Methicillin Resistant Staphylococcus Aureus In Raw Meat Biology Essay

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Humans have enjoyed an intimate relationship with domestic animals for thousands of years, sharing habitats, many a times encroaching on animal habitats, which gave birth to zoonoses by sharing infections with each other. In the UK, one of the best known infections is caused by Methicillin Resistant Staphylococcus aureus (MRSA). CA-MRSA in fit healthy people is not a particular problem, is a much greater problem for hospitalized patients. Recently MRSA (animal and human) have been isolated from raw meat. Different studies show prevalence of positive samples ranging from 2.5% to 11.9% with indications showing rapid spread of MRSA across the European countries. Sixty percent of all meat consumed in the UK is imported from European countries where the presence of MRSA is already confirmed. According to The Department for Environment, Food and Rural Affairs (DEFRA) there is no UK legislation to control or detect MRSA in meat. There is little evidence for the occurrence of MRSA in food producing animals and also Staphylococcus aureus is not generally considered to be major pathogen in livestock species other than cattle where it is responsible for causing mastitis which causes heavy economic losses.

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The current research aimed to determine the prevalence of MRSA in retail meat sold in and around Manchester. A double enrichment method was used to isolate and detect MRSA from chicken, pork and beef (30 samples of each). Samples were collected from supermarkets and retail butchers shops. The result showed that MRSA was present in meat with a prevalence of 3.3%(3) from chicken, 1.1%(1) from pork and 1.1%(1) from beef samples. Total 5 MRSA isolates obtained were of the UK origin and isolated from the packed supermarket meat.

Further, study is recommended for typing of isolates to trace back the origin of MRSA, whether human or animal and to determine the route of transmission of MRSA in to the meat.

List of Abbreviations

BTM

Bulk Tank Milk

CA-MRSA

Community Associated or Community Acquired MRSA

CCs

Cassette Chromosomes

DEFRA

Department for Environment, Food and Rural Affairs

EMRSA

Epidemic Strian of MRSA

HA-MRSA

Healthcare Associated or Healthcare Acquired MRSA

HPA

Health Protection Agency

IgG

Immunoglobulin G

LA-MRSA

Livestock Associated MRSA

MHB

Mueller-Hinton Broth

MRSA

Methicillin resistant Staphylococcus aureus

MSSA

Methicillin susceptible Staphylococcus aureus

NT

Non-Typable

NT-MRSA

Non-Typable MRSA

PBP

Penicllin Binding Protein

PBP2a

mecA bound or altered PBP

PFGE

Pulsed-Field Gel Electrophoresis

PHMB+

Phenol Red Mannitol Broth containing Ceftizoxime (5 μg/ml) and Aztreonam (75 μg/ml)

SCCmec

Staphylococcal Cassette Chromosome

spa

polymorphic X region of the protein A gene

SPSS

Statistical Package for the Social Sciences

ST398

Sequence Typing Cluster 398

TSA

Tryptone Soya Agar

List of Figures

Figure 1: Resistance Mechanism of MRSA

Page 8

Figure 2: Prevalence of MRSA isolates according to type of meat.

Page 24

Figure 3: Prevalence of MRSA isolates according to packed meat or retail meat.

Page 24

Figure 4: Prevalence of MRSA isolates according to origin of meat from different countries.

Page 25

Figure 5: Typical denim blue colored colonies on BrillianceTM MRSA.

Page 25

List of Tables

Table 1: Prevalence of MRSA in beef, pork and chicken from different studies conducted so far

Page 17

Table 2: Prevalence of MRSA isolates according to type of meat

Page 23

Chapter I: Introduction

1.0 Introduction:

Humans have enjoyed an intimate relationship with domestic animals for many years, sharing habitats and, encroaching animal habitats, which gave birth to zoonoses by sharing infections with each other. 'Any disease or infection that is naturally transmissible from vertebrate animals to humans and vice-versa is classified as a zoonoses' (Anon, 2010b). There are 61% of human infectious diseases which are zoonotic, 75% of human emerging infectious diseases are zoonotic, 33% of zoonoses are transmissible between humans (Taylor & Latham, 2001). Zoonotic diseases are an understudied aspect of global health, despite their potential to cause significant disease burden in wild and domestic animal populations and affect global economies. One of the key potential zoonotic pathogens is methicillin-resistant Staphylococcus aureus, MRSA (Epstein & Price, 2009) which is a variant of Staphylococcus aureus (S. aureus) commonly found on the skin and mucosa of anterior nares of up to 25% of healthy humans and animals, S. aureus produces at least seven different types of toxins causing food poisoning (Anon, 2006, 2010a).

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S. aureus is considered to be the most virulent of the Staphylococcus genus due to the wide spectrum of disease it can cause, ranging from localized and systemic infections to toxin-mediated illness. S. aureus infection was a major source of morbidity and mortality, before the introduction of penicillin in early 1940s. Only two years later resistant strains of S. aureus were reported in the UK, and by 1960 almost 80% of S. aureus isolates were resistant to most common antibiotics used in hospitals giving birth to international epidemic across many countries (Lowy, 2003; Vanderhaeghen et al., 2010b). MRSA was first identified as a nosocomial pathogen in human hospitals. These organisms cause same type of infection as of S. aureus. These strains are termed as Healthcare Associated or Healthcare Acquired MRSA, HA-MRSA (Lee, 2003; Middleton et al., 2005). MRSA has since emerged as a serious concern in human medicine (Fitzgerald et al., 2001; Weese et al., 2005).

Since the 1990s, MRSA has also become a concern in people who have not been hospitalized at all or recently had an invasive procedures; the strains that cause such infections are termed as CA-MRSA, Community Associated or Community Acquired MRSA, (Lee, 2003; Weese et al., 2005; Cuny et al., 2006; Otter & French, 2010). CA-MRSA was reported in the high risk population such as drug users, people in nursing homes, and people who were chronically ill (Duquette & Nuttall, 2004). Outbreaks have also been reported in the community e.g. one outbreak involved five members of a UK rugby football team found positive for MRSA (Stacey et al., 1998). These strains have been termed as community acquired MRSA (CA-MRSA).

