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New broad-spectrum β-lactam antibiotics are widely used in clinical and animal husbandry and aquaculture, subject to its highly selective pressure, bacteria produce a new class of β-lactamase (β-lactamase, bla), its hydrolysis enzyme substrate ratio broader spectrum, called extended-spectrum β-lactamases (extended-spectrum-beta lactamase, ESBL), it can quickly hydrolyze β-lactam antibiotics, allowing bacteria to such antibiotics resistance. ESBL mostly intestinal bacteria produce rod Branch, including TEM, SHV, CTX-M and OXA and many other types, including CTX-M-is our nation's most prevalent genotype. ESBL-producing strains of bodies of water, such resistance genes in pathogenic strains and conditions spread between pathogens, if the wound is water ESBL-producing strains of infection, the treatment will be very difficult, which is engaged in the crossing, crossing battle wading operations forces or other groups constitutes a new health hazard. Although people ESBL-producing strains resistant problem has long been concerned, but on the water environment ESBL-producing bacteria proliferation and Hazards of very little. My room on the Yangtze River pollution resistant for a long study found ESBL-producing thermotolerant coliforms in river pollution, plasmid carrying rate, showed multiple drug resistance. bla gene plasmid mediated primarily by which conjugative plasmid majority in the bla gene transfer plays a dominant role in non-conjugative plasmid smaller proportion, only under certain conditions can be achieved bla gene transfer, regardless of conjugative plasmid or non-conjugative plasmid, all in bla horizontal gene transfer play a role. My room Chen Hao confirmed ESBL-producing strains by means of drinking water delivered to the bla gene level default mouse intestinal colonization of Escherichia coli. There is no water in the wound ESBL-producing strains of horizontal transfer of resistance genes and colonization areas reported. Therefore, it is necessary to water environment ESBL-producing strains bla horizontal gene transfer and harm-depth study and exploration. Based on the consideration in the bla gene plasmid propagation and the important role played by non-ESBL-producing strains obtained bla gene of the potential hazards, the subject of ESBL-producing E. coli as a starting point to conduct research in four parts, the first part of the study Pets can conjugative plasmid of ESBL-producing E. coli bla CTX-M horizontal gene transfer factors; second part of the study of the non-conjugative plasmid carrying the ESBL-producing E. coli capture aminoglycoside modifying enzyme gene (aminoglycoside modifying enzyme, AME) and bla CTX-M horizontal gene transfer; Part III observed the burn wounds were ESBL-producing and non-ESBL-producing E. coli mixed infection of bla CTX-M horizontal gene transfer; fourth part of the study of non- ESBL-producing E. coli bla CTX-M genes obtained before and after the burn wound colonization metastasis. Experimental methods a water sample collection and identification of strains isolated from the Yangtze River and Jialing River water samples, cryopreservation, and in the water samples within 4 h to the laboratory, with the membrane assay bacterial count, coliform and heat coliforms; watery dilution and filtration, containing cefotaxime (CTX) of M-FC media screening strains blue colonies were picked with bioMerieux automatic biochemical analyzer to identify bacteria; using standard disk diffusion Determination of resistant strains spectrum; using double disc method of ESBL confirmation. (2) temperature and pH on the bonding of ESBL-producing strains in vitro effects of the Yangtze River water to two observations will carry conjugative plasmid of ESBL-producing E. coli (E. coli 425 number and E. coli 452) and non-ESBL-producing but Lee Fuping highly resistant E. coli NK5449 mixed inoculated in LB liquid medium, temperature and pH was observed in two factors ESBL-producing E. coli bla CTX-M gene transfer efficiency. 3 ESBL-producing E. coli genes for AME capture and bla CTX-M gene transfer to non-conjugative plasmid carrying the two ESBL-producing strains (No. E. coli 293 and E. coli 310, producing ESBL, for the big celebration neomycin-sensitive) and the recipient strain (No. E. coli 295, non-producing ESBL, carrying conjugative plasmid, gentamicin resistance) are joined to form a zygote transfer, zygote transfer again with the E. coli NK5449 be joined to observe the AME gene capture and bla CTX-M horizontal gene transfer; detected by PCR analysis of donor and recipient zygote fungi and transfer test strain bla genotype, AME gene and integration of sub-carrier status, and use random amplified polymorphism analysis (random amplified polymorphic DNA, RAPD) technology to determine the relevant strains homology. 4 burn wound bacterial resistance to horizontal gene transfer and fluid resistance with horizontal gene transfer compared to 10 20 ~ 22g male BALB / c mice were constructed as part of deep Ⅱ degree burn model (burn in hip back, burn area 3% of the body surface area) were randomly divided into mixed infection group (7) and blank control group (3). Mixed infection of mice to burn zone subcutaneous infusion broth mix, ESBL-producing E. coli 452 and non-ESBL-producing E. coli NK5449 each 0.1 ml, bacteria concentration 1.5 × 10 8 CFU / ml; burn to blank control group of mice injected subcutaneously 0.2 ml sterile area PBS. Infection after 2 d, take muscle tissue burns district, weighed and homogenized by serial dilutions containing cefotaxime (CTX, 64μg/ml) and rifampicin (RIF, 160μg/ml) of M-FC Flat screened transfer zygote containing rifampicin (RIF, 160μg/ml) M-FC medium screening recipient strain, calculated engage frequency and detect metastasis zygote homology. The E. coli 452 and E. coli NK5449 in liquid medium with 37 ℃ mixed cultures 6 h, 18 h and 48 h, the joint frequency for comparison. 