ABSTRACTS
This study carried out aimed at determine the multi drug resistance patterns of Listeria species in frozen and live chicken sold in Owerri. Isolated Listeria spp. where identified using biochemical and bacteriological identification scheme. The antimicrobial susceptibility was tested for each isolates. The following antibiotics tested using Bauer–Kirby technique (Anon. 1997) are, 10 μg ampicillin (A10), 30 μg chloramphenicol (C30), 15 μg erythromycin (E15), 10 μg gentamicin (CN10), 10 units penicillin G (P10), 10 μg streptomycin (S10), 30 μg tetracycline (TE30) or 30 μg vancomycin (VA30). After inoculation (organisms and antibiotics) and incubation of the plates with the antibiotics, the diameter (in mm) of the zone around each disc was measured and interpreted in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Form the study, Chloramphenicol, Gentamycin, Vanconmycin and Erthromycin were 100% sensitive. Streptomycin and Penicillin were 75% sensitive and Tetracyclin and Ampicillin showed 50% sensitive.
CHAPTER ONE
1.0 INTRODUCTION/LITERATURE REVIEW
1.1 INTRODUCTION
Listeria species are ubiquitous bacteria, well adaptable in the environment, in animal and vegetable foods. The genus Listeria comprises seven species. Six of them ( L. grayi, L. innocua, L. ivanovii, L. welshimeri, L. murrayi and L. seeligeri) are not usually pathogenic for humans, while L. monocytogenes is considered one of the major foodborne pathogens that can induce listeriosis in humans and animals
(McLauchlin, 1997). Human listeriosis is associated with consumption of contaminated milk, soft cheese, undercooked meat, and unwashed raw vegetables and cabbage (Oliver et al., 2005; Aygun & Pelivanlar, 2006; Colak et al., 2007). It may range from mild flu2like sickness to severe manifestations. Groups at highest risk are pregnant women, neonates, adults with underlying disease, elderly and immunocompromised individuals (McLauchlin et al.,2004).
The excessive use of antimicrobials has led to antibiotic resistance and particularly multiresistance, which are important public health concerns since they may cause failure of therapeutic treatment. Furthermore, antimicrobials used as growth promoters in animal feed have resulted in the dissemination of antimicrobial to resistant bacteria into the environment (Jansen et al., 2003). Monitoring the antimicrobial resistance of L. monocytogenes in humans and animals is important to control the use of antimicrobial agents and prevent the spread of multi2drug resistant bacteria (Harakeh et al., 2009).
Currently, there is limited information regarding the prevalence and antimicro2
bial susceptibility patterns of Listeria spp. in foods in Iran. Therefore, the present
study was undertaken to determine the prevalence and the antimicrobial resistance rate of Listeria strains isolated from traditional dairy product samples in Chahar Mahal & Bakhtyari province, Iran.
1.2 AIM AND OBJECTIVES
This study aim at determining the multi drug resistance patterns of listeria species in frozen and live chicken sold in Owerri and its objectives is as stated;
- To isolates Listeriia spp. from poultry meat
- Identification of Listeria spp. from poultry meat
- Evaluation of the antimicrobial profile of isolated Listeria spp.
1.3 LITERATURE REVIEW
1.3.1 Background of study
Listeria is a genus of bacteria that up to 1992 contained 10 known species, each containing two subspecies. As of 2014 another five species were identified. Named after the British pioneer of sterile surgery Joseph Lister, the genus received its current name in 1940. Listeria species are Gram-positive, rod-shaped, facultatively anaerobic, and nonspore-forming. The major human pathogen in the Listeria genus is L. monocytogenes. It is usually the causative agent of the relatively rare bacterial disease listeriosis, a serious infection caused by eating food contaminated with the bacteria. The disease affects pregnant women, newborns, adults with weakened immune systems, and the elderly (Singleton, 1999).
Listeriosis is a serious disease for humans; the overt form of the disease has a case-fatality rate around 20%. The two main clinical manifestations are sepsis and meningitis. Meningitis is often complicated by encephalitis, when it is known as meningoencephalitis, a pathology that is unusual for bacterial infections. L. ivanovii is a pathogen of mammals, specifically ruminants, and has rarely caused listeriosis in human (Christella Guillet, et al., 2010). The incubation period can vary between 3 and 70 days.
