal CBHI enzymes belonging to glycosyl hydrolase family 7.

Chemical compositions, antifungal and antioxidant activities of essential oil and various extracts of Melodorum fruticosum L. flowers.

This research presents the chemical composition antifungal and antioxidant activities of essential oils and various extracts from Melodorum fruticosum flowers. The essential oil composition of M. fruticosum flowers were investigated by GC-MS with 88 identified volatile constituents. Phenyl butanone, linalool, benzyl alcohol, alpha-cadinol, globulol and viridiflorol were found to be the major components, respectively. The dichloromethane extract played a major role as a remarkable fungicide according to their inhibition action against all tested pathogens followed by hexane extract, essential oil and methanol extract, respectively, along with their respective MIC values ranging from 125 to 1000 microg/ml. The dichloromethane extracts were also evaluated to be superior to all extracts tested with an IC(50) value of 87.6 microg/ml whereas other extracts showed their IC(50) values ranging from 100.13 to 194.50 microg/ml.