Annals of Burns and Fire Disasters - vol. XII - n. 1 - March 1999

SUMMARY. Certain strains of Bifidobacteria may promote recovery from acute diarrhoea. Bifidobacteria are of human origin and are natural colonizers of the gastrointestinal tract. We therefore investigated the possible role of Bifidobacteria preparation in the treatment of children with post-burn diarrhoea. Thirty-one severely burned children with diarrhoeal complications were studied. The children received 10' colony-forming units of Bificlobacteria preparation daily for six days. The clinical outcome of diarrhoea and stool microflora was evaluated. The results showed that the intestinal flora of all children with diarrhoea changed greatly before treatment and that the proportions of Bifidobacteria and bacteroid decreased significantly, whereas those of aerobes and Candida were relatively increased. The ratio of anaerobes to aerobes decreased. As a result, the children's intestinal flora was altered. After six days of treatment with Bifidobacteria preparation, the cultures of Bifidobacteria from stool samples demonstrated that administration of Bifidobacteria resulted in their colonization of the gastrointestinal tract. The diarrhoea in most of the children ceased and the intestinal microflora was restored. It is thus concluded that Bifidobacteria preparation is effective as a therapeutic reagent in post-burn diarrhoea in children.

Normal gastrointestinal microflora is very important in the protection of the host against diseases of the gastrointestinal tract."' Several studies have indicated that the administration of probictic agents attenuates acute episodes of diarrhoea." Diarrhoea is a common complication in burned children.' Bifidobacteria sp. are a natural component of the dominant colonic anaerobic flora within the intestinal lumen and play a critical role in maintaining the equilibrium among normal intestinal microflora.' The purpose of this study was to investigate the possible effect of Bifidobacteria preparation on the treatment of diarrhoea in burned children.

Clinical data
TREATMENT GROUP: The thirty-one severely burned children (22 males, 9 females) enrolled in the study were admitted to our burn ward between November 1995 and October 1997 with an average total burn surface area of 8.7% (range, 3-37%) and an average age of 6.4 yr (range, 13-10.0 yr). The causes of the burns were fire (11 cases), hot liquid (19 cases), and electricity (1 case). All the patients survived. The enrolment criteria for the children were:

1. aged below 10 yr;
2. free of renal dysfunction or failure;
3. stable dynamic blood circulation;
4. eligible for oral or nasal feeding;
5. no indication of bloody purulent stool on laboratory examination.

NORMAL GROUP: Twenty normal children (17 male, 3 female) were taken as a normal group (average age, 6.1 yr; range, 2-10 yr). None of these normal children had any recent history of taking antibiotics, hormones, other immunosuppressive agents, or radio- and chemotherapeutic agents. Sample taking was the same as that in the treatment group.

Experimental procedures
All the children were treated according to our routine methods. Bifidobacteria preparation was given to the diarrhoeal children immediately after the occurrence of diarrhoea, at a dose of 10 ml per dose three times a day. One millilitre of agent contained 2.6 x 10' of live Bifidobacteria. The faecal samples were collected prior to Bifidobacteria preparation feeding, and 3 and 6 days later. During the period of study there was no oral administration of antibiotic or antidiarrhoeal drugs.

Analysis of Jaecal microflora
All specimens were taken immediately to the laboratory and processed within 1 h. Half a gram of specimen was placed in an anaerobic chamber, diluted 10fold in reduced 0.05% yeast extract solution, and emulsified in a vortex mixer. Serial 10-fold dilutions were made in a reduced 0.05% yeast extract solution. Samples (0.1 ml) of selected dilution were plated on selective and non-selective aerobic, anaerobic, and Candida media for enumeration of members of the intestinal microflora. The plates were incubated at 37 'C for 24 h for aerobic cultures or for 48 h in anaerobic chamber for anaerobic cultures. Plates that showed 30 to 300 colonies were used for bacterial counts. The incubated bacteria or Candida were counted and identified.

Clinical observation
The frequency of faecal discharge was monitored every day, as well as its appearance and whether or not there was diarrhoea. Body weight and gastrointestinal reactions were also recorded.

The results were expressed as mean + SD. The level of significance (p) was calculated by Student's t-test.

Changes in faecal bacterial microflora
The Table shows that the intestinal microflora changed remarkably during the course of diarrhoea. For example, there was an obvious decrease in the total amount of intestinal bacterial. The rates of Bifidobacteria and bacteroid clearly decreased, as also the E. coli rate. The Candida rate, however, clearly increased. After administration to the children of Bifidobacteria preparation, their intestinal Bifidobacteria and bacteroid rates increased, while the Candida rate decreased.

Clinical observation
Diarrhoea occurred 4-8 days post-burn in 31 children in the group. The stool appeared watery and increased in frequency to 3-4 times per day. However, experimental examination of the stool revealed no specific pathogenic germs. After oral administration of Bifidobacteria preparation, the diarrhoea ceased in two days in 19 children, in three days in eight children, and in four days in two children. Stool appearance returned to normal simultaneously. Stool production returned to once a day. The effective rate reached 93.5% (29/31). The children's appetite recovered satisfactorily and no side-effects were observed.

