Whole Genome Sequencing and Tn5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Triclosan: antimicrobial mechanisms, antibiotics interactions, clinical applications, and human health.

The large-scale applications of Triclosan in industrial and household products have created many health and environmental concerns. Despite the fears of its drug-resistance and other issues, Triclosan is still an effective drug against many infectious organisms. Knowing the cross-interactions of Triclosan with different antibiotics, bacteria, and humans can provide much-needed information for the risk assessment of this drug. We review the current understanding of the antimicrobial mechanisms of Triclosan, how microbes become resistant to Triclosan, and the synergistic and antagonistic effects of Triclosan with different antibiotics. Current literature on the clinical applications of Triclosan and its effect on fetus/child development are also summarized.

Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus in long-term care facilities in eastern Taiwan.

OBJECTIVE: The prevention of infections is crucial in long-term care programs. Investigations of the occurrence and sources of pathogens in long-term care facilities (LTCFs) are still lacking, especially in eastern Taiwan. In this study, we conducted a surveillance of two common pathogens, Acinetobacter baumannii (AB) and methicillin-resistant Staphylococcus aureus (MRSA), in LTCFs in Hualien. MATERIALS AND METHODS: Pathogenic assays including isolation, identification, and antimicrobial susceptibility tests were conducted for AB and MRSA at LTCFs in Eastern Taiwan. Staphylococcal cassette chromosome mec typing assays were done to understand the relatedness of clonal strains of MRSA. RESULTS: All AB-positive samples in the LTCFs were mainly from water-rich samples and were drug susceptible. Our data indicated that the AB strains from LTCFs were similar to those from Puzi River watersheds in Taiwan, which were not drug resistant to commonly used antibiotics. On the other hand, the drug resistance analysis of MRSA indicated that the genotypes from the LTCFs were similar to those from nearby hospitals. Eight strains of MRSA were isolated from four LTCFs, of which five were identified as hospital-acquired strains according to SSCmed typing assays. CONCLUSION: These findings suggest that MRSA in LTCFs might propagate from hospitals and could be transmitted between hospitals and LTCFs. Health authorities should be aware of this risk. The long-term follow-up of MRSA is recommended in local medical institutions as well as in LTCFs for correlative analysis.

Involvement of type VI secretion system in secretion of iron chelator pyoverdine in Pseudomonas taiwanensis.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

Characterization of an insecticidal toxin and pathogenicity of Pseudomonas taiwanensis against insects.

Pseudomonas taiwanensis is a broad-host-range entomopathogenic bacterium that exhibits insecticidal activity toward agricultural pests Plutella xylostella, Spodoptera exigua, Spodoptera litura, Trichoplusia ni and Drosophila melanogaster. Oral infection with different concentrations (OD = 0.5 to 2) of wild-type P. taiwanensis resulted in insect mortality rates that were not significantly different (92.7%, 96.4% and 94.5%). The TccC protein, a component of the toxin complex (Tc), plays an essential role in the insecticidal activity of P. taiwanensis. The ΔtccC mutant strain of P. taiwanensis, which has a knockout mutation in the tccC gene, only induced 42.2% mortality in P. xylostella, even at a high bacterial dose (OD = 2.0). TccC protein was cleaved into two fragments, an N-terminal fragment containing an Rhs-like domain and a C-terminal fragment containing a Glt symporter domain and a TraT domain, which might contribute to antioxidative stress activity and defense against macrophagosis, respectively. Interestingly, the primary structure of the C-terminal region of TccC in P. taiwanensis is unique among pathogens. Membrane localization of the C-terminal fragment of TccC was proven by flow cytometry. Sonicated pellets of P. taiwanensis ΔtccC strain had lower toxicity against the Sf9 insect cell line and P. xylostella larvae than the wild type. We also found that infection of Sf9 and LD652Y-5d cell lines with P. taiwanensis induced apoptotic cell death. Further, natural oral infection by P. taiwanensis triggered expression of host programmed cell death-related genes JNK-2 and caspase-3.

A proteome analysis of the tetracyanonickelate (II) responses in Klebsiella oxytoca.

