MORE ABOUT LISTERIA (by F. S. Southwick & D. L. Purich)

Listeria monocytogenes, an aerobic and facultatively anaerobic gram-positive bacillus, can be readily isolated from soil, dust, fertilizer, sewage, stream water, plants, and even processed foods stored at 4°c. This organism is also carried within the intestinal tract of numerous mammals, birds, fish, and crustaceans. Recent epidemiological studies provide strong evidence that both sporadic and common-source outbreaks of listeriosis are food-borne. Despite this pathogen's pervasiveness in the environment, the annual incidence of listeriosis is only 0.7 cases per 100,000. However, persons over age 70 years have a three times higher (2.1/100,000/yr) and pregnant women a 17 times higher rate of infection (12/100,000/yr). The risk of listeriosis is markedly increased in immuno-compromised patients, particularly among those undergoing renal transplantation, receiving high doses of corticosteroids, or suffering with AIDS or cancer. AIDS patients are estimated to have a 100 to 300 times higher risk of Listeria infection than the general population. At a major referral hospital Listeria was the third-most frequent cause of community-acquired bacterial meningitis (11 percent of cases) in adults. Listeriosis also concerns public health physicians, because Listeria infection is associated with a 23 percent mortality; by contrast, other food-borne diseases such as Salmonella are rarely fatal. Given the increasing number of elderly and immunocompromised patients, physicians are increasingly likely to encounter patients with listeriosis. Understanding how Listeria survives in host cells and spreads from cell to cell provides new insights into the unique epidemiology, clinical manifestations, and treatment of listeriosis.

INTRACELLULAR SURVIVAL AND SPREAD OF LISTERIA IN HOST CELLS---

Listeria monocytogenes has an unusual lifestyle (Figure 1). It can be phagocytosed and has a family of bacterial cell wall surface proteins called the internalins that enhance its internalization into epithelial cells and hepatocytes. After phagocytosis, the bacterium becomes enclosed in a subcellular organelle called a phagolysosome, a hostile and toxic environment for most bacteria. The low pH of this organelle activates listeriolysin-O, an exotoxin that can lyse its membrane within 30 min and allow the escape of Listeria into the cytoplasm. All pathogenic strains of Listeria produce listeriolysin-O, and escape into cytoplasm of the host cell is required for pathogenesis. Once within the growth-permissive cytoplasm, the bacteria proliferate (with doubling times of about one hour) and become surrounded by an electron-dense material. About two hours later, this material polarizes at one end of the bacterium. Many bacteria then migrate to the periphery of the cytoplasm where they push against the host cell's outer membrane to form elongated protrusions or filopods that can be ingested by adjacent cells. The exact mechanisms by which adjacent cells recognize and ingest bacteria-laden filopods remain uncharacterized. Once the bacterium enters the adjacent cell, the life cycle begins anew. Listeria therefore spread from cell to cell without directly contacting the extracellular environment. Pursuing the hypothesis that the host cell material surrounding the bacterium was the cytoskeletal protein actin, one of us (F.S.S.) infected cells grown in tissue culture and used rhodamine-labeled phallacidin to localize actin filaments. Wherever a bacterium was seen, actin filaments were also present, and bacteria frequently had actin filament structures extending from one end. It has become evident that Listeria efficiently usurps the host cell's contractile system to facilitate cell-to-cell spread and cause disease. Two other important pathogens, Shigella flexneri and rickettsia, likewise use the host cell's actin-contractile system to survive and spread.

CLINICAL CONSEQUENCES OF ACTIN-BASED LISTERIA MOTILITY---

The information presented above indicates how Listeria invades and spreads within the host cells. At the same time, this characteristic intracellular life style provides the clinician with a useful framework for understanding many of the clinical consequences of listeriosis.

