Journal of Antimicrobial Chemotherapy (1979) 5, 201-210
Activity of metronidazole against Escherichia coli in experimental infra-abdominal sepsis A. B. Onderdonk, T. J. Louie, F. P. Tally and J. G. Bartlett
Metronidazole treatment was found to reduce both mortality rates and the incidence of abscess formation in experimental animals challenged with an intraperitoneal implant of pooled caecal contents. This was not anticipated since previous studies of this model indicated that death was due to Escherichia coli bacteraemia. This suggested that metronidazole was active against coliforms in vivo, despite in vitro resistance. Using an intraperitoneal challenge off. coli plus Bacteroides fragilis or E. coli alone, metronidazole treatment reduced mortality rates, but only in animals which received E. coli plus B. fragilis. There was no significant decrease in mortality with animals challenged with E. coli alone, indicating that metronidazole may be active against E. coli only in the presence of other susceptible bacteria. In chemostat cultures of E. coli metronidazole produced a reduction in the growth rate of E. coli, only in the presence of B. fragilis. Metronidazole has in vivo activity against bacteria which are highly resistant according to conventional in vitro susceptibility tests but only when a susceptible microbe is also concurrently present. Introduction
Previous studies from this laboratory have employed an experimental animal model tc define the role of various bacteria in intra-abdominal infections. Rats challenged with an inoculum of pooled faecal contents initially developed an acute peritonitis with E. col bacteraemia and a 40% mortality. Survivors developed the second stage of disease, characterized by intra-abdominal abscesses in which B. fragilis was the predominant isolate (Onderdonk, Weinstein, Sullivan, Bartlett & Gorbach, 1974). Studies using pure cultures showed that E. coli caused early death in this model, whereas B. fragilis produced intra-abdominal abscesses (Onderdonk, Bartlett, Louie, Sullivan-Sigler & Gorbach, 1976; Onderdonk, Kasper, Cisneros & Bartlett, 1977). Antimicrobials active against coliforms prevented death, whereas agents active against anaerobes prevented abscess formation (Weinstein, Onderdonk, Bartlett & Gorbach, 1975). We concluded that coliforms caused early death, and anaerobes abscess formation. The present studies were prompted by the unexpected observation that metronidazole prevented mortality as well as abscess formation in this model despite the fact that metronidazole is inactive in vitro versus coliforms. The drug may, therefore, have in vivo activity against E. coli not detected with the usual in vitro susceptibility assays. We have now tested metronidazole against E. coli using several modifications of the previously described Wistar rat model, and in continuous culture. 0305-7453/79/020201 +10 J01.00/0
201 © 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Infectious Diseases Research Laboratory, Boston Veterans Administration Hospital and Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts
202
A. B. Onderdonk et al Materials and Methods
Metronidazole treatment Male, Wistar rats weighing 180 to 200 g (Charles River, Wilmington, Mass.) were used for all experiments. The animals were housed two per cage and received chow (Ralston Purina, St Louis, Mo.) and water ad libitum. Preliminary studies were performed to determine the appropriate dose of metronidazole based on serum level determinations. Healthy rats were given arbitrarily selected doses by intramuscular injection followed by percutaneous transthoracic cardiac blood samplings at 0-5, 1, 4 and 8 h. Serum levels were determined by the clostridial haemolysis inhibition assay (Louie, Tally, Bartlett & Gorbach, 1976), using appropriate standards.
Inocula ofE. coli alone and in combination with B. fragilis The second type of inoculum consisted of E. coli (BVA 1-2) alone and in combination with B. fragilis (ATCC No. 23745). These inocula examined the effect of metronidazole treatment on these two species in the absence of other bacterial components of pooled caecal contents.
