The Change Of Intestinal Microecology And The Spreading Of Drug-resistant Gene In Sepsis Rats Under The Pressure Of Broad-spectrum Antibiotic

Sepsis is a common complication after a serious trauma or burn injuries, severe shock, and major surgery. Furthermore, it may lead to septic shock and multiple organ dysfunction syndromes (MODS). MODS have become one of the important causes of death in clinical critically ill patients. The gastrointestinal tract has been considered as a stress central organ, and gut barrier failure played an important role in sepsis and MODS. The theory was emphasized that gut bacteria and their products could be an important trigger for the development of sepsis and MODS in critically injured or burned patients. In the pathogenesis of traumatic stress that the gastrointestinal tract involved, the intestinal bacterial translocation is the main pathological link. The endogenous infection caused by opportunistic pathogen that stored in the gastrointestinal tract becomes a formidable problem. Under the pressure of the trauma and antibiotics, nosocomial pathogens could colonize in the intestine and give rise to resistance gene transfer among different kinds of bacteria, causing intestinal origin bacteria to acquire new resistance features, so that the intestine become the largest spreading platform of drug resistance genes. In addition to causing own endogenous infection, patients with diarrhea and fecal incontinence will lead to the spread of resistant bacteria in the hospitals. There is no clear research results and understanding of intestinal colonization of alien bacteria caused by the trauma and propagation of antibiotic resistance genes under the pressure of antibiotics, making the lack of prevention and treatment on this issue. The hypothesis of this study is that the trauma can damage intestinal micro-ecological environment or barrier, following the foreign bacteria colonization and bacterial translocation. Broad-spectrum antibiotics kill a great quantity of sensitive intestinal flora and pick out opportunistic pathogen and drug-resistant strains. Resistance genes can be further passed to the gastro-intestinal origin bacteria and continue to exist in the gut. Rhubarb can protect gut ecological environment of critical illness, diminish the choice of antibiotics on intestinal flora, reduce bacterial translocation and explore some new way to prevent the nosocomial infections caused by intestinal resistant. Avoiding colonization of alien drug-resistant strains and limiting on the use of broad-spectrum antibiotics are the important means to reduce acquired drug resistance in the gut.PARTâ… Effect of Burn and "Second Hit" by Endotoxin on Gut Microflora and Bacterial Translocation in RatsObjective:To approach the impact of different degrees of trauma on the intestinal flora and bacterial translocation and the effect of rhubarb on intestinal microecology in sepsis rats.Methods:32 SD rats with 30%â…¢degree burns on back were hit by endotoxin at a dose of 20 mg/kg 24 hours after the burn injury. The rhubarb was given at a dose of 100mg (50mg/kg, q12h) orally just after the second hit. At the end of the treatment, the bacteria and fungi in colon were cultured and the incidence of bacterial translocation of rats in each group was detected.Results:1) The impact of different degrees of trauma on the number of the intestinal flora in colon:The number of the enterobacteriaceae and enterococci did not change significantly after burn, but the number of fungi significantly increased (P<0.05) and the number of lactobacilli decreased from 108 cfu/g to 106-107 cfu/g (P<0.05). With the "second-hit" by endotoxin, the number of enterobacteriaceae and enterococci were increased 103 and 102 orders of magnitude, which were significantly different to those of burn group (P<0.01), and the number of fungi was slightly higher than that of the normal control group, but the difference was no statistical significance, the number of lactobacilli significantly less than that of the normal control group (P<0.05).2) The impact of different degrees of trauma on the species of the intestinal flora in colon:In normal control group, escherichia coli was the sole species of Enterobacter, Enterococcus faecalis and geotrichum capitatum accounted for the majority of enterococci and fungi respectively. After the burn and "second hit" by endotoxin, escherichia coli, enterococcus faecalis and geotrichum capitatum remained the dominant bacterial strains in gut. The significant change after trauma was that the opportunistic pathogen such as pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii or bacillus proteus appeared.3) Bacterial translocation ratio in normal control group was 12.5%, bacterial translocation only occurs in mesenteric lymph nodes, translocation to blood and distant organs was not found. There is no increase in the rate of intestinal bacterial translocation after burn. With the "second-hit" by endotoxin, the rate of intestinal bacterial translocation was significantly increased (compared with the burn group, the individual translocation rate P<0.05; overall organs translocation rate P<0.01) and intestinal bacteria widely translocated to the blood, liver, lungs and other distant organs.4) The effect of rhubarb on intestinal microecology in sepsis rats:Rhubarb weakened the harm of burn and endotoxin on lactobacilli. Rhubarb can significantly reduce overall organs translocation rate in septic rats, especially reduce the translocation of opportunistic pathogen and intestinal bacterial translocation to the distant organs.Conclusion:1) Trauma can destroy the intestinal microecology and the decrease of probiotics in gut lead to the lost of colonization resistance that allow proliferation of opportunistic pathogen (such as pseudomonas aeruginosa, klebsiella