Background
Bacterial meningitis is a life-threatening infectious disease of the central nervous system, which is most commonly caused by
Streptococcus pneumoniae and
Neisseria meningitidis [
1,
2].
Listeria monocytogenes is the third most common pathogen causing bacterial meningitis in adults, and is found in 5–10% of cases [
2‐
4]. Listeria distributes easily in the environment and can be found in soil, ground water, and feces of animals [
5,
6]. Main source for human infection is food, and it primarily affects elderly and immunocompromised persons, [
7] in whom it can cause up to 40% of community-acquired bacterial meningitis cases [
8,
9]. A nation-wide prospective cohort study on
L. monocytogenes meningitis described an increasing mortality rate over time, from 17 to 36% over the past decade [
10]. This trend was also reported by a French cohort study including 252 patients with listerial meningitis [
7].
In the immune response neutrophils, monocytes and macrophages are activated by pro-inflammatory cytokines such as IL-1α, IL-1, IL-6, IL-12, TNF-α, and IFN-γ [
11‐
14]. Anti-inflammatory cytokine IL-10 plays an important role in limiting immune-mediated damage and at the same time antagonizes IFN-γ activity which makes the host more vulnerable for an invasive listerial infection [
14]. Several mouse and rat listeria models have been developed to study invasive
L. monocytogenes diseases including cerebral and meningeal infection, using oral [
15‐
17], intravenous [
18], intracerebral [
19‐
23], or intracisternal inoculation methods [
24‐
26]. Problems with reproducibility, limited disease progression, or iatrogenic structural damage, combined with a need for a single model in which most pathological features seen in human listerial meningitis can be measured, have created the need for development of a new animal model. We developed a listerial meningitis mouse model to counter these problems and compared infection with listerial ST1 with ST6.
Methods
Bacterial strain L. monocytogenes sequence type 1 (ST1) was used for the experiments, time point studies were repeated with a ST6 strain. Both strains were obtained from human positive cerebrospinal fluid (CSF) isolates stored at the Netherlands Reference Laboratory for Bacterial Meningitis (NRLBM). The isolates were grown to mid-log phase in 1–1.5 h at 37 °C in BHI to an optical density (OD600) of 0.45–0.55, then, centrifuged at 2000 rpm for 20 min at 4 °C. Supernatant was removed, and sterile 0.9% NaCl was added to yield the needed concentration. Before and after inoculation, the dose was determined by serial dilution method and plated on blood agar plates overnight at 37 °C.
Experiments were performed with eight- to ten-week-old C57BL/6 mice (Charles River Laboratories, Germany). In the non-treatment survival experiments, both sexes were used; in the non-treatment time point experiments and all treatment experiments, male C57BL/6 mice were used. The mice were kept to a controlled 12-h light/dark cycle, and food and water were provided ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of the Academic Medical Center, Amsterdam and performed according to the institution and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [
27].
Mouse model of listerial meningitis: non-treatment
Survival experiments were performed to determine clinical course of disease with the aim to achieve a median lethal dose for 50% of mice (LD50) after 36–48 h. Mice were inoculated with 1 μl bacterial suspension
L. monocytogenes ST1 into the cisterna magna using a 32-gauge needle and syringe to dispense 1–10 μl. During inoculation, mice received short-term anesthesia using 2% isoflurane (Baxter). Five inoculum sizes were tested between 10
5 and 10
9 CFU/ml (
n = 6 in 10
5 and 10
6 CFU/ml,
n = 12 in 10
7 and 10
9 CFU/ml groups). After inoculation, mice were checked according to a clinical scoring list for direct neurological deficits which could indicate puncture failure (such as occipital bleeding). If direct neurological deficits were found, mice were euthanized and excluded from the experiment. Mice were monitored every 4–6 h (starting from 12 h after inoculation), and the clinical score (Table
1) and the portion of surviving mice in each group was determined up to 90% mortality [
28]. Mice were sacrificed by intraperitoneal injection (i.p.) with dexmedetomidine (0.3 mg/kg) in combination with ketamine (190 mg/kg) when a clinical score of ≥ 15 was reached. Subsequently, time point experiments were performed (
n = 12 per time point) with 10
8 CFU/ml
L. monocytogenes ST1 or ST6 which were compared to a control group (
n = 6) receiving 1 μl 0.9% NaCl intracisternally. At 6 and 24 h post-inoculation, mice were sacrificed, and blood, CSF, and organs were collected, processed, and stored [
28,
29].
