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Journal of Wound Care. Volume 16, №4, April 2007

Главная » Зарубежные публикации » Journal of Wound Care. Volume 16, №4, April 2007

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

BREAKING THE BOUNDARIES

Breaking the boundaries articles present reports on preliminary research data, novel treatments and unusual conditions and therapies that may be out with the main stream. They are not always peer-reviewed.

Can translocated bacteria reduce wound infection?

Swab cultures from acute surgical wounds showed that Bacillus subtilis was the most common bacteria present. The investigators propose, that bacteria which originate in the gastrointestinal tract, play an anti-infective role in wound site.

Bacteria : suppuration, translocation.

The composition of purulent wounds exudates is being studied on an ongoing basis, but as far as we are aware there are no published papers on the identification of bacteria in the wound in the early stages of healing.1 However, it has been suggested that if a wound swab indicates that bacteria are present, they should be eliminated and sterile environment created.2 This study set out to determine the most common bacteria present in human wound swabs. In vitro and animal studies were undertaken to identify their source and determine if they are capable of exogenous antibiotic activity.

Human study

Method

All patients undergoing surgery for open limb fractures (n=64) or osteosynthesis of closed fractures (n=73) from February 2001 to December 2006 were included. The age range was 18-86 years ( mean 41).

Ethical approval was given by Orenburg State Medical Academy. All patients gave informed consent. The periwound skin of all wounds was cleansed preoperatively with three antiseptics (1% povidoneiodine, 70% alcohol solution and 0.5 % dioxydine) in rapid succession.

At the end of each operation the wounds were swabbed with sterilized tampons. In the first three postoperative days wound exudate from between the sutures wasswabbed using a microbiological loop.

Results

The first wound swab, taken at the end of surgery, showed there were no microorganisms in 52/64 patients with open fractures and in 58/73 patients with closed fractures. Full results are given in Table 1.

Open fractures

In spite of debridement of non-viable tissue and antiseptic lavage, microorganisms were identified in almost one-fifth of the patients (19%) with open fractures following wound excision. Most were monolayer cultures of Staphylococcus epidermidis, Staphylococcus aureus, Staphylococcus saprophyticus, Proteus and other Gram-negative bacillus.

There was a marked increase in the presence of bacteria in the first three post-operative days as, in spite of the use of antiseptic bandages, wounds were sterile in only 13/64 patients (20%). This was primarily due to the onset of sporogenous Gram-positive microflora of genus Bacillus, which occurred in 41% of cases as a monoculture and in 6% as a polyculture.Samples from 15 patients were cultured in anaerobic conditions, with positive results two cases. Bacteroides were identified in one patient.

Closed fractures

Microorganisms were found in wound swabs taken at the end of surgery in only 15 of the 73 patients with closed fractures. In the first three postoperative days the presence of bacteria increased , with only eight wounds (11%) not having any microorganisms. The most frequently identified bacterium was Bacillus subtilis (strains 36 and 73); in four patients it coexisted with other bacteria ( Table1).

Suppuration

None of the wounds of the patients in either group showed any clinical signs of suppuration in the first three postoperative days. Only a small number of bacteria was present in the wounds – no more than 103-4 per gram of wound exudate. Genus Bacillus were more often identified in patients with closed fractures. Of the 73 patients, 35 were not given antibiotics during surgery and the first two postoperative days. We believe these two findings are related. The identified strains of Bacillus subtilis were highly sensitive to antibiotics such as penicillin, chloramphenicol, tetracycline and cephalosporin.
On the fifth postoperative day, 11patients with open fractures and two patients with closed fractures had clinical signs of suppuration. Four patients with suppurating wounds had monocultures of Staphylococcus aureus, three had Gram0negative Enterococcus spp. And one had Streptococcus spp. and Bacteroide spp. Bacterial combinations were found in four patients.
We analyzed the relationship between the number of wounds that suppurated and the presence of the various microorganisms in the first three postoperative days. The results are given in Table 2.
Wounds in which genus Bacillus was identified in the first three post-operative days of the wound-healing process were less prone to suppuration. Of the 70 patients in whom genus Bacillus were identified, only two (3%) had suppurating wounds. Of the 46 patients in whom other strains of bacteria were identified seven (15%) had suppurating wounds. Similar results were recorded in the 21 patients with no bacteria in their wounds in the first three postoperative days – only four (19%) suppurated.

