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Effects of atmosphere of incubation on recovery of bacteria and yeasts from blood cultures in tryptic soy broth
DIAGNMICROBIOLINFECTDIS 1983;1:215-219
215
Effects of Atmosphere of Incubation on Recovery of Bacteria and Yeasts from Blood Cultures in Tryptic Soy Broth Duane M. Ilstrup and John A. Washington II
A comparison was made of the results of blood cultures between 1974 and 1981 in unvented and transiently vented bottles of Tryptic soy broth under vacuum with CO2. A total of 14,646 isolates were available for statistical analysis. Significantly more isolates of Staphylococcus epidermidis, Staphylococcus aureus, Bacillus, Eseheriehia, Pseudomonas, Klebsiella, Serratia, Acinetobacter, Alcaligenes, Neisseria, and Candida were recovered from the vented bottle. Significantly more isolates of Corynebacterium, Haemophilus, Flavobacterium, Moraxella, Bacteroidoceae, and Peptostreptococcus were recovered from the unvented bottle.
INTRODUCTION The necessity for all blood culture media to be incubated aerobically and anaerobically in every clinical setting has recently been questioned by Tenney et al. {1082}. On the basis of their analysis of two prior studies (Blazevic et al., 1075; Washington, 1978}, and their own study of the effects of atmosphere of incubation on recovery of bacteria and yeasts from blood cultures, Tenney and coworkers found that a substantial percentage of clinically important, frequently isolated organisms would be missed without aerobic incubation and that, therefore, aerobic incubation conditions were routinely necessary. On the other hand, anaerobic incubation, principally for isolation of the Bacteroidaceae, appeared to be medium- and/or systemdependent. Tenney et al. therefore concluded, on the basis of their studies with supplemented peptone broth, that the very small number of isolates of Bacteroidaceae missed without anaerobic incubation would be outweighed by the number of additional isolates of Pseudomonas detected by the additional volume of blood available for aerobic incubation. Although the advantages of aerobic incubation conditions, regardless of medium (Tenney et al., 1082}, for isolation of Pseudomonas and Candida and of anaerobic incubation of Columbia (Blazevic et al., 1975) and Tryptic soy {Washington, 1078} broths for isolation of Bacteroidaceae seem clearcut, less attention has been given to the effects of atmosphere of incubation on the isolation of other genera, due in part to practical limitations on the size of controlled studies of variables affecting blood cultures and, therefore, the variety of genera and number of isolates with genera available for statistical analysis.
From the Mayo Clinic/Mayo Foundation, Rochester, Minnesota. Address reprint requests to: Duane M. Ilstrup, Section of Medical Research Statistics, Mayo
Clinic, Rochester, MN 55905. Received February 28, 1983; accepted March 23, 1983.
216
D.M. Ilstrup and J.A. Washington II"
The purpose of this study was to compare isolation rates of organisms from unvented and vented vacuum blood culture bottles containing Tryptic soy broth.
MATERIALS AND METHODS
During the period of study (1974-1981), blood for culture was collected aseptically by trained phlebotomists from patients with suspected septicemia at the Mayo Clinic and affiliated hospitals and inoculated in equal amounts into each of three or, in instances in which only small amounts of blood could be obtained, two blood culture bottles. Throughout the period of study, it was standard practice to culture two separately collected 30 ml samples of blood from adults (Washington, 1978). The study was limited to those cultures in which both or two of the three bottles inoculated consisted of 100 ml of Tryptic soy broth under vacuum with 0.025% sodium polyanetholsulfonate and 5-10% CO2 (Difco Laboratories, Detroit, MI). Upon arrival in the laboratory, one of these two bottles was transiently vented to release its vacuum; the other remained unvented. Both bottles were incubated at 35°C and inspected for macroscopic evidence of growth later in the day of their receipt in the laboratory, daily for 7 days, and finally after 13 days of incubation. Routine subcultures were performed between 6 and 17 hrs and after 48 hr of incubation by removing aseptically an aliquot of broth from each bottle with a needle and syringe and inoculating it onto a quadrant of a chocolate blood agar plate which was incubated for 48 hr at 35°C in room air atmosphere plus 5-10% CO2. Anaerobic incubation of routine subcultures was not performed. The results of all positive cultures were analyzed by statistical methods described elsewhere by Ilstrup (1978). The significance of the difference in positivity rates between the vented and unvented bottles was assessed with the sign test. Estimates of the proportions of isolates that would be missed without anaerobic incubation were the ratios of the number of isolates detected only in the unvented bottle to the total number of isolates detected by either the vented or the unvented bottles, or both. The 95% confidence intervals for these proportions were derived from the cumulative binomial distributions and were taken from the Geigy Scientific Tables (1970).
