Objective: This validation study investigates the treatment of cornea organ culture medium (Modified Eagle Medium, Biochrom GmbH, Berlin, Germany) with RESEP, a new medical device for antibiotics removal, before microbiological testing with BACTEC^TM blood culture bottles.

Methods and analysis: 10–100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtillis, Aspergillus brasiliensis, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9mL of cornea organ culture medium.
In group A, the medium was withdrawn with RESEP and treated for 20 min at room temperature, and then inoculated in BACTEC Plus Aerobic/F/Anaerobic/F blood culture bottles.
In group B, the medium, spiked by the inoculation of microorganism, was injected directly. For each strain, a growth control was performed, by direct inoculation of the microorganisms in BACTEC^TM vials (positive control). All samples were incubated in the automated BACTEC^TM blood culture system at 36°C ±1°C for maximum of 14 days or until a positive reading. The elimination of antibiotics from the medium by RESEP was determined by high-performance liquid chromatography.

Results: After 20 min of RESEP treatment, 100% (n=9) of streptomycin, 100% (n=9) of amphotericin B and 99.7% (n=9) of penicillin G were eliminated.
In group A , all microorganisms were detected within 3 days of incubation with a sensitivity of 100% (n=99) and showed no significant delay compared with the positive controls.
In group B, the overall sensitivity was 67.9% (n=96) with a significant delay until detection of microbial growth for all tested microorganisms except for A. brasiliensis.

Conclusion: The use of RESEP to eliminate the antibiotics from cornea organ culture medium increases the sensitivity of the microbiological testing with BACTEC^TM Plus blood culture bottles significantly and fulfils the requirements of the European Pharmacopoeia method suitability test.

Purpose: This study aimed at validating the method for sterility testing of the corneal culture medium, TISSUE-C (AL.CHI.MI.A. S.r.l.), and the transport/deswelling medium, CARRY-C (AL.CHI.MI.A. S.r.l.), according to the method suitability test, as defined by the European Pharmacopoeia (EP), using RESEP (AL.CHI.MI.A. S.r.l), which is a new medical device for removal of antimicrobial agents, and HB&L (Alifax) automated culture system.

Materials and Methods: The six EP reference strains were inoculated in TISSUE-C and CARRY-C. Half of the samples were treated with RESEP (RESEP+ group) prior to the sterility testing, whereas the remaining samples were untreated (RESEP− group). Growth controls were obtained by direct inoculation of the microorganisms in the culture broths. Microbial growth was read by HB&L automated light scattering culture system within 48 h.

Results: The use of RESEP allowed detection of microbial growth in 100% of the tested samples, with a mean time to detection (TTD) comparable with that of the growth control group. Significantly lower sensitivity (38.83% ± 20.03% for both media, p < 0.05) and TTD variability, depending on the tested microorganism, were observed in the RESEP− group. The method specificity was 100% for both groups.

Conclusion: The use of RESEP increased the sensitivity of the sterility testing method to 100% and, for the first time, allowed validation of the method for sterility testing of corneal storage media according to the EP method suitability test. This further increases the safety of the corneas intended for transplantation.

Purpose: The aim of this study was to validate the method for sterility testing of corneal storage media Tissue-C and Carry-C according to the “Method suitability test” (EP) using BACTEC (Becton Dickinson) automated system in a multicentric study.

Material & Methods: The validation study was performed at the Eye Bank of Rome and Eye Bank of Monza, Italy. Samples of organ culture medium (Tissue-C, AL.CHI.MI.A. S.r.l.), deswelling/transport medium (Carry-C, AL.CHI.MI.A. S.r.l.), and optimal growth media (growth control) were inoculated with 6 EP reference strains to obtain final microbial concentration of 10 cfu/ml, and tested at least in triplicate with BACTEC automatized system.
Method sensitivity, specificity and robustness were determined for each medium, with and without antibiotic removal from samples with RESEP (AL.CHI.MI.A. S.r.l.).

