OCCURRENCE OF LISTERIA MONOCYTOGENES IN FRESH MEAT AND OFFAL SOLD FOR HUMAN CONSUMPTION IN ZARIA METROPOLIS, NIGERIA

ABSTRACT

Listeriosis is a disease caused by a gram positive, non-spore forming intracellular pathogenic bacteria of the genus Listeria, commonly associated with food. To determine the presence of Listeria monocytogenes and some of its associated virulence genes in fresh meat samples sold for human consumption, 240 samples were obtained comprising of 156 beef, 26 liver, 25 reproductive tract and 33 intestinal parts sold at various locations in Zaria from July to November, 2013. Sixty six (27.5%) isolates of Listeria species were obtained and characterised through conventional biochemical tests, out of which, 8 (3.3%) were Listeria monocytogenes, but only one was subsequently confirmed using the Microgen^R 12L ID kit. Beef had the highest occurrence of 66.7% followed by reproductive tract (13.6%), intestines (10.6%) and liver (9.1%). Antibiotic susceptibility testing of the eight isolates by Kirby-Bauer disk diffusion method revealed that 100% of the L. monocytogenes isolates were resistant to ampicillin, 87.5% to tetracycline and cefixime, 75% to amoxicillin/clavulanic acid, 62.5% to erythromycin, 50% to chloramphenicol, nitrofurantoin and sulfamethoxazole,

37.5% to ciprofloxacin and kanamycin and 12.5% to gentamicin,. The least resistance frequency was to gentamicin 12.5%. However, all the isolates were resistant to at least 3 antimicrobial agents. Antibiotic resistance profiles of the isolates revealed 6 different antimicrobial resistance patterns. Isolates were subjected to a multiplex PCR to detect the presence of the haemolysin (hylA), internalin (inlA) and invasiveness associated protein (iap) virulence genes. None (0%) of the eight isolates was found to harbour haemolysin hylA, 1(12.5%) was positive for both internalin inlA and invasiveness associated proteins iap while 5(62.5%) harboured iap gene. The findings of this study showed the occurrence of Listeria monocytogenes in fresh meat samples in Zaria metropolis which displayed multiple antibiotic resistance and possessed virulence genes inlA, and iap. This finding is of public health significance.

CHAPTER ONE

INTRODUCTION

1.1 Background of the Study

Listeriosis is a disease caused by a gram positive, non-spore forming intracellular pathogenic bacteria of the genus Listeria, commonly associated with food. Listeria is widely distributed in nature. The bacteria are ubiquitous saprophytes that live in plant-soil environments and have been isolated from about 42 species of domestic and wild mammals and 22 species of birds, as well as fish, crustaceans, insects, sewage, water, feedstuffs, milk, cheese, meconium, faeces and soil (Kirkan et al., 2006). Although several species are recognised (Listeria monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, L. marthii, L. rocourthiae, L. grayii, L. fleisehmanni, L. coloradonensis, L. decoloradonensis, L. aquatic sp. nov., L. floridinensis nov., L. cornellensis sp. nov., L. gradensis sp. nov., L. riparia sp. nov., and L. murrayi), only two, Listeria monocytogenes and L. ivanovii, are pathogenic; the former is responsible for disease in both humans and animals, while the latter causes disease mostly in ruminants but also in other animals. The other Listeria species are generally considered to be non-pathogenic (Low and Donachie, 1997; Brugere-Picoux, 2008). There are reports however of L. seeligeri and L. ivanovii causing illness in humans (Cummins et al., 1994; Cocolin et al., 2002), and L. innocua occasionally associated with encephalitis in ruminants (Walker et al., 1994) Individuals who are particularly susceptible to this condition are those who are immune-compromised (as in HIV/AIDS infection), pregnant women, new-born babies, and the elderly (Farber and Peterkin, 1991; McLauchlin et al., 2004). Only Listeria monocytogenes and Listeria ivanovii were found pathogenic in test animals causing death within five days. Listeria monocytogenes induced pathological changes characterised by abscesses in the liver, pericarditis and frank haemorrhage in the kidneys. The most notable change was the accumulation of mononuclear cells in the loose connective tissue surrounding the bile ducts. The generic name was given in honour of a British Surgeon, Sir Joseph Lister. Pirie changed the name of the bacterium to L. monocytogenes from Bacterium monocytogenes or Listerella hepatolytica in 1940, and in 1948 the organism was recognised by the same name in the 6th edition of Bergey‟s Manual of Determinative Bacteriology (Breed et al., 1948). L. monocytogenes was first isolated from sheep in 1929 and the first human case was reported in the same year by Nyfeldt (1929). By 1961 L. monocytogenes was the only known species in the genus Listeria.

Listeria survive wide ranges of temperature (-7⁰ C to -45⁰C), pH (4.3-9.6) (Buchanan et al., 1993), salt concentrations (up to 10%) (Robert and Wiedmann, 2003), grow in gas or vacuum-packaged products at refrigeration temperatures (Duffy et al., 1994); and survive low water activity (Nolan et al., 1992). The ability to survive and multiply under conditions frequently used for food preservation makes the bacteria particularly problematic to the food industry (Roberts and Wiedmann, 2003). All Listeria species are psychotrophic and widely spread in the environment. L. monocytogenes has indeed emerged as a foodborne pathogen and a contaminant of various foodstuffs such as milk and dairy products, meat and meat products, vegetables and seafood.

1.2   Statement of Research Problem

It has been shown by various studies that listeriosis is a food-mediated illness (Embarek, 1994; Slutsker and Schuchat, 1999). A wide range of foods such as salads, sea-foods, meat, and dairy have been implicated in listeriosis, which follows the oral ingestion of the contaminated food (Finlay, 2001). L. monocytogenes has only recently been recognised as causing foodborne disease with major public health consequences. The pathogen gained prominence in the 1980s after several large outbreaks in the United States, Canada, and Europe established conclusively that contaminated foods are the primary means by which this microorganism affects human populations. The Centers for Disease Control and Prevention (CDC‟s) baseline estimate is that foods contaminated with L. monocytogenes caused approximately 2,500 cases of illness, including approximately 500 fatalities, in the United States each year. At the recommendation of CDC, the risk assessment used 2,078 cases of listeriosis and 390 fatalities as the baseline values for the national annual public health impact of the disease (CDC). This pathogen has been implicated in major outbreaks and sporadic cases in different countries. The annual endemic disease rate varied from 2 to 15 cases per million populations, with published rates varying from 1.6 to a high rate of 14.7 in France for 1986. Zambia had 85 reported cases of meningitis due to Listeria. In Ethiopia, an incidence of 18.3% was recorded in raw meat (Firehiwot, 2007). In 2000, the CDC reported that of all the foodborne pathogens tracked by them, L. monocytogenes had the second highest case fatality rate (21%) and the highest hospitalisation rate (90.5%) after E. coli (El-Shamy et al.,1993). In Northern Nigeria, 27% mortalilty rate due to L monocytogenes (serovar 4) was reported (Onyemelukwe et al., 1983). There are other products, traditionally considered of low risk, which have recently been linked to listeriosis transmission, such as the large listeriosis outbreak reported in Italy due to the consumption of corn. Though no fatalities occurred, more than 1500 people were affected (Aguado et al., 2004). Outbreaks have been reported in cattle in Nigeria (Akpavie and Ikheloa., 1992) and Oni et al (1989) isolated L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, 3a, and 4b from eight species of domestic animals including horses (68.4%), pigs (36.1%), cattle (20.8%), goats (20%), dogs (20%), free range chickens (6.8%), sheep from Zaria (14.7%) and submissions to Ahmadu Bello University Veterinary Teaching Hospital (13%). The manifestations of listeriosis include septicaemia, meningitis, encephalitis and intrauterine or cervical infections in pregnant women which may result in spontaneous abortions during the 2^nd or 3^rd trimester or still birth. The onset of the aforementioned disorders is usually preceded by influenza-like symptoms including persistent fever followed by nausea, vomiting and diarrhoea, particularly in patients who use antacid (Tominaga et al., 2006). Once the bacterium enters the host‟s monocytes, macrophages or polymorphonuclear leukocytes, it is blood-borne and can grow in body cells (Sebelius et al., 1999).

1. monocytogenes also produces an exotoxin Listeriolysin (LLO) which is a key agent in human neutrophil activation. The stimulation of these phagocytes, however, requires additional listerial virulence factors such as internalin (inlA) and haemolysin (hylA). Internalin (inlA) causes the internalisation of the bacteria; haemolysin (hylA) causes haemolysis of red blood cells which are both regulated by prfA gene. Invasivenes associated protein (iap), is useful for invasion of nonprofessional phagocytic cells, cell division and intracellular uptake of L. monocytogenes by mammalian cells. It has also been known to produce catalase, superoxide dismutase and enables the organism to neutralize the effects of phagocytic oxidative burst. It is however not regulated by prfA (Todar, 2004).

A variety of food products have been involved in most outbreaks, including soft cheese (Bille et al., 1990) and cooked meat products (Aguado et al., 2004). These are considered of special risk due to the ability of Listeria to grow and survive in them (Aguado et al., 2004).

1.3 Justification

1. monocytogenes is a bacterium that occurs widely in both agricultural (soil, plants and water) and food processing environments. In 1998, among populations tracked by FoodNet, listeriosis resulted in higher rates of hospitalisation than illnesses caused by any other foodborne pathogen and caused over one third of the reported deaths associated with food-borne illnesses.

The organism has been isolated in faecal samples of cattle, sheep, goats, chickens and pigs (Umeh and Okpokwasili, 2009) and water samples obtained from lakes in Nigeria (Nwachukwu et al., 2010). Faeces from these animals are used as fertilizers for plants and also contaminate lakes that are consumed by these animals as food and source of drinking water, respectively. There is paucity of information on the prevalence of this organism in fresh beef in Zaria metropolis. Listeriosis is considered a serious health problem due to the severity of symptoms and its high mortality rate. Inspite of the high mortality rate and the increasing resistance of L. monocytogenes to antibiotics, there are limited studies on the occurrence of the organism in fresh meat in Zaria metropolis.

Due to the fact that consumption of meat has increased tremendously especially due to improved conditions of living, which reveal an upward trend (Anonymous, 2013) adequate control and inspection of meat purchased and consumed is required in a country confronted with challenges of increasing human population and shortage of animal protein.

Therefore, the purpose of this study was to determine the occurrence, distribution, antibiotic resistance and molecular detection of L. monocytogenes virulence genes isolated from fresh meat and offal samples in Zaria metropolis. Data on antibiograms will be useful in the empiric management of patients whereas molecular profiles will provide templates for epidemiological applications.

1.4 Research Questions

1. Is Listeria monocytogenes present in raw meat and offal sold for human consumption in Zaria metropolis?
2. What are the antimicrobial resistance patterns for the isolates obtained?
3. Do isolates possess virulence genes such as hyl A, inl A, iap?

1.5 Aim and Objectives:

1.5.1    Aim

To determine the occurrence of L. monocytogenes in fresh meat sold for human consumption in Zaria metropolis.

1.5.2    Objectives

Specific objectives are to:

1. determine the occurrence of monocytogenes in fresh meat and offal sold for human consumption in Zaria metropolis.
2. ascertain the antimicrobial susceptibilities of Listeria to commonly used antimicrobial agents.
3. detect hylA, inlA, and iap virulence genes in monocytogenes isolates using PCR.

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