Microbiology

Thermal Response Of Listeria Monocytogenes On Different Culture Media: Microbiology ABSTRACT Cellular responses against environmental stresses are the most highly regulatory responses among all organisms. Exposure of cells to stresses such as heat shock leads to the accumulation of partly and fully denatured proteins that interfere with normal cellular function. Present study was designed to examine the growth and physiology of Listeria monocytogenes under different temperatures ranging from 25° to 45°C using patching, spot test, and spectroscopic measurement of cell turbidity and to determine the critical growth temperature of Listeria monocytogene on different media. The results showed that the elevation of temperature inhibited the normal phenotypic colony characteristics. According to the spot test, slower growth was observed upon increase of temperature.

1.1 Background Microorganisms live in a permanently changing environment. The temperature variation is one of the most important existing stress factors. For instance, a temperature increase induces the bacterial heat shock response which allows cells to adapt and survive thermal stress conditions (Bukau, 1993; Connolly, et al.,1999;Geogopoulos,et., 1994). Nowadays the heat shocks response is of importance for many scientific and industrial applications, i.e.in processes where temperature-induced heterogonous protein production takes place.(Han et al.,2004).The general heat shock response was first discovered in Drosophila sp. By Rotissa, who suggested that cells exposed to heat induce the synthesis of a well-define number of protein (Rotissa et al., 1963). The proteins induced by heat and cold shock in Listeria monocytogenes (pathogenic for humans) and Listeria innocua (nonpathogenic) strains were analyzed by two-dimensional (2-D) electrophoresis with the help of a computerized 2-D gel analysis system. Heat (49°C) and cold (4°C) shock repressed roughly half the number of proteins synthesized at normal temperature (25°C) and decreased the level of numerous other proteins. Conversely, the synthesis of a great number of proteins was enhanced and novel proteins appeared upon temperature stress. There were more proteins induced in the Listeria monocytogenes strain than in the Listeria innocua strain. Each stress induced a set of specific proteins. There was overlap between these sets of proteins induced by heat and cold shock.

Furthermore, a number of heat or cold shock proteins were found to be induced in both Listeria species and by both heat and cold shock in both species. The induction by heat shock was more intense than that by cold shock. The most strongly induced common stress a molecular protein of Listeria had mass of 17.6 kDa and an isoelectric point of 5.

Misfolding, and aggregation of proteins are major damaging consequences of stress situation such as heat shock and pathophysiological states (mormoto et.at al.,1994; Horwich and Weissman 1997; Lindquist and Schirmer 1999.) The cellular defense against such damage is moleculer chaperones , whice prevent aggregation, assist refolding and mediate degradation of misfolded proteins (morimoto et al.,1994;Hart 1996;Bukau,1999). Chaperones can cooperate in vitro as part of functional network in which folder chaperones actively assist aggregation of misfolded proteins, whereas” folder” chaperones actively assist refolding (Langer et al.,1992; Buchberger et al.,1996;Freeman and morimoto,1996.Ehrsperger et al.,1997; Johnson Craig 1997;veinger et al., 1998).

The contribution of individuals chaperones to this folding network in vivo and the identity of the stress sensitive cellular proteins remain unknown. Moreover, since cells have only a limited chaperone capacity to prevent protein aggregation under stress condition (Craig and

Gross, 1991; Tatsuta et al.,1998), it becomes important to determine the extent to which chaperones can resolubilize aggregates of proteins that escaped the protective function of holder chaperones.

All living organisms have developed sophisticated strategies to respond to several environmental stresses , such as osmolarity, pH ,nutrition deprivation and thermal stresses .

In terms of thermal stress , heat shock stress (temperature up shift) has been extensively studied and a common strategy how to respond to it has been emerged from bacteria to human (Henrdricks and Hartl,1993;Gottesman et al .,1997). Whereas the activities and transcriptional regulation of the heat shock proteins have been well characterized comparatively little is known about the exact nature of the signal generated by stress and how this signal is conveyed to the transcriptional regulators of the stress response.