Results: Both eye banks obtained the same method sensitivity and specificity results. The method for sterility testing of Tissue-C and Carry-C samples after RESEP-treatment using BACTEC system showed 100% sensitivity and specificity. Samples treated with RESEP showed similar times to detection as compared to growth controls.

Conclusions: BACTEC system can be considered validated with 100% sensitivity and specificity, and robustness for samples of corneal storage media contaminated with 1-10 cfu/ml, and treated with RESEP.

Purpose: To evaluate the use of RESEP device (ALCHIMIA S.r.l.), intended for antibiotic removal from liquid samples, on tissue homogenates.

Material and method
RESEP treatment of tissues:
– Homogenization: 1 gr in 10 ml of BASE solution with IKA ULTRA TURRAX homogenizer
– 2 passages in RESEP, 20 min/each at RT RESEP was evaluated for the following parameters:
1. Bacterial retention: sterile human fresh aorta fragments were homogenized, contaminated with P.aeruginosa (3 doses) and treated with RESEP.
2. Interference with bacterial growth: sterile human fresh aorta and pericardium fragments were contaminated with P.aeruginosa and S.aureus (3 doses).
3. Antibiotic removal ability: decontamination (BASE 128_125ml/g: 14 h, 37°C) washing (BASE 6,5 ml/g. 2×5 mln, 1x6h, 4°C) and homogenization procedures were applied on sterile porcine cardiovascular tissues. Microbial strains were spiked on RESEP treated/untreated homogenates.
All the homogenates were tested on agar plates for microbal quantification and in BactAlert.

Results: Two sequential passages of the homogenates on RESEP only slightly reduced P.aeruginosa load tested on agar plates. Samples were positive in BacAlert.
P.aeruginosa and S.aures in RESEP treated and untreated homogenates showed similar growth capability on agar plates and were positive in BacAlert.
RESEP-treated homogenates showed a slightly better bacterial recovery than RESEP-untreated samples both in agar plates and in BactAlert.

Conclusions: RESEP device is compatible with homogenized tissues ensuring a good bacterial recovery and absence of interference with bacterial growth. Preliminary data suggest also the capability of RESEP to remove antibiotic in tissue homogenates.

Purpose: Establish a method to decontaminate, wash and freeze cardiovascular tissues.

Methods: Samples sterile porcine cardiovascular tissues, decontaminationantibioticsolutionBASE128_125ml/g 14h 37°C, washing: BASE 6.5 ml/g. 2×5 min. 1x6h. 4°C, RESEP treatment of hormogenates (antibiotic removal device): 2×20 min. RT.

Evaluation of:
1. Decontamination efficiency: samples were immersed in contaminated solutions (103-108 cfu/ml: 10 bacteria and 2 fungi strains), decontaminated, washed and homogenised. Homogenates (RESEP treated/untreated) were tested for contamination on agar plates and BactAlert.
2. Capability of washing procedure to remove antibiobiotic: decontamination and washing procedure was applied. 12 microbial strains were spiked on RESEP treated/untreated homogenates, tested on agar plates and BactAlert.
3. Freezing method: several profiles were tested on aortic, pulmonary and pericardium specimens.

Results:
1. Decontamination resulted in 6 Log 10 reduction for 5 strains (P.aeruginosa, B.atrophaeus, S.marcescens, P.acnes, E.coli), 5 Log 10 for 4 strains (S.epidermidis, K.pneumoniae, C.sporogenes, B.fragilis), 4 Log 10 for S.pyogenes, 1.0 Log 10 for C.albicans and A.brasiliensis.
2. Microbial growth was demonstrated on:
– RESEP-treated tissues: 12/12 strains (agar plates and BactAlert)
– RESEP-untreated tissues: 11/12 strains on agar plates (at less extent for 4 strains), 10/12 strains on BactAlert.
3. A freezing profile was defined leading to optimal freezing curve (-1°C/min to -40°C, -3°C/min to -120°C).

