Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid

An established infant model of bacterial meningitis was used as described previously [7, 8]. The animal studies were approved by the Animal Care and Experimentation Committee of the Canton of Bern, Switzerland, and followed National Institutes of Health guidelines for the performance of animal experiments. Briefly, nursing Wistar rats with their dams were purchased (Charles River, Germany). For each test organism, four animals (weighing 23.6 ± 1.1 g) were infected on postnatal day 11 by direct intracisternal injection of 10 μl saline containing 1 × 10⁶ cfu/ml S. pneumoniae, 3 × 10⁸ cfu/ml N. meningitidis and 2 × 10⁵ cfu/ml L. monocytogenes using a 32-gauge needle. The pathogens investigated in the study show significant differences in virulence and pathogenicity. Therefore the number of bacteria used for infection needed to be adapted to generate reproducible infection and associated disease severity. Thus, for each pathogen the inoculum (cfu/ml) used for intracisternal infection was based on previously established disease models [9, 10]. Control animals were injected with 10 μl sterile saline (n = 2) or sterile saline containing heat-inactivated (80°C for 20 min) S. pneumoniae at a concentration corresponding to 2 × 10⁸ cfu/ml (n = 2).

Clinical isolates of S. pneumoniae (serogroup 3), N. meningitidis (type C) and L. monocytogenes (serotype 4b) were used. The infecting organisms were previously passaged through infant rats in the course of experimental studies [9]. Organisms were grown on 5% sheep blood agar plates (Becton Dickinson, Heidelberg, Germany), cultured overnight in 10 ml of trypticase soy broth (TSB) for S. pneumoniae and N. meningitidis, or brain heart infusion (BHI) for L. monocytogenes, diluted in fresh medium, and grown to logarithmic phase at 37°C in 5% CO₂. The culture broth was centrifuged for 10 minutes at 5,000 × g and the pellet was resuspended in sterile saline to the desired density for intracisternal injection. The accuracy of the inoculum dose was confirmed by quantitative cultures by serial dilution for each experiment. In addition, 10-fold of serial dilutions of test organisms were prepared in concentrations from 10⁶ to 10^-1 cfu/ml as quantitative standards for comparison with CSF samples.

At 18 h after infection, approx. 15 - 20 μl CSF was obtained by puncture of the cisterna magna and used for quantitative cultures and calorimetry. For quantitative culture, CSF was plated in 10-fold dilutions on 5% sheep blood agar plates (Becton Dickinson). For calorimetry, 10 μl and 1 μl CSF were added to 90 μl and 99 μl saline, respectively (total volume of 100 μl), and inoculated in calorimetry ampoules containing 3 ml TSB. Culture broth with 100 μl saline was used as control. Animals were sacrificed after CSF sampling with an overdose of pentobarbital (100 mg/kg intraperitoneally).

An isothermal calorimetry instrument (Thermal Activity Monitor, Model 3102 TAM III, TA Instruments, New Castle, DE, USA) equipped with 48 channels was used to measure the heat flow at 37°C precisely controlled within 0.0001°C. The calorimetric sensitivity according to the manufacturer is ± 0.2 μW. Heat generated or absorbed was continuously measured in air-tightly sealed 4-ml glass calorimetry ampoules, containing the test or control samples; the gas phase consisted of ambient air. Ampoules were sequentially introduced into the calorimetry instrument and remained 15 minutes in the thermal equilibration position before lowering into the measurement position. Heat flow was measured for up to 48 hours at 10 s intervals. After the measurement was completed, the content of each calorimetry ampoule was cultured to confirm the inoculated microorganisms in infected animals or sterility of the negative controls.

Calorimetric detection time was defined as the time from insertion of the ampoule into the calorimeter until the growing culture produced an exponentially rising heat flow signal exceeding 10 μW. Peak heat flow was defined as the highest value of the heat power-time curve. Total heat was determined by integration of the area below the heat flow-time curve. Data analysis was accomplished using the manufacturer's software (TAM Assistant, TA Instruments, New Castle, DE, USA) and Origin 7.5 (Microcal, Northampton, MA, USA).

At 18 hours after intracisternal injection, all infected rats suffered from meningitis as evidenced by lethargy, diminished weight gain and positive CSF cultures. Table 1 shows the bacterial density in CSF at 18 hours after infection. In control animals, injected with sterile saline or heat-inactivated bacteria, no signs of meningitis were observed at 18 h after infection and no bacteria grew in CSF cultures.

Heat signal was detected in CSF samples from all infected animals, whereas those from non-infected animals generated no detectable heat (<10 μW). Figure 1 shows heat power-time curves from 10-μl CSF samples (bold lines) in correlation with heat produced by serial 10-fold bacterial dilutions ranging from 10⁶ to 10^-1 cfu/ml (grey lines). The heat curves of CSF from different individual animals infected by the same organism showed similar characteristics. For example, S. pneumoniae showed a discrete initial peak heat flow at 40 μW, followed by a second peak at about 350 μW. N. meningitidis demonstrated a clear initial peak, followed by a higher peak at approximately 80 μW. Replicates of L. monocytogenes showed three peaks, at ≈ 80 μW, ≈ 300 μW and ≈ 350 μW. The shape of the curves was dependent on the growth medium in the calorimetry ampoule, but independent from the initial bacterial concentration (data not shown). The bacterial titer in CSF at 18 hours after infection correlated with the standard dilutions for S. pneumoniae, but was two orders of magnitude lower for N. meningitidis and three orders of magnitude lower for L. monocytogenes. The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). CSF from animals that had been injected with heat-killed bacteria resulted in power-time curves very similar to those of the medium-controls. The values always remained below the 10 μW positivity limit and continuously decreased towards baseline.

Table 1 summarizes the calorimetric detection times for the different pathogens generated from 10 μl and 1 μl of CSF. The detection time is defined by the time elapsed from the start of measurement until the detected heat flow exceeds 10 μW. In 10-μl CSF samples, S. pneumoniae was detected after a mean ± SD of 1.5 ± 0.2 hours, followed by N. meningitidis after 3.9 ± 0.7 hours and by L. monocytogenes after 9.1 ± 0.5 hours.

As determined from standard bacterial dilutions by linear regression analysis, the lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes using TSB (Figure 2). The lowest bacterial titer was detected after 15.2, 19.7 and 12.2 hours of incubation, respectively.

In addition to TSB, BHI was used as an alternative growth medium for the detection of S. pneumoniae and L. monocytogenes. CSF drawn from animals was split into identical volumes of 10 μl and inoculated in parallel in calorimetry ampoules containing TSB or BHI. The mean detection time (± SD) in TSB was 1.71 ± 0.37 hours shorter for S. pneumoniae and 1.17 ± 0.87 hours shorter for L. monocytogenes compared to BHI. Similarly, the peak heat flow of CSF in TSB was 174 μW higher for S. pneumoniae and 69 μW higher for L. monocytogenes as compared to BHI.