pneumoniae, acinetobacter baumannii or bacillus proteus).2) In septic state, intestinal barrier function was severely inhibited, intestinal bacilli (especially pseudomonas aeruginosa, bacillus proteus, klebsiella pneumoniae and enterobacter cloacae) widely translocated to the blood flow, liver and lungs, which lead to intestinal infections.3) The rhubarb can prevent the loss of the number of anaerobic bacteria. In some extent, the rhubarb can protect gut biogeocoenosis and reduce the incidence of intestinal infection.PARTâ…¡Effect of Broad-spectrum Antibiotics on Gut Microflora and Bacterial Translocation in Scalded Rats and Sepsis RatsObjective:To approach the selection of broad-spectrum antibiotics on the intestinal flora and the effect on bacterial translocation in scalded rats and sepsis rats.Methods:120 SD rats with 30%â…¢degree burns on back were hit by endotoxin at a dose of 20 mg/kg 24 hours after the burn injury. The rhubarb was given at a dose of 100mg (50mg/kg, q12h) orally just after the second hit. The animals were treated with intraperitoneal injection of ceftriaxone or imipenem 24h after the injury (60mg/kg, q12h). At the end of the treatment, the bacteria and fungi in colon were cultured and the incidence of bacterial translocation of rats in each group was detected. Results:1) The impact of broad-spectrum antibiotics on the intestinal flora in colon: Ceftriaxone obviously reduced the number of enteric bacilli in scalded rats (P<0.01). On the contrary, the number of enteric bacilli significantly increased after nine-day treatment of imipenem (P<0.05). In sepsis rats, ceftriaxone and imipenem both obviously reduced the number of enteric bacilli (P<0.01). After the rats received antibiotics treatment, opportunistic pathogen became the dominant enteric bacilli, ceftriaxone showed the high selectivity for pseudomonas aeruginosa and acinetobacter baumannii and imipenem showed the selectivity for klebsiella pneumoniae and proteus. The antibiotics significantly increased the number of Enterococcus and fungi, E.faecium become the predominant flora in the gut and Candida albicans, Candida glabrata, Candida krusei and etc have emerged in gut. There is no significant change in the number of lactobacilli after antibiotics treatment.2) The impact of broad-spectrum antibiotics on the intestinal bacterial translocation:The rate of intestinal bacterial translocation was significantly increased and persistence of bacterial translocation to blood and distant organs was been seen after antibiotics treatment. Species of translocated bacteria is also change from bacilli enterococcus, specifically enterococcus faecium.3) The effect of rhubarb on intestinal microecology and bacterial translocation in scalded and sepsis rats:Rhubarb did not relieve the intestinal flora disturbance and can not effectively inhibit bacterial translocation caused by broad-spectrum antibiotics.Conclusion:1) Broad-spectrum antibiotics further increase the degree of intestinal flora disorder in scald and sepsis rats. Broad-spectrum antibiotics kill a large number of the sensitive intestinal flora and left space to drug-resistant strains to become dominant microorganisms in gut.2) The rate of intestinal bacterial translocation was significantly increased and persistence of bacterial translocation to blood and distant organs was been seen after antibiotics treatment. Species of translocated bacteria is also changing from bacilli enterococcus, specifically enterococcus faecium, further aggratated the enterogenic infections.3) Long-term use of antibiotics weakens the effects of rhubarb on gastrointestinal protective action of micro-ecological environment and intestinal bacterial translocation in vivo.PARTâ…¢The Transmission of Drug Resistance Gene in Gut of Sepsis Rats under the Pressure of Broad-spectrum AntibioticsObjective:To establish intestinal colonization model of rats by Klebsiella pneumoniae resistant strains and continuously observe the drug resistance change of Klebsiella pneumoniae resistant strains and original E. coli under the pressure of broad-spectrum antibiotics.Methods:40 SD rats with 30%â…¢degree burns on back were hit by endotoxin at a dose of 10 mg/kg 24 hours after the burn injury. Two hours after burn or injection of endotoxin, esophageal inoculation of 108 CFU of ESBL-producing K.pneumoniae suspended in 1ml of phosphate buffered saline was performed. The animals were treated with intraperitoneal injection of ceftriaxone 24h after the lavage. At 1,3,6,9 day of the treatment and 2, 7,14,21,28 day of drug discontinuance, stool samples were collected and detected.Results:1) Intestinal colonization by Klebsiella pneumoniae resistant strains happened in scald and sepsis rats but not rats in normal control group. The amount of colonized Klebsiella pneumoniae resistant strains is higher in sepsis rats than that in scald rats.2) Colonized Klebsiella pneumoniae resistant strains disappeared soon from intestine in rats without broad-spectrum antibiotics use. Under the pressure of broad-spectrum antibiotics, Colonized Klebsiella pneumoniae resistant strains repeatedly appeared in the intestinal tract and the change of resistance to ceftriaxone occurred with part of the strains.3) Original E.coli in sepsis rats was found acquired the resistance gene from Klebsiella pneumoniae resistant strains on day 9 of the treatment with ceftriaxone.Conclusion:Intestinal colonization by external resistant strains can occur after trauma. Due to the use of broad-spectrum antibiotics, the loss of colonization resistance permit external resistant strains continuing presence in the intestinal tract and cause simultaneous induction of antibiotic resistance. Most dangerous hazard is that intestinal original bacteria can acquire the resistance gene from external resistant strains and persist in the gut.