Citrex612 e-en-presentacion final listeriamonocytogenes

IN VITRO EVLUATION OF THE CITREX®

MOLECULE AGAINST Listeria monocytogenes

DETERMINING THE MINIMUM INHIBITORY CONCENTRATION (MIC)

OF THE CITREX® DISINFECTANT

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.

CITREX® was tested at the following concentrations:

400 ppm 200 ppm 100 ppm 50 ppm 25 ppm 12.5 ppm 6.25 ppm 3.125 ppm 1.562 ppm 0.781 ppm

For test purposes, an organism suspension equivalent to McFarland’s pattern of 105 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.

The assay was carried out by adding to 1 ml of the relevant CITREX® solution per tube, 1 ml of the 105 CFU/ml standardized organism suspension (in accordance with McFarland’s turbidity pattern).

Pertinent controls were prepared in each case for test validation, as follows:- 1. Control test tube 1: Organism livability control. One (1) ml sterile BHI broth plus 1 ml organism suspension, for each case.- 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.- 3. Control test tube 3: BHI broth sterility control. Two (2) ml of the BHI broth used for each assay..

Table 1. Test 1:

MicroorganismCITRE

X400 ppm

CITREX

200 ppm

CITREX

100 ppm

CITREX

50 ppm

CITREX

25 ppm

CITREX12.5 ppm

CITREX

6.25 ppm

CITREX3.125 ppm

CITREX

1.562 ppm

CITREX

0.781 ppm

L. monocytogenes ATCC 19115 - - - - - + + + + +

L. monocytogenes Wild Strain - - - + + + + + + +

Microorganism CITREX400 ppm

CITREX200 ppm

CITREX100 ppm

CITREX

50 ppm

CITREX

25 ppm

CITREX12.5 ppm

CITREX6.25 ppm

CITREX3.125 ppm

CITREX1.562 ppm

CITREX0.781 ppm

L. monocytogenes ATCC 19115 - - - - - + + + + +

L. monocytogenes Wild Strain - - - + + + + + + +

Table 2. Test 2:

Table 3. Test 3:

MicroorganismCITREX400 ppm

CITREX200 ppm

CITREX100 ppm

CITREX

50 ppm

CITREX

25 ppm

CITREX12.5 ppm

CITREX6.25 ppm

CITREX3.125 ppm

CITREX1.562 ppm

CITREX0.781 ppm

L. monocytogenes ATCC 19115 - - - - - + + + + +

L. monocytogenes Wild Strain - - - + + + + + + +

Table 4. Result summary from all 3 assays

MicroorganismMinimum inhibitory concentration

(ppm CITREX ®)

L. monocytogenes ATCC 19115

25

L. monocytogenes Wild Strain

100

RESULT DISCUSSION

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.

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.

MicroorganismCITREX400 ppm

CITREX200 ppm

CITREX100 ppm

CITREX50 ppm

CITREX25 ppm

CITREX12.5 ppm

CITREX6.25 ppm

CITREX3.125 ppm

CITREX1.562 ppm

CITREX0.781 ppm

L. monocytogenes ATCC 19115 - - - - - + + + + +

L. monocytogenes Wild Strain - - - + + + + + + +

MIC RESULTS

Blood Agar

Blood Agar

Blood Agar

Blood Agar

6.- MINIMUM BACTERICIDAL CONCENTRATION

6.1.- RESULTS

Microorganism Minimum bactericidal concentration

(ppm)

L. monocytogenes ATCC 50

L. monocytogenes Wild Strain

200

DIRECT IN VITRO VISUALIZATION OF THE

CITREX® MOLECULE ACTION ON

Listeria monocytogenes

Bacterial suspensions (105 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.

CITREX® PERFORMANCE IN THE DIRECT PROPHYLACTIC DISINFECTION OF FOODS.

EVALUATION OF CITREX® PERFORMANCE IN THE DISINFECTION OF HIGHLY L. monocytogenes-CONTAMINATED SURFACES.

Surface sampling: 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.

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 .

Once the 6-hour period elapsed, the surface was pre-cleaned then cleaned with regular liquid detergent.

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 cm2 treated surface area.

The clean surface area was then treated with 400 or 600 ppm Citrex®, respectively.

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 cm2 treated surface area.

EVALUATION OF CITREX® AS A DISINFECTANT ON HIGHLY L. monocytogenes-CONTAMINATED SURFACES

SURFACE ANALYSIS AFTER A 6-HOUR ADAPTATION PERIOD

AREA 1 COUNTLESS

AREA 2 COUNTLESS

POST-INOCULATION SURFACE ANALYSIS AFTER CLEANSING TREATMENT

AREA 1 48 CFUs/25 cm2

AREA 2 36 CFUs/25cm2

RESULTS AFTER 15 AND 30 MINUTES OF EXPOSURE TO CITREX® AS A SURFACE DISINFECTANT

AREA 1: 400 ppm Citrex® 0 CFUs/25 cm2 (no isolation)

AREA 2: 600 ppm Citrex® 0 CFUs/25cm2 (no isolation)

Our research showed that, under normal conditions, Citrex® is able to disinfect L. monocytogenes-contaminated surfaces, within a standardized sanitation program.

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.