IN VITRO EVLUATION OF THE CITREX ® MOLECULE AGAINST Listeria monocytogenes
<ul><li>DETERMINING THE MINIMUM INHIBITORY CONCENTRATION  (MIC)  </li></ul><ul><li>OF THE CITREX® DISINFECTANT </li></ul>
<ul><li>MICs for two serotype B Listeria monocytogenes strains, i.e.: ATCC 19115 and a wild strain were determined. These ...
CITREX® was tested at the following concentrations: <ul><li>400 ppm </li></ul><ul><li>200 ppm </li></ul><ul><li>100 ppm </...
<ul><li>For test purposes, an organism suspension equivalent to McFarland’s pattern of 10 5  colony-forming units [CFUs]/m...
<ul><li>The assay was carried out by adding to 1 ml of the relevant CITREX® solution per tube, 1 ml of the 10 5  CFU/ml st...
 
 
 
 
 
 
 
Table 1. Test 1: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CI...
Table 2. Test 2: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CI...
Table 3. Test 3: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CI...
Table 4. Result summary from all 3 assays Microorganism Minimum inhibitory concentration (ppm CITREX ®) L. monocytogenes  ...
RESULT DISCUSSION <ul><li>The performance of Citrex® was consistent with expected results, within the margins established ...
MIC RESULTS Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CITREX ...
Blood Agar
Blood Agar
Blood Agar
Blood Agar
6.- MINIMUM BACTERICIDAL CONCENTRATION 6.1.- RESULTS Microorganism Minimum bactericidal concentration (ppm) L. monocytogen...
DIRECT  IN VITRO  VISUALIZATION OF THE CITREX® MOLECULE ACTION ON   Listeria monocytogenes
<ul><li>Bacterial suspensions (10 5  CFUs/ml) were mixed with 400 ppm CITREX® then allowed to react for 15, 30, or 60 minu...
 
 
 
 
 
 
<ul><li>CITREX® PERFORMANCE IN THE DIRECT PROPHYLACTIC DISINFECTION OF FOODS . </li></ul><ul><li>EVALUATION OF CITREX® PER...
Surface sampling: <ul><li>For this research, chicken offal was pooled with ground beef, for a total of 100 g mix. The mix ...
 
 
 
Product samples (meats):   <ul><li>For the meat inoculation test, 10-g pieces of both chicken meat and beef were used.   <...
 
 
Inoculum preparation : <ul><li>For this assay, 100 CFU  L. monocytogenes/ ml were used in accordance with WHO/FAO, which i...
Sample Preparation <ul><li>Samples were inoculated with 100 CFUs  L. monocytogenes/ ml, using sterile Petri dishes, then k...
Analysis:   <ul><li>Samples were washed with 10 ml sterile saline. The resulting washing solution was diluted (1:100) thre...
 
 
 
TEST RESULTS PRIOR TO USING CITREX® ON MEAT   Results with samples inoculated with  L. monocytogenes , not disinfected wit...
<ul><li>L. monocytogenes CFUs/g PRODUCT: CONCENTRATION VS. EXPOSURE TIME   </li></ul>
SAMPLE TYPE: CHICKEN MEAT TIME CONCENTRATION COUNT, CFUs 0 hours 600 ppm Countless 0 hours 400 ppm Countless 6 hours 600 p...
SAMPLE TYPE: BEEF TIME CONCENTRATION COUNT, CFUs 0 hours 600 ppm Countless 0 hours 400 ppm Countless 6 hours 600 ppm 4.7 X...
 
 
 
 
 
 
 
 
<ul><li>EVALUATION OF CITREX® AS A DISINFECTANT ON HIGHLY L. monocytogenes-CONTAMINATED SURFACES   </li></ul>
SURFACE ANALYSIS AFTER A 6-HOUR ADAPTATION PERIOD   POST-INOCULATION SURFACE ANALYSIS AFTER CLEANSING TREATMENT RESULTS AF...
 