Table 1
Clinical scoring list for bacterial meningitis mouse model
Weight loss | Normal 5% | 0 | |
5–10% | 1 | |
10–15% | 2 | |
15–20% | 3 | |
20–25% | 4 | 4 |
Activity | Normal | 0 | |
Increased/aggressive | 1 | |
Mildly diminished | 1 | |
Diminished | 2 | |
Severely diminished | 3 | 3 |
Condition | Normal, does not lay on back | 0 | |
Upright within 5 s | 2 | |
Upright within 30 s | 4 | |
Does not turn upright | 6 | 6 |
Coat | Normal | 0 | |
Diminished grooming | 1 | |
Soiled | 1 | |
Piloerection | 1 | 3 |
Posture | Normal | 0 | |
Slightly hunched back | 1 | 2 |
Severe hunched back | 2 | |
Eyes | Normal | 0 | |
Protruding | 1 | |
Closed eyelids | 1 | |
Discharge | 1 | 4 |
Respiration rate (per min) | > 150 | 0 | |
100–150 | 1 | |
75–100 | 2 | |
50–75 | 3 | |
< 50 | 4 | 4 |
Breathing | Irregular | 2 | |
Labored | 2 | 4 |
Neurological examination | Normal | 0 | |
Coordination problem | 2 | |
Paresis/paralysis | 2 | |
Epileptic seizure | 2 | |
Status epilepticus | 6 | 10 |
Humane endpoints | Total score ≥ 15 | | |
Status epilepticus | | |
≥ 2 seizures in 15 min | | |
Hemiparalysis | | |
≥ 25% weight loss | | |
Mouse model of listerial meningitis: treatment
Three survival experiments with 84 male mice (12 mice per subgroup) were performed to test dosage and frequency of intraperitoneal amoxicillin treatment, and the potentially beneficial effect of adding intraperitoneal gentamicin, as is the preferred antibiotic treatment used in human listerial meningitis [
30]. After each experiment, brains of one or two surviving mice per subgroup were harvested to measure bacterial outgrowth. In the first experiment, mice were inoculated with 10
9 CFU/ml
L. monocytogenes ST1 and treated 16 h post-inoculation with 50 or 100 mg/kg amoxicillin every 24 h. In the second experiment, higher doses (100 vs. 200 mg/kg/24 h amoxicillin) and shorter treatment interval (100 mg/kg amoxicillin every 12 h vs. every 24 h) was tested, starting 16 h post-inoculation. To determine the effect of additional gentamicin, 20 mg/kg/24 h of gentamicin was administered concomitant to 100 mg/kg/24 h amoxicillin 16 h post-inoculation compared to 100 mg/kg/24 h amoxicillin only in mice who were inoculated with 10
8 CFU/ml. In the time point treatment experiments, mice were inoculated with 10
9 CFU/ml
L. monocytogenes ST1 or ST6 per strain. Twelve mice were sacrificed 16 h after infection, and 12 mice were treated with 100 mg/kg amoxicillin i.p. 16 h after infection and sacrificed after 24 h.
Scoring, harvesting, and cytokine analyses
Clinical scoring was performed by two observers according to a previously developed scoring list for a pneumococcal meningitis mouse model (Table
1) [
28]. Each scoring parameter ranges from zero, corresponding to no abnormalities, to a variable maximum score. Animals reaching humane endpoint (HEP) criteria were humanely killed. After anesthetizing the mice, cardiac puncture was performed for blood collection, and intracisternal puncture for CSF collection. Brain, spleen, liver, and lungs were harvested and processed as described previously [
28]. Supernatant, plasma, and CSF were stored at − 80 °C until further use. Cytokine analyses were performed using Luminex technology Bio-plex Pro Mouse Cytokine 6-plex Assay (Bio-Rad Laboratories, Veenendaal, the Netherlands). We analyzed levels of IL-1β, IL-6, IL-10, IL-17A, TNF-α, and IFN-γ to include early and late pro- and anti-inflammatory cytokines. For cytokine values below the lower limit of detection (LLOD), the LLOD was used in the calculation of median and interquartile range. Histopathology was performed on the left hemisphere of the brain. Brain was fixed in 4% paraformaldehyde and paraffin embedded in seven coronal plaques. In all mice, a hematoxylin and eosin (H and E) staining and Gram staining were performed. Histopathology was scored (blinded) in six categories by a neuropathologist as previously described. An additional table shows this in more detail (Additional file
1) [
29].
Comparison of survival curves between groups within each model was calculated using the log-rank test. Clinical scores were compared using a linear mixed model, assuming an exponential and group-specific time effect. Comparisons of cytokine levels between groups were calculated using the Mann–Whitney U test, and for dichotomous variables in histopathology scoring, the Fisher’s exact test was used. All statistical tests were two-tailed, and a p value of < 0.05 was considered to be significant.
Discussion
We developed and validated a murine model of listerial meningitis
. We used
Listeria sequence types [
18,
32] that commonly cause invasive disease [
18,
31]. Previously described intracisternally inoculation mice and rat studies used a serotype 4b strain with unknown sequence type [
24‐
26]; intracerebral inoculation mice studies [
22,
23] used a less virulent laboratory EGD strain compared to the ST1 and ST6 strains [
18,
33]. We used intracisternal inoculation aiming for a reproducible meningitis model. Previous studies using oral and intravenous inoculation reported difficulties with respect to neuro-invasion reproducibility [
15‐
17,
34‐
36], while intracerebral injection primarily causes cerebritis. Intracerebral inoculation methods have been used successfully previously to study the role of macrophage inflammatory protein and TNF-α in listerial meningitis [
22,
23]. Intracisternal inoculation has been used to study heat production [
24], compare effectiveness of antibiotics [
26], and the role of reactive oxygen and nitric oxide in listeria growth [
25]. Our model allows evaluation of multiple features including bacterial growth, host immune response, clinical severity, and histopathological damage.
