Biofilms

Favorable conditions with respect to the development of biofilm by Listeria onocytogenes were studied on model system that was represented by microtiter plates as well as certain useful disinfectant agents were found. Listeria monocytogenes Lm-24, an industrial isolate as well as Listeria monocytogenes ATCC 13932 were compared in terms of their biofilms’ forming abilities.
0.5 McFarland was the reproducible result that was lead by the initial concentration of the cells. The testes temperatures were between 8°C to 37°C and the best values to create biofilms in buffered peptone water (BPW) with glucose of 0.05% was between 25°C and 30°C. The constant strain L. monocytogenes Lm-24 was compared with the reference strain and it was concluded that L. monocytogenes Lm-24 had more massive biofilms. A numbe of disinfectants such as Merades Alco, Savo, benzalalkonium chloride were also applied. L. monocytogenes Lm-24 were used as organisms’ model in a persistent industry and were used for such tests. The most effective way to destroy the biofilm was recognized as treatment of benzalalkonium chloride. 500 mg/l treatment and 125 mg/l treatment for planctonic cells was dangerous for the biofilm cells. The viability was suppressed by Savo by 20% on average of biofilms whereas it was dangerous for planctonic cells. Weak impact was showcased by Merades Alco.
In the external environment, Listeria monocytogenes is present everywhere as it is a Gram-positive foodborn pathogenic bacterium. Listeriosis is caused by Listeria monocytogenes and has an global mortality rate of about 20-30% and it can exist for several years in environments of foodprocessing which is a significant food contamination cause.
Biofilms are formed by attaching L. monocytogenes which are defensive polysaccharides and protein layers which cover the bacteria and cling onto various equipments and surfaces. A number of bacteria can to stick to the colonized environmental surfaces by developing a matrix of three dimensions of extracellular polymeric substances (EPS) known as biofilms. A group of microorganisms covered by the slime they secrete are known as biofilms which are connected to the living or inert surfaces. In contrast, planctonic cells are free individual liquid living things.
The arrangement of biofilm permits the microorganisms to exist in the environment as well as resist resiccation, antimicrobial treatment, UV light and sanitizing agent. It is hard to control biofilms because they develop wherever there is plenty of water and where there is no proper cleaning. Filling/packaging equipment, walls, conveyors, floor drains, racks for transporting products, freezers, cooling pipes, and hand tools or gloves are the usual isolation sites of L. monocytogenes from processing plants of dairy industry.
A number of indirect and direct procedures of experiment are established to enable studies of reproducible bacteria attachment and colonization. The development of biofilm is a complicated procedure which is controlled by different characteristics of substrate, cell surface and growth medium.
Indirect methods for the purpose of amount of bacteria estimation in situ fits in right by the procedure of microtitier plate and can be change for a number of biofilm formation assays. A process of using the microtitier plates was used in this study. The main purpose of the experiment was to obtain an appropriate medium for forming biofilm by means of a reference strain as compared to the industrial isolate and also to examine the efficiency of the disinfectants generally used against the biofilm.
Material Required and Methods:
Culture preparation. Throughout the process L. monocytogenes which was industrial isolated and labeled as Lm.24 and Listeria monocytogenes ATTC 13932 were used. At -80°C in a brain-heat infusion (BHI) in presence of 15% glycerol, stock culture were kept.
At 4°C for about 30 days, cultures which were working kept on plate count agar (PCA) slants. For about 18 hours at 37°C in 10ml of BHI, cultures were grown well before the experiment.
Media: Following media were used for bio film production assays:
Buffered peptone water (BPW) – Different combination of NaCl and glucose were used. 0.5% NaCl in BPW, BPW with 5.0% NaCl, 0.05% glucose in BPW, 0.5% glucose in BPW, 0.5% glucose and 5.0% NaCl in BPW, 0.05% glucose and 0.5% NaCl in BPW.
Brain heart infusion - BHI and physiological saline – PS, 10 time diluted BHI
Microtiter plate biofilm production assay:
Manufactured in USA , Microtiter plates COSTAR 3796 were selected as standard tool for performing of all the experiments. In BHI, overnight cultures were diluted to the media which is mentioned above and was equaled to 0.5 McGarland values which is approx. 107 CFU/ml. As compared to previous tests this gave improved results.

