Listeria monocytogenes

Listeria monocytogenes

8

Listeriamonocytogenes

Summary

The significance thebacterium in medicine, food processing,microbiology and other related sciences cannot be ignored. Althoughthe bacterium is not one of the most common among food bornediseases, it is responsible for 19% deaths from foodborne disease inthe US and 17% in France. The bacterium has been in existence forclose to a century and its classification and changes with timecapture the continued need to understand microorganisms better andtheir associated features such as their morphology and pathogenesisinforms scientists on the adaptation of such a microorganism to itsenvironment and the possible approaches in treating infections anddiseases caused by the bacterium. Most importantly, the clinicalsignificance of the bacterium has been on the rise. This is the faceof increased cases of outbreaks such as in Canada, Greece and Indiahave increased. The threat that the bacterium poses to humans andanimals, more so, in relation to pregnant women and the food industryunderlines the need to better understand the bacterium and the bestways of managing it. Through continued research, different fieldscontinue to investigate additional methods through which thebacterium can be managed. Some of the new approaches beingexperimented are medical based on new understanding of the bacterium.Another approach that has the potential to gain a wider following isrelated to nutritional approaches in managing the bacterium. Thisapproach is not only to attract certain group of people but is alsomore sustainable in the long run. Furthermore, changes I behavioralpractices especially in handling foods can be utilized in managingthe bacterium and fighting its related infections.

Bacteria are some of the most widely occurring organisms in theenvironment with a wide ranging effect on other living organisms. is just of the many types of bacteriawith disease causing capabilities. It is a gram positive bacteriumthat is taxonomically classified in the division of firmicutes, whichmay go by different names for medical doctors, veterinarians andpeople involved in food science and processing. The most commondiseases associated with the bacterium are listeriosis, meningitis,and gastroenteritis in humans while in animals the diseases includesilage sickness and tiger river disease. The bacterium has in thelast few years emerged as a mild medical concern especially after theoutbreak in Canada and France a few years ago. Thus, due to thegrowing significance of the bacterium, this paper seeks to increasethe understanding of the organism by examining its source organism,taxonomic classification, cellular morphology, clinical significance,parthenogenesis, virulence, isolation and identification, treatmentand antibiotic resistance, habitat and current experimental therapiesbeing employed and being examined.

Source oforganism and details of the original description and taxonomy

L. monocytogenes was first described by Prof. GustavHülphers on March 30 1910 and named it bacillus hepatis. He isolatedthe bacteria from a colony of rabbits but did not preserve thebacterial strains (1). This led to the strain being rediscoveredagain several years later among dead rabbits by Murray and hiscolleagues who named it Bacteriummonocytogenes(1,2,32). The description of the strain presented by Murray matchedthe previous one presented by Hülphers but it is Murray who iscredited with having discovered the bacteria simply because hemanaged to isolate and preserve the strain that can still be accessedtoday (ATCC no. 15313 ATCC no. 4428). Harvey Pirie also managed toisolate the same microorganism from gerbils in a place near TigerRiver in South Africa (12). The disease was thus named after theriver as Tiger River disease while the bacterium was named Listerellahepatolytica in referenceto a British surgeon, Lord Joseph Lister (12, 2). In 1940, thebacterium was named (1,2). Atthat time, there was only one species of the bacterium but otherscame to be discovered in the 1960s and 1970s. Others have been addedand removed and added again in what has come to characterize theproblems associated with classifying the bacterium (1,3).

Taxonomy andcellular morphology

Taxonomy

As a bacterium, falls under thefirmicutes, then Bacili, then Bacillales, then Listeriaceae.As per scientific taxonomic classification, the name listeriastands for the genus while the name monocytogenes denotes thebacterium’s species. As aforementioned, the modern classificationof this bacterium has undergone various changes. Forinstance, previously the genus category of Listeriawas categorized under the family of Corynebacteriaceae butthis changed with the 8thedition of Bergey`s Manual of Determinative Bacteriology groupingListeriatogether with Erysipelothrixand Caryophanonunder uncertain affiliation. Due to differences noted in some speciesand lacking in others following a numerical taxonomic study of 123strains of L and other genera by some researchers, there weresuggestions to create a new family name, Listeriaceae,to accommodate the generaListeria and Morraya(32). However, thesesuggestions were not adopted and Listeriaremains to be a genus from the sub-branch Clostridium thatalso includes Staphylococcus, Streptococcus, Lactobacillus andBrochothrix (3,7).

