Nikitenko,V.I., DM, Professor, Department of Trauma Surgery, Orthopaedics and Military Surgery, Orenburg State Medical Academy, Russia. E-mail – bacoren@rambler.ru

Infection prophylactics of gunshot wounds using probiotics.

Objective – to report a new method of surgical infection prophylactics for postoperative gunshot wounds to the extremities. Method – gunshot wounded animals were divided into 3 groups : treatment (probiotic Sporobacterin), antibiotic ( cephalosporin cefamezin) and control ( no treatment). Histological studies of wound-bed tissue were taken on days 1,3,5 and 10 of the study.

Results – the probiotic administrated per os was more effective than antibiotics for prophylaxis of surgical infection. Conclusion – the probiotic`s effect is based on the natural defence mechanism activated after injury – the bacterial translocation of saprophytic bacteria from the gut to the wound.

Declaration of interest – None.

Surgical infection prophylaxis, bacterial translocation

Infection prophylaxis in surgery, particularly in war conditions and peacetime major accidents, is a high priority.1 In up to 50% of traumatic injuries, purulent inflammatory diseases of soft tissues occur. This is due to a number of factors – poor hygiene, inadequate feeding, delay n receiving treatment and the nature of wounds produced by modern weapons.

Prevention of infection is thus one of the main aims of gunshot wound management. According to military guidelines, antibiotics such as Cefazolin ( Kefzol/Cefalozolin), ampicillin and tetracycline are used prophylactically.2 However, cumulative clinical experience and published data testify, that these are denerally ineffective3,4d issues such as allergic or anaphylactic reactions, dysbacteriosis ( pathological alteration in the composition of gut microorganisms) and impaired resistance have to be considered.

Using probiotic bacteria

Investigations into the use of probiotic bacteria began in 1978. Initial bacteriological studies into animal wound healing by primary intention found bacteria in the deep tissues, commonly Bacillus subtilis, Bacillus licheniformis, Bacillus coagulants and Bacillus circulans. Subsequent studies revealed these bacteria originated in the gastrointestinal tract. Their relocation to the wound site is a phenomenon called bacterial translocation5 , which occurs trauma, burns and during shock and surgical manipulation.6 In a orevious study involving healthy animals7 , having traced the path of orally administrated bacteria in the gastrointestinal tract using radionuclide and bacteriological and morphological techniques, we found that some bacteria survived in tissues and could prevent surgical- site infection.

In a wound, translocated bacteria such as Bacillus subtilis, Lactobacillus acidophilius and Bifidobacterium bifidum can produce broad spectrum, antibacterial, biologically active substances that supperss growth of pathogenic and opportunistic microorganisms, promote cleansing and protect against necrosis.7 There was a chance discovery, so we set out to investigate this phenomenon in more detail. We have developed a new bacterial medicine, Sporobacterin ( Bacoren, Orenburg, Russia) which has been used in clinical practice in Russia since 1992. It is based on a live culture of Bacillus subtilis 534, isolated from human wounds healing by primary intention.

This study explores a new method of preventing surgical infection in gunshot wounds, based on bacterial translocation via topical application of probiotic medicine.

Method

This randomized blind experimental animal study compared efficacy of infection prophylactic agents in animal gunshot wounds. In vitro and in vivo, we also studied properties such s microbial antagonism of Bacillus subtilis 534, a component of Sporobacterin.

A small-bore rifle was used to create gunshot wounds in the soft tissue of the rabbit’s back. Forty – eight rabbits were wounded under total barbiturate anesthesia. Wounds were left exposed and not derided. The rabbits were divided into four groups :

Intramuscular injection of cetamezin ( licensed as ceftazidime in the UK) twice daily ( 0,1 g per 1 kg of weight of the animal) ( n=12)

Sporobacterin wound irrigation ( 0,2 ml twice daily) (n=12)

Control group ( no treatment) (n=12)

Clinical observation included:

Assessment of the animal’s general condition

Body temperature

Oedema ( inflammatory infiltrates)

Hemoglobin and white and red-blood cell counts

Histological study of the wound-bed tissue.

Three rabbits from each group were sacrificed with a lethal injection of sodium pentobarbital on days 1,3,5 and 10, when the wound-bed tissue was taken for histological study.

Results

Differences in the healing process were observed on the day after wounding and on day 3.