Listeria monocytogenes is a Gram-positive, nonspore - forming, motile, facultatively anaerobic, rod-shaped bacterium. It is catalase-positive and oxidase-negative, and expresses a beta hemolysin, which causes destruction of red blood cells. This bacterium exhibits characteristic tumbling motility when viewed with light microscopy. Although Listeria monocytogenes is actively motile by means of peritrichous flagella at room temperature (20−25°C), the organism does not synthesize flagella at body temperatures (37°C).

The genus Listeria belongs to the class, Bacilli, and the order, Bacillales, which also includes Bacillus and Staphylococcus. The genus Listeria includes seven different species (Listeria monocytogenes, Listeria ivanovii, Listeria innocua, Listeria welshimeri, Listeria seeligeri, Listeria grayi, and Listeria murrayi). Both Listeria ivanovii and Listeria monocytogenes are pathogenic in mice, but only Listeria monocytogenes is consistently associated with human illness. There are 13 serotypes of Listeria monocytogenes that can cause disease, but more than 90 percent of human isolates belong to only three serotypes: 1/2a, 1/2b, and 4b. Listeria monocytogenes serotype 4b strains are responsible for 33 to 50 percent of sporadic human cases worldwide and for all major food borne outbreaks in Europe and North America since the 1980s.

Listeria monocytogenes was first described by E.G.D. Murray in 1926 based on six cases of sudden death in young rabbits Murray referred to the organism as Bacterium monocytogenes before Harvey Pirie changed the genus name to Listeria in 1940 Although clinical descriptions of Listeria monocytogenes infection in both animals and humans were published in the 1920s, not until 1952 in East Germany was it recognized as a significant cause of neonatal sepsismeningitis Listeriosis in adults would later be associated with patients living with compromised immune systems, such as individuals taking immunosuppressant drugs and corticosteroids for malignancies or organ transplants, and those with HIV infection.

Not until 1981, however, was Listeria monocytogenes identified as a cause of food borne illness. An outbreak of listeriosis in Halifax, Nova Scotia involving 41 cases and 18 deaths, mostly in pregnant women and neonates, was epidemiologically linked to the consumption of coleslaw containing cabbage that had been treated with Listeria monocytogenes-contaminated raw sheep manur Since then, a number of cases of food borne listeriosis have been reported, and Listeria monocytogenes is now widely recognized as an important hazard in the food.

The present research work is designed to investigate the growth response of Listeria monocytogenes on different media at different temperature ranging from 25°C to 45°C and to determine their critical temperature 1.2 Review of the literature

1.2.1 Listeria Listeria is a bacterial genus that contains seven species. Named after the English pioneer of sterile surgery Joseph Lister, the genus received its current name in 1940. Listeria species are gram-positive bacilli. The major human pathogen in the Listeria genus is Listeria monocytogenes. It is usually the causative agent of the relatively rare bacterial disease, listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects primarily pregnant women, newborns, adults with weakened immune systems, and the elderly.

Listeriosis is a serious disease for humans; the overt form of the disease has a mortality rate of about 20 percent. The two main clinical manifestations are sepsis and meningitis.

Meningitis is often complicated by encephalitis, a pathology that is unusual for bacterial infections. Listeria ivanovii is a pathogen of mammals, specifically ruminants, and has rarely caused listeriosis in humans.

The first documented case of Listeria was in 1924. In the late 1920s, two researchers independently identified Listeria monocyctogenes from animal outbreaks. They proposed the genus Listerella in honor of surgeon and early antiseptic advocate Joseph Lister; however, that name was already in use for a slime mold and a protozoan. Eventually, the genus Listeria was proposed and accepted. All species within the Listeria genus are Gram-positive, nonsporeforming, catalase-positive rods. The genus Listeria was classified in the family Corynebacteriaceae through the seventh edition of Bergey’s Manual of Systematic Bacteriology. The 16S rRNA cataloging studies of Stackebrandt, et al. demonstrated that L. monocytogenes is a distinct taxon within the Lactobacillus-Bacillus branch of the bacterial phylogeny constructed by Woese. In 2001, the genus was placed in the newly created Family Listeriaceae. The only other genus in the family is Brochothrix.