 
 
 
DISCUSSION   <ul><li>This research shows that Citrex® exerts a prophylactic disinfectant effect, since it substantially de...
<ul><li>We must state that, even though the values found in our research significantly exceed tolerance values  (WHO/FAO) ...
<ul><li>Our research showed that, under normal conditions, Citrex® is able to disinfect  L. monocytogenes- contaminated su...
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Citrex612 e-en-presentacion final listeriamonocytogenes

  1. 1. IN VITRO EVLUATION OF THE CITREX ® MOLECULE AGAINST Listeria monocytogenes
  2. 2. <ul><li>DETERMINING THE MINIMUM INHIBITORY CONCENTRATION (MIC) </li></ul><ul><li>OF THE CITREX® DISINFECTANT </li></ul>
  3. 3. <ul><li>MICs for two serotype B Listeria monocytogenes strains, i.e.: ATCC 19115 and a wild strain were determined. These organisms are important for both the food industry and human health. </li></ul>
  4. 4. CITREX® was tested at the following concentrations: <ul><li>400 ppm </li></ul><ul><li>200 ppm </li></ul><ul><li>100 ppm </li></ul><ul><li>50 ppm </li></ul><ul><li>25 ppm </li></ul><ul><li>12.5 ppm </li></ul><ul><li>6.25 ppm </li></ul><ul><li>3.125 ppm </li></ul><ul><li>1.562 ppm </li></ul><ul><li>0.781 ppm </li></ul>
  5. 5. <ul><li>For test purposes, an organism suspension equivalent to McFarland’s pattern of 10 5 colony-forming units [CFUs]/ml was prepared. Both L. monocytogenes strains (ATCC and wild strain), were originally activated at 8°C for 6 hours, then seeded in blood agar and Oxford agar (35 ± 2°C for 24 hours) for isolation. Inocula from these media were seeded in BHI broth and incubated for 24 hours at 35 ± 2°C, as to have the strains in their log-growth phase. From these cultures, the organism suspensions were prepared. </li></ul>
  6. 6. <ul><li>The assay was carried out by adding to 1 ml of the relevant CITREX® solution per tube, 1 ml of the 10 5 CFU/ml standardized organism suspension (in accordance with McFarland’s turbidity pattern). </li></ul><ul><li>Pertinent controls were prepared in each case for test validation, as follows: </li></ul><ul><li>- 1. Control test tube 1: Organism livability control. One (1) ml sterile BHI broth plus 1 ml organism suspension, for each case. </li></ul><ul><li>- 2. Control test tube 2: CITREX® solution sterility control. One (1) ml sterile BHI broth plus 1 ml CITREX® preparation, to be discarded from the last dilution tube. </li></ul><ul><li>- 3. Control test tube 3: BHI broth sterility control. Two (2) ml of the BHI broth used for each assay . </li></ul>
  7. 14. Table 1. Test 1: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CITREX 6.25 ppm CITREX 3.125 ppm CITREX 1.562 ppm CITREX 0.781 ppm L. monocytogenes ATCC 19115 - - - - - + + + + + L. monocytogenes Wild Strain - - - + + + + + + +
  8. 15. Table 2. Test 2: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CITREX 6.25 ppm CITREX 3.125 ppm CITREX 1.562 ppm CITREX 0.781 ppm L. monocytogenes ATCC 19115 - - - - - + + + + + L. monocytogenes Wild Strain - - - + + + + + + +
  9. 16. Table 3. Test 3: Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CITREX 6.25 ppm CITREX 3.125 ppm CITREX 1.562 ppm CITREX 0.781 ppm L. monocytogenes ATCC 19115 - - - - - + + + + + L. monocytogenes Wild Strain - - - + + + + + + +
  10. 17. Table 4. Result summary from all 3 assays Microorganism Minimum inhibitory concentration (ppm CITREX ®) L. monocytogenes ATCC 19115 25 L. monocytogenes Wild Strain 100
  11. 18. RESULT DISCUSSION <ul><li>The performance of Citrex® was consistent with expected results, within the margins established for disinfectants of this nature. ATCC strains are clinically used as controls in sensitivity tests performed by microbiological laboratories. They are also used in the evaluation of drugs for clinical antibiotic therapy. We evaluated the performance of Citrex® against both ATCC 19115 strain and a wild strain of L. monocytogenes . The latter was isolated from fresh cheese, and we used it as a representative organism causing problems in the food industry. </li></ul><ul><li>Probably the different MICs for both strains can be explained by the fact that wild strains are continually subjected to disinfectants and other chemicals used in the food industry that make organisms to express survival mechanisms, therefore they successfully develop adaptation processes. </li></ul>
