Decreased Antibiotic Susceptibility in Pseudomonas aeruginosa Surviving UV Irradition.

Given its excellent performance against the pathogens, UV disinfection has been applied broadly in different fields. However, only limited studies have comprehensively investigated the response of bacteria surviving UV irradiation to the environmental antibiotic stress. Here, we investigated the antibiotic susceptibility of Pseudomonas aeruginosa suffering from the UV irradiation. Our results revealed that UV exposure may decrease the susceptibility to tetracycline, ciprofloxacin, and polymyxin B in the survival P. aeruginosa. Mechanistically, UV exposure causes oxidative stress in P. aeruginosa and consequently induces dysregulation of genes contributed to the related antibiotic resistance genes. These results revealed that the insufficient ultraviolet radiation dose may result in the decreased antibiotic susceptibility in the pathogens, thus posing potential threats to the environment and human health.

Chlorine disinfection promotes the exchange of antibiotic resistance genes across bacterial genera by natural transformation.

Chlorine disinfection to drinking water plays an important role in preventing and controlling waterborne disease outbreaks globally. Nevertheless, little is known about why it enriches the antibiotic resistance genes (ARGs) in bacteria after chlorination. Here, ARGs released from killed antibiotic-resistant bacteria (ARB), and culturable chlorine-injured bacteria produced in the chlorination process as the recipient, were investigated to determine their contribution to the horizontal transfer of ARGs during disinfection treatment. We discovered Escherichia coli, Salmonella aberdeen, Pseudomonas aeruginosa and Enterococcus faecalis showed diverse resistance to sodium hypochlorite, and transferable RP4 could be released from killed sensitive donor consistently. Meanwhile, the survival of chlorine-tolerant injured bacteria with enhanced cell membrane permeabilisation and a strong oxidative stress-response demonstrated that a physiologically competent cell could be transferred by RP4 with an improved transformation frequency of up to 550 times compared with the corresponding untreated bacteria. Furthermore, the water quality factors involving chemical oxygen demand (CODMn), ammonium nitrogen and metal ions (Ca2+ and K+) could significantly promote above transformation frequency of released RP4 into injured E. faecalis. Our findings demonstrated that the chlorination process promoted the horizontal transfer of plasmids by natural transformation, which resulted in the exchange of ARGs across bacterial genera and the emergence of new ARB, as well as the transfer of chlorine-injured opportunistic pathogen from non-ARB to ARB. Considering that the transfer elements were quite resistant to degradation through disinfection, this situation poses a potential risk to public health.

Inactivation of Poliovirus by Ozone and the Impact of Ozone on the Viral Genome.

OBJECTIVE: To investigate the mechanisms underlying ozone-induced inactivation of poliovirus type 1 (PV1). METHODS: We used cell culture, long-overlapping RT-PCR, and spot hybridization assays to verify and accurately locate the sites of action of ozone that cause PV1 inactivation. We also employed recombinant viral genome RNA infection models to confirm our observations. RESULTS: Our results indicated that ozone inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ozone specifically damaged the 80-124 nucleotide (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 lacking this region was non-infectious. CONCLUSION: In this study, we not only elucidated the mechanisms by which ozone induces PV1 inactivation but also determined that the 80-124 nt region in the 5'-NCR is targeted by ozone to achieve this inactivation.

Chlorine injury enhances antibiotic resistance in Pseudomonas aeruginosa through over expression of drug efflux pumps.

Adaption to adverse environments plays an important role in bacterial survival and is receiving increasing globe attention now. Here, cultivable chlorine-injured Pseudomonas aeruginosa, produced on the chlorination process, was investigated about their resistance to antibiotics. Then, global transcriptional analyses, quantitative PCR (qPCR) validation and antioxidant enzymes measurement were performed to explore the underlying mechanisms. The results showed that chlorine injury enhanced antibiotic resistance in P. aeruginosa and cultivable chlorine-injured P. aeruginosa exposed to 4 mg/L sodium hypochlorite (half of the lethal dose) improved antibiotic resistance against ceftazidime, chloramphenicol and ampicillin by 1.4-5.6 fold. This increase in antibiotic resistance was not hereditable and over expression of the MexEF-OprN efflux pump resulting from oxidative stress contributed to it. These results demonstrate temporal physiological persistence to antibiotics in cultivable chlorine-injured pathogens, suggesting their survival from adverse environments with antibiotic exposure and thereby posing lasting hazards to human health.

