A PRELIMINARY STUDY OF BIFIDOBACTERIA FEEDING ON THE PREVENTION OF BACTERIAL TRANSLOCATION IN SEVERELY BURNED RATS

Chen J., Zhang Y., Xiao G., Li A.(N.)

Institute of Burn Research, Southwestern Hospital, Third Military Medical University
Chongqing, People's Republic of China

SUMMARY. Disruption of the ecological balance of the normal indigenous microflora is one of the major pathogenic factors responsible for bacterial translocation. This study was conducted to evaluate the role of Bifidobacteria feeding in the prevention of bacterial translocation in severely burned rats. Antibiotics and Bifidobacteria were orally administered to rats which had sustained 30% T13SA scalding on the back, maintaining the intestinal biological barrier. The rats were randomly divided into a Bifidobacteria feeding group, a burn group, and a normal group. In the first two groups, the bacterial contents in the caecum, the incidence of bacterial translocation, caecal secretory immunoglobulin A (slgA), and endotoxin level in the vena cava were monitored respectively 24, 48, 72 and 96 hours post-burn. The results showed that the content of bacterial flora in the caecum changed considerably, i.e. the number of anaerobes decreased, while that of Enterobacteria and Candida increased significantly 24 hours post-burn, which suggests that the ecological balance of the normal indigenous microflora was disrupted in the post-burn period. In the burn group, caecal sIgA decreased significantly, but the bacterial translocation rate and the plasma toxin level increased. In contrast, the plasma endotoxin level and the bacterial translocation rate in the Bifidobacteria feeding group decreased, whereas caecal sIgA content increased and caecal content changed slightly. These results suggest that Bifidobacteria could in some measure prevent the translocation of both endotoxin and bacteria from the gut lumen to the internal organs.

Bacterial translocation has generally been defined as the transportation of viable enteric bacteria passing through the intestinal epithelial cell barrier to the mesenteric lymph nodes (MLN) and other distant organs. It has been widely accepted that alteration of intestinal flora is one of the major pathogenic factors responsible for bacterial translocation. Bacterial translocation has been reported in several animal burn models. Bifidobacteria sp. are a natural component of the dominant colonic anaerobic flora within the intestinal cavity.'They play a critical role in keeping the balance of normal intestinal microflora. The purpose of this study was to investigate the possible role of Bifidobacteria feeding in the prevention of bacterial translocation in severely burned rats.

Adult Wistar rats (weight 190-220 g) in equal sex ratio were used in the study. The animals were quarantined for one week in standard laboratory conditions so that they could adapt to experimental conditions and be identified and excluded if they presented pre-existing diseases.

The rats were intraperitoneally anaesthetized with pentobarbital sodium (30 mg/kg). The backs of the rats were shaved and each rat was placed in a wooden holder containing a window exposing the back. The rats'backs were then immersed in hot water (92 'C) for 18 seconds, causing a full skin-thickness burn in approximately 30% total body surface area (TBSA). Eight nal of sterile normal saline were injected intraperitoneally as a routine measure to prevent burn shock.

The rats were randomly divided into three groups. In group 1 (n = 40), the treated group, the rats were given 2 mI doses of Bifidobacteria every 8 hours. In group 11 (n = 40), the burn group, the rats were given water (2 mI every 8 hours). In group 111 (n = 10), the normal control group, nothing was given. After the day of scalding, the rats received in their drinking water 500 units/mI of penicillin and streptomycin. The drinking water pot was renewed every 24 h and the rats continued to receive penicillin and streptomycin throughout the experiment.

Faecal samples were collected 24, 48, 72 and 96 hours post-burn, placed in sterile tubes, weighed and diluted ten times (10-1-1011) with reduced 0.05% yeast extract solution. One-tenth mI of each dilution was placed in selective and non-selective media and the plates were incubated under aerobic and anaerobic conditions for a period of 24-72 hours. The media employed contained 5% sheep blood agar for the isolation of aerobic gram-positive organisms, MacConkey agar for the isolation of aerobic gram-negative enteric organisms, BBL agar for Bifidobacteria, GAM agar for anaerobic organisms, and Soubro agar for Candida.

The peritoneal cavity was exposed in an aseptic operation. The MI-N, liver, spleen, and kidneys were removed aseptically in serial time schedule. Each organ was homogenized in sterile conditions and diluted serially (wt/vol 1/10) in normal saline. The suspensions were then cultured on 5% sheep blood agar and incubated aerobically at 37 'C. Bacteria colonies on each plate were counted after 24 hours. Positive results were identified by the growth of bacteria on the plates, which suggested the existence of translocated bacteria in these organs.

A modification of the chromogenic limulus amoebocyte lysate technique was employed to measure plasma endotoxin levels, which were expressed as pg/ml.

Measurement of caecal secretory immunoglobulin A (sIgA) The radioimmunoassay method was employed to determine sIgA faecal content, and the results were expressed as mg/g. All data were processed by the chi-square test. A p value less than 0.05 was considered significant.

The results showed an obvious difference of rat caecal microflora between the two groups of rats , i.e., a 100-fold increase of the populations of Enterobacteria and Candida in the caecum were found in the burn group. In the treated group the amount of bacteria changed only slightly, but to a greater extent than in the normal group. The amount of Bifidobacteria in the treated group was much higher than in the burned group but lower than in the normal group.