Oedema/wound infiltration (day 10)

Control group All wounds contained dark grey tissue and creamy pus, and had an unpleasant odour ( Fig.1)

Fig. 1. Control group: wound on day 10.

Intramuscular cefamezine All rabbits had skin hyperemia and infiltrates 4-7 cm in diameter, with scabs over the center. Dissection of infiltrates revealed abscesses containing creamy pus. ( Fig.2)

Fig. 2. Intramuscular cefamezin group: wound on day 10.

Irrigation with Sporobacterin These rabbits had purulent wounds, but these were narrower and produced less purulent discharge than the control group ( Fig.3)

Fig.3 Sporobacterin-irrigation group: wound on day 10.

Sporobacterin per os These wounds healed without surgical infection ( Fig.4) ( Table1)

Fig.4. Sporobacterin-per-os group: wound on day 10.

Temperature

Normal body temperature values were 38.7+ 0.30 C. On the first day post-wounding, rabbits in all groups were running temperatures 1.3-1.7 0 C higher than before wounding. By the third day temperatures continued to rise in all groups except the Sporobacterin-per-os group, where it dropped to normal. By the fifth and tenth days the highest temperature was recorded in rabbits treated with intramuscular cefamezin, due to abscess formation.

Blood values

Hemoglobin Normal hemoglobin concentration was 66.1+0.7g/l. This dropped by 12-15% in all animals on the first day after wounding and continued to decrease until day 3. By day 4 a gradual increase was observed, although this was most marked in the Sporobacterin-per-os group. On this day, average hemoglobin concentrations were :

• 54.3+ 1.2 g/l in the control group
• 55.9+ 0.9g/l in the intramuscular cefamezin group
• 4.1 + 0.8g/l in the Sporobacterin- irrigation group
• 56.1 + 0.6 g/l in the Sporobacterin-per-os-group
• Erythrocytes The normal erythrocyte value was 3.49 + 0.05 x 1012 per litre. Fluctuations in erythrocytes values were similar to those for hemoglobin. Values fell over three days in all groups. By the tenth day there was a clear tendency to normalization in the Sporobacterin-per-os group, which we presume had a beneficial effect on healing. Anemia was recorded in the other groups.

Leucocytes Normal leucocyte values were 9.1-9.25 x 109 per litre. This increased by 5-8% on the day after wounding in all groups. By the third day this had further increased by:

• 19% in the control group
• 10% in the intramuscular cefamazine group
• 11% in the Sporobacterin – irrigation group
• 3.8 % in the Sporobacterin – per-os-group
• By the fifth day leucocytosis was presenting in the control group, the intramuscular cefamezin group and the Sporobacterin – irrigation group. By the tenth day leucocyte levels had normalized in the Sporobacterin – per-os-group, but remained higher than normal in the other groups. They were highest in the intramuscular cefamezin group because of the abscess formation.
• Tissue histology

By the end of day 1 characteristics of moderate post-traumatic inflammation such as hyperaemia, haemorrage, necrosis of the adjacent tissues and serofibrinous exudation with low numbers of leucocytes were observed in all groups.

By the second day the anabolic process had stopped and catabolism and denaturation of proteins had increased as confirmed by the eosinophilla of cells and nuclei of muscle fibres. The pershing fibres broke up and were exposed to phagocytosis, which was confirmed by the index of fibroblasts and the amount of degenerated myosymplasts. A myosymplasts is a multinucleated cell that produces muscle fibre.

By the third day the inflammatory process in the wound and adjacent tissues had become more intense, with destructive changes in the muscle tissue and derma. Granulation tissue and microvessel gems began to form, most noticeably in the Sporobacterin – per-os-group ( Fig.5). In this group the reproductive activity in the perivascular cell and endotheliocyte activity as well as tissue basophils, leucocytes and lymphocyte function.

Fig. 5. Area of the wound channel. Sporobacterin-per-os group: day 3. A=arteriole ready for proliferation

By the fifth day granulation tissue growth was observed, and collagen fibres were apparent between fibroblasts in all groups. The repair of connective and muscle tissue in the wound and restoration of vessels in the microcirculation system increased, but again mainly in the Sporobacterin – per-os-group. The index of fibroblast destruction in this group was 2.5 times lower than in the control group. The myosatellitocye is a mononuclear cambial cell found under the membrane of the muscle fibre.