In vitro studies

The morphological and biochemical behaviours of the microorganisms identified in the swabs, along with their antibacterial properties, we studied using standard methodology.3 We also set out to determine whether any of the bacteria present showed a potential for exogenous antibiotic activity.
The identify the source of Bacillus subtilis in the wounds under antiseptic bandage, we seeded bacteria from the wound swabs, periwound tissue, urine, faeces and blood of 17 patients, as well as from the treatment-room air( the latter was achieved by exposing beef-extract brooth to open air for 15 minutes and then heating it to 370 C)
The results indicated that genus Bacillus penetrated the wound from the gastrointestinal tract: the biochemical and physiological features of the bacterial colony from the wound swabs were the same as those from blood, urine and faeces.
We then isolated strains of genus Bacillus from 10 wounds healing by primary intention and sent them to the Moscow Research Institute of Antibiotics, which verified that all 10 strains had antimicrobial properties. These antimicrobial properties were associated with proteinaceous structures with widely varying molecular weights.
In addition, we identified 17 strains of Bacillus from the wounds4:

  1. Bacillus subtilis – six srains

  2. Bacillus circularis – three strains

  3. Bacillus cereus – two stains

  4. Bacillus alvei – two strains

  5. Bacillus laterosporus – one strain

  6. Bacillus macquariensis – one strain

These were grown separately in beef-extract broth, and the presence of proteolytic ferments was determined by mixing the corresponding substratum with filtered liquid.5
The strains produced large numbers of proteolytic ferments that broke down albumin, protein, collagen, fat, carbohydrates and vegetable cellulose. One of the natural strains ( whose number and characteristics we are unable to name as we have not yet patented it) synthesized and eliminated human fibroblast growth factors ( FGF). We were unable to find any reference to such a bacterium in the literature; genetically modified strains of bacteria are used to produce fibroblast growth factors. We believe that genus Bacillus produces substances used in the prophylaxis and treatment of purulent inflammation.

Animal studies

Method

We undertook an animal study to identify the dosage at which the administration f genus Bacillus would be lethal. In all, 206 mice were injected intramuscularly and subcutaneously with 10 strains of genus Bacillus in doses ranging from 0.5 milliard to 170 milliards in 1 ml of 0.9% sodium chloride. ( Both intramuscular and subcutaneous injections were used as it was thought the method of administration might affect toxicity). The aim was to determine the dosage that would kill 50% of the animals. A stain is considered harmless if two milliards of bacteria or more fail to kill a mouse.
Intravenous (IV) and intraperitoneal infusion of the 10 strains at the above dosages did not have a toxic effect. All of the mice remained active, and did not experience any weight loss. All six mice given an IV injection of 170 milliard remained healthy. We conclude therefore that genus Bacillus can be considered harmless.
In a separate animal study involving 20 rabbits, we set out to determine the source of the bacteria.
To determine a safe dosage, Bacillus subtilis strains 534 and 538, marked with radioactive H3 – leucine, were given subcutaneously at 1-5 milliards of bacteria in 1 ml of 0.9% sodium chloride solution to 12 rabbits weighing 2-2.5 kg each. No adverse effects occurred.
Bacillus subtilis, again marked with radioactive H3 – leucine, were then administrated orally into the intragastric region of eight other rabbits (four rabbits received strain 534 and four rabbits strain 538). Dosage was 1 milliard in 103 of sodium chloride. Dermal wounds 4 cm long were created under inhalation anaesthesia in four of the eight rabbits ( two had been given strain 534 and two strain 538). The wounds were closed and hermetically sealed with glue BF-6 to insulate them from the external environment. After a second administration of bacteria 48 hours later, the rabbits were killed with inhalation anesthesia. Blood was collected from an ear vein shortly before this. Tissue samples were taken from the wound or skin ( if the rabbits were not wounded), liver, spleen, lymph nodes from the mesentery of the small intestine, brain, lungs, stomach and large and small intestines. These samples were dissected as recommended by Ginkin6.
Histological analysis of the eight rabbits showed numerous radiolabels in the lymph nodes of the large and small intestines lymphoid follicle of the spleen ( Fig.1a) and liver. Wounded rabbits had greater amounts of marked bacteria in the connective tissues of the wound area (Fig.1b). Some radiolabels were found in blood smears ( Fig.1c).
In our opinion, the bacteria penetrate into blood through the desquamation areas and intercellular slots in the area of the healthy stomach and small intestine. Bacterial translocation through the mucous coat of the large intestine was not observed.

