Annals of
Burns and Fire Disasters - vol. XIII - n° 1 - March 2000
EFFECTS OF TREATMENT
WITH IMMUNOGLOBULIN ON BACTERIAL TRANSLOCATION IN BURN WOUND INFECTION
Herek O., Ozturkk H., Ozyurt M., Albay A., Cetinkursun S.
Department of Paediatric Surgery, Pamukkale
University Faculty of Medicine, Denizii,
Turkey and Departments of Paediatric Surgery and Microbiology,
Gfflhane Military Academy Faculty of Medicine, Ankara, Turkey
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.
Introduction
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.
Material and methods
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.
Results
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).
Groups |
Incidence
of BT |
Mesenteric
lymph nodes |
Spleen |
Liver |
Blood |
I. Sham
II. Burn + |
0/11(0%) |
0/11 (0%) |
0/11 (0%) |
0/11 (0%) |
0/11 (0%) |
Infection |
9/11 (82%) * |
9/11 (82%) * |
8/11 (72%) ++ |
6/11 (55%) ¶ |
1/11 (9%) |
Ill. Burn +
infection +
immunoglobulin |
7/11 (64%)
* |
6/11 (55%)
+ |
1/11 (9%) |
0/11 (0%) |
0/11 (0%) |
* p <
0.01 compared with sham group
+ p < 0.05 compared with sham group
+ + p < 0.91 compared with sham and burn + infection + immunoglobulin group
¶ p < 0.05 compared with sham and burn + infection + immunoglobulin group |
|
Table I
- Incidence and sites of bacterial translocation (BT) |
|
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.
Group
II |
Group III |
1406
± 105* |
1041 ± 80 |
Mean ± SEM
colony-forming units/g tissue
* p < 0.05 |
|
Table II -
Quantitative results of mesenterie lymph node cultures |
|
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).
|
Mesenteric
lyrnph nodes |
Spleen |
Liver |
Blood |
Group II |
1/11 (9%) |
2/11 (18%) |
2/11 (18%) |
8/11 (73%)* |
Group III |
0/11 (0%) |
0/11 (0%) |
0/11 (00/0 |
2/11 (18%) |
* p <0.05
compared with Group III |
|
Table III - Incidence and
sites of invasion of Pseudomonas infection |
|
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).
Group I |
Group 11 |
Group III |
23 ± 6.6 |
25300 ± 13522* |
3350 ± 1222*+ |
Mean ± SEM x 104
colony-forming units/g caecal contents
* p < 0.01 compared with Group I
+ p >0.05 compared with Group II |
|
Table IV -
Quantitative results of total bacteria in caecal cultures |
|
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. |
|
Discussion
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.
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This paper was received on
22 November 1999. Address
correspondence to:
Ozkan Herek, MD
Pamukkale Oniversitesi Tip FakUltesi Cocuk Cerrahisi Anabilint Dali Hastane Caddesi
20100 Denizli, Turkey. |
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