Annals of Burns and Fire Disasters - vol. XIII - n° 1 - March 2000

EFFECTS OF TREATMENT WITH IMMUNOGLOBULIN ON BACTERIAL TRANSLOCATION IN BURN WOUND INFECTION

SUMMARY. The aim of this study was to evaluate the effects of inummotherapy on bacterial translocation in burn wound infection. Thirty-three male albino rats in three equal groups were used in the study. A sham burn group (Group 1) was exposed to 21 "C water. A burn + infection group (Group 11) and a burn + infection + immunoglobulin group (Group 111) were exposed to 95 'C water for 10 see, producing full-thickness bums in 30% total body surface area; Pseudomonas aeruginosa (I X 108) was inoculated immediately after the burn injury. Saline (I ml) was injected intramuscularly in Groups I and 11 as a placebo for four days. Immunoglobulin (500 mg/kg) was administered for four days in Group III after burn injury. All rats were sacrificed on day 5 post-bum. Cultures were performed of the mesenteric lymph nodes, liver, spleen, blood, and caecal contents. Blood samples (1.5 md) were taken for the determination of immunoglobulin levels. Bacterial translocation was not observed in the sham group. A significant increase in bacterial translocation was present in Groups 11 (9/11) and 111 (6/11) on the mesenteric lymph nodes compared with Group I (p < 0.0 1). In contrast, significant decreases in translocation to the spleen (1/11) and liver (0/11) were observed in Group III compared with Group 11 (8/11, 6/11) (p < 0.01, p < 0.05). A significant increase in the total number of bacteria was found in caecal stool cultures in Groups 11 and III compared with Group I ~p < 0.01). These results suggested that the incidence of bacterial translocation to the mesenteric lymph nodes does not significantly change with immunotherapy in burn wound sepsis, while the administration of immunoglobulin protects the immunological and intestinal equilibrium. Immunotherapy therefore prevents the spread of translocated bacteria from the mesenteric lymph nodes to other sites.

Bacterial translocation has been defined as the passage of viable or non-viable bacteria and their products, including endotoxin, across an anatomically intact intestinal barrier to the mesenteric lymph nodes and beyond.` Recent studies have shown that bacterial translocation is caused and enhanced by haemorrhagic shock, burn injury, intestinal obstruction, total parenteral nutrition, antibiotic therapy, malnutrition, and trauma. In these conditions, failure of the gut barrier, the imbalance of intestinal flora, and impaired host immune defences are identified as major causes of bacterial translocation.
Animal studies have shown that thermal injury enhances bacterial translocation. Clinical and experimental studies have demonstrated that mesenteric blood flow significantly decreases and that intestinal ischaemia occurs after burn injury. The intestinal mucosal barrier is damaged by this intestinal ischaernia, which causes bacterial translocation In uncomplicated burn injury the intestinal mucosa generally heals and the gut barrier is repaired within four days. Thus, in uncomplicated burn injury, bacterial translocation does not occur or is not enhanced four days post-burn Conversely, bacterial translocation is enhanced and prolonged by insufficient fluid resuscitation, the use of H2-receptor blockers, and burn wound sepsis." Jones et al. demonstrated that bacterial translocation is enhanced and prolonged by burn wound infection due to intestinal atrophy and deficiency of the gut barrier. However, the effects of immunological alterations on bacterial translocation have not been well defined in infected burn injury. This study investigated the effects of itnammoglobulin therapy during burn wound infection on bacterial translocation.

This study was performed in the Research Centre of G51hane Military Academy Faculty of Medicine (Ankara, Turkey) with the approval of the Faculty's ethical committee.
Thirty-three male albino rats (weight 170-220 g) were used. All the rats were obtained from the Faculty Research Centre. The animals were randomly distributed in three groups: a sham burn group (Group 1, no. 11), a. burn + infection group (Group II, no. 11), and a burn + infection + inummoglobulin group (Group Ill, no. 11). The total body surface areas (TBSA) were calculated by the formula described by Horst et al. The backs of all the rats were shaved, on which 30% TBSA was determined. The burn trauma was performed using standard animal burn models, as described by Walker and Mason. The rats were anaesthetized with an intramuscular injection of 50 mg/kg ketamine sulphate. Group 1 was exposed to 21 °C water for control purposes. Group 11 and 111 animals were exposed to 95 'C hot water for 10 see in order to produce a fullthickness burn injury in 30% TBSA. A 1 x 108 colonyforming unit/ml of Pseudomonas aeruginosa was immediately inoculated after full-thickness burn injury in Groups 11 and Ill, as described by Yurt et al.' All the rats were resuscitated with 25 ml/kg 0.9% NaCI via subcutaneous injection. Intramuscular injections of immurroglobulin (Globuman, Berne) (500 mg/kg/day) were initiated immediately in Group 111 and the same volume of NaCI 0.9% was injected as a placebo in Groups 1 and 11 for four days.
All the rats were sacrificed on day 5 post-bum. Laparotomy was performed under intramuscular ketamine anaesthesia and a 2-mI blood sample was taken from the inferior vena cava for quantitative blood cultures and determination of immunoglobulin levels. The mesenteric lymph nodes and spleen, liver, and caecal contents were removed and homogenized for aerobic quantitative cultures. Swab cultures from burn wound infection were taken in Groups 11 and 111. The types and quantitative amounts of bacteria per g tissue/ml were determined 48 h later. The presence of 100 or more bacteria per g tissue/ml (except for Pseudomonas aeruginosa, which is accepted as a bacterial invasion from burn wound infection) was accepted as the criterion for translocation.
The Fisher exact and chi square tests were used for statistical analysis of the incidence of bacterial translocation and bacteraemia by Pseudomonas aeruginosa. The quantitative results of the cultures and the levels of immunoglobulin were analysed by One-Way ANOVA, Kruskal-Wallis, and Mann-Whitney U.