RESULTS
The number of isolates detected in unvented and vented vacuum bottles of Tryptic soy broth are given separately by organism detected in Table 1. Significantly more isolates were detected in the vented bottle than in the unvented bottle for Staphylococcus epidermidis, Staphylococcus aureus, Bacillus, Escherichia, Pseudomonas, Klebsiella, Serratia, Acinetobacter, Alcaligenes, Neisseria, and
Candida. Significantly more isolates were detected in the unvented bottle than in the vented bottle for Corynebacterium/Propionibacterium, Haemophilus, Flavobacterium, Moraxella, Bacteroidaceae, and Peptostreptococcus. Estimates of the percent of and 95% confidence intervals for the true percent of isolates that would have been missed without the unvented bottle for the organisms favored by the unvented bottle are given in Table 2. At least one-fourth of the isolates in each group of these microorganisms would have been missed if anaerobic incubation had not been used.
T A B L E 1. N u m b e r of I s o l a t e s i n U n v e n t e d a n d V e n t e d V a c u u m B o t t l e s of T r y p t i c Soy Broth
Organism
Total
Both bottles
Unvented bottle only
Vented bottle only
1748 1636 1438 818 663 378 126 116 106 103 29
557 1116 237 514 407 260 3 68 88 74 21
498 229 710 144 82 53 36 24 13 15 4
693 291 491 160 87 65 87 24 5 14 4
29 26 17 5
15 20 17 3
9 4 O 1
5 2 0 1
2559 930 817 364 361 266 248 69 67 57 33 29 29 23 15 10 8 8 8 13
1603 495 514 220 216 97 170 49 40 30 10 26 9 11 10 3 1 0 1 5
441 123 119 71 78 104 29 12 9 5 6 1 4 3 3 4 7 8 2 1
515 312 184 73 67 65 49 8 18 22 17 2 16 9 2 3 0 0 5 7
2 1
2 1
0 0
0 0
810 193 31 27 27 10 1
435 118 6 12 12 4 0
224 43 15 12 8 3 0
151 32 10 3 6 3 1
~0.001 NS NS ~0.05 NS NS NS
397 8
52 0
13 2
332 8
~0.001 NS
P
Aerobic and facultatively anaerobic Gram-positive
Staphylococcus epidermidis Staphylococcus a u r e u s Corynebacterium/Propionibacterium Streptococcus, viridans Streptococcus, g r o u p D Streptococcus pneumoniae Bacillus Streptococcus, g r o u p B Streptococcus, unclassified Streptococcus, g r o u p A Streptococcus, g r o u p s other than
~0.001 ~0.01 ~0.0001 NS NS NS ~0.0001 NS (0.05 ~ P ~ .10) NS NS
A-F
Lactobacillus Listeria Streptococcus, g r o u p C Streptococcus, g r o u p F
NS NS NS NS
Gram-negative
Escherichia Pseudomonas Klebsiella Proteus Enterobacter Haemophilus Serratia Citrobacter Salmonella Acinetobacter Alcaligenes Aeromonas Neisseria Campylobacter Providencia Actinobacillus Flavobacterium Moraxella Pasteurella Cardiobacterium and Eikenella Shigella Arizona
~0.02 ~0.001 ~0.001 NS NS ~0.01 ~0.05 NS NS ~0.01 ~0.05 NS ~0.05 NS NS NS ~0.05 ~0.01 NS (0.05 ~ P ~ .10) NS NS
Anaerobic bacteria
Bacteroidaceae Clostridium Peptococcus Peptostreptococcus Eubacterium Veillonella Bifidobacterium Yeasts
Candida Cryptococcus
218
D.M. Ilstrup and J.A. Washington II
TABLE 2. Percent of Isolates Missed Without Anaerobic Incubation Isolates unvented Total Percent 95% Confidence interval alone isolates missed for percent missed
Corynebacterium/Propionibacterium Haemophilus Flavobacterium Moraxella Bacteroidaceae Peptostreptococcus
710 104 7 8 224 12
1438 266 8 8 810 27
49.4 39.1 87.5 100.0 27.7 44.4
46.8-52.0 33.2-45.0 43.4-99.7 63.1-100.0 24.6-30.7 25.5-64.7
