Listeria monocytogenes

Listeria monocytogenes

Clinical Mlcrobmlogy Newsletter • • Vol. 7, No. 9 May 1, 1985 ill Listeria Monocytogenes Jill E. Clarridge, Ph.D. Department of Microbiology and ...

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Clinical Mlcrobmlogy Newsletter •

Vol. 7, No. 9

May 1, 1985


Listeria Monocytogenes Jill E. Clarridge, Ph.D. Department of Microbiology and Immunology and Pathology Bm'lor College of Medichze Chief, Microbiology Section VA Medical Center Houston, Texas 77211 Alice S. Weissfeld, Ph.D. Department of Microbiology attd Immunology Baylor College of Medicine Director. Microbiology Specialists Inc. tlouston, Texas 77005

In 1926, Murray, Webb, and Swann (5) described small, gram-positive rods they called Bacterium monocytogenes. The organism produced a striking monocytosis in laboratory rabbits which had acquired an epizootic infection. The first human case reported involved a boy with an infectious mononucleosis syndrome, subsequ6ntly shown to be a rare presentation of listeriosis. This organism, which has also been known as Listerella hepatolytica, Listerella monocytogenes, and Erysipelothrix monocytogenes, is a gram-positive, facultatively anaerobic, asporogenous bacillus that produces a characteristic tumbling motility at 20-25°C, beta-hemolysis on blood agar, and intraceilular growth. It is the only recognized human pathogen in a genus that presumptively contains four other species. The Genus Listeria In the eighth edition of Bergey's Manual (12), species in the genus Listeria are described as gram-positive,

CMNEEJ 7(9)59-66,1985

asporogenous, rod-shaped bacteria of uncertain affiliation. Four species, L. monocytogenes, L. grayi, L. murrayi, and L. denitrificans, are included in the genus. Ongoing investigations, however, have led to several provisional modifications in this scheme. A list of the proposed species in the genus Listeria is shown in Table I. The subcommittee of the International Committee on Systematic Bacteriology has suggested removal of L. denitrificans from this genus and designated it as Listeria denitrificans @omen generum perplexum) (21). L. murrayi and L. grayi are closely related and have been tentatively assigned to a new genus, Murraya (18). L. innoctta, which has been isolated from environmental sources, is considered nonpathogenic although under special experimental conditions it has produced encephalitis in animals (11). Previous infection with L. hmoctta protects against subsequent challenge with virulent strains of L. monocytogenes (20). Two additional species, L. welshhneri and L. seeligeri, formerly classified as nonpathogenic L. monocytogenes, have been recognized on the basis of DNA-hybridization studies (7). L. ivanovii, formerly called L. monocytogenes serovar 5 or L. bulgarica, is associated with disease in sheep and produces a pronounced beta-hemolysis on blood agar. Although these taxonomic changes may seem confusing, L. monocytogenes, as now defined, contains the serovars l/2a, l/2b, and 4a that are associated most often with disease, and is still the


species of concern to clinical microbiologists. Epidemiology L. monoo'togenes is ubiquitous in nature; it has been isolated from both wild and domestic mammals, birds, fish, plants, and soil throughout the world,. L. monocytogenes is a soil and plant saprophyte that most people are probably exposed to from many sources throughout their lifetimes. Why certain groups of individuals have an increased risk of active disease is unknown. In mice, both immunologic status and genetic predisposition appear to be important (15). In most human infections, the source of the organism is usually obscure. Raw cabbage, however, as used in cole slaw, was implicated as the source of an epidemic that occurred in the Maritime Province of Canada in 1980 and 1981 (9). The cabbage had been sold to a commer-

In T h i s Issue Listeria monocytogenes . . . . . . . . . . . 59 Current taxonomy and laboratory diagnosis Microbiology Laboratory Director-Supervisor Relationships .. 62 Two viewpoints of laboratory management Abstracts of Recent Literature .. 64

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Table 1 Biochemical Reactions of Provisional and F o r m e r Species in the Genus Listeria Hemo~'sis Species

on S B A

with S. attreus

L. monocytogenes

+ (narrow beta)


L. h m o c u a

L. ivanovii L. welshimeri L. seeligeri L. denitrificans M. murrayi M. grayi


+ (2 zones) D



Xylose --




arrow shaped









+ + -

Laboratory Diagnosis In direct smears, L. monocytogenes may appear as gram-positive coccobacilli. Unlike other causes of bacterial meningitis, however, organisms are usually not seen on the cerebral spinal fluid Gram-stained smear. L. monocytogenes grows well on 5% sheep blood or chocolate agar. Colonies are small, smooth, translucent, moist, and betahemolytic on blood agar following 24-

