У coli salmonella and listeria monocytogenes

Authors O'Bryan, Crandall, and Johnson are with Dept. of Food Science, Univ. of Arkansas, 2650 Young Ave., Fayetteville, AR 72704. Authors Martin and Griffis are with Dept. of Biological and Agricultural Engineering, Univ. of Arkansas, Fayetteville, Ark. Direct inquiries to author O'Bryan (E‐mail: cobryan@uark.edu).

ABSTRACT: The heat‐resistance data in meat and poultry for various strains of Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7 as well as Listeria innocua M1 are summarized. Heat resistance of these organisms is affected by many factors. Different strains of the same organism have different responses to heat. Heat resistance can also be influenced by the age of the culture, growth conditions, pH, and numerous other factors. Data from this review may prove useful to processors in validating their times and temperatures for thermal processing of meat and poultry. The obvious gaps in the data will provide researchers opportunities to fill those gaps. In addition, it will encourage the development of surrogates, whether biological or otherwise, that will be able to be used in an actual processing environment.

When someone swallows bacteria that cause food poisoning, there is a delay (incubation period) before symptoms begin. This is because most bacteria that cause food poisoning need time to multiply in the intestine. The length of the incubation period depends on the type of bacteria and how many are swallowed. It could be hours or days.

The bacteria stick to the lining of the intestine and destroy those cells, either by sheer weight of numbers or by the toxins (poisons) they produce.

Sometimes these toxins are absorbed and cause damage elsewhere in the body. Some bacteria produce toxins when they grow in food. Because the toxins themselves are harmful, the bacteria don’t need to multiply in the intestine to make someone ill, so the symptoms come on very quickly.

Because the bacteria enter the body through the digestive system, symptoms will generally be in this part of the body – nausea, vomiting, abdominal cramps and diarrhoea. In some cases, food poisoning can cause very serious illness or even death.

How bacteria grow

Bacteria need warmth and moisture to grow. They reproduce by dividing themselves, so one bacterium becomes two and then two become four and so on. In the right conditions one bacterium could become several million in 8 hours and thousands of millions in 12 hours.

This means that if a food is contaminated with a small number of bacteria and you leave it out of the fridge overnight it could be seriously contaminated by the next day. Then just one mouthful could make someone ill. If you put food in the fridge it will stop bacteria from multiplying.

Since you can’t see, taste or smell bacteria, the only way that you can be sure that food is safe is to follow good food hygiene at all times. See the Keeping food safe section.

The Food Hygiene Campaign is part of the UK Food Agency’s strategy to reduce food poisoning. The success of this strategy is being measured by a reduction in the number of laboratory-confirmed cases of the following five bacteria; Campylobacter, Salmonella, Listeria, E.coli O157, Clostridium perfringens.

Clostridium Perfringens

Clostridium perfringens is found in low numbers in many foods, particularly meat and poultry and their products. It is also found in the soil, the intestines of humans and animals, in sewage and in animal manures.

Infection with Clostridium perfringens normally causes diarrhoea and severe abdominal pain. It may occasionally cause nausea but it rarely causes vomiting or fever.

Unlike many other types of bacteria that cause foodborne disease, clostridium perfringens isn’t completely destroyed by ordinary cooking. This is because it produces heat-resistant spores.

The bacteria are killed at cooking temperatures, but the heat-resistant spores they produce are able to survive and may actually be stimulated to germinate by the heat. If the food is not eaten at once but is allowed to cool slowly, the bacteria produced when the spores germinate multiply rapidly. Unless the food is reheated so that it is piping hot (at least to 60 o C and preferably to 75 o C), the bacteria will survive. After ingestion, if there are sufficient numbers present, the bacteria will produce toxins and the toxins will cause symptoms.

Foods most likely to be associated with Clostridium perfringens food poisoning are those that are cooked slowly in large quantities and left to stand for a long time at room temperature.

Salmonella

Salmonella is the second most common cause of food poisoning after Campylobacter. It has been found in unpasteurised milk, eggs and raw egg products, meat and poultry. It can survive if food is not cooked properly.

Salmonella can grow in food. If a small number of bacteria are present in a food, they will multiply unless it is chilled.

