Reduction of Aeromonas hidrophyla biofilm on stainless …

Abstract

This study demonstrates the possibility of using sanitizing detergents based on natural products for the elimination and/or reduction of Aeromonas hydrophila biofilm formed on stainless steel surfaces. The goal of this work was to determine the reduction effect of sanitizing detergents containing essential oils of Thymus vulgaris (thyme) and Cymbopogon citratus (lemongrass) on biofilm formed by A. hydrophila on AISI 304 stainless steel coupons, using UHT skimmed milk as substratum. There was adhesion and biofilm formation by A. hydrophila at 28 C, presenting 7.60 log cfu.cm2 after the fourth day of cultivation. There was no significant difference between the lemongrass treatment and that of the thyme oil (p < 0.05). However, both treatments significantly reduced the biofilm, differing significantly from the NaOH control (p > 0.05). The treatment with lemongrass solution reduced the biofilm by 4.51 log cfu cm2 at 25 C. The thyme detergent also reduced the number of cfu cm2 by 3.84 log cycles at 25 C. The use of the lemongrass and thyme solutions efficiently reduced the A. hydrophila biofilm.

Keywords: Thymus vulgaris, Cymbopogon citratus, natural disinfectants, sanitation

Aeromonas hydrophila is recognized for its capacity to produce several virulence factors such as cytotoxins and enterotoxins and adhesion capacity. These factors can cause hemolysis and allow the invasion of epithelial cells (Prigent-Combaret and Lejeune, 1999) causing gastroenteritis in humans. Due to its aquatic origin, it presents a ubiquitous distribution in the environment, being able to present itself in the most varied types of animal products and vegetables, such as fish and meat and its derivatives or any food that comes in contact with water (Trombetta et al., 2005). A. hydrophila is a gram-negative, facultative aerobic, not spore former, catalase-positive, oxidase-positive and has an optimum development temperature of 28 C (Joseph and Carnahan, 2003; Lafarge et al., 2004). Due to presence of polar and lateral flagella, this bacterium presents mobility and adhesion and biofilm formation capacity (Gavn et al., 2002).

Biofilms are complex microbiologic ecosystems embedded in matrixes of organic polymers adhered to a surface. Once established, sessile bacteria express genes in a model that differs greatly from that of planktonic bacteria, leading to phenotypic changes (Prigent-Combaret and Lejeune, 1999). One of those outstanding properties is the increase of sessile cell resistance to host defenses, biocides, antibiotics and various physiochemical agents (Costerton, 2005; Rota et al., 2008). Therefore, biofilm cells can persist and survive even after sanitization processes, representing a possible source of food contamination and subsequent food poisoning in humans and animals (Chavant et al., 2007). A large number of studies aiming to find effective elimination and biofilm control strategies have been published (Gandhi and Chikindas, 2007), demonstrating the necessity of obtaining new products with biocide action, seeking the elimination of microbial biofilms and their effective control in food industries.

The interest in natural antimicrobial compounds has been growing due to the changes of consumer attitude toward the use of synthetic food preservation, detergents and sanitizers which possess negative impacts on the environment (Danielsen et al., 2008; Lebert et al., 2007). Thus, the use of essential oils stands out. The action mechanisms of essential oils are little known. Considering the great number of different groups of chemical compounds present in such oils, it is observed that their antibacterial activity is not attributed to a single specific mechanism, which leads to the conclusion that they target several processes in the microbial cell (Mondello et al., 2003; Ogunwande et al., 2005; Suen et al., 2003).

The species Cymbopogon citratus (A.D.) Stapf., belonging to the family Poaceae (Gramineae), is popularly known by more than 20 names, among these lemongrass, grass-saint lemon verbena, fragrant grass, cidreira grass, lesser citronella and true cidreira herb (Cardoso et al., 2000). The most expressive economical use of C. citratus is the production of its essential oil, rich in citral and widely used in the food, pharmaceutical and cosmetic industries (Cardoso et al., 2000). Studies suggest that the antibacterial activity of C. citratus oil is mainly due to the and -citral components present in it (Oliveira et al., 2010). Besides C. citratus, the genus Thymus encompasses numerous species and varieties, which have had their essential oils studied (Martins et al., 2004; Onawunmi et al., 1984; Sotomayor et al., 2008). Its essential oil is rich in timol, presenting traces of carvacrol, scientifically recognized potent bactericides and fungicides (Essawi and Srour, 2000). More recent studies prove the antibacterial activity of T. vulgaris as being effective against gram-positive and Gram negative bacteria (Barbosa et al., 2009).

Aimed at discovering alternate forms of eliminating biofilms formed by A. hydrophila on stainless steel surfaces, this work evaluated the sanitizing capacity of sodium hydroxide solutions enhanced with the essential oils of C. citratus and T. vulgaris.

Aeromonas hydrophila ATCC 27853 was employed. The growth curves were prepared for standardization of the colony-forming units count. Aliquot of pure cultures (10 L) were transferred to Erlenmeyer flasks containing 200 L of BHI (Brain Heart Infusion) broth and incubated at 28 C. Growth was monitored every hour through spectrophotometry (620 nm). The absorbance readings were taken periodically, until 109 cfu mL1 were obtained.

Dried leaves of C. citratus were used, obtained from the Medicinal Plant Nursery of the Federal University of Lavras, (UFLA). The collection was conducted from 8 to 9 oclock in the morning, on sunny days, in September of 2009, under a temperature of approximately 20 C. The T. vulgaris dry leaves were acquired from the local commerce of Lavras, MG, Brazil.

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Reduction of Aeromonas hidrophyla biofilm on stainless ...