The genus Listeria currently contains seven species: L. grayi, L. innocua, L. ivanovii, L. monocytogenes, L. murrayi, L. seeligeri, and L. welshimeri. Listeria dinitrificans, previously thought to be part of the Listeria genus, was reclassified into the new genus Jonesia. Under the microscope, Listeria species appear as small, Gram-positive rods, which are sometimes arranged in short chains. In direct smears, they may be coccoid, so they can be mistaken for streptococci. Longer cells may resemble corynebacteria. Flagella are produced at room temperature, but not at 37°C. Hemolytic activity on blood agar has been used as a marker to distinguish L. monocytogenes among other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization may be necessary to distinguish between the different species of Listeria.

Listeria can be found in soil, which can lead to vegetable contamination. Animals are most common carriers. Listeria has been found in uncooked meats, uncooked vegetables, fruit such as cantaloupes unpasteurized milk, foods made from unpasteurized milk, and processed foods. Pasteurization and sufficient cooking kill Listeria; however, contamination may occur after cooking and before packaging. For example, meat-processing plants producing ready-toeat foods, such as hot dogs and deli meats, must follow extensive sanitation policies and procedures to prevent Listeria contamination. Once an infection occurs in humans, one out of five people die. Once the infection begins in a processing plant, it is almost impossible to eradicate it. Listeria monocytogenes is commonly found in soil, stream water, sewage, plants, and food. Listeria are responsible for listeriosis, a rare but potentially lethal food-borne infection. The case fatality rate for those with a severe form of infection may approach 25% (Salmonella, in comparison, has a mortality rate estimated at less than 1% Although Listeria

monocytogenes has low infectivity, it is hardy and can grow in temperatures from 4⁰C (39.2⁰F) (the temperature of a refrigerator), to 37⁰C (98.6⁰F), (the body's internal temperature). Listeriosis is a serious illness, and the disease may manifest as meningitis, or affect newborns due to its ability to penetrate the endothelial layer of the placenta.

1.2.2 Pathogenesis Listeria uses the cellular machinery to move around inside the host cell: It induces directed polymerization of actin by the ActA transmembrane protein, thus pushing the bacterial cell around.

Listeria monocytogenes, for example, encodes virulence genes that are thermoregulated. The expression of virulence factor is optimal at 37 degrees Celsius and is controlled by a transcriptional activator, PrfA, whose expression is thermoregulated by the PrfA thermoregulator UTR element. At low temperatures, the PrfA transcript is not translated due to structural elements near the ribosome binding site. As the bacteria infect the host, the temperature of the host melts the structure and allows translation initiation for the virulent genes.

The majority of Listeria bacteria are targeted by the immune system before they are able to cause infection. Those that escape the immune system's initial response, however, spread through intracellular mechanisms and are, therefore, guarded against circulating immune factors (AMI).

To invade, Listeria induces macrophage phagocytic uptake by displaying D-galactose in their teichoic acids that are then bound by the macrophage's polysaccharide receptors. Other important adhesins are the internalins. Once phagocytosed, the bacterium is encapsulated by the host cell's acidic phagolysosome organelle. Listeria, however, escapes the phagolysosome by lysing the vacuole's entire membrane with secreted hemolysin, now characterized as the exotoxin listeriolysin. The bacteria then replicate inside the host cell's cytoplasm.Listeria must then navigate to the cell's periphery to spread the infection to other cells. Outside the body, Listeria has flagellar-driven motility, sometimes described as a "tumbling motility."