Tetracyanonickelate (II) (TCN) has been proved to be degraded by Klebsiella oxytoca. In order to examine the physiological responses of TCN degradation by this bacterium, two-dimensional (2-DE) electrophoresis approach and Matrix-assisted laser desorption/ionization-time of flight-mass spectrometry allow us to identify 91 proteins spots that were significantly altered in the presence of 1 mM TCN in relative to that in 1 mM ammonia when K. oxytoca grown at the late-log phase. Among them, 43 proteins were successfully identified. Fractions enriched in hydrophobic proteins were obtained with a specific extraction method based on temperature-dependent phase partitioning with Triton X-114, with the successful identification of 26 proteins out of 41 differential proteins. Some proteins were related with TCN metabolism. OsmC-like protein, molecular chaperone DnaK, glutathione S-transferase, alkyl hydroperoxide reductase, DNA protection during starvation conditions and DNA binding ferritin-like protein can counteract the oxidative stress from TCN biodegradation. The nitrogenase had been suggested to participate in TCN degradation by K. oxytoca, and was upregulated in TCN-treated cells as expected. The induction of glutamine synthetase could enhance the assimilation of limited nitrogen source produced from the bioconversion of TCN into ammonia as the alternate nitrogen source for bacteria growth. These findings could provide new insights into the inducible mechanisms underlying the capacity of K. oxytoca to tolerate TCN stress.

Health care needs of patients during early recovery after total knee-replacement surgery.

AIM: The aim of this study was to understand the health care needs and related factors for patients with knee osteoarthritis during the early recovery period after total knee-replacement surgery. BACKGROUND: Knee-replacement surgery effectively relieves pain and improves mobility for patients with knee osteoarthritis. With shorter hospital stays, postoperative knee-replacement patients need guidance from medical personnel about self-care, but little is known about these patients' health care needs. DESIGN: This descriptive, correlational study used a longitudinal design. METHOD: A convenience sample of 85 patients undergoing total knee-replacement surgery in northern Taiwan was interviewed before and 1-2 weeks after hospital discharge. Data were collected on participants' demographic characteristics, disease characteristics, symptom distress and health care needs. RESULTS: Participants' health care needs diminished significantly after hospital discharge. Before hospital discharge, the most important need was medical personnel to help relieve postoperative pain, and health care needs were predicted by symptom distress and age. After hospital discharge, the most important need was medical personnel to help understand the conditions requiring a return visit to hospital, and health care needs were predicted by symptom distress, health care needs before discharge, age and gender. CONCLUSIONS: The results of this study provide a comprehensive understanding of health care needs before and after discharge, as well as predictive factors for patients undergoing total knee-replacement surgery. RELEVANCE TO CLINICAL PRACTICE: Given today's shorter hospital stays, clinicians need to prioritise health care needs indicated by predictive variables. This strategy would help optimise assessment and care management by focusing on patients' greater health care needs and by tailoring care information and skills to patients' individual needs.

Pain experiences, control beliefs and coping strategies in Chinese elders with osteoarthritis.

AIM: To explore pain experiences, pain control beliefs and pain coping strategies among elders with osteoarthritis in Taiwan. BACKGROUND: Osteoarthritis, a degenerative disease that commonly affects older people, is manifested by pain and disability. Despite the increasing number of older people, few studies have explored their chronic pain experiences, pain beliefs and pain coping strategies. DESIGN: Survey. METHODS: Participants (n = 205) were recruited by convenience sampling from three outpatient clinics at a medical centre in northern Taiwan. Data were collected by questionnaire on pain sites, pain intensity, pain interference with activity, pain control beliefs, pain coping strategies, depressive tendency and demographic variables. Scores of worst pain intensity were used to divide participants into three groups: mild, moderate and severe pain. RESULTS: Participants' average number of pain sites was 2.9 (SD 2.2). Most participants (70%) suffered moderate to severe worst pain. Participants perceived average pain control beliefs (mean 1.7, SD 0.7). Most used pharmacological coping strategies about half of the time (mean 1.8, SD 0.7) and non-pharmacological coping strategies about one-quarter of the time (mean 1.0, SD 0.5). Scores on satisfaction with living situation, depressive tendency, pain intensity, pain interference, pain control beliefs and frequency of using pharmacological coping strategies differed significantly among the three pain groups. Results of regression analysis showed that the intensity of average pain, pain interference with walking, pain interference with sleeping and pain control beliefs significantly predicted intensity of worst pain, explaining 54% of the variance in intensity of worst pain. CONCLUSION: This study highlights the importance of dealing with pain in this population. RELEVANCE TO CLINICAL PRACTICE: Since health care providers play an important role in helping elders to manage pain, the authors recommend training physicians and nurses to regularly assess pain and to provide current knowledge about pain assessment and management strategies for elders with osteoarthritis.

Validation testing in thorough QT/QTc clinical trials.