Following other minor modifications in the classifications, the genusListera as of today comprises of six species namely:L. innocua, L. monocytogenes , L. seeligeri , L.welshimeri, L. grayi and L.ivanovii. L. ivanovii is further split into two subspecies namelyivanovii andlondoniensis. L. monocytogenesis the most potent in terms of its pathological effect as it cancause disease in both animals and plants (2,3,21). However, L.ivanovii and L.seeligeri have been reported in animals while L. ivanovii isattributed to cases of spontaneous abortion in sheep. L.monocytogenes infection in humans is largely associated incontamination through consumption of infected foods. It must be notedthat animal-based foods such as cheese, meat and milk are most likelyto be contaminated during processing or storage (26).

Morphology

L. monocytogenes which is gram positive and facultivelyanaerobic appears as regular short rods with rounded ends measuring0.4-0.5 micrometer in diameter and 0.5-2 micrometer in length (4,31). In most cases, they occur in singular in pairs or threes to formletter V of Y. When they occur as a cluster of more than three, theyusually form short chains. The bacterium is motile and when culturedin room temperature (20-22oC) develops one to fiveperitrichous flagel which enables it to rotate around is long axis atan average of 507±106 micrometer per sec and average distance perrotation being 29.4±11.8 (4, 23, 9, 16). However, the growth andmorphology of the bacterium is largely influenced by the environment.One such study revealed that in the presence of 5% NaCl and 7% KCl,the filament was observed to grow at higher rates with higher saltconcentration (5). This implies that the morphological traits of thebacterium reported may change with the environment. Normally, thefilaments in older cultures can grow to a length of up to 20 mu,which is several times the length of the body (5, 22). However, theseolder cultures may lose the ability to stain gram-positive. Thebacterium is very versatile in regards to temperature of environment.Optimal temperature is 30-37oC but can thrive intemperatures of below 0oC (5, 6).

Clinicalsignificance of this bacteria and parthenogenesis

The bacterium is distributed inthe environment in many ways. They include natural methods such aswater and soil and also man-made methods such as silage, walls,floors etc. The bacterium can also exist in intestines of animals andhumans without necessarily causing illness (28,3). In fact, may typesof foods, especially that which has been stored or refrigerated forlong periods, contain trace amounts of L. monocytogenes.It is estimated that there is a minimum amount of L.monocytogenes that must be ingested to represent a significantrisk of diseases in humans. It is estimated to stand at 1,000 cells(7). Upon ingestion, the illness is not immediate as the organismtakes about 21 days to incubate (7,8). The bacterium is responsiblefor a number of infections in humans and animals as discussed next.

FebrileGastroenteritis

This is a medical condition affecting the gastrointestinal tract inhumans commonly attributed to a wider range of bacteria and viruses.Norovirus, adenovirus, astovirus and rotavirus are most commonviruses while Escherichia coli and Campylobacter are the commonbacteria that cause the infection (2, 26, 9). However, only recentlythat research revealed that L. monocytogenes has a causativerole in the infection. Common symptoms may range from mild to severewhich include fever, vomiting, diarrhea, abdominal pain,arthromyalgia and headache (9,22,25). However, FebrileGastroenteritis associated with L. monocytogenes may registeradditional symptoms such as fever, malaise, headache, vomiting anddiarrhea (1,5). Nonetheless, the presence of L. monocytogenesin diarrhea stool should not be linked to the causative agent of thediarrhea because the bacterium has been observed in 5% of samplestool in some studies (9, 10).