The normal bacterial microflora in the human intestinal lumen is an important microbiological species. The variable aerobes and anaerobes in normal human intestinal lumen maintain a relatively constant balance between their ratios. The microecological balance is maintained by the interaction between the host and these normal physiological bacterial species. Our results showed that total faecal flora decreased significantly (Bifidobacteria decreasing by nearly 1000 times), while E. coli and Candida clearly increased. This suggested an obvious disturbance of the intestinal bacterial flora, of which where would appear to be three major causes.
The first explanation is that the biological and microecological condition, which previously was adequate for colonization of normal intestinal bacterial flora, altered as a result of a series of post-burn pathological modifications. Consequently, the recessive flora dominated.
The second explanation is that enterokinesis increased because of diarrhoea, which accelerated the passing of intestinal content through the bowel, thus hampering the growth, multiplication, and colonization of intestinal microbes. Intestinal mucosal fluid was simultaneously lost in large quantities, leading to alteration of the intestinal flora. The microbiological barrier of the intestinal mucosa was destroyed, which provided more opportunities for the colonization of non-conventional bacteria.
The third explanation is that abuse of copious antibiotics killed large quantities of exogenous flora, which induced the overgrowth of some drug-resistant strains and of Candida. After oral administration of Bifidobacteria preparation, the quantity of intestinal Bifidobacteria increased, while that of Candida decreased. The altered intestinal flora therefore returned to near normal conditions. The ratio of aerobes to anaerobes decreased.
Oral administration of Bifidobacteria preparation therefore restored the microecological balance of bacterial flora and reconstructed the microbiological barriers that prevented the colonization of exogenous bacteria in the treatment of diarrhoea. Broad-spectrum antibiotics such as Tienam and Forturn have often been applied clinically to severely burned children in order to manage infection, but this has led to the generation of many drug-resistant strains, the death of normal flora, the destruction of the microecological balance of human intestinal flora, and bacterial translocation.'In addition, the beneficial clinical effect of Bifidobacteria preparation may result from other mechanisms, such as stimulation of the intestinal immune reponses. Diarrhoeal children in the trial ceased to manifest the effects after taking Bifidobacteria preparation, without any side-effects. We therefore recommend the use of Bifidobacteria preparation in burned children in order to restore the intestinal microfloral balance when this been disturbed by abuse of antibiotics.

RESUME. Certaines souches des Bifidobactères peuvent accélerer la guérison de la diarrhée aiguë. Les Bifidobactères sont d'origine humaine et sont des colonisateurs naturels de l'appareil gastrointestinal. Les Auteurs ont étudié le rôle possible de la préparation des Bifidibactères dans le traitement des enfants atteints de la diarrhée à la suite d'une brûlure. Ils ont pris en considération 31 enfants gravement brûlés atteints de complications diarrhéiques qui ont reçu quotidiennement pendant 6 jours la préparation des Bifidobactères capables de former des colonies. Les résultats cliniques de la diarrhée et de la microflore des fèces ont été évalués et ils ont démontré que la flore intestinale de tous les enfants diarrhéiques se sont modifiés notablement avant le traitement et que les proportions des Bifidobactères et des bactéroides ont diminué en manière significative, tandis que les proportions des aérobies et de la Candida augmentaient relativement. Le rapport aérobies/anaérobies diminuait. Par conséquence, la flore intestinale des enfants était altérée. Après six jours de traitement avec la préparation de Bifidobactères, les cultures de Bifidobactères obtenues des fèces démontraient que l'administration des Bifidobactères conduisait à leur colonisation de l'appareil gastrointestinal. Dans la plupart des enfants la diarrhée a cessé et la microflore intestinale a été réintegrée. Les Auteurs concluent que la préparation des Bifidobactères est efficace comme réactif thérapeutique dans la diarrhée des enfants brûlés.

BIBLIOGRAPHY

1. Fuller R.: Probiotics in human medicine. Gut, 32: 439-42, 1991.
2. Salminen S., Deighton M.: Lactic acid bacteria in the gut in normal and disordered states. Dig. Dis., 10: 227-38, 1992.
3. Branser 0., Araya M., Espinoza J., Guesry P.R., Secretin M.C., Pacheco I.: Effect of an acidified milk on diarrhea and the carrier state in infants of low socio-economic stratum. Acta Paediatr. Scand., 78: 259-62, 1989.
4. Boudraa G., Touhami M., Pochart P., Soltana R., Mary J.Y., Desjeux J-F.: Effect of feeding yogurt versus milk in children with persistent diarrhea. J. Pediatr. Gastroenterol. Nutt., 11: 509-12, 1990.
5. Li Ao: "Burn Treatment" (2nd ed.), People's Health Publishing Co., 228-36, 1995.
6. Kang Bai: Microecology in human gastrointestine. In: "Microecology" (Ist ed.), Da Lian Publishing Co., 203-7; 1988.
7. Dai Li-ren: The intestinal microflora change in acute bacillary dysentery and the observation of therapeutic effects of Bifidobacteria preparation. Chinese J. Microecology, 3: 234-7, 199 1.
8. Perdigon G., Alvares S., Nader De Macias M., Roux M., Pesce de Ruiz Holgado A.: The oral administration of lactic acid bacteria increases the mucosal intestinal immunity in response to enteropathogens. J. Food Protec., 53: 404-10, 1990.
9. Perdigon G., Medici M., Bibas Bonet de Jorrat M.E., Valverde de Budeguer M., Pesce de Ruiz Holgado A.: Immunomodulating effects of lactic acid bacteria on mucosal and Immoral immunity. Int. J. Immunother., 9: 29-52, 1993.