By the tenth day the formation of connective tissue resulted in contraction. In the control group the new connective tissue did not show such sighs of cellular differentiation and collagen formation. In this group, the intramuscular cefamezin group and the Sporobacterin – irrigation group there were signs of fibrinopurulent inflammation, and necrotic tissue was evident in the wound borders, starting in the deep layers and spreading to the surface.

Also, on day 10, wounds in the Sporobacterin – per-os-group filled with granulation tissue, with an orderly formation of collagen fibres in the periphery. On the wound border the incrassate, stratified, flat keratinising epithelium crawled under a crust of a granulation tissue-like rim 3mm wide. By this time, the inflammatory process had calmed down and granulation had ceased to occur.

Microbiology

In the control group, within 24 hours of wounding we isolated Staphylococcus species from seven animals, Gram-negative bacteria ( Escherichia coli, Proteus, Pseudomonas) from five rabbits, other microorganisms from four animals and Bacillus subtilis from 1 rabbit. Microbial associations were found in 50% of cases.

In the cefamezin group there were Staphylococcus species in eight cases, Gram-negative bacteria in four cases and other bacteria in four cases.

In the Sporobacterin – irrigation group we isolated Staphylococcus species in eight animals, Gram-negative bacteria in five rabbits, other microorganisms in two animals and Bacillus subtilis in 11 rabbits.

In the Sporobacterin – per-os-group there were Staphylococcus species in eight cases, Gram – negative bacteria in three cases , other bacteria in five cases and Bacillus subtilis in seven cases.

Results in the microbiological study were available on the fifth day. By the tenth day we isolated common agents of surgical infection, such as Staphylococcus , Escherichia coli, Proteus, Pseudomonas from the purulent wounds and abscesses in the control, intramuscular cefamezin and Sporobacterin – irrigation groups. At the same time the wounds in the Sporobacterin – per-os-group had healed by primary intention.

We investigated the antagonism of Bacillus subtilis 534 microorganisms discharged from wounds. A total of 143 strains of bacteria and fungi were studied. Almost all common agents of surgical infection, both aerobes and anaerobes ( Staphylococci, Proteus and Clostridiae), were very sensitive ( 95% and more) to the antibiotic secreted by Bacillus subtilis 534. Growth of all 36 investigated Staphylococcus strains was depressed.

Ninety-four per cent of the dysentery agents were sensitive. This is important because dysentery often complicates an epidemiological situation in war conditions and in peacetime major accidents.

Less sensitive to the antibiotic secreted by Bacillus subtilis 534 were Escherichia coli (75%), Klebsiella (75%) and Pseudomonas ( 60%). Bacillus subtilis 534 depressed growth of all four studied strains of Candida.

Discussion

The results showed that live bacteria Bacillus subtilis 534 administrated per os were more effective prophylaxis for surgical infection following gunshot wounds that cefamezin.

This is a mechanism for delivering bacteria from the gastrointestinal tract into an inflamed or damaged area. Bacteria pass through the gut barrier, presenting in the gastrointestinal tract in small quantities and preventing the development of acute enteric infection. Bacterial translocation occurs in various pathological conditions : traumas, burns, shock and after operations.8-12 Mane researches have considered bacterial translocation as pathology because it leads to bacteremia and tissue contamination. 13-14

To my knowledge, I am the first to demonstrate, that bacterial translocation is a natural protective mechanism which can be applied to clinical practice.15 In the inflamed area certain bacteria, such as Bacillus subtilis and other saprophytes, produce broad- spectrum, antibacterial biologically active substances that suppress the growth of pathogenic and opportunistic microorganisms, promote wound cleansing and protect against necrosis.

Finally, Sporobacterin was used locally as bacteria do not permeate deep into wound.

Conclusion

Probiotics can be used for surgical infection prophylaxis. We consider that they can used in the treatment of military wounds and those incurred during peacetime large-scale incidents.

Table 1 .Oedema/ wound infiltrate

Group Inflammatory infiltration ( cm) day 1 post-wounding ( average) Inflammatory infiltration ( cm) day 3 post-wounding ( average)

Intramuscular cefamzine 1.75+0.11 3.26 + 0.14

Sporobacterin irrigation 1.62+0.07 2.33 + 0.16

Sporobactern per os 1.17 + 0.08 Constant

Control 2.53 + 0.13 3.56 + 0.25

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