Controlled Comparison of Original Vented Aerobic FAN Medium with New Nonvented BacT/ALERT FA Medium for Culturing Blood

Abstract

To evaluate the performance of BacT/ALERT FA (FA) medium, a new aerobic BacT/ALERT FAN (FAN) medium (Organon Teknika Corporation, Durham, N.C.) that does not require the added cost and inconvenience of a venting unit, we inoculated blood specimens from adult patients with suspected sepsis into an original FAN aerobic culture bottle and an FA bottle. Of 7,745 blood culture sets containing both bottles, 5,256 (68%) met the criteria for adequacy of filling. A total of 466 isolates judged to represent the causes of true infections were recovered from 276 patients; 271 isolates were recovered from both bottles, 82 were recovered from the FAN bottle only, and 113 were recovered from the FA bottle only (P < 0.05). More Burkholderia cepacia isolates (P < 0.01), Candida albicans isolates (P < 0.001), Cryptococcus neoformans isolates (P < 0.01), yeasts overall (P < 0.001), and total microorganisms (P < 0.05) were recovered from FA bottles. Of cultures found to be positive within the first 72 h of incubation, the mean times to detection were almost identical for FAN (20.4 h) and FA (20.7 h) bottles. Of 263 isolates that caused monomicrobic episodes of bloodstream infections, 180 were detected in both bottles, 32 were detected in FAN bottles only, and 51 were detected in FA bottles only (P < 0.05). Of 186 isolates considered to be contaminants, 63 were detected in both media, 64 were detected in FAN bottles only, and 59 were detected in FA bottles only (P was not significant). The number of false-positive results were comparable: 69 (1.3%) in FAN bottles and 56 (1.1%) in FA bottles. However, there were 14 isolates with false-negative results (6 yeasts, 6 nonfermenters, and 1 isolate each of Propionibacterium acnes and coagulase-negative staphylococci) in FAN bottles, whereas there were none in FA bottles. On the basis of these results, we conclude that the new nonvented FA bottle is superior to the original vented FAN medium for the recovery of B. cepacia and yeasts, especially C. albicans and C. neoformans, and is comparable to FAN medium for other microorganisms.

The detection of bacteremia and fungemia is one of the most important functions of the clinical microbiology laboratory. Modern automated detection systems have increased the rate of isolation of pathogenic microorganisms with improved formulations of media and computerized algorithms to detect evidence of growth. Organon Teknika Corporation (Durham, N.C.) developed an aerobic BacT/ALERT FAN (FAN) medium that increases the rate of detection of microorganisms compared with that obtained with standard medium in the BacT/ALERT microbial detection system. However, in the current BacT/ALERT instrument, the bottles containing the aerobic FAN medium require transient manual venting before incubation. The venting unit requires additional time for the insertion and transient venting of each bottle, requires care for avoidance of the potential hazards due to the use of sharps, and adds additional cost.

Organon Teknika has developed a new aerobic FAN blood culture medium that incorporates additional oxygen, thereby allowing the bottle to be incubated without transient venting. The study described here was designed to compare this new formulation (designated the BacT/ALERT FA [FA] formulation) with the current FAN formulation for the detection of bacteremia and fungemia in patients with suspected sepsis.

(This work was presented in part at the 99th General Meeting of the American Society for Microbiology, Chicago, III., 1999 [S. Mirrett, L. B. Reller, and R. J. Everts, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. C-495. p. 206, 1999].)

MATERIALS AND METHODS

Blood culture and collection.

Samples for blood culture were collected from adult patients hospitalized at Duke University Medical Center. Institutional review board approval was obtained prior to the study, and all blood cultures were performed as part of routine patient care. Venipuncture sites were disinfected with alcohol and then povidone iodine and were allowed to dry. Up to 30 ml of blood was obtained with a sterile needle and syringe. Needles were not changed before or between inoculation of blood culture bottles. Ten milliliters of blood was placed into each of three blood culture bottles: an aerobic FAN bottle, the new nonvented FA bottle, and a BACTEC LYTIC/10 Anaerobic/F bottle (which was not part of the evaluation but which was included for optimal recovery of microorganisms). A comparison of the two medium formulations is shown in Table 1.

TABLE 1.

