Research paper Degradation of parabens by Pseudomonas beteli and Burkholderia latens Aeshna Amin a , Sateesh Chauhan b , Manish Dare b , Arvind Kumar Bansal a, * a National Institute of Pharmaceutical Education and Research (NIPER), Punjab, India b Promed Exports Pvt. Ltd., Haryana, India article info Article history: Received 29 October 2009 Accepted in revised form 1 March 2010 Available online 3 March 2010 Keywords: Methyl paraben Propyl paraben Microbial degradation Antimicrobial preservative Preservative effectiveness test abstract p-Hydroxybenzoic acid esters (parabens) are commonly used antimicrobial preservatives in pharmaceu- tical formulations. Two microorganisms, isolated from non-sterile methyl paraben (MP) and propyl para- ben (PP) solutions, were found to degrade the respective parabens. Identification by 16S rRNA partial gene sequencing revealed them to be Pseudomonas beteli and Burkholderia latens, respectively. The present work describes a previously unreported interaction of the parabens with P. beteli and B. latens. Degradation of MP at various concentrations by P. beteli, followed a logarithmic pattern, while that of PP by B. latens was found to be linear. It was subsequently observed that P. beteli could degrade only MP, while B. latens could degrade both the parabens. Absence of HPLC chromatogram peaks of expected degradation products indicated that the parabens were used up as a carbon source. The behaviour of pathogens (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Aspergillus niger) of the pharmacopoeial preservative effectiveness test (PET), towards MP, showed that none had the ability to degrade the paraben. It was concluded that, for a paraben-preserved multi-dose ophthalmic formula- tion, the sole use of the four pathogens that are recommended by the pharmacopoeia for PET can falsely indicate the formulation to be effective against ‘in-use’ contamination. Ó 2010 Elsevier B.V. All rights reserved. 1. Introduction Non-sterile and multi-dose sterile pharmaceutical formulations contain antimicrobial agents to kill or limit the growth of any micro- organism that are present initially or gain entry during use of the product. The presence of unwanted microorganisms in pharmaceu- tical products can lead to either spoilage of product or transmission of disease to the consumer. Spoilage can either be in terms of chem- ical degradation of drugs/excipients by the enzymes produced by the microorganisms or a physical breakdown of the product leading to visible, olfactory, taste or tactile changes, thus rendering the prod- uct unfit for its intended use [1]. It is also possible that the microor- ganisms, without causing spoilage, induce disease in the consumer [2]. This assumes further criticality in case of multi-dose sterile products like ophthalmic solutions where contaminated eye drops can pose a hazard to the patients’ vision [3]. Ophthalmic solutions are formulated essentially as other phar- maceutical solutions [4] with the additional requirement of sterility [5–7]. Antimicrobial preservatives are a pharmacopoeial require- ment for multi-dose ophthalmic solutions. Although the sterility is assured in the multi-dose ophthalmic product by using aseptic pro- cesses or terminal sterilization processes, additionally, antimicro- bial preservatives need to be incorporated to prevent growth or destroy microbes introduced due to accidental ‘in-use’ contamina- tion [1,8]. Incidentally, preservative-free multi-dose eye drops have been previously used and have shown contamination with poten- tially pathogenic microorganisms [9]. Some of the common antimi- crobial preservatives for ophthalmic use include benzalkonium chloride, chlorhexidine digluconate, benzyl alcohol, chlorbutol and p-hydroxybenzoic acid esters (also known as parabens). The latter mainly include methyl paraben (MP) and propyl paraben (PP) and are essentially used in the concentrations of 0.015–0.2% w/v and 0.005–0.01% w/v, respectively [10,11]. A study in our lab was conducted to determine the effect of autoclaving on paraben content (0.015% w/v MP and PP) during in-house ophthalmic formulation development. The study revealed a substantial reduction in the concentration of parabens in non- sterile formulations after one month of accelerated stability stud- ies (40 °C). Autoclaved formulations, however, did not show any loss of paraben content. Based on the published literature, it was postulated that presence of microbial contamination in the non- sterile formulations might have interacted with the parabens lead- ing to their loss. Microbes from the above-mentioned MP and PP formulation were isolated. The present work was delineated with the aim to understand the effect of these two isolated organisms on parabens in solution and to comprehend the implications that resulted from it. 0939-6411/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejpb.2010.03.001 * Corresponding author. Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar, Mohali, Punjab 160 062, India. Tel.: +91 172 2214682; fax: +91 172 214692. E-mail address: akbansal@niper.ac.in (A.K. Bansal). European Journal of Pharmaceutics and Biopharmaceutics 75 (2010) 206–212 Contents lists available at ScienceDirect European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb