Characterization of integrons and novel cassette arrays in bacteria from clinical isloates in China,2000-2014

Rapid dissemination of antibiotic resistance genes among bacterial isolates is an increasing problem in China.Integron,a conserved DNA sequence,which is carried on episomal genetic structures,plays a very important role in development of antibiotic resistance.This systematic analysis was based on MEDLINE and EMBASE databases.We summarized the distribution and proportion of different types of gene cassette arrays of integrons(including class 1,2,3 and atypical class 1 integron) from clinical bacteria isolates in China.Fifty-six literatures were included in this study.Most of the strains were Gram-negative bacteria(94.1%,7,364/7,822) while only 5.9% strains were Grampositive bacteria.Class 1 integrons were detected in 54.2%(3956/7295) Gram-negative strains.aadA2 was the most popular gene cassette array detected from 60 Gram-positive bacteria while dfrA17-aadA5 were detected in 426 Gramnegative bacteria.This study identified 12 novel gene cassette arrays which have not been previously found in any species.All the novel gene cassette arrays were detected from Gram-negative bacteria.A regional characteristic of distribution of integrons was presented in this study.The results highlight a need for continuous surveillance of integrons and provide a guide for future research on integron-mediated bacteria resistance.     

Establishment of an animal model of open tibial fractures with infection in New Zealand rabbits北大核心

BACKGROUND: Open facture is easy to induce infection, which is an urgent problem in clinic. Establishing a reliable animal model of open fracture with infection is of great significance for drug and instrument development and application. OBEJCTIVE: To develop an open fracture with infection model in New Zealand white rabbits, and to identify the available number of bacteria that can cause infection. METHODS: The amount of bacteria was determined by establishing open fracture structure and verifying the concentration of bacterial colonies. Thirty New Zealand white rabbits were randomly divided into control group and four experimental groups, and a transverse fracture at the middle part of tibia was established in all rabbits, followed by the injection of 1 mL of normal saline or 1mL of Staphylococcus aureus suspension at the concentrations of 1×105, 1×106, 5×106, and 1×107 CFU/mL. Afterwards, the optimal concentration of 1 mL of bacteria liquid causing infection was determined by gross observation, body temperature analysis and body mass measurement, white blood cell and C-reactive protein detection, bacterial culture and pathological observation. RESULTS AND CONCLUSION: Rabbits in the 5×106 CFU/mL group were all infected and had higher survival rate. In the 1×105 and 1×106 CFU/mL groups, some rabbits showed no infection. One rabbit died due to infection in the 1×107 CFU/mL group. In summary, the reliable infection model of open fracture can be induced by injected with 1 mL of Staphylococcus aureus at the concentration of 5×106 CFU/mL in New Zealand white rabbits, which can be used as an effective model to guide drugs and instruments related anti-infective research. © 2018, Journal of Clinical Rehabilitative Tissue Engineering Research. All rights reserved.     

Parallel Detection of Bacterial Pathogens in Cerebrospinal Fluid with 16S rRNA Probe Microarray

The most commonly used method for detection ofpathogenic bacteria in cerebrospinal fluid (CSF) speci mens of clinical laboratories is isolation and identificationof the causative agents by cultural method, biochemicaland serological t…     

Segmentation of Bacteria Image Based on Level Set Method

In biology ferment engineering, accurate statistics of the quantity of bacteria is one of the most important subjects. In this paper, the quantity of bacteria which was observed traditionally manuauy can be detected automatically. Image acquisition and processing system is designed to accomplish image preprocessing, image segmentation and statistics of the quantity of bacteria. Segmentation of bacteria images is successfully realized by means of a region-based level set method and then the quantity of bacteria is computed precisely, which plays an important role in optimizing the growth conditions of bacteria.     

Functions of the MMR system and special roles of mutL in bacterial evolution

DNA mismatch repair guards the integrity of the genome of almost all organisms by correcting DNA biosynthetic errors and by ensuring the fidelity of homologous genetic recombination. MutL is one of the important proteins involved in mismatch repair system. It has been suggested to function as a master coordinator or molecular matchmaker because it interacts physically with MutS, the endonuclease MutH, and DNA helicase UvrD. It also binds to DNA and has an ATPase activity. MutL defective bacteria strains have elevated mutation rates and it has been reported recently that MutL defect may have an important impact on bacterial evolution.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

