Developing a Novel Murine Meningococcal Meningitis Model Using a Capsule-Null Bacterial Strain

Abstract

Background: Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that colonises the nasopharynx of about 10% of the healthy human population. Under certain conditions, it spreads into the body, causing infections with high morbidity and mortality rates. Although the capsule is the key virulence factor, unencapsulated strains have proved to possess significant clinical implications as well. Meningococcal meningitis is a primarily human infection, with limited animal models that are dependent on a variety of parameters such as bacterial virulence and mouse strain. In this study, we aimed to develop a murine Neisseria meningitidis meningitis model to be used in the study of various antimicrobial compounds.

Method: We used a capsule-deficient Neisseria meningitidis strain that was thoroughly analysed through various methods. The bacterial strain was incubated for 48 h in brain-heart infusion (BHI) broth before being concentrated and injected intracisternally to bypass the blood-brain barrier in CD-1 mice. This prolonged incubation time was a key factor in increasing the virulence of the bacterial strain. A total of three more differently prepared inoculums were tested to further solidify the importance of the protocol (a 24-h incubated inoculum, a diluted inoculum, and an inactivated inoculum). Antibiotic treatment groups were also established. The clinical parameters and number of deaths were recorded over a period of 5 days, and comatose mice with no chance of recovery were euthanised.

Results: The bacterial strain was confirmed to have no capsule but was found to harbour a total of 56 genes coding virulence factors, and its antibiotic susceptibility was established. Meningitis was confirmed through positive tissue culture and histological evaluation, where specific lesions were observed, such as perivascular sheaths with inflammatory infiltrate. In the treatment groups, survival rates were significantly higher (up to 81.25% in one of the treatment groups compared to 18.75% in the control group).

Conclusion: We managed to successfully develop a cost-efficient murine (using simple CD-1 mice instead of expensive transgenic mice) meningococcal meningitis model using an unencapsulated strain with a novel method of preparation.

Keywords: Neisseria meningitidis; capsule deficient meningococcus; capsule null locus; meningococcal meningitis; murine model.

Figure 1

Latex agglutination results of the Neisseria meningitidis strain showed non-specific positivity for both serogroups A and C and negative results for serogroup B. Testing for serogroup Y/W135 was not performed as the manufacturer does not recommend using bacterial isolates from agar media. No testing was performed for Hi.b-R3, Strep.Pneum-R4, and Strep B-R5, as these are designated for other bacterial species. (CTRL R9: negative polyvalent control, Nm.A-R6: serogroup A Neisseria meningitidis, Nm.C-R7: serogroup C Neisseria meningitidis, Nm.B/E.coli K1-R1: serogroup B Neisseria meningitidis/Escherichia coli K1 antigen, CTRL R2: negative control for serogroup B Neisseria meningitidis/Escherichia coli K1 antigen, Nm.Y/W135-R8: serogroup Y/serogroup W135 Neisseria meningitidis, Hi.b-R3: Haemophilus influenzae serotype b, Strep. Pneum-R4: Streptococcus pneumoniae, Strep B-R5: group B Streptococcus).

Figure 2

Survival of mice inoculated with the 24-h and 48-h incubated inoculums.

Figure 3

Survival of mice in the inactive inoculum, diluted inoculum (10⁻²), and 48-h incubation group.

Figure 4

Survival of mice in the two antibiotic treatment groups compared to the 48-h incubated control group* (data pooled from 16 mice from the two 48-h incubated inoculum groups).

Figure 5

Colony-forming units per gramme of brain tissue in the 48-h incubated control group (No treatment) and the ciprofloxacin and ceftriaxone groups (Treatment).

Figure 6

Histological sections of the brains of the animals treated with PBS, stained with haematoxylin and eosin stain: Severe inflammation in the brain parenchyma is characterised by neutrophilic and lymphocytic infiltrate ((A–D), black arrow) along with haemorrhagic foci both in the brain parenchyma and at its border and at the border with the cerebellum ((A–E), orange arrow), with perivascular inflammation. Abscess formation is accompanied by inflammation of the vascular endothelium ((A), green arrow) and phagocytosis of neutrophils ((F), blue arrow).

Figure 7

Histological sections of the brains of the ceftriaxone-treated (A–C) and ciprofloxacin-treated (D–F) animals that succumbed to the disease, stained with haematoxylin and eosin stain: maintenance of neutrophilic and lymphocytic inflammatory infiltrate ((A,C), black arrow) or diffuse (black arrow), inflammation of vascular tissues and abscesses delimited by inflammatory reaction ((B,D), green arrow) together with haemorrhagic foci ((D–F), orange arrow).