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Caecal content inoculum Two types of inocula were used. The first inoculum consisted of pooled, frozen caecal contents from meat-fed rats mixed with 10 % w/v barium sulphate (Weinstein, Onderdonk, Bartlett and Gorbach, 1974). 0-5 ml amounts of this material were placed in gelatin capsules for intraperitoneal implantation following injection of rats with Nembutal. Following challenge, the animals received one of three treatment regimens; metronidazole, clindamycin or gentamicin. A fourth group served as untreated controls. [The results in this model using clindamycin and gentamicin treatment have been previously reported (Weinstein, Onderdonk, Bartlett & Gorbach, 1975); these data are included here for the purpose of comparison with metronidazole]. Antimicrobial dosages were 16,15 and 2 mg per animal for metronidazole, clindamycin and gentamicin, respectively. The drugs were given intramuscularly beginning 4 h after implantation and then at 8 h intervals, for 10 days. Survivors were sacrificed 12 days after implantation for necropsy examination. Two parameters were used to assess results: (1) mortality rates of animals which expired during the 12 days of observation and (2) the incidence of intra-abdominal abscesses at necropsy examination on the 12th day. The criterion for denning an abscess was a loculated collection of grossly purulent exudate which showed bacteria and a predominance of polymorphonuclear leukocytes on Gram stain. Additional rats were challenged with the same inoculum of pooled caecal contents, to obtain blood cultures, by percutaneous transthoracic cardiac puncture. The results of these experiments were not included in abscess or mortality data described above, as the methods used to obtain blood cultures might alter the outcome. There were two experimental groups, rats treated with metronidazole, as previously described, and untreated controls. Blood was obtained by percutaneous transthoracic cardiac puncture during light ether anaesthesia. 01 ml of blood was placed into 19 ml of molten brain-heart infusion agar, mixed and poured into sterile petri plates. An additional 0-1 ml amount was diluted 1 : 100 with VPI dilution salts and plated in an identical manner. The plates were incubated in an anaerobic chamber for 48 h, and colony types were enumerated and identified.
Activity of metronidazole against E. eoB
203
Each of the test strains was grown in peptone-yeast glucose broth for 48 h in an anaerobic chamber, and five ml aliquots were frozen at — 40°C for future use. The inocula consisted of gelatin capsules containing 0-5 ml of one of two bacterial suspensions: (1) E. coli broth culture 1 : 1 with peptone yeast glucose broth or (2) E. coli culture diluted 1 : 1 with a B. fragilis culture. All inocula contained 50% V/V sterile caecal contents and 10 % W/V barium sulphate. Animals receiving these intraperitoneal challenges were divided into two groups. One was treated with metronidazole as described above, and the second group served as untreated controls.
Mortality rates in animals challenged with E. coli and B. fragilis This experiment was designed to evaluate the relative roles of B. fragilis and E. coli on mortality rates in animals challenged with these organisms alone and in combination. No treatment was employed, since the purpose was to assess the possibility that B.fragilis caused mortality or potentiated lethality due to E. coli. Inocula consisted of gelatin capsules containing 10% w/v barium sulphate, 50% v/v autoclaved caecal contents and serial PYG broth dilutions of E. coli (BVA 1-2), B. fragilis (ATCC 23745), or both organisms in combination. Continuous cultures The effect of metronidazole on growth rates of E. coli (BVA 1-2) alone or in the presence of B. fragilis (ATCC 23745) was tested using continuous culture techniques. The organisms were grown in a C-30 bioflo fermenter (New Brunswick Scientific Co., New Brunswick, New Jersey), modified to maintain anaerobic conditions. All experiments were conducted using a minimal medium (Varel & Bryant, 1974) containing glucose as a fermentable carbon source. A dilution rate of 016 h " 1 was used to establish continuous culture following inoculation of the fermenter vessel with the test organism(s). Oxidationreduction potential (Eh), pH, dissolved oxygen concentration, OD 550 and viable cell density were monitored as described previously (Onderdonk, Johnston, Mayhew & Gorbach, 1976). Metronidazole was added to established continuous culture via the
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Antimicrobial susceptibility testing Antimicrobial susceptibilities were determined by the broth dilution technique using the Microtiter system (Cooke Laboratory Products, Alexandria, Virginia). Organisms tested were representative strains of the four isolates which were most consistently present and recovered in highest counts from infections in untreated animals challenged with the inoculum of pooled caecal contents: B. fragilis, F. varhtm, E. coli (BVA 1-2) and enterococci. B.fragilis (ATCC No. 23745) was also tested, since this organism was used in the experiments utilizing inocula of B. fragilis plus E. coli described above. The test strains were grown for 6 to 8 h in supplemented Brain Heart Infusion broth (Scott Laboratories, Fiskeville, Rhode Island) to a density corresponding to a No. 1 McFarland nephelometer standard and then diluted 1000 fold (final concentration = 106"8 CFU/ml.) E. coli and enterococci were tested in Mueller-Hinton Broth (BBL), incubated aerobically for 24 h. E. coli was also tested in the anaerobic chamber to compare susceptibility results with anaerobic and aerobic incubation. Plates for testing obligate anaerobes were incubated in an anaerobic chamber for 36 h. The minimum inhibitory concentration (MIC) was the lowest concentration of antibiotic showing no macroscopic growth.