In the UK, MRSA is probably the most well known infections. MRSA in fit healthy people is not a particular problem at present but, MRSA is a much greater problem for hospitalized patients. At present 30-40% of S. aureus bloodstream infections in hospitals in England are caused by MRSA. The resistance to common antibiotics can make these infections more difficult to treat. There are a number of strains of MRSA. Those responsible for most infections in the UK are well adapted to spread between patients. A higher proportion of patients are now susceptible to these infections. "Improved medical care prolongs life but can leave patients with weakened immune systems" (Anon, 2004). Mortality in patients diagnosed with MRSA bacteraemia in England during 2004-2005 was found to be high, with 38% of individuals dying within 30 days of diagnosis, rising to 57% in patients aged ≥85 years (Lamagni et al., 2010). This shows the severity of present human colonization of MRSA in the UK.

MRSA has become an emerging public health problem worldwide, no longer only associated with healthcare-associated infections. With the exception of some recent reports confirming infections in cattle, cats, dogs and horses, infections with MRSA in companion animals have been infrequently reported (Cuny et al., 2006). MRSA has been isolated from dogs and cats (Duquette & Nuttall, 2004). MRSA has also been isolated from farm animals mostly pigs (De Neeling et al., 2007). MRSA have been detected in foods such as bovine milk, cheese, meat products and raw chicken meat (Vanderhaeghen et al., 2010b). It was first recognized as zoonosis in the Netherlands (Voss et al., 2005; Cuny et al., 2008) when same strain of MRSA was isolated from the pigs, pig farmers, veterinarian, veterinarian's son and the nurse who treated veterinarian's son; all of them were directly or indirectly related to pig farming (Voss et al., 2005). Since then MRSA has been detected in number of countries across Europe (Battisti et al., 2009; Denis et al., 2009; Van den Eede et al., 2009). MRSA can be transmitted between people and animals during close contact (Seguin et al., 1999b; Duquette & Nuttall, 2004; Weese et al., 2005; Cuny et al., 2006; Weese et al., 2006).

Recently, MRSA has also been isolated from meat products in Netherlands, especially from pork (van Loo et al., 2007a; De Boer et al., 2009). The MRSA strain associated with pig is ST398 (Cuny et al., 2006). This ST398 type was emerged from the livestock i.e. pig (Armand-Lefevre et al., 2005; Van Loo et al., 2007b) so termed as LA-MRSA Livestock Associated MRSA (Vanderhaeghen et al., 2010b).

As both human and animal MRSA strains have been isolated from meat products in Netherlands. Two studies have reported ST398 from retail meat. The prevalence of positive samples ranged from 2.5% to 11.9% (van Loo et al., 2007a; De Boer et al., 2009). Human MRSA has also been reported from chicken meat in Japan and Korea (Lee, 2003; Kitai et al., 2005).

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Most of the pork meat consumed in UK, approximately 60% is imported from European countries and much of it comes from Netherlands, Denmark, Spain and Germany where MRSA in pigs has been confirmed. There is little evidence for the occurrence of MRSA in food producing animals and also S. aureus is not generally considered to be major pathogen in livestock species other than cattle where it is responsible for causing mastitis which causes heavy economic losses (Cóilín & Young, 2007; Vanderhaeghen et al., 2010a).

This continuous emergence of MRSA in the community is a public-health problem that warrants increased vigilance for the diagnosis and control of MRSA (Diederen & Kluytmans, 2006) in whatever means it is possible, which should be monitored for its presence in both food and pet animals (Saleha et al., 2010). There is a real risk that MRSA may be transmitted in to the community through raw meat and bovine milk and has the potential to cause human infections. Although, the precise cause and route of MRSA into the veterinary field and community has not been fully determined, but MRSA does exist in this habitat and is transmitted regardless of antibiotic use (Kwon et al., 2006).

The aim of this study was to investigate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in raw meat sold across the city of Manchester.

1.1 Objectives of the study:

To undertake a literature review of MRSA epidemics across the globe.

To use 'Double Enrichment method' for isolation and detection of MRSA isolates from raw meat.

To analyze the prevalence of MRSA isolates using SPSS software.

To correlate positive samples with factors such as origin of meat, whether meat is packed or retail from butcher shop.

To give recommendations for further research and gene sequencing of isolates to determine origin of MRSA isolates whether it is human or an animal origin.

1.2 Methodology:

A literature review will be undertaken to understand epidemiology of MRSA and best method to isolate and detect MRSA from meat. Data will be collected from the library sources such as journals articles, books, organization and government websites to support relevant arguments.

Chapter II: Literature Review

2.0 Introduction:

S. aureus is considered to be the most virulent of the Staphylococci genus due to its wide spectrum of disease, ranging from localized and systemic infections to toxin-mediated illness, before the introduction of penicillin in early 1940s. Only then two years later the resistant strain of S. aureus was reported in UK, and by 1960 almost 80% of S. aureus isolates were resistant to most common antibiotics used in hospitals giving birth to international epidemic of methicillin resistant S. aureus (MRSA) across of many countries (Lowy, 2003; Vanderhaeghen et al., 2010b). This chapter will discuss the origin of MRSA and its journey so far and how it had entered the food chain across the globe and poses public health risk.

2.1 Staphylococcus aureus:

Microbiology of Staphylococcus aureus:

Staphylococcus aureus (S. aureus) is a Gram-positive bacterium. The name 'Staphylo' is derived from ancient Greek, meaning 'bunch of grapes', reflecting the appearance of the micro-organism under the microscope (Trusts & Trusts, 2008). S. aureus are catalase positive cocci (Forbes et al., 2002). S. aureus have cell wall of peptidoglycan which may act as an endotoxin, and have important structural components such as polysaccharide and surface proteins (Lowy, 1998).

S. aureus expresses many potential virulence factors.

Surface proteins that promote colonization of host tissues.