With the help of 70% ethanol micortiter plates were cleaned and then air dried. For each well of different microtiter plated extectly 100 μl of individual culture dilution which taken form the tested media were transferred. 16 parallels that is 8 wells and 2 rows were diluted for single type of dilution and medium. Both after as well as before 22hr of incubation, with the help of Tecan-Spectra 9440012 spectrophotometer at 20 nm, microtiter plates were calculated. After incubation of 22hr, medium is removed from wells and then wells are washed with sterile distilled water for 5 times to remover any bacteria. Then for 45min plates are air dried and then each well is stained with the help of 150 μl of 1% crystal violet solution mixed in water for another 45mins.
The plates were scrubbed five times by using infertile condense water, this is done after maculation. For quantitative analysis 200 μl of 95% of ethanol is added to distain well so that the analysis can be performed easily and when 45 minutes have passed 100 μl was conveyed to micro titer plate and the level of crystal violet that was already present in the solution was at about 620 nm at Tecan-Spectra 9440012. The test was performed in 8, 25, 30 or 37 °C temperature.
Disinfectant attempts
From many disinfectants three were tested which includes Savo S, a mixture of NaClO and NaOH, Prague, Penta and Czech Republic, used in household and food industry, other is Merades Alco (MA) used in dairy industry, composed of Merak, Czech Republic, Prague Ethanol and Proponal and third one is Benzalalkonium Cloride (BC) comprises of QUAT group, St. Loius, Ammonium Salt, Fluka Analytical and USA, used in food industry. The time incurred in treatments and strength of solutions is mention in table 1. Micro titer plates were checked by using 100 μl of the agent after 22 h and this is done to pretend the real circumstances. While using A-assessment for checking the disinfectant effect on biofilm, the plates were washed and deal in same manner as discussed above after the mention time has passed.
After this process the measurement of desolation of biofilm that occurred due to this agent becomes easy. For testing the B-viability of biofilm cells sterile water was used to wash the micro titer plates after the occurrence of disinfectants procedure and after this 100 μl of BPW which had 0.05% of glucose in it was place into the wells.
The making of biofilm and density produced were compared with those that obtained by disinfectant handling while the micro titer plates needed 30 °C for about 22 h to be hatched.


Many innovative mechanisms have been developed by intracellular pathogens to replicate and compartmentalize in the cells of host. On the other hand, innate immunity of mammals has developed such innate immune system which can identify the infections caused by microorganisms via different cytosolic, vacuolar and surface receptors which can identify protected microbial molecules. Taking this in account, it wasn’t unexpected that to continue with the pathogenesis, pathogens evolved counteracting mechanisms against innate immunity of the host. Although the whole idea of innate immunity of mammals has attracted massive interest of those who are concerned with the study of adaptive immunity, inflammation and infectious diseases, but our main knowledge is still based on interaction between purified components taken out from microorganisms and host cells. Interaction between host and pathogens which is the hot topic of now days is still undefined and striving to answer the question of how the host cells differentiate and react to live and replicate to protect against non virulent microbes and pathogenic microorganisms.
There are two main categories of intracellular pathogens: those who exist in vacuole-like compartment and those who live directly in cytosol of host cell. The reference organism being used for studies various characteristics of acquired and innate immunity is, Listeria monocytogenes which is an example of latter category. Once the organism i.e. Listeria monocytogenes has entered in the cytosol of host cell, strains known as wild-type (w.t.) activate a IRF-3/TBK1-dependent and MyD88/TRIF-independent response of host transcription leading to transcription of many genes which include production of cytokine IFN and robust expression.
Dangerous strains of various other pathogens like, Mycobacterium tuberculosis, Francisella tularensis, Legionella pneumophila and Brucellae also initiate this pathway. On the other hand avirulent pathogens do not do this. At present, although flavones associate compounds and nucleic acids can induce a very same response but host receptors and character of bacterial ligand(s) involved in appreciation of bacteria in cytosol of host is unclear. To completely understand the relationship between immunity of host and infection, an unbiased screening of genetic makeup to evaluate surveillance pathways of organism was done and it showed that strains of replicating cytosolic L. monocytogenes that stimulate reduced or elevated response of host, expressed multidrug resistance transporters (MDRs) of super family of MRDs of bacteria and the fact that they control the cytosolic surveillance passageway both in vivo and in vitro.