Conclusions: Decontamination was effective (4-6 Log 10) for 10/12 strains. The extensive washing procedure, able to remove antibiotic from the solutions and tissues. In combination with the RESEP ensures a reliable sterility assay. An optimal freezing profile was set-up.

Purpose: The aim of this study was to validate the sterility testing of tissue samples according to the “Method suitability test” defined by the European Pharmacopoeia (EP, chapter 2.6.1), using the MEB buffer (AL.CHI.MI.A. S.r.l.) for extraction of microorganisms from tissue samples and RESEP (AL.CHI.MI.A. S.r.l.) for elimination of antimicrobials before direct inoculation of growth media.

Materials and methods: Samples consisting of one gram of sterile porcine aortic valve were immersed in BASE.128 (AL.CHI.MI.A. S.r.l.) at 4°C for 24 h to simulate the tissue decontamination process with an antibiotic cocktail. The samples were then contaminated with 10-100 cfu of EP reference strains (Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes), and introduced in a vial containing MEB extraction buffer and stirred at room temperature for 20 min in order to extract microorganisms from the tissues. The buffer was then treated with RESEP syringe for removal of antimicrobial residues and inoculated in Tryptic Soy Broth (TSB) or Thioglycolate (TG). The turbidity of the growth media was determined visually after 5 days of incubation at 22°C (TSB) or 33°C (TG).

Results: MEB buffer extracted the whole 10-100 cfu of all tested microorganisms from aortic valve samples. All microorganisms showed growth in TG or TSB media after RESEP treatment indicating vitality and absence of BASE.128 antimicrobial residues. Turbidity of growth media was detected within 5 days after inoculation in all tested conditions.

Conclusions: The sterility test of the tissue samples, including the extraction of microbial contaminants from tissues using MEB buffer and removal of antimicrobials using RESEP, before direct inoculation, was successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).

Background: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to the European Pharmacopoeia (chapter 2.6.27.) using RESEP (ALCHIMIA) a new medical device for the removal of antibiotics and blood culture bottles with the automatic BACTEC system (Becton Dickinson, Franklin Lakes, New Jersey, USA).

Methods: For the validation 10-100 colony forming units of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis (ATCC 12228) were inoculated in 9 ml of cornea organ culture medium (MEM with 2% fetal australian calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml); Merck, Biochrom GmbH, Germany). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC™ Plus Aerobic/F and BACTEC™ Plus Anaerobic/F blood culture bottles and incubated until a positive reading was shown or for at least 14 days at 36 ±1°C. Medium control samples, without RESEP treatment, containing the same microorganisms, were inoculated in BACTEC vials directly. Microbial growth was controlled by direct inoculation of BACTEC flasks (growth control). The decrease of antibiotics from the medium by RESEP was determined by high performance liquid chromatography (HPLC).

Results: After 20 minutes of treatment at room temperature with RESEP (confirmed by HPLC) the antibiotics were completely eliminated . In samples treated with RESEP, the growth of all microorganisms could be detected within 3 days of incubation in the BACTEC system and showed no significant delay compared to the growth controls. In contrast, untreated medium samples showed no reproducibility of Staphylococcus aureus, Enterobacter cloacae, Candida albicans and Bacillus subtilis.

Conclusions: The microbiological testing of cornea organ culture medium with the automatic BACTEC blood culture system using RESEP for the removal of antibiotics has been successfully validated according to the European Pharmacopoeia (Chapter 2.6.27.)

