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Rapid detection of positive blood cultures with bioluminescence assay in comparison to gram staining
Zbl. Bakt. Hyg. A 258, 464-471 (1984)
Rapid Detection of Positive Blood Cultures with Bioluminescence Assay in Comparison to Gram Staining H. R. M. LANG! and B. BECKERS 2 ! Department of Medical Chemistry, University of Vienna, Vienna, Austria Department of Medical Microbiology, Medical Faculty, RWTH Aachen, Federal Republic of Germany
2
With 6 Figures· Received May 5, 1984 . Accepted August 8, 1984
Summary A bioluminescence assay for the rapid detection of bacteria in blood culture bottles was developed and the results compared with the findings of microscopic examination. For this purpose 50 culture bottles supplemented with blood were inoculated with bacteria. After 4, 6 and 7 h of incubation at 37°C samples were removed and examined microscopically and with bioluminescence technique measuring bacterial ATP in whole blood. Colony counts were performed at each interval. The limit of detection in bioluminescence assays was reached at a bacterial density of 1 x 105 CFU/ml (p < 0.001) using Escherichia coli ATCC 25923 and Staphylococcus aureus ATCC 25922. At the same time 19 out of 20 microscopic examinations were positive. The results indicate that the bioluminescence technique has an equal sensitivity in detecting bacterial growth in blood culture bottles as the microscopic examination using stained smears. The bioluminescence technique has the potential of automation.
Zusammenfassung Ein Biolumineszenzverfahren zum schnellen Bakteriennachweis in Blutkulturflaschen wurde entwickelt und die Ergebnisse mit mikroskopischen Befunden verglichen. Fur die Untersuchungen wurde eine definierte Bakterienmenge und Blut 50 Kulturflaschen zugegeben. Nach einer Inkubation von 4, 6 und 7 Std. bei 37°C wurden aliquote Anteile entnommen und sowohl mikroskopisch als auch mit qem Biolumineszenzverfahren untersucht. Diese Methode dient dem Nachweis von bakteriellem ATP in den Proben. Zu gleichen Zeiten wurde die Anzahl kolonienbildender Einheiten bestimmt. Die Nachweisgrenze fur das Biolumineszenzverfahren liegt bei einer Bakteriendichte von 1 x 105 KBE/ml (P < 0,001), wenn die Blutkulturflaschen mit Escherichia coli ATCC25923 und Staphylococcus aureus ATCC 25922 beimpft wurden. Zu diesem Zeitpunkt ergaben auch 19 von 20 mikroskopischen Untersuchungen ein positives Ergebnis. Die Ergebnisse zeigen eine gleiche Empfindlichkeit zum Nachweis von Bakterien in Blutkulturflaschen zwischen mikroskopischer Beurteilung gefiirbter Ausstriche und dem Biolumineszenzverfahreno Letzteres hat den Vorteil einer m6glichen Automatisierung.
Bioluminescent Assay in Blood Cultures
465
Introduction Despite recent progresses in clinical microbiology, rapid detection of septicemia remains one of the most important tasks in diagnostic bacteriology. Early positive results can significantly improve the therapy and final outcome of the patient. Recently several new methods for the early detection of bacteria in blood cultures were introduced (3, 4, 5, 6, 9). One of these is the bioluminescence assay, which bases on the detection of bacterial ATP in blood cultures (1). The same technique has been already used for bacteriuria screening (7, 8) and rapid susceptibility testing of mycobacteria (2). In this paper two variations of the bioluminescence technique using blood cell free supernatant and blood culture directly were described. The results were compared with findings of microscopic examinations and determinations of colony forming units.