Factors that probably inhibit phagocytosis (capsule, immunoglobulin binding protein A).

Toxins that damage host tissues and cause disease symptoms. Coagulase-negative Staphylococci are normally less virulent and express fewer virulence factors. S. epidermidis readily colonizes implanted devices, however (Anon, 1996).

S. aureus is a common colonizer of the skin and mucosa of noses of up to 25% of healthy humans and animals. It can cause disease if the organism gains entry in the body. It is one the most important organisms as it can produce seven different types of toxins which frequently causes food poisoning (Anon, 2006, 2010a). S. aureus remains a versatile and dangerous pathogen in humans and there is a steady increase in frequencies of both community-acquired and hospital-acquired staphylococcal infections, with little change in overall mortality rate (Lowy, 1998).

In animals, S. aureus is one of the three major pathogenic Staphylococcus species, together with S. (pseudo) intermedius and S. hyicus. The scale of infections it may be involved in, is as broad as the number of animal species suffering from it, ranging from pneumonia, joint infections, osteomyelitis and septicemia in poultry, subcutaneous abscesses, mastitis and pododermatitis in rabbits, dermatitis and cellulitis in horses to septicemia in pigs. However, S. aureus plays its most significant animal pathogenic role as cause of intramammary infections in cattle and small ruminants, leading to considerable economic losses in cattle farming (Vanderhaeghen et al., 2010b). S. aureus is without question the most virulent of the Staphylococci, due to its wide spectrum of disease, ranging from localized and systemic infections to toxin-mediated illness (Forbes et al., 2002).

S. aureus has ability to gain resistance to almost all antibiotics to which it was previously sensitive. Penicillin was introduced in early 1940s which allowed dramatic improvement in prognosis of the patients with staphylococcal infection. But in late 1960s more than 80% of the cases were almost resistant to penicillin. In 1961, 'methicillin' the first of the semisynthetic penicillinase-resistant penicillins was introduced. This followed by the rapid reports of the methicillin resistant isolates. Since then methicillin resistant strains are the critical ones for the clinician (Lowy, 2003).

2.2 Methicillin Resistant Staphylococcus aureus (MRSA):

As stated above, methicillin was used for the treatment of S. aureus infection in patients in early 1960s. Massive use of methicillin, lead to the development of the resistance in S. aureus which have the acquired resistance to the β-lactamase, stable β-lactam antibiotics also, are called as methicillin resistant S. aureus i.e. MRSA.

Resistent Mechanism of MRSA.jpg

Figure 1 Resistance Mechanism of MRSA (Larson & Reah, 2010)

Bimodal resistance is exhibited by S. aureus

By producing Beta-lactamase enzyme,

By acquiring mecA gene which alters Penicillin Binding Protein-PBP2a in bacterial cell membrane which shows low affinity to all β-lactam antibiotics.

This makes MRSA resistant to other penicillins, cephalosporins and other beta-lactam antibiotics, such as meropenem, imipenem and aztreonam.

Resistance can be bimodal due to presence of mecA gene. This gene alters penicillin-binding protein (PBP) 2a. PBP are important cell membrane components involved in peptidoglycan synthesis, which β-lactam antibiotics bind to. In normal strains methicillin binds to and inhibits PBP2, which makes them methicillin sensitive. In MRSA strains PBP" is replaced by PBP2a, which shows a very low affinity for almost all β-lactam antibiotics, methicillin-resistant S. aureus (MRSA) is resistant to other penicillins, cephalosporins and other beta-lactam antibiotics, such as meropenem, imipenem and aztreonam (Georgopapadakou et al., 1982; Hartman & Tomasz, 1984; Chambers, 1997; Trusts & Trusts, 2008; Vanderhaeghen et al., 2010b). The mecA gene resides on a large heterogeneous mobile genetic element called the (SCCmec) staphylococcal cassette chromosome (Ito et al., 1999; Katayama et al., 2000). Studies also show that most methicillin-resistant Staphylococcus aureus (MRSA) strains also produce beta-lactamase (Norris et al., 1994). Both mecA and enzymatic type of resistance is shown in Figure 1.

Analysis of genetic background of MRSA describes SCCmec variations which include ST1, ST8, ST30, ST59, ST80 and ST93 these are relatively large and are typically found in strains associated with hospitals and other healthcare facilities (Vandenesch et al., 2003), while other SCCmec types IV and V are smaller in size and are usually found in MRSA associated with community-acquired infections (Okuma et al., 2002; Ito et al., 2004; Tristan et al., 2007). Molecular analyses of numerous MRSA strains indicate that resistance genes have been transferred to various methicillin-susceptible S. aureus (MSSA) strains on multiple occasions (Robinson & Enright, 2004).

2.3 History/ Epidemiology of MRSA:

2.3.1 MRSA in Hospitals:

In 1961, the first isolation of MRSA was reported in London hospital (Cóilín & Young, 2007; Trusts & Trusts, 2008; Vanderhaeghen et al., 2010b). After this MRSA was found in many countries, there was epidemic of MRSA worldwide (Panlilio et al., 1992; Johnson et al., 2001; Diekema et al., 2004; Styers et al., 2006; Vanderhaeghen et al., 2010b). The international epidemic which belonged to only five Cassette Chromosomes (CCs) i.e. CC5, CC8, CC22, CC30 and CC45 strains (Enright et al., 2002; McCarthy et al., 2010; Vanderhaeghen et al., 2010b). Use of large and inappropriate quantities of antimicrobials in humans is assumed to be the cause, which triggered the emergence of resistance in micro-organisms against the antimicrobial agents (Vanderhaeghen et al., 2010b). But MRSA remained rare during 1960s and 1970s, and only increased slightly in 1980s. Then further, it increased dramatically from very low to very high levels (Johnson et al., 2005; Cóilín & Young, 2007). Higher acquisition of MRSA was due to prolonged exposure of antimicrobials, prolonged hospitalization, surgical techniques, and close proximity to the patient in the hospital who were infected or colonized with MRSA (Monnet et al., 2004; Monnet et al., 2005).

MRSA was first identified as a nosocomial pathogen in human hospitals. These organisms are termed as Healthcare Associated or Healthcare Acquired MRSA, HA-MRSA (Lee, 2003; Middleton et al., 2005). MRSA has since emerged as a serious concern in human medicine (Fitzgerald et al., 2001; Weese et al., 2005).

2.3.2 MRSA in Community:

Since the 1990s, second phase was unfolding. MRSA has also become a concern in people who have not been hospitalized at all or recently had invasive procedures; the strains that cause such infections are termed as CA-MRSA, Community Associated or Community Acquired MRSA (Udo et al., 1993; Herold et al., 1998; Bukharie et al., 2001; Chambers, 2001; Lee, 2003; Weese et al., 2005; Cuny et al., 2006; Otter & French, 2010).

In UK, outbreaks of infection caused by MRSA were common in hospitals and nursing homes, but none were reported in the community until one outbreak which involved five members of a rugby football team (Stacey et al., 1998). The Health Protection Agency (HPA) says approximately 100 cases with one fatality have been detected. But a recent study found that 1,981 CA-MRSA infections were recorded by primary-care institutions between 2000 and 2004 (Schneider-Lindner et al., 2007). In the UK, probably the most well known infection is MRSA. MRSA in fit healthy people is not a particular problem. But MRSA has become more of a problem for a number of interrelated reasons. At present 40% of S. aureus bloodstream infections in hospitals in England are caused by MRSA. The resistance can make these infections more difficult to treat. There are a number of strains of MRSA responsible for most infections in the UK, which are well adapted to spread from patient to patient. A higher proportion of patients are now susceptible to these infections.

"Improved medical care prolongs life but can leave patients with weakened immune systems" (Anon, 2004).

Mortality in patients diagnosed with MRSA bacteraemia in England during 2004-2005 was found to be high, with 38% of individuals dying within 30 days of diagnosis, rising to 57% in patients aged ≥85 years (Lamagni et al., 2010).

2.3.3 MRSA in Animals:

MRSA is no longer only associated with healthcare-associated infections (Cuny et al., 2006). Third phase of MRSA had started, as the first animal isolate of MRSA was reported from Belgian cow, as a cause of mastitis, which was later confirmed to be of human origin (Devriese et al., 1972; Devriese & Hommez, 1975). Soon MRSA of human origin was reported in pets and later pets were confirmed to be the reservoirs of antimicrobial resistant bacteria (Cefai et al., 1994; Seguin et al., 1999a; Duquette & Nuttall, 2004; Guardabassi et al., 2004; van Duijkeren et al., 2004; Loeffler et al., 2005; Hanselman et al., 2008).

Detecting this strain was relatively easy with pulsed-field gel electrophoresis (PFGE) since it is non-typable (NT), this is the method used for surveillance of MRSA at the National Reference Centre for MRSA (National Institute of Public Health and the Environment, Bilthoven, the Netherlands). Further typing of NT-MRSA showed that almost all strains belonged to 1 multilocus sequence typing cluster, ST398 (Van Loo et al., 2007b). This ST398 type is emerged from the livestock i.e. pig (Armand-Lefevre et al., 2005; Van Loo et al., 2007b) so termed as LA-MRSA, Livestock Associated MRSA (Vanderhaeghen et al., 2010b).

MRSA drew attention once again, when a new type of MRSA was confirmed from the Netherlands (Van Loo et al., 2007b). An unexpected isolation of MRSA from a pig farmer's family and some of their pigs were of the same origin. A study showed that almost 40% of the pigs were colonized with a comparable strain of MRSA (ST398) and that ≈80% of the pig farms were affected (De Neeling et al., 2007). The pig farmers from the same geographical region were carrying MRSA in a >760 x higher carriage rate than the general Dutch population. Further analysis proved that all strains were closely related to each other. Later a pig farmer, a veterinarian and his son along with the nurse treating this boy were found colonized with this MRSA, but one thing was common they were all directly or indirectly related to pig farming (Voss et al., 2005). A recent study reported concurrent skin infection in Belgian swine worker (Denis et al., 2009). MRSA was first recognized as zoonosis in Netherlands, posing risk of human to animal and animal to human as well as human to human transmission, indirectly concluding that pig farming might possess a risk for MRSA carriage in humans (Voss et al., 2005; De Neeling et al., 2007; Cuny et al., 2008; Van Duijkeren et al., 2008).

Human colonization of LA-MRSA is commonly associated with livestock farms such as cow, horse and poultry farms (Juhász-Kaszanyitzky et al., 2007; Van Belkum et al., 2008) including veterinarians who are in close contact with domestic animals (Weese et al., 2005; Wulf et al., 2006). MRSA can be transmitted between people and animals during close contact (Seguin et al., 1999b; Duquette & Nuttall, 2004; Weese et al., 2005; Cuny et al., 2006; Weese et al., 2006) but the potential role of environment in spreading MRSA infection is unclear (Hsieh et al., 2008) due to lack of information on routes of transmission, further research is required (Vanderhaeghen et al., 2010b).

Recent study in Netherlands have reported, hospital based outbreak of LA-MRSA confirming five patients positive for ST398 MRSA strain (Wulf et al., 2006) and another study confirmed animals in long term care facility infected with the human forms of MRSA (Coughlan et al., 2010). Following these studies, ST398 have been detected in 14 countries by healthcare related surveys (Smith & Pearson, 2010). MRSA had also been detected in number of countries across Europe (Denis et al., 2009; Van den Eede et al., 2009).

In UK a study provides an evidence of EMRSA-15 (epidemic strain of MRSA) mucosal carriage in veterinary staff and hospitalized dogs, with the risk of MRSA carriage in veterinary staff being significantly higher than reported for the UK healthy community. EMRSA-15 was predominant in the hospital environment, including humans, dogs, and inanimate objects, but the mode by which the strain was introduced and spread remains uncertain (Loeffler et al., 2005). EMRSA is a well known predominant clone of MRSA across UK since 1991 (O'Neill et al., 2001).