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nutrient reservoir, in a concentration of 30 ug/ml. The concentration of metronidazole in the reservoir and the fermentation vessel was assayed periodically to calculate the amount of biologically active drug present. The effect of metronidazole on the test species was compared to the theoretical washout rate for a static, but viable population within the fermenter. A slope of viable cell density versus time which exceeded the calculated washout rate indicates a bactericidal effect, while a slope less than the washout rate suggests a decrease in growth rate but not a bacteriostatic effect. A slope equal to the washout rate indicates a net bacteriostatic effect.
Antimicrobial treatment of animals challenged with caecal contents Results of the three antimicrobial treatment regimens compared to untreated control animals are summarized in Table I. Untreated animals had a mortality rate of 37 %, and all deaths occurred during the first five days following challenge. All surviving animals in the group receiving no treatment had intra-abdominal abscesses when sacrificed at 12 days. Treatment with gentamicin Teduced mortality rates, but 54 of 55 surviving animals had abscesses. By contrast, clindamycin treatment had no significant effect on the mortality rate, but only 5 % of surviving animals had abscesses. Metronidzole treatment resulted in a mortality rate of only 10%, while 15% of surviving animals had intra-abdominal abscesses. Chi square analysis using two by two contingency tables to compare treatment groups, show that the reduction in mortality with metronidazole was significantly greateT than for untreated controls (P< 001), and not significantly different compared to gentamicin (/ > >01). Blood cultures on a separate group of animals challenged with the caecal inoculum revealed significant differences with metronidazole treatment compared to untreated controls. Nineteen of twenty blood cultures in untreated animals yielded E. coli. Only one of ten blood cultures was positive in the metronidazole treatment group. The mean Table L Results with antimicrobial treatment of animals challenged with caecal contents Antimicrobial
Mortality
(Untreated controls) 58/157(37%)* Metronidazole 5/50 (10%) Clindamycin 21/60 (35%) Gentamicin 2/57 (4%) •No. died/no, tested (%) t No. with abscesses/no, of survivors tested (%)
Abscess 99/99 (100%)t 6/45 (13%) 2/39 (5%) 54/55 (98%)
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Results Antimicrobial serum levels and in vitro susceptibility tests Mean serum levels in five healthy rats given 16 mg doses of metronidazole intramuscularly were 26, 33, 12 and 4 mg/ml at 0-5, 1, 4 and 8 h, respectively. The mean peak serum level with clindamycin was 5 5 mg/ml, and 6-9 (ig/ml for gentamicin. The MICs for metronidazole were: B.fragilis—4 jig/ml; E. coli (BVA 1-2)—1024 ug/ ml (aerobic and anaerobic); F. varium—1 ug/ml and enterococcus 64 ug/ml.
Activity of metronidazole against £. coB
205
Table EL Treatment of animals challenged with E. coli alone and in combination with B. fragilis Antimicrobial treatment None Metronidazole None Metronidazole
Inoculum
j , tested/"/"!