Wound infection was observed in all the rats in Groups II and III. Bacterial translocation was not observed in the sham burn group (Group I). The incidence of bacterial translocation was 9/11 (82%) in Group II and 7/1 1 (64%) in Group III. Bacterial translocation was significantly increased in Groups II and III compared with Group I (p < 0.01). The results for translocation were not significant with regard to Groups II and III (p > 0.05) (Table I).

The main site of translocation was only the mesenteric lymph nodes (6/11, 55%) in rats treated with immunoglobulin (Group III), while bacterial translocation was detected on the mesenteric lymph nodes (9/11, 82%), spleen (8/11, 72%), and liver (6/11, 55%) in Group II The incidence of bacterial translocation was significantly increased on the mesenteric lymph nodes in Groups II and 111 (p < 0.01, p < 0.05). There was no difference in the incidence of bacterial translocation on the mesenteric lymph nodes between Group II and Group III (p > 0.05); the incidence of bacterial translocation to the spleen and liver was however statistically significant in Group II compared with Groups I and III (p < 0.0 1 in the spleen and p < 0.05 in the liver). Translocation to blood was found in only one rat in Group II (l/1 1) (p > 0.05). These detta indicate that bacterial translocation did not spread beyond the mesenteric lymph nodes in burned infected rats treated with immunoglobulin. The results of quantitative mesenteric, lymph node cultures are shown in Table II.

These findings indicate that the quantitative results of positive mesenteric lymph node cultures were significantly increased in Group II compared with Group III (p < 0.05). E. coli, Proteus and Klebsiella were isolated from specimens as mainly translocated bacteria.
The incidence of bacteraemia of Pseudomonas was found to be significantly decreased in GroupIII  (2/11, 18%), unlike Group II (8/11, 73%) (p < 0.05) (Table III).

The total bacteria count of caecal flora significantly increased in Groups 11 and 111 compared with Group 1 (p < 0.01). There were no statistical differences between Groups II and III (p > 0.05) (Table IV).

The levels of immunoglobulin G and M are shown in Fig. 1. IgG was significantly increased in Group 111 compared with Groups I and II (p < 0.05 and < 0.01, respectively). Although IgG was slightly decreased in Group II, it was not statistically significant from the sham group (p < 0.05).

Fig. 1 - Levels of immunoglobulin in the three groups.