Hepatitis C virus infection induces endoplasmic reticulum stress and apoptosis in human fetal liver stem cells.

The cellular mechanisms by which hepatitis C virus (HCV) replication might mediate cytopathic effects are controversial and not entirely clear. In this study, we found that blood-borne HCV (bbHCV) infection could lead to endoplasmic reticulum (ER)-stress and mitochondria-related/caspase-dependent apoptosis at the early stages of infection based on use of the highly efficient bbHCV cell culture model established previously. Sections of bbHCV-infected human fetal liver stem cells (hFLSCs) revealed convolution and nonlinear ER, cell vacuolization, swelling of mitochondria, and numerous double membrane vesicles (DMVs). The percentage of apoptotic hFLSCs infected by bbHCV reached 29.8% at 16 h postinfection, and the amount of cytochrome c increased remarkably in the cytosolic protein fraction. However, over time, apoptosis was inhibited due to the activation of NF-κB. The expression of NF-κB-p65, Bcl-xL, XIAP, and c-FLIPL in hFLSCs was increased significantly 24 h after in infection by bbHCV. The accelerated cell death cycles involving apoptosis, regeneration and repair by bbHCV infection might give rise to the development of cirrhosis, and ultimately to hepatocellular carcinogenesis. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Profiling of intracellular and extracellular antibiotic resistance genes in tap water.

Antibiotic resistance genes (ARGs) have gained global attention due to their public health threat. Extracelluar ARGs (eARGs) can result in the dissemination of antibiotic resistance via free-living ARGs in natural environments, where they promote ARB transmission in drinking water distribution systems. However, eARG pollution in tap water has not been well researched. In this study, concentrations of eARGs and intracellular ARGs (iARGs) in tap water, sampled at Tianjin, China, were investigated for one year. Fourteen eARG types were found at the highest concentration of 1.3 × 105 gene copies (GC)/L. TetC was detected in 66.7% of samples, followed by sul1, sul2, and qnrA with the same detection frequency of 41.7%. Fifteen iARGs (including tetA, tetB, tetM, tetQ, tetX, sul1, sul2, sul3, ermB, blaTEM, and qnrA) were continuously detected in all collected tap water samples with sul1 and sul2 the most abundant. Additionally, both eARG and iARG concentrations in tap water presented a seasonal pattern with most abundant prevalence in summer. The concentration of observed intracellular sulfonamide resistance genes showed a significantly positive correlation with total nitrogen concentrations. This study suggested that eARG and iARG pollution of drinking water systems pose a potential risk to human public health.

Chlorine disinfection increases both intracellular and extracellular antibiotic resistance genes in a full-scale wastewater treatment plant.

The emergence and spread of antibiotic resistance has posed a major threat to both human health and environmental ecosystem. Although the disinfection has been proved to be efficient to control the occurrence of pathogens, little effort is dedicated to revealing potential impacts of disinfection on transmission of antibiotic resistance genes (ARGs), particularly for free-living ARGs in final disinfected effluent of urban wastewater treatment plants (UWWTP). Here, we investigated the effects of chlorine disinfection on the occurrence and concentration of both extracellular ARGs (eARGs) and intracellular ARGs (iARGs) in a full-scale UWWTP over a year. We reported that the concentrations of both eARGs and iARGs would be increased by the disinfection with chlorine dioxide (ClO2). Specifically, chlorination preferentially increased the abundances of eARGs against macrolide (ermB), tetracycline (tetA, tetB and tetC), sulfonamide (sul1, sul2 and sul3), ß-lactam (ampC), aminoglycosides (aph(2')-Id), rifampicin (katG) and vancomycin (vanA) up to 3.8 folds. Similarly, the abundances of iARGs were also increased up to 7.8 folds after chlorination. In terms of correlation analyses, the abundance of Escherichia coli before chlorination showed a strong positive correlation with the total eARG concentration, while lower temperature and higher ammonium concentration were assumed to be associated with the concentration of iARGs. This study suggests the chlorine disinfection could increase the abundances of both iARGs and eARGs, thereby posing risk of the dissemination of antibiotic resistance in environments.

Development of a novel and highly efficient method of isolating bacteriophages from water.