Materials and Methods
Inoculated blood cultures: Ten seperate assays consisting each of 5 blood cultures containing 70 ml tryptic soy broth ("Liquoid", La Roche) were run. The inoculum was prepared starting from an 18 h culture of E. coli ATCC 25923 and of Staphylococcus aureus ATCC 25922. Group 1 of blood cultures was inoculated with 1 ml of a 10-2 diluted S. aureus culture. A dilution of 10-3 was used as inoculum for group 2. Group 3 and group 4 were contaminated with 1 ml of a 10-3 and 10-4 diluted culture of E. coli. Group 5 consisted of sterile blood cultures and served as negative control. Before incubation at each bottle was supplemented with 10 ml human blood. After 4, 6 and 7 h 5 ml samples were removed and examined for the presence of bacteria with two different methods. Assays were performed with blood cell free supernatant obtained after 10 min centrifugation at 100 g from 2.5 ml blood culture and with uncentrifuged blood culture.
3rc
5 ml sample
1
I
2.5 milO' 100 9
supernatant
.--
1 ml 10'
1000 g
1
sediment
Gram staining
+
1 ml • 1 ml NR5 R 10' 1000g
1
sediment R • 100 III NR5 R • 20 III 50mase
45' 37'C
1
AlP extraction
1 ml
10'
1000 g
1
sediment Gram staining
1 ml • 1 ml NR5 R
10'
1000g
1
0.5 ml
1
sediment R 10 fold serial • 100 III NR5 R d'l . • 20 III 50mase I ut.on
45' 37'C
1
AlP extraction
Fig. 1. Way of processing samples from experimental blood cultures.
18 h 37 C 0
1
colony counts
466
H. R. M. Lang and B. Beckers
Bioluminescence assay: From both samples 1 ml each was mixed with 1 ml NRS® (a nucleotide releasing reagent for somatic cells, Lumac/3M) in measuring cuvettes and centrifuged for 10 min at 1000 g. The initial NRS® treatment and centrifugation removes the majority of somatic ATP. The pellets were resuspended and supplemented with 100 Il1 NRS® and 20 I-tl Somase® (ATPase enzyme). During the following incubation at 37°C for 45 min the remaining somatic cells are lysed and the so liberated somatic ATP degraded enzymatically (Fig. 1). Thereafter the bacterial cells are destroyed by addition of 100 I-tl NRB® (a nucleotide releasing reagent for bacterial cells, Lumac/3M). After 10 sec the cuvettes were placed in the counting chamber of a Biocounter model M2000 (Lumac/3M) and ATP logCFU/ml
7
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t1 V •
" 4<
3,
l/l)
l~l/l
1T•--------- j I
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I
4
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• 1
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670
i i i
4
6
7
h
Fig. 2. Results of determination of CFU/ml in experimental blood cultures. Left curves (group 1, • and group 2,0) represent blood cultures which were inoculated with 1 ml of a 10-2 resp. 10-3 culture of S. aureus ATCC 25922 and corresponds to 106 resp. 10 5 bacterial bottle. Right curves (group 3, • and group 4, 0) represent blood cultures which were inoculated with 1 ml of a 10-3 resp. 10-4 diluted culture of E. coli ATCC 25923 and corresponds to 105 resp. 104 bacteria/bottle.
Bioluminescent Assay in Blood Cultures
467
determined immediately by addition of 100 !-II Lumit PM (Iuciferine-Iuciferase reagent, Lumad3M). During an integration period of 10 sec the light production of luciferine luciferase reaction was recorded and expressed as relative light units (RLU). Microscopic examination: Parallel to the bioluminescence assay 1 ml of blood cell free supernatant and 1 ml not centrifuged blood cultures were centrifugated at 1000 g for 10 min. The supernatant was discarded and from 20 !-II pellet resuspended by shaking a smear prepared (Fig. 1). After Gram staining, the slides were examined by 2 technicians for the presence of bacteria in 20 visual fields. As each group of blood cultures consisted of 10 bottles which were examined by 2 persons a total of 20 reports were obtained. Determination of colony forming units (CPU): From 0.5 ml samples a 10-fold serial dilution in sterile saline was performed up to a dilution of 10-6 (Fig. 1). From each dilution step 2 drops of 20 !-II were dropped on the surface of nutrient agar plates. After an incubation of 18 h at 37°C the colonies were counted and the number of CFU/ml blood culture calculated. The determination of the number of CFU/ml was done immediately after inoculation and also after 4, 6 and 7 h of incubation.