Invasive Disease,Septicaemia and Meningitis

Studies have linked L. monocytogenes with Septicaemiaand meningitis especially in severely immunocompromised individualsand the elderly. L. monocytogenes is responsible forapproximately a third of all cases of meningitis especially amongadults (8,9). This type infection poses a more serious threat becausepatients rarely present atypical clinical features associated withmeningitis though it is common identify it through cerebrospinalfluid findings (8,26).

PregnancyAssociated Disease

Listeriosis has a greater impact on pregnant women and the fetus. Thebacterium can infect the unborn infant which can be commonlydiagnosed during the third trimester of pregnancy (26). Mothers mayregister symptoms that are difficult to differentiate from atypicalpregnancy systemptoms such as vomiting, backache and diarrhea.However, other additional symptoms specific to listeriosis areinfluenza-like illnesses, malaise, fever, diarrhea, and chills. Inrare cases, premature births may occur (8).

Listeriosis inanimals

Listeriosis in animals has been reported in many cases in animalssince the early identification by Hülphers, (1911) and Murray et al(1926). In most cases, listeriosis infections in animals haveoccurred as sporadic cases and occasional outbreaks especially in theIndian subcontinent (20). It has been reported in a many types ofanimals including sheep, goat, rabbits, chicken, cows and evenbuffalos. Some farming practices such as debeaking poultry areconsidered to increase predisposition to listeric infections (10)

Parthenogenesis

During this period, there is rapid growth. As a facultative pathogen,it grows in a wide range of tissue cells. Colonization takes place inthe intestine before intestinal translocation and replication inmajor organs such as liver and spleen (20). This process largelyrelies both on host factors and the bacterial factors. Some of thedetermining host factors include genetic history of the host,behavior, age and the interaction between microorganism and the host.Bacterial factors factor mainly comprise of the proteins released bythe bacterium (7). Two proteins hly (previously called hlyA andlis4). The L. monocytogenes hemolysin, listeriolysin O (LLO),which are a family of sulfhydryl-activated pore-forming cytolysins,of which streptolysin O is the prototype, are the most significantdeterminants of the pathogenesis. Studies have hypothesized that thefunction of LLO is to lyse the host vacuole. However, there are otherdeterminants which may contribute to the lysis of the host vacuoleboth initially and during cell-to-cell spread (7, 18).

The growth process of the L. monocytogenes is relativelysimple. Once the bacterium enters the body of the host throughcontaminated food, there are chances that the bacterium isphagocytosed by the gastrointestinal cells meaning that it can remainresent without causing an infection in the gastrointestinal tract.Once inside the cytoplasm of the host’s cells, it rapidly growspushing up again the cell membrane which makes it easier for theadjacent cells to absorb it. Through this process, the bacterium isable to multiply and spread throughout the body tissue without beingexposed to antibodies or antibiotic treatment located in theextracellular fluid. The same mechanism is applied by the bacteriumto cross the placental barrier to infect the fetus and also theblood-brain barrier that can lead to meningitis or encephalitis (11).

Virulence factor

L. monocytogenes virulence is driven by a highly complex andwell organized life cycle denoted by several key steps. Thecompletion of each step is determined by the activity andcoordination of specific set of regulators (10,7).

Step 1: bacterial contact and adherence- L. monocytogenesprovokes internalization into nonphagocytic cells such as epithelialcells and hepatocytes through two main mechanisms inlA and inlBgenes. Proteins InlA or InlB initiate the binding to their respectivehost receptors.

Step 2: The formation of the first ligand-receptor complexessends out transient signals that induce actin polymerization andmembrane extension in host cells. Actin depolymerisation occurs afterthe closure and withdrawal of the phagocytic cup.

Step 3: L. monocytogenes gene such as hly instructspore formation through cytolysin listeriolysin O (LLO) that helps thebacterium to break out from the phagocytic vacuole. The bacteriumthen secretes two types of phospholipase C (PLC),Phosphatidylinositol PLC and phosphatidycholine-PLC, which worktogether with LLO is lysis of the vacuole (12).