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hr incubation at 35°C in 5% CO 2. The zone of beta-hemolysis may be so narrow that colonies may need to be scraped off the surface of the medium to be visible. The Gram stain, catalase, and motility tests are useful for screening purposes because Listeria may be confused with group B betahemolytic streptococci, enterococci, or Corynebacterium spp. A positive catalase test distinguishes Listeria from the streptococci. Although L. monocytogenes resembles diphtheroids microscopically, the combination of beta-hemolytic colonies and a positive motility test excludes corynebacteria. To test for motility, several suspicious colonies may be suspended in brainheart infusion or tryptic soy broth and incubated at room temperature for 4 to 6 hr. L. monocytogenes exhibits tumbling motility when viewed microscopically using a wet-mount or hanging drop preparation. Alternately, colonies may be inoculated into two tubes of semisolid motility medium; one tube is incubated at 35°C and the other at 25°C. L. monocytogenes produces characteristic umbrella-type motility at 25°C (Figure 1) but not at 35°C. Columbia colistin-nalidixic acid (CNA) agar is a good selective medium for isolation of Listeria spp. from polymicrobic specimens. Positive tests for esculin hydrolysis, growth in 6% NaC1 broth, and acetoin production (Voges-Proskauer test) are important in differentiating Listeria (Table 1) from other bacteria. Both

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only confirmed human pathogen plants/soil, human, animal feces pathogenic for mice, shecp, possibly humans plants/soil plants, soil, sheep feces, possibly human reduces nitrate fcrments mannitol ferments mannitol





cial distributor by a farmer who had fertilized his field with raw manure from sheep infected with L. monocytogenes (called "circling disease" in sheep). Listeriosis cases have also been traced to the ingestion of raw milk or vegetables (9). Fecal-oral transmission may be important as well; L. monocytogenes has been isolated from stool cultures of between 1 - 5 % of healthy humans (2). Except for vertical transmission from mother to fetus or child, person-to-person spread of Listeria is not known, although transmission from animals to humans (particularly veterinarians) does occur. Males acquire meningeal and centi-al nervous system (CNS) forms of disease about four times as frequently as females (6). In temperate climates most human iisteriosis cases occur in the summer and autumn; listeriosis is rare in the tropics. Although a cold enrichment procedure for recovery of isolates from clinical specimens has been used for some time (2), the effect of cold on environmental organisms is unknown.


with R. equi

nitrate and mannitol tests are negative for the species presently considered as Listeria.

Although xylose, rhamnose, and hemolysis are used to distinguish between the Listeria spp. (3, 7), augmentation of hemolysis is the most valuable test to differentiate L. monocytogenes from the other species (except L. seeligeri, which has been reported only once as isolated from a clinical specimen). This phenomenon can be demonstrated with the unknown strain of Listeria inoculated perpendicularly to a beta-lysin producing Staphylococcus attreus. If Rhodococcus (CorynebacteriunO equi is used in place of the S. attreus (13, 14), L.

Figure 1. Motility o f L. m o n o c y t o g e n e s at

24 hrs.

Clinical Microbiology Nev.sletter 7:9.1985

monocytogenes and L. ivanovii will

show hemolysis augmentation. As shown in Figures 2 and 3, a positive result with R. equi may be more dramatic. S. aureus (the same strain used in the CAMP test, i.e., ATCC 25923), however, grows faster than R. equi, which usually takes two days to grow enough for a valid test. Skalka and Smola (13) have developed a diagnostic medium using an R. equi extract that can distinguish between L. innoctta, L. monocytogenes, and L. ivanovii by the relative size of the hemolytic zones produced. Serotyping of epidemiologically significarit strains can be done by a reference laboratory but may be of limited use because 70% of clinical isolates are serotype 4b or l/2b.

Figure 3. Same as Figure 2, except that the vertical streak is Rhodococcus equi.