Results The results of determination of CFU/ml are presented in Fig. 2. An early increase in viable counts was observed in each group. Growth curves of both species run in a
log RLU/ml
2
7
6
1 1
1
9
1
19 18
11 1
4
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3
2
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r/i 1---1 T 10______
1
4
---0
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o
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67
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1
•
I IL-----i"I 1 4
67h
Fig. 3. Bioluminescence assay (RLU/ml) and microscopic examination in group 1 (e) of experimental blood cultures being inoculated with 10 6 S. aureus in comparison to negative controls (group 5,0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examinations. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
H. R. M. Lang and B. Beckers
468
log RLU/ml
J
o
1
1
3
11
4
1
11
14
11
I
I/o
3
o
l/~I
2
T
.-~~T
I
7
4
ll-----.:.i.l-Lf 1 4
6
lL----- I
•T
o
o
6
7
h
Fig. 4. Bioluminescence assay (RLU/ml) and microscopic examination in group 2 (e) of experimental blood cultures being inoculated with 105 S. aureus in comparison to negative controls (group 5, 0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
parallel fashion. The differences between each groups are due to the different generation time and the size of inoculum used. In Figs. 3 and 4 the results of bioluminescence assay with S. aureus blood cultures are shown compared with the negative controls (group 5). The results of S. aureus (group 1) blood cultures are shown in Fig. 3. During incubation an increasing amount of bacterial ATP expressed in RLU/ml blood culture is measured. This increase is comparable to the growth curve shown in Fig. 2. The difference in ATP levels between seeded cultures and sterile controls is significant (p < 0.001) after 6 h of incubation. At this time the bacterial density of the blood culture is 1 x 105 cfu/ml. The microscopic examination gives in 7 cases of a total of 20 a positive result if blood cell free supernatant is analysed, whereas the microscopy fails only in 1 case of uncentrifuged blood culture. Results of S. aureus blood cultures (group 2) using a lower inoculum are represented in Fig. 4. Also in this group a slight increase in RLU values can be observed during incubation, after 7 h the values show a significant difference to sterile controls (p < 0.05). After 7 h of incubation only 3 resp. 14 cases were positive by Gram staining. Results of blood cultures inoculated with E. coli are shown in Fig. 5 and 6. Even after 4 h of incubation increasing amounts of bacterial ATP can be observed in group 3 of blood cultures. A significant difference (p < 0.001) between seeded blood cultures of
Bioluminescent Assay in Blood Cultures
469
group 4 in comparison to negative controls can be observed after 6 h of incubation. The bacterial density at this time is 2.4 X 10 5 CFU/ml blood culture. The microscopic examination reveals 12 resp. 16 positive cases of a total of 20. Discussion The aim of this investigation was to develop a screening test for the rapid detection of bacteria in blood cultures. Previous studies (1) revealed that the bioluminescence technique is a suitable method for measuring bacterial ATP under these conditions. To discriminate between high levels of somatic ATP derived from blood cells and low amounts of ATP derived from bacteria, a centrifugation step at 100 g for 10 min in order to obtain a crude separation of bacteria from blood cells had been performed (1). In this study an imptoved method was elaborated in order to make this test more suitable for routine analysis. The method was modified obtaining degradation of non101 RLU/ml
5
9
18
18
111
1
13
1
1/1
4
19 19
11 1
1/'
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o
1
3
o
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0______ T
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T__To
L-----j"'I
1
j
67
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----.0
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.----
4
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I
67
h
Fig. 5. Bioluminescence assay (RLU/ml) and microscofic examination in group 3 (e) of experimental blood cultures being inoculated with 10 E. coli in comparison to negative controls (group 5, 0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
470
H.R.M.Lang and B.Beckers
log RLU/ml
4
1
12 17
11
4
7
1
r
I
/ •
2
]
1/1
4
•
[Ill
n------l-r 1
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T
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16 18
1
~i'--1
1
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67
4
67h
Fig. 6. Bioluminescence assay (RLU/ml) and microscofic examination in group 4 (e) of experimental blood cultures being inoculated with 10 E. coli in comparison to negative controls (group 5,0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly. bacterial ATP by lysis of blood cells followed by enzymatic degradation of liberated ATP with ATPase. The comparison of these methods is shown in the left resp. right parts of figures 3, 4, 5 and 6. The ATP measurement in the latter case shows a greater deviation. This may be due to optical and chemical quenching by lysed blood cells; sensitivity, however, is better. This is supported by results of microscopic examination. Six positive smears were found in group 1 after 7 h when supernatant was analysed. From uncentrifuged blood cultures 18 cases were positive. In group 2 similar results were obtained. The reason for the diminished sensitivity is that there is a loss of 60 to 70 per cent of staphylococci in the supernatant of blood cultures when the material is centrifuged for 10 min at 100 g. If blood cultures are inoculated with E. coli only 20 per cent of bacteria are lost by this centrifugation step. The microscopic examination of stained smears and the determination of bacterial ATP are both suitable for the detection of bacteria in blood cultures. The bioluminescence method seems to be more sensitive. This fact is shown in Fig. 6, where after 6 h of incubation the ATP values indicate bacterial growth with high significance (p < 0.001), whereas Gram staining is positive only in 12 resp. 16 cases of a total of 20. The bioluminescence method could be introduced as a screening test for rapid detection of bacteremia. It has the advantage of being rapid and sensitive, independant from subjective factors and suitable for automation. But until the moment fully automated instruments are not available and costs for reagents are high.
Bioluminescent Assay in Blood Cultures
471
As the test system for reasons of improved reproducibility was performed starting with unphysiological high densities of bacteria/ml of blood, it cannot be concluded that detection of bacteremia with bioluminescence assay can always be performed within 4-6 h after inoculation of blood cultures. For this purpose further investigations under routine conditions using blood cultures from patients are needed.
References
1. Beckers, B. and H. R. M. Lang: Bioluminescence measurement of ATP for the rapid detection of positive blood cultures. Naturwissenschaften 69 (1982) 145-146 2. Beckers, B., H. R. M. Lang, and D. Schimke: Susceptibility testing for Mycobacteria within 7 days. Abstract: 83rd Annual Meeting of the American Society for Microbiology, March 1983, New Orleans 3. Doern, G. V. and L. I. Robbie: Direct Identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test. ]. din. Microbiol. 16 (1982) 1048-1051 4. Fossieck, B. and B. Fedorko: Counter-immuno-electrophoresis of blood cultures: Temporal relationship of positive Gram stains to positive counter-immuno-electrophoresis. Amer. J. din. Path. 71 (1979) 326-329 5. Fung, J. C. and K. Wicher: Minimum number of bacteria needed for antigen detection by counter-immuno-electrophoreses: in vivo and in vitro studies. J. din. Microbiol. 13 (1981) 681-687 6. Herlich, M. W., R. F. Schell, M. Francisco, and J. L. Le Frock: Rapid detection of simulated bacteriemia by centrifugation and filtration. ]. din. Microbiol. 16 (1982) 99-102 7. McWalter, P. W. and C. A. Sharp: Evaluation of a commercially available semi-automated bioluminescence system for bacteriuria screening. Europ. ]. din. Microbiol. 1 (1982) 223-227 8. Ruokonen, A., M. Koskinen, M. Leinonen, A. Tilikainen, and R. Vihko: Screening of bacteria in urine using luciferine-luciferase assay of microbial ATP, a comparative study. Ann. din. Biochem. 6 (1982) 416-420 9. Wetkowski, M. A., E. M. Peterson, and G. Frankel: Counter-immuno-electrophoresis for early detection and rapid identification of Hemophilus inf/uenzae type B and Streptococcus pneumoniae in blood cultures. J. din. Microbiol. 12 (1980) 614-616 Dr. H. R. M. Lang, Dept. of Medical Chemistry, University of Vienna, Wiihringer Str. 10, A-1090 Vienna, Austria
Rapid Detection of Positive Blood Cultures with Bioluminescence Assay in Comparison to Gram Staining H. R. M. LANG! and B. BECKERS 2 ! Department of Medical Chemistry, University of Vienna, Vienna, Austria Department of Medical Microbiology, Medical Faculty, RWTH Aachen, Federal Republic of Germany
2
With 6 Figures· Received May 5, 1984 . Accepted August 8, 1984
Summary A bioluminescence assay for the rapid detection of bacteria in blood culture bottles was developed and the results compared with the findings of microscopic examination. For this purpose 50 culture bottles supplemented with blood were inoculated with bacteria. After 4, 6 and 7 h of incubation at 37°C samples were removed and examined microscopically and with bioluminescence technique measuring bacterial ATP in whole blood. Colony counts were performed at each interval. The limit of detection in bioluminescence assays was reached at a bacterial density of 1 x 105 CFU/ml (p < 0.001) using Escherichia coli ATCC 25923 and Staphylococcus aureus ATCC 25922. At the same time 19 out of 20 microscopic examinations were positive. The results indicate that the bioluminescence technique has an equal sensitivity in detecting bacterial growth in blood culture bottles as the microscopic examination using stained smears. The bioluminescence technique has the potential of automation.
Zusammenfassung Ein Biolumineszenzverfahren zum schnellen Bakteriennachweis in Blutkulturflaschen wurde entwickelt und die Ergebnisse mit mikroskopischen Befunden verglichen. Fur die Untersuchungen wurde eine definierte Bakterienmenge und Blut 50 Kulturflaschen zugegeben. Nach einer Inkubation von 4, 6 und 7 Std. bei 37°C wurden aliquote Anteile entnommen und sowohl mikroskopisch als auch mit qem Biolumineszenzverfahren untersucht. Diese Methode dient dem Nachweis von bakteriellem ATP in den Proben. Zu gleichen Zeiten wurde die Anzahl kolonienbildender Einheiten bestimmt. Die Nachweisgrenze fur das Biolumineszenzverfahren liegt bei einer Bakteriendichte von 1 x 105 KBE/ml (P < 0,001), wenn die Blutkulturflaschen mit Escherichia coli ATCC25923 und Staphylococcus aureus ATCC 25922 beimpft wurden. Zu diesem Zeitpunkt ergaben auch 19 von 20 mikroskopischen Untersuchungen ein positives Ergebnis. Die Ergebnisse zeigen eine gleiche Empfindlichkeit zum Nachweis von Bakterien in Blutkulturflaschen zwischen mikroskopischer Beurteilung gefiirbter Ausstriche und dem Biolumineszenzverfahreno Letzteres hat den Vorteil einer m6glichen Automatisierung.
Bioluminescent Assay in Blood Cultures
465
Introduction Despite recent progresses in clinical microbiology, rapid detection of septicemia remains one of the most important tasks in diagnostic bacteriology. Early positive results can significantly improve the therapy and final outcome of the patient. Recently several new methods for the early detection of bacteria in blood cultures were introduced (3, 4, 5, 6, 9). One of these is the bioluminescence assay, which bases on the detection of bacterial ATP in blood cultures (1). The same technique has been already used for bacteriuria screening (7, 8) and rapid susceptibility testing of mycobacteria (2). In this paper two variations of the bioluminescence technique using blood cell free supernatant and blood culture directly were described. The results were compared with findings of microscopic examinations and determinations of colony forming units.
Materials and Methods
Inoculated blood cultures: Ten seperate assays consisting each of 5 blood cultures containing 70 ml tryptic soy broth ("Liquoid", La Roche) were run. The inoculum was prepared starting from an 18 h culture of E. coli ATCC 25923 and of Staphylococcus aureus ATCC 25922. Group 1 of blood cultures was inoculated with 1 ml of a 10-2 diluted S. aureus culture. A dilution of 10-3 was used as inoculum for group 2. Group 3 and group 4 were contaminated with 1 ml of a 10-3 and 10-4 diluted culture of E. coli. Group 5 consisted of sterile blood cultures and served as negative control. Before incubation at each bottle was supplemented with 10 ml human blood. After 4, 6 and 7 h 5 ml samples were removed and examined for the presence of bacteria with two different methods. Assays were performed with blood cell free supernatant obtained after 10 min centrifugation at 100 g from 2.5 ml blood culture and with uncentrifuged blood culture.
3rc
5 ml sample
1
I
2.5 milO' 100 9
supernatant
.--
1 ml 10'
1000 g
1
sediment
Gram staining
+
1 ml • 1 ml NR5 R 10' 1000g
1
sediment R • 100 III NR5 R • 20 III 50mase
45' 37'C
1
AlP extraction
1 ml
10'
1000 g
1
sediment Gram staining
1 ml • 1 ml NR5 R
10'
1000g
1
0.5 ml
1
sediment R 10 fold serial • 100 III NR5 R d'l . • 20 III 50mase I ut.on
45' 37'C
1
AlP extraction
Fig. 1. Way of processing samples from experimental blood cultures.
18 h 37 C 0
1
colony counts
466
H. R. M. Lang and B. Beckers
Bioluminescence assay: From both samples 1 ml each was mixed with 1 ml NRS® (a nucleotide releasing reagent for somatic cells, Lumac/3M) in measuring cuvettes and centrifuged for 10 min at 1000 g. The initial NRS® treatment and centrifugation removes the majority of somatic ATP. The pellets were resuspended and supplemented with 100 Il1 NRS® and 20 I-tl Somase® (ATPase enzyme). During the following incubation at 37°C for 45 min the remaining somatic cells are lysed and the so liberated somatic ATP degraded enzymatically (Fig. 1). Thereafter the bacterial cells are destroyed by addition of 100 I-tl NRB® (a nucleotide releasing reagent for bacterial cells, Lumac/3M). After 10 sec the cuvettes were placed in the counting chamber of a Biocounter model M2000 (Lumac/3M) and ATP logCFU/ml
7
!
•I
t1 V •
" 4<
3,
l/l)
l~l/l
1T•--------- j I
o
I
4
1 r
• 1
1""'--
670
i i i
4
6
7
h
Fig. 2. Results of determination of CFU/ml in experimental blood cultures. Left curves (group 1, • and group 2,0) represent blood cultures which were inoculated with 1 ml of a 10-2 resp. 10-3 culture of S. aureus ATCC 25922 and corresponds to 106 resp. 10 5 bacterial bottle. Right curves (group 3, • and group 4, 0) represent blood cultures which were inoculated with 1 ml of a 10-3 resp. 10-4 diluted culture of E. coli ATCC 25923 and corresponds to 105 resp. 104 bacteria/bottle.
Bioluminescent Assay in Blood Cultures
467
determined immediately by addition of 100 !-II Lumit PM (Iuciferine-Iuciferase reagent, Lumad3M). During an integration period of 10 sec the light production of luciferine luciferase reaction was recorded and expressed as relative light units (RLU). Microscopic examination: Parallel to the bioluminescence assay 1 ml of blood cell free supernatant and 1 ml not centrifuged blood cultures were centrifugated at 1000 g for 10 min. The supernatant was discarded and from 20 !-II pellet resuspended by shaking a smear prepared (Fig. 1). After Gram staining, the slides were examined by 2 technicians for the presence of bacteria in 20 visual fields. As each group of blood cultures consisted of 10 bottles which were examined by 2 persons a total of 20 reports were obtained. Determination of colony forming units (CPU): From 0.5 ml samples a 10-fold serial dilution in sterile saline was performed up to a dilution of 10-6 (Fig. 1). From each dilution step 2 drops of 20 !-II were dropped on the surface of nutrient agar plates. After an incubation of 18 h at 37°C the colonies were counted and the number of CFU/ml blood culture calculated. The determination of the number of CFU/ml was done immediately after inoculation and also after 4, 6 and 7 h of incubation.
Results The results of determination of CFU/ml are presented in Fig. 2. An early increase in viable counts was observed in each group. Growth curves of both species run in a
log RLU/ml
2
7
6
1 1
1
9
1
19 18
11 1
4
Iii
3
2
1
r/i 1---1 T 10______
1
4
---0
T_ T
1
o
1
67
1/
1
•
I IL-----i"I 1 4
67h
Fig. 3. Bioluminescence assay (RLU/ml) and microscopic examination in group 1 (e) of experimental blood cultures being inoculated with 10 6 S. aureus in comparison to negative controls (group 5,0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examinations. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
H. R. M. Lang and B. Beckers
468
log RLU/ml
J
o
1
1
3
11
4
1
11
14
11
I
I/o
3
o
l/~I
2
T
.-~~T
I
7
4
ll-----.:.i.l-Lf 1 4
6
lL----- I
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o
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6
7
h
Fig. 4. Bioluminescence assay (RLU/ml) and microscopic examination in group 2 (e) of experimental blood cultures being inoculated with 105 S. aureus in comparison to negative controls (group 5, 0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
parallel fashion. The differences between each groups are due to the different generation time and the size of inoculum used. In Figs. 3 and 4 the results of bioluminescence assay with S. aureus blood cultures are shown compared with the negative controls (group 5). The results of S. aureus (group 1) blood cultures are shown in Fig. 3. During incubation an increasing amount of bacterial ATP expressed in RLU/ml blood culture is measured. This increase is comparable to the growth curve shown in Fig. 2. The difference in ATP levels between seeded cultures and sterile controls is significant (p < 0.001) after 6 h of incubation. At this time the bacterial density of the blood culture is 1 x 105 cfu/ml. The microscopic examination gives in 7 cases of a total of 20 a positive result if blood cell free supernatant is analysed, whereas the microscopy fails only in 1 case of uncentrifuged blood culture. Results of S. aureus blood cultures (group 2) using a lower inoculum are represented in Fig. 4. Also in this group a slight increase in RLU values can be observed during incubation, after 7 h the values show a significant difference to sterile controls (p < 0.05). After 7 h of incubation only 3 resp. 14 cases were positive by Gram staining. Results of blood cultures inoculated with E. coli are shown in Fig. 5 and 6. Even after 4 h of incubation increasing amounts of bacterial ATP can be observed in group 3 of blood cultures. A significant difference (p < 0.001) between seeded blood cultures of
Bioluminescent Assay in Blood Cultures
469
group 4 in comparison to negative controls can be observed after 6 h of incubation. The bacterial density at this time is 2.4 X 10 5 CFU/ml blood culture. The microscopic examination reveals 12 resp. 16 positive cases of a total of 20. Discussion The aim of this investigation was to develop a screening test for the rapid detection of bacteria in blood cultures. Previous studies (1) revealed that the bioluminescence technique is a suitable method for measuring bacterial ATP under these conditions. To discriminate between high levels of somatic ATP derived from blood cells and low amounts of ATP derived from bacteria, a centrifugation step at 100 g for 10 min in order to obtain a crude separation of bacteria from blood cells had been performed (1). In this study an imptoved method was elaborated in order to make this test more suitable for routine analysis. The method was modified obtaining degradation of non101 RLU/ml
5
9
18
18
111
1
13
1
1/1
4
19 19
11 1
1/'
III
o
1
3
o
j )
2
0______ T
1
T__To
L-----j"'I
1
j
67
4
----.0
1
.----
4
I
I
67
h
Fig. 5. Bioluminescence assay (RLU/ml) and microscofic examination in group 3 (e) of experimental blood cultures being inoculated with 10 E. coli in comparison to negative controls (group 5, 0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly.
