BIOFILM FORMATION BY STAPHYLOCOCCUS AUREUS ISOLATED FROM FOOD CONTACT SURFACES

ABSTRACT

This study was to determine the incidence of biofilm formation among Staphylococcus aureus isolated from food contact surfaces in Michael Okpara University of Agriculture, Umudike eateries. In this study a total of one hundred (100) food contact surfaces were swabbed with a swab stick before and after use. The samples were cultured on mannitol salt agar using streak plate method. A total of forty seven (47) staphylococcal strains and nineteen (19) Coagulase Negative staphylococcal strains were isolated from 100 samples of food contact surfaces. The prevalence rate in this study showed that the highest number and percentage of staphylococcal isolates was observed to be 47%, followed by Coagulase Negative staphylococcal isolate which recorded 19%. About 38% of samples from food contact surface showed no growth of Staphylococcal strains. However, the drug susceptibility profile of bacterial isolate from the food contact surfaces revealed varying percentage of sensitivity and resistance to the antibiotics. From this study, the Staphylococcus aureus exhibited high degree of sensitivity against Ofloxacin and Gentamicin at 41(87.2%) and 38(80.9%) respectively. Cefuroxime (30mcg), Ceftiaxone (30mcg), Erythromycin (30mcg), Cloxacillin (5mcg), Amoxicillin (30mcg) and Ceftazidime (30mcg) showed high level of resistance against the Staphylococcus isolates. The incidence of biofilm reveals that out of the forty seven (47) Staphylococcus aureus isolated from food contact surfaces, 28(58.6%) was positive to biofilm formation, while 19(40.4%) of the Staphylococcus aureus was negative to biofilm formation. These results therefore point towards the need to improve hygiene conditions during the production of food.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of contents                                                                                                                    vi

Lists of Table                                                                                                                          viii

Abstract                                                                                                                                  ix

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    4

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       Brief Description of Biofilm                                                                                      5

2.2       Biofilm Formation Processes in Food                                                                        5

2.2.1    Initial Reversible Attachment                                                                                    5

2.2.2    Irreversible Attachment                                                                                              6

2.2.3    Early Development of Biofilm Structure                                                                   6

2.2.4    Maturation                                                                                                                  6

2.2.5    Dispersion                                                                                                                   6

2.3       Foodborne Pathogens in Mixed Biofilms                                                                   7

2.4       Food Borne Bacteria in Food Processing Environments                                           8

2.4.1    Listeria monocytogenes                                                                                              9

2.4.2    Salmonella spp                                                                                                            9

2.4.3    Escherichia coli                                                                                                          10

2.4.4    Pseudomonas spp                                                                                                       10

2.4.5    Bacillus spp                                                                                                                10

2.6       Biofilm Sanitizer Tolerance                                                                                       10

2.7       Biofilm Cell Transfer from Contact Surface to Food Product                                 12

2.8       Potential Involvement of Biofilms in Meat Contamination                                       14

2.9       Biofilm Mediated Infections and Pathogenesis                                                          15

2.9.1    Device related infections                                                                                            15

2.9.2    Central venous catheter infection                                                                               16

2.9.3    Prosthetic heart valves                                                                                                16

2.9.4    Urinary catheters                                                                                                        17

2.10     Prevention and Inactivation of Biofilms in Food                                                       17

2.10.1  Physical Methods                                                                                                        18

2.10.2  Chemical Methods                                                                                                      18

2.11     Staphylococcus aureus                                                                                               19

2.11.1  Methicilline Resistant Staphylococcus aureus                                                           21

2.11.2  Epidemiology of Staphylococcus aureus                                                                   21

2.11.3  Modes of Transmission                                                                                              22

2.11.4  Colonization                                                                                                               22

2.12     Infection Caused by Staphylococcus aureus                                                              23

2.12.1  Bacteremia                                                                                                                 23

2.12.2  Endocarditis                                                                                                                23

2.12.3  Metastatic Infections                                                                                                  24

2.12.4  Sepsis                                                                                                                          24

CHAPTER THREE

3.0       Materials and Methods                                                                                               25

3.1       Sample Collection                                                                                                      25

3.2       Media and Reagents                                                                                                   25

3.3       Microbiological Investigations                                                                                   25

3.3.1    Sterilization Method                                                                                                   25

3.4       Sample Preparation and Isolation of Staphylococcus aureus                                     25

3.4.1    Sample Inoculation                                                                                                     25

3.5       Purification of Isolates                                                                                               26

3.6       Identification of Staphylococcus aureus Isolates                                                      26

3.7       Gram Staining                                                                                                            26

3.8       Biochemical Test                                                                                                        27

3.8.1    Catalase Test                                                                                                               27

3.8.2    Coagulase Test                                                                                                           27

3.9       Detection of Biofilm by Congo red Agar (CRA) Method                                          27

3.10     Antibiotic Susceptibility Testing                                                                                27

CHAPTER FOUR

4.0       Results                                                                                                                        29

CHAPTER FIVE

5.0       Discussion and Conclusion                                                                                        35

5.1       Discussion                                                                                                                   35

5.2       Conclusion                                                                                                                  39