E. coli & B. fragilis E. coli & B. fragilis E.coli E.coli
10/10 (100 %) 2/10 (20%) 20/20(100%) 17/20 (85%)
Metronidazole treatment of animals challenged with E. coli and E. coli in combination with B. fragilis All untreated animals challenged with E. coli plus B.fragilis died (Table II). By contrast, with metronidazole treatment the mortality rate was only 20 %. This difference is statistically significant by chi square analysis (P
Activity of metronidazole against Escherichia coli in experimental infra-abdominal sepsis A. B. Onderdonk, T. J. Louie, F. P. Tally and J. G. Bartlett
Metronidazole treatment was found to reduce both mortality rates and the incidence of abscess formation in experimental animals challenged with an intraperitoneal implant of pooled caecal contents. This was not anticipated since previous studies of this model indicated that death was due to Escherichia coli bacteraemia. This suggested that metronidazole was active against coliforms in vivo, despite in vitro resistance. Using an intraperitoneal challenge off. coli plus Bacteroides fragilis or E. coli alone, metronidazole treatment reduced mortality rates, but only in animals which received E. coli plus B. fragilis. There was no significant decrease in mortality with animals challenged with E. coli alone, indicating that metronidazole may be active against E. coli only in the presence of other susceptible bacteria. In chemostat cultures of E. coli metronidazole produced a reduction in the growth rate of E. coli, only in the presence of B. fragilis. Metronidazole has in vivo activity against bacteria which are highly resistant according to conventional in vitro susceptibility tests but only when a susceptible microbe is also concurrently present. Introduction
Previous studies from this laboratory have employed an experimental animal model tc define the role of various bacteria in intra-abdominal infections. Rats challenged with an inoculum of pooled faecal contents initially developed an acute peritonitis with E. col bacteraemia and a 40% mortality. Survivors developed the second stage of disease, characterized by intra-abdominal abscesses in which B. fragilis was the predominant isolate (Onderdonk, Weinstein, Sullivan, Bartlett & Gorbach, 1974). Studies using pure cultures showed that E. coli caused early death in this model, whereas B. fragilis produced intra-abdominal abscesses (Onderdonk, Bartlett, Louie, Sullivan-Sigler & Gorbach, 1976; Onderdonk, Kasper, Cisneros & Bartlett, 1977). Antimicrobials active against coliforms prevented death, whereas agents active against anaerobes prevented abscess formation (Weinstein, Onderdonk, Bartlett & Gorbach, 1975). We concluded that coliforms caused early death, and anaerobes abscess formation. The present studies were prompted by the unexpected observation that metronidazole prevented mortality as well as abscess formation in this model despite the fact that metronidazole is inactive in vitro versus coliforms. The drug may, therefore, have in vivo activity against E. coli not detected with the usual in vitro susceptibility assays. We have now tested metronidazole against E. coli using several modifications of the previously described Wistar rat model, and in continuous culture. 0305-7453/79/020201 +10 J01.00/0
201 © 1979 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Infectious Diseases Research Laboratory, Boston Veterans Administration Hospital and Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts
202
A. B. Onderdonk et al Materials and Methods
Metronidazole treatment Male, Wistar rats weighing 180 to 200 g (Charles River, Wilmington, Mass.) were used for all experiments. The animals were housed two per cage and received chow (Ralston Purina, St Louis, Mo.) and water ad libitum. Preliminary studies were performed to determine the appropriate dose of metronidazole based on serum level determinations. Healthy rats were given arbitrarily selected doses by intramuscular injection followed by percutaneous transthoracic cardiac blood samplings at 0-5, 1, 4 and 8 h. Serum levels were determined by the clostridial haemolysis inhibition assay (Louie, Tally, Bartlett & Gorbach, 1976), using appropriate standards.
Inocula ofE. coli alone and in combination with B. fragilis The second type of inoculum consisted of E. coli (BVA 1-2) alone and in combination with B. fragilis (ATCC No. 23745). These inocula examined the effect of metronidazole treatment on these two species in the absence of other bacterial components of pooled caecal contents.