Infection and sepsis are the most important causes of mortality and morbidity, despite advances in burn injury care. Burn injury impairs both immoral and cellular immunity in addition to causing defects in local barriers This immune suppression increases the susceptibility to infection, and infection, endotoxaemia, and inflammatory responses to the burn wound are the cause of systemic immunosupression. Studies have demonstrated that immunosuppression promotes bacterial translocation due to bacterial overgrowth. Jones et al. observed in an experimental study that bacterial translocation is prolonged at the level of the mesenteric lymph nodes with the elevation of corticosteroids, in burn injury.
Bacterial translocation may lead to septic complications in severely burned patients, especially in the early period of burn trauma when blood endotoxin levels rise without wound infection. A significant decrease in mesenteric blood flow was demonstrated 4 h after a 30% scald burn in experimental studies. Mesenteric blood flow returns to baseline levels 24 h post-bum. This low-flow state causes damage to intestinal mucosa and gut barrier function. For these reasons, bacterial translocation significantly increases during the first 24-48 h post-bum. In general, intestinal mucosa and gut barrier function heals within 4 days post-bum. Bacterial translocation does not occur after 4 days post-burn in uncomplicated burn injury.
Although Jones et al. demonstrated that bacterial translocation in burn wound sepsis is enhanced and prolonged owing to continued intestinal atrophy and gut barrier defect, the effects of immunological responses on bacterial translocation with burn wound infection have not yet been well defined.` The effects of immunological alterations on bacterial translocation have recenlly been studied in other experimental models. Dijkstr:a et al. showed that bacterial translocation was not decreased with an increase of IgG titres against intestinal Neomycinresistant Escherichia coli.` Eaves-Pyles and Alexander recently demonstrated that murine granulocyte colonystimulating factor (NIG-CSF) enhanced the ability to kill translocated organisms and improved survival in gutderived sepsis models. It was also found in the same study that MG-CSF did not improve gut barrier function or the incidence of bacterial translocation.` In our study we found no difference between the burn infected group (Ciroup 11) and the immunoglobulin group (Group 111) as regards the incidence of bacterial translocation (82% and 64%, respectively). However, bacterial translocation was detected predominantly on the mesenteric lymph ~nodes in the immunotherapy group, and was found on mesenteric lymph nodes, spleen, and liver in the burn infected group. The increase in bacterial translocation in the spleen and liver was significant in Group 11 (p < 0.01, p < 0.05). These data suggest that the administration of immunoglobulin in burn wound sepsis does not decrease bacterial translocation at the level of the mesenteric lymph nodes.
However, immunotherapy protects the ecological equilibrium in the intestinal microflora, decreases the number of translocated bacteria, and prevents the spread of bacterial translocation from the mesenteric lymph nodes. On the other hand, the incidence of bacterial invasi[on from burn wound infection is significantly decreased and infection is localized by immunotherapy. Immunotherapy thus prevents the other side effects of bacterial invasion. For these reasons immunotherapy effectively prevents bacterial translocation from spreading beyond the mesenteric lymph nodes.
Gamma globulin therapy has been proposed and used for the treatment of sepsis and infection in bums. Kefalides et al. reported that gamma globulin administration reduced mortality from sepsis in children with burn injury. Holder et al. demonstrated that the use of intravenous immunoglobulin reduced bacterial counts in the skin and liver of rats infected with Pseudomonas aeruginosa. They suggested that intravenous immunoglobulin could be used successfully to treat Pseudomonas aeruginosa infections in burns. Jones et . al. also demonstrated that mortality was lower (0%) in burned children treated with Pseudomonas immunoglobulin than in controls (21%). We could not use anti-Pseudomonas immunoglobulin in our study. Therapy using Pseudomonas inummoglobulin in burn wound infection may be more effective on bacterial translocation than treatment using general immunoglobulin.
In conclusion, the administration of inummoglobulin does not decrease bacterial translocation at the level of the mesenteric lymph nodes and does not improve gut barrier function. However, the use of inimunoglobulin treatment decreases the incidence of bacteraemia from burn wound infection, protects the intestinal ecological equilibrium by decreasing bacterial overgrowth in the intestinal microflora, decreases the number of translocated bacteria, and prevents bacterial translocation spread beyond the mesenteric lymph nodes. For these reasons, the administration of inummoglobulin in burn wound infection effectively prevents sepsis duto to bacterial translocation.

RESUME. Le but des Auteurs dans cette étude était d'évaluer les effets de l'immunothérapie sur la translocation bactérienne dans l'infection des brûlures. Trente-trois rats mâles albinos ont été utilisés. Les rats du premier groupe (groupe 1, 11 rats) ont été exposés à l'eau à 21 'C. Dans le deuxième groupe (groupe 11, 11 rats) et le troisième groupe (groupe 111, 11 rats), définis respectivement groupe brûlures + infection et groupe brûlures + infection + immunoglobuline, les animaux ont été exposés à l'eau à 95 'C pour 10 sec pour produire des brûlures à toute épaisseur dans 30% de la surface corporelle totale, et une quantité de 1 x 108 Pseudomonas aeruginosa a été injectée en manière intramusculaire pour 4 jours comme placebo. Le groupe 111 a reçu 500 mg/kg d'immunoglobuline après la brûlure pour 4 jours. Tous les rats ont été sacrifiés 5 jours après la brûlure. Les cultures des ganglions lymphatiques mésentériques, du foie, de la rate, du sang et du contenu cécal ont été effectuées, et du sang (1,5 ml) a été prélevé pour déterminer les niveaux de l'immunoglobuline. Les Auteurs n'ont pas relevé la translocation bactérienne dans le groupe 1 (fausses brûlures) mais ils ont observé une intensification significative dans la translocation bactérienne dans les groupes 11 (9/11) et 111 (6/11) sur les ganglions lymphatiques mésenteriques par rapport au groupe 1 (p < 0,01). Par contre, ils ont observé une diminution de la translocation vers la rate (1/11) et le foie (0/11) dans le groupe Il par rapport au groupe Il (8/11 et 6/11) (p < 0,01 et p < 0,05). Ils ont en outre observé une augmentation du numéro total des bactéries dans les cultures des fèces cécales dans les groupes Il et 111 par rapport au groupe 1 (p < 0,01). Les résultats semblent indiquer que l'incidence de la translocation bactérienne n'est pas modifiée en manière significative par l'immunothérapie à la suite de l'infection due aux brûlures. Par contre, l'administration de l'immunoglobuline protège l'équilibre immunologique et intestinal. Pour ces raisons l'immunothérapie prévient la diffusion des bactéries transloquées par les ganglions lymphatiques mésentériques vers des autres sites.