Bacteroides fragilis and Escherichia coli

J. Med. Microbiol. - Vol. 35 (1991), 18-22

01991 The Pathological Society of Great Britain and Ireland

Early events after intra-abdominal infection with Bacteroides fragilis and Escherichia coli W. R. VERWEIJ, F. NAMAVAR, W. F. SCHOUTEN and D. M. MACLAREN Research Groupfor CommensalInfections,Department of Medical Microbiology, School of Medicine, Vrije Universiteit, v.d. Boechorststraat 7, P.O.B. 7 7 61, 1007 MC Amsterdam, The Netherlands

Summary. Growth of Bacteroides fragilis and Escherichia coli was monitored during early

stages of single (mono-) and mixed intra-abdominal infection in a rat fibrin clot model. When B. fragilis and E. coli were together involved in the infection, B. fragilis numbers increased about 6 h after an initial decline. This increase was not found with B. fragilis mono-infections. The numbers of E. coli increased rapidly in both mono- and mixed infections and stayed high for several days, but only mixed infection resulted in abscesses that persisted for more than 7 days. Macrophages, the main component of the peritoneal cellular defence mechanism, were outnumbered by polymorphonuclear leucocytes during the first 6 h of infection. Further characterisation of the macrophage population by means of monoclonal antibodies showed a shift from resident to exudate macrophages as the result of influx of the latter.

Introduction Bacteroidesfragilis and Escherichia coli are common pathogens in intra-abdominal infections.19 The contribution of each to the pathogenesis of mixed infections has been well demonstrated, particularly in animal models of intra-abdominal and subcutaneous infections.3-5 On the basis of several experimental animal studies, it has been postulated that these organisms act synergically, leading to either enhanced mortality4 or increased abscess formation.69 In these experimental studies, abscesses developed after 3-7 days. However, there is little information on the kinetics of bacterial growth during the early stages of infection. It is well known that free bacteria are readily removed from the peritoneal cavity into the bloodstream by the host peritoneal translymphatic system.* Therefore, we studied the growth kinetics of the bacteria and the reaction of the cellular components of the peritoneal defences at early stages of infection. A rat fibrin clot model’ with some modifications was used. This model is analogous to the clinical situation in which bacteria spill into the peritoneal cavity after visceral perforation and are rapidly walled off by fibrinous exudate, mobile viscera and omentum.

’

Material and methods Bacterial strains The B. fragilis (BE1) and E. coli (EB1) strams studied had been isolated from the wound infection of Received 4 Sep. 1990; accepted 15 Oct. 1990.

a patient at the Academic Hospital of the Vrije Universiteit. They had been used in earlier studies.5. 10-12 Techniques of storage, growth conditions, ingredients of the minimal growth medium and method of enumeration have been previously described. For inoculation into the fibrin clot, B. fragilis and E. coli cultures (16-1 8 h) were pelleted by centrifugation, washed and resuspended in Hanks’s Buffered Salts Solution (HBSS, Gibco) supplemented with cysteine hydrochloride 0-5 mg/L, and saline 0.9%solution. The concentrations of the bacterial suspensions were adjusted to 1 x lo9 cfu/ml for B.fragi1i.s and 1 x lo6 cfu/ml for E. coli.

Incorporation of bacteria intofibrin clots Infected fibrin clots were made by a modification of a previously described method.l 4 Briefly, human fibrinogen (Sigma) was suspended in calcium-free phosphate-buffered saline (PBS) to a concentration of 2 mg/ml and sterilised by ultraviolet irradiation for 10 min; 1.5-ml volumes were frozen at - 80°C and thawed on ice before use. Thrombin (Sigma) was suspended in sterile distilled water to a concentration of 30 NIH units/ml and 250-p1 samples were kept at - 80°C. For the preparation of infected clots, 1.4 ml of fibrinogen was placed in a polystyrene tube and 100 p1 of either B. fragilis or E. coli suspension, or both, were added. In monomicrobial clots, the appropriate control was used to replace the synergicbacterial partner. After gently mixing the tube, 200 pl of the thrombin solution was added and the tubes were gently mixed again. Fibrin clots formed almost

Downloaded from www.microbiologyresearch.org by 18 IP: 107.174.36.21 On: Wed, 02 Nov 2016 09:50:09

INTRA-ABDOMINAL INFECTION WITH B. FRQGZLZS AND E. COLZ

instantly. Serial 10-fold dilutions of the two bacterial suspensions were plated on blood agar and nutrient agar plates to verify the starting inocula.