It has been stated that, Listeria monocytogenes, which is a Gram-positive bacillus, is pathogenic for animals and human beings. It has been found out and confirmed recently in various food-borne listeriosis which are epidemic. L. monocytogenes is extensively distributed in various components of environment, sewage, soil, uncooked and decaying vegetables. The tendency of the bacterium to enter the units of food handling and manufacture is very vast. Main diseases caused by L. monocytogenes include encephalitis, meningitides, septicemia and abortion. Many researches carried out in the past evidently point out towards the fact that the likelihood of L. monocytogenes to cause disease is dangerously vast. Most of the strains of L. monocytogenes express clinical manifestation and most of the strains cause disease only when they enter a human or animal host. Various sporadic cases of L. monocytogenes infecting the vegetables of daily use and farms have been identified in Ado-Ekiti; and this type of contaminated food which has to be distributed is the main source of transmission. Rate of the outbreaks of these diseases happening again and again is still not known because reoccurrence of these infections caused by L. monocytogenes because mostly the infections like these are not conveyed to officials of public health.
The L. monocytogenes have proved to be in bold opposition to the environmental issues including the low pH value, the pasteurization or heating process, the osmotic pressure and the refrigeration degree (4oC). Researchers notice that he susceptibility of the antibiotics have been generally stable and systematic over the years, even though certain antibiotics were found to be becteriostatic. In recent years, some researchers have laid a lot of emphasis and researched on the level of resistance provided by the antibiotics of L. monocytogenes. The waste of animals and their feces are used as a source of organism on the agricultural land so as to apply the manure to make the land more fertile for plantation. These are then tranferred to the healthy animals as well as humans with the vegetables that are extracted from this piece of land.

RESULTS AND DISCUSSION
The cultural and morphological features aided in the classification and identification of the Isolates. The nature and characteristics of these isolates were observed and biochemical tests were carried out to find more about them. It was derived from the studies that there were traces of Pathogen present in the samples. The results also showed the presence of cow dung, agricultural soil and vegetables. The vegetables obtained from the Enu-Odi Market have the highest level of pathogen and Amarathus cruetus. These vegetables showed the highest proportion of L. monocytogenes. It was also shown that the vegetables had differing levels of resistance to the antibiotics. Results showed that chloramphenicol has the least effect on the vegetables and only 52.29% of the isolates showed resistance to it. Augmetin also proved to e quite ineffective. However, cloxacillin proved to have the highest resistance level. Some researchers found that the L. monocytogenes isolates were quite responsive to chloramphenicol and gentamicin. Certain extrinsic factors played a role in influencing the extent of resistance. These factors included the level of exposure, the antibiotic type used to in the local area and the occurrence of plasmids in the isolates. These have been observed in various L.monocytogenes and these plasmids may prove to be either pheromones-responsible or broad-host-range. The differing antibiotic pressures also tend to influence the results to a great extent.
The level of threat to the resistence power from the cow wastes varies between 41.18-70.59%. This variation is a result of the contact supervision of the antiobiotics that are sublethal and are used to cause an increase in the growth rate and cause a fall in the level of feed used to bring the animal to the optimum slaughter size.
It has been observed that vegetables are one of the highest transmission mediums for L.monocytogenes. The sample taken showed traces of the organism. This evidence strengthens the previous findings that the vegetables harbor the antibiotic resistant organisms.
A study was conducted to examine the level of resistance put up by eight different types of antibiotics. The results differed in many ways to the earlier findings and highlighted the reasons for the occurrence of these changes. There are varying reasons for the changes such as genetic modification and the selective patterns of antibiotics. The multiple resistant bacterial strains were capable of being transferred to humans as well by coming in contact with it or directly consuming them. These occurrence were able to further complicate the management by the clinical staff due to the Pathogen and transfer of the resistance levels.