2.4 MRSA in Pigs:

Most studies reported LA-MRSA as a predominant strain in pigs, but only two studies show non-LA-MRSA strain in pigs. Overall, four MRSA isolates were cultured from three pigs and from a clinician/scientist. Two were ST22-MRSA-IV, a human strain type which was associated with epidemic spread. This was reported in Singapore (Sergio et al., 2007) it is also known as EMRSA-15. This is one of the two predominant hospital clones of MRSA which emerged in 1991 in the UK commonly known as epidemic strain (O'Neill et al., 2001), this shows human contamination of pig herds (Lewis et al., 2008). Another study in Canada showed 14% MRSA isolates from pig were of human clone origin and remaining strains were LA-MRSA and other rare strains. The same study also demonstrated transmission between human and pig (Khanna et al., 2008). There are as many as reports of LA-MRSA isolated from pigs in the Netherlands with almost 40% of pig population affected (Huijsdens et al., 2006; De Neeling et al., 2007; Van Duijkeren et al., 2007; Van Duijkeren et al., 2008). MRSA ST398 has been reported across all Europe including Austria, Belgium, Denmark, France, Germany, Italy, the Netherlands, Poland, Portugal, Spain and Switzerland (Guardabassi et al., 2007; Witte et al., 2007; Schwarz et al., 2008; Anon, 2009; Huber et al., 2010; Hunter et al., 2010; Johnson, 2011). It has also been reported out of Europe from Canada (Khanna et al., 2008) Singapore (Sergio et al., 2007) and USA (Smith et al., 2009).

2.5 MRSA in Cattle:

MRSA was first reported from Belgian cow as a cause of mastitis, which was later confirmed to be of human origin (Devriese et al., 1972; Devriese & Hommez, 1975). MRSA is occasionally considered to be the cause of mastitis in cattle (Vanderhaeghen et al., 2010a). In 2007, a study concluded that several cases of subclinical mastitis in cows on a farm in Hungary were caused by MRSA and that these strains were indistinguishable from the MRSA isolated from a carrier working in close contact with the cows. This suggests the transmission of these isolates between humans and cows, although the direction of transfer (cow to human or human to cow) was not proven. In cattle MRSA is mostly related to mastitis, human transmission can be considered but route is unknown yet (Juhász-Kaszanyitzky et al., 2007). Recent studies have reported that ST398 is present in German cows (Fessler et al., 2010), Dutch veal calves (Graveland et al., 2010) and Belgian cows (Vanderhaeghen et al., 2010a).

2.6 MRSA in Chicken:

In Korea, three mecA positive MRSA isolates were reported, of which one was isolated from a suppurative region in chicken meat and the other two isolates were from joints of the chicken, which had signs of arthritis (Lee, 2003) and another study later in 2006 reported two mecA positive MRSA from two chicken samples (Kwon et al., 2006). In Japan, two strains of MRSA isolate reported were of human origin, suggesting transfer between workers involved in poultry processing and raw chicken meat (Kitai et al., 2005). A Belgian study reported spa-type t1456 of MRSA strain from broiler chickens, which was different from ST398 MRSA strains reported in other studies in other food animals so far. Whether this spa type is typically associated with poultry still needs to be confirmed (Persoons et al., 2009).

2.7 MRSA in Meat:

MRSA has been detected both in animals and human. Now the fourth phase has also been started which shows evidences that both animal and human MRSA has been detected on meat. Such as studies in Japan and Korea reported human MRSA from chicken meat (Lee, 2003; Kitai et al., 2005). Taiwanese study reported MRSA in pork and chicken carcasses (Lin et al., 2009). MRSA have also been detected from the foods such as bovine milk, cheese, meat products and raw chicken meat (Vanderhaeghen et al., 2010b). Prevalence of MRSA in different type of meat is summarized in Table 1, which shows highest prevalence of MRSA reported from Germany 33.3% from beef, 10.5% from pork and 20.5% from chicken (Knodl et al., 2010) followed by Netherlands reporting 10.6% from beef, 10.7% from pork and 16.0% from chicken (De Boer et al., 2009). But previous study reported 3.1% of MRSA only from pork in 2007 (van Loo et al., 2007a). And the lowest prevalence of MRSA is reported in the study from Korea showing 1% from beef, 0.3% from pork and 0.3% from chicken (Lim et al., 2010).

Table 1: Prevalence of MRSA in beef, pork and chicken from different studies conducted so far.

Prevalence of MRSA in different type of meat (%)

Country

Beef

Pork

Chicken

Canada

5.6%

9.6%

1.2%

Germany

33.3%

10.5%

20.5%

Korea

1%

0.3%

0.3%

Korea

5%

0

0

Netherlands

0

3.1%

Netherlands

10.6%

10.7%

16%

Poland

0

3.9%

0

Spain

1.8%

0.7%

Taiwan

-

4.3-11.3%

0.3-7.8%

USA

3.3%

5.6%

-

One important thing to note is MRSA can be transmitted between people and animals during close contact (Seguin et al., 1999b; Duquette & Nuttall, 2004; Weese et al., 2005; Cuny et al., 2006; Weese et al., 2006). Food and meat contaminated with MRSA may constitute a risk for consumers and especially for immune-compromised individuals. In immune-compromised persons the specific and non-specific immune responses are not able to act as barriers to prevent colonization of the gastrointestinal tract and ingestion of food contaminated by MRSA may lead to sometimes lethal disease (Kluytmans et al., 1995; Normanno et al., 2007). 