However, at 37°C, flagella cease to develop and the bacterium instead usurps the host cell's cytoskeleton to move. Listeria, inventively, polymerizes an actin tail or "come", from actin monomers in the host's cytoplasm with the promotion of virulence factor ActA. The comet forms in a polar manner and aids the bacteria's migration to the host cell's outer membrane.

Gelsolin, an actin filament severing protein, localizes at the tail of Listeria and accelerates the bacterium's motility. Once at the cell surface, the actin-propelled Listeria pushes against the cell's membrane to form protrusions called filopodsor "rockets". The protrusions are guided by the cell's leading edge to contact adjacent cells, which then engulf the Listeria rocket and the process is repeated, perpetuating the infection. Once phagocytosed, the Listeria is never again extracellular: it is an intracytoplasmic parasite like Shigella flexneri and Rickettsia.

1.2.3 Epidemiology The Center for Science in the Public Interest has published a list of foods that have sometimes caused outbreaks of Listeria: hot dogs, deli meats, raw milk, cheeses (particularly soft-ripened cheeses like feta, Brie, Camembert, blue-veined, or Mexican-style “queso blanco”), raw and cooked poultry, raw meats, ice cream, raw vegetables, raw and smoked fish. Cantaloupe has been implicated in an outbreak of listeriosis from a farm in Colorado,and the Australian company GMI Food Wholesalers were fined AU$236,000 for providing Listeria monocytogenes contaminated chicken wraps to the airline Virgin Blue.

1.2.4 Prevention Preventing Listeria as a food illness requires effective sanitation of food contact surfaces.

Alcohol is an effective topical sanitizer against Listeria. Quaternary ammonium can be used

in conjunction with alcohol as a food contact safe sanitizer with increased duration of the sanitizing action. Refrigerated foods in the home should be kept below 4⁰C (39.2⁰F) to discourage bacterial growth. Preventing listeriosis also can be done by carrying out an effective sanitation of food contact surfaces.

1.2.5 Treatment Antibiotics effective against listeria species include ampicillin, vancomycin (unclear effectiveness), ciprofloxacin, linezolid, azithromycin. Mixtures of bacteriophages have also proven effective in the treatment of Listeria.

1.2.6 Bacterial stress response and role of alternative sigma factors: Bacteria in natural environments are constantly challenged by the need to adapt to changes in nutrient availability and stress condition. A range of bacteria ,including E.coli(Jenkins et al.,1988;Matin 1990 ;Ferenci 2001),Salmonella spp (Foster & spector 1998) pseudomonas spp (Givskov et al .,1994 0and Vibrio spp (Kjelleberg et al.,1993; Ostling et al, Flardh et al., 1994) have now been shown to elicit sophisticated intracellular reorganization programmes in response to such changes. Typically ,these programmes are characterized by a series of physiological and genetic changes that facilitate the development, of multi –stress resistant cells capable of long-term survival as well as immediate recovery and outgrowth (Ostling et al.,1993 ; Mcdouglad et al;.,1999).

All organisms possess mechanisms of stress responses that allow them to survive under changing condition by altering the gene expression of genomics gene (Hayden &Ades,2008).In bacteria ,many stress responses are mediated by alternative sigma factors that can rapidly reprogram gene expression against various signals by recruiting RNA polymerase to specific subsets of prompters in the cell (Gruber & Gross ,2003).

1.2.7 Activation pathway of σB: To measure σB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σB-dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the Listeria monocytogenes wild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and σB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and σB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σ B dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. σB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σ B regulation (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

Figure 1.1: Model of σB activation pathway 1.3 Aims and objectives of the study: The present study was designed with the following aims and objectives: • To distinguish among the growth patterns of Listeria monocytogenes at lower (25°C ), optimum (37°C) and higher (45°C) temperatures .

• Establishing the growth characteristics through spot tests, patching at different temperatures together with conventional approaches i.e determination of culture turbidity ,enumeration of colony forming units (CFUs).

