Listeria monocytogenes
	1. Introduction The facultative, intracellular, gram-positive rod that causes listeriosis is Listeria monocytogenes. Listeria only produces a self-limited gastrointestinal illness in immunocompetent individuals, but it can cause serious infections in the elderly, newborns, and immunocompromised individuals.[1]
	2. Habit and Habitat The bacterium grows well at temperatures ranging from -0.4 to 45 ℃ [2]. L. monocytogenes is abundant in soil, water, and decomposing plants. The bacteria can also be found in human digestive tracts. The foods with the highest incidence of L. monocytogenes infections are raw sprouts, unpasteurized milk, and soft cheeses, cold deli meats, cold hot dogs, and smoked seafood.[1] Species and Serotypes The Listeria family has ten distinct species, with L. monocytogenes being the most commonly seen in humans. 13 serotypes have been discovered based on somatic (O) and flagellar (H) antigens that includes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e, and 7 respectively. However, only three serotypes (1/2a, 1/2b, and 4a) cause illness in humans [3,4].
	3. History of outbreaks L. monocytogenes has been linked to a number of foodborne disease outbreaks in the past. It was discovered in 1981 that L. monocytogenes was a foodborne pathogen associated with a wide range of foods. A soft cheese brand was linked to a significant epidemic of L. monocytogenes in 1985 that resulted in 142 cases, 28 fatalities, and 20 fetal losses [5].
	4. Epidemiology According to WHO, listeriosis has an annual incidence of 0.1 to 10 cases per million [6]. According to the Centres for Disease Control and Prevention (CDC), roughly 1,600 individuals get listeriosis each year, with approximately 260 dying from the condition [7]. According to studies, L. monocytogenes is the third greatest cause of mortality from foodborne diseases in the United States, accounting for roughly 260 deaths per year. The mortality rate for the confirmed cases of listerosis has been reported to be 15% but can go higher depending upon the condition of patients and comorbidities [8].
	5. Risk groups L. monocytogenes infection (listeriosis) can cause sepsis, meningitis, encephalitis, spontaneous abortion, fever, and self-limiting gastroenteritis in a healthy adult. Pregnant women, newborns, immunocompromised people, and the elderly are among the populations most vulnerable to L. monocytogenes infections [1]. Cadherin is an epithelial attachment protein found in abundance in both the blood-brain and placental-fetus barriers, which may explain why the bacteria may infect newborns and cause meningitis. Listeria meningitis causes fever, neck stiffness, headache, altered mental state, neurological impairments, and other typical meningitis symptoms [1]. The patient may have a positive Brudzinski's or Kernig's sign during the physical exam. The patient's mental state may be disturbed, and they may be unaware of their identity, location, or date. Inquiring about the usage of stomach acid suppressants may help to alleviate the increased risk of L. monocytogenes infection [1,9].
	6. Cultural and Biochemical Characteristics Listeria spp. are tiny gram-positive bacilli measuring 0.5-4 μm in diameter and 0.5-2 μm in length. They are non-spore former, facultatively anaerobic bacteria with catalase-positive and oxidase-negative reactions. Listeria exhibits tumbling motility corresponding to 25°C to 37°C owing to its peritrichous flagella in wet mount light microscopy after inoculation in Trypticase Soy Broth with 0.6% Yeast Extract (TSBYE) [10,18]. When cultivated on blood agar, L. monocytogenes is a facultative intracellular rod bacterium that is gram-positive, catalase positive, and beta-hemolytic [1]. Culture Media PALCAM Media: PALCAM (Polymyxin Acriflavine Lithium Chloride Ceftazidime Aesculin Mannitol) media is a selective and differential medium used primarily for the isolation and differentiation of Listeria species, particularly Listeria monocytogenes. This medium is widely used in food microbiology and clinical diagnostics due to its effectiveness in identifying Listeria in contaminated samples. When Listeria species grow on PALCAM media, they hydrolyze aesculin to produce a black precipitate around the colonies. Additionally, Listeria monocytogenes ferments mannitol, causing a change in the pH of the medium and leading to a color change due to the phenol red indicator. The selective agents in the medium (lithium chloride, acriflavine, polymyxin B, and ceftazidime) suppress the growth of competing bacteria, making it easier to isolate and identify Listeria species. Composition The key components and their functions in PALCAM media are: Peptone: Provides nutrients such as nitrogen, vitamins, minerals, and amino acids essential for bacterial growth. Yeast Extract: Supplies additional growth factors, including vitamins and amino acids. Mannitol: A fermentable carbohydrate source. Aesculin: A glycoside used to detect Listeria species, as they hydrolyze aesculin to produce a black precipitate. Ferric Ammonium Citrate: Reacts with the hydrolysis product of aesculin to form a black precipitate. Lithium Chloride: Inhibits the growth of gram-positive bacteria other than Listeria. Acriflavine: An antimicrobial agent that inhibits gram-positive bacteria. Polymyxin B: An antibiotic that inhibits gram-negative bacteria. Ceftazidime: A broad-spectrum antibiotic that inhibits a wide range of gram-negative bacteria. Phenol Red: A pH indicator that changes color to indicate mannitol fermentation. Oxford Agar (also known as Modified Oxford Medium) is another selective and differential medium designed for the isolation and differentiation of Listeria species, particularly Listeria monocytogenes. It shares a similar purpose to PALCAM media but has a different formulation and mode of action. When Listeria species grow on Oxford Agar, they hydrolyze aesculin to produce a black precipitate around the colonies due to the reaction with ferric ammonium citrate. The selective agents in the medium (lithium chloride, acriflavine, colistin, cefotetan, phosphomycin, and cycloheximide) suppress the growth of competing bacteria and fungi, facilitating the isolation and identification of Listeria species. Composition The key components and their functions in Oxford media are: Peptone: Provides essential nutrients for bacterial growth, including nitrogen, vitamins, minerals, and amino acids. Yeast Extract: Supplies additional growth factors, including vitamins and amino acids. Glucose: A fermentable carbohydrate source. Aesculin: A glycoside used to detect Listeria species by the production of a black precipitate when hydrolyzed. Ferric Ammonium Citrate: Reacts with the hydrolysis product of aesculin to form a black precipitate. Lithium Chloride: Inhibits the growth of gram-positive bacteria other than Listeria. Cycloheximide: Inhibits the growth of fungi. Colistin: An antibiotic that inhibits gram-negative bacteria. Acriflavine: An antimicrobial agent that inhibits gram-positive bacteria. Cefotetan: A broad-spectrum antibiotic that inhibits a wide range of gram-negative bacteria. Phosphomycin: Inhibits the growth of a variety of bacteria by interfering with cell wall synthesis. Both PALCAM and Oxford media are used for the isolation and differentiation of Listeria species, but they differ in their formulations and selective agents: Selective Agents: Both media contain lithium chloride and acriflavine. PALCAM uses polymyxin B and ceftazidime, while Oxford uses colistin, cefotetan, and phosphomycin. Differential Components: Both media use aesculin and ferric ammonium citrate for differential purposes. PALCAM also includes mannitol and phenol red to detect mannitol fermentation. These differences may influence the choice of medium based on the specific requirements of the laboratory or the nature of the samples being tested.
	7. Virulence Factors The capacity to replicate at refrigerator temperatures and intracellular movement through actin polymerization are two of L. monocytogenes' pathogenicity factors. Control by the food businesses is hampered by this. The fecal-oral route is the mode of transmission for the bacteria, and foods like cold deli meats and unpasteurized dairy items are frequently involved. Recent years have seen a decline in L. monocytogenes cases as a result of advancements in treatment, detection, and prevention [1,11]. The use of proton pump inhibitors or other stomach acid reduction techniques facilitates L. monocytogenes passage through the stomach and invasion of enterocytes [1]. Howeover, four primary virulence factors can be attributed for pathophysiology [1,10,11,12,13,14]: (i) Internalins (InlA and InlB): L. monocytogenes contains cell-surface galactose residues, lipoteichoic acids, and surface proteins known as "internalins" (internalin A and B), which attach predominantly to gastrointestinal epithelial cells via host protein cadherin, allowing entrance into the cell. After invading host cells, the bacterium has a proclivity to elicit a cell-mediated immune response. Cadherin is an epithelial attachment protein found in abundance in both the blood-brain and placental-fetus barriers, which may explain why the bacteria may infect newborns and cause meningitis [1,12]. (ii) Listeriolysin O (LLO): Phagocytized L. monocytogenes may lyse the internalised vacuole using a pore-forming cytotoxic protein known as "listeriolysin O" (LLO), as well as non-pore-forming phospholipase proteins. LLO is also responsible for the beta-hemolysis observed on blood agar [1,12]. (iii) Actin polymerization (ActA): Once released from the vacuole, the bacteria can disrupt normal cellular processes by migrating across the cell using actin polymerization. ActA, the bacterium's surface protein, is driven by host intracellular proteins that naturally control actin filaments. These actin monomers are linked asymmetrically (only at one end) via host intracellular cytoskeleton filaments. This tail, sometimes known as a rocket tail, enables the bacteria to travel quickly across the intracellular cytosol, between cells, and hematogenously [1,13]. (iv) Phosphatidylinositol-specific phospholipase C (PI-PLC): Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) is essential for the human pathogen's egress from host cell vacuoles. Unlike conventional bacterial PI-PLCs, the L. monocytogenes enzyme has relatively low action on glycosylphosphatidylinositol (GPI)-anchored proteins. [1,12].