  12. 19. MIC RESULTS Microorganism CITREX 400 ppm CITREX 200 ppm CITREX 100 ppm CITREX 50 ppm CITREX 25 ppm CITREX 12.5 ppm CITREX 6.25 ppm CITREX 3.125 ppm CITREX 1.562 ppm CITREX 0.781 ppm L. monocytogenes ATCC 19115 - - - - - + + + + + L. monocytogenes Wild Strain - - - + + + + + + +
  13. 20. Blood Agar
  14. 21. Blood Agar
  15. 22. Blood Agar
  16. 23. Blood Agar
  17. 24. 6.- MINIMUM BACTERICIDAL CONCENTRATION 6.1.- RESULTS Microorganism Minimum bactericidal concentration (ppm) L. monocytogenes ATCC 50 L. monocytogenes Wild Strain 200
  18. 25. DIRECT IN VITRO VISUALIZATION OF THE CITREX® MOLECULE ACTION ON Listeria monocytogenes
  19. 26. <ul><li>Bacterial suspensions (10 5 CFUs/ml) were mixed with 400 ppm CITREX® then allowed to react for 15, 30, or 60 minutes. Both direct smears and Gram stained smears were prepared. Microscope images were photographed. </li></ul>
  20. 33. <ul><li>CITREX® PERFORMANCE IN THE DIRECT PROPHYLACTIC DISINFECTION OF FOODS . </li></ul><ul><li>EVALUATION OF CITREX® PERFORMANCE IN THE DISINFECTION OF HIGHLY L. monocytogenes -CONTAMINATED SURFACES . </li></ul>
  21. 34. Surface sampling: <ul><li>For this research, chicken offal was pooled with ground beef, for a total of 100 g mix. The mix was inoculated with 10 ml of a 100 CFU/ml L. monocytogenes suspension, for a final content of 1,000 CFU in the total mix. </li></ul><ul><li>The mix was applied over a surface with delimited boundaries and allowed to act for 6 hours, for bacterial adaptation. The first sampling was then performed in order to test bacterium survival . </li></ul><ul><li>Once the 6-hour period elapsed, the surface was pre-cleaned then cleaned with regular liquid detergent. </li></ul><ul><li>The second sample was obtained in order to determine the presence of the organism, in accordance with W. Mannheimer and T. Ibáñez (1971), covering a 25 cm 2 treated surface area. </li></ul><ul><li>The clean surface area was then treated with 400 or 600 ppm Citrex®, respectively. </li></ul><ul><li>After 15 and 30 minutes of Citrex® treatment, samples were similarly obtained using the technique described by W. Mannheimer and T. Ibáñez (1971), covering a 25 cm 2 treated surface area. </li></ul>
  22. 38. Product samples (meats): <ul><li>For the meat inoculation test, 10-g pieces of both chicken meat and beef were used. </li></ul>
  23. 41. Inoculum preparation : <ul><li>For this assay, 100 CFU L. monocytogenes/ ml were used in accordance with WHO/FAO, which is the tolerance limit established by some countries that allow for the presence of L. monocytogenes in non-growth promoting foods. </li></ul><ul><li>For inoculum preparation purposes, 1 ml of a 10 3 CFU (as per McFarland’s technique) L. monocytogenes suspension in its exponential growth phase was mixed with 9 ml saline (0.85% sodium chloride water solution), for a final suspension containing 100 CFUs/ml . </li></ul><ul><li>Chicken meat and beef samples were inoculated with 1 ml of the L. monocytogenes inoculum. </li></ul>
  24. 42. Sample Preparation <ul><li>Samples were inoculated with 100 CFUs L. monocytogenes/ ml, using sterile Petri dishes, then kept refrigerated at 4 ± 1°C for 6 hours, for adaptation to occur, and for bacterial growth activation. </li></ul><ul><li>After 6 hours of refrigerated incubation, samples were treated with 400 or 600 ppm Citrex® disinfectant solutions, respectively. Samples were constantly subjected to their respective Citrex® concentrations. </li></ul><ul><li>A few similarly-processed samples were not treated, to be used as bacterial livability controls. </li></ul><ul><li>Likewise, chicken meat and beef samples were previously processed in agreement with the traditional Listeria research technique (Draft International Standard, ISO/DIS 11290-2 [1995]), as to assure that samples were not previously contaminated with the organism . </li></ul>