Main histopathological characteristics of listerial meningitis were meningeal inflammation, ventriculitis, and abscesses. This is in line with previous studies [
37,
38]. We also observed a high rate of cerebral hemorrhages, an uncommon feature in human listerial meningitis (2% of cases) [
10]. This is consistent with the observed difference in pneumococcal meningitis in a human and mouse model [
28,
39]. In human bacterial meningitis, it has been suggested that dysregulation of coagulation and fibrinolytic pathways, vascular endothelial cell swelling, and vasculitis plays a role in the pathophysiology of hemorrhages [
40‐
44].
IL-1β, IL-6, IL-10, IL-17A, TNF-α, and IFN-γ mediated the hosts immune response against
L. monocytogenes. Previous listerial mouse models showed that monocyte recruitment to the brain is triggered by pro-inflammatory cytokines in particular IFN-γ- , TNF- , and IL-6-related immune response [
45,
46]. These cytokines and IL-1β and IL-17A are able to mobilize phagocytes and activate other cytokines [
11‐
14,
46‐
54], whereas IL-10 limits the immune-mediated injury; nonetheless can increase severity of
L. monocytogenes disease by reducing the immune response [
55‐
57]. Since IL-6 and IFN-γ were elevated in the brain and blood of both our treatment and non-treatment mouse models, these cytokines are relevant outcome measures in our model to study changes in the inflammatory response. An interesting aspect of IFN-γ is its ambiguous role in listerial infections. It is known for its protective and controlling role in the early immune response, though it seems to promote susceptibility for
L. monocytogenes later on. Study models with interferon-deficient mice showed protective effects during systemic listerial infections [
58‐
60]. In CSF of patients with listerial meningitis, elevation of IFN-γ, IFN-α2, and interferon-related cytokines IL-18, CX3CL1, and CCL20 were associated with an unfavorable outcome [
61]. The use of amoxicillin, a bacteriolytic antibiotic, in our model did not lead to a significant increase in cytokine levels after therapy, as observed in other experimental models of bacterial meningitis also using bacteriolytic antibiotic.
Infection with
Listeria strain of the ST1 type led to a more rigorous inflammatory response and more brain damage as compared to infection with ST6. Both STs have been marked as hypervirulent strains with a tropism for neuro-invasion [
18]. ST1 has been among the most common genotypes causing listerial meningitis in the Netherlands over the last 25 years [
31]. ST6 has been emerging over the last years and has been associated with an increasing rate of unfavorable outcome among adults with listerial meningitis, from 27 to 61% over a 14-year period [
10]. The increased incidence of ST6 listerial meningitis in the Netherlands has been associated with the introduction of a novel plasmid, carrying the efflux transporter emrC [
62]. Although speculative, differences in virulence between ST1 and ST6 found in our model could be explained by (i) degree of cell-to-cell spread from infected phagocytes to endothelial cells [
63,
64]; (ii) the interaction with macrophages, neutrophils, and subsequently the cytokine signaling [
65]; (iii) the proportion of
Listeria bacteria residing in the brain parenchyma rather than extracellularly in the CSF, and thereby causing different degrees of histopathological damage [
66]; (iv) degree of expression of the specific neuro-invasive internalin InlF and its binding to the filament protein vimentin [
67,
68]; (v) presence of certain genetic elements in the bacteria such as LIPI-3 (in both ST1 and ST6) [
69], LGI 2 (found in ST1) [
70], or pLMST6 (found in ST6) [
62]; (vi) other yet unknown factors influencing and differentiating the virulence of
L. monocytogenes strains. Since ST1 and ST6 are clinically relevant strains, these unknown factors should be investigated and might help to unravel the pathophysiology of
L. monocytogenes.
Our model has several limitations, of which some are inherent to the use of modeling of human disease in animals. First, we infected mice by inoculating directly into the cisterna magna, while the route of infection in humans mainly is through the digestive system. However, meningitis is difficult to evoke unless bacteria are injected directly intracranial, partially because animals tend to die due to systemic illness before meningitis develops [
71]. Furthermore, the amount of bacteria reaching the brain cannot be controlled using digestive tract or intravenous inoculation. Second, in the treatment survival experiments, we observed that
Listeria could be cultured from the murine brains despite high doses antibiotic treatment. This can be explained as the pathogen is intracellular and has a relatively slow growth rate. Patients with listerial meningitis are therefore treated for at least 3 weeks. To make sure we did not use insufficient dosage or type of antibiotics, we increased the dose and frequency of the amoxicillin and added gentamycin, but these changes did not influence outcome or bacterial outgrowth at the end of the experiment. Therefore, we feel that we achieved an optimal amoxicillin dose to perform the experiments with. We did observe that bacterial counts decreased in all treated mice. It could be argued that other antibiotics with a previously suggested effect and/or had a synergism in treatment of listerial meningitis should have been tested [
30]. However, amoxicillin with or without gentamicin is the most commonly used treatment in human listerial meningitis, and therefore testing other antibiotics is beyond the scope of this article.