BACTERIAL CONTAMINATION OF MOUTH CONTACT POINT FOR CONSUMPTION OF CANNED SOFT DRINKS

ABSTRACT

Bacteria contamination of mouth contact point for consumption of canned soft drinks sold in Umuahia were carried out in Umuahia metropolis. About 100 samples of canned soft drink were used in this study, they includes beta-malt, Grand malt, Amstel malt, Malta Guiness and Dubic malt. Five (5) bacterial genera were isolated which include Bacillus sp., Staphylococcus aureus, Streptococcus sp., Escherichia coli, Bacillus sp, COANS, Micrococcus sp, Enterococcus faecalis, Klebsiella sp and α-haemolytic Streptococcus. Occurrence of bacterial isolates showed that CoANS has the highest occurrence of 30(42.86%) while Pseudomonas aeruginosa 2(2.85%), Micrococcus sp., α-haemolytic Streptococcus 1(1.42%), Enterococcus faecalis 1(1.42%) had the lowest occurrence. CoANS had the highest occurrence of 30(42.86%) while Pseudomonas aeruginosa 2(2.85%), Micrococcus sp., α-haemolytic Streptococcus 1(1.42%), Enterococcus faecalis 1(1.42%) had the lowest occurrences.

TABLE OF CONTENTS

Certification                                                                                                                            i

Dedication                                                                                                                              ii

Acknowledgements                                                                                                                iii

Table of Contents                                                                                                                   iv

List of Tables                                                                                                                          v

Abstract                                                                                                                                  vi

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1              Aim                                                                                                                             4

1.2              Objectives                                                                                                                   4

CHAPTER TWO

2.0       Literature review                                                                                                         5

2.1       Biofilm formation                                                                                                       7

2.2       Factors that affect microbial attachment to abiotic surfaces                                      9

2.2.1    Properties of food contact surfaces                                                                            9

2.2.2    Topography of food contact surfaces                                                                         9

2.2.3    Contact time                                                                                                               10

2.2.4    Adhesive properties of the bacterial cell surface                                                        11

2.2.5    Substratum preconditioning                                                                                        13

2.2.6    Microcolony formation                                                                                               14

2.2.7    Maturation of the biofilm                                                                                           15

2.3       Factors influencing biofilm development                                                                   16

2.3.1    Detection methods                                                                                                      19

2.3.2    Factors influencing detachment                                                                                  20

2.4       Foodborne pathogens and spoilage organisms in biofilms                                         22

2.4.1    Listeria monocytogenes                                                                                               22

2.4.2    Pseudomonas spp.                                                                                                      22

2.4.3    Bacillus spp.                                                                                                                22

2.4.4    Salmonella spp.                                                                                                           22

2.5       Biofilm removal and control                                                                                       23

2.5.1    Cleaning                                                                                                                      23

2.5.2    Sanitizing                                                                                                                    25

2.5.3    Equipment design                                                                                                       28

2.5.4    Biofilm detection                                                                                                        29

2.6       Consequences of biofilm development                                                                       30

CHAPTER THREE

3.0       Materials and methods                                                                                                32

3.1       Study location                                                                                                             32

3.2       Media used                                                                                                                 32

3.3       Sample collection                                                                                                        32

3.4       Microbiological analysis                                                                                              33       

3.4.1    Inoculation                                                                                                                  33

3.4.2    Characterization of bacteria                                                                                        33

3.5       Gram staining reaction                                                                                                33

3.6       Biochemical identification of bacterial isolates                                                         34

3.6.1    Catalase                                                                                                                       34

3.6.2    Coagulase test                                                                                                             34

3.6.3    Citrate test                                                                                                                  34

3.6.4    Indole test                                                                                                                   34

3.6.5    Carbohydrate (sugar) utilization test                                                                          35

3.6.6        Antibiotic sensitivity testing                                                                                       35

CHAPTER FOUR   

4.0       Results                                                                                                                        36

CHAPTER FIVE

5.0       Discussion and conclusion                                                                                          41

5.1       Discussion                                                                                                                   41

5.2       Conclusion                                                                                                                  43

5.3       Recommendation                                                                                                        44

            References

LIST OF TABLES

Table                                                           Title of tables                                                    Page

 1                                 List of canned soft drinks                                                                 37

 2                                 Biochemical characterization of bacteria isolates                           38

 3                                 Percentage occurrence of bacteria isolates                         39

 4                                 Antibiotic sensitivity test                                                                   40