	8. Infectious Dose, Clinical Manifestations, Diagnosis and Prevention In general, the infectious dosage of L. monocytogenes is high, which means that a significant number must be consumed to produce clinical illness; yet, because growth and division may persist at refrigerator temperatures, these levels can be attained despite standard food storage procedures. The host's immunological response also influences the infectious dosage [1]. Meningitis caused by Listeria is frequent in the young (neonates), elderly, and immunocompromised patient populations. L. monocytogenes infection in healthy people usually results in a self-limiting gastrointestinal illness with fever and diarrhoea [1,16]. Diagnosis L. monocytogenes must be identified using a culture of bacteria from the blood, cerebral spinal fluid, or placental fluid. Listeria species grow in the lab on a specific form of agar known as Meuller-Hinton agar. Culture will reveal gram-positive rods and beta-hemolytic colonies [1]. According to the CDC, Stool culture has not been studied as a screening technique and is not suggested for diagnosing listeriosis. Because the bacteria is often found in the environment, it is usual to consume Listeria-contaminated food [19]. However, in the suspicion of meningitis, it has been advised on considering the followings [1]: • Blood cultures • Lumbar punctures • Antibiotics • Non-contrast CT scan of the head. Prevention To prevent transmission of L. monocytogenes, avoid foods that are often contaminated with L. monocytogenes and use adequate hand-washing techniques. [1,20] The FDA has also approved a number of food additives, including bacteriophage (Listeria Phage P100) sprays, to inhibit the spread of L. monocytogenes in the general population. The spray includes a bacteriophage virus capable of eliminating L. monocytogenes. The spray will be mostly used on deli meats and cheeses [1,21]. Antibiotic Treatment Early detection and treatment with ampicillin, penicillin G, or trimethoprim-sulfamethoxazole are highly successful against L. monocytogenes [1,22]. The recommended antibiotic therapy is intravenous (IV) ampicillin or penicillin G. Trimethoprim-sulfamethoxazole is an alternate therapy option if the patient is allergic to penicillin. L. monocytogenes is naturally resistant to all cephalosporin drugs. Because other causes of meningitis include gram-positive organisms such as Streptococcus pneumoniae and gram-negative organisms such as Neisseria meningitidis, Ceftriaxone is included in the treatment regimen. Gentamicin is primarily used to treat gram-negative meningitis caused by Escherichia coli in infants. Finally, Vancomycin is given to treat Methicillin-resistant Staphylococcus aureus (MRSA) [1,23].