People infected with Salmonella should be particularly careful with personal hygiene because they could infect another person who comes into direct contact with them. For example, if a carrier doesn’t wash their hands properly after going to the toilet, they could have bacteria on their hands.

Listeria

Listeria monocytogenes is present all around in the environment. It has also been found in low numbers in many foods. In certain foods, such as soft mould-ripened cheeses and pâtés, it may be present in higher numbers. Eating foods containing high levels of listeria monocytogenes is generally the cause of illness.

Listeria monocytogenes usually causes illness in vulnerable groups such as pregnant women, babies, the elderly and people with reduced immunity. Among these groups, the illness is often severe and life threatening.

E.coli 0157

Most strains of E.coli are harmless, but those that produce verocytotoxin (called verocytotoxin-producing E.coli, or VTEC) can cause severe illness. In the UK, the most common type is E.coli 0157.

E.coli or Escherichia coli are bacteria that normally live in the intestines of humans and animals. Although, most strains of these bacteria are harmless, several are known to produce toxins that can cause diarrhea. One particular E.coli strain called 0157 can cause severe diarrhea and kidney damage.

Campylobacter

Campylobacter is the most common identified cause of foodborne disease. It has been found mainly in poultry, red meat, unpasteurised milk and untreated water. Although it doesn’t grow in food it spreads easily, so only a few bacteria in a piece of undercooked chicken could cause illness.

Campylobacter infections don’t usually cause vomiting, but diarrhoea can be severe and bloody with abdominal cramps.

Please contact Accepta to learn more about our Poultry Farm Water Treatment Services.

If you require lab testing for water quality or chemical dosing solutions such as chlorine dioxide, please contact us.

By Maria Teresa Mascellino

chapter and author info

Sapienza University of Rome, Italy

*Address all correspondence to: mariateresa.mascellino@uniroma1.it

Edited by Mitra Ranjbar, Marzieh Nojomi and Maria T. Mascellino

1. Introduction

The present book deals with the following microorganisms: E. coli, Salmonella, and Listeria. The first two are Gram-negative bacteria belonging to the group of Enterobacteriaceae with the characteristic of becoming resistant to the most common antibiotics; whereas, the last one is a Gram-positive bacterium belonging to Corynebacterium, Erysipelothrix, and other Gram-positive microorganisms showing an involvement in pathologies as newborn meningitis and gynecological infection which may interfere with the pregnancy outcome. The peculiarity of all these bacteria is that they can be transmitted by contaminated food.

2. E. coli

Class: Gamma Proteobacteria

The bacteria, in fact, can be found in the gastrointestinal tract (GI) of humans and animals, but they are mainly considered as ubiquitous microorganisms.

This bacterium includes a single species (E. coli) and is divided into 171 serotypes, aerobic-anaerobic Gram-negative rods with flagella fimbriae, and able to ferment glucose and lactose.

The most important serotype is Escherichia coli O157:H7 or enterohemorrhagic Escherichia coli (EHEC), which often leads to enterohemorrhagic diarrhea and is also able to induce hemolytic uremic syndrome (HUS) which is characterized by acute renal failure, hemolytic anemia, and thrombocytopenia that are more common in children and in elderly people [1].

Serotype O157-H7 causes numerous outbreaks and sporadic cases of bloody diarrhea. Foodborne pathogenic E. coli contamination, such as that with E. coli O157 and O104, is very common even in developed countries. Bacterial contamination may occur from environmental, animal, or human sources and cause foodborne illness [2].

The three main diseases, depending on each particular serotype involved, are urinary tract infections, intestinal diseases, and neonatal meningitis [3].

Many different mechanisms of action are reported regarding the virulence of E. coli. Although most strains are saprophytic colonizing the large bowel, some types of them are involved in different pathologies such as traveler’s and childhood diarrhea (ETEC and EPEC also in Mexico and North Africa EAEC), hemorrhagic colitis (EHEH), and a Shiga-like disease (EIEC). As far as this last point is concerned, it is reported that the differentiation between Shigella and E. coli is quite more complicated when we consider enteroinvasive E. coli (EIEC). In fact, EIEC are strains that are similar to E. coli but are able to cause dysentery using the same method of invasion as Shigella. In fact, in this specific situation, EIEC is more related to Shigella than to non-invasive E. coli [4]. This strain is among the most common cause of foodborne diseases other than of neurological and renal complications, especially in children.