Discussion

Following surgery for open or closed limb fractures most of the patients had a moderate number of Bacillus subtilis in their wounds in spite of the antiseptic lavage and antibiotic treatment. As far as I am aware, this is the first time this has been described in the scientific literature. As expected, the nonexuding wounds that closed up were sterile. In my opinion, bacteria, living in the lumen and the periwound tissue play a key-role in the healing process. This led to conclude that the presence of Bacillus subtilis 534 reduces the possibility of suppuration.
The phenomenon of bacterial penetration from the gastrointestinal tract into blood and tissues has been called « translocation». Most researches regard this as a pathologic process resulting from factors such as stress, immunodeficiency and shock. 1-7,10. In contrast, I consider that translocation from the gastrointestinal tract through uninjured mucous coats plays an anti-infective role. As far as I am aware, these results are the first to indicate that the presence of microorganisms in the wound is not always harmful. It is possible that they could be used in the development of cheap prophylactic and anti-inflammatory drugs.

1a

1b

1c

References.

  1. Nikitenko V.I. Infection prophylactics of gunshot wounds using probiotics. J Wound Care 2004; 13:9, 363-366
  2. Kucuk, C.,Souzuer, E., Gursoy, S. et al. Treatment with Met – RANTES decreases bacterial translocation in experimental colitis. Am J Surg 2006; 191: 1, 77-83
  3. Birger, M.O.Handbook of Methods of Microbiology and Virology ( in Russian). Moscow Medicine, 1982
  4. Breed, R.S., Murray, E.G.D., Smith, N.R. (eds). Bergey`s Manual of Determinative Bacteriology. Williams and Wilkins. 1974.
  5. Pokrovskii, A.A. The biochemical studies in clnic. Moscow medicine 1969; page 632.
  6. Ginkin, L.N. The usage of the radionuclides in histology. The radioactibve indicators in histology. Leningrad, IEM AMN USSR, 1959; 5-33.
  7. Kuzin, M.I., Kostuchenok, B.M. Wounds and Wound infection ( in Russian). Moscow , 1981.
  8. Kane, T.D., Alexander, J.W., Johannigram, J.A. The detection of microbial DNA in the blood : a sensitive method for diagnosing bacteremia and/or bacterial translocation in surgical patients. Ann Surg 1988; 227:1, 1-9.
  9. Sakamoto, H., Naito, H., Ohta, Y. et al. Isolation of bacteria from cervical lymph nodes inpatients with oral cancer. Arch Oral Biol 1999; 44: 10, 789-793.
  10. Song, D., Shi, B., Xue, H. et al. Green fluorescent protein labeling Escherichia coli TGI confirms intestinal bacterial translocation in a rat model of chemotherapy. Curr Microbiol 2006; 52: 1, 69-73.

Table 1
Wound microflora identified in patients during the first three postoperative days.

Open fractures (n=64)

Closed fractures ( n=73)

At the end of surgery №(%)

1-3 days post - wounding № (%)

At the end of surgery №(%)

1-3 days post - wounding № (%)

No growth

52(81)

13(20)

58(80)

8(11)

Genus Bacillus

1(2)

26(41)

2(3)

36(49)

Staphylococcus aureus

1(2)

2(6)

2(3)

3(4)

Staphyl. epidermidis

2(3)

3(5)

3(4)

4(6)

Staphyl. saprophyticus

1(2)

5(8)

1(2)

3(4)

Streptococcus spp.

1(2)

1(2)

1(1)

2(3)

Enterococcus spp.

0

1(2)

1(1)

2(3)

Escherichia coli

1(2)

2(3)

1(1)

3(4)

Pseudomonas aeruginosa

0

1(2)

1(1)

2(3)

Klebsiella spp.

0

1(2)

1(1)

1(1)

Proteus spp.

1(2)

1(2)

0

1(1)

Candida

0

1(2)

0

1(1)

Staphylococcus aureus with Genus Bacillus

0

1(2)

0

1(1)

Staphylococcus aureus with Gram-negative coccus

0

0

0

1(11)

Staphylococcus saprophyticus or Staphylococcus epidermidis in association with genus Bacillus

1(2)

3(5)

2(3)

3(4)

Staphylococcus saprophyticus or Staphylococcus epidermidis

0

0

0

1(1%)

Anaerobe*

0

2(3)

-

-

Others

3(5)

1(2)

0

1(1)

Total

64

64

73

73

Table 2
Relationship between the number of wounds that suppurated and the composition of the microorganisms in the wound in the first three postoperative days.

Open fractures

Closed fractures

№ of wounds

№ that suppurated

№ of wounds

№ that suppurated

Sterilized seedings

13

3

8

1

Staphylococcus spp.
Enterococcus spp.etc.

21

6

25

1

Genus Bacillus

30

2

40

0


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