CHAPTER ONE

1.0       INTRODUCTION

Contact surfaces are considered a serious factor contributing to contamination of mouth contact point if not properly cleaned (WHO, 2008). In addition, surface contamination may lead to bacteria biofilm formation which enhances the capacity of food-borne bacteria to survive stress conditions encountered within food processing environments (Giaouris et al., 2012). Surface contamination by pathogenic bacteria results in serious food-borne outbreaks generating a considerable disease burden and also economic losses (Sofos and Geornaras, 2010). The economic cost of food-borne outbreaks is highly affecting the US economy at a cost of 50 to 80 billion US dollar annually. Other statistics has estimated that the total burden of FBDs was 152 billion US dollar. In Australia and New Zealand, the cost of food-borne outbreaks has been estimated at 1,289 billion and 86 million US dollar respectively per year. In Sweden, the annual cost of food-borne outbreaks was estimated to be 171 million US dollar (Toljander et al., 2012). In this regard, globalizing of food market with worldwide transportation makes food safety a major priority in order to prevent spreading of pathogenic bacteria and bacteria poisoning outbreaks worldwide. In England and Wales, FBDs cause more than 2 million cases, 21,138 hospitalizations and 718 deaths per year. Pathogenic bacteria are able to adhere and form biofilms on various food contact surfaces (Di Bonaventura et al., 2008). It is now established that the persistence of pathogenic bacteria on food contact surfaces, equipment and processing environments, is a contributing factor in food-borne outbreaks, especially those involving L. monocytogenes, B. cereus, S. aureus, E. coli and Salmonella spp. (Gounadaki et al., 2008). Equipment, utensils and cutting boards are likely to be the key cross contamination routes as they become contaminated with pathogens from the handlers, sewage, water and condensation caused by the faulty ventilation. Therefore, it has been reported that in the United Kingdom, 14 % of all food-borne illnesses involving S. aureus, E. coli, Salmonella enterica and L. monocytogenes, may be due to inadequately cleaned cutting boards and knives (de Jong et al., 2005). According to the French national health monitoring institute (InVS), 1,380 FBD outbreaks were reported in 2014, affecting 12,109 people, including 649 hospitalizations and 2 deaths. The three most frequently suspected pathogens were S. aureus (30%), B. cereus (22%) and Salmonella spp. (15%). The French available data showed also that contact surfaces were up to 60 % involved in FBD outbreaks (2011) in collective and home catering. In fact, stainless steel and aluminium represent a favorable environment for bacterial adhesion and biofilm formation (Donlan, 2002). Contact surfaces are often contaminated by pathogenic bacteria including L. monocytogenes, S. aureus, Salmonella spp., B. cereus and E. coli. Moreover, it has been reported that even after cleaning, E. coli bacterial densities up to 10⁵CFU/cm² could be recovered on food processing surfaces (Marouani-Gadri et al., 2010). The physicochemical properties of both bacteria cell and material surfaces (Stainless steel, aluminum) are very critical proprieties affecting the adhesion of bacteria and the formation of biofilm (Renner and Weibel, 2011). Moreover, bacterial adhesion is an extremely complicated process that is affected by many other factors including the environmental conditions (pH, temperature, bacterial concentration, nutrient availability and the associated flow conditions) that need to be controlled in order to find strategies against biofilm formation. The number of attached bacteria is significantly affected by the flow conditions and generally the number of attached bacteria decreases when shears rates are high. Moreover, variations in pH value in the culture environment also influence bacterial adhesion and the growth of biofilm. The pH influences the cell surface hydrophobicity and better adhesion to hydrophobic surfaces was found at pH in the range of the isoelectric point when bacteria are uncharged (Bunt et al., 1993). Therefore, pH influences bacterial adhesion by influencing the surface charge and changing surface characteristics of the bacteria. Moreover, variations in external pH can disturb the trans-membrane electrochemical gradient and have a biocidal effect on the microorganisms. The growth temperature is also an important condition for bacterial adhesion and biofilm formation as well as the presence of nutrient. High growth temperature was found to increase the biomass and the attachment ability of bacteria probably, due to the production of heat stress proteins associated with the cell surface. Otherwise, different studies concerning S. aureus biofilm formation have shown that temperature variation has no clear effect on the biomass. Thus, optimum temperature enhances the biofilm formation. Temperature also affects the bacterial surface polymer composition which decreases at low temperature and reduces the adhesive properties of bacteria. Another important factor in biofilm formation is nutrient availability. In fact, nutrients influence the surface charge of bacteria. For instance, glucose and lactic acid in the growth medium decreased the bacterial cell wall electro-negativity through the neutralization of the surface charge. Thus, a synergistic effect between the environmental factors may occur and affect biofilm formation. Modified abiotic surfaces expected to be used inside or in contact with human body have to meet the demands required for both their surface and bulk properties (Maillard, 2005). Several studies have indicated that various bacteria, including Escherichia coli, Staphylococcus aureus and Salmonella spp., survive on hands, sponges/cloths, utensils and currency for hours or days after initial contact with the microorganisms (Scott and Bloomfield, 1990). Kusumaningrum et al., (2003) showed that the presence of residual foods and the level of contamination on stainless steel surfaces may have an important role as it may improve the survival of Salmonella enteritidis, Staphylococcus aureus and Campylobacter jejuni for several hours or even days. Haeghebaert et al., (2001) suggested that 40.5% of all food-borne infection outbreaks registered in 1998 in France were linked to contamination by equipment with biofilms. Biofilm formation is a well-known bacterial mode of growth and survival on food surfaces as Reuter et al. described for Campylobacter jejuni in the food chain and during transfer between hosts (Reuter et al., 2010). Barnes et al. (1999) suggested that surface roughness may play an important role in the adhesion of microorganisms by protecting them from shear forces and increasing the available surface area. In this study, greater numbers of S. aureus adhered to untreated steel (with the rougher surface). In the same study, the authors showed cross-contamination caused by contact surfaces such as stainless steel. Today, quick and cheap methods have to be defined and standardized which are especially easy to perform in the field. Innovative methods are needed to better control microbiological hazardous events on abiotic (non-living) materials.

1.1       AIM

i. To evaluate the bacterial contamination at the mouth contact point of canned soft drinks sold in Umuahia, Abia state.

1.2       OBJECTIVES

i.                    To isolate and identify bacteria found at the mouth contact point of canned soft drinks;

ii.                  To determine the percentage occurrence of pathogenic bacteria observed at the mouth contact point of canned soft drinks;

iii.                To determine the antibiotic sensitivity pattern of isolates in the study

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