Bacteriophages are widely used to the treatment of drug-resistant bacteria and the improvement of food safety through bacterial lysis. However, the limited investigations on bacteriophage restrict their further application. In this study, a novel and highly efficient method was developed for isolating bacteriophage from water based on the electropositive silica gel particles (ESPs) method. To optimize the ESPs method, we evaluated the eluent type, flow rate, pH, temperature, and inoculation concentration of bacteriophage using bacteriophage f2. The quantitative detection reported that the recovery of the ESPs method reached over 90%. The qualitative detection demonstrated that the ESPs method effectively isolated 70% of extremely low-concentration bacteriophage (100 PFU/100L). Based on the host bacteria composed of 33 standard strains and 10 isolated strains, the bacteriophages in 18 water samples collected from the three sites in the Tianjin Haihe River Basin were isolated by the ESPs and traditional methods. Results showed that the ESPs method was significantly superior to the traditional method. The ESPs method isolated 32 strains of bacteriophage, whereas the traditional method isolated 15 strains. The sample isolation efficiency and bacteriophage isolation efficiency of the ESPs method were 3.28 and 2.13 times higher than those of the traditional method. The developed ESPs method was characterized by high isolation efficiency, efficient handling of large water sample size and low requirement on water quality.

Efficient replication of blood-borne hepatitis C virus in human fetal liver stem cells.

The development of pathogenic mechanisms, specific antiviral treatments and preventive vaccines for hepatitis C virus (HCV) infection has been limited due to lack of cell culture models that can naturally imitate the entire HCV life cycle. Here, we established an HCV cell culture model based on human fetal liver stem cells (hFLSCs) that supports the entire blood-borne hepatitis C virus (bbHCV) life cycle. More than 90% of cells remained infected by various genotypes. bbHCV was efficiently propagated, and progeny virus were infectious to hFLSCs. The virus could be passed efficiently between cells. The viral infectivity was partially blocked by specific antibodies or small interfering RNA against HCV entry factors, whereas HCV replication was inhibited by antiviral drugs. We observed viral particles of approximately 55 nm in diameter in both cell culture media and infected cells after bbHCV infection. CONCLUSION: Our data show that the entire bbHCV life cycle could be naturally imitated in hFLSCs. This model is expected to provide a powerful tool for exploring the process and the mechanism of bbHCV infection at the cellular level and for evaluating the treatment and preventive strategies of bbHCV infection. (Hepatology 2017;66:1045-1057).

Culture-dependent enumeration methods failed to simultaneously detect disinfectant-injured and genetically modified Escherichia coli in drinking water.

Underestimation of Escherichia coli in drinking water, an indicator microorganism of sanitary risk, may result in potential risks of waterborne diseases. However, the detection of disinfectant-injured or genetically modified (GM) E. coli has been largely overlooked so far. To evaluate the accuracy of culture-dependent enumeration with regard to disinfectant-injured and GM E. coli, chlorine- or ozone-injured wild-type (WT) and GM E. coli were prepared and characterized. Then, water samples contaminated with these E. coli strains were assayed by four widely used methods, including lactose tryptose broth-based multiple-tube fermentation (MTF), m-endo-based membrane filtration method (MFM), an enzyme substrate test (EST) known as Colilert, and Petrifilm-based testing slip method (TSM). It was found that MTF was the most effective method to detect disinfectant-injured WT E. coli (with 76.9% trials detecting all these bacteria), while this method could not effectively detect GM E. coli (with uninjured bacteria undetectable and a maximal detection rate of 21.5% for the injured). The EST was the only method which enabled considerable enumeration of uninjured GM E. coli, with a detection rate of over 93%. However, the detection rate declined to lower than 45.4% once the GM E. coli was injured by disinfectants. The MFM was invalid for both disinfectant-injured and GM E. coli. This is the first study to report the failure of these commonly used enumeration methods to simultaneously detect disinfectant-injured and GM E. coli. Thus, it highlights the urgent requirement for the development of a more accurate and versatile enumeration method which allows the detection of disinfectant-injured and GM E. coli on the assessment of microbial quality of drinking water.

[Changes of the mitochondrial DNA copy number and the antioxidant system in the PBMC of hepatocellular carcinoma].