In order to validate the results of a thorough QT/QTc (the duration of depolarization and repolarization of ventricles or the duration corrected for heart rate) clinical trial, ICH E14 recommended to include a concurrent positive control treatment in the trial. It further recommended that validation is achieved if the positive control has an effect on the mean QT/QTc interval of about 5 ms. Zhang (2008) discussed the intersection-union test approach for testing the validation hypotheses and an alternative global average test approach. In this article, we further discuss the difference and relationship of the two sets of hypotheses and the difference in the efficiencies of the two approaches. We conclude that validation can be achieved if either one test rejects the null hypotheses without inflating the family-wise Type I error rate. However, using both approaches may improve the efficiency in validation assessment.

Noninferiority testing beyond simple two-sample comparison.

In order to fulfill the requirement of a new drug application, a sponsor often need to conduct multiple clinical trials. Often these trials are of designs more complicated than a randomized two-sample single-factor study. For example, these trials could be designed with multiple centers, multiple factors, covariates, group sequential and/or adaptive scheme, etc. When an active standard treatment used as the control treatment in a two-arm clinical trial, the efficacy of the test treatment is often established by performing a noninferiority test through comparison of the test treatment and the active standard treatment. Typically, the noninferiority trials are designed with either a generalized historical control approach (i.e., noninferiority margin approach or delta-margin approach) or a cross-trial comparison approach (i.e., synthesis approach or lambda-margin approach). Many of the statistical properties of the approaches discussed in the literature were focused on testing in a simple two sample comparison form. We studied the limitations of the two approaches for the consideration of switching between superiority and noninferiority testing, feasibility to be applied with group sequential design, constancy assumption requirements, test dependency in multiple trials, analysis of homogeneity of efficacy among centers in a multi-center trial, data transformation and changing analysis method from the historical studies. Our evaluation shows that the cross-trial comparison approach is more restricted to simple two sample comparison with normal approximation test because of its poor properties with more complicated design and analysis. On the other hand, the generalized historical control comparison approach may have more flexible properties when the variability of the margin delta is indeed negligibly small.

Significance levels for stability pooling test: a simulation study.

Shelf life of a drug product is defined as the length of time under specific conditions of storage that the product will remain within specifications established to ensure its identity, strength, quality, and purity. The objective of an new drug application (NDA) stability study is to collect and evaluate the evidence in support of the sponsor-proposed shelf life. The proposed shelf life is supported when each batch of the drug products in the study has shelf life no shorter than the proposed shelf life. When the value of the batch mean changes linearly, sometimes batches of the same product may share the same slope or regression line. In practice, batches are pooled to have a common estimate of slope or regression line when there is no significant difference in slope or regression line. Such practice is often applied in pooling across levels of a design factor such as package or strength in a stability study designed with multiple factors. However, falsely pooling different slopes or intercepts may increase false positive rates on the decision of approval for the proposed shelf life. The proposed algorithm used with the simulation technique tries to reduce false pooling rates for testing slope or intercept differences in order to bring down the false positive rates on the decision of approval for the proposed shelf life to the prespecified Type-I error rate of 5 or 10%.

ANCOVA approach for shelf life analysis of stability study of multiple factor designs.

For a traditional multiple batch stability design with no other factor, the conventional analysis is analysis of covariance (ANCOVA) modeling using F-tests based on type I sum of squares to determine whether the batches may be pooled for a common estimate of the linear regression line(s). In the last decade, many multiple factor designs were proposed in stability studies. With the objective of model selection, the generalization of the conventional ANCOVA model using type I sum of squares to designs with multiple factors requires a prespecified hierarchical pooling test ordering to determine whether any of the factors may be eliminated. Different shelf life estimates may be derived using different hierarchical pooling test orderings. On the other hand, setting the hierarchical ordering can be subjective and controversial. The stepwise modeling based on F-tests using type III sum of squares for model determination and factor elimination is proposed to eliminate such difficulties.

Shelf life determination based on equivalence assessment.

In a regular analysis of covariance (ANCOVA) approach to stability analysis, the decision for pooling data from different batches plays a key role in the determination of the shelf life of the drug product. Conventionally, the decision to pool data for the estimate of slope and intercept of common or individual regression lines is made by "no evidence to reject the null hypothesis of no difference." With typically limited observations, a significance level of much higher than 0.05 was recommended for the pooling tests in order to avoid inflation of type-I error rate of the shelf life testing. This logic of the pooling test decision making discouraged the use of replicates to improve power of testing and precision of estimation. The concept of pooling by equivalence test was originally proposed by Ruberg and Hsu in their 1990 article "Multiple comparison procedures for pooling batches in stability studies" Such a concept has evolved to pooling batches based on the shelf life equivalence test by Yoshioka et al. in their 1996 article "Power of analysis of variance for assessing batch-variation of stability data of pharmaceuticals." In this article, an approximation test of shelf life equivalence and a test of chemical value equivalence for the data pooling decision are proposed as an alternative to the conventional ANCOVA approach.