The role of bifidobacteria in gut barrier function after thermal injury in rats

BACKGROUND: Early multiple organ dysfunction syndrome appears to be facilitated with bacterial translocation in severe burn injury, yet the mechanisms of bacterial translocation remain in dispute. The aim of this study was to characterize the potential role of intestinal bifidobacteria in the pathogenesis of gut-derived bacterial translocation after burns and to analyze the effects of bifidobacterial supplement on gut barrier function. METHODS: Wistar rats were randomly divided into burn group (Burn, n = 60), sham burn group (SB, n = 10) in experiment 1, and burn + saline group (BS, n = 30), burn + bifidobacteria group (BB, n = 30), and sham-burn + saline group (SS, n = 30) in experiment 2. Animals in BB group were fed bifidobacterial preparation (5 x 10(9) CFU/mL) after burns, 1.5 mL, twice daily. Animals in BS and SS were fed saline. Samples were taken on postburn days 1, 3, and 5. The incidence of bacterial translocation and counts of Bifidobacterium, fungi and Escherichia coli in gut mucosa, as well as the sIgA levels in mucus of the small intestine were determined. The positive sIgA expression in lamina propria and ileum mucosal injury were evaluated light microscopically by blinded examiners. RESULTS: The incidence of bacterial translocation was increased after burns, which was accompanied by significant decrease in number of bifidobacteria but significant increase in E. coli and fungi in gut mucosa, and elevation of levels of plasma endotoxin and IL-6 (p < 0.001). The incidence of bacterial translocation was markedly reduced after 3- and 5-day supplementation of bifidobacteria compared with control group (p < 0.05). The counts of mucosal bifidobacteria were increased by 4- to 40-fold, whereas E. coli and fungi were decreased by 2- to 30-fold and 10- to 150-fold, respectively, after bifidobacterial supplementation. The damage of mucosa tended to be less pronounced after 3-day bifidobacteria-supplemented formula compared with control group (grade 2 [0-6] versus grade 4 [3-6], p < 0.05). Moreover, the expression and release of sIgA was markedly augmented after 3-days of bifidobacteria-supplementation formula and it returned to normal range on postburn day 5. CONCLUSIONS: The decrease in counts and proportion of bifidobacteria to other flora in gut may play an important role in the development of bacterial translocation after thermal injury. Supplementation of exogenous bifidobacteria could improve gut barrier function, and attenuate bacterial/endotoxin translocation secondary to major burns.     

补充双歧杆菌可促进烫伤大鼠肠道分泌型sIgA合成与分泌

目的　探讨严重烫伤后补充双歧杆菌与肠道sIgA合成、分泌的关系。　 方法　Wistar大鼠随机分为烫伤对照组 (BC组 ,30只 )、烫伤治疗组 (BT组 ,30只 )、假伤组 (NC组 ,10只 )。BT组大鼠烫伤后灌胃双歧杆菌悬液 (5 0× 10 9CFU/ml) 1 5ml,2次 /d。观测大鼠细菌移位、肠黏膜菌群双歧杆菌量、肠道sIgA分泌和表达情况等。　 结果　 (1)伤后 3d ,BC组与BT组大鼠脏器细菌移位率分别为 4 2 %和 16 % (P =0 0 0 4 ) ;伤后 5d分别为 30 %和 8% (P =0 0 0 2 )。 (2 )伤后大鼠肠黏膜菌群中双歧杆菌减少 10～ 6 0倍 ,应用悬液后双歧杆菌明显增多。 (3)BC组大鼠肠黏液sIgA平均减少 30 % ,伤后 3d达最低 ;BT组 3d后基本恢复正常。伤后大鼠肠道sIgA表达减弱 ,补充双歧杆菌后sIgA表达显著增强。 (4)肠膜菌群中双歧杆菌量与肠黏液sIgA浓度呈显著正相关。　 结论　大鼠严重烫伤后肠道sIgA产生明显受抑制 ,补充外源性双歧杆菌可促进肠道sIgA合成与分泌。     