Research of the Effect of the Shear Stress on Endothelial Cells

1 IntroductionCellular mechanism is one of the foundations of regenerating medicine and tissue engineering, which is also an advanced subject in cell mechanism in recent years~([1]). The form and function of a cell, and the growing, reproducing and death, even canceration are related to the characteristics of cell mechanism. While the research of the shear stress on endothelial cells is an important field in cell mechanism. The main bio-functions of endothelial cells are as follows: anti-cruor…     

Molecular mechanism of the qnrA genemediated quionlone resistance in Gram-negative bacteria

To explore the prevalence of the plasmid-mediated quinolone resistance gene qnrA in Gramnegative bacteria and to investigate its molecular genetic background and resistance profile in isolates harboring this gene, a total of 629 nalidixic acid-resistant isolates of non-repetitive Gram-negative bacteria were collected from clinical specimens between April 2004 and April 2006 and these isolates were screened for qnrA gene by PCR using specific primers combined with DNA sequencing. The extended spectrum β-1actamase （ESBL） or AmpC-producing isolates were distinguished by the phenotypic confirmatory test combined with DNA sequencing, and the antibiotics susceptibility test for qnrA-positive isolates was carried out by Kirby-Bauer and E-test method. To detect the location of the qnrA gene, plasmid conjugation and Southern hybridization were performed and the integron structure containing the qnrA gene was cloned by PCR strategy and sequenced by primer walking. It was demonstrated that the incidence of the qnrA-positive strains in nalidixic acid-resistant bacteria was 1.9% （12/629）, in which the detection rates for Klebiesiella pneumoniae. Enterobacter cloacae, Enterobacter aerogenes, Citrobacterfreundii and Salmonella choeraesuis were 2.2% （3/138）, 17. 1% （6/35）, 9. 1% （1/11）, 12.5% （1/8）, and 14.3% （1/7）, respectively. The qnrA gene was found to be embedded in the complex sull-type integron located on plasmids with varied size （80-180 kb）. Among them, 4 qnrA-positive isolates carried integron In37 and 8 isolates carried a novel integron, temporarily desig- nated as InX. All the qnrA-positive isolates were ESBL-producing and transferable for the multi-drug resistance. It is concluded that the plasmid-mediated drug-resistance mechanism exists in the quinolone resistant strains of isolates from hospitals in Guangdong area, but the incidence was rather low. Nevertheless, it is still possible that the horizontal transfer of the resistant qnrA gene might lead to the spreading of drug-resista     

Correlation of the antiseptic resistance in Acinetobacter baumannii and the antiseptic resistance gene qacE Δ 1 located in class Ⅰ integron

In the past decade, uses of antiseptics and disinfectants in hospitals and other health care centers are rather common, but the chance to develop resistance to antiseptics and disinfectants is also increased. Acinetobacter baumannii is one of the opportunistic bacteria involving in the nosocomial infection . In the present study, the correlation of the antiseptic resistance in A . baumannii and the antiseptic resistance gene qacEΔ1 was investigated by means of determination of MICs. Meanwhile, the MICs of glutaraldehyde, chlorhexidine, benzalkonium bromide, iodophor and trichloroisocyanurate to 80 clinical isolates of A. baumannii were detected by tube dilution assay and the resistance genes intI1 and qacEΔ1 in these isolates were amplified by PCR and verified by DNA sequencer. It was found that the MIC50 for these 5 antiseptics tested were 32, 8, 8, 4 and 1μg/ml respectively, and the detection rates of intI1 and qacEΔ1 gene were 60.0% and 77.6% respectively. In addition, 55% of the 80 isolates simultaneously possessed both intll and qacEΔ1 gene, and the percentage of antiseptic resistance of A . baumannii carring both genes to benzalkonium bromide were higher than that without these two genes, however, there was no significant difference between intll and qacEΔ1 gene. The result in bactericidal efficiency assay indicated that chlorhexidine could still produce rapid and strong bactericidal effect at concentration of 1 MIC after 10 min exposure. These results suggest that the antiseptic resistance of A . baumannii to various antiseptics is correlated with the presence of the antiseptic resistance genes qacEΔ1 in bacteria, thus warning that the increase of the antiseptic resistance should not be ignored and the relative high concentration or prolonged application time is required to achieve a sufficient bactericidal effect.     

