Antimicrobial Activity of ReNu MultiPlus Against Pseudomonas
Michael J. Miller, PhD;
Robert J. Manchester, AAS; and Denise Callahan, AAS
A new study shows that ReNu MultiPlus multi-purpose solution effectively kills Pseudo-monas aeruginosa.
In article published in the January 2000 issue, "How Dangerous is Noncompliance with Multipurpose Solutions," purported to investigate the antimicrobial activity of a number of multi-purpose solutions against two strains of Pseudomonas aeruginosa that were isolated from patients' eyes. This article reported experimental results differing dramatically from those obtained using proper standardized test methods, prompting further review and inquiry. Additionally, a competing chemical lens disinfecting solutions firm put the article to immediate commercial use. Since the study design and methods were not appropriately provided, other laboratories were unable to validate the report through further testing.
Although the authors referenced the current U.S. Food and Drug Administration (FDA) Premarket Notification (510(k)) Guidance Document for Contact Lens Care Products as the basis for their experimentation, they informed us that they did not conduct their study in accordance with the FDA test methodology. Several specific deviations from the standard methodology were identified:
- The authors used 10 times more challenge organisms than what is required in the FDA Guidance Document
- The authors did not use a standardized stock culture of P. aeruginosa ATCC 9027 that is required in the FDA procedure
- The authors evaluated only one lot of each product, rather than three, as is required under the FDA Guidance Document
- The procedure was not validated for each test solution
Also, no information was provided as to how the test was actually carried out and whether the remainder of the FDA procedure was followed. There is no information on what volume of test material was used, the culture conditions for the challenge organisms after exposure to the test material or if the procedure was validated for each test solution, which is a requirement of the FDA.
Materials and Methods
We obtained three separate lots of ReNu MultiPlus Multi-Purpose Solution (Bausch & Lomb) from commercial sources. The lots were within expiration dating and were evaluated according to the manufacturer's label instructions for minimum disinfection time.
The methods used were according to the U.S. FDA Guidance for Industry: Premarket Notification (510(k)) Guidance Document for Contact Lens Care Products, except that the challenge organisms tested were three strains of P. aeruginosa, and an additional study was performed using an inoculum concentration approximately 10 times greater than what is specified in the document.
The following test organism is specified in the FDA Guidance Document and was obtained from the American Type Culture Collection (ATCC): P. aeruginosa 9027. Two additional P. aeruginosa clinical strains, #6294 and #6206, were provided by Drs. Suzanne Fleiszig and Carol Lakkis, University of California at Berkley, and, according to Drs. Fleiszig and Lakkis, were identical to the two P. aeruginosa strains used in the January 2000 article. All P. aeruginosa strains were prepared according to the FDA Guidance Document.
We conducted the stand-alone disinfectant efficacy evaluation using 10ml of test solution for each challenge organism. The test was carried out in polypropylene tubes that were compatible with the solution being evaluated. Test solutions were inoculated with 0.1 ml of 1 x 107 - 1 x 108 CFU/ml suspensions and mixed for 5 seconds to evenly disperse the organisms, resulting in a final concentration of 1 x 105 - 1 x 106 CFU/ml. To simulate the January study, separate test solutions were inoculated with 0.1 ml of 1 x 108 - 1 x 109 CFU/ml suspensions and mixed for 5 seconds to evenly disperse the organisms, resulting in a final concentration of 1 x 106 - 1 x 107 CFU/ml. Inoculated test solutions were stored at 20-25C throughout the test period.
We sampled test solutions for viable organisms at 100 percent of the manufacturers' minimum disinfection time, which is four hours for ReNu MultiPlus Solution. Test solutions were mixed and a 1 ml aliquot was withdrawn from each test vessel, added to separate tubes containing 9.0 ml of Dey-Engley neutralizing broth (DEB), mixed and permitted to stand at ambient temperature for at least 10 minutes. This step neutralizes the preservative to allow the growth of surviving organisms. The samples in DEB were then serially diluted and pour plated in triplicate with TSA tempered to 40-50C. The media were allowed to solidify, and the plates were inverted and incubated at 30-35C for two to four days. Following incubation, the number of viable organisms were enumerated. Normally, the FDA stand-alone procedure requires that a contact lens disinfecting solution be evaluated at 25, 50, 75 and 100 percent of the manufacturers' minimum disinfection time. For the purposes of comparing our results with those reported in the January paper, we have presented data obtained for only the four-hour time point. We validated the neutralizer efficacy of DEB for each P. aeruginosa strain as required in the FDA Guidance Document.
Tables 1 and 2 summarize the number of survivors and mean log reductions for each strain of P. aeruginosa after a 4 hour exposure to ReNu MultiPlus Solution (soak only).
|Mean Log Reduction of Pseudomonas aeruginosa Following Exposure To ReNu MultiPlus Solution for 4 Hours (soak only). Initial challenge concentration of 1 x 105 - 1 x 106 CFU/ml.|
|Pseudomonas aeruginosa|| Mean Initial Concentration:
CFU/ml (Log value)
|Mean Log Reduction|
|ATCC 9027 (FDA/ISO strain)||8.7 x 105 (6.0)||0||>5.0|
|Clinical strain #6206||9.0 x 105 (6.0)||0||>5.0|
|Clinical strain #6294||8.3 x 105 (5.9)||0||>4.9|
|* Mean survivors on the highest dilution plate.|
|Mean Log Reduction of Pseudomonas aeruginosa Following Exposure To ReNu MultiPlus Solution for 4 Hours (soak only). Initial challenge concentration of 1 x 106 - 1 x 107 CFU/ml.|
|Pseudomonas aeruginosa|| Mean Initial Concentration:
CFU/ml (Log value)
| Mean Survivors*:
|Mean Log Reduction|
|ATCC 9027 (FDA/ISO strain)||8.2 x 106 (6.9)||0||>5.9|
|Clinical strain #6206||8.8 x 106 (6.9)||0.3||5.9|
|Clinical strain #6294||8.6 x 106 (6.9)||0.2||5.9|
* Mean survivors on the highest dilution plate.