Cell-to-Cell Spread--- Although an association between contaminated foods and listeriosis has been well documented, evidence for gastrointestinal pathology was absent in the majority of cases. The mechanism by which Listeria gained entry into the gastrointestinal tract without associated erosive lesions was uncertain. Our current understanding of Listeria's mechanism of entry into host cells and its cell-to-cell spread helps to explain these clinical observations. Because Listeria can be phagocytosed by cultured gastrointestinal cells and macrophages, entry into the host can be achieved while still maintaining the integrity of the gastrointestinal tract. Subsequently, Listeria commandeers the host cell contractile proteins actin, VASP and profilin to spread from cell-to-cell and eventually enter the blood stream either in monocytes and neutrophils or as free organisms following cell lysis.

Epidemiology--- Listeria's intracellular lifestyle may also explain the higher incidence of listeriosis in immunocompromised patients, neonates, and pregnant women. Listeria manages to avoid the extracellular environment; hence, immunoglobulins and complement would not be expected to play prominent roles in defending against this pathogen. There is no known association between Listeria infection and a deficiency in immunoglobulin or complement. Accordingly, cell-mediated immunity is likely to serve as the host's primary defense against this pathogen, and those clinical conditions and therapies (particularly the use of corticosteroid therapy) that impair cell-mediated immunity increase the risk of listeriosis. AIDS patients most commonly develop Listeria infection when their CD4 T-lymphocyte count falls below 40 cells/cubic mm. Treatment of patients having chronic lymphocytic leukemia with fludarabine and prednisone markedly lowers their CD4 T-lymphocyte counts and increases the incidence of listeriosis.

Listeria can cause a number of clinical syndromes including sepsis and focal infections of bones, joints, eye, endocardium, spinal cord, peritoneum, and gall bladder. Two syndromes that may result in manifestations which are unique to Listeria include meningitis-meningoencephalitis and granulomatosis infantiseptica. Meningitis and Meningoencephalitis Although the clinical presentation of Listeria meningitis is similar to that of other forms of bacterial meningitis, several characteristics distinguish Listeria. Tremor and grand mal and focal motor seizures are more frequent in patients with Listeria meningitis, suggesting more extensive invasion of the nervous system. A review of 38 cases of Listeria meningitis revealed that a significantly higher percentage had seizures as compared with cases of community-acquired bacterial meningitis at the same hospital4 (39 versus 23 percent, P= 0.02) (F. S. Southwick MD, unpublished observations).

Central Nervous System Infection--- Direct invasion of the cerebral cortex can result from Listeria infection, but it is not a recognized complication of other common forms of bacterial meningitis. This organism most commonly invades the brain stem causing a syndrome that has been called rhomboencephalitis. Listeria meningoencephalitis is generally associated with multiple cranial nerve deficits, particularly of the VIth and VIIth nerves, as well as hemiparesis, ataxia and respiratory abnormalities often leading to respiratory arrest. The ability of Listeria to cross the meninges and blood-brain barrier is also likely to be the result of endothelial cell or macrophage phagocytosis of the organisms and utilization of the host cell contractile system to migrate to and grow within the brain. The cerebrospinal fluid response may also reflect the intracellular nature of Listeria. In most forms of bacterial meningitis the cellular response consists of greater than 80 percent neutrophils. In Listeria meningitis, however, the percentage of neutrophils is reduced. In two series, 53 and 70 percent of patients had less than 80 percent neutrophils in their cerebrospinal fluid. In cases of meningoencephalitis, cerebrospinal fluid monocyte counts may reach 80 to 90 percent. The mechanisms by which intracellular pathogens stimulate the recruitment of monocytes to the site of infection are poorly understood. Monocytes exposed to Mycobacterium tuberculosis release monocyte chemoattractant protein (MCP-1), a protein that can attract monocytes as well as lymphocytes. Whether exposure to Listeria can elicit a similar response is not known.