肠道双歧杆菌与烫伤大鼠肠源性细菌/内毒素移位

目的　观察肠道双歧杆菌在肠源性细菌 /内毒素移位中的变化和作用。　方法　制作严重烫伤大鼠模型 ,同时设假伤组。检测细菌和内毒素 (LPS)移位及盲肠膜菌群变化 ,ELISA法检测血浆白细胞介素 6(IL 6)浓度。　结果　大鼠严重烫伤后脏器细菌移位明显增多 (P <0 .0 1) ;血LPS水平在致伤 1、3、5d后分别为 (0 .2 3 6± 0 148)Eu/ml、(0 .197± 0 .15 6)Eu/ml、(0 10 4± 0 .0 90 )Eu/ml,显著高于假伤组的 (0 .0 72± 0 .0 49)Eu/ml(P <0 .0 5 ) ;盲肠膜菌群中双歧杆菌数剧减 2 0～2 5 0倍、真菌数剧增至 5～ 60倍、大肠杆菌数增加 0 .5～ 3 0倍 ,双歧杆菌与大肠杆菌比值由假伤组的2 5 0 0 0∶1降为伤后的 4～ 80 0∶1;血浆IL 6水平显著增高。经分层统计 ,与未发生肠道细菌移位大鼠相比 ,盲肠膜菌群移位大鼠的双歧杆菌量减少约 12 0倍 ,真菌数增加约 5 0倍 ,大肠杆菌数增加约 3 0倍。盲肠膜菌群中双歧杆菌数量与血浆中IL 6、LPS浓度呈负相关 (r1=- 0 .4817,r2 =- 0 .4912 ,P <0 .0 1) ,血IL 6和LPS浓度间存在显著正相关 (r =0 .82 5 8,P =0 .0 0 0 1)。　结论　严重烫伤可导致大鼠盲肠膜菌群紊乱 ,细菌和LPS移位增加 ;盲肠膜菌群中双歧杆菌的比例和数量的减少 ,可能促使了严重烫伤后肠源性细菌 /内毒素移位     

严重烧伤后大鼠肠道生物屏障损害的初步研究

目的　观察严重烧伤大鼠肠道生物屏障的动态变化。　方法　以 30 %TBSAⅢ度烫伤大鼠为模型并设对照组。于伤后 2 4、4 8、72、96h分别采用微生物分析、生化、放射免疫等方法观察盲肠膜菌群、肠内容物粘蛋白、分泌型免疫球蛋白A(SIgA)、内毒素、肠道细菌移位率及定量分析腔静脉内毒素含量等。　结果　严重烧伤后早期 ,大鼠肠道生物屏障发生明显变化 :肠道膜菌群总量减少 ,其中以双歧杆菌为主的厌氧菌群显著减少 ,需氧菌群略有增加 ,酵母样真菌迅速过度生长 ,需、厌氧菌比例严重失调 ;肠道生物屏障被破坏 ,定植抗力减小 ;肠道细菌移位率明显增加 ,肠内容物、腔静脉内毒素含量增高 ,粘蛋白、SIgA含量降低。　结论　严重烧伤可致大鼠肠道生物屏障受损 ,成为烧伤后肠源性感染发生的原因之一。     

Adrenalin tolerance does not prevent bacterial translocation in a murine burn model

Burn injury causes mesenteric vasoconstriction and bacterial translocation. Since catecholamines are powerful vasoconstrictors and elevated immediately after burn injury, we hypothesised that adrenaline tolerance might decrease burn-induced mesenteric vasoconstriction and bacterial translocation. Adrenaline tolerance was developed in Swiss albino mice. Adrenaline tolerant and control animals were subdivided into sham-burn and burn subgroups. 24 h after sham-burn or burn injury, specimens were obtained for microbiological evaluation. Also, in a separate group of adrenaline tolerant and control animals, superior mesenteric blood flow was measured. Burn injury increased bacterial translocation rate in both control (P = 0.001) and adrenaline tolerant groups (P = 0.0351). The caecal bacterial level increase was significant after burn injury in control groups (P = 0.0004) but was not significant in adrenaline tolerant animals (P = 0.743). Mesenteric blood flow was decreased significantly by burn injury in both control and adrenaline tolerant animals (P < 0.00001). The results showed that catecholamines do not mediate postburn mesenteric vasoconstriction or bacterial translocation.     

Distribution and survival of Escherichia coli translocating from the intestine after thermal injury.