Comparison of medium formulations of FAN and FA bottles

Ingredient	FAN bottles	FA bottles
Brain heart infusion solids (% [wt/vol])	2.8	0.1
Casein-soybean solids (% [wt/vol])	0.0	2.0
Sodium polyanetholsulfonate (% [wt/vol])	0.05	0.05
Pyridoxine HCl (% [wt/vol])	0.001	0.001
Menadione (% [wt/vol])	0.00005	0.00005
Hemin (% [wt/vol])	0.0005	0.0005
Activated charcoal (% [wt/vol])	8.5	6.5
l-Cysteine and other amino acids	Yes	Yes
Atmosphere of oxygen and carbon dioxide under vacuum	Yes	Yes
Headspace (ml)	33	50
Total medium vol (ml)	40	30
Detection sensor	Embedded disk	Liquid emulsion

Adequacy of blood volume.

Upon receipt in the laboratory, the volume of fluid in each bottle was measured against a volume standard to determine how many milliliters of blood had been inoculated into each of the FAN bottles. All bottles were processed regardless of the volume of blood received. A bottle pair was included in the data analysis if the blood volume of the bottle with the smaller volume was within 20% of the blood volume in the bottle with the larger volume.

Bottle processing.

After transient vent of the aerobic FAN bottle, the two bottles were loaded into the BacT/ALERT Classic instrument. Bottles flagged by the instrument as positive were removed, and an aliquot of the blood-broth mixture was removed from the bottle with a sterile needle and syringe. A portion was used for a Gram stain, and the remainder was subcultured onto solid plate medium according to the results of the Gram stain. Subsequent microbial isolation, identification, and antimicrobial susceptibility testing were performed by standard techniques (2). Gram-stain-negative bottles were returned to the instrument for the remainder of the 5-day incubation period or until they were reflagged by the instrument. These Gram-stain-negative bottles that were flagged by the instrument were considered to have false-positive results if no microorganisms were isolated on subculture. Bottles that were not flagged by the instrument were incubated for a total of 5 days, and 20% of the first 2,000 FA bottles were selected at random for terminal subculture to detect false-negative instrument readings. Negative bottles for which the companion bottle was positive were subcultured to determine whether the instrument failed to detect the microorganism. Bottles that were instrument negative but that grew a microorganism on subculture were considered to have false-negative results.

Clinical assessment.

All isolates were reviewed by an infectious disease physician or a pathologist and categorized as clinically important, indeterminate, or a contaminant. These assessments were made in accord with published criteria (5). An episode of bacteremia or fungemia was defined as a period beginning with the first positive blood culture and ending when 7 days (2 days for coagulase-negative staphylococci) had passed without another positive blood culture with the same microorganism, regardless of whether samples negative by culture were drawn in the intervening days. When a second clinically significant isolate was detected within 3 days of isolation of the first isolate, the episode was considered polymicrobial. Patients were considered to be on therapy at the time that the blood sample for culture was drawn if the antimicrobial agent given was either known or presumed, if susceptibility testing was not routinely done, to inhibit the clinically significant microorganism subcultured from the positive blood culture bottle.

Data analysis.

Comparison of recovery rates from the bottles was done by the chi-square test. Yates' correction was used when n was less than 20 (1). When both bottles were positive within 72 h, the times to detection were compared by the paired t test.

RESULTS

The laboratory received a total of 7,745 blood culture sets containing both bottles. Of these, 5,256 (68%) met the criteria for adequacy of filling. Among the isolates from adequately filled bottle pairs, 466 isolates from 276 patients were classified as clinically significant (Table 2). The FA bottle detected more Burkholderia cepacia (P < 0.01), Candida albicans (P < 0.001), and Cryptococcus neoformans (P < 0.01) isolates and all microorganisms combined (P < 0.05). Results for subset of blood cultures from patients who were on antimicrobial agents at the time that the blood sample for culture was obtained are summarized in Table 3; Staphylococcus aureus (P < 0.005) was detected more often in the original FAN bottles, but yeasts were detected more often in FA bottles (P < 0.005).

TABLE 2.