• To determined the critical temperature of Listeria monocytogenes on different media (listeria media , luria –berteni and minimal media).

2. Materials & methods 2.1 Working place All the experiments of this project work were carried out in the Laboratory of Bacteriology of the Department of Microbiology, Stamford University Bangladesh.

2.2 General procedures and equipments 2.2.1 Sterilization All equipment and glassware were sterilized by autoclaving at 15 p.s.i for 20 minutes or by heating in a hot air oven at 170ºC for 2 hr. All growth media add solution was sterilized by autoclaving at 15 p.s.i for 15 minutes in the autoclave. Fresh agar plates were prepared at room temperature, stored at 4ºC unit use, and dried at room temperature under aseptic condition before incubation of microorganism.

2.2.2 Preparation of solution Accurate weight of the components of various solutions were using a balance and for approximate weights a standard laboratory balance was used .A pH meter was used for determining pH values of different buffer solution, media, etc.

2.2.3 Maintenance of culture, reagents and solution

Preservation and maintenance of different materials including microbial stains, heat labile chemicals or reagents, were carried out in a refrigerator at 4ÂşC. 2.3 Growth media 2.3.1 Listeria identification Agar Base (PALCAM): This medium is especially used for the isolation of Listeria from foods and dairy materials. It may also be used for the isolation from clinical specimens such as stools in epidemiologic studies of carriage rates of Listeria or for other clinical specimens strongly contaminated with normal flora. On BD PALCAM Listeria Agar, colonies of Listeria appear gray-green with a black halo. On this medium, all Listeria species will grow and will have the same colony appearance. Therefore, all isolates must be identified with biochemical and/or serological procedures to the species level. Incubation of this medium in an aerobic atmosphere enriched with carbon dioxide or in a micro aerobic atmosphere may result in a color change of the phenol red pH indicator from red to yellow or orange, mimicking a false positive mannitol fermentation. Therefore, an aerobic atmosphere is preferred.

2.3.2 Luria-bertani Agar A subtype of nutrient agar, this is the general medium for microbiology studies and may be used for routine cultivation of not particularly fastidious microorganisms. Also, does not preferentially grow one kind of bacteria over another.

2.3.3 Minimal Media Minimal Media contains the essentials for bacterial species to grow. The media is often used to define if a particular microbial species is a heterotroph, namely an organism that does not have any nutritional requirements beyond core sources of carbon (sugars) and nitrogen (to synthesize amino acids and nucleic acid bases). Auxotrophs, or organisms with nutritional requirements will not be able to grow on minimal media.

2.4 Sample preparation 2.4.1 Sample collection The bacterial strain used in the experiment was a previously preserved stock culture of listeria. the medium used for the growth and subsequent subculture of the bacteria are listeria identification agar base medium.

2.4.2 Isolation of the bacteria Colonies present in the media were observed carefully. To acquire pure culture each type of colony, was picked up properly by using sterile needle and streaked onto same type of media.

As the results were same as that of the master plate, the colonies were again picked up aseptically by a sterile needle and streaked onto a new nutrient agar plate to get pure culture.

At last, stock culture was preserved in small vials containing a non-selective media having buffering capacity, with sterile paraffin oil and preserved at 24°C for future use.

2.4.3 Serial dilution of the samples The serial dilution was done in sterile test tube, each containing 9ml of sterile normal saline.

The conical flask containing the samples was shaken and allowed to stand for sometime. I ml from the original samples was taken carefully using an auto pipette with sterile tip and transferred to the test tube containing 9 ml of sterile saline, gave to 10-1 dilution. In this manner, ten fold dilution of samples were made up to 10-6 dilution in sterile normal, ten saline. These dilutions were made mixed using a vortex mixer prior to plating.