Associated Diseases Listeriosis is the term used to describe the various clinical manifestations of L. monocytogenes infection. Specific individuals at increased risk of infection include: 1) pregnant women; 2) fetuses; 3) newborns (in the first three weeks after premature or full-term delivery); 4) individuals over 50 yr of age; and 5) immunosuppressed patients such as those undergoing therapy for lymphomas and leukemias; renal transplant recipients; alcoholics; patients with liver diseases

Figure 2. Two different strahzs of L. monocytogenes streaked horizontally, with S. aureus streaked vertically.

such as hepatitis; and those undergoing long-term hemodialysis (6, 16). Pregnant women with listeriosis usually have a relatively mild, influenzalike illness; however, the effect of the bacterium on the fetus is devastating. In the first trimester, the fetus is usually aborted; during the later stages of gestation, the fetus may be stillborn or the newborn baby may become acutely ill. Neonatal listeriosis parallels group B streptococcal infections. Early-onset disease is apparent at birth or shortly thereafter and is manifest by fulminant and often fatal septicemia. Late-onset disease appears after the first week of life with sepsis often associated with meningitis. In adults, the principle presentations are meningitis followed by primary bacteremia (6). Endocarditis, peritonitis, local abscess formation, and endophthalmitis occur infrequently. Granuloma formation is associated with listeriosis. Organisms can be seen in and isolated from inflammatory cells at the area of peripheral necrosis.


with disease, although an experimentally produced encephalitis due to L. innocua has been reported (I 1). Recent experimental evidence using chicken embryos links the level of hemolysin production with the severity of disease (I). In the mouse, hemolysin is a lethal cardiotoxin, causing damage to myocardial tissue. The hemolysin, however, is not always correlated with pathogenicity. L. ivanovii is more hemolytic than L. monocytogenes, yet rarely causes human disease (I0). A soluble lipase and Listeria lipopolysaccharide that is antiphagocytic may also be involved in the disease process. The tropism towards CNS tissue may also be a virulence factor. Because Listeria is an intracellular parasite, infection also depends upon the organism's successful entry and growth in mononuclear cells, a phenomenon that can be modulated by both host and parasite. Resistance to L. monocytogenes infection is thought to be mediated by T-lymphocyte-activated macrophages. Pregnancy alters humoral and cellular immune function, thus decreasing both resistance to L. monocytogenes and the capacity to develop immunity to Listeria (4). Therapy In vitro, L. monocytogenes is susceptible to penicillin, ampicillin, gentamicin, erythromycin, tetracycline, and chloramphenicol (19). Chloramphenicbl appears to be ineffective in vivo (17), therefore penicillin or ampicillin with or without an aminoglycoside is usually recommended for therapy (6). Adults allergic to penicillins may be treated with tetracycline; erythromycin is substituted in treatment of pregnant women or children with a penicillin allergy. Although potentially useful, trimethoprim-sulfamethoxazole and rifampin require more clinical study before they can be recommended (8).

The hemolytic properties of L. monocytogenes have been correlated cir-

cumstantially to pathogenicity. Essentially only the isolates hemolytic on sheep blood agar plates and showing augmentation of hemolysis with S. auretts or R. equi have been associated


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1. Basher, ti. A. et ai. 1984. Pathogenesis and growth of Listeria nlonocytogenes in fertile hens' eggs. Zentralbl. Bakteriol. Mikrobiol. Hyg. [A] 256:497-509.

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2. Bojsen-M~ller, J. 1972. Human listeriosis. Diagnostic, cpidemiological and clinical studies. Acta Pathol. Microbiol, lmmunol. Scand. [B] 229:1155. 3. Groves, R. D., and II. J. Welslfimer. 1977. Separation of pathogenic from non-pathogenic Listeria monoo'togenes by three in vitro reactions. J. Clin. Microbiol. 5:559. 4. Luft, B. J., and J. S. Remington. 1982. Effect of pregnancy on resistance to Listeria monocytogenes and Toxoplasma gondii infections in mice. Infect. lmmun. 38:1164-71. 5. Murray, E. G. D., R. A. Webb, antl M. B. R. Swann. 1926. A disease of rabbits characterized by a large mononuclear leukocytosis, caused by a hitherto undescribed bacillus Bacterium monocytogenes (n. sp.). J. Pathol. Bactcriol. 29:407-439. 6. Nieman, R. E., and B. Lorber. 1980. Listeriosis in adults: a changing pattern. Report of eight cases and review of the literature, 1968-1978. Rev. Infect. Dis. 2:207-227. 7. Rocourt, J., and P. A. D. Grimont. 1983. Listeria welshimeri sp. nov. and Listeria seeligeri sp. nov. Int. J. Syst. Bactcriol. 33:866-869. 8. Scheld, W. M. 1983. Evaluation of rifampin and other antibiotics against