Abstract: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to “Method Suitability Test” defined by the European Pharmacopoeia, using blood culture bottles and the automated BACTEC system (BD), after removal of antibiotics with the new medical device RESEP (AL.CHI.MI.A. Srl, Italy).
10-100 CFU of Staphylococcus Aureus, Pseudomonas Aeruginosa, Candida Albicans, Bacillus Subtilis, Aspergillus Niger, Clostridium Sporogenes, Enterobacter Cloacae and Staphylococcus Epidermidis were inoculated in 9 ml cornea organ culture medium (MEM with 2% fetal calf serum and Streptomycin (130 µg/ml), Penicillin (60 µg/ml) and Amphotericin B (2,5 µg/ml), Biochrom). The medium was treated with RESEP for 20 minutes, inoculated in BACTEC plus vials (BD) and incubated until a positive reading or for at least 14 days at 36 ± 1°C. Untreated medium, positive and negative control samples were also tested. The elimination of antibiotics from the medium by RESEP was determined by HPLC.
The antibiotics were completely eliminated after 20 min. of treatment with RESEP at room temperature. In RESEP treated samples, the growth of all microorganisms was detected within 3 days, and showed no delay compared to the positive control samples. In contrast, the untreated medium samples showed no repeatable results of Staphylococcus Aureus, Enterobacter Cloacae, Candida Albicans and Bacillus Subtilis.
The microbiological testing of cornea organ culture medium with the automated BACTEC blood culture system using RESEP for the removal of antibiotics, could be successfully validated according to the “Method Suitability Test” of the European Pharmacopoeia (chapter 2.6.1.).

Abstract: The aim of this study was to validate the microbiological testing of cornea organ culture medium according to the European Pharmacopoeia using blood culture bottles and the automatized BACTEC system (BD) after removal of antibiotics with the new medical device RESEP (AL.CHI.MI.A. Srl, Italy).
For the validation, 10-100 CFU of Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Clostridium sporogenes, Enterobacter cloacae and Staphylococcus epidermidis were inoculated in 9 ml of cornea organ culture medium (MEM with 2% fetal calf serum and antibiotics, Biochrom). The medium was treated with RESEP for 20 min., inoculated in BACTEC blood culture bottles and incubated until a positive reading or for at least 14 days at 36 ± 1°C. Medium control samples, containing the same microorganisms, were inoculated in BACTEC vials without RESEP treatment. Microbial growth was controlled by direct  inoculation of BACTEC flasks (growth control).
The antibiotics were completely eliminated after 20 min. of treatment with RESEP at room temperature (confirmed by HPLC). In RESEP treated samples, the growth of all microorganisms was detected within 3 days of incubation in BACTEC system and showed no significant delay in comparison with the growth controls. Without the use of RESEP, several tests with Staphylococcus aureus, Enterobacter cloacae and Bacillus subtilis samples were not detected and other strains showed a prolonged detection time compared to treated RESEP samples and growth controls.
The microbiological testing of cornea organ culture medium with automatized BACTEC blood culture system could be successfully validated using RESEP for the removal of antibiotics.

Purpose: aim of the study was to compare the direct inoculum method of corneal preservation media in the presence of antibiotics with the same method after elimination of antibiotics with the RESEP device.

Methods: 42 samples of cold storage media (EUSOL-C, AL.CHI.MI.A. S.r.l.) were obtained from the Eye Bank of Rome after donor cornea storage for up to 7 days. Aliquots of 3 ml of the media were either inoculated directly in trypton soy broth and fluid thioglycollate medium or treated with RESEP to eliminate antibiotic residues and then inoculated in growth media. 10 samples of sterile broth, treated with RESEP and tested by direct inoculum method, served as controls. To evaluate the initial contamination, bioburden was performed for each of the tested EUSOL-C by membrane filtration (MILLIFLEX SENSOR II – MILLIPORE). Positive samples were identified genetically.

Results: 19 out of 42 tested samples (45.2%) resulted contaminated when assessed by the direct inoculum method without RESEP treatment. The same samples treated with RESEP showed 25 of 42 (59.5%) positive samples. The RESEP positive samples, which were not detected without antibiotic elimination, were contaminated with Staphylococcus spp, Bacillus spp, Propionibacterium acnes. Turbidity was detected up to 5 days in advance using RESEP. None of the control samples was contaminated.

Conclusions: The elimination of antibiotic residues from samples allowed to reveal an additional 14% of contaminated samples as compared to the direct inoculum method without elimination of antibiotics. Our study demonstrated that the antibiotics present in the corneal preservation media may interfere with the microbial growth during microbiological analyses by direct inoculum method and lead to false negatives.