The first documented case of listeriosis was in 1924. In the late 1920s, two researchers independently identified L. monocytogenes from animal outbreaks. They proposed the genus Listerella in honor of surgeon and early antiseptic advocate Joseph Lister, but that name was already in use for a slime mold and a protozoan. Eventually, the genus Listeria was proposed and accepted. All species within the Listeria genus are Gram-positive, nonspore-forming, catalase-positive rods. The genus Listeria was classified in the family Corynebacteriaceae through the seventh edition of Bergey’s Manual of Systematic Bacteriology. The 16S rRNA cataloging studies of Stackebrandt, et al. demonstrated that L. monocytogenes is a distinct taxon within the Lactobacillus-Bacillus branch of the bacterial phylogeny constructed by Woese. In 2004, the genus was placed in the newly created family Listeriaceae. The only other genus in the family is Brochothrix (Elliot, et al., 1999).
The genus Listeria currently contains 17 species: L. aquatica, L. booriae, L. cornellensis, L. fleischmannii, L. floridensis, L. grandensis, L. grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. newyorkensis, L. riparia, L. rocourtiae, L. seeligeri, L. weihenstephanensis, and L. welshimeri. Listeria dinitrificans, previously thought to be part of the Listeria genus, was reclassified into the new genus Jonesia (Collins, et al., 1991). Under the microscope, Listeria species appear as small, rods, which are sometimes arranged in short chains. In direct smears, they may be coccoid, so they can be mistaken for streptococci. Longer cells may resemble corynebacteria. Flagella are produced at room temperature, but not at 37°C. Hemolytic activity on blood agar has been used as a marker to distinguish L. monocytogenes from other Listeria species, but it is not an absolutely definitive criterion. Further biochemical characterization may be necessary to distinguish between the different species of Listeria.
Listeria can be found in soil, which can lead to vegetable contamination. Animals can also be carriers. Listeria has been found in uncooked meats, uncooked vegetables, fruit such as cantaloupes and apples, pasteurized or unpasteurized milk, foods made from milk, and processed foods. Pasteurization and sufficient cooking kill Listeria; however, contamination may occur after cooking and before packaging. For example, meat-processing plants producing ready-to-eat foods, such as hot dogs and deli meats, must follow extensive sanitation policies and procedures to prevent Listeria contamination. Listeria monocytogenes is commonly found in soil, stream water, sewage, plants, and food. Listeria is responsible for listeriosis, a rare but potentially lethal foodborne illness. The case fatality rate for those with a severe form of infection may approach 25%. (Salmonellosis, in comparison, has a mortality rate estimated at less than 1%.) Although Listeria monocytogenes has low infectivity, it is hardy and can grow in temperatures from 4°C (39.2°F) (the temperature of a refrigerator), to 37°C (98.6°F), (the body’s internal temperature). Listeriosis is a serious illness, and the disease may manifest as meningitis, or affect newborns due to its ability to penetrate the endothelial layer of the placenta (“Todar’s Online Textbook of Bacteriology”, 2013).
1.3.2 Listeria monocytogenes
Listeria monocytogenes is a bacterium that causes listeriosis, a disease that can have severe consequences for particular groups of the population. It can cause miscarriages in pregnant women and be fatal in immunocompromised individuals and the elderly. In healthy people, listeriosis generally only causes a mild form of illness. L. monocytogenes can be found throughout the environment. It has been isolated from domestic and wild animals, birds, soil, vegetation, fodder, water and from floors, drains and wet areas of food processing factories.
1.3.2.1 Description of the organism
- monocytogenes is a Gram-positive, non-spore forming rod-shaped bacterium. It belongs to the genus Listeria along with L. ivanovii, L. innocua, L. welshimeri, L. selligeri and L. grayi (Rocourt and Buchrieser 2007). Of these species, only two are considered pathogens: L. monocytogenes which infects humans and animals, and L. ivanovii which infects ruminants (although there have been rare reports of
- ivanovii being isolated from infected humans) (Guillet et al. 2010). There are thirteen known serotypes of L. monocytogenes: 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4ab, 4b, 4c, 4d, 4e and 7. The serotypes most often associated with human illness are 1/2a, 1/2b and 4b (FDA 2012).