DISCUSSION It is clear that, excluding Corynebacterium/Propionibacterium, which are presumed contaminants, an unvented bottle is necessary for the isolation of Haemophilus, Flavobacterium, Moraxella, Bacteroidaceae, and Peptostreptococcus, and that at least one-fourth of these isolates would have gone undetected without one. Tenney et al. (1982) estimated the percent of isolates missed without anaerobic incubation from the ratio of the difference between the discordant pairs of the matched aerobic and anaerobic bottles to the total isolates detected by either or both bottles. For example, they showed that for Bacteroidaceae 34 isolates were recovered from both bottles, 10 from the vented bottle only, and 17 from the unvented bottle only. They then estimated the percent missed without the unvented bottle as (17 - 10)/ 7 (34 + 17 + 10) - 11%. We feel that this formulation has no clinical basis and 61 is a meaningless figure. The true estimate of the percent of Bacteroidaceae missed should have been 1 7 or 27.9%, which is remarkably close to our own result of 27.7%. 61 In effect, Tenney and coworkers underestimated the percent of strains missed both without unvented and without vented bottles. Although favored by the vented bottle, the following proportions of clinically significant organism groups were detected only in the unvented bottle: 13% of Pseudomonas; 14% of Staphylococcus aureus; 17% of Escherichia; 15% of Klebsiella; 12% of Serratia; 9% of Acinetobacter; 18% of Alcaligenes; and 14% of Neisseria. Only 3% of Candida were detected only in the unvented bottle. The conjecture by Tenney et al. that the additional isolates from an additional vented bottle of Pseudomonas and other organisms favored by the vented bottle would outweigh the isolates of Bacteroidaceae and other organisms favored by the unvented bottle seems unsupported by the data. To test such a hypothesis one would have to conduct a study of at least three bottles, two vented and one unvented. Even then, it is our impression from our data that a significant number of isolates would be detected only in the unvented bottle. One must ask for each organism: "What percent of the isolates of this organism can I clinically tolerate being missed if the unrented bottle is not used?" It is our feeling that for the organisms favored by the unvented bottle, and even for the organisms favored by the vented bottle, the unrented bottle is clinically necessary. It should be emphasized, however, that this recommendation is based on our analysis of results obtained with a specific blood culture system. Different results and conclusions might be obtained with another system because of differences among manufacturers' products in the composition of media, the amounts of vacuum present within bottles, and the composition of the gases used to partially replenish the
Incubation A t m o s p h e r e for Blood Cultures
219
v a c u u m w i t h i n bottles. The resulting redox potential of the m e d i u m may, therefore, vary w i d e l y among different products and either m i n i m i z e or accentuate differences in the recovery of various groups of organisms in vented and u n v e n t e d pairs of the same blood culture bottle. REFERENCES Blazevic DJ, Stemper JE, Matsen JM (1975) Effect of aerobic and anaerobic atmospheres on isolation of organisms from blood cultures. J Clin Microbiol 1:154. Geigy Scientific Tables (1970) 7th Edition. Ardsley, New York: Geigy Pharmaceuticals. Ilstrup DM (1978) Statistical methods employed in the study of blood culture media. In The Detection of Septicemia. Ed., JA Washington II, West Palm Beach, Florida: CRC Press Inc. pp. 31-39. Tenney JH, Relier LB, Mirrett S, Weinstein MP, Wang W-LL (1982) Controlled evaluation of the effect of atmosphere of incubation on detection of bacteremia and fungemia in supplemented peptone broth. J Clin Microbial 16:437. Washington JAII (1978) Conventional approaches to blood culture. In: The Detection of Septicemia. Ed. JA Washington II. West Palm Beach, Florida: CRC Press Inc. pp. 41-87.
215
Effects of Atmosphere of Incubation on Recovery of Bacteria and Yeasts from Blood Cultures in Tryptic Soy Broth Duane M. Ilstrup and John A. Washington II
A comparison was made of the results of blood cultures between 1974 and 1981 in unvented and transiently vented bottles of Tryptic soy broth under vacuum with CO2. A total of 14,646 isolates were available for statistical analysis. Significantly more isolates of Staphylococcus epidermidis, Staphylococcus aureus, Bacillus, Eseheriehia, Pseudomonas, Klebsiella, Serratia, Acinetobacter, Alcaligenes, Neisseria, and Candida were recovered from the vented bottle. Significantly more isolates of Corynebacterium, Haemophilus, Flavobacterium, Moraxella, Bacteroidoceae, and Peptostreptococcus were recovered from the unvented bottle.
INTRODUCTION The necessity for all blood culture media to be incubated aerobically and anaerobically in every clinical setting has recently been questioned by Tenney et al. {1082}. On the basis of their analysis of two prior studies (Blazevic et al., 1075; Washington, 1978}, and their own study of the effects of atmosphere of incubation on recovery of bacteria and yeasts from blood cultures, Tenney and coworkers found that a substantial percentage of clinically important, frequently isolated organisms would be missed without aerobic incubation and that, therefore, aerobic incubation conditions were routinely necessary. On the other hand, anaerobic incubation, principally for isolation of the Bacteroidaceae, appeared to be medium- and/or systemdependent. Tenney et al. therefore concluded, on the basis of their studies with supplemented peptone broth, that the very small number of isolates of Bacteroidaceae missed without anaerobic incubation would be outweighed by the number of additional isolates of Pseudomonas detected by the additional volume of blood available for aerobic incubation. Although the advantages of aerobic incubation conditions, regardless of medium (Tenney et al., 1082}, for isolation of Pseudomonas and Candida and of anaerobic incubation of Columbia (Blazevic et al., 1975) and Tryptic soy {Washington, 1078} broths for isolation of Bacteroidaceae seem clearcut, less attention has been given to the effects of atmosphere of incubation on the isolation of other genera, due in part to practical limitations on the size of controlled studies of variables affecting blood cultures and, therefore, the variety of genera and number of isolates with genera available for statistical analysis.
From the Mayo Clinic/Mayo Foundation, Rochester, Minnesota. Address reprint requests to: Duane M. Ilstrup, Section of Medical Research Statistics, Mayo
Clinic, Rochester, MN 55905. Received February 28, 1983; accepted March 23, 1983.
216
D.M. Ilstrup and J.A. Washington II"
The purpose of this study was to compare isolation rates of organisms from unvented and vented vacuum blood culture bottles containing Tryptic soy broth.
MATERIALS AND METHODS
During the period of study (1974-1981), blood for culture was collected aseptically by trained phlebotomists from patients with suspected septicemia at the Mayo Clinic and affiliated hospitals and inoculated in equal amounts into each of three or, in instances in which only small amounts of blood could be obtained, two blood culture bottles. Throughout the period of study, it was standard practice to culture two separately collected 30 ml samples of blood from adults (Washington, 1978). The study was limited to those cultures in which both or two of the three bottles inoculated consisted of 100 ml of Tryptic soy broth under vacuum with 0.025% sodium polyanetholsulfonate and 5-10% CO2 (Difco Laboratories, Detroit, MI). Upon arrival in the laboratory, one of these two bottles was transiently vented to release its vacuum; the other remained unvented. Both bottles were incubated at 35°C and inspected for macroscopic evidence of growth later in the day of their receipt in the laboratory, daily for 7 days, and finally after 13 days of incubation. Routine subcultures were performed between 6 and 17 hrs and after 48 hr of incubation by removing aseptically an aliquot of broth from each bottle with a needle and syringe and inoculating it onto a quadrant of a chocolate blood agar plate which was incubated for 48 hr at 35°C in room air atmosphere plus 5-10% CO2. Anaerobic incubation of routine subcultures was not performed. The results of all positive cultures were analyzed by statistical methods described elsewhere by Ilstrup (1978). The significance of the difference in positivity rates between the vented and unvented bottles was assessed with the sign test. Estimates of the proportions of isolates that would be missed without anaerobic incubation were the ratios of the number of isolates detected only in the unvented bottle to the total number of isolates detected by either the vented or the unvented bottles, or both. The 95% confidence intervals for these proportions were derived from the cumulative binomial distributions and were taken from the Geigy Scientific Tables (1970).