Step 4: Hexose phosphate transporter (Hpt) is responsible forintercellular replication while bacterial movement is facilitated bythe listeria-induced polymerization of actin filaments in cytosol. Atransmembrane design in the carboxyl-terminal domain also aids inbacterial movement. Propulsion capability of the bacterium is gainedby distributing protein ActA on the bacterium’s surfaceasymmetrically (2,12).

Step 5: bacterial movement is necessary to avoid humoral andcellular immune responses. Exploitation of the host actinpolymerization is used in spreading from cell to cell.

Step 6: Thisstep involves the movement of the bacterium across a double-walledvacuole created when the bacterium is propelled from one cell intoanother. Phosphatidylinositol PLC and phosphatidycholine-PLC areusually employed in this process.

Isolation andidentification

Since discovery of the L. monocytogenes in rodents and sheep,the bacterium has been isolated from sample stools in humans, storedfoods, vegetation, soil, water, and a wide range of animal species.Human beings excrete the bacterium symptomatically andasymptomatically. The bacterium has also been isolated fromeffluences associated with animals and sewage sludge and has alsobeen detected during sewage treatment and storage all in levelsgreater than 1800 cfu/L. For most of the treated sewage sludge, thebacterium exists in less that 3-7cfu/L and is often deposited inlands as manure which can lead to contamination of crops and animals(10).

Treatment andantibiotic resistance

Given that L. monocytogenes infections are relatively rare,there are no prospective studies on antibiotic regimens. For anantibiotic to be successful against L. monocytogenes, it mustbe capable of penetrating host cell and maintain high concentrationwithout affecting the pH that may also affect its efficacy. Theamitotic should then bind to the penicillin-binding protein 3(PBP3)of the microorganism that is charged with cell death. In the case oftreating pregnant mothers, the antibiotic used must be capable ofmoving across the placenta in adequate concentration (11).

Antibiotic treatment of L. monocytogenes has employedPenicillin, ampicillin, and amoxicillin extensively. They work byinhibiting PBPs and do not penetrate intracellular walls. To ensurepenetration through the umbilical cord, higher concentrations areused (11, 28). However, some in vitro studies have suggested asynergetic effect when gentamicin is used alongside the three drugs.However, the toxic capabilities of gentamicin have discouraged thisapproach among some practitioners. Another issue that can affect theefficacy pertains to the dosage offered to infected individuals inwhichever antibiotic chosen. A daily dosage of 6g daily or 2g every 8hours is recommended. The dosage maybe increased in case ofpregnancies especially for ampicillin (11).

Alternativetreatments for patients allergic to penicillin are available.Trimethoprim/sulfamethoxazole (TMP/SMX) is the most commonalternative. TMP/SMX has the best oral biovilability but may poseside effects on kidney function and platelets during high doses.Furthermore, the antifolate capability of the drug, it should not beused on patients on the early stages of a pregnancy o in patients atrisk of neural tube defects (11).

There are rare cases of L. monocytogenes resistance toantibiotics especially from animal isolates. However, certainantibiotics which are usually not used in antibiotic therapies for L.monocytogenes cases have recorded resistance from the bacteriumisolates from food. These antibiotics include streptomycin,kanamycin, erythromycin, gentamicin, trimethoprim, and rifampin (2).Again resistance is also rare when it comes to alternative treatments(11). Additionally, most cleaning agents with disinfectants have thecapacity to eliminate the bacterium effectively (13).

Habitat, nichephysiology

The environment is the natural habitat of L. monocytogenes.This means that the bacterium can be isolated from soil, sewage,soil, dust, water, processed foods, raw meat, and herbage. Althoughthe bacterium can thrive in adverse environmental conditions,aerobically spoiled silage provides the best niche conditions for itto thrive (10, 13). This means that silage is the best method ofinfection of animals. The exposure of herbage and fodder to L.monocytogenes from the soil and sewage sludge used asbio-fertilizer remains relatively small (10, 24). However, exposureof the same to niche conditions in spoilt silage significantlyincreases the presence of L. monocytogenes. A study in Germanyon crops leaves and grass before harvesting tested for L.monocytogenes with none of the ten samples showing anypresence of the bacterium. However, after harvesting the same cropsand storing in silages, 9/10 samples gave positive tests for L.monocytogenes (10,6).