The present investigation was performed to study the kinetics of tissue distribution and deposition of Escherichia coli and endotoxin translocating from the intestine after thermal injury. Escherichia coli was grown in the presence of 14C glucose and both labeled bacteria and endotoxin prepared from the labeled bacteria were used as translocation probes. Escherichia coli (10(8) to 10(10) bacteria) and E. coli endotoxin (100 micrograms per animal) were gavaged into the stomach immediately before a 30% burn injury was inflicted in mice. Animals were killed 1, 4 and 24 hours after burn injury. Translocation occurred extensively within 1 hour after burn injury. Expressed as amount of radioactivity per gram of tissue, translocation was greatest in the mesenteric lymph node (MLN) followed by spleen, lung, and liver. Translocation of endotoxin was similar to translocation of intact bacteria, with the exception that less radioactivity could be found in the peritoneal cavity and more in the liver. Both intact E. coli and endotoxin translocated directly through the intact bowel wall. Killing of bacteria was greatest in the MLN and spleen, approximating 95% to more than 99% of translocating bacteria. Killing efficiency was lowest in the lungs. It is concluded that estimation of translocation by viable bacterial counts in tissues grossly underestimates the extent of translocation of bacteria and ignores the extent of translocation of endotoxin. Translocation of endotoxin may have biologic significance that is independent of and in addition to translocation of intact bacteria.     

喹吖因对肠道缺血再灌注损伤后肠道细菌/内毒素移位的影响

目的 探讨磷脂酶A2（PLA2）抑制剂喹吖因对大鼠肠道缺血再灌注（gut ischemia reperfusion,GIR)损伤后肠源性细菌／内毒素移位的影响。方法 42只wistar大鼠随机分为正常组（6只）和GIR损伤组（36只）。GIR损伤组又分为GIR损伤对照组、再灌注后3h喹吖用药组和12h喹吖用药组,每组12只。取正常对照组、GIR损伤对照组及用药组48、72h门、腔静脉血测定内毒素含量、腔静脉血浆TNFα含量和肠系膜淋巴结、肝、肺、肾等肠道外器官组织进行细菌培养。结果 早期应用喹吖因,无论是GIR损伤后3h用药组还是12h用药组,均可明显降低GIR损伤后血浆内毒素和TNFα水平（P＜0．01）;显降低GIR损伤后肠系膜淋巴结和肺、肝、肾组织器官中的细菌移位发生率（P＜0．05－0．01）。结论 GIR损伤后早期应用喹吖因可显地降低肠源性细菌／内毒素移位,减轻促炎性介质和细菌因子的释放,减轻肠道外脏器损伤。     

Endotoxin but not malnutrition promotes bacterial translocation of the gut flora in burned mice

Previously we have shown that under certain conditions, bacteria can pass through the intact epithelial mucosa to the mesenteric lymph nodes (MLN), liver, spleen, and bloodstream to cause infection, a process termed bacterial translocation. To extend these studies, we determined the influence of protein malnutrition and endotoxemia on bacterial translocation in burned (25% TBSA) and unburned mice. The results of these experiments documented that protein malnutrition did not promote bacterial translocation from the gut in either burned or unburned animals, although it did disrupt the normal indigenous gut flora. In contrast, a nonlethal dose of endotoxin (IP) promoted bacterial translocation to the mesenteric lymph nodes in burned and unburned mice, but only in burned mice did the bacteria translocate from the gut to other systemic organs (p less than 0.01). Furthermore, the mortality rate of mice receiving only endotoxin or burn was less than 10%, while the combination of endotoxin plus a thermal injury increased the mortality rate to 100% (p less than 0.01). These studies support the concept that bacteria may translocate from the gut to other organs and be a potential source of lethal infections after thermal injury.     