Negative CSF Gram Stain--- Also in keeping with Listeria's intracellular lifestyle, organisms are rarely found in gram stains of cerebrospinal fluid. In one series of patients with Listeria meningitis, only 2 of 40 had a positive gram stain,41 and in another series, 2 of 16 patients had positive tests. A monocytic response in the cerebrospinal fluid and a negative cerebrospinal fluid gram stain can lead the clinician to confuse Listeria meningitis and meningoencephalitis with Herpes and other forms of viral encephalitis, viral meningitis, tuberculous meningitis, Lyme disease, syphilis, cryptocococcal meningitis, Wegener's granulomatosis or central nervous system sarcoidosis. Granulomatosis Infantiseptica Although Listeria bacteremia causes few symptoms in pregnant women, such infections can be devastating in the fetus. Listeria can readily invade the placenta and precipitate premature labor and fetal death. Transplacental transmission causes the unique clinical syndrome, granulomatosis infantiseptica, associated with disseminated Listeria abscesses or granulomas involving the fetal liver, spleen, lung, kidney, brain and skin, and 35 to 55 percent of infected newborn infants die. Cell-to-cell spread of Listeria, again mediated by host cell actin-based motility, probably accounts for Listeria's ability to cross the placenta and to invade fetal tissue.

Antibiotic Treatment--- Bacteriostatic drugs, such as chloramphenicol and tetracycline, are associated with high failure rates in patients with listeriosis and cannot be recommended. Ampicillin or penicillin has generally been recommended as the treatment of choice. Nonetheless, in immunosuppressed patients, relapse has been reported after two weeks of penicillin therapy. The poor response to bacteriostatic drugs and the slow response to penicillin therapy are likely to result from Listeria's ability to survive and grow within cells. The intracellular levels of ampicillin or penicillin may not be sufficient for complete sterilization. Immunosuppression reduces the host's ability to clear infected cells, thereby allowing Listeria to survive and spread for prolonged periods in a protected intracellular environment. Thus, antibiotic treatment for 3 to 4 weeks is highly recommended in immunosuppressed patient. Antibiotics that penetrate cells poorly, such as aminoglycosides, may be synergistic in vitro but are unlikely to prove efficacious in the living host. Although some experts have recommended an aminoglycoside be added to ampicillin, Listeria grows in cells in the presence of extracellular gentamicin concentrations of 10 to 20 µg/ml. Therefore, aminoglycosides are unlikely to be efficacious in listeriosis, and certainly should be avoided in kidney transplant recipients and other patients with renal dysfunction. On the other hand, trimethoprim-sulfamethoxazole, a drug combination that readily enter cells and is bactericidal for Listeria, may prove to be the most effective agent for treating Listeria infection. Trimethoprim-sulfamethoxazole has proved effective in patients with listeriosis and penicillin hypersensitivity.46 In a preliminary study of Listeria meningitis, treatment with ampicillin combined with trimethoprim-sulfamethoxazole was associated with a lower failure rate and fewer neurological sequelae, as compared with ampicillin combined with an aminoglycoside.

Conclusions--- Actin filament assembly clearly plays a central role in the ability of Listeria to avoid extracellular antibiotic, antibody, and complement action. By usurping the host cell contractile system, Listeria can survive and thrive within the host. Actin assembly is essential for the cell-to-cell spread of Listeria, and the oligoproline-containing protein ActA is a major virulence factor for listeriosis. The organisms ability to move through the cytoplasm of host cells and to be transferred from one host cell to another accounts for the following characteristic clinical aspects of Listeria infection: invasion of the gastrointestinal tract without producing erosive lesions; a predisposition to infect patients with defective cell-mediated immunity; an increased propensity to induce a monocytic response in the cerebrospinal fluid; the low sensitivity of gram stains of cerebrospinal fluid; the pathogen's invasiveness into the cerebral cortex; invasion of the placenta and fetus during maternal bacteremia; and the persistence of infection despite antibiotic treatment. The recent studies of the pathogenesis of Listeria serve as an excellent illustration of how a pathogen subverts the host cell's normal biochemical pathways to cause disease, and this new understanding provides a conceptual framework for effective diagnosis and treatment of listeriosis.