5 non-ESBL-producing E. coli bla CTX-M genes obtained before and after the burn wound colonization compared to 21 male BALB / c were built as part of deep Ⅱ degree burn model and randomly divided into three groups, namely: control Groups (3); E. coli C452 (E. coli 452 and E. coli NK5449 formed zygote transfer) infection (9); E. coli NK5449 infected group (9). Respectively, to the burned area subcutaneously infected mice tested bacterial suspension 0.2 ml, blank group of mice injected with PBS 0.2 ml OK. After infection 1 d, 4 d and 7 d, each group were randomly selected in each 1/3 the number of mice were sacrificed, aseptic, take subcutaneous muscle tissue burns zone, upper jejunum, lung and liver sections, called After re-homogenized by serial dilutions containing cefotaxime and rifampicin M-FC medium screening E. coli C452 strain containing rifampicin M-FC medium screening strain E. coli NK5449 and analyzed using SPSS 18.0 software for each group of muscle tissue and organ growth of the number of colonies within the statistical analysis. The experimental results a water sample test results Yangtze River water samples of bacterial contamination was significantly lower than its tributary Jialing River and Jialing River water samples total bacterial count, coliform and thermotolerant coliforms and other indicators of the Yangtze River water samples were 11 times, 144 times and 5 times. Donor strain used in the experiments were bla CTX-M-type genotype, the performance of multi-drug resistance. (2) temperature and pH on the impact of bonding strain E. coli 425 and E. coli NK5449 occurrence frequency of engagement and increased with decreasing temperature from 35 ℃ to 15 ℃, at pH6.1, pH7.1 and pH8 .1 respectively increased by 16.6 times, 33.8 times and 6.4 times; at 35 ℃ and 25 ℃, when the pH value of 8.1 occurs more frequently than engaging high pH value of 6.1, but there is at 15 ℃ The changes, pH value of 6.1, the joint frequency than the high pH of 8.1. Same temperature, pH7.1 best suited for bonding occurs. ESBL-producing E. coli 452 and non-ESBL-producing E. coli NK5449 bonding frequency decreased significantly with temperature from 35 ℃ to 15 ℃, when the pH value of 6.1, 7.1 and 8.1, its bonding frequencies were 620-fold lower , 3220-fold and 15,999-fold. At the same temperature, pH7.1 best place for engagement. 35 ℃ and 25 ℃ at two temperatures, when the pH value is 8.1, the two ESBL-producing E. coli bacteria and receptor engagement frequency than high pH of 6.1, and are subject to change at 15 ℃ , at pH 6.1, pH 8.1 engagement frequencies above the engagement frequency. 3. Producing ESBL E. coli and horizontal gene transfer captured AME bla CTX-M genes strains carrying non-conjugative plasmid can be used as the recipient strain carrying AME genes with non-ESBL producing bacteria donor engagement, engagement frequencies were 1.6 × 10 -6 and 7.2 × 10 -6 . With foreign movable genetic elements, which itself can be transformed into donor strain, the bla CTX-M genes horizontal transfer of bacteria to other receptors, bonding frequencies were 3.7 × 10 -6 and 7.4 × 10 -6 . After a strain carrying Ⅰ integron variable regions were sequenced and compared with Blastn, Ⅰ integron variable region excluding bla CTX-M genes. E. coli 293 295 NK5449, E. coli 310 295 NK5449 plasmid and plasmids E. coli 295 NK5449 consistent with E. coli 293 and E. coli 310 plasmid carried a significant difference. 4 burn wound bacterial bla CTX-M horizontal gene transfer produced ESBL E. coli 452 and the non-producing ESBL E. coli NK5449 mixed after 2 d infected wounds, burns zone in seven mice were screened within the muscle tissue of the transfer of engagement son, after testing homology with E. coli NK5449, double-disk method confirmed as ESBL-producing strains, bla genotype CTX-M, engagement frequency (3.0 ± 0.8) × 10 -1 , a minimum of 2.0 × 10 -1 , up to 4.3 × 10 -1 . Control group did not develop a colony. 5 non-ESBL-producing E. coli bla CTX-M genes obtained before and after the burn wound colonization compared E. coli NK5449 and E. coli C452 two infection after infection, 1 d, liver and upper jejunum All tissue culture positive, which colonize the upper jejunum largest number of infected bacteria, reaching 2.6 × 10 5 CFU / g tissue and 2.7 × 10 5 CFU / g tissue. After infection 4 d, liver and upper jejunum tissue cultures were negative colonies. Two groups of mice infected with bacteria on burn wound colonization ability and the ability to transfer to other organs was not statistically significant. Infection 4 d and 7 d, the above two groups of mice infected organs were no detectable bacteria. Bacterial lung infection is always without detection. Blank control group without organ colony growth. Conclusion water temperature and pH on ESBL-producing E. coli bla CTX-M gene transfer has an important influence in the pH value, the near-neutral environment most suitable for bonding occurs. Different