超声对金黄色葡萄球菌生物膜的体外破坏作用

BACKGROUND: Studies have shown that the ultrasound enhances the on bactericidal activity of antibiotics in an intensity-dependent manner, that is, the higher the ultrasound intensity, the greater its effectiveness.OBJECTIVE: To study the destructive effect of different intensities of ultrasound on Staphylococcus aureus and its biofilm.METHODS: Staphylococcus aureus biofilms were cultured in vitro using guide sheet culture method and divided into three groups for intervention. The biofilm in the control group received no treatment. The biofilm in the low-intensity ultrasound group was intervened by pulsed ultrasound with an intensity of 500 mW/cm and frequency of 200 kHz for 10 minutes. The biofilm in the high-intensity ultrasound group was intervened by continuous ultalsound with an intensity of 40 W and frequency of 1 MHz for 10 minutes. Bacterial colonies were counted using ultrasonic oscillation-live bacteria counting method. DNA and polysaccharide of the bacteria were respectively marked using propidium iodide and FITC-ConA. The molding of the bilfilm was determined using laser scanning confocal microscope.RESULTS AND CONCLUSION: The number of bacterial colonies in the high-intensity ultrasound group were lower than that in the control and low-intensity ultrasound groups (P < 0.05), and there were no significant differences between control and low-intensity ultrasound groups. There were no significant differences in the number and intensity of red fluorescence and green fluorescence between low-intensity ultrasound and control groups; however, the number and intensity of red fluorescence and green fluorescence in the high-intensity ultrasound group were significantly decreased compared with the low-intensity ultrasound and control groups. These results demonstrate that the low-intensity ultrasound cannot kill the bacteria and it has a tiny destructive effect on the biofilm of bacteria; however, the high-intensity ultrasound can effectively kill the bacteria and has a strong destructive effect on the bilfilm of bacteria.  中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

Genetic variants of the class A scavenger receptor gene are associated with coronary artery disease in Chinese

The class A scavenger receptor,encoded by the macrophage scavenger receptor 1(MSR1) gene,is a pattern recognition receptor(PPR) primarily expressed in macrophages.It has been reported that genetic polymorphisms of MSR1 are significantly associated with the number of diseased vessels and coronary artery narrowing greater than 20% in Caucasians.However,whether it links genetically to coronary artery disease(CAD) in Chinese is not defined.Here,we performed an independent case-control study in a Chinese population consisting of 402 CAD cases and 400 controls by genotyping ten single nucleotide polymorphisms(SNPs) of MSR1.We found that rs416748 and rs13306541 were significantly associated with an increased risk of CAD with per allele odds ratio(OR) of 1.56 [95% confidence interval(CI) = 1.28-1.90;P < 0.001] and 1.70(95% CI = 1.27-2.27;P < 0.001),respectively.Our results indicate that genetic variants of MSR1 may serve as predictive markers for the risk of CAD in combination with traditional risk factors of CAD in Chinese population.     

Detection of Hantavirus gene from peripheral blood of patients with HFRS in Heilongjiang province and the epidemiological significance

The aim of this study is to further understand the genotype of Hantavirus(HV) from peripheral blood of patients with hemorrhagic fever with renal syndrome (HFRS) and the epidemiological significance of this disease in Heilongjiang province in recent years. Thirty-one serum samples of clinically diagnosed patients with HFRS were examined by RT-PCR to decide the genetic subtype. On the basis of infection season, the serum samples were divided into two groups: winter (Nov, 2003—Feb, 2004) , spring and summer (April, 2004—Sep, 2004). Further analysis was performed in combination with clinical symptoms. It was found that among the total 31 samples, 22 were sero-positive. Among 14 serum samples in winter, 8 were sero-positive, of which 5 cases were of typeⅠ(Hantaan virus, HTNV) and 3 of typeⅡ(Seoul virus, SEOV). Among 17 samples in spring and summer, 14 were sero-positive, of which 5 cases were of typeⅠand 9 of typeⅡ. So it concludes that both of the two types of Hantavinis exist in Heilongjiang. The typeⅠis the main pathogen of HFRS in winter, and typeⅡis the main in spring and summer.     

Structure of Middleware in RFID Based System： Application to the Infant Protection

This paper introduces a middleware design and implementation for an infant protection system-maternal infant safety and surveillance （MISS）, using radio-frequency identification （RFID） technology. The middleware is located between application level and hardware-device level in the hierarchical software structure, which is in further divided into three levels, i.e. data, logical and service respectively. The rationale for the design is based on the consideration of the performance, organization, maintenance, and upgrade factors.     