Animals and operation Male Wistar rats weighing 200g on arrival were allowed to adapt for 5-7 days before operation. Fibrin clots containing bacteria were implanted intra-peritoneally (i.p.) via a 2-3-cm midline incision under ether anaesthesia. The incision was closed in two layers with four sutures each. Total anaesthesia time was less than 10 min. When examined within the first 8 h after operation, rats were kept in the operating room under a lamp to avoid cooling, otherwise they were placed in standard housing conditions. All animals were provided with food and water ad libitum.

Bacterial counts For each animal, the number of bacteria in both fibrin clot and peritoneal cavity was determined. Animals were killed in a sterile cabinet with C 0 2 after 2, 4, 6 and 8 h. After i.p. injection of 5 ml of saline supplemented with bovine serum albumin (BSA) 0.2% and gentle massage of the belly for 1 min, 3-4 ml of peritoneal fluid was collected from each rat and divided between two glass tubes, one of which was placed on ice to avoid adherence of cells from the peritoneal cavity. The peritoneal cavity of the animal was then opened and fibrin clots were transferred to a glass tube with 5 ml of sterile saline, weighed and homogenised in a grinder (Heidolph, Electro Abi, Haarlem, The Netherlands). Serial 10-fold dilutions of both peritoneal fluid and homogenised clot were seeded on to blood-agar plates (Blood Agar Base No. 2, Oxoid) supplemented with horse blood 5%, haemin (BDH) 5 pg/ml and menadione (Merck) 2 pg/ ml and incubated anaerobically for 48 h. Nutrient agar (Bacto Agar, Difco) plates were used for aerobic culture.

Quant$cation and characterisation of the peritoneal cell population Peritoneal cell samples were counted in a Burker chamber and viability was determined by trypan blue exclusion. The total numbers of cells were calculated by correcting for the volume that could be withdrawn from the peritoneal cavity. Differential cell counts were made from cytocentrifugepreparations (Shandon Southern Instruments Ltd, UK) stained with MayGrunwald Giemsa in which 500 cells were counted. Further characterisation of the macrophage population was done with monoclonal antibodies (MAbs) ED1 and ED2 against rat macrophages, kindly provided by Dr C. D. Dijkstra (Department of Cell biology, Faculty of Medicine, Vrije Universiteit Amsterdam, The Netherlands). MAb ED1 has been shown to react with the total monocyte population and

19

MAb ED2 with more than 50% of the resident macrophage population of the peritoneal cavity. l 6 Macrophages expressing antigens recognised by MAbs ED1 and ED2 were detected by an immunoperoxidase procedure in cytospin centrifuge preparations with diaminobenzidine-tetra-HC1 (DAB) as chromogen. Briefly, cytospin preparations, dried overnight on silica gel, were fixed in formalin 1% v/v in acetone, washed with PBS and incubated for 3060 min with MAbs diluted in PBS supplemented with BSA 0.2% w/v. After washing three times with PBS, samples were incubated with rabbit anti-mouse IgG horseradish peroxidase (DAKO) diluted in PBS with BSA 0.2% and normal rat serum 1% for 30 min. Samples were washed again three times and incubated for 15 min with the chromogen DAB (0-5 mg/ml in 0.05 M Tris-HC1, pH 7.6) and H 2 0 20.03%v/v. After another three wash steps, the samples were counterstained with haematoxylin for 1 min. Sometimes an additional incubation in CuSO, 0.5% was used to “amplify” the staining. At least 400 cells were examined for the presence of the different antigens. 59

Results Counts of bacteria in mono-infectedfibrin clots To obtain information about the early events after intra-abdominal infection, the bacterial contents of both peritoneal cavity and fibrin clot were monitored at 2-h intervals during the first 8 h and 5-7 days after implantation of the clot. When clots were infected with 2 x lo5 cfu of E. coli EB1 alone, an increase to (4-5) x lo7 cfu/clot was found within the first 8 h. Thereafter, the numbers of E. coli in the clots did not change during the first 5 days but after more than 7 days there were less than 50 cfu/clot. When the clot was infected with B. fragilis BE1 alone, the bacteria (and clot) disappeared within 24 h (fig. 1). Monoinfections with E. coli EB1 led to small peritoneal abscesses, which disappeared after 7 days. Monoinfections with B.fragilis BE1 did not lead to the development of abscesses. Bacterial content of the peritoneal cavity was highest 2 h after implantation and then decreased rapidly. The total number of free bacteria never exceeded 1%of the clot inoculum.