Escherichia coli K1 strains are major causative agents of invasive disease of newborn infants. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to blood stream infections (BSI). Indeed, these microorganisms are the cause of life-threatening infections that are acquired from the mother at birth thus colonizing the small intestine, from where they invade the blood and central nervous system.

E. coli is increasingly present as a MDR (multi-drug resistant) bacterium, in fact its genomic outfit has acquired various antibiotic resistances through the production of ESBL [5] and carbapenemases as well as metallo-beta lactamases (NDM = New Delhi metallo-beta lactamases) making the infections of this bacterium extremely worrying [6] (Figures 1 and 2).

3. Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, mobile, rod-shaped bacterium that is ubiquitous in the environment. It can be isolated in soil and wood and decays in the natural environment; however, the principal acquisition of Listeria is through the ingestion of contaminated food products. Listeria is a foodborne pathogen that contaminates food-processing environments and persists within biofilms in the surroundings. The peculiar characteristic of this microorganism is its ability to grow even in extreme situations, such as under high salt conditions and refrigeration temperatures, maintaining its vitality in various food products [7]. Even though the incidence of listeriosis is lower than other enteric illnesses, infections caused by L. monocytogenes are more serious and may lead to hospitalizations and fatalities. These infections mainly affect women and children who acquire the disease through vertical transmission from mother to infant during pregnancy or childbirth. Nosocomial infections between children are rare but anyhow they were reported. The most important disease for the newborns is the neonatal meningitis, which shows a high degree of mortality (higher in the developing countries which can reach 40–58% of cases). Listeriosis requires rapid treatment with antibiotics and most drugs suitable for Gram-positive bacteria are effective against L. monocytogenes. Generally, the Listeria clinical strains are susceptible to the common antibiotics because only a minority results as being resistant to antimicrobial agents. In the same way, several strains detected from food exhibited resistance to antimicrobials not suitable against listeriosis [8]. Pregnant women can carry Listeria asymptomatically in their gastrointestinal tract or vagina and the risk of transmitting this infection to their babies is high. The consequence of listeriosis to human health is a very important issue due to its virulence mainly in children with an underlying immunodeficiency. Symptoms include fever, headache, abdominal pain, diarrhea, vomiting, and convulsions. The complications can be appendicitis and Meckel’s diverticulitis [9].

Listeria which is saprophyte in the environments such as water, soil, and food, once internalizes into the mammalian host, shows its virulence through the expression of many gene products reported in Figure 3 [10].

Phagocytosis of Listeria. Legenda: (internalins InlA and InlB), phagosome lysis (listeriolysin O (LLO)), phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine ((PC)-PLC), cell-to-cell spread (actin assembly-inducing protein (ActA)), intracellular growth (hexose-6-phosphate transporter (Hpt)) [10].

4. Salmonella

Salmonella is the most commonly isolated bacterial agent of foodborne and epidemic infections. It was reported for the first time in 1886, in a case of swine fever by the American doctor Daniel Elmer Salmon.

The genus Salmonella is characterized by Gram-negative facultative anaerobic bacilli without spores. They are mobile through peritrichous flagella with the exception of S. gallinarum and S. pullorum. The serotypes are diversified according to the somatic antigen “O,” the flagellar antigen “H” and the surface antigen “Vi.” The Vi antigen is exclusively expressed by S. typhi and is able to circumvent the innate immune response by repressing flagellin and LPS expression [11]. The “O” antigens are distinguished in the serogroups A, B, C1, C2, D, and E.

Salmonella is present in the environment and can be either commensal or pathogen for men and various animals; some serotypes are exclusively pathogen for humans (i.e., S. typhi and S. paratyphi A and C), others infect both humans and animals such as S. typhimurium [12].

In humans, there are two kind of infectious diseases:

typhoid and paratyphoid fever [13]

Salmonella infection is transmitted through fecal route by the ingestion of contaminated food and drink. Salmonella typhi is responsible for typhoid fever, and its transmission can occur, especially in developing countries, by water and food infected or with direct contact among people, especially in poor hygienic conditions. The minimum infectious dose can be less than 15–20 cells. Individual sensitivity depends on the patients’ age and on the nature of Salmonella strains.