2.8 Conclusion:

S. aureus is the most virulent of the Staphylococcus spp. Responsible for a wide spectrum of disease, ranging from localized and systemic infections to toxin-mediated illness. Heavy uses of antibiotics lead to the development of methicillin resistant S. aureus (MRSA) strains reported in UK, which has resistance to all common antibiotics used so far. Since then MRSA epidemic is has been reported. Further MRSA had also been reported in animals firstly in cow mostly responsible for mastitis, then horses and pet animals. But the reports of MRSA in food animals and bovine milk, confirms that the MRSA has entered food chain and continues to spread. Other reports of MRSA transmission between human and food animals, originated from both human and animals, confirms zoonosis. This wider host range provides favorable circumstances for development of new strains. This warrants there is a need of continuous monitoring for MRSA.

Chapter III: Methodology

3.0 Introduction:

The rapid method for isolation and detection of MRSA from meat as suggested by De Boer et al., (2009) and Van Loo et al., 2007a, we call it as a 'Double Enrichment Method' was used for isolation and identification of MRSA from meat. This chapter will describe the research methodology used to fulfill the objective of this research.

3.1 Sample collection:

A total 30 samples each of beef, pork and chicken (n = 90) were randomly collected from local super-markets and butcher shops, between 1st March, 2011 and 1st April, 2011. Meat samples were packed meat from supermarket and retail meat from butcher shop. Meat from supermarket was purchased as available packets ranging from 200 g to 500 g while the retail meat was purchased approximately 25 g to 35 g each and origin of meat was noted down. All samples collected were unprocessed raw and fresh meat. After collection meat was kept overnight under refrigeration. Approximately 25 g of each meat sample was separated in the sterile stomach bag and numbered depending upon the type of meat for example, B1, P1 and C1to B30, P30 and C30 for respective samples of beef, pork and chicken.

3.2 Culture Media:

Two enrichment media were used as follows Mueller-Hinton broth (MHB) with 6.5%NaCl (Oxoid, UK) and Phenol red mannitol broth (PHMB+) containing ceftizoxime (5 μg/ml) and aztreonam (75 μg/ml), (Media Products BV, Netherlands). Selective isolation media, BrillianceTM MRSA Agar (Oxoid, UK) and Tryptone Soya Agar (TSA) (Oxoid) were used for further culture.

3.3 Diagnostic Kit:

Staphaurex Plus® test kits (Remel, UK) were used to confirm isolates from meat. It is a latex agglutination test for the detection of clumping factor, Protein A and certain polysaccharides found in MRSA.

Principle of the Procedure: Staphaurex Plus® Test Latex consists of yellow latex particles which have been coated with fibrinogen and rabbit immunoglobulin G (IgG) specific for S. aureus. When drop of the reagent is mixed on a card with S. aureus organisms, rapid agglutination occurs through the interaction of (i) fibrinogen and clumping factor, (ii) the Fc portion of IgG and protein A or (iii) specific IgG and cell surface antigens.

Some strains of Staphylococcus spp. particularly S. saprophyticus, may cause non-specific aggregation of latex particles. Therefore a Control Latex is provided to assist with the identification of non-specific reactions.

Staphaurex Plus® Test Kit: Staphaurex Test Reagent, Staphaurex Control Reagent.

3.5 Processing of Samples:

Approximately 25 g of meat sample was homogenized in 9 ml of Peptone water to prepare a suspension, using a Stomacher blender for around 2 min. Then 1 ml of suspension was introduced to 9 ml of MHB + 6.5%NaCl an enrichment medium followed by incubation for 16 to 24 h at 37°C. After incubation 1 ml of enriched culture was added to PHMB+ and incubated for 16 to 24 h at 37°C. After incubation, loopful of the PHMB+ was streaked on the surface selective isolation media BrillianceTM MRSA agar using sterile wire loop to obtain single colonies, followed by incubation for 24 h at 37°C. For plates with no growth, incubation was extended for another 24 h. These plates were examined for the typical denim colored colonies, 5 of such colonies from each plate, were selected and sub-cultured on TSA plates, for further confirmation and incubated for 16 to 24 hours at 37°C. Staphaurex Plus® test kit was used to confirm these isolates, which is a latex agglutination test used for the detection of clumping factor, Protein A and certain polysaccharides found in S. aureus microorganisms to differentiate S. aureus from other Staphylococcus spp.

3.6 Statistical Analysis:

SPSS 17.0 version software was used to statistically analyze the data obtained. For statistical analysis, all samples were coded, considering the type of meat, country of origin of meat and whether meat is packed or retail depending upon purchased from supermarket or retail butcher shop. Isolates obtained were also coded. Below is the sample coding considering the type of meat and isolates used for statistical analysis.

Chicken = 1 MRSA positive isolates = 1

Beef = 2 Staphylococcus spp. = 2

Pork = 3 Negative isolates = 3

These values were used to cross-tabulate the isolates against type of meat, country of origin of meat, packed and retail meat.

Chapter IV: Results and Analysis

4.0 Research Findings:

Total 30 samples of each beef, pork and chicken (n = 90) were randomly collected from local super-markets and butcher shops, within the period of 1st March, 2011 to 1st April, 2011. All samples collected were unprocessed raw and fresh meat. Approximately 25 g of each meat sample was separated in the sterile stomach bag and numbered depending upon the type of meat for example, B1, P1 and C1to B30, P30 and C30 for respective samples of beef, pork and chicken. The rapid method for isolation and detection of MRSA from meat was used as suggested by De Boer et al., (2009) and Van Loo et al., (2007a), we call it as a 'Double Enrichment Method' for isolation and identification of MRSA from meat. The growth of Staphylococcus aureus on this selective isolation medium is typical denim blue colored as shown in Figure 5. BrillianceTM MRSA Agar plates were examined for such typical denim blue colored colonies. These isolates were further confirmed using Staphaurex Plus® test kits protein A and clumping factor which are characteristic of Staphylococcus aureus. After processing of all samples, isolation and detection of the MRSA and Staphylococcus spp., data was analyzed using SPSS statistical software. Below are the results.