Counts of bacteria in mixed infectedfibrin clots Mixed infected clots were investigated at the same time intervals as with mono-infections. An inoculum of (1-2) x lo8 cfu of B. fragilis and (1-2) x lo5 cfu of E. coli in fibrin clots resulted in a rapid increase in numbers of E. coli (fig. 2). Numbers of E. coli remained high even after 7 days. On the other hand, numbers of B. fragilis declined during the first 4-6 h after implantation of the clot. After 8 h, the numbers of B. fragilis had increased to (0.5-1) x lo8 cfu/clot. This decrease in the count of B. fragilis followed by a rise during the

Downloaded from www.microbiologyresearch.org by IP: 107.174.36.21 On: Wed, 02 Nov 2016 09:50:09

20

W. R. VERWEIJ ET AL.

97

(fig. 2). Animals with mixed infected clots developed larger abscesses than when E. coli alone was used. As with mono-infected clots, free bacteria were detectable

Fig. 1. Bacterial growth kinetics after implantation of monomicrobial fibrin clots containing (1-2) x lo8 cfu of B.fragilis BE1 ( 0 )or (1-2) x lo5 cfu of E. coli EBl

a).

9 1

T

T

z0

n 0

t5 0

0

Y

population further, two MAbs directed against rat macrophages were used. Resident macrophages are the predominant type of peritoneal macrophages without stimulation. Fig. 3 shows the results of macrophage characterisation with these two MAbs. The decrease in the absolute number of macrophages during the early stages of infection was due to a decrease in resident (ED1 + /ED2 +) macrophages possibly due in part to leakage from the wound. On the other hand, the exudate (ED1 /ED2 -) macrophages increased and at 8 h after implantation compensated for the loss of resident macrophages. The absolute number of (1-2) x lo7 macrophages in the peritoneal cavity of the rats corresponds well with values reported by others.

+

cd

. I

L

a

4-

Discussion

0

cd

P YI

0

G

P

E

3

z

4

0 2 4 6 8 20 60 100 140 180 Time (h)

Fig. 2. Bacterial growth kinetics of B.fragilis (m)and E. coli (A) after implantationof mixed infected fibrin clots containing (1-2) x 108 cfu of B.fragilh BE1 and (1-2) x 105 cfu of Em coli EB1, Values are expressed as means and 5D of four independent experiments.

Several animal models have been used previously to study mixed aerobic-anaerobic infections. In this study the rat fibrin clot model was adopted because it seemed the most suitable model for the study of intraabdominal infections. It mimics very well the clinical situation in which bacteria spilled into the peritoneal cavity after visceral perforation are rapidly walled off by a fibrinous exudate, mobile viscera and omentum. This model was used to study the first 8-h period of a synergic infection with E. coli and B. fragilis and the results were compared with those obtained with the murine subcutaneous model. other investigators, who used the fibrin clot model, focused On abscess formation and abscess size after 57 days? They showed that these two parameters


INTRA-ABDOMINAL INFECTION WITH B. .FRAGILIS AND E. COLI

1 \

--5

*’

0 2 4 6 8 20 60 100140 180 Time (h)

Fig. 3. Cellular response in the peritoneal cavity to implantationof mixed infected fibrin clots containing (1-2) x lo8 cfu of B. frugilis and (1-2) x lo5 cfu of E. coli; ( 0 )total peritoneal cells, (*) PMNL, (0)total macrophages, resident (ED1 +/ED2 +) macrophages, and (0)exudate (ED1 + /ED2 -) macrophages.

a)

were influenced by bacterial inocula as well as bacterial strains used. We used E. coli and B. fragilis strains isolated from a wound infection. The inocula of (12) x lo8 cfu of B.fragilis and (1-2) x lo5 cfu of E. coli resulted in persistent abscesses only when used in combi nation. While studying the bacterial growth kinetics within the fibrin clot we observed what we regard as the “decisive moment” in abscess formation, about 6 h after infection. In contrast to the situation with monoinfected B. fragilis clots, the number of B. fragilis in mixed clots began to increase from 6 h after implantation after an initial decline to about 10% of the starting inoculum. The presence of E. coli, or conditions created by its presence and metabolism, enabled B. fragilis to survive instead of being cleared from the peritoneal cavity. The effects of E. coli on local conditions could be changes of pH, reduction of the oxygen level or excretion of “nutrients” for B. fragilis, or they might involve interference with chemotaxis or the activities of host defence cells. In this fibrin clot model the growth kinetics of E. coli in mono- and mixed infected clots are very similar ;irrespective of the presence of B. fragilis. The numbers of E. coli increased rapidly and stayed at a high level for several days. The effect of the presence of B. fragilis on E. coli was seen only after 5 days when mono-abscesses with E. coli disappeared whereas