In most cases, Salmonella infection occurs in mild form and resolves on its own within a few days. In these situations, the advice is not to consider the diarrheal phenomenon, since it is the natural defense mechanism used by the organism to expel germs. Normally, for Salmonella, it should be enough to adopt a supportive therapy: administration of oral rehydration solutions (which are used to compensate for water and salts lost with vomiting and diarrhea), lactic ferments, and probiotics.

Although salmonellosis is a bacterial infection, the use of antibiotics is not recommended as it could lengthen the persistence time of Salmonella in feces or induce antimicrobials resistance [15]. Hospitalization and the use of antibiotics are indicated only in severe cases (with extra-intestinal symptoms), in infants under 3 months and in subjects with chronic-degenerative diseases.

In recent times, Salmonella has changed its characteristics worldwide, becoming the etiologic agent of many peculiar pathological processes such as cancer development, inflammatory process, and immune-pathogenesis [16, 17] (Figure 4).

Salmonella infection pathogenesis. The ingestion of contaminated food or water begins the infective processes (gastroenteritis or systemic infection) depending on the species of Salmonella involved (minor and major Salmonellae). The microorganisms reach the intestinal epithelial cells and migrate to the lamina propria invading the liver from where Salmonella reaches the gall bladder and can cause chronic carriage which gives rise to healthy carriers [18].

Abstract

Escherichia coli O157:H7 and Listeria monocytogenes were able to grow for a period of 2 days in fresh chicken manure at 20°C with a resulting 1–2 log units increase in CFU; Salmonella typhimurium remained stable. Prolongation of the storage time to 6 days resulted in a 1–2 log decrease of S. typhimurium compared to the initial count and a 3–4 log decrease of E. coli O157:H7; the number of L. monocytogenes did not decrease below the initial. These changes were accompanied by an increase in pH and accumulation of ammonia in the manure. The destruction of the three microorganisms was greatly increased by drying the manure to a moisture content of 10% followed by exposure to ammonia gas in an amount of 1% of the manure wet weight; S. typhimurium and E. coli O157:H7 were reduced by 8 log units, L. monocytogenes by 4.

1 Introduction

Poultry as well as other animals shed Salmonella and other enteric microorganisms in the feces if they become colonized. This may facilitate horizontal transmission and has also caused concern in connection with the practice of feeding chicken manure to cattle or applying it as a fertilizer for growing produce for human consumption; this may result in disease unless the manure is sterilized or pasteurized. Composting or other processing procedures can reduce the number of viable pathogens in manure but only a limited amount of manure is composted and the process may not always be under strict control. Drying in itself reduces the number of Salmonella in manure and litter [1–3] but it has been found that drying is only effective at certain intermediate levels of water activity; when most of the water has been removed Salmonella will survive for long periods of time [4]. Ammonia above a certain concentration also has a killing effect [2]. However, there does not seem to be any published report related to the application of ammonia and drying to reduce human pathogens in manure. The aim of this report is to provide an evaluation of the effect of drying and/or exposure to ammonia on Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in chicken manure.

2 Materials and methods

Fresh chicken manure was obtained from the Department of Avian Science facility, University of California, Davis. It was found to be free of natural contamination with the three test pathogens. E. coli O157:H7 was obtained from Dr. B. Walsh, Department of Population Health and Reproduction, University of California, Davis; S. typhimurium was isolated in a chicken house in California; L. monocytogenes was obtained from Dr. D. Hirsh, Department of Pathology, Microbiology and Immunology, University of California, Davis.

Inocula to be added to manure were grown on blood agar at 37°C for 24 h and the cells were suspended in sterile saline. The numbers of microorganisms in the manure before and after treatments were determined by suspending a 10-g manure sample in 100 ml sterile saline and surface plating on brilliant green novobiocin agar (Difco plus 20 mg novobiocin per liter) for Salmonella, violet red bile agar (Difco) for E. coli and lithium chloride phenyl ethanol moxalactam agar [5] for L. monocytogenes. Quantification of low numbers was done by a most probable number procedure [6] where four 10-fold dilutions, two units per dilution level, were cultured in pre-enrichment broth followed by confirmation using the plating media mentioned above. Pre-enrichment of Salmonella and E. coli took place in lactose broth (16 h at 37°C); for Salmonella this was followed by selective enrichment in tetrathionate broth (Difco) for 24 h at 37°C. Pre-enrichment of Listeria took place in Brucella broth (Difco) for three weeks at 4°C.

