Isolation of Antibiotic-Resistant Micrococcus Aloeverae from Milk Powder
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obiolog icr y , M S : a d f e Journal of Food: Microbiology, Safety o t Sankarankutty and Damani, J Food Microbiol Saf o y F & f Hyg 2018, 3:1 H o l y a g n ISSN: 2476-2059i & Hygiene DOI: 10.4172/2476-2059.1000134 r e n u e o J Research Article Open Access Isolation of Antibiotic-Resistant Micrococcus aloeverae from Milk Powder Meera Sankarankutty* and Juhi Damani Department of Food Science and Nutrition, Post-Graduate Studies and Research in Home Science, S.N.D.T Women’s University, Juhu, Mumbai-400049, Maharashtra, India *Corresponding author: Meera Sankarankutty, Faculty, Department of Food Science and Nutrition, Department of Post-Graduate Studies and Research in Home Science, S.N.D.T Women’s University, Juhu, Mumbai-400049, Maharashtra, India, E-mail: [email protected] Received date: January 26, 2018; Accepted date: February 23, 2018; Published date: February 26, 2018 Copyright: © 2018 Sankarankutty M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The emergence of antibiotic resistance in humans via the food chain has become an area of great concern. Antibiotic resistance gene transfer from a food-borne bacteria or probiotic agent to a human gut microbe can lead to a serious food safety issue. Such seemingly harmless bacteria or probiotics can potentially become a threat to human health if they carry mobile genes conferring antibiotic resistance. The recent trend of probiotic application in powdered infant formula and milk products and the presently known contamination of these products by certain opportunistic Enterobacteriaceae bacteria serves as the rationale for evaluating their safety. In this study, 25 milk powder samples and 25 powdered infant formula samples were analyzed for the presence of antibiotic resistant bacteria of family Enterobacteriaceae using an isolation technique. Out of the 50 samples screened, an isolate was detected in one milk powder sample. Its biochemical characteristics, Gram character, antibiotic susceptibility, and motility were analyzed. It was found to be a Gram-positive, non-motile, catalase-positive and oxidase-negative microbe. The isolate was resistant to Rifampicin and Vancomycin, but susceptible towards Streptomycin, Ciprofloxacin, Ceftriaxone and Gentamycin. Molecular characterization by 16S rDNA sequencing identified the isolate as Micrococcus aloeverae. The unique occurrence of Micrococcus aloeverae in milk powder elicits an area of food safety concern for such samples. To the best of our knowledge, this study is the first to report antibiotic resistance by Micrococcus aloeverae. Although it may be a non-pathogenic microbe, its resistance towards antibiotics cannot be ignored. The possibility of Micrococcus aloeverae serving as a potential vehicle for transfer of antibiotic resistance genes to human gut microbiota represents a prospective food safety concern. Keywords: Micrococcus aloeverae; Antibiotic resistance; Milk and in milk powder in Turkey [14]. Likewise, in a study from Iran, an powder; Gene transfer; Food safety antibiotic resistant opportunistic microbe Pantoea (Enterobacter) agglomerans was isolated from a consumed powdered infant formula Introduction [15]. Hence, it is necessary to screen such products for investigation of Enterobacteriaceae bacteria. Antibiotic resistance is one of the major threats to food security and human health [1]. Bacterial resistance towards antibiotics could The use of beneficial probiotic agents in powdered infant formula is endanger the current medicinal advancements in antibiotic therapy becoming increasingly popular [16]. The application of probiotics as [1]. The transmission of antibiotic resistance from an external source antimicrobial agents in powdered infant formula has been suggested as to humans can occur via a number of routes but the food-borne route a promising approach to improve the microbiological safety of the is considered to be the most important [2]. Hence, reports of antibiotic product [17]. Similarly, probiotic use in dairy products can serve as an resistance in food-borne bacteria and probiotic agents used in the food innovative alternative to improve the quality of the product [18]. In a industry shed a great concern with respect to food safety. The World certain study, the viability of probiotic agents added to whole milk Health Organization, the Food and Agricultural Organization and the powders was evaluated [18]. From the microbiological point of view, World Animal Health Organization have stated that there is evidence the milk powders with added probiotics showed satisfactory results. It of unfavourable human health consequences