            References

LIST OF TABLES

CHAPTER ONE

1.0       INTRODUCTION

The surfaces that come into contact with foods are important sources for the transmission of microorganisms in food processing plants. Many pathogenic or spoilage bacteria can be found attached to surfaces in the form of planktonic cells or sessile cells forming a biofilm (Braga et al., 2005). Worldwide there is a concern about the impact of microbial foodborne diseases on the human behalf (White et al., 2002). The importance of contaminated surfaces in spreading pathogenic microorganisms to foods is already well established in food processing, catering and domestic environment (Vasseur et al., 2008). One of the most common ways for bacteria to live is adhering onto surfaces and forming organized communities named biofilms (Malheiros et al., 2010). Stainless steel, glass, rubber and polypropylene surfaces can be contaminated either by spoilage or pathogenic bacteria, which under certain conditions adhere to these surfaces, initiating the cell growth and leading to the biofilm formation. According to Costerton et al. (2009) biofilms are cell aggregates embedded in an organic extracellular polymeric matrix that confers resistance to involved microorganisms. Bacteria aggregated to form biofilms have greater resistance to the environmental stress than the planktonic counterparts, including the sensitivity to sanitizers (Fux et al., 2004). Bacterial aggregates detached from biofilms retain the high level of resistance to antimicrobials and may contain enough number of cells to represent a potential infectious dose. The formation of biofilms on food-contact surfaces is known as a potential risk to the consumer’s health, particularly, if the cross contamination of food occurs after a bactericidal procedure (Spoering and Lewis, 2001).

Staphylococcus aureus has been frequently found in surfaces of food processing plants being responsible for outbreaks related to the consumption of fresh and processed foods worldwide (Braga et al., 2005). The establishment of the food poisoning caused by S. aureus depends on the ability of the strain to survive in/on a colonized substrate, multiply under a variety of conditions and produce several extracellular substances (Pastoriza et al., 2002). Although some researchers have observed the ability to adhere and form biofilm by Staphylococcus genera, the most studies have been addressed to clinical aspects related to the biofilm formation by Staphylococcus intermedius on medical implants and materials (Marques et al., 2007). Currently, there is a lack of information about the capacity of S. aureus from food service surfaces of adhering and forming biofilm when exposed to different environmental conditions, and about the efficacy of sanitizers in removing the cells forming the biofilm. Regarding these aspects, this study was carried out with the aim of evaluating the ability of S. aureus isolates from food services surfaces to adhere and form biofilms on stainless steel and polypropylene surfaces when cultivated in a vegetable-based broth under different temperatures (7 and 28^oC). Still, it was observed the effect of the sanitizers peracetic acid and sodium hypochlorite in reducing the number of bacterial viable cells on a preformed biofilm.

The ability of Staphylococcus aureus to form biofilms provides it an important virulence factor. The bacteria surrounded by a biofilm are more difficult to be removed than those in the planktonic form and, once a biofilm is established, it becomes a source of contamination for products and surfaces. In vitro studies indicated that bacterial strains growing in biofilms may become 10-1,000 times more resistant to the effects of sanitizers than the same strain in planktonic form. Moreover, biofilms are capable of releasing planktonic cells from the outer layers, enabling persistent bacterial infection (Clutterbuck et al., 2007). Microorganisms embedded in biofilms can catalyze chemical and biological reactions that cause metal corrosion in the pipelines and bulk tanks, besides interfering with the efficiency of heat transfer. The time necessary for biofilm formation depends on the frequency of equipment cleaning. Surfaces that are in contact with food products must be cleaned several times a day, and other surfaces in the food production environment, such as walls, may be cleaned at least only once a week. The surface of finished products may be contaminated by direct contact, and the food production environment may indirectly contaminate the finished products via vectors, ventilation and cleaning systems, and food handlers.

The ability of strains isolated from mastitis-causing pathogens to adhere to stainless steel, glass, rubber and polypropylene surfaces has been widely studied. In dairy farms, a recent investigation showed that 42% and 39% of 31 Staphylococcus aureus strains isolated from milking parlor environments were biofilm producers on stainless steel and rubber, respectively, indicating a possible persistence of this pathogen in the milking environment. These findings are of major concern in dairy farms, taking into account the association between the occurrence of biofilms and bovine mastitis (Melchior et al., 2012). Staphylococcus aureus biofilm-producing strains have shown greater ability than non-biofilm-producing strains to adhere to the mucosa of the mammary gland. Moreover, Staphylococcus aureus strains with phenotypically active genes encoding biofilm components may have the ability to start biofilm production, causing persistent intramammary infections (Baselga et al., 2003).

The mechanism for formation of Staphylococcus aureus biofilms on surfaces is a complex process, resulting from physical-chemical interactions between different components, including material surface properties, surface properties of bacteria and environmental factors. Therefore there is a need for further studies for an effective control of undesirable biofilms in the environment of dairy farms. The main issues should include the initial investigation of the prevalence and identification of Staphylococcus aureus strains with the ability to produce biofilms on materials commonly used in the dairy industry, the evaluation of different concentrations of new and commonly used sanitizers in milk handling and processing lines, and how the natural mixed microbiota influences pathogen reduction during disinfection (Lee et al., 2014). Importantly, these studies should be carried out on a regional basis using local dairy herds, since Staphylococcus aureus strains found in the milking environments show considerable variability in relation to various parameters of growth and metabolic activity.

1.1       AIM AND OBJECTIVES

The aim of this study is to determine incidence of biofilm formation among Staphylococcus aureus isolated from food contact surfaces in Michael Okpara University of Agriculture, Umudike eateries, while the specific objectives are;

·       To isolate and identify Staphylococcus aureus species from food contact surfaces.

·       To determine the biofilm formation potential of Staphylococcus aureus species isolated from the food contact surface.

·       To determine the antibiotic susceptibility profile of the Staphylococcus aureus isolates.

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