Each of the P. aeruginosa strains evaluated far exceeded the stand-alone primary acceptance criteria. A 3.0 log reduction is required at the recommended disinfection time of four hours. There was no recovery of any of the three P. aeruginosa strains that had an initial inoculum concentration of 1 x 105 - 1 x 106 CFU/ml at the four hour time point. This represents a mean log reduction of greater than 4.9. There was no recovery of P. aeruginosa strain 9027, the FDA/ISO required organism, with a higher initial inoculum concentration of 1 x 106 - 1 x 107 CFU/ml at the four hour time point, a mean log reduction of greater than 5.9. At the four hour time point, P. aeruginosa strains 6206 and 6294 at the higher concentration were reduced by a mean log reduction of 5.9. Even if the FDA procedure required testing at these higher concentrations, these results far exceed the criteria for bacteria for a stand-alone disinfectant: the number of organisms recovered per ml were reduced by a mean value of not less than 3.0 logs within the minimum recommended disinfection period, four hours.
The validity of the study published in the January 2000 issue is highly suspect. The authors did not follow the FDA procedure for disinfecting products, which simulates the soak-only disinfecting phase of a lens care regimen. Not only was 10 times the amount of challenge organism utilized, but the FDA-required strain of P. aeruginosa was never employed in their testing. More importantly, it is impossible to repeat the testing performed by the authors because detailed methods were never provided in their paper.
We performed two separate evaluations employing the two clinical strains of P. aeruginosa identical to those used in the January 2000 paper, as well as the FDA-required strain (ATCC 9027). One evaluation used an inoculum challenge concentration similar to what was used in the January 2000 study, and the second evaluation used a challenge concentration as specified in the FDA stand-alone procedure. Side-by-side testing using three separate organism preparations and three separate lots of ReNu MultiPlus Solution clearly demonstrate that there is no difference in antimicrobial activity against the two clinical P. aeruginosa strains and the FDA reference strain. Furthermore, there was no recovery of any of the strains when tested according to the FDA procedure, and a mean of less than 1 CFU/ml recovered for the two clinical strains at the higher challenge concentration. The results of these studies show that each of the P. aeruginosa strains were killed during a four-hour disinfecting soak. This was true whether using a 106 or a 107 CFU/ml inoculum concentration. In all cases, the FDA stand-alone primary criteria (a mean value of not less than 3.0 logs) was far exceeded.
The results of our evaluations do not agree with the conclusions presented in the January 2000 paper. The January 2000 paper reported that "all solutions resulted in the kill rate required by the FDA except ReNu MultiPlus Solution." This statement is incorrect because the FDA procedure was never followed. Furthermore, we have previously demonstrated that P. aeruginosa is rapidly and completely killed by most multi-purpose lens care products, including ReNu MultiPlus Solution.
The January 2000 paper also suffers from other technical limitations. The authors state that their data may provide a more accurate picture of how lens care solutions interact with pathogenic organisms; however, they did not provide any comparative data to demonstrate that the two clinical strains were significantly more resistant to chemical disinfection than the FDA standardized strain (ATCC 9027). This study proves that there is no difference in microbial kill when exposed to ReNu MultiPlus for four hours. Next, the authors imply that a higher challenge concentration is more stringent than the FDA test. Certainly, if the FDA believed that a 106 CFU/ml challenge level was not appropriate for this type of evaluation, then a different challenge inoculum would have been required. In actuality, the FDA Guidance Document states that the "size of the microbial challenge chosen in this test is not intended to be representative of the likely challenge in practice but to provide countable numbers from which estimation of the rate and extent of viability loss can be determined." The authors imply that one reason why their results may be more clinically relevant than the FDA test is that they used ocular isolates, and that "stock strains are organisms that have been passed on artificial media for many generations, and often, these organisms lose their disease producing ability." The authors failed to mention that the FDA procedure specifies that "challenge cultures should be no greater than five passes removed from the depository stock." It is interesting to note that in order for the authors to conduct their experiments, they would be required to grow their clinical strains on artificial media in order to obtain sufficient quantity of organisms for the product inoculations. Unfortunately, this was never addressed. Additionally, the authors did not specify how many times the clinical strains were passed on laboratory media, nor did they disclose the type of media that was used or under what conditions the organisms were cultivated. Specific media and growth requirements are provided in the FDA Guidance Document. The lack of this information calls into question the validity of their entire study.
ReNu MultiPlus Multi-Purpose Solution is a FDA-approved lens care product that exceeds the requirements of the stand-alone primary criteria for disinfecting solutions. This product provides superior antimicrobial efficacy against P. aeruginosa and the other required challenge organisms as measured against the U.S. FDA's Guidance Document. The results of these studies prove that standardized and clinical strains of P. aeruginosa are effectively killed during the disinfecting (soak-only) phase of the total recommended lens care regimen.
Acknowledgement: We wish to thank Dr. Suzanne Fleiszig and Dr. Carol Lakkis, University of California at Berkley, for providing the Pseudomonas aeruginosa clinical strains that were used in these studies.
To receive references via fax, call (800) 239-4684 and request document #60. (Have a fax number ready.)
Dr. Miller is Director of Biological & Sterilization Sciences at Bausch & Lomb, and is a United States Delegate to ISO Ophthalmic Standards Committees.
Mr. Manchester is a technician in the Research Microbiology Department at Bausch & Lomb.
Ms. Callahan is a technician in the Analytical Microbiology Department at Bausch & Lomb.