Comparative yields of clinically important microorganisms in FAN versus FA aerobic blood culture bottles

Microorganism	No. of isolates detected by:			P value
	Both bottles	FAN bottles only	FA bottles only
Gram-positive cocci
Staphylococcus aureus	94	28	19	NSa
Coagulase-negative staphylococci	43	16	14	NS
Streptococcus spp.b	14	3	4	NS
Enterococcus faecalis	7	3	4	NS
Enterococcus faecium	9	4	2	NS
Micrococcus spp.	1	0	1	NS
Gram-positive bacillic	2	0	2	NS
Gram-negative bacilli
Members of the family Enterobacteriaceaed	40	13	5	NS
Pseudomonas aeruginosa	12	2	6	NS
Burkholderia cepacia	2	0	9	<0.01
Other gram-negative bacteriae	13	0	2	NS
Anaerobic bacteria
Bacteroides fragilis	0	3	0	NS
Veillonella spp.	0	1	0	NS
Propionibacterium spp.	0	1	0	NS
Yeasts
Candida albicans	8	1	20	<0.001
Cryptococcus neoformans	1	0	9	<0.01
Other yeastsf	25	7	16	NS
All microorganisms	271	82	113	<0.05

^aNS, not significant (P > 0.05). 

^bIncludes 10 viridans group streptococci, 6 Streptococcus pneumoniae isolates, 4 group B beta-hemolytic streptococcal isolates, and 1 group G beta-hemolytic streptococcus. 

^cIncludes three coryneform bacteria and one Corynebacterium jeikeium isolate. 

^dIncludes 27 Escherichia coli, 14 Klebsiella pneumoniae, 10 Enterobacter cloacae, 2 Enterobacter aerogenes, and 2 Serratia marcescens isolates and 1 isolate each of Klebsiella oxytoca, Citrobacter freundii, and an unidentified gram-negative rod. 

^eIncludes seven Acinetobacter baumanii and three Stenotrophomonas maltophilia isolates and one isolate each of Acinetobacter haemolyticus, Chryseobacterium meningosepticum, Flavimonas oryzihabitans, a Neisseria sp., and an unidentified glucose-nonfermenting gram-negative rod. 

^fIncludes 24 Candida glabrata, 11 Candida tropicalis, 7 Candida parapsilosis, and 4 Candida kefyr isolates, 1 Candida kruseii isolate, and 1 Trichosporon beigelii isolate. 

TABLE 3.

Comparative yields of clinically important microorganisms from patients on antimicrobial therapy in FAN versus FA aerobic blood culture bottles

Microorganism	No. of isolates detected by:			P value
	Both bottles	FAN bottles only	FA bottles only
Staphylococcus aureus	23	16	2	<0.005
Coagulase-negative staphylococci	3	2	0	NSa
Group B streptococci	0	0	1	NS
Enterococcus faecalis	2	2	0	NS
Members of the family Enterobacteriaceaeb	8	1	0	NS
Other gram-negative bacteriac	2	0	1	NS
Yeastsd	4	1	9	<0.005
All microorganisms	42	22	13	NS

^aNS, not significant (P > 0.05). 

^bIncludes three Escherichia coli, three Klebsiella pneumoniae, and two Serratia marcescens isolates and one Enterobacter cloacae isolate. 

^cIncludes one isolate each of Acinetobacter baumanii, Burkholderia cepacia, and Stenotrophomonas maltophilia. 

^dIncludes nine Candida glabrata, three Candida albicans, and two Cryptococcus neoformans isolates. 

The mean times to detection by the instrument of 261 isolates for which both bottles were positive within 72 h are shown in Table 4. The overall mean times to detection were almost identical for FAN (20.4 h) and FA (20.7 h) bottles. The largest discrepancies were for coagulase-negative staphylococci (3.4 h in favor of FAN bottles [P = 0.0043]), nonfermentative gram-negative rods (3.5 h in favor of FA bottles [P was not significant]), and yeasts (3.2 h in favor of FA bottles [P was not significant]).

TABLE 4.

Comparative times to detection in FAN versus FA blood culture bottles when both bottles were positive within 72 h

Microorganism	No. of isolates	Avg (range) time to detection (h)		P value
		FAN bottles	FA bottles
Staphylococcus aureus	92	21.3 (5.0–64.8)	21.8 (4.5–64.8)	NSa
Coagulase-negative staphylococci	42	22.7 (7.0–57.6)	26.1 (7.3–72.0)	0.0043
Streptococcus and Enterococcusb	30	13.0 (8.0–30.7)	13.1 (8.3–26.5)	NS
Other gram-positive microorganismsc	3	35.7 (21.7–47.2)	45.3 (23.7–72.0)	NS
Members of the family Enterobacteriaceaed	40	11.3 (2.0–45.3)	12.6 (1.2–55.7)	NS
Nonfermentative gram-negative rodse	26	21.2 (6.3–72.0)	17.7 (4.8–47.2)	NS
Neisseria spp.	1	14.8	15.3
Yeastsf	27	32.9 (13.0–64.8)	29.7 (12.2–64.8)	NS
All microorganisms	261	20.4 (2.0–72.0)	20.7 (1.2–72.0)	NS

^aNS, not significant (P > 0.05). 

^bIncludes nine Enterococcus faecium, seven Enterococcus faecalis, seven viridans group streptococcus, five Streptococcus pneumoniae, and two group B streptococcus isolates. 

^cIncludes one isolate each of Corynebacterium jeikeium, a coryneform bacterium, and a Micrococcus sp. 

^dIncludes 18 Escherichia coli, 9 Enterobacter cloacae, 8 Klebsiella pneumoniae, 2 Serratia marcescens, and 2 Enterobacter aerogenes isolates and 1 Citrobacter freundii isolate. 

^eIncludes 12 Pseudomonas aeruginosa, 7 Acinetobacter baumanii, 2 Burkholderia cepacia, and 2 Stenotrophomonas maltophilia isolates and 1 isolate each of Acinetobacter haemolyticus, Flavimonas oryzihabitans, and an unidentified nonfermenting gram-negative rod. 

^fIncludes seven Candida albicans, six Candida tropicalis, five Candida glabrata, four Candida kefyr, and four Candida parapsilosis isolates and one Cryptococcus neoformans isolate. 

The FA medium detected more episodes of bacteremia and fungemia caused by all microorganisms combined than FAN medium did, and specifically, episodes caused by B. cepacia, C. albicans, and C. neoformans were detected more frequently in the FA medium (Table 5). Samples from an additional 36 polymicrobial episodes were not included in the analysis.

TABLE 5.

Comparative yields of clinically important episodes in FAN versus FA aerobic blood culture bottles

Microorganism	No. of episodes detected by:			P value
	Both bottles	FAN bottles only	FA bottles only
Staphylococcus aureus	65	7	5	NSa
Coagulase-negative staphylococci	30	5	3	NS
Streptococcus spp.b	8	1	2	NS
Enterococcus spp.c	11	4	3	NS
Unidentified coryneform bacteria	3	0	1	NS
Members of the family Enterobacteriaceaed	31	8	3	NS
Pseudomonas aeruginosa	9	1	4	NS
Burkholderia cepacia	2	0	6	<0.05
Other gram-negative bacteriae	5	0	2	NS
Bacteroides fragilis	0	2	0	NS
Candida albicans	5	1	9	<0.05
Cryptococcus neoformans	1	0	6	<0.05
Other yeastsf	10	3	7	NS
All microorganisms	180	32	51	<0.05

^aNS, not significant (P > 0.05). 

^bIncludes seven viridans group streptococci and four Streptococcus pneumoniae isolates. 

^cIncludes 10 Enterococcus faecium and 7 Enterococcus faecalis isolates and 1 Enterococcus sp. 

^dIncludes 21 Escherichia coli, 9 Klebsiella pneumoniae, 6 Enterobacter cloacae, 2 Serratia marcescens, and 2 Enterobacter aerogenes isolates, 1 Citrobacter freundii isolate, and 1 Klebsiella oxytoca isolate. 

^eIncludes three Stenotrophomonas maltophilia and two Acinetobacter baumanii isolates, one Flavimonas oryzihabitans isolate, and one Chryseobacterium meningosepticum isolate. 

^fIncludes eight Candida glabrata, five Candida parapsilosis, and four Candida tropicalis isolates and one isolate each of Candida kefyr, Candida krusei, and Trichosporon beigelii. 