due to resistant was observed that milk powders provide optimum conditions for organisms arising from non-human usage of antimicrobials [3]. maintaining the viability of probiotics [18]. Antibiotic genes exist in food animals, fish, plants and vegetables. Although probiotic use in powdered infant formula and milk These sources serve as reservoirs of genes that can be potentially powders may be beneficial, the safety of these food products should be transferred to human pathogens by indirect contact via the assessed and the safety of the probiotic strain should be evaluated for consumption of contaminated food [4]. their ability of acquiring and transferring resistant determinants [19]. Powdered infant formula is not a completely sterile product [5,6]. In certain bacteria and probiotics, specific antibiotic resistant The pasteurization process during the manufacture of powdered infant determinants present on mobile genes exist and they serve as formula eliminates any pathogenic bacteria. Thus, reports of reservoirs of resistance for potential food or gut pathogens [20]. contamination in powdered infant formula are most likely attributed to Transfer of antibiotic resistance genes from probiotic Lactobacillus spp. poor hygiene standards post pasteurization [7]. There have been to Enterococcus faecalis has been demonstrated [21]. Transmissible reports of isolation of a pathogenic and opportunistic bacterium of antibiotic resistance genes in Bifidobacterium spp. have also been Enterobacteriaceae family in powdered infant formula in Mexico [8], observed [21]. Such genes conferring antibiotic resistance can be South Africa [9], Jordan [10], India [11], Iraq [12] and Nigeria [13] J Food Microbiol Saf Hyg, an open access journal Volume 3 • Issue 1 • 1000134 ISSN:2476-2059 Citation: Sankarankutty M, Damani J (2018) Isolation of Antibiotic-Resistant Micrococcus aloeverae from Milk Powder. J Food Microbiol Saf Hyg 3: 134. doi:10.4172/2476-2059.1000134 Page 2 of 5 transferred to other bacteria by conjugative plasmids, transposons, inoculum of the isolate was prepared using sterile Buffered Peptone integrons and insertional elements [16]. water and standardized by adjusting its density to match the 0.5 McFarland turbidity standard. Using a sterile cotton swab, the Hence, it is imperative to screen powdered infant formula and milk inoculum was spread onto the entire surface of the agar. These powders for antibiotic resistant Enterobacteriaceae bacteria. The aim of inoculated plates were allowed to stabilize for 5 min, after which the this study was to evaluate powdered infant formula and milk powder antibiotic discs were laid aseptically on the surface. Sterile discs of 6 samples for the presence of antibiotic resistant bacteria of family millimeter diameter were prepared using Whatman paper no. 1 and Enterobacteriaceae. they served as ‘Blank’ controls. The plates were incubated at 37°C for 18 h. The zones of inhibition were measured and recorded. The results Materials and Methods were interpreted in accordance to the criteria set by the Clinical and Laboratory Standards Institute [23]. Sample collection A total number of 50 samples, comprising 25 powdered infant Gram staining formula samples of an international brand and 25 milk powder The Gram character of the isolate was assessed by the Gram staining samples of international and local brands were procured from markets technique [24]. A primary Crystal violet stain was applied to a heat- in Mumbai city in Maharashtra, India. The typical composition of the fixed smear of the bacterial culture for 1 min. The slide was washed powdered infant formula samples used in this study include milk with water, followed by application of the mordant (Gram’s iodine) for solids, sucrose, maltodextrin, dried glucose syrup, soyabean oil, corn 1 min. The slide was washed with water, followed by decolorizing it oil, vitamins and minerals. The milk powders contained milk solids, with alcohol. A counter-stain Safranin was applied to the smear for 1 sugar and stabilizer. The media used were obtained from HiMedia min. The slide was washed with water and then observed under oil- Laboratories (India). immersion objective. Isolation Bile salt tolerance test The samples were evaluated using the isolation technique described The ability of any Gram-positive isolate to grow on media such as by the United States Food and Drug Administration [22]. Pre- Enterobacteriaceae Enrichment Broth and Violet Red Bile Glucose enrichment was carried out by adding 10 g of sample to 90 ml of Agar containing bile salts suggests that it exhibits bile salt tolerance. If Buffered Peptone water. Following incubation at 37°C for 24 h, 1ml of a Gram-positive isolate was detected, it was