OBJECTIVE: To investigate the relationship between the changes of the copy numbers of mtDNA in peripheral blood mono-nucle- ar cell(PBMC) and the disordered of antioxidant capacity of hepatocellular carcinoma (HCC) patients. METHODS: The Ficoll Hypaque method was used to isolate the PBMC from blood specimens. The ND1 gene of the mitochondrial was amplified by real-time PCR; meantime ß-actin was served as a quantitative standard marker; the difference of mtDNA copy number in PBMC was compared between HCC and healthy control group. The level of reactive oxygen species (ROS) in PBMC was determined by flow cytometry. The change of total antioxidant capacity (T- AOC) of plasma was detected by the biochemistry examination. RESULTS: The copy numbers of ND1 gene in PBMC of HCC was 73% that of the healthy control group,which suggested a decrease of the copy numbers of mtDNA in HCC. The levels of ROS of PBMC in HCC was (417. 82 ± 110.62) and (301.82 ± 75.54) in control group, which showed that the levels of ROS of PBMC in HCC were significant higher than that in control group (P < 0.01).Plasma T-AOC in HCC was (1.30 ± 0.85), and (3.20 ± 1.62) in control. The T-AOC of plasma of HCC was significantly lower than in control group (P < 0.01). CONCLUSION: There was a certain relationship between the decrease of the copy numbers of mtDNA and the disordered antioxidant capacity in hepatocellular carcinoma, which may be associated with the development of hepatocellular carcinoma.

A new adsorption-elution technique for the concentration of aquatic extracellular antibiotic resistance genes from large volumes of water.

Extracellular antibiotic resistance genes (eARGs) that help in the transmission and spread of antibiotic-resistant bacteria are emerging environmental contaminants in water, and there is therefore a growing need to assess environmental levels and associated risks of eARGs. However, as they are present in low amounts, it is difficult to detect eARGs in water directly with PCR techniques. Here, we prepared a new type of nucleic acid adsorption particle (NAAP) with high capacity and developed an optimal adsorption-elution method to concentrate eARGs from large volumes of water. With this technique, we were able to achieve an eARG recovery rate of above 95% from 10 L of water samples. Moreover, combining this new method with quantitative real-time PCR (qPCR), the sensitivity of the eARG detection was 10(4) times that of single qPCR, with the detection limit lowered to 100 gene copies (GCs)/L. Our analyses showed that the eARG load, virus load and certain water characteristics such as pH, chemical oxygen demand (CODMn), and turbidity affected the eARGs recovery rate. However, high eARGs recovery rates always remained within the standard limits for natural surface water quality, while eARG levels in water were lower than the detection limits of single qPCR assays. The recovery rates were not affected by water temperature and heterotrophic plate counts (HPC). The eARGs whatever located in the plasmids or the short-length linear DNAs can be recovered from the water. Furthermore, the recovery rate was high even in the presence of high concentrations of plasmids in different natural water (Haihe river, well water, raw water for drinking water, Jinhe river, Tuanbo lake and the Yunqiao reservoir). By this technology, eARGs concentrations were found ranging from (2.70 ± 0.73) × 10(2) to (4.58 ± 0.47) × 10(4) GCs/L for the extracellular ampicillin resistance gene and (5.43 ± 0.41) × 10(2) to (2.14 ± 0.23) × 10(4) GCs/L for the extracellular gentamicin resistance gene in natural water for the first time, respectively. All these findings suggest that NAAPs have great potential for the monitoring of eARGs pollution in water.

A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.

Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

Seroprevalence and evolutionary dynamics of genotype 4 hepatitis E virus in Shandong Province, China.