2.5 Spectrophotometry A Single colony was inoculated in 5 ml luria-bertani broth and minimal broth. luria broth, minimal broth were incubated for 24 hours, 48 hours and 72 hours consecutively. The culture was incubated at different temperatures rang(25°C,37°C, and 45°C). The cell growth was monitored by measuring OD at 600 nm. Results were recorded.

2.6 Spot test A method of micro chemical, qualitative, or semi quantitative analysis in which both the solution to be analyzed and the reagents are used in amounts of several drops.

After 6 hours growth in luria-bertani broth, minimal broth,theOD600nm of respective culture was adjusted to 0.1.The bacteria were serially diluted in. 85% normal saline up to 10 ¯3 dilutions.1 ml from the original bacterial suspension(0D600nm 0.1 ) was transferred to 9 ml normal saline for the first dilution and continued up to 10¯3 dilution. then from each of the dilution,10 µL of the bacterial suspension was spotted on luria-bertani agar , and minimal agar plates and was incubated for 24 hours. Results were recorded.

2.7 Patching A single colony was patched by toothpick on Luria-Bertani Agar, and Minimal Agar plates and then overnight incubation was done at the above mentioned temperature for 24 hours.

Finally, the plates were observed and results were recorded.

3. Results 3.1 Isolation of Listeria monocytogenes Listeria monocytogenes was isolated on Listeria identification agar base medium. After 24 hours of incubation at 37°C, the plates were observed carefully for the characteristic black gray color colonies surrounded by black halos.

Figure 3.1 Plate showing black gray colonies on Listeria identification agar base medium 3.2 Spectrophotometry: Optical Density (OD) at 600nm was determined after incubating the Listeria cells at various temperatures at different medium. Figure 2, 3 & 4 show graphical presentation of the OD observed after 24h, 48h & and 72h respectively. Here it can be seen that at 37⁰C a satisfactory growth was obtained after all the incubation period compared to other temperatures. Lowest growth was found at 45⁰C; however the growth tends to increase with the extension of incubation period.

Optical density

Temperature Figure

Optical density

3.2: Optical density (OD at 600mm) observed after 24 hours incubation of Listeria at various temperatures in different culture media.

emperatur

T e

Optical density

Figure 3.3: Optical density (OD at 600mm) observed after 48 hours incubation of Listeria at various temperatures in different culture media.

Te Temperature Figure 3.4: Optical density (OD at 600mm) observed after 72 hours incubation of Listeria monocytogenes at various temperatures in different culture media.

3.3 Spot test After spoting , incubation was done at various temperatures (25-45⁰C).The results are recorded in Table-3.1. After overnight incubation visible growth was observed all three temperatures. However, a small growth was seen at 45⁰C. In other two temperatures acceptable growth was observed. Growth rates tend to decrease with increasing temperature.

The growth response of Listeria monocytogenes was more positive towards 25°C rather than 45°C. Table 3.1. Spot test at 45⁰C, 37⁰C and 25⁰C. Temperature(⁰C)

Growth on different media Luria-bertani Agar

Minimal Agar

10-1

10-2

10-3

10-1

10‾2

10‾3

25

+

+++

++

+++

++

+

37

+

+++

++

+ ++

+

45

_

+

_

++ +++

_

+++ Indicates Rapid Growth ++ Indicates Moderate Growth + indicates slow Growth - Indicates No Growth

Luria-Bertani Agar A.

25 ⁰C

37 ⁰C

45⁰C

_

Minimal Agar 25 ⁰C

B.

37 ⁰C

45 ⁰C

Figure 3.5 : Spot test on Luria-Bertini agar & minimal agar respectively at different dilutions 3.4 Patching at different temperature After patching, incubation was done at various temperatures (25-45⁰C). The results are recorded in(Table:1).After overnight incubation visible growth was observed in luria-bertani agar and minimal agar at 45⁰C.However,a small growth was seen at the same in other three considerable growths was observed in all three medium.