血友病B的基因诊断

The haemophilia B(HB)is caused by the mutations in the factⅨ gene, and is known as an X-linked recessive disease. At present, due to lacking of the eradicative therapy, the gene diagnosis and detection of the carriers is an effective method to prevent the infant patients to be born, block the transmission of harmful gene, and improve the population quality. This should be actively encouraged for its pratical usefulness.     

Genetics of T cell defects in lupus

Systemic lupus erythematosus （SLE） is an autoimmune disease characterized by anti-nuclear autoantibodies that cause damage to multiple organs and tissues. Intrinsic defects have been demonstrated in the lymphoid and myeloid cellular compartments, including T cells. Lupus susceptibility is mediated through the interplay of a large number of genes, most of which are still unidentified. Most of the genetic studies in both human patients and mouse models have addressed lupus susceptibility as a whole. More recently however, more attention has been paid to the inheritance of specific lupus-associated phenotypes. In this review, we summarized our results obtained with the Slel locus in the NZM2410 mouse model, which mediates the generation of anti-histone autoreactive T cells. Slel, which is constituted of multiple genes, is the only known genomic region that is sufficient for the generation of autoreactive T cells. The identification of the corresponding genes will constitute a landmark for our understanding of the mechanisms of autoimmunlty. Our results are discussed in the context of candidate genes and the role of T cells in systemic autoimmunlty. Cellular ＆ Molecular Immunology. 2005;2（6）：403-409.     

Metabolic Induction of Lactic Acid Bacteria for Urea Removal

Objective：This study aims to induce nonpathogenic bacteria for urea removal as a potential treatment in renal failure. Methods：Lactococus lactis MG1363 was induced by repeated exposure to urea-rich culture media, the ability to remove urea from the media was evaluated. The effect of gastroenteric environment, such as low pH, bile salt and antiagonistic properties were investigated. The antimicrobial activities on pathogenic E. coli and S. aureus in the intestinal tract and the antibiotic tolerance of the induced bacteria were also studied. Results ： Induced bacteria of 50 generations could decrease the urea level from 40.01 mg/dL to 32.99 mg/dL after 24 h. The bacteria could grow after treatment at pH3.0 for 2 h and in 0.1% bile salt for 6 h, and the urea removal activity was retained in such simulated gastroenteric environment. The removal of urea was significantly enhanced to 35.8% by addition of Ni^2＋ to the culture medium at neutral pH. It was also found that the induced bacteria could inhibit the growth of E. coli and S. aureus, and tolerate ampicillin, gentamicin, roxithromycin, tetracycline and cefradine. The safety tests were performed by feeding normal rats with either Lactococus lactis MG1363 or induced Laetococus lactis MG1363. The two materials did not cause any changes in blood cells, blood biochemical indexes and body weight. Conclusion： These results ＂suggest that the induced Laetoeocus lactis MG1363 has the potential as an oral therapy for the removal of urea in patients with renal failure.     

Left ventricular myxoma：a case report

Cardiac myxoma,the most common primary heart tumor,is located mainly in the left atrium.We reported a rare case of left ventricular myxoma incidentally found on echocardiography in an asymptomatic 60-year-old male.The tumor was carefully resected without fragmentation.The patient had an uneventful recovery and was discharged home on the 4th postoperative day.Surgical resection of this type of cardiac myxoma is recommended due to the rarity of tumor location.     

Quality control and evaluation of human immunodeficiency virus antibody assays used for screening donated blood in China

During 2004, a total of 124 batches of HIV antibody ELISAs from domestic and overseas manufacturers, comprising approximately 60 million tests, were tested for quality and released for screening blood in China. The inter- and intra-batch variation, specificity, and sensitivity were evaluated using a laboratory panel and clinical samples. The inter-batch variation was less than 15% and only 2 of 12 assays had intra-batch variation of less than 20% for 4 dilutions of a control specimen. 257 samples confirmed positive for HIV antibody and 4826 negative samples from different regions in China were used to evaluate the sensitivity and specificity of the assays. The results showed that the sensitivity is in the range from 93.7% to 100% for assays sampled directly from the manufacturers, and 91. 4%-99. 6% for those retrieved from the consumers; the specificity was in the range from 97.88% to 99.97% . The testing environment may vary in different regions of China. Therefore, manufacturers should provide robust assays to satisfy the requirements of these diverse environments, and especially reduce the intra-assay variation and improve the stability of the kits.     

Rapid Identification of Pathogenic Bacteria by means of Two Conservative Gene Loci＇ Specific PCR-CE-RFLP

To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis-Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined.The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease Hae Ⅲ in-completely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.