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.

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.

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.

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.

An immunosensor based on magnetic relaxation switch and polystyrene microparticle-induced immune multivalency enrichment system for the detection of Pantoea stewartii subsp. Stewartii.

A rapid, sensitive, and simple immunosensor has been developed for the detection of Pantoea stewartii subsp. Stewartii (Pss). This immunosensor combines magnetic relaxation switch (MRS) assay with polystyrene microparticle-induced immune multivalency enrichment system. Comparing to conventional enzyme-linked immunosorbent assay (ELISA), the immunosensor developed in this study provides higher sensitivity and requires less analysis time. The detection limit of Pss obtained by immunosensor was determined to be 10(3)cfu/mL, 50 times lower than that by ELISA (5×10(4)cfu/mL), while the analysis time required by immunosensor is 30min much shorter than that by ELISA. The average recoveries studied with Pss at various spiking levels ranged from 85.5% to 93.4% with a relative standard deviation (RSD) below 6.0%. No cross-reaction with the other five strains was found, demonstrating a good specificity of Pss detection. The results showed that the MRS immunosensor combined with PS-induced immune multivalency enhancement system is a promising platform for the determination of large biological molecules due to its high sensitivity, specificity, homogeneity, and speed.

Immunosensor based on magnetic relaxation switch and biotin-streptavidin system for the detection of Kanamycin in milk.

A rapid, sensitive, and simple immunosensor was developed for the detection of Kanamycin (KM) in milk. This immunosensor is based on magnetic relaxation switch (MRS) assay and biotin-streptavidin system (B-SA system). The target analyte (KM) competed with those on the surface of the superparamagnetic iron oxide (SPIO) nanoparticles and hence affected the formation of SPIO aggregates. The dispersed and aggregated states of SPIO can modulate the spin-spin relaxation time (T(2)) of the neighboring water molecule. T(2) was then changed as an effect of the target analyte. The B-SA system was used to amplify the SPIO binding, thus enhance the sensitivity. The detection working was 1.5 to 25.2ng mL(-1) and limit of detection (LOD) was determined to be 0.1ng mL(-1). The LOD of the immunosensor decreased tenfold, and its analysis time (45min) was much shorter than that of enzyme-linked immunosorbent assay (6h to 8h). The average recoveries of the KM at various spiking levels ranged from 80.2% to 85.6% with a relative standard deviation (RSD) below 4.0%. The results showed that the MRS immunosensor was a promising platform for the determination of small molecular residues because of its high sensitivity, specificity, homogeneity, and speed.

Quantum dots capped with dummy molecularly imprinted film as luminescent sensor for the determination of tetrabromobisphenol A in water and soils.

Molecularly imprinted film with diphenolic acid (DPA) as dummy template molecule has been grafted on the surface of Mn-doped ZnS quantum dots (QDs) to develop a selective and sensitive sensor for rapid determination of tetrabromobisphenol A (TBBPA) in water and soils. The obtained DPA-MIP-QDs sensor has distinguished selectivity and high binding affinity to TBBPA. The fluorescence quenching fractions of the sensor presented a satisfactory linearity with the concentrations of TBBPA in the range of 0.1-100 µM, and its limit of detection can reach 0.015 µM. The sensor has been successfully applied to determine the TBBPA in water and soil samples, and the average recoveries of the TBBPA at various spiking levels ranged from 80.2% to 96.5% with relative standard deviation below 8.0%. The results provided a clue to develop sensors for rapid determination of hazardous materials from complex matrixes.

[Effect of taurine on NOS activity in hippocampus of rat exposed lead].

OBJECTIVE: To study the effect of taurine on lead-induced damage to the ability of learning and memory. METHODS: Using NADPH-d histochemistry method to study the change of rat NOS positive neurons in hippocampus. Rats in experimental groups were fed with different doses of lead in drinking water (0.011, 0.110 g/L), and different doses of taurine (5, 10 g/kg). RESULTS: Taurine (10 g/kg) could increase the number of NOS positive neurons in CA1 and dentate gyrus subregion in hippocampus of rats exposed to lead. The number of NADPH-d positive neurons in CA1 and dentate gyrus subregion for low lead (0.011 g/L) and high taurine (10 g/kg) group (51.80 +/- 4.68, 47.40 +/- 4.20, respectively) were higher than those in the low lead (0.011 g/L) group (41.20 +/- 5.32, 39.87 +/- 3.81, respectively, P < 0.05). CONCLUSION: Taurine may antagonize lead-induced damage to the ability of learning and memory.