谷氨酰胺对严重烧伤小型香猪肠道免疫功能的影响

为研究烧伤后肠道分泌Ｓ－ＩｇＡ的变化，利用３０％Ⅲ度小型香猪烧伤动物模型，动态观察了早期肠道营养液中加入谷氨酰胺对肠道分泌Ｓ－ＩｇＡ的影响，结果显示：未补充谷氨酰胺的动物空肠和回肠液中Ｓ－ＩｇＡ的含量在伤后明显降低，动脉血中ＩｇＡ的含量也明显降低，而补充谷氨酰胺的动物空肠、回肠液中的Ｓ－ＩｇＡ，以及动脉血中的ＩｇＡ均明显高于未补充的动物，且与伤前无明显差别。提示早期肠道营养液中加入谷氨酰胺能明显维护肠粘膜细胞分泌Ｓ－ＩｇＡ的功能，对防治烧伤后细菌或毒素移位有重要意义。     

Endotoxin promotes the translocation of bacteria from the gut

Experiments were performed in mice to determine whether endotoxin could cause bacteria normally colonizing the gut to spread systemically, a process termed bacterial translocation. Endotoxin given intraperitoneally promoted bacterial translocation in a dose-dependent fashion from the gut to the mesenteric lymph node (MLN). The incidence of bacterial translocation to the MLN was similar whether the endotoxin was administered intramuscularly or intraperitoneally, although the number of bacteria colonizing the MLN was greater with intraperitoneal endotoxin. The incidence and magnitude of endotoxin-induced bacterial translocation were similar between CD-1 and C3H/HeJ (endotoxin-resistant) mice, indicating that bacterial translocation is not prevented by genetic resistance to endotoxin. Thus, it appears that the gut may serve as a reservoir for bacteria causing systemic infections during endotoxemia.     

Mesenteric lymphadenectomy prevents postburn systemic spread of translocated bacteria.

We investigated the role of mesenteric lymph nodes in postburn systemic spread of intestinal bacteria. Group 1 minipigs (n=8) had a 40% third-degree burn. Group 2 minipigs (n=7) had the same burn injury, but their mesenteric lymph nodes were removed immediately after burn. Group 3 minipigs (n=8) had sham burn, and group 4 minipigs (n=6) had mesenteric lymph node removal under anesthesia. All minipigs were killed at 48 hours, and tissues were harvested for bacteriological culture. Group 1 showed a large number of positive cultures from several of the systemic organs. Group 2 demonstrated no positive cultures in any of the tissues except the peritoneal fluid. These data suggest that bacterial translocation occurs mainly via mesenteric lymphatics to mesenteric lymph nodes and, thence, into other systemic tissue. After major burns, mesenteric lymph nodes may become an additional focus of infection.     

Effects of anesthesia, surgery, fluid resuscitation, and endotoxin administration on postburn bacterial translocation

The aim of the study reported here was to assess the effects of some clinically relevant factors on the incidence and outcome of postburn bacterial translocation. Miniature pigs in 8 groups (n = 6 in each) underwent: (1) general anesthesia (GA); (2) operation (insertion of Swan-Ganz, arterial, and portal catheters) under GA; (3) burn (40% total body surface area, third degree, under GA); (4) burn and operation; (5) burn, operation, and resuscitation (Parkland); (6) burn, operation, and resuscitation plus endotoxin (100 micrograms/kg IV bolus, 2nd day). Groups 1-6 were killed at 48 hours and tissue samples were harvested for bacteriologic culture. Groups 7 and 8 were the same as 2 and 5, respectively, but were killed at 96 hours. Resuscitation and endotoxin increased postburn bacterial translocation but only endotoxin promoted systemic sepsis. In the absence of additional trauma, translocated bacteria were cleared by 96 hours postburn.     

Bacterial translocation to the thoracic duct in a setting of ischemia, partial resection and reperfusion of the porcine liver.

BACKGROUND/AIMS: Bacterial translocation is postulated as a risk factor in the development of a systemic inflammatory response syndrome (SIRS). Research on this topic has focused on the detection of bacteria and endotoxin in blood or mesenteric lymph nodes (MLNs). We investigated whether bacterial translocation occurs beyond the MLNs into the thoracic duct in a setting of ischemia, partial resection and reperfusion of the porcine liver. METHODS: A porcine model of severe, extra-intestinal tissue injury, consisting of prolonged hepatic ischemia and reperfusion, in combination with hemihepatectomy, was used (experimental group, n = 5 pigs). To prevent venous congestion of the gut during ischemia, a temporary portal-caval shunt was created. In 5 animals (sham group) a sham portal-caval shunt was constructed while liver ischemia, partial resection and reperfusion were not induced. Thoracic duct lymph, portal blood and systemic blood were collected, and analyzed for the presence of bacteria and endotoxin. RESULTS: In the experimental group, the incidence of bacterial translocation to the thoracic duct was significantly higher during early reperfusion compared to the sham group (5/5 animals versus 1/5 animals, p < 0.05). CONCLUSION: This study demonstrates bacterial translocation into the thoracic duct. Translocation at this level leads to direct discharge of bacteria and endotoxin into the systemic circulation and therefore, may potentially enhance the development of SIRS.     