Ten grams of manure was distributed evenly on the bottom of a standard petri dish (100 mm×15 mm) and five 20-µl volumes of bacterial suspensions, containing approximately 10 10 CFU per ml, were distributed on the surface of the manure. For counting bacteria, pH measurements and determination of ammonia the entire manure sample was suspended in 60 ml sterile saline.

Manure was dried by placing the open petri dish on the laboratory bench at 20°C and under normal ventilation (relative air humidity was about 50%); This resulted in a drop of the moisture content in the manure from 60–70% to about 10% in 24 h, this corresponds to water activity levels of 0.91–0.99 and 0.37 respectively (unpublished data).

Exposure to ammonia gas was done by placing a small petri dish with calculated amounts of ammonium sulfate and potassium hydroxide and water in the dish containing the manure sample; a matching half of a standard petri dish was then used to close the dish with the manure sample and sealed in place with electric tape. After the unit, which had an air space of 122 cm 3 , had been closed it was tilted to allow the potassium hydroxide to react with water and ammonium sulfate to release the desired amount of ammonia. When combinations of drying and exposure to ammonia was used the drying was done first.

Total moisture was determined by drying a manure sample at 105°C for 2 h; pH was determined with a glass electrode. Ammonia was determined by using an improvised diffusion chamber; 2 ml of the manure suspension was placed in a small, open petri dish (3.5 cm diameter), 1 ml diluted sulfuric was placed in another open, small petri dish. Both were placed in a standard petri dish, potassium hydroxide pellets were put into the manure suspension and the standard petri dish was immediately closed with a matching half of a petri dish that was sealed in place with electric tape. After 24 h at room temperature the amount of volatile bases (ammonia) was determined by titrating the sulfuric acid with 1 N sodium hydroxide using bromocresol purple as an indicator.

3 Results

L. monocytogenes and E. coli O157:H7 grew for a limited time in the manure and reached levels 10–100 times higher than the initial level in 2 days (Fig. 1). Following that a decline set in and S. typhimurium and E. coli O157:H7 declined to below the initial level in 6 days, L. monocytogenes was more persistent. The decline was accompanied by an increase in pH to close to 9.5, probably due to an increase in ammonia (Fig. 2) that reached levels of 3.4 mg g −1 in 48 h.

pH changes and growth/survival of pathogens at 20°C in fresh chicken manure with 71% moisture content.

pH and ammonia levels (mg g −1 ) in fresh chicken manure.

The separate and combined effects of drying and gassing with ammonia were tested in a factorial experiment with two replications. Drying reduced the water content of the manure to 10%, gassing was done by generating ammonia in amounts corresponding to 1% of the wet manure weight. The results, expressed as changes in log CFU, are shown in Fig. 3. The untreated controls exhibited growth of the microorganism which is in agreement with the results for fresh manure (Fig. 1). Drying alone and gassing with ammonia alone resulted in less than two log reductions. Drying followed by gassing with ammonia resulted in a 4-log reduction of E. coli O157:H7, 3 logs for S. typhimurium and 2.5 logs for L. monocytogenes. These results were obtained with a 24-h treatment and a partial replication of the experiment was done where the treatment with ammonia gas was extended to 72 h (Fig. 4). This resulted in an almost 8-log reduction of S. typhimurium and E. coli O157:H7 and more than 4-log reduction of L. monocytogenes from an initial number of 10 9 CFU g −1 .

Reduction of log CFU g −1 of pathogens in manure exposed to drying to 10% moisture in 24 h at 20°C and/or exposed to 1% ammonia gas for 24 h at 20°C.

Survival of pathogens in chicken manure after drying at 20°C to a moisture level of 10% followed by exposure to 1% ammonia gas for 24 h, 48 h and 72 h