Out of a total 90 (n) samples processed Staphylococci spp. were isolated from 25 samples. Total 5 (5.6%) isolates were confirmed as MRSA positive and 20 (22.2%) were confirmed as Staphylococcus spp. This applied method for testing of MRSA from raw meat is qualitative method, indicating the presence or absence of MRSA in 25 g of raw meat sample which is summarized in Table 1 and in Figure's 2, 3 and 4. Although limited number of MRSA isolates were obtained from only 5 raw meat samples. The highest prevalence of MRSA was found in chicken 3/30 (3.3%) followed by beef 1/30 (1.1%) and pork 1/30 (1.1%) which is summarized in Table 1 and Figure 2. Positive MRSA isolates were obtained from packed meat 5/45 (5.6%) and meat which had originated from the UK rather than retail meat and the meat which is imported from other countries which is summarized in Figures 3 and 4 respectively.

Table 2: Prevalence of MRSA isolates according to type of meat

MRSA Isolates

Total

MRSA Positive

Staphylococcus spp

Type of Meat

Chicken

% within Type of Meat

10.0%

26.7%

63.3%

100.0%

% of Total

3.3%

8.9%

21.1%

33.3%

Beef

Count

1

6

23

% within Type of Meat

3.3%

20.0%

76.7%

% of Total

1.1%

6.7%

25.6%

Pork

Count

1

6

23

% within Type of Meat

3.3%

20.0%

76.7%

% of Total

1.1%

6.7%

25.6%

Total

Count

5

20

65

% within Type of Meat

5.6%

22.2%

72.2%

% of Total

5.6%

22.2%

72.2%

100.0%

Figure 2: Prevalence of MRSA isolates according to type of meat:

6.7%

1.1%

6.7%

1.1%

8.9%

3.3%

Figure 2, shows highest prevalence of MRSA positive isolates in chicken 3/30 (3.3%) meat followed by beef 1/30 (1.1%) and pork 1/30 (1.1%) meat.

Figure 3: Prevalence of MRSA isolates according to packed meat or retail meat.

12.2%

5.6%

10.0%

Figure 4: Prevalence of MRSA isolates according to origin of meat from different countries.

1.1%

10%

1.1%

6.7%

10%

5.6%

Figure 5: Typical denim blue colored colonies of MRSA on BrillianceTM MRSA Agar

MRSA ID 5.JPG

4.1 Conclusion:

The results obtained in this study showed a 5.6% prevalence of MRSA from raw meat sold across the city of Manchester, with highest prevalence in chicken (3.3%) followed by beef (1.1%) and pork (1.1%). Other criteria such as whether the meat had been packed meat from a supermarket or retail meat sold across the counter of different butchers shops across the city of Manchester, it was only packed meat from supermarket which was contaminated with MRSA. Surprisingly MRSA was found in the meat which had originated from UK itself, rather than meat which had been imported from different European countries, where MRSA in meat and food animals is already confirmed and further research is ongoing.

As the literature suggests contamination of raw meat by the butchers and meat handlers may occur during the process of slaughtering of an animal, however this study shows that there is possibly contamination of meat production facilities or the contamination carcass during the slaughtering of an animal. This MRSA contamination of meat is of human origin or animal origin can be confirmed only by typing of MRSA isolates obtained in this study.

Chapter V: Discussion

This study was designed to find the prevalence of MRSA in meat sold across the city of Manchester. Out of a total 90 (n) samples processed Staphylococci were isolated from 25 samples, of which 5 (5.6%) isolates were confirmed as MRSA positive and 20 (22.2%) were confirmed as Staphylococcus spp. The highest prevalence of MRSA was found from chicken 3/30 (3.3%) followed by beef 1/30 (1.1%) and pork 1/30 (1.1%) which is summarized in Table 2 and Figure 2.

There are different studies conducted to detect the prevalence of MRSA from meat (Table1). There are differences in the results of different studies which may be because the methods used for isolation of MRSA were different. But different methods used to isolate and detect MRSA, consistently recover small amount of MRSA from meat, although there is no particular standardized method for the isolation and detection of MRSA from meat (Weese et al., 2010). Many studies considered different factors for collection of meat samples, such as different meat cuts, swabs from carcasses, meat pieces or ground meat, etc., these factors can give different results as it is very clear, different studies show different prevalence of MRSA from meat Table 1. This study show, highest prevalence of MRSA from chicken 3.3% while other studies reported highest prevalence from beef in Germany 33.3% (Knodl et al., 2010) followed by Netherlands 10.6% from beef (De Boer et al., 2009).

The present study considered different factors such as meat packaging - whether meat is packed or retail and the country of origin of meat. This is the first study of its kind in which we considered such factors to differentiate prevalence of MRSA from meat. As the rationale of this study says approximately, 60% of meat is imported in the UK from different European countries and much of the meat comes from Netherlands, Denmark, Spain and Germany where MRSA in pig has been already confirmed. But, this study showed presence of MRSA from meat which had originated from the UK, rather than imported meat. This may be due to limited sample size of 90, further research with wide sample size is recommended.

Another thing to note is prevalence of MRSA from 5/45 (5.6%) packed meat rather than retail meat. A recent study conducted in USA had reported MRSA in pig production facilities, concluding that meat gets contaminated in the meat production facilities (Larson & Reah, 2010). This study shows that, there is possibility of contamination of meat from production facilities in the UK. This can be confirmed by typing of isolates obtained, to know the origin of MRSA, whether it is of human or an animal origin. As MRSA is also reported from intestinal tract (Bhalla et al., 2007) these intestinal contents can possibly contaminate carcass during slaughtering process. There is also, a chance of contamination of carcass from the slaughtering environment or by the MRSA colonized personnel working in slaughter house (De Boer et al., 2009). So slaughter house personnel should be screened for MRSA and MRSA testing should be included in the routine surface testing of meat production facilities.

A qualitative method to test the presence of MRSA in raw meat using 'Double Enrichment Method' as suggested by De Boer et al., (2009) and Van Loo et al., 2007a was used. Double enrichment is a use of enrichment broth to enhance the growth of desired bacteria. As raw meat can have small amount of MRSA within the numerous other micro-flora present in meat. But our aim is to isolate MRSA from meat, so we used enrichment broth to enhance the growth of MRSA from within the other undesired micro-flora. The primary culture medium used was MHB with 6.5% NaCl, normally MHB is used for the antimicrobial susceptibility testing of bacteria. High salt concentration tolerance is reported in Staphylococcus spp. This will allow and favor growth of only Staphylococcus spp. in high salt concentration medium. After the use of first enrichment, the second enrichment medium PHMB+ was used which contains ceftizoxime (5 μg/ml) and aztreonam (75 μg/ml). This will further favor the growth of Staphylococcus spp. which are resistant to these antibiotics. After this double enrichment, we used BrillianceTM MRSA Agar to get the typical denim blue colored colonies of MRSA, which is a characteristic of S. aureus. These typical denim blue colored colonies are shown in Figure 5.

Some of the BrillianceTM MRSA Agar plates reported to have grown typical beige or white colored colonies which were not S. aureus isolates but were methicillin resistant Staphylococcus spp. A recent study had confirmed such beige colored colonies are of MR-CNS, methicillin resistant - coagulase negative Staphylococcus. This study was conducted by Huber et al., (2011) who identified the different Staphylococcus spp. and found 48.2% prevalence of MR-CNS isolates from samples such as livestock, chicken carcasses, BTM (bulk tank milk) and minced meat, as well as persons in contact with livestock (Huber et al., 2011). It is assumed that methicillin-resistance genes had evolved in coagulase-negative Staphylococci and were then horizontally transferred among Staphylococci (Archer et al., 1994; Barbier et al., 2010). This shows that mecA gene is now distributed among both coagulase-positive and coagulase-negative staphylococcal species; this high prevalence of MR-CNS in livestock production might become an emerging problem for veterinary medicine in near future (Huber et al., 2011).

This study also reported such beige or white colored colonies on BrillianceTM MRSA Agar, which were confirmed not to be MRSA by Staphaurex Plus® test kit, but other species of Staphylococci, we termed those isolates as Staphylococcus spp. showing prevalence as follows 8/30 (8.9%) from chicken, 6/30 (6.7%) from beef and 6/30 (6.7%) from pork, which is summarized in Table 1 and Figure 2.

Now another criteria under consideration shows the prevalence of Staphylococcus spp. from packed and retail meat is as follows 11/45 (12.9%) and 9/45 (10.0%), respectively which is summarized in Figure 3. We have not confirmed other species of Staphylococcus from these isolates due to limitations of this study, further research is recommended.

Although the prevalence of MRSA reported from the meat was low, the possible contamination of meat can lead to colonization in humans. Touching ones nose after handling MRSA contaminated meat, may lead to nasal colonization. Ingestion of MRSA contaminated meat can cause gastro-intestinal colonization and the potential for subsequent extra-intestinal infection or transmission. Likewise contact of MRSA contaminated meat with skin lesion can cause infection (Weese et al., 2010). Chances of MRSA food poisoning is low but it is reported that MRSA can cause food poisoning leading to staphylococcal enterotoxin-associated diarrhea (Jones et al., 2002). As the possible ways of contamination of meat with MRSA is unknown but, good meat handling practices, including prevention of cross-contamination, adequate cleaning and disinfection and good personal hygiene practices can reduce risk of infection.

In the UK, there is no evidence of MRSA in meat but, little evidence of MRSA from food producing animals is reported. This is the first study from the UK, which shows prevalence of MRSA from meat sold across the city of Manchester.

Chapter VI: Conclusion and Recommendations

6.0 Conclusion:

This study shows that, the occurrence of MRSA from meat is not uncommon. As raw meat sold across the city of Manchester, contains 5.6% of MRSA, with highest prevalence reported is from chicken 3/30 (3.3%) followed by beef 1/30 (1.1%) and pork 1/30 (1.1%). In consideration, with other criteria such as whether the meat is packed meat of supermarket or a retail meat sold across the different butcher shops the city of Manchester, highest prevalence of MRSA was reported in packed meat 5/45 (5.6%) from supermarket. MRSA was found in the meat which had originated from UK itself, rather than the meat which had been imported from different European countries, where MRSA in meat and food animals is already confirmed and further research is ongoing.

In the UK, MRSA is hugely colonized in humans, but when it comes to food animals it is not considered as a potential risk pathogen as there is little evidence. Although the prevalence of MRSA from meat is low but, chances of infection cannot be denied and there are reports of MRSA food poisoning in humans. This shows MRSA had already entered the food chain, which can cause food poisoning. Transfer of mecA gene from coagulase-positive to coagulase-negative staphylococcal species, increases risk of developing resistance to methicillin by all the staphylococcal species. As this study shows, out of 25 Staphylococcus spp. 5 isolates were confirmed as MRSA. There is need of further study. Along with MRSA other species of Staphylococci group should be given importance. As we know, an immune-compromised individual is at constant risk of infection due to the common micro-flora, but it can get worse, when the common micro-flora possess mecA gene which will make them resistant to methicillin, making it very difficult to treat such infections. So this study warrants continuous monitoring of emergence MRSA in the UK with respect to veterinary public health and typing of the isolates obtained and confirming their origin.

6.1 Recommendations:

There are several limitations of this study which recommends further research in particular aspects -

Limited sample size

Limited area coverage

Limited type of meat considered for study (beef, pork and chicken)

No funds for study to cover typing of isolates obtained

This study recommends research to be conducted on large scale covering whole country with specific zone system, specifying large number of sample size to be included covering most type of meat consumed in the UK. This study can detect MRSA at different stages/phases such as -

Screening of food animals on farm level before introducing them to slaughtering environment,

Screening of animals once they are introduced in the slaughtering environment,

Screening of carcasses after slaughter,

Screening of meat when it in the production facilities,

And also screening of packed and retail meat with due respect to origin of country.

Screening studies should be conducted at different stages in different phases and typing of possible isolates obtained, to trace back the origin of MRSA. This large scale study will confirm, whether there is need to revise the meat import policies in the UK. This study can confirm possible routes of contamination of meat with MRSA.