AIM: To investigate the seroprevalence and evolutionary dynamics of hepatitis E virus (HEV) and assess the ancestor of HEVs in China's Shandong Province. METHODS: A total of 2028 serum, 60 fecal and 82 bile samples were collected from the general human population, patients and swine, respectively. This seroepidemiological study was conducted using an immunnosorbent assay and HEV RNA was detected by the reverse transcription-nested polymerase chain reaction (RT-nPCR) method. Complete genome sequences of the prevalent strains (CH-YT-HEV01, CH-YT-HEV02 and CH-YT-sHEV01) were determined, and the sequences were analyzed phylogenetically. In addition, the evolutionary dynamics of three HEV isolates were determined using the framework of coalescent analysis in the program package BEAST, and the time of the most recent common ancestors (TMRCAs) of China-indigenous genotype 4 HEV isolates was calculated. RESULTS: The overall viral burden in the general human population was 0.1%, and the positive rates of anti-HEV IgG and IgM in the serum specimens were 25.1% (509/2028) and 2.3% (51/2028), respectively. In addition, IgG positivity increased with age. The phylogenetic analysis based on the full-length nucleotide sequences showed that the strain CH-YT-HEV02 was directly related to CH-YT-sHEV01 with a 94% identity, suggesting that they were involved in cross-species transmission. The isolate CH-YT-HEV01 was close to HB-3 and CHN-SD-sHEV with a bootstrap value of 100%, sharing a 96.1%-96.4% identity with each other. Surprisingly, the HB-3 strain was a representative strain prevalent in swine in Hubei, and the isolate CHN-SD-sHEV was obtained from swine in Shandong in a previous report. TMRCA for the clade of CH-YT-HEV01 and HB-3 was 2003, which was consistent with the TMRCA for the clade of CHN-SD-sHEV and HB-3, and they were both earlier than the TMRCA for the clade of CH-YT-HEV01 and CHN-SD-sHEV (2004). CONCLUSION: The strains CH-YT-HEV01, CHN-SD-sHEV and HB-3 are involved in trans-regional transmission, and the ancestors of HEVs in Shandong come from Hubei Province.

Development of a novel filter cartridge system with electropositive granule media to concentrate viruses from large volumes of natural surface water.

Exposure to various infectious viruses in environmental drinking water can constitute a public health risk. However, it is difficult to detect viruses in water due to their low concentration. In this study, we have developed a novel filter cartridge system containing electropositive granule media (EGM). Viruses present in large volumes of environmental samples were adsorbed onto the EGM, and then recovered by elution and poly(ethylene glycol) (PEG) concentration. To evaluate the system's efficiency in viral recovery, poliovirus (PV-1), a surrogate for enteric viruses, was used to artificially contaminate river water samples which were then assayed by quantitative real-time PCR. To optimize the concentration procedure, the eluent type, water flow rate and properties (e.g., pH, bacterial, and viral loads), were evaluated. The highest virus recovery was obtained by pumping river water at a flow rate of 300 mL/min and then pushing 3 L of an eluent containing 3× broth [1.5% (w/v) NaCl, 3% (w/v) tryptone, 1.5% (w/v) beef powder] with 0.05 mol/L glycine through the filter. Using this procedure, the recovery efficiencies of PV-1 from 10 to 100 L of spiked river water were up to 99%. In addition, this method is virus load and pH dependent. Virus recovery was maximal at a load of between 10(3.5) and 10(5.5) TCID50 and a pH ranging from 5 to 7. The bacterial load in the water has no effect on virus recovery. Different types of viruses and surface water were tested to validate the system's applicability. Results revealed that the EGM filter cartridge was able to concentrate PV-1, human adenoviruses (HAdVs) and noroviruses (HuNoVs) with high efficiency from river, lake, and reservoir water. Furthermore, it showed more efficient recovery than glass wool and 1MDS filters. These data suggest that this system provides rapid and efficient virus recovery from a large volume of natural surface water and, as such, could be a useful tool in revealing the presence of viruses in surface water.

A survey of drug resistance bla genes originating from synthetic plasmid vectors in six Chinese rivers.

Antibiotic resistance poses a significant challenge to human health and its rate continues to rise globally. While antibiotic-selectable synthetic plasmid vectors have proved invaluable tools of genetic engineering, this class of artificial recombinant DNA sequences with high expression of antibiotic resistance genes presents an unknown risk beyond the laboratory setting. Contamination of environmental microbes with synthetic plasmid vector-sourced antibiotic resistance genes may represent a yet unrecognized source of antibiotic resistance. In this study, PCR and real-time quantitative PCR were used to investigate the synthetic plasmid vector-originated ampicillin resistance gene, ß-lactam antibiotic (blá), in microbes from six Chinese rivers with significant human interactions. Various levels of blá were detected in all six rivers, with the highest levels in the Pearl and Haihe rivers. To validate the blá pollution, environmental plasmids in the river samples were captured by the E. coli transformants from the community plasmid metagenome. The resultant plasmid library of 205 ampicillin-resistant E. coli (transformants) showed a blá-positive rate of 27.3% by PCR. Sequencing results confirmed the synthetic plasmid vector sources. In addition, results of the Kirby-Bauer disc-diffusion test reinforced the ampicillin-resistant functions of the environmental plasmids. The resistance spectrum of transformants from the Pearl and Haihe rivers, in particular, had expanded to the third- and fourth-generation of cephalosporin drugs, while that of other transformants mainly involved first- and second-generation cephalosporins. This study not only reveals environmental contamination of synthetic plasmid vector-sourced blá drug resistance genes in Chinese rivers, but also suggests that synthetic plasmid vectors may represent a source of antibiotic resistance in humans.