	REFERENCES 1. Rogalla D, Bomar PA. Listeria Monocytogenes. [Updated 2023 Jul 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2024 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK534838/ 2. Bucur FI, Grigore-Gurgu L, Crauwels P, et al. Resistance of Listeria monocytogenes to Stress Conditions Encountered in Food and Food Processing Environments. Front Microbiol 2018;9:2700. 10.3389/fmicb.2018.02700 3. Meloni D. Focusing on the main morphological and physiological characteristics of the food-borne pathogen Listeria monocytogenes. Journal of Veterinary Science and Research. 2014;1:1–2. 4. Ranjbar R, Halaji M. Epidemiology of Listeria monocytogenes prevalence in foods, animals and human origin from Iran: a systematic review and meta-analysis. BMC Public Health. 2018 Aug 23;18(1):1057. 5. Jackson KA, Gould LH, Hunter JC, Kucerova Z, Jackson B. Listeriosis Outbreaks Associated with Soft Cheeses, United States, 1998-2014. Emerg Infect Dis. 2018 Jun;24(6):1116-1118. 6. https://www.who.int/news-room/fact-sheets/detail/listeriosis#:~:text=It%20is%20a%20relatively%20rare,a%20significant%20public%20health%20concern. 7.https://www.cdc.gov/listeria/index.html#:~:text=An%20estimated%201%2C600%20people%20get,people%20with%20weakened%20immune%20systems.&text=Pregnant%20women%20are%2010%20times,to%20get%20a%20Listeria%20infection. 8. Choi MH, Park YJ, Kim M, Seo YH, Kim YA, Choi JY, Yong D, Jeong SH, Lee K. Increasing Incidence of Listeriosis and Infection-associated Clinical Outcomes. Ann Lab Med. 2018 Mar;38(2):102-109. 9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548367/ 10. Jamshidi A, Zeinali T. Significance and Characteristics of Listeria monocytogenes in Poultry Products. Int J Food Sci. 2019 Apr 18;2019:7835253. doi: 10.1155/2019/7835253. PMID: 31139641; PMCID: PMC6500651. 11. Shamloo E, Hosseini H, Abdi Moghadam Z, Halberg Larsen M, Haslberger A, Alebouyeh M. Importance of Listeria monocytogenes in food safety: a review of its prevalence, detection, and antibiotic resistance. Iran J Vet Res. 2019 Fall;20(4):241-254. PMID: 32042288; PMCID: PMC6983307. 12. Lindén S.K., Bierne H., Sabet C., Png C.W., Florin T.H., McGuckin M.A., Cossart P. Listeria monocytogenes internalins bind to the human intestinal mucin MUC2. Arch. Microbiol. 2008;190:101–104. doi: 10.1007/s00203-008-0358-6. 13. Roe JM, Seely K, Bussard CJ, Eischen Martin E, Mouw EG, Bayles KW, Hollingsworth MA, Brooks AE, Dailey KM. Hacking the Immune Response to Solid Tumors: Harnessing the Anti-Cancer Capacities of Oncolytic Bacteria. Pharmaceutics. 2023 Jul 21;15(7):2004. doi: 10.3390/pharmaceutics15072004. PMID: 37514190; PMCID: PMC10384176. 14. Robbins JR, Barth AI, Marquis H, de Hostos EL, Nelson WJ, Theriot JA. Listeria monocytogenes exploits normal host cell processes to spread from cell to cell. J Cell Biol. 1999 Sep 20;146(6):1333-50. doi: 10.1083/jcb.146.6.1333. PMID: 10491395; PMCID: PMC1785326. 15. https://www.pnas.org/doi/full/10.1073/pnas.0501725102 16.https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/listeria-meningitis 17. Le Monnier A, Abachin E, Beretti JL, Berche P, Kayal S. Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene. J Clin Microbiol. 2011 Nov;49(11):3917-23. doi: 10.1128/JCM.01072-11. Epub 2011 Sep 14. PMID: 21918022; PMCID: PMC3209115. 18. Girma, L., Geteneh, A., Amenu, D. et al. Isolation and characterization of Listeria monocytogenes among women attending Jimma University medical center, Southwest Ethiopia. BMC Infect Dis 21, 564 (2021). https://doi.org/10.1186/s12879-021-06254-w 19. https://www.cdc.gov/listeria/outbreaks/ice-cream-03-15/health-professionals.html#:~:text=culture%20for%20L.-,monocytogenes.,commonly%20present%20in%20the%20environment. 20. https://www.cdc.gov/listeria/prevention.html 21. Kawacka I, Olejnik-Schmidt A, Schmidt M, Sip A. Effectiveness of Phage-Based Inhibition of Listeria monocytogenes in Food Products and Food Processing Environments. Microorganisms. 2020 Nov 10;8(11):1764. doi: 10.3390/microorganisms8111764. PMID: 33182551; PMCID: PMC7697088. 22. Ma Y, Hu W, Song W. A case report of oral sulfamethoxazole in the treatment of posttransplant Listeria monocytogenes meningitis. Transl Androl Urol. 2023 Mar 31;12(3):524-529. doi: 10.21037/tau-23-83. Epub 2023 Mar 27. PMID: 37032758; PMCID: PMC10080344. 23. Magiar O, Vulpie S, Musuroi C, Marincu I, Murariu A, Turaiche M, Musuroi SI, Muntean D, Licker M. Listeria Monocytogenes Meningitis in an Immunocompetent Patient. Infect Drug Resist. 2022 Mar 10;15:989-994. doi: 10.2147/IDR.S351132. PMID: 35299851; PMCID: PMC8921837.