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Microbiological Quality and Occurrence of Antibiotic Resistant Bacteria in Ready-To-Eat Salad

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Microbiological Quality and Occurrence of Antibiotic Resistant Bacteria in Ready-To-Eat Salad Microbiological quality and occurrence of antibiotic resistant bacteria in ready-to-eat salad Tamara Lupan Master thesis for a diploma in Food Technology and Nutrition, 30 ECTS credits Department of Food Technology, Engineering and Nutrition, Faculty of Engineering, Lund University Abstract An increase in demand for fresh vegetables resulted in an increased production of minimally processed, ready-to-eat salad in Sweden. That also brought new food safety challenges that are yet to be addressed. To assess whether ready-to-eat leafy green vegetables present a threat in terms of food borne outbreaks, aerobic bacteria, as well as bacteria belonging to the Enterobacteriaceae family, were recovered, using selective media, from 3 different sets (n=18) of ready-to-eat rocket salad. Bacterial investigation showed that most bacteria retrieved from ready-to-eat rocket salad belongs to the Pseudomonadaceae family, which are typical spoilage bacteria commonly associated with vegetables. No food-borne pathogens (e.g. Clostridium botulinum, Escherichia coli O157:H7, Salmonella spp., Shigella spp., Listeria monocytogenes) were isolated from any of three sets of rocket salad. Following the assumption that microbiological quality of the salad could change during the storage period, as well as a result of salad bags being opened, sealed, stored and opened again in the household, the total aerobic count as well as the total amount of Enterobacteriaceae were assessed every day before the best-before date. Following the NSW food Authority guidance on the microbiological status of ready-to-eat food, it can be confirmed, that each of three sets of salad were from the first day after packaging, unsatisfactory in regards to total aerobic count, showing values higher than 5 log CFU/g. The bacterial investigation also concluded that opening the bags and storing them sealed in the household will produce little, if any effect on the microbiological quality and will not challenge the validity of the expiration dates. To asses weather ready-to-eat rocket salad available for sale in Sweden, could be a possible reservoir of antibiotic resistance, the isolated bacteria from each bag of salad were tested for susceptibility to eight antibiotics, representative of the six classes of antibiotic compounds commonly used in clinical settings in Sweden. The cases of possible acquired antibiotic resistance were rare, however, the bacteria, suspected to be immune to the drug action, should be subject for further investigations aimed to discover the mechanism of such resistance. 2 Table of Contents Abstract ............................................................................................................................. 2 Acknowledgment ............................................................................................................... 4 1 Introduction .................................................................................................................... 5 1.1 Background ......................................................................................................................... 5 1.2 Salad microbiology .............................................................................................................. 6 1.3 Antibiotic resistance in bacteria present in food .................................................................. 8 1.4 Aim ........................................................................................................................................ 9 2. Materials and Methods ................................................................................................ 10 2.1 Choice of material ............................................................................................................... 10 2.2 Preparations ........................................................................................................................ 10 2.3 Bacterial investigation ......................................................................................................... 11 2.4 Sequencing bacteria isolates ................................................................................................ 11 2.5 Screening for antibiotic resistance ....................................................................................... 12 2.6 Statistical analysis ............................................................................................................... 13 3 Results ........................................................................................................................... 14 3.1 Validity of expiration dates .................................................................................................. 14 4 Discussion ..................................................................................................................... 32 4.1 Validity of expiration dates .................................................................................................. 32 4.2 Isolated bacterial strains ...................................................................................................... 33 4.3 Screening for antibiotic resistance ....................................................................................... 33 5 Conclusions ................................................................................................................... 35 References ....................................................................................................................... 36 7. Appendix ...................................................................................................................... 39 7.1 Recipes ................................................................................................................................ 39 7.2 Detailed working procedure of investigating bacterial content depending on the opening day 39 7.3 Detailed sampling plan for the investigation of bacterial content ........................................ 41 7.4 Detailed working procedure of investigating bacterial susceptibility to clinically relevant antibiotics ................................................................................................................................. 42 7.5 P-values from Mann-Whitney U tests .................................................................................. 42 3 Acknowledgment I am grateful for the opportunity to implement my Master Thesis at the Food Hygiene department of the Faculty of Engineering at Lund University. I am particularly thankful to Elisabeth Uhlig, my master thesis supervisor, that was helping and guiding me throughout this project. I am also extremely grateful for the assistance given by Bharath Prasad, whom has been my co-worker during this project. His work, patience and support has allowed us to carry out this study. This publication is part of my research work at Lund University, thanks to a Swedish Institute scholarship. 4 1 Introduction 1.1 Background Fruits, berries and vegetables play a major role in a healthy diet. They contain fibre, vitamins, minerals and a range of other substances essential for a proper nutrition. The Swedish national agency recommends to consume as much as 500 grams of fruits and vegetables daily, and also points out the importance of consuming organically produced products in order to minimize the adverse effects of agriculture on the environment (The National Food Agency, Sweden 2015). Thus, during last years in Sweden, the sales of ready-to eat and on-the-go products were rising (Irish Food Board 2013). They are sold in almost every grocery or convenience store; they are served from fast food chains and restaurants to hospitals and school canteens and seen by consumers as a convenient and healthy part of a meal. There is a whole variety of ready-to-eat salads, including cut or integral bite size leafy green vegetables or their mixes. These products are fresh, previously washed by the manufacturer, dried and packed in bags with modified atmosphere. Having a ready-to eat status, salads don’t require any heat treatment step meant to kill pathogens and consumers perceive them as safe to consume raw (The Commission of the European Comunities 2005). However, there are increasing concerns regarding the microbiological safety of leafy green vegetables. According to the European Food Safety Authority, from 2008 to 2011, an increase in numbers of food-borne outbreaks was recorded: cases, hospitalisations and deaths associated with products of non-animal origin (FoNAO). During the stated period, in European Union, FoNAO were associated with 10 % of the outbreaks, 26 % of the cases, 35 % of the hospitalizations and 46 % of the deaths. The general picture from 2011, if not taking in consideration VTEC O104 related outbreaks, looks the following way: 10 % of the outbreaks, 18 % of cases, 8 % of the hospitalizations and 5% of the deaths. (European Food Safety Authority 2013) In almost every case bacteria belonging to the family of Enterobacteriaceae have been involved, where the most often met combination of food/pathogen was raw leafy greens vegetables contaminated with Salmonella spp. (European Food Safety Authority 2013). It is crucial to mention that in case salad leaves are contaminated, the pathogens cannot be reduced to an acceptable level using washing, even with addition of sanitizing agents (M.F. Lynch 2009). This can be explained by the fact that pathogens are able to adhere to plant surfaces, or become internalised through the stomata,
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