RESULTS
The number of isolates detected in unvented and vented vacuum bottles of Tryptic soy broth are given separately by organism detected in Table 1. Significantly more isolates were detected in the vented bottle than in the unvented bottle for Staphylococcus epidermidis, Staphylococcus aureus, Bacillus, Escherichia, Pseudomonas, Klebsiella, Serratia, Acinetobacter, Alcaligenes, Neisseria, and
Candida. Significantly more isolates were detected in the unvented bottle than in the vented bottle for Corynebacterium/Propionibacterium, Haemophilus, Flavobacterium, Moraxella, Bacteroidaceae, and Peptostreptococcus. Estimates of the percent of and 95% confidence intervals for the true percent of isolates that would have been missed without the unvented bottle for the organisms favored by the unvented bottle are given in Table 2. At least one-fourth of the isolates in each group of these microorganisms would have been missed if anaerobic incubation had not been used.
T A B L E 1. N u m b e r of I s o l a t e s i n U n v e n t e d a n d V e n t e d V a c u u m B o t t l e s of T r y p t i c Soy Broth
Organism
Total
Both bottles
Unvented bottle only
Vented bottle only
1748 1636 1438 818 663 378 126 116 106 103 29
557 1116 237 514 407 260 3 68 88 74 21
498 229 710 144 82 53 36 24 13 15 4
693 291 491 160 87 65 87 24 5 14 4
29 26 17 5
15 20 17 3
9 4 O 1
5 2 0 1
2559 930 817 364 361 266 248 69 67 57 33 29 29 23 15 10 8 8 8 13
1603 495 514 220 216 97 170 49 40 30 10 26 9 11 10 3 1 0 1 5
441 123 119 71 78 104 29 12 9 5 6 1 4 3 3 4 7 8 2 1
515 312 184 73 67 65 49 8 18 22 17 2 16 9 2 3 0 0 5 7
2 1
2 1
0 0
0 0
810 193 31 27 27 10 1
435 118 6 12 12 4 0
224 43 15 12 8 3 0
151 32 10 3 6 3 1
~0.001 NS NS ~0.05 NS NS NS
397 8
52 0
13 2
332 8
~0.001 NS
P
Aerobic and facultatively anaerobic Gram-positive
Staphylococcus epidermidis Staphylococcus a u r e u s Corynebacterium/Propionibacterium Streptococcus, viridans Streptococcus, g r o u p D Streptococcus pneumoniae Bacillus Streptococcus, g r o u p B Streptococcus, unclassified Streptococcus, g r o u p A Streptococcus, g r o u p s other than
~0.001 ~0.01 ~0.0001 NS NS NS ~0.0001 NS (0.05 ~ P ~ .10) NS NS
A-F
Lactobacillus Listeria Streptococcus, g r o u p C Streptococcus, g r o u p F
NS NS NS NS
Gram-negative
Escherichia Pseudomonas Klebsiella Proteus Enterobacter Haemophilus Serratia Citrobacter Salmonella Acinetobacter Alcaligenes Aeromonas Neisseria Campylobacter Providencia Actinobacillus Flavobacterium Moraxella Pasteurella Cardiobacterium and Eikenella Shigella Arizona
~0.02 ~0.001 ~0.001 NS NS ~0.01 ~0.05 NS NS ~0.01 ~0.05 NS ~0.05 NS NS NS ~0.05 ~0.01 NS (0.05 ~ P ~ .10) NS NS
Anaerobic bacteria
Bacteroidaceae Clostridium Peptococcus Peptostreptococcus Eubacterium Veillonella Bifidobacterium Yeasts
Candida Cryptococcus
218
D.M. Ilstrup and J.A. Washington II
TABLE 2. Percent of Isolates Missed Without Anaerobic Incubation Isolates unvented Total Percent 95% Confidence interval alone isolates missed for percent missed
Corynebacterium/Propionibacterium Haemophilus Flavobacterium Moraxella Bacteroidaceae Peptostreptococcus
710 104 7 8 224 12
1438 266 8 8 810 27
49.4 39.1 87.5 100.0 27.7 44.4
46.8-52.0 33.2-45.0 43.4-99.7 63.1-100.0 24.6-30.7 25.5-64.7