A total of 186 contaminant isolates were detected equally in the two bottles, with 63 detected in both bottles, 64 detected in FAN bottles only, and 59 detected in FA bottles only (P was not significant). Isolates in 14 bottles with false-negative results were detected when the negative companion bottle to a positive culture bottle was subcultured after 5 days. All 14 isolates (4 Pseudomonas aeruginosa, 3 Candida glabrata, 2 C. albicans, and 2 B. cepacia isolates and 1 isolate each of C. neoformans, a coagulase-negative staphylococcus, and Propionibacterium acnes) from 11 patients were detected from FAN bottles with false-negative results. All isolates except the coagulase-negative staphylococcus and P. acnes were considered clinically significant.

There were 69 (1.3%) FAN bottles with false-positive results and 56 (1.1%) FA bottles with false-positive results. No microorganisms were isolated from the random sample of 402 negative FA bottles that were subcultured at the end of 5 days of incubation.

DISCUSSION

Conclusions about the performances of diagnostic media formulated for culture of blood are best based on clinical trials with control of known variables and adequate numbers of comparisons to enable detection of differences. These criteria were met in the current study.

The new nonvented formulation in the FA bottle showed improved sensitivity for detection of B. cepacia and yeasts compared with that of the original vented FAN formulation (Table 2). This seemingly paradoxical improved yield from a nonvented bottle over a vented bottle for microorganisms that are primarily aerobic may be accounted for by the increased headspace in the bottle. Although the nonvented FA bottles and the vented FAN bottles are both backfilled with similar concentrations of oxygen (concentrations higher than the 20% oxygen found in ambient air) during production, the larger headspace of the FA bottles results in an increased absolute volume of oxygen. A recent report comparing nonvented with vented standard media from Organon Teknika, however, did not show an improved yield in the nonvented bottle, despite the increased oxygen level and the increased headspace (3). This suggests that the FA medium, which differs both in charcoal content and in the base medium used from the original FAN formulation (Table 1), also plays a role along with the increased volume of oxygen in the larger headspace to provide improved recovery of microorganisms. Our experience with B. cepacia bacteremia may differ from those of other institutions owing to our lung transplant program's acceptance of patients colonized with B. cepacia as potential candidates for transplantation; however, yeasts are frequently isolated at many tertiary-care centers.

The improved rates of isolation of S. aureus in FAN medium compared with that in standard (non-FAN) medium have been documented previously, especially from those patients receiving antimicrobial therapy (4). In the present study, S. aureus isolates from patients on antimicrobial therapy were detected more frequently in the original vented formulation of the FAN medium than in FA medium. The original formulation of FAN medium contains 8.5% (wt/vol) charcoal, whereas FA medium contains only 6.5% (wt/vol) charcoal (Table 1). This suggests that one of the several possible explanations for the differences may be either that S. aureus grows more readily with the higher concentration of charcoal in the original formulation or that the higher concentration is more effective in interfering with antimicrobial activity or other inhibiting substances. Another explanation may be the increased dilution of growth-inhibiting factors that could result from the addition of blood to 40 ml of broth in FAN medium versus that from the addition of blood to 30 ml of broth in FA medium (Table 1). Detection of episodes of bacteremia with S. aureus, however, was not significantly decreased by the lower concentration of charcoal in FA medium (Table 5). The improved rate of recovery of yeasts observed in the FA bottle for the subset of patients on antifungal therapy (Table 3) suggests that the presence of these agents had little effect on the isolation of yeasts from this medium.

As shown in Table 1, there are many differences between FAN and FA bottles. The potential individual influences of the differences in headspace, concentration of charcoal, base medium, and volume of broth between these two bottles have been discussed. The relative role of each is impossible to assess, as is the difference between the sensors for detection of growth. However, the principle behind both sensors is the same, namely, the colorimetric detection of CO₂ produced by growing microorganisms. The new liquid emulsion sensor was designed to streamline the manufacturing process for the FA bottle.

The safety of not having to introduce a venting unit and the decreased risk for injuries from sharps with the FA bottle are important benefits, in addition to its increased rates of detection of B. cepacia and yeasts. Moreover, use of the FA bottle decreases the amount of setup time required and reduces the additional costs associated with the vented bottle. Therefore, we conclude that the FA bottle offers important safety benefits with an improved yield compared with that of the original formulation of the FAN medium.