BIOFILM FORMATION BY STAPHYLOCOCCUS AUREUS ISOLATED FROM MEAT CONTACT TABLE SURFACES

ABSTRACT

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. In the present investigation, Staphylococcus aureus species isolated from meat contact table surfaces was evaluated for their antibiotic susceptibility profile and biofilm production.  A total of 100 swabbed meat contact table surfaces was inoculated into Mannitol salt agar plates. From the 100 swab surfaces analyzed, 61 representing 61% of the total sample Harbored S. aureus. The 61 isolates were tested for their antimicrobial susceptibility, Levofloxacin gave the highest zone of inhibition with (83.6%), followed by Ofloxacin (81.9%), Ciprofloxacin (78.7%), Gentamicin (68.9%), Erythromycin (19.7%), Ceftriaxone (14.8%), Cefepine (4.9%), Augmenting (0.0%), Cefazidime (0.0%), Cefuroxine (0.0%). The biofilm detection test was carried out using Congo red agar (CRA) method.  A total of 26 (42.6%) of the isolates appeared as black crusty colonies on the CRA which indicates them as strong biofilm producers while 35 (57.4) which appeared as smooth pink colonies were non biofilm producers.

TABLE OF CONTENTS

Title page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          ix

Abstract                                                                                                                                  x

CHAPTER ONE

Introduction                                                                                                                            1

1.1       Background of Study                                                                                                  1

1.2       Aim and Objective                                                                                                     4

CHAPTER TWO: LITERATURE REVIEW

2.1       Staphylococcus aureus                                                                                               5

2.1.1    Etiology                                                                                                                      5

2.1.2    Scientific identification                                                                                              6

2.1.3    History                                                                                                                        6

2.1.4    Discovery                                                                                                                    6

2.1.5    Evolution                                                                                                                    7

2.1.6    Pathogenicity                                                                                                              8

2.1.7    Virulence factor                                                                                                          9

2.1.8    Epidemiology                                                                                                             10

2.1.9    Control and Prevention                                                                                               10

2.2       Colonization of Meat and food contact table surfaces by S. aureus                                    11

2.2.1    Sources of meat contamination                                                                                  11

2.2.2    Risks Associated with Consumption of S. aureus Contaminated Meat                12

2.2.3    Antibiotics Resistance/susceptibility profile of staphylococcus aureus from

meat contact table surfacess                                                                                       12

2.3       Biofilms                                                                                                                      13

2.4       Development of formation of biofilm                                                                        16

2.4.1    Initial attachment                                                                                                        17

2.4.2    Irreversible attachment                                                                                               17

2.4.3    The cell matrix                                                                                                            17

2.4.4    Maturation                                                                                                                  18

2.4.5    Dispersal                                                                                                                     18

2.5       Staphylococcus aureus biofilm                                                                                  19

2.5.1    PIA-dependent biofilm formation                                                                              19

2.5.2    PIA-independent biofilm formation                                                                           20

2.5.3    eDNA and biofilm formation                                                                                     21

2.6       S. aureus biofilm related diseases                                                                              21

2.6.1    Osteomyelitis                                                                                                              22

2.6.2    Indwelling medical device infection                                                                          22

2.6.3    Periodontitis and peri-implantitis                                                                               22

2.6.4    Chronic wound infection                                                                                            23

2.6.5    Chronic rhinosinusitis                                                                                                 23

2.6.6    Endocarditis                                                                                                                23

2.6.7    Ocular infection                                                                                                          23

2.7       Therapy and prophylaxis of S. aureus biofilm infections                                          23

2.7.1    Antimicrobial therapy                                                                                                24

2.7.2    Inclusion of antimicrobial agents at the site of infection                                           25

CHAPTER THREE

MATERIALS AND METHODS

3.1       Sources of samples                                                                                                     26

3.2       Sterilization of materials                                                                                            26

3.3       Media used                                                                                                                  26

3.4       Sample preparation and isolation of microorganism                                                  26

3.4.1    Sample inoculation                                                                                                     26

3.5       Identification of the isolate                                                                                         27

3.5.1    Gram staining                                                                                                             27

3.6       Biochemical test                                                                                                         27

3.6.1    Coagulase test                                                                                                             27

3.6.2    Catalase test                                                                                                                28

3.6.3    DNAse test                                                                                                                 28

3.7       Susceptibility of isolates to antibiotics                                                                       28

3.7.1    Susceptibility testing                                                                                                  28

3.8       Biofilm formation assay                                                                                             29

CHAPTER FOUR

Results                                                                                                                                    30

CHAPTER FIVE: DISCUSSION, CONCLUSION, RECOMMENDATION

5.1       Discussion                                                                                                                   35

5.2       Conclusion                                                                                                                  36

5.3       Recommendation                                                                                                        36

References                                                                                                                  37

LIST OF TABLES

Table                              Title                                                    Page

4.1       The occurrence of S aureus in meat contact table surfaces in Umuahia                     31

4.2       Colonial and biochemical features of the isolates                                                      32

4.3       Drug susceptibility profile of S aureus isolates from meat contact table surface    33

4.4       Biofilm formation by the S aureus isolates (Congo red method)                                    34

CHAPTER ONE

INTRODUCTION

1.1       BACKGROUND OF STUDY

In the food industry, biofilms increase bacterial resistance to environmental stresses including cleaning, disinfection, and inhibition, enabling these microorganisms to persist on surfaces and processing equipment, compared to planktonic cells (Kostski et al., 2012; Laird et al., 2012; Bridier et al., 2015). Formation of biofilms can occur on all types of surfaces of technological systems in food contact surfaces. The detection of biofilms in the food industry can be related to the presence of pathogenic microorganisms in the industrial settings.

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). Biofilms are aggregates of microbial cells surrounded by a matrix of exopolymers, which confers resistance to these microorganisms (Costerton et al., 1999). Bacteria that aggregate to form biofilms are known to possess greater resistances to stress conditions than their planktonic counterparts, which are dispersed in the environment, including the susceptibility to sanitizers and other antimicrobials (Fux et al., 2004).

Staphylococcus aureus is a Gram-positive, ubiquitous bacterial species. In the human population, approximately 20–25% has become persistently colonized and 75–80% intermittently or never colonized. Previous studies have shown that there is a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection. Nasal carriage provides a staging ground for S. aureus to disseminate to other areas of the body where, once transmitted to the circulatory system through an epithelial breach, planktonic growth and upregulation of adherence factors occurs. Invading staphylococci are then either removed by the host innate immune response or attach to host extracellular matrix proteins and form a biofilm. The cellular physiology is then quickly transformed into one reflective of a biofilm. Owing to the escalating involvement of Staphylococcus aureus in foreign body-related infections, the swift development and exhibition of multiple-antibiotic resistance, and their predilection to transform from an acute infection to one that is persistent, chronic and recurrent, this pathogen continues to receive considerable attention.

Poor hygiene practices in food processing plants may result in the contamination of food products with pathogens, which means a serious risk for the health of consumers. Moreover, the complete elimination of pathogens from food processing environments is a difficult task, in part because bacteria can attach to food contact surfaces and form biofilms, where they survive even after cleaning and disinfection (Brooks and Flint, 2008). Biofilms are the most common bacterial lifestyle in nature. Biofilms are a serious problem in many food industry sector Staphylococcal food poisoning is caused by an infection with Staphylococcus aureus bacterium (Case, 2004). This bacterium Staphylococcus aureus, which can be carried by food causes food poisoning and other food-borne diseases (Foskett et al., 2003).Staphylococcal food poisoning is an illness caused by a toxin or poison released by bacteria from the staphylococcus group (Lennox et al., 2012). It is a food borne intoxication that develops in people who ingest food that has been improperly stored or cooked (particularly food such as processed meats, chicken, pastries, and hollandaise sauce) in which Staphylococcus aureus has grown (Prescott et al., 2008).

S. aureus is found in the environment and is also found in normal human flora, located on the skin and mucous membranes (most often the nasal area) of most healthy individuals (Rasigade and Vandenesch, 2014). S. aureus does not normally cause infection on healthy skin; however, if it is allowed to enter the bloodstream or internal tissues, these bacteria may cause a variety of potentially serious infections (Rasigade and Vandenesch, 2014). Transmission is typically from direct contact. However, some infections involve other transmission methods (Tong et al., 2015).

Staphylococcus aureus is a food-borne pathogen that can cause staphylococcal food poisoning. In the USA, staphylococcal food poisoning is estimated to account for 241,188 illnesses, 1,064 hospitalizations, and six deaths, annually (Scallan et al., 2011). S. aureus can adhere to and develop biofilms on food contact surfaces, thereby affecting the quality and safety of food products (Marques et al 2007; Srey et al., 2013).

Staphylococcus aureus including methicillin-resistant S. aureus (MRSA) has the propensity to form biofilms, and causes significant mortality and morbidity in individuals. S. aureus biofilm mode of growth is tightly regulated by complex genetic factors. Host immune responses against persistent biofilm infections are largely ineffective and lead to chronic disease. However, current research has taken biofilm formation into account in terms of elucidating host immunity toward infection, and may lead to the development of efficacious anti-biofilm S. aureus therapies.

The extracellular matrix of S. aureus biofilms is usually composed of exopolysaccharide (PIA/PNAG), but the proteinaceous and extracellular DNA matrix can also be present in staphylococcal biofilms (Boles et al., 2010). Depending on the environment in which the biofilm was developed, the biofilm matrix can also contain blood components or non cellular materials such as mineral crystals, corrosion particles, and clay or silt particles (Donlan, 2002). PIA is linked to the irreversible attachment phase (Szczuka et al., 2013). The formation of biofilm of Staphylococcus aureus is not only mediated by the PIA-dependent biofilm formation, but it can exist in PIA-independent biofilm. In the PIA-independent biofilm, despite the importance of the ica gene locus in biofilm development, biofilms can occur in an ica-independent fashion where biofilm-associated protein (Bap) and Bap-related proteins of S. aureus can confer biofilm development independently or PIA production through cell-to-cell aggregation and are characterized by their high molecular weight, presence of the bacterial surface, role as a virulence factor, and occasional containment in mobile elements (Lasa and Penades, 2006;  Archer et al., 2011).

•       Aim and Objectives

The aim of this work is to evaluate the biofilm-forming ability of S. aureus isolates, recovered from meat contact table surfaces in Umuahia.

Specific objectives

•       Isolation and identification of S. aureus from the samples.

•       Determination of the antibiogram of the S. aureus isolates

•       Determination of the biofilm forming ability of S. aureus

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