DISCUSSION It is clear that, excluding Corynebacterium/Propionibacterium, which are presumed contaminants, an unvented bottle is necessary for the isolation of Haemophilus, Flavobacterium, Moraxella, Bacteroidaceae, and Peptostreptococcus, and that at least one-fourth of these isolates would have gone undetected without one. Tenney et al. (1982) estimated the percent of isolates missed without anaerobic incubation from the ratio of the difference between the discordant pairs of the matched aerobic and anaerobic bottles to the total isolates detected by either or both bottles. For example, they showed that for Bacteroidaceae 34 isolates were recovered from both bottles, 10 from the vented bottle only, and 17 from the unvented bottle only. They then estimated the percent missed without the unvented bottle as (17 - 10)/ 7 (34 + 17 + 10) - 11%. We feel that this formulation has no clinical basis and 61 is a meaningless figure. The true estimate of the percent of Bacteroidaceae missed should have been 1 7 or 27.9%, which is remarkably close to our own result of 27.7%. 61 In effect, Tenney and coworkers underestimated the percent of strains missed both without unvented and without vented bottles. Although favored by the vented bottle, the following proportions of clinically significant organism groups were detected only in the unvented bottle: 13% of Pseudomonas; 14% of Staphylococcus aureus; 17% of Escherichia; 15% of Klebsiella; 12% of Serratia; 9% of Acinetobacter; 18% of Alcaligenes; and 14% of Neisseria. Only 3% of Candida were detected only in the unvented bottle. The conjecture by Tenney et al. that the additional isolates from an additional vented bottle of Pseudomonas and other organisms favored by the vented bottle would outweigh the isolates of Bacteroidaceae and other organisms favored by the unvented bottle seems unsupported by the data. To test such a hypothesis one would have to conduct a study of at least three bottles, two vented and one unvented. Even then, it is our impression from our data that a significant number of isolates would be detected only in the unvented bottle. One must ask for each organism: "What percent of the isolates of this organism can I clinically tolerate being missed if the unrented bottle is not used?" It is our feeling that for the organisms favored by the unvented bottle, and even for the organisms favored by the vented bottle, the unrented bottle is clinically necessary. It should be emphasized, however, that this recommendation is based on our analysis of results obtained with a specific blood culture system. Different results and conclusions might be obtained with another system because of differences among manufacturers' products in the composition of media, the amounts of vacuum present within bottles, and the composition of the gases used to partially replenish the
Incubation A t m o s p h e r e for Blood Cultures
219
v a c u u m w i t h i n bottles. The resulting redox potential of the m e d i u m may, therefore, vary w i d e l y among different products and either m i n i m i z e or accentuate differences in the recovery of various groups of organisms in vented and u n v e n t e d pairs of the same blood culture bottle. REFERENCES Blazevic DJ, Stemper JE, Matsen JM (1975) Effect of aerobic and anaerobic atmospheres on isolation of organisms from blood cultures. J Clin Microbiol 1:154. Geigy Scientific Tables (1970) 7th Edition. Ardsley, New York: Geigy Pharmaceuticals. Ilstrup DM (1978) Statistical methods employed in the study of blood culture media. In The Detection of Septicemia. Ed., JA Washington II, West Palm Beach, Florida: CRC Press Inc. pp. 31-39. Tenney JH, Relier LB, Mirrett S, Weinstein MP, Wang W-LL (1982) Controlled evaluation of the effect of atmosphere of incubation on detection of bacteremia and fungemia in supplemented peptone broth. J Clin Microbial 16:437. Washington JAII (1978) Conventional approaches to blood culture. In: The Detection of Septicemia. Ed. JA Washington II. West Palm Beach, Florida: CRC Press Inc. pp. 41-87.