  25. 43. Analysis: <ul><li>Samples were washed with 10 ml sterile saline. The resulting washing solution was diluted (1:100) three times in order to perform viable bacterial cell counts (CFUs). </li></ul><ul><li>All dilutions (10 1 , 10 2 , and 10 3 ) were seeded on agar medium in agreement with Oxford’s technique, with a chromogen. Two additional Oxford agar (a selective, differentiating medium) plates were inoculated by surface seeding then incubated at 35 ± 1°C for up to 48 hours. Finally, bacterial concentration was determined in terms of CFUs/g food.. </li></ul><ul><li>Follow up was performed as follows: </li></ul><ul><li>Zero (0) minutes after prophylactic disinfection with 400 and 600 ppm Citrex®, respectively. </li></ul><ul><li>Six (6) hours after prophylactic disinfection with 400 and 600 ppm Citrex®, respectively. </li></ul><ul><li>Twenty four (24) hours after prophylactic disinfection with 400 and 600 ppm Citrex®, respectively. </li></ul><ul><li>Forty eight (48) hours after prophylactic disinfection with 400 and 600 ppm Citrex®, respectively. </li></ul>
  26. 47. TEST RESULTS PRIOR TO USING CITREX® ON MEAT Results with samples inoculated with L. monocytogenes , not disinfected with Citrex® SAMPLE RESULT Chicken meat Negative Beef Negative SAMPLE COUNT Chicken meat COUNTLESS Beef COUNTLESS
  27. 48. <ul><li>L. monocytogenes CFUs/g PRODUCT: CONCENTRATION VS. EXPOSURE TIME </li></ul>
  28. 49. SAMPLE TYPE: CHICKEN MEAT TIME CONCENTRATION COUNT, CFUs 0 hours 600 ppm Countless 0 hours 400 ppm Countless 6 hours 600 ppm 10 X 10 2 6 hours 400 ppm 1.5 X 10 2 24 hours 600 ppm 8.4X10 1 24 hours 400 ppm 1.5 X 10 2 48 hours 600 ppm 23 x 10 2 48 hours 400 ppm 50 x 10 2
  29. 50. SAMPLE TYPE: BEEF TIME CONCENTRATION COUNT, CFUs 0 hours 600 ppm Countless 0 hours 400 ppm Countless 6 hours 600 ppm 4.7 X 101 6 hours 400 ppm 7 X10 1 24 hours 600 ppm 4 x 10 2 24 hours 400 ppm 51 x 10 2 48 hours 600 ppm 3 x 10 2 48 hours 400 ppm 30 x 10 2
  30. 59. <ul><li>EVALUATION OF CITREX® AS A DISINFECTANT ON HIGHLY L. monocytogenes-CONTAMINATED SURFACES </li></ul>
  31. 60. SURFACE ANALYSIS AFTER A 6-HOUR ADAPTATION PERIOD POST-INOCULATION SURFACE ANALYSIS AFTER CLEANSING TREATMENT RESULTS AFTER 15 AND 30 MINUTES OF EXPOSURE TO CITREX ® AS A SURFACE DISINFECTANT AREA 1 COUNTLESS AREA 2 COUNTLESS AREA 1 48 CFUs/25 cm 2 AREA 2 36 CFUs/25cm 2 AREA 1: 400 ppm Citrex® 0 CFUs/25 cm 2 (no isolation) AREA 2: 600 ppm Citrex® 0 CFUs/25cm 2 (no isolation)
  32. 65. DISCUSSION <ul><li>This research shows that Citrex® exerts a prophylactic disinfectant effect, since it substantially decreased bacterial levels from countless to extremely lower counts. Some countries exist with stringent (zero tolerance) L. monocytogenes legislations, while others tolerate up to 100 CFUs/25 g in foods that do not promote L. monocytogenes growth . </li></ul><ul><li>Results clearly show an important property of L. monocytogenes , i.e.: its high adaptation ability once surfaces or foods are contaminated/colonized. Its fast growth rates together with its property of activating its growth under refrigeration conditions, make L. monocytogenes a potential food-born pathogen . </li></ul>
  33. 66. <ul><li>We must state that, even though the values found in our research significantly exceed tolerance values (WHO/FAO) after using Citrex®, its disinfecting action was able to decrease counts substantially, from countless to quantifiable values . </li></ul><ul><li>It is important to notice that under normal conditions the actual contamination found in food companies is very low, frequently 1-10 bacteria in rather large areas . </li></ul><ul><li>Our research showed that, under normal conditions, Citrex® is able to disinfect L. monocytogenes- contaminated surfaces, within a standardized sanitation program . </li></ul><ul><li>Results show that Citrex® was able to remove 100% L. monocytogenes from the surfaces studied between 15 and 30 minutes, at the concentrations of 400 and 600 ppm. </li></ul>
  34. 67. <ul><li>Our research showed that, under normal conditions, Citrex® is able to disinfect L. monocytogenes- contaminated surfaces, within a standardized sanitation program. </li></ul><ul><li>Results show that Citrex® was able to remove 100% L. monocytogenes from the surfaces studied between 15 and 30 minutes, at 400 and 600 ppm concentrations. </li></ul>

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