strains of the effects of water temperature on the opposite effect, some ESBL-producing E. coli engage frequency at elevated temperatures, increased frequency of engagement, while others did ESBL-producing E. coli engage frequency but increased as the temperature decreases. While engaging in the natural frequency of the case will be reduced, but the Yangtze River water pH of 7.1 to 8.3, temperature 11 ~ 26 ℃, still suitable for the occurrence of horizontal gene transfer bla. And carrying conjugative plasmid of ESBL-producing E. coli compared to non-conjugative plasmid carrying the ESBL-producing E. coli missing bla CTX-M gene transfer capability is temporary, when there are foreign conjugative plasmid, integration, or other assistance movable genetic elements, such strains can be achieved on other resistance genes bla gene capture and transfer of non-conjugative plasmid carrying the ESBL-producing E. coli, can be captured by engaging manner other resistance genes, such as the AME gene and bla gene can be transferred to a foreign conjugative plasmid, the realization of horizontal transfer, as this process can not move with the conjugative plasmid lure, Ⅰ integron initiative to capture and transfer of existing transposon be explained, suggesting that there may be other transfers way. Murine infection model by building burn, burn wounds were observed among strains mixed infections can be achieved bla CTX-M horizontal gene transfer, the frequency of its engagement with the engagement in vitro for 6 h engage frequency equivalent, but lower than in vitro for 48 h engagement engagement frequency, which is mixed with different bacterial infections also have reference value; under the experimental conditions, access to water non-ESBL-producing E. coli strains of ESBL-producing bla CTX-M genes before and after the burn wound colonization and metastasis was not statistically significant , indicating that the recipient strain in only won bla CTX-M genes, in addition to resistance spectrum to expand outside its colonization and metastasis will not change significantly. The innovation of this study 1 was found in some ESBL-producing E. coli at a lower ambient temperatures, but increased the frequency of its engagement, which in the previous literature have not been reported, although the mechanism is not clear, but illustrates the bla gene transfer breadth and complexity. (2) In carrying non-conjugative plasmid of ESBL-producing E. coli bonding experiments, observed its AME gene capture and bla CTX-M gene transfer, the transfer mechanism is different inducers of conjugative plasmid, also ruled out Ⅰ integrons, transposons and insertion sequences common effect, so there may be other transfers inferred way. 3 model of the burn wound observed bla CTX-M gene transfer to the recipient strain level, which seen reported in the literature, in the sense that, by multiple bacterial infection of the wound or undue delay in handling the case, may cause bla CTX-M genes rapidly to other opportunistic pathogens or pathogen transfer, treatment difficulty increases. 4 pairs of recipient strain obtained bla CTX-M genes before and after the burn wound colonization model compared with the ability to transfer, although no significant difference, but observed that bacterial infection to organ metastasis, and to the highest concentration of intestinal tissue . In severe infection or trauma the body, the bacteria was found in burn wounds migration phenomenon may be related to the phenomenon of migration of intestinal bacteria exist, these two migration pathway in clinical treatment and prevention should also be taken seriously. Experiments deficiencies 1 This experimental study relates only to the Yangtze River and Jialing River water ESBL-producing E. coli, and the river still exist in Klebsiella pneumoniae, Acinetobacter baumannii and other ESBL-producing strains, and they are Pets resistance plasmid, so need to expand the scope of the study. (2) In addition to pH and temperature are two important factors, but also take into account the water β-lactam antibiotic residues, velocity, nutrition, turbidity and other physical and chemical factors. 3 wound colonization in burned mice within the ESBL-producing E. coli can be bla CTX-M gene level is passed to the non-ESBL-producing E. coli. Since many types of wounds, injuries have their own characteristics, for wound bla CTX-M gene transfer needs further study, such as increased cuts, chemical burns and other studies to obtain a general conclusion. Prospect 1 in the aquatic environment ESBL-producing strains of drug-resistant horizontal gene transfer problems, increase dissolved β-lactam antibiotic residues concentration, flow rate, nutrient conditions and other physical and chemical factors, build bla horizontal gene transfer mathematical model to predict water resistant genetic pollution levels. (2) an increase in follow-up experiments in a variety of wound types, researchers mixed bacterial infection bla CTX-M horizontal gene transfer and its hazards. 3 Elimination of the plasmid is to deal with ESBL-producing strains bla CTX-M gene transfer of more promising approach, but has not yet developed a safe and efficient plasmid remover, therefore, rational drug and developed a simple and reliable removal from water resistant , drug resistance genes and β-lactam antibiotics, a method should be more important.
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