470
H.R.M.Lang and B.Beckers
log RLU/ml
4
1
12 17
11
4
7
1
r
I
/ •
2
]
1/1
4
•
[Ill
n------l-r 1
1l
T
1/1 3
16 18
1
~i'--1
1
j
67
4
67h
Fig. 6. Bioluminescence assay (RLU/ml) and microscofic examination in group 4 (e) of experimental blood cultures being inoculated with 10 E. coli in comparison to negative controls (group 5,0). The arrows above the curves indicate number of positive Gram stains out of a total of 20 examination. The left part of the figure was performed using blood cell free supernatant. The right part shows results obtained using blood culture directly. bacterial ATP by lysis of blood cells followed by enzymatic degradation of liberated ATP with ATPase. The comparison of these methods is shown in the left resp. right parts of figures 3, 4, 5 and 6. The ATP measurement in the latter case shows a greater deviation. This may be due to optical and chemical quenching by lysed blood cells; sensitivity, however, is better. This is supported by results of microscopic examination. Six positive smears were found in group 1 after 7 h when supernatant was analysed. From uncentrifuged blood cultures 18 cases were positive. In group 2 similar results were obtained. The reason for the diminished sensitivity is that there is a loss of 60 to 70 per cent of staphylococci in the supernatant of blood cultures when the material is centrifuged for 10 min at 100 g. If blood cultures are inoculated with E. coli only 20 per cent of bacteria are lost by this centrifugation step. The microscopic examination of stained smears and the determination of bacterial ATP are both suitable for the detection of bacteria in blood cultures. The bioluminescence method seems to be more sensitive. This fact is shown in Fig. 6, where after 6 h of incubation the ATP values indicate bacterial growth with high significance (p < 0.001), whereas Gram staining is positive only in 12 resp. 16 cases of a total of 20. The bioluminescence method could be introduced as a screening test for rapid detection of bacteremia. It has the advantage of being rapid and sensitive, independant from subjective factors and suitable for automation. But until the moment fully automated instruments are not available and costs for reagents are high.
Bioluminescent Assay in Blood Cultures
471
As the test system for reasons of improved reproducibility was performed starting with unphysiological high densities of bacteria/ml of blood, it cannot be concluded that detection of bacteremia with bioluminescence assay can always be performed within 4-6 h after inoculation of blood cultures. For this purpose further investigations under routine conditions using blood cultures from patients are needed.
References
1. Beckers, B. and H. R. M. Lang: Bioluminescence measurement of ATP for the rapid detection of positive blood cultures. Naturwissenschaften 69 (1982) 145-146 2. Beckers, B., H. R. M. Lang, and D. Schimke: Susceptibility testing for Mycobacteria within 7 days. Abstract: 83rd Annual Meeting of the American Society for Microbiology, March 1983, New Orleans 3. Doern, G. V. and L. I. Robbie: Direct Identification of Staphylococcus aureus in blood culture fluid with a commercial latex agglutination test. ]. din. Microbiol. 16 (1982) 1048-1051 4. Fossieck, B. and B. Fedorko: Counter-immuno-electrophoresis of blood cultures: Temporal relationship of positive Gram stains to positive counter-immuno-electrophoresis. Amer. J. din. Path. 71 (1979) 326-329 5. Fung, J. C. and K. Wicher: Minimum number of bacteria needed for antigen detection by counter-immuno-electrophoreses: in vivo and in vitro studies. J. din. Microbiol. 13 (1981) 681-687 6. Herlich, M. W., R. F. Schell, M. Francisco, and J. L. Le Frock: Rapid detection of simulated bacteriemia by centrifugation and filtration. ]. din. Microbiol. 16 (1982) 99-102 7. McWalter, P. W. and C. A. Sharp: Evaluation of a commercially available semi-automated bioluminescence system for bacteriuria screening. Europ. ]. din. Microbiol. 1 (1982) 223-227 8. Ruokonen, A., M. Koskinen, M. Leinonen, A. Tilikainen, and R. Vihko: Screening of bacteria in urine using luciferine-luciferase assay of microbial ATP, a comparative study. Ann. din. Biochem. 6 (1982) 416-420 9. Wetkowski, M. A., E. M. Peterson, and G. Frankel: Counter-immuno-electrophoresis for early detection and rapid identification of Hemophilus inf/uenzae type B and Streptococcus pneumoniae in blood cultures. J. din. Microbiol. 12 (1980) 614-616 Dr. H. R. M. Lang, Dept. of Medical Chemistry, University of Vienna, Wiihringer Str. 10, A-1090 Vienna, Austria