Other common sources of the bacterium include food and food industryequipments. A recently as 2008, Canada experienced a Literiosisoutbreak that saw many packed foods being recalled from the market.Several other countries in Europe have also recorded mild outbreaksof the infections. In all these cases, the source of the bacteria hasbeen identified as food. One study in Sweden examined a wide range ofready to eat food types for L. monocytogenes. They includedcheese, a variety of fish and meat products. The samples were testedat the end of their shelf-lives assunming that the bacterium mighthave been growing under harsh conditions of storage. Both cold-smokedand gravad fish tested positive for L. monocytogenes withSwedish samples testing only 8% while samples processed in foreigncountries recorded higher positive levels at 45%. In both Swedish andforeign processed food samples, both gravad and cold-smoked fishsamples recorded the highest L. monocytogenes prevalencelevels at 14% (13).

Food processing and kitchen equipments are also a major source of L.monocytogenes contamination (15,16,29). However, a low initialbacterial charged is required for an infection to occur and thesource may vary. However, poor cleaning practices and presence ofharborage sites creates a suitable environment that the bacterium canthrive. On the good side, regular cleaning agents and disinfectantsin the food processing industry have proved effective in fightingthis infection and no persistence has been recorded in the face ofsuch cleaning agents (6).

Experimentaltherapies

The increased incidence of L. monocytogenes has increasedresearch in the management of the bacterium. Additionally,experimental therapies are driven by the need to identify effectivealternative treatments to the existing ones especially for personswith allergic reactions to antibiotics. There are a number of studiesassessing the effectiveness various therapies in fighting L.monocytogenes (9,22,21). One such study explores the role ofcytokine IL-6 in immune responses against bacterial infections. Thecytokine is key regulator of pro-inflammation and anti-inflammationprocesses in the body as a response to infection. The cytokine isproduced by a number of cells. However, the particular mechanisms ofthis cytokine are not well understood. Therefore, it is throughexperimentation with that researchers are seeking to prove IL-6deficient mice have impaired control of L. monocytogenes.Furthermore, L. monocytogenes infection causes intensesystemic IL-6 release and rapid loss of IL-6Rα surface expression onneutrophils, inflammation inducing monocytes, and various types oflymphocyte. This study is relevant in L. monocytogenesmanagement because IL-6 might induce protective mechanisms inhepatocytes, for example, via the induction of the acute phaseproteins (14).

One experiment in Brazil explored the management of L.monocytogenes on fresh vegetables. The study experimented with anumber of fresh vegetables such as cucumbers and parsley immersed inwashing solutions and acetic acid. The experiment revealed that whiletemperatures, acidity levels and period of immersion influenced theL. monoctyogenes count significantly, the impact different from onefood to the other. Resistance to treatment in cucumbers was higher.The researchers thus conclude that there is need to consider hostfactors as significant determinants in management of the bacterium(15).

Another experiment explored the effectiveness of Puroindoline A andpuroindoline B sourced from plant seeds, bovine lactoferrin obtainedfrom a select animals and chicken eggs lysozyme. The study was basedon the knowledge that these foods have antimicrobial proteins thatlyse invading organisms. Research results indicated significantreduction in L. monocytogenes count after 5 days of infection.Although the research was carried out on mice, it suggests that thesefoods have the capacity to control L. monocytogenes infectionsin humans (17).

Conclusion

From the discussion, it is clear that given niche habitat of theorganism, it is bound to proliferate based on current development inlifestyle changes more so on increased periods of food refrigerationand continued waste and sewage management methods. The bacterium canonly grow in form of the risks it poses to humans and animals. Theonly comforting thing at the moment is that the bacterium has notrecorded cases of resistance to current antibiotic treatmentprograms. Nonetheless, there is need to focus research onnon-antibioctic approaches to managing the bacterium as suggested bya therapy of certain foods with antibacterial properties.

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