21

abscesses caused by the combination of bacteria persisted (figs. 1 and 2). With the subcutaneous model1 there was a dose-dependent, reciprocal requirement of E. coli and B. fragilis to avoid clearance early after the injection. The differences in time and dose at which effects of B. fragilis on E. coli were seen in the fibrin clot model might be explained by the protective barrier provided by the fibrin clot. In the experimental approach described here, whether the mixed infection progressed to an abscess or not seemed to be determined during the very early stages of infection. The importance of the first hours of an infection with respect to abscess formation can also be deduced from other experiments” in which tissue plasminogen activator (t-PA) was used. It was shown, with the same rat fibrin clot model, that intraabdominal abscess formation could be completely prevented when t-PA was given i.p. during, or by means of open lavage within a very short period after, surgery. More recently it was shown that an increased concentration of t-PA (0-1 mg/ml) prevented abscess formation after a 6-h delay.20A decisive period in the early stages of infection with regard to antibiotic prophylaxis has previously been postulated2’*22 with a guinea-pig model. A decisive moment or period leading to an increase of B. fragilis in mixed infected clots after 6 h in this study might be explained by a change in clot structure or by decreased peritoneal host defence in consequence of an altered peritoneal cell population. Firstly, clot contraction alone or in combination with host fibrin deposition could interfere with clot infiltration by host defence cells and bacterial killing. Secondly, the altered peritoneal population might be less effective with regard to fibrinolytic and phagocytic capacities. To investigate the latter possibility, an inventory of the peritoneal cell population was made during the early stages of infection. The results (table and fig. 3) show changes in both the total number of peritoneal

Table. Peritoneal subpopulationsafter implantation of fibrin clots infected with B.fragiZis and E. coli

Peritoneal cells

Mean* cell count ( x lo’) and (range)t at time (h) after clot implantation 0

Eosinophils PMNL$ Macrophages Rest Total

0-6 (20-30) 0.0 (0) 1.8 (67-79) 0.1

2

0.2 (8-12) 0.8 (43-58) 0.6 (34-39) 0-0 (4-6) (2-3) 2.4 1.6

4


8

0.2 0.1 0.1 (8-12) (1-3) (1-3) 3.7 4.3 3-1 (62-68) (64-67) (59-67) 1-7 2.4 0-9 (16-20) (22-26) (33-36) 0.1 0.1 0.0 (0-1) (1-3) (1-3) 4-4 6.8 5.6

* Mean of four independentexperiments. t Ranges as percentagesof the total population. 1PMNL minus eosinophils.

6

22

W. R. VERWEIJ ET AL.

cells and their distribution in several subpopulations. An influx of PMNL was found as a response to both surgical trauma and the presence of bacteria in the peritoneal cavity. The influx of these cells, mainly neutrophils, is in agreement with the observations of others who used a bacterial containment chamber in mice23and after an i.p. injection of 2 x lo8 dead E. coli cells.24 It was observed that macrophages, the most common cell type in the peritoneal cavity at the start of the infection, were outnumbered by PMNL within 2 h after implantation of the mixed infected clot. However, although the total number of macrophages did

not change markedly, it was possible to detect a shift from one subpopulation to another by means of MAbs ED1 and ED2. The increasing contribution of exudate macrophages (ED1 /ED2-) (fig. 3) may have important consequences for bacterial clearance and abscess formation. Resident macrophages (ED1 + / ED2 ) are differentiated phagocytotic cells adjusted to the peritoneal environment. Exudate macrophages, on the other hand, are fresh blood monocytes; though these have phagocytic activity, they are less differentiated. Separation and characterisation of these cell types combined with functional assays may provide more information on their role in abscess formation.