As shown in Table H the bacterial translocation rate in the burn group was much higher than in the Bifidobacteria treated group, except for 24 hours post-burn, and higher than in the normal control group.

The data presented show that the levels of plasma endotoxin in rats of both the treated and the untreated groups increased during the early post-burn period. The plasma endotoxin concentration was however significantly lower in the Bifidobacteria treated group than in the burn group 48, 72 and 96 hours post-burn, yet higher than in the normal control group.

The data revealed that sIgA levels decreased during the early post-burn stage in both the treated and the untreated groups, compared with the normal group, but were significantly higher in the treated group than in the burn group 48, 72 and 96 hours post-burn.

In order to simulate clinical conditions as closely as possible, the rats received a low dose (500 units/ml) of penicillin and streptomycin in their drinking water from the time of the burn injury. Our data showed that the anaerobic flora in the burn group decreased 1000-fold and that the number of Bifidobacteria also dropped markedly. There was however a significant increase in the number of Enterobacteria and Candida. These results demonstrate the alteration of intestinal bacterial microflora. The overgrowth of Enterobacteria may be one of the major mechanisms of bacterial translocation. Previous studies have already shown that Bifidobacteria sp. belong to the dominant anaerobic flora in the colon in normal conditions. The anaerobic bacteria form a kind of paste on the intestinal surface, and their presence has been shown to inhibit the epithelial cell receptors from adhering to gram-negative bacteria, thus preventing bacterial translocation.1,1 The data obtained from the Bifidobacteria treated group showed that the quantity of anaerobic flora was significantly higher than in the burn group, while the number of Enterobacteria and Candida in the treated group was lower than in the burn group. These results suggest that Bifidobacteria feeding could to some extent correct the abnormal alteration of intestinal flora.
Our experiments show that the incidence of bacterial translocation to the XILN and other organs as also the level of plasma endotoxin in the control groups was significantly higher than in the Bifidobacteria treated group. Post-burn, the drop in anaerobic bacteria and the overgrowth of gram-negative bacteria inevitably lead to bacterial translocation. The administration of antibiotics and the overgrowth of gram-negative bacteria may cause the bacteria to release large amounts of endotoxin, which then affect the intestinal mucosa - thus leading to the disruption of the mechanical barrier." As a consequence, many inflammatory cells are induced to secrete a vast variety of inflammatory fators, leading to multiple organ failure.
SIgA is the most abundant immunoglobulin in the mucosal immune system," and it has been demonstrated that it selectively coats gram-negative enteric flora." Our data indicate that burn injury causes a significant decrease in the sIgA of caecal contents, and bacterial translocation to MLN and other internal organs. In the Bifidobacteria treated group, there was a clear increase in sIgA content. Also, the incidence of bacterial translocation was significantly lower than in the burn group, which indicates that Bifidobacteria feeding could increase the sIgA level in the gastrointestinal tract, help to coat gram-negative bacteria, and prevent sIgA-coated bacteria from translocation.
We may thus conclude that the intestinal bacterial microflora is disrupted following a burn lesion, and that Bifidobacteria feeding could inhibit gram-negative bacteria overgrowth, maintain the balance of normal flora, and prevent gut bacteria from translocating to other organs.

RESUME. Le dérangement de l'équilibre écologique de la microflore indigène normale est un des principaux facteurs pathogènes responsables de la translocation bactérienne. Les Auteurs ont effectué cette recherche pour évaluer le rôle de l'alimentation avec les Bifidobacteria dans la prévention de la translocation bactérienne dans les rats atteints de brûlures sévères. Les antibiotiques et les Bifidobacteria ont été administrés par voie orale à des rats soumis à des brûlures d'ébouillantement dans le dos équivalentes à 30% de la surface corporelle, conservant la barrière biologique intestinale. Les rats ont été divisés au hasard en trois groupes: un premier qui a reçu l'alimentation avec les Bifidobacteria, un deuxième soumis à la brûlure, et un troisième groupe normal. Dans les premiers deux groupes, les Auteurs ont contrôlé le contenu bactérien du caecum, l'incidence de la translocation bactérienne, l'immunoglobuline sécrétoire caecale A (slgA), et le niveau de l'endotoxine dans la veine cave respectivement à 24, 48, 72 et 96 heures après la brûlure. Les résultats ont montré que le contenu de la flore bactérienne dans le caecum changeait considérablement, c'est-à-dire le numéro des anaérobies diminuait, tandis que les Enterobactéries et la Candida augmentait en manière significative 24 heures après la brûlure, ce qui ferait penser que l'équilibre écologique de la microflore indigène normale était disturbé dans la période à la suite de brûlure. Dans le groupe brûlé la sIgA caecale dirnin4ait en manière significative, mais le taux de la translocation bactérienne et le niveau de la toxine plasmatique augmentaient. Par contraste, le niveau de l'endotoxine plasmatique ' et le taux de la translocation bactérienne dans le groupe alimenté avec les Bifidobacteria augmentaient, tandis que le contenu de la sIgA caccale changeait légèrement. Ces résultats semblent indiquer que les Bifidobacteria pourraient dans une certaine mesure prévenir la translocation des endotoxines et des bactéries de la lumière de l'intestin jusqu'aux organes internes.

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