[Characteristics of the extracellular polymeric substances of a heterotrophic nitrifying bacterium strain].

To study the variation of extracellular polymers substances (EPS) composition of the heterotrophic denitrifying bacteria (Acinetobacter sp. YY-5) at different growth stages, The EPS of YY-5 was extracted by thermal, and the composition of protein, polysaccharides, nucleic acid and free amino acids were analyzed at different growth stages. Subsequently, the proteins in extracts were hydrolyzed into amino acids, the amino acid's changes and effect on physical and chemical properties of proteins were investigated. The results showed the strain has a high EPS contents which mainly consist of proteins, and the EPS content achieved the highest value at stable stage. The amount of proteins increased from 14.599 mg x g(-1) to 28.489 mg x g(-1), then declined to 15.139 mg x g(-1). Polysaccharides content increased from 6.757 mg x g(-1) to 10.199 mg x g(-1), then declined to 7.857 mg x g(-1). The nucleic acid contents increased from 1.56 mg x g(-1) to 6.287 mg x g(-1) in the whole growth stages. The free amino acids contents increased from 3.713 mg x g(-1) to 4.374 mg x g(-1), then obviously declined to 1.299 mg x g(-1). After the proteins were hydrolyzed into amino acids, the amount of polar amino acids showed the trend that increased earlier and declined later, the contents of nonpolar amino acids increased at all growth stages. The amino acids with negative charges were more than that with the positive.

Isolation and characterization of Bacillus sp. producing broad-spectrum antibiotics against human and plant pathogenic fungi.

A strain of bacterium producing antifungal antibiotic was isolated and identification of the strain was attempted. We could identify the bacterium as being a Bacillus sp., based on morphological observation, physiological characteristics, and 16S rDNA sequence analysis, thus leading us to designate the strain as Bacillus sp. AH-E-1. The strain showed potent antibiotic activity against phytopathogenic and human pathogenic fungi by inducing mycelial distortion and swelling and inhibiting spore germination. The antibiotic metabolite produced by the strain demonstrated excellent thermal and pH (2-11) stability, but was labile to autoclaving. From these results, we could find a broader antifungal activity of Bacillus genus. Isolation and characterization of the active agent produced by the strain are under progress.

A rapid subtractive immunization method to prepare discriminatory monoclonal antibodies for food E. coli O157:H7 contamination.

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1:10(6) with E. coli O157:H7 and affinity constants ranged from 1.57 × 10(10) to 2.79 × 10(10) L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food.

The 40-80 nt region in the 5'-NCR of genome is a critical target for inactivating poliovirus by chlorine dioxide.

Chemical disinfection is the most common method used to inactivate viruses from drinking water throughout the world. In this study, cell culture, ELISA, RT-PCR, and spot hybridization were employed to investigate the mechanism underlying chlorine dioxide (ClO(2) )-induced inactivation of Poliovirus type 1 (PV1), which was also confirmed by recombinant viral genome RNA infection models. The results suggested that ClO(2) inactivated PV1 primarily by disrupting the 5'-non-coding region (5'-NCR) of the PV1 genome. Further study revealed that ClO(2) degraded specifically the 40-80 nucleotides (nt) region in the 5'-NCR. Recombinant viral genome RNA infection models confirmed that PV1 RNA lacking this 40-80 nt region was not infectious. This study not only elucidated the mechanism of PV1 inactivation by ClO(2), but also defined the critical genetic target for the disinfectant to inactivate Poliovirus. This study also provides a strategy by which rapid, accurate, and molecular methods based on sensitive genetic targets may be established for evaluating the effects of disinfectants on viruses.