References

13. Namavar F, Verweij AMJJ, Bal Myvan Steenbergen TJM, de Graaff J, MacLaren DM. Effect of anaerobic bacteria on killing of Proteus mirabilis by human polymorphonuclear leukocytes.Infect Immun 1983;40:930-935. 14. Dunn DL, Rotstein OD, Simmons RL. Fibrin in peritonitis. IV. Synergistic intraperitoneal infection caused by Escherichia coli and Bacteroides fragilis within fibrin clots. Arch Surg 1984; 119; 139-144. 15. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology 1985;54: 589-599. 16. Beelen RHJ, Eestermans IL, Dopp EA, Dijkstra CD. Monoclonal antibodies ED1, ED2 and ED3 against rat macrophages : expression of recognized antigens in different stages of differentiation. Transplant Proc 1987; 19: 31663170. 17. Bos HJ, Meyer F, de Veld JC, Beelen RHJ. Peritoneal dialysis fluid induces change of mononuclear phagocyte proportions. Kidney Int 1989; 36 : 20-26. 18. MacLaren DM, Namavar F, Verweij-Van Vught AMJJ Vel WAC, Kaan JA. Pathogenic synergy: mixed intraabdominal infections. Antonie Van Leeuwenhoek 1984;50: 775-787. 19. Rotstein OD, Kao J. Prevention of intra-abdominal abscesses by fibrinolysis using recombinant tissue plasminogen activator. J Infect Dis 1988; 158: 766-772. 20. McRitchie DI, Cummings D, Rotstein OD. Delayed administration of tissue plasminogen activator reduces intraabdominal abscess formation. Arch Surg 1989; 124: 14061410. 21. Miles AA, Miles EM, Burke J. The value and duration of defence reactions of the skin to the primary lodgement of bacteria. Br J Exp Pathol 1957;38: 79-96. 22. Shapiro MySacks T. A decisive period in cefoxitin prophylaxis of experimental synergistic wound infection produced by Bacteroidesfragilis and Escherichia coli. Isr J Med Sci 1982; 18: 863-865. 23. Onderdonk AB, Cisneros RL, Crabb JH, Finberg RW, Kasper DL. Intraperitoneal host cellular responses and in vivo killing of Bacteroides fragilis in a bacterial containment chamber. Infect Immun 1989;57: 3030-3037. 24. Dunn DL, Barke RA, Knight NB, Humphrey EW, Simmons RL. Role of resident macrophages, peripheral neutrophils, and translymphaticabsorption in bacterial clearance from the peritoneal cavity. Infect Immun 1985;49: 257-264.

1. Swenson RM, Lorber ByMichaelson TC, Spaulding EH. The bacteriology of intra-abdominal infections. Arch Surg 1974; 109: 398-399. 2. Swenson RM, Lorber B. Clindamycin and carbenicillin in treatment of patients with intra-abdominal and female genital tract infections. J Infect Dis 1977; 135: S40-S45. 3. Onderdonk AB, Bartlett JG, Louie T, Sullivan-Seigler N, Gorbach SL. Microbial synergy in experimental intraabdominal abscess. Infect Immun 1976; 13: 22-26. 4. Rotstein OD, Pruett TL, Simmons RL. Lethal microbial synergism in intra-abdominal infections. Arch Surg 1985; 120: 146-151. 5. Verweij-Van Vught AMJJ, Namavar F, Sparrius MyVel WAC, MacLaren DM. Pathogenic synergy between Escherichia coli and Bacteroides fragilis : studies in an experimental mouse model. J Med Microbioll985 ; 19: 325-33 1. 6. Rotstein OD, Kao J. The spectrum of Escherichia coliBacteroidesfragilis pathogenic synergy in an intraabdominal infection model. Can J Microbioll988; 34: 352-357. 7. Rotstein OD, Kao J, Houston K. Reciprocal synergy between Escherichia coli and Bacteroides fragilis in an intraabdominal infection model. J Med Microbiol 1989; 29: 269-276. 8. Dunn DL, Barke RAYEwald DC, Simmons RL. Macrophages and translymphatic absorption represent the first line of host defence of the peritoneal cavity. Arch Surg 1987; 122: 105-110. 9. AhrenholzDH, SimmonsRL. Fibrin in peritonitis. I. Beneficial and adverse effects of fibrin in experimental E. coli peritonitis. Surgery 1980; 88: 41-47. 10. Vel WAC, Namavar F, Verweij-Van Vught AMJJ, Pubben ANB, MacLaren DM. Interactions between polymorphonuclear leucocytes, Bacteroides sp., and Escherichia coli: their role in the pathogenesis of mixed infection. J Clin Patholl986; 39: 376-382. 11. Verweij-Van Vught AMJJ, Namavar F, Vel WAC, Sparrius My MacLaren DM. Pathogenic synergy between Escherichia coli and Bacteroidesfragilis or B. vulgatus in experimental infections: a non-specific phenomenon. J Med Microbioll986; 21: 43-47. 12. Namavar F, Verweij-Van Vught AMJJ, Bal My MacLaren DM. Effect of Bacteroides fiagilis cellular components on chemotactic activity of polymorphonuclear leukocytes towards Escherichia coli. J Med Microbiol 1987; 24: 119124.

+

+