益生菌与核黄素联用对烫伤大鼠肠道屏障的保护作用

目的　观察益生菌与核黄素联合应用对烫伤大鼠细菌移位的防治效果 ,探讨其可能的作用机制。　方法　将Wistar大鼠随机分为烫伤对照组 (SC组 ,30只 )、烫伤治疗组 (ST组 ,30只 )、正常对照组 (NC组 ,10只 )。SC、ST组大鼠作 30 %ⅢTBSA度烫伤 ,ST组大鼠伤后立即向胃中灌注含双歧杆菌 5× 10 12 个集落形成单位 /L、蜡样芽孢杆菌 5× 10 10 个集落形成单位 /L和核黄素 5 0 0mg/L的等渗盐水混悬液 1.5ml,2次 /d。SC、NC组于相同时间灌注等量等渗盐水。观察细菌移位、肠道膜菌群、回肠分泌型免疫球蛋白A(SIgA)合成分泌及肠黏膜损伤修复等变化。　 结果　与SC组比较 ,ST组大鼠各脏器细菌移位率显著下降 (P =0.0 0 0～ 0.0 2 5),血浆内毒素水平在伤后 3d内降低显著 (P <0 0 5 ),回盲部膜菌群中双歧杆菌量升高 2 0～ 4 0倍 ,大肠杆菌和真菌量显著降低 ( P <0.0 1),致伤后 5d内黏膜损伤评分为 0～ 3(P <0.0 5),小肠黏液SIgA含量伤后 5d可恢复正常 ( P <0 0 1)。结论　益生菌与核黄素联合应用 ,可减轻烫伤大鼠细菌 /内毒素移位程度 ,有效保护肠道屏障     

Experimental study of the effects of splenectomy and partial splenectomy on bacterial translocation.

BACKGROUND: The aim of this study is to evaluate the effect of splenectomy and partial splenectomy in a burn-induced bacterial translocation model and to study Kupffer cell (KC) morphology and number. METHODS: Mice were divided into sham-burn and burn groups. Each group was also subdivided to sham-splenectomy, partial-splenectomy, and splenectomy subgroups. At day 0, operations were performed. At the postoperative 10th day, a sham burn or burn injury was made in all animals. Twenty-four hours later, cultures for bacterial translocation were obtained and livers were evaluated for the quantity and morphology of KCs. RESULTS: Burned-splenectomized animals had significantly decreased bacterial translocation when compared with sham-splenectomized animals (p = 0.031). Interestingly, in both the sham-burned and burned groups, splenectomy subgroups had significantly higher numbers of KCs compared with partial-splenectomy and sham-splenectomy subgroups (p<0.00000). Burn injury caused a significant decrease of KC numbers in all subgroups compared with their correspondent sham-burned subgroups (p<0.05). CONCLUSION: Results revealed that splenectomy decreases bacterial translocation and also increases the number of KCs.     

原位肝移植大鼠肠道微生态状况研究

目的 探讨肝移植术后大鼠肠道微生态的变化.方法 将雄性Brown-Norway(BN)大鼠40只随机分成肝移植组(BN→BN,n=16,共8对)、模拟移植组(n=8)、假手术组(n=8)和对照组(n=8),24 h后处死,分析肠道菌群构成、回肠末端超微结构变化、血浆内毒素水平以及细菌易位至肝、脾、肾和肠系膜淋巴结的比例.结果 肝移植术后24 h存在明显的肠道菌群紊乱,表现为肠杆菌科细菌和肠球菌数量增加(P<0.05),乳杆菌和双歧杆菌数量下降(P<0.05),移植组与模拟移植组存在肠黏膜上皮细胞微绒毛的损伤;肝移植组血浆内毒素水平升高(P<0.01),细菌易位至肝脏、脾脏和肠系膜淋巴结的阳性率增加(P值均<0.05);与模拟移植组比较,肝移植组细菌易位至肝脏的阳性率增加(P<0.05).结论 肝移植术后存在一定程度的肠道微生态紊乱和肠道屏障功能损伤,可能与肝移植手术过程中所经历的缺血再灌注过程有关.     