1.3.2.2 Growth and survival characteristics
The growth and survival of L. monocytogenes is influenced by a variety of factors. In food these include temperature, pH, water activity, salt and the presence of preservatives. The temperature range for growth of L. monocytogenes is between -1.5 and 45°C, with the optimal temperature being 30–37°C. Temperatures above 50°C are lethal to L. monocytogenes. Freezing can also lead to a reduction in L. monocytogenes numbers (Lado and Yousef , 2007). As L. monocytogenes can grow at temperatures as low as 0°C, it has the potential to grow, albeit slowly, in food during refrigerated storage. L. monocytogenes will grow in a broad pH range of 4.0–9.6 (Lado and Yousef, 2007). Although growth at pH <4.0 has not been documented, L. monocytogenes appears to be relatively tolerant to acidic conditions. L. monocytogenes becomes more sensitive to acidic conditions at higher temperatures (Lado and Yousef, 2007). Like most bacterial species, L. monocytogenes grows optimally at a water activity (aw) of 0.97. However, L. monocytogenes also has the ability to grow at a aw of 0.90 (Lado and Yousef, 2007). Johnson et al., (1988) demonstrated that L. monocytogenes can survive for extended periods of time at a value of 0.81. L. monocytogenes is reasonably tolerant to salt and has been reported to grow in 13–14% sodium chloride (Farber et al., 1992). Survival in the presence of salt is influenced by the storage temperature. Studies have indicated that in concentrated salt solutions, the survival rate of L. monocytogenes is higher when the temperature is lower (Lado and Yousef, 2007). L. monocytogenes can grow under both aerobic and anaerobic conditions, although it grows better in an anaerobic environment (Sutherland et al., 2003; Lado and Yousef, 2007).
The effect of preservatives on the growth of L. monocytogenes is influenced by the combined effects of temperature, pH, salt content and water activity. For example, sorbates and parabens are more effective at preventing growth of L. monocytogenes at lower storage temperatures and pH. Also, adding sodium ch
loride or lowering the temperature enhances the ability of lactate to prevent L. monocytogenes growth. At decreased temperatures (such as refrigeration storage) sodium diacetate, sodium propionate and sodium benzoate are more effective at preventing growth of L. monocytogenes (Lado and Yousef, 2007).
1.3.2.3 PATHOGENESIS
Listeria uses the cellular machinery to move around inside the host cell: It induces directed polymerization of actin by the ActA transmembrane protein, thus pushing the bacterial cell around (Smith and Portnoy, 1997).
- monocytogenes, for example, encodes virulence genes that are thermo regulated. The expression of virulence factor is optimal at 39°C, and is controlled by a transcriptional activator, PrfA, whose expression is thermo regulated by the PrfA thermo regulator UTR element. At low temperatures, the PrfA transcript is not translated due to structural elements near the ribosome binding site. As the bacteria infect the host, the temperature of the host melts the structure and allows translation initiation for the virulent genes.
The majority of Listeria bacteria are targeted by the immune system before they are able to cause infection. Those that escape the immune system’s initial response, however, spread through intracellular mechanisms and are, therefore, guarded against circulating immune factors (AMI) (“Todar’s Online Textbook of Bacteriology”, 2013).
To invade, Listeria induces macrophage phagocytic uptake by displaying D-galactose in their teichoic acids that are then bound by the macrophage‘s polysaccharide receptors. Other important adhesins are the internalins. Listeria uses internalin A and B to bind to cellular receptors. Internalin A binds to E-cadherin, while internalin B binds to the cell’s Met receptors. If both of these receptors have a high enough affinity to Listeria‘s internalin A and B, then it will be able to invade the cell via an indirect zipper mechanism. Once phagocytosed, the bacterium is encapsulated by the host cell’s acidic phagolysosome organelle. Listeria, however, escapes the phagolysosome by lysing the vacuole’s entire membrane with secreted hemolysin, now characterized as the exotoxin listeriolysin O. The bacteria then replicate inside the host cell’s cytoplasm (“Todar’s Online Textbook of Bacteriology”, 2013).
Listeria must then navigate to the cell’s periphery to spread the infection to other cells. Outside the body, Listeria has flagellar-driven motility, sometimes described as a “tumbling motility”. However, at 37°C, flagella cease to develop and the bacterium instead usurps the host cell’s cytoskeleton to move. Listeria, inventively, polymerizes an actin tail or “comet”, from actin monomers in the host’s cytoplasm with the promotion of virulence factor ActA (“Todar’s Online Textbook of Bacteriology”, 2013). The comet forms in a polar manner and aids the bacteria’s migration to the host cell’s outer membrane. Gelsolin, an actin filament severing protein, localizes at the tail of Listeria and accelerates the bacterium’s motility. Once at the cell surface, the actin-propelled Listeria pushes against the cell’s membrane to form protrusions called filopods or “rockets”. The protrusions are guided by the cell’s leading edge to contact adjacent cells, which then engulf the Listeria rocket and the process is repeated, perpetuating the infection. Once phagocytosed, the bacterium is never again extracellular: it is an intracellular parasite like S. flexneri, Rickettsia spp., and C. trachomatis (“Todar’s Online Textbook of Bacteriology”, 2013).
1.3.2.4 Symptoms of disease
There are two main forms of illness associated with L. monocytogenes infection. Non-invasive listeriosis is the mild form of disease, while invasive listeriosis is the severe form of disease and can be fatal (FDA 2012). The likelihood that invasive listeriosis will develop depends upon a number of factors, including host susceptibility, the number of organisms consumed and the virulence of the particular strain (WHO/FAO, 2004).
Symptoms of non-invasive listeriosis can include fever, diarrhoea, muscle aches, nausea, vomiting, drowsiness and fatigue. The incubation period is usually 1 day (range 6 hours to 10 days) (Painter and Slutsker 2007; FDA 2012). Non-invasive listeriosis is also known as listerial gastroenteritis or febrile listeriosis. Invasive listeriosis is characterised by the presence of L. monocytogenes in the blood, in the fluid of the central nervous system (leading to bacterial meningitis) or infection of the uterus of pregnant women. The latter may result in spontaneous abortion or stillbirth (20% of cases) or neonatal infection (63% of cases). Influenza-like symptoms, fever and gastrointestinal symptoms often occur in pregnant women with invasive listeriosis. In non-pregnant adults, invasive listeriosis presents in the form of bacterial meningitis with a fatality rate of 30%. Symptoms including fever, malaise, ataxia, seizures and altered mental status (Painter and Slutsker 2007). The incubation period before onset of invasive listeriosis ranges from 3 days
to 3 months (FDA 2012).
1.3.2.5 Virulence and infectivity
When L. monocytogenes is ingested, it may survive the stomach environment and enter the intestine where it penetrates the intestinal epithelial cells. The organism is then taken up by macrophages and non-phagocytic cells. The L. monocytogenes
surface protein internalin is required for this uptake by non-phagocytic cells, as it binds to the receptors on the host cells to instigate adhesion and internalization. The bacterium is initially located in a vacuole after uptake by a macrophage or non-phagocytic cell. L. monocytogenes secrete listeriolysin O protein, which breaks down the vacuole wall and enables the bacteria to escape into the cytoplasm. Any bacteria remaining in the vacuole are destroyed by the host cell. Once located in the cytoplasm of the host cell, L. monocytogenes is able to replicate. L. monocytogenes is transported around the body by the blood, with most L. monocytogenes being inactivated when it reaches the spleen or liver.
- monocytogenes is able to utilise the actin molecules of the host to propel the bacteria into neighbouring host cells. In the case of invasive listeriosis, this ability to spread between host cells enables L. monocytogenes to cross the blood-brain and placental barriers (Montville and Matthews 2005; Kuhn and Goebel 2007; Bonazzi et al., 2009).
1.3.2.6 Mode of transmission
The most common transmission route of L. monocytogenes to humans is via the
consumption of contaminated food. However, L. monocytogenes can be transmitted directly from mother to child (vertical transmission), from contact with animals and through hospital acquired infections (Bell and Kyriakides 2005). Healthy individuals can be asymptomatic carriers of L. monocytogenes, with 0.6–3.4% of healthy people with unknown exposure to Listeria being found to shed
- monocytogenes in their faeces. However, outbreak investigations have shown that listeriosis patients do not always shed the organism in their faeces (FDA/USDA/CDC 2003; Painter and Slutsker 2007). Therefore the role of healthy carriers in the transmission of L. monocytogenes is unclear.
1.3.2.7 Incidence of illness and outbreak data
Listeriosis is a notifiable disease in all Australian states and territories. The incidence of listeriosis notified in Australia in 2012 was 0.4 cases per 100,000 population (93 cases). This is a slight increase from the previous 5 year mean of 0.3 cases per 100,000 population per year (ranging from 0.2–0.4 cases per 100,000 population per year) (NNDSS 2013). In Australia the fatality rate in 2010 was 21%, which was an increase from the 14% fatality rate of the previous year (OzFoodNet 2010; OzFoodNet , 2012). The notification rate for listeriosis in New Zealand in 2011 was 0.6 cases per 100,000 population (26 cases). This was an increase from the 2010 rate of 0.5 cases per 100,000 population. The fatality rate in New Zealand in 2011 was 3.8% (Lim et al., 2012). In the United States (US) the notification rate for listeriosis in 2010 was 0.27 cases per 100,000 population. This was similar to the 2009 rate of 0.28 cases per 100,000 population (CDC, 2012). In the European Union (EU) there were 0.32 confirmed cases of listeriosis per 100,000 population in 2011 (ranging from 0.04–0.88 cases per 100,000 population between countries). This was a 7.8% decrease in the number of cases from 2010. The reported fatality rate in the EU in 2011 was 12.7% (EFSA 2013). Invasive
- monocytogenes infections can be life threatening, with average fatality rates
being 20–30% among hospitalized patients (WHO/FAO 2004; Swaminathan and Gerner-Smidt 2007).
Most cases of listeriosis are sporadic. Despite this, foodborne outbreaks due to
- monocytogenes have been associated with cheese, raw (unpasteurised) milk, deli meats, salad, fish and smoked fish, ice cream and hotdogs (Montville and Matthews 2005; Swaminathan and Gerner-Smidt 2007) (refer to Table 2).
1.3.2.8 Occurrence in food
- monocytogenes has been isolated from various ready-to-eat products. In a study by Meldrum et al. (2010) the prevalence of L. monocytogenes was 4.1% in crustaceans (n=147), 6.7% in smoked fish (n=178), 2% in sushi (n=50) and 0.9% in green salad (n=335) samples in Wales. Wong et al. (2005) isolated L. monocytogenes from 1% of ham (n=104) and 1.7% of pate (n=60) samples in New Zealand. L. monocytogenes has also been isolated from dairy products. For example, L. monocytogenes was detected in 1.3% of fresh cheese samples in Spain (n=78), 0.2% of hard cheese samples in the United Kingdom (n=1242) and 0.3% of ice creams in Italy (n=1734) (Busani et al., 2005; Cabedo et al., 2008; Little et al.,
2009). The prevalence of L. monocytogenes in bulk milk tank internationally is 1–60% (FSANZ 2009). The presence of L. monocytogenes in ready-to-eat products is probably due to contamination occurring after the product has been processed. This contamination may occur during additional handling steps such as peeling, slicing and repackaging. Also, in the retail and food service environment, contamination may be transferred between ready-to-eat products (Lianou and Sofos 2007). The type of handling that ready-to-eat meat receives may also influence the level of L. monocytogenes contamination. In a survey of retail packaged meats there was a significantly higher prevalence of L. monocytogenes reported in products cut into cubes (61.5%) (n=13), compared with sliced products (4.6%) (n=196) (Angelidis and Koutsoumanis 2006). Host factors that influence disease People at risk of invasive listeriosis include pregnant women and their foetuses, newborn babies, the elderly and immunocompromised individuals (such as cancer, transplant and HIV/AIDS patients). Less frequently reported, but also at a greater risk, are patients with diabetes, asthma, cirrhosis (liver disease) and ulcerative colitis (inflammatory bowel disease) (FDA, 2012).
1.3.2.9 Dose response
Investigations of foodborne outbreaks of non-invasive listeriosis have concluded that consumption of food with high levels of L. monocytogenes (1.9 x 105/g to 1.2 x 109/g) is required to cause illness in the general healthy population (Sim et al. 2002). The number of L. monocytogenes required to cause invasive listeriosis depends on a number of factors. These include the virulence of the particular serotype of L. monocytogenes, the general health and immune status of the host, and attributes of the food (for example fatty foods can protect bacteria from stomach acid). Some L. monocytogenes serovars are more virulent than others; this may be attributed to differences in the expression of virulence factors which could influence the interactions between the bacterium and the host cells and cellular invasion (Severino et al., 2007). The FDA and WHO have developed separate models for both healthy and susceptible populations to predict the probability that an individual will develop listeriosis (FDA/USDA/CDC 2003; WHO/FAO 2004). The probability that a healthy person of intermediate age will become ill from the consumption of a single L. monocytogenes cell was estimated to be 2.37 x 10-14. For more susceptible populations the probability that illness will occur was estimated to be 1.06 x 10-12. A more recent assessment on invasive listeriosis in
susceptible populations was performed which took into account the different serotypes of L. monocytogenes (Chen et al., 2006). This study showed that the probability of a susceptible individual developing invasive listeriosis ranged from 1.31 × 10-8 to 5.01 × 10-11, suggesting that there are large differences in virulence between L. monocytogenes serotypes.
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