Table 3.2 Observation of patching morphology at 45⁰C, 37⁰C & 25⁰C. Temperature(◦C)

Growth on different media Luria-bertani Agar

Minimal Agar

45

+++

+++

37

+++

+++

25

++

++

+++ Indicates Rapid Growth ++ Indicates Moderate Growth Luria-Bertani Agar A.

25 ⁰C

37 ⁰C

45⁰C

Minimal Agar B.

25 ⁰C

37 ⁰C

45⁰C

Figure 3.6: Patching morphology on Luria-Bertini agar & minimal agar respectively at different dilutions 4. Discussion The complete heat-shock regulation of Listeria monocytogenes that acts in response to a temperature increase has, apparently, not been investigated before, even though heating is an important preservation strategy for the food industry during minimal processing (Desneus et al.,2003;Dukan &Nystrom,1999) The present study was carried out to determine the response of Listeria monocytogenes to heat stress. Exposure to elevated temperatures triggers the classical heat-shock genes, and, in addition, a transient effect on expression of genes involved in the cell replication machinery was observed (Ahmed et al.,2008). Another novel finding is that heat shock triggers the SOS response in Listeria monocytogenes.

Long known as an animal pathogen, Listeria monocytogenes has recently been recognized as a important food borne agent in human disease (Mackey et al.,1991). The widespread distribution of Listeria. monocytogenes and other Listera spp. in nature and an association with domestic livestock makes the occasional presence of these bacteria on raw meats almost unavoidable. Contamination of ready-to-eat meat products with Listeria. monocytogenes poses a special threat to public health because of the organism's ability to grow at refrigeration temperatures and its pathogenicity within certain segments of the population (Mecsas et al.,1993;Missiakas et al.,1996b;De Las Penas et al.,1997).

The present study focused to distinguish among the growth patterns of Listeria monocytogenes low temperature 25⁰C,37⁰C, optimum 45⁰C and at high 45⁰C temperature and to determined to the growth characteristics through the spot test , patching at different temperature together with conversional approaches ,such as determination of culture turbidity.

In this regard, first of all , some experimentation were performed to observe some phenotypic indications of cells lysis with elevated temperature . To understand the size and shape of the cells grown at different temperature.

All these result clearly indicate that at higher temperature the cells undergo some sort of morphological alteration due to misfolded or lysed proteins and other cellular materials and it may be the reason of more aggregation at high temperature . It is already shown that high temperature cause abnormalities in cell shape, size, staining behavior, sporulation and germination (Nitta et al., 2000).

Further, at high temperature lysis and degeneration of cells is more pronounced. To authenticate these results, colony morphology test was also done to observed the same effect of increased temperature from cellular morphology to colony characteristics. It was found that, with the increase in temperature, the Listeria showed variation in their characteristics. At 45ºC, the colonies became irregular shaped , smaller flat and there was little growth which means defective colonies. At 30ºC the colonies were small and round at 37ºC ,the colonies were round , gummy , convex and there was considerable growth. It was previously shown that variation in membrane lipid of bacteria (Rouviere & Gross,1996; Walsh et al.,2003), as well as changes in membrane protein composition has been reported with increased temperature . Then the test organisms were subjected to spot test revealed that the growth of the test organisms became gradually lesser with the increase in temperature especially when the cells were transferred to 45ºC consecutively.

Since, elevated temperature has its effects on the cell morphology and colony characteristics, eventually Listeria cells were than examined for their colony forming ability along with their OD at 600nm at in contrast to different temperature. The OD observed after 24hr, 48hr, and 72 hr respectively. Here it can be seen that at 37⁰C satisfactory growth was obtained after all the incubation period compared to other temperatures .It clearly depicts that with the increase in temperature, the Listeria lose its culture ability . The cells were examined at three temperatures (25⁰C, 37⁰C, 45⁰C) and it was found that the OD at 600nm remained almost constant throughout the incubation period at all the temperature considered in this study.

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