Listeria monoo'togenes. Rev. Infect. Dis. 5. Suppl. 3:5593-5599. Sehleeh, W. F. III el al. 1983. Epidemic of listeriosis--cvidence for transmission by food. N. Engl. J. Mcd. 308:203-206. Seeliger, ti. P. R. et al. 1984. Listeria ivanovii sp. nov. Int. J. Syst. Bacteriol. 34:336-337. Seeliger, II. P. R. 1981. Apathogcne listcricn: L. imlocue sp. n. (Sccliger ct Schoofs, 1977). Zcntralbl. Bakteriol. Mikrobiol. Hyg. I. Abt. Orig. A. Med. Mikrobiol. Infektonskr. 249:487-493. Seeliger, II. P. R., and II. J. Welshimet. 1974. Listeria, pp. 593-596. hi R. E. Buchanan and N. E. Gibbons (eds.), Bergey's manual of determinative bacteriology. 8th cd. Williams and Wilkins, Baltimore. Skalka, B., and J. Smola. 1983. Selective media for pathogenic Listeria spp. J. Chn. Mtcrob,ol. 18:14321433. Skalka, B., J. Smola, and K. Elischerova. 1982. Routine test for in vitro differentiation of pathogenic and apathogenic Listeria monocytogenes strains. J. Clin. Mierobiol. 15:503507. Skamene, E., L. Kongshavn, and D. II. Sachs. 1979. Resistance to Lis..












teria monoo'togenes in mice: genetic control by genes that are not linkcd to the H-2 complex. J. Infect. Dis. 139:228-23 I. Stature, A. 51. el al. 1982. Listcriosis in renal transplant recipients: report of an outbreak and review of 102 cases. Rev. Infect. Dis. 4:665-682. Stature, A. M. 1982. Chloramphenicol: ineffective for treatment of listcria meningitis. Am. J. Mcd. 72:820. Stuart, S. E., anti II. J. Welshimer. 1974. Taxonomic reexamination of Listeria pirie and transfer of Listeria grayi and Listeria murravi to a new genus Murraya. Int. J. Syst. Bacteriol. 24:177-185. Tuazon, D. U., D. Shamsuddin, and tl. Miller. 1982. Antibiotic susceptibility and synergy of clinical isolates of Listeria monoo'togenes. Antimicrob. Agents Chemother. 21:535-537. Von Koenig, C. II. W. et al. 1983. Course and development of immunity in experimental infections of mice with listeria serotypes. Infect. lmmun. 40:1170-1177. Welshimer, 1I. J. 1981. The genus Listeria and related organisms, p. 1680. In M. P. Starr et al. (eds.), The prokaryotes: a handbook on habitats, isolation and identification of bacteria. Springer-Verlag, New York.

Editorial The Microbiology Laboratory Director-Supervisor Relationship: Complementary or Conflicting? Richard R. Clark, M.S. Supervisor, Microbiology Laboratory Department of Laboratot T Medicine University Hospital Boston, Massachusetts 02118 Cynthia A. Needham, Ph.D. Director, Microbiology Laboratory Department of Laboratory Medicine University Hospital Boston, Massachusetts 02118

The Supervisor's Viewpoint We have been asked to discuss the nature o f the professional relationship between a clinical microbiology laboratory director (usually a P h . D . / M . D . / Sc.D.) and a supervisor (usually an M . S . / B . S . ) . Although any o f these re-


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lationships will be as unique as the individual personalities and styles involved, the same basic qualities will determine the relative success or failure o f this important relationship, which in turn will have a profound effect on the operation o f the laboratory. Perhaps another question is implied: does anything about clinical microbiology make this relationship unique? Having personally experienced or observed the styles of several director and supervisor relationships, four characteristics stand out that appear to determine their success: 1) mutual communication, 2) mutual trust, 3) mutual respect, and 4) mutual support or loyalty.

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A m o n g the four qualities listed above, communication is the most critical (1). Although both the director and the supervisor must initiate and be receptive, the supervisor must be the active conduit between the administrative, idea-driven universe o f the director and the practical, hands-on, procedure-oriented world of the staff technologist. The supervisor must transmit information o f appropriate importance to both technologists and director. The revolutionary principles o f " a c tive l i s t e n i n g , " pioneered by Dr. Thomas Gordon in Parent Effectivehess Trahling (2) and applied to the work environment in Leader Effectiveness Training (3), have become standard fare in management courses and bear directly on improved communication between employer and employee. Mutual trust is the basis for any relationship and is a quality that devel-