Bacteriology at UW-Madison
Figure 1. Neisseria meningitidis scanning EM
Neisseria meningitidis is usually cultivated in a peptone-blood base medium in a moist chamber containing 5-10% CO2. All media must be warmed to 37 degrees prior to inoculation as the organism is extremely susceptible to temperatures above or below 37 degrees. This trait is rather unique among bacteria. Also, the organism tends to undergo rapid autolysis after death, both in vitro and in vivo. This accounts for the dissemination of lipopolysaccharide (endotoxin) during septicemia and meningitis.
The organism tends to colonize the posterior nasopharynx of humans, and humans are the only known host. Individuals who are colonized are carriers of the pathogen who can transmit disease to nonimmune individuals. The bacterium also colonizes the posterior nasopharynx in the early stages of infection prior to invasion of the meninges. Most individuals in close contact with a case of meningococcal meningitis become carriers of the organism. This carrier rate can reach 20 percent of the contact group before the first case is recognized, and may reach as high as 80 percent at the height of an epidemic.
Marchiafava and Celli were the first to report observing Gram-negative diplococci in cerebrospinal fluid of a fatal case of meningitis in 1884. In 1887, Weichselbaum isolated the bacterium from six cases of meningitis and established the isolates as a distinct species and proven to be the cause of meningitis.
Infection is by aspiration of infective bacteria, which attach to epithelial cells of the nasopharyngeal and oropharyngeal mucosa, cross the mucosal barrier, and enter the bloodstream. If not clear whether blood-borne bacteria may enter the central nervous system and cause meningitis.
The mildest form of disease is a transient bacteremic illness characterized by a fever and malaise; symptoms resolve spontaneously in 1 to 2 days. The most serious form is the fulminant form of disease complicated by meningitis. The manifestations of meningococcal meningitis are similar to acute bacterial meningitis caused by other bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and E. coli. Chills, fever, malaise, and headache are the usual manifestations of infection. Signs of meningeal inflammation are also present.
In older children and adults, specific symptoms and signs are usually present, with fever and altered mental status the most consistent findings. Headache is an early, prominent complaint and is usually very severe. Nausea, vomiting, and photophobia are also common symptoms.
Neurologic signs are common; approximately one-third of patients have convulsions or coma when first seen by a physician. Signs of meningeal irritation such as spinal rigidity, hamstring spasms and exaggerated reflexes are common.
Petechiae (minute hemorrhagic spots in the skin) or purpura (hemorrhages into the skin) occurs from the first to the third day of illness in 30 to 60% of patients with meningococcal disease, with or without meningitis. The lesions may be more prominent in areas of the skin subjected to pressure, such as the axillary folds, the belt line, or the back.
Fulminant meningococcemia occurs in 5 to 15% of patients with meningococcal disease and has a high mortality rate. It begins abruptly with sudden high fever, chills, myalgias, weakness, nausea, vomiting, and headache. Apprehension, restlessness, and delirium occur within the next few hours. Widespread purpuric and ecchymotic skin lesions appear suddenly. Typically, no signs of meningitis are present. Pulmonary insufficiency develops within a few hours, and many patients die within 24 hours of being hospitalized despite appropriate antibiotic therapy and intensive care.
Figure 3. The characteristic skin rash (purpura) of meningococcal septicemia, caused by Neisseria meningitidis
The human nasopharynx is the only known reservoir of N. meningitidis. Meningococci are spread via respiratory droplets, and transmission requires aspiration of infective particles. Meningococci attach to the nonciliated columnar epithelial cells of the nasopharynx. Attachment is mediated by fimbriae and possibly by other outer membrane components. Invasion of the mucosal cells occurs by a mechanism similar to that observed with gonococci. Events involved after bloodstream invasion are unclear and how the meningococcus enters the central nervous system is not known.
Purified meningococcal LOS is highly toxic and is as lethal for mice as the LOS from E. coli or Salmonella typhimurium; however, meningococcal LOS is 5 to 10 times more effective than enteric LPS in eliciting a dermal Shwartzman phenomenon (a characteristic type of inflammatory reaction) in rabbits. Meningococcal LOS has been shown to suppress leukotriene B4 synthesis in human polymorphonuclear leukocytes. The loss of leukotriene B4 deprives the leukocytes of a strong chemokinetic and chemotactic factor.
The integrity of the pharyngeal and respiratory epithelium appears to be important in protection from invasive disease. Chronic irritation of the mucosa due to dust or low humidity, or damage to the mucosa resulting from a concurrent upper respiratory infection, may be predisposing factors for invasive disease.
The presence of serum bactericidal IgG and IgM is probably the most important host factor in preventing invasive disease. These antibodies are directed against both capsular and noncapsular surface antigens. The antibodies are produced in response to colonization with carrier strains of N. meningitidis, as well as N. lactamica, and other nonpathogenic Neisseria species that are normal inhabitants of the upper respiratory tract. Protective antibodies are also stimulated by cross-reacting antigens on other bacterial species such as Escherichia coli. The role of bactericidal antibodies in prevention of invasive disease explains why high attack rates are seen in infants from 6 to 9 months old, the time at which maternal antibodies are being lost. Individuals with complement deficiencies (C5, C6, C7, or C8) may develop meningococcemia despite protective antibody. This emphasizes the importance of the complement system in defense against meningococcal disease.
Meningococcal disease is contracted through association with infected individuals, as evidenced by the 500- to 800-fold greater attack rate among household contacts than among the general population. Because such household members are at high risk, they require chemoprophylaxis. Sulfonamides were the chemoprophylactic agent of choice until the emergence of sulfonamide-resistant meningococci. At present, approximately 25 percent of clinical isolates of N. meningitidis in the United States are resistant to sulfonamides; nowadays, rifampin is the chemoprophylactic agent of choice.
There is an obvious need for a universal vaccine for meningococcal meningitis, but the development of an effective vaccine against all forms of N. meningitidis has been hampered by the high degree of variation in the proteins on the surface of the bacterium which leads to the occurrence of many different antigenic types.
More than 10% of the population may be carrying the bacterium at any one time on the mucosal surfaces of the nose and throat. The majority of these carriers will not have any symptoms of the disease, but this continual exposure to the immune system puts pressure on the bacterium to mutate its surface components in order to survive. Thus, natural selection is the driving force for the emergence of new antigenic variants.
Among the class 2 and 3 outer membrane proteins of N. meningitidis, Por A has been considered a primary target for a vaccine-induced antibody. PorA is a major component of the outer membrane of N. meningitidis, and anti-PorA antibodies are thought to be a critical component in immunity. Interactions between antibodies and PorA have been studied. Different strains of the bacterium have different PorA amino acid sequences within the region of the protein that specifically binds to antibody molecules. PorA has several large amino acid "loop" regions that protrude from the surface, and it is these loops that are targets for antibody binding.
In the laboratory, the antigen-binding fragment (Fab) of anti-PorA antibodies can be crystallized and reacted with the antigenic loop regions of PorA in order to determine the specificity of binding between antigen and antibody. Slight changes in PorA amino acid sequence have been shown to cause loss in the ability to bind to antibody molecules. In nature, the bacterium mutates to insert new amino acid residues into the tip of the loop, which alters or eliminates many of the interactions with antibody and allows the bacterium to bypass previous immune responses.
Figure 4. Image of the antibody (Fab) molecular surface, with the PorA antigen superimposed. The dark colored groove on the surface of the antibody matches precisely the shape of the PorA antigen; hence any changes in the sequence of PorA in this region can disrupt antibody binding. Jeremy Derrick, UMIST. SRS ANNUAL REPORT 1999-2000
Hence, by introducing changes into portions of the PorA protein that are exposed at the surface, the bacterium can evade the attention of the immune system. These alterations are apparently introduced without compromising the biological function of PorA, as a pore-forming protein. Designing vaccines that are able to take into account these changes is a huge challenge, but as more information of this type becomes known, it leads to a more rational approach to design of a universal vaccine for meningococcal meningitis.