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Caecal content inoculum Two types of inocula were used. The first inoculum consisted of pooled, frozen caecal contents from meat-fed rats mixed with 10 % w/v barium sulphate (Weinstein, Onderdonk, Bartlett and Gorbach, 1974). 0-5 ml amounts of this material were placed in gelatin capsules for intraperitoneal implantation following injection of rats with Nembutal. Following challenge, the animals received one of three treatment regimens; metronidazole, clindamycin or gentamicin. A fourth group served as untreated controls. [The results in this model using clindamycin and gentamicin treatment have been previously reported (Weinstein, Onderdonk, Bartlett & Gorbach, 1975); these data are included here for the purpose of comparison with metronidazole]. Antimicrobial dosages were 16,15 and 2 mg per animal for metronidazole, clindamycin and gentamicin, respectively. The drugs were given intramuscularly beginning 4 h after implantation and then at 8 h intervals, for 10 days. Survivors were sacrificed 12 days after implantation for necropsy examination. Two parameters were used to assess results: (1) mortality rates of animals which expired during the 12 days of observation and (2) the incidence of intra-abdominal abscesses at necropsy examination on the 12th day. The criterion for denning an abscess was a loculated collection of grossly purulent exudate which showed bacteria and a predominance of polymorphonuclear leukocytes on Gram stain. Additional rats were challenged with the same inoculum of pooled caecal contents, to obtain blood cultures, by percutaneous transthoracic cardiac puncture. The results of these experiments were not included in abscess or mortality data described above, as the methods used to obtain blood cultures might alter the outcome. There were two experimental groups, rats treated with metronidazole, as previously described, and untreated controls. Blood was obtained by percutaneous transthoracic cardiac puncture during light ether anaesthesia. 01 ml of blood was placed into 19 ml of molten brain-heart infusion agar, mixed and poured into sterile petri plates. An additional 0-1 ml amount was diluted 1 : 100 with VPI dilution salts and plated in an identical manner. The plates were incubated in an anaerobic chamber for 48 h, and colony types were enumerated and identified.
Activity of metronidazole against E. eoB
203
Each of the test strains was grown in peptone-yeast glucose broth for 48 h in an anaerobic chamber, and five ml aliquots were frozen at — 40°C for future use. The inocula consisted of gelatin capsules containing 0-5 ml of one of two bacterial suspensions: (1) E. coli broth culture 1 : 1 with peptone yeast glucose broth or (2) E. coli culture diluted 1 : 1 with a B. fragilis culture. All inocula contained 50% V/V sterile caecal contents and 10 % W/V barium sulphate. Animals receiving these intraperitoneal challenges were divided into two groups. One was treated with metronidazole as described above, and the second group served as untreated controls.
Mortality rates in animals challenged with E. coli and B. fragilis This experiment was designed to evaluate the relative roles of B. fragilis and E. coli on mortality rates in animals challenged with these organisms alone and in combination. No treatment was employed, since the purpose was to assess the possibility that B.fragilis caused mortality or potentiated lethality due to E. coli. Inocula consisted of gelatin capsules containing 10% w/v barium sulphate, 50% v/v autoclaved caecal contents and serial PYG broth dilutions of E. coli (BVA 1-2), B. fragilis (ATCC 23745), or both organisms in combination. Continuous cultures The effect of metronidazole on growth rates of E. coli (BVA 1-2) alone or in the presence of B. fragilis (ATCC 23745) was tested using continuous culture techniques. The organisms were grown in a C-30 bioflo fermenter (New Brunswick Scientific Co., New Brunswick, New Jersey), modified to maintain anaerobic conditions. All experiments were conducted using a minimal medium (Varel & Bryant, 1974) containing glucose as a fermentable carbon source. A dilution rate of 016 h " 1 was used to establish continuous culture following inoculation of the fermenter vessel with the test organism(s). Oxidationreduction potential (Eh), pH, dissolved oxygen concentration, OD 550 and viable cell density were monitored as described previously (Onderdonk, Johnston, Mayhew & Gorbach, 1976). Metronidazole was added to established continuous culture via the
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Antimicrobial susceptibility testing Antimicrobial susceptibilities were determined by the broth dilution technique using the Microtiter system (Cooke Laboratory Products, Alexandria, Virginia). Organisms tested were representative strains of the four isolates which were most consistently present and recovered in highest counts from infections in untreated animals challenged with the inoculum of pooled caecal contents: B. fragilis, F. varhtm, E. coli (BVA 1-2) and enterococci. B.fragilis (ATCC No. 23745) was also tested, since this organism was used in the experiments utilizing inocula of B. fragilis plus E. coli described above. The test strains were grown for 6 to 8 h in supplemented Brain Heart Infusion broth (Scott Laboratories, Fiskeville, Rhode Island) to a density corresponding to a No. 1 McFarland nephelometer standard and then diluted 1000 fold (final concentration = 106"8 CFU/ml.) E. coli and enterococci were tested in Mueller-Hinton Broth (BBL), incubated aerobically for 24 h. E. coli was also tested in the anaerobic chamber to compare susceptibility results with anaerobic and aerobic incubation. Plates for testing obligate anaerobes were incubated in an anaerobic chamber for 36 h. The minimum inhibitory concentration (MIC) was the lowest concentration of antibiotic showing no macroscopic growth.
204
A. B. Onderdonk et aL
nutrient reservoir, in a concentration of 30 ug/ml. The concentration of metronidazole in the reservoir and the fermentation vessel was assayed periodically to calculate the amount of biologically active drug present. The effect of metronidazole on the test species was compared to the theoretical washout rate for a static, but viable population within the fermenter. A slope of viable cell density versus time which exceeded the calculated washout rate indicates a bactericidal effect, while a slope less than the washout rate suggests a decrease in growth rate but not a bacteriostatic effect. A slope equal to the washout rate indicates a net bacteriostatic effect.
Antimicrobial treatment of animals challenged with caecal contents Results of the three antimicrobial treatment regimens compared to untreated control animals are summarized in Table I. Untreated animals had a mortality rate of 37 %, and all deaths occurred during the first five days following challenge. All surviving animals in the group receiving no treatment had intra-abdominal abscesses when sacrificed at 12 days. Treatment with gentamicin Teduced mortality rates, but 54 of 55 surviving animals had abscesses. By contrast, clindamycin treatment had no significant effect on the mortality rate, but only 5 % of surviving animals had abscesses. Metronidzole treatment resulted in a mortality rate of only 10%, while 15% of surviving animals had intra-abdominal abscesses. Chi square analysis using two by two contingency tables to compare treatment groups, show that the reduction in mortality with metronidazole was significantly greateT than for untreated controls (P< 001), and not significantly different compared to gentamicin (/ > >01). Blood cultures on a separate group of animals challenged with the caecal inoculum revealed significant differences with metronidazole treatment compared to untreated controls. Nineteen of twenty blood cultures in untreated animals yielded E. coli. Only one of ten blood cultures was positive in the metronidazole treatment group. The mean Table L Results with antimicrobial treatment of animals challenged with caecal contents Antimicrobial
Mortality
(Untreated controls) 58/157(37%)* Metronidazole 5/50 (10%) Clindamycin 21/60 (35%) Gentamicin 2/57 (4%) •No. died/no, tested (%) t No. with abscesses/no, of survivors tested (%)
Abscess 99/99 (100%)t 6/45 (13%) 2/39 (5%) 54/55 (98%)
Downloaded from http://jac.oxfordjournals.org/ at Russian Archive on December 11, 2013
Results Antimicrobial serum levels and in vitro susceptibility tests Mean serum levels in five healthy rats given 16 mg doses of metronidazole intramuscularly were 26, 33, 12 and 4 mg/ml at 0-5, 1, 4 and 8 h, respectively. The mean peak serum level with clindamycin was 5 5 mg/ml, and 6-9 (ig/ml for gentamicin. The MICs for metronidazole were: B.fragilis—4 jig/ml; E. coli (BVA 1-2)—1024 ug/ ml (aerobic and anaerobic); F. varium—1 ug/ml and enterococcus 64 ug/ml.
Activity of metronidazole against £. coB
205
Table EL Treatment of animals challenged with E. coli alone and in combination with B. fragilis Antimicrobial treatment None Metronidazole None Metronidazole
Inoculum
j , tested/"/"!
E. coli & B. fragilis E. coli & B. fragilis E.coli E.coli
10/10 (100 %) 2/10 (20%) 20/20(100%) 17/20 (85%)
Metronidazole treatment of animals challenged with E. coli and E. coli in combination with B. fragilis All untreated animals challenged with E. coli plus B.fragilis died (Table II). By contrast, with metronidazole treatment the mortality rate was only 20 %. This difference is statistically significant by chi square analysis (P