急性坏死性胰腺炎时肠屏障损害及肠源性细菌和内毒素移位的实验研究

目的 观察急性坏死性胰腺炎（ANP）时肠粘膜屏障的改变和肠源性细菌和内毒素移位。方法 将Wistar大鼠随机分为正常对照组（n=6)、假手术组（n=30)和ANP组（n=39)。采用人工胆汁胰管逆行灌注法制作ANP模型。观察胰腺、小肠病理改变和小肠粘膜上皮细胞间紧密连接（冷冻蚀刻电镜）变化。动态测定血浆D-乳酸、内毒素水平,以及腹腔脏器细菌移位率。结果 ANP后小肠粘膜损伤,皮皮细胞间紧密连接破坏甚至消失,血浆D-乳酸水平上升,发病早期即出现内毒素血症;ANP发生后72h脏器细菌移位率达到59.5%。结论 ANP早期肠粘膜屏障功能受损。导致肠道细菌和内毒素移位,成为全身炎症反应和胰腺继发感染的根源。     

中药清胰汤及双歧杆菌合剂对急性坏死性胰腺炎肠道细菌移位影响的比较研究

目的 观察中药清胰汤(CM)及双歧杆菌合剂(BM)对犬急性坏死性胰腺炎(ANP)肠道细菌移位(BT)的影响。方法 杂种犬31只,分对照组(n=7)、ANP组(n=8)、中药治疗组(CM,n=8)和双歧杆菌合剂治疗组(BM,n=8)。结照组犬仅行剖腹术;经主胰管注入5％牛磺胆酸钠(0.5ml/kg)和胰蛋白酶(3000U/kg)复制ANP模型,CM组和BM组犬术后每天分别经胃管灌服中药清胰汤及双歧杆菌合剂。结果 与ANP组比较,CM及BM组肠粘膜损害明显减轻;肠粘膜菌群中大肠杆菌、类杆菌数量减少,双歧杆菌、乳杆菌数量显著增加(P<0.05),肠道微生态趋于平衡;CM组脏器细菌移位率减少SO％,BM组减少37.5％,两组移位细菌数量减少10～40倍:两组血培养阳性率均由100％降为37.5％;两组血中内毒素水平下降1～2倍,淀粉酶水平下降2～3倍,磷脂酶A_2活性亦显著下降,并均于第7天后接近正常;肠通透性下降。结论 中药清胰汤及双歧杆菌合剂具有减轻ANP后肠粘膜损害,调节肠道菌群微生态平衡、保护肠屏障功能,从而减少BT致肠源性感染的作用     

The gut as a portal of entry for bacteremia. Role of protein malnutrition.

The current studies were performed to determine the influence of malnutrition alone or in combination with endotoxemia in promoting bacterial translocation from the gastrointestinal tract. Bacterial translocation did not occur in control, starved (up to 72 hours), or protein-malnourished (up to 21 days) mice not receiving endotoxin. Bacterial translocation to the mesenteric lymph nodes (MLNs) occurred in 80% of control mice 24 hours after receiving endotoxin (p less than 0.01). However, the combination of malnutrition plus endotoxin was associated with a higher incidence of translocation to the systemic organs (p less than 0.01), and higher numbers of bacteria per organ (p less than 0.01), than was seen in normally nourished mice receiving endotoxin. Additionally, mice that were protein malnourished were more susceptible to the lethal effects of endotoxin than were control animals, and the mortality rate was directly related to the degree of malnutrition (R2 = 0.93) (p less than 0.05). Histologically, endotoxin in combination with protein malnutrition resulted in mechanical damage to the gut mucosal barrier to bacteria. Thus, in the mice that were protein malnourished the spread of bacteria from the gut could not be controlled nor could translocated bacteria be cleared as well as normally nourished mice receiving endotoxin. These results support the concept that under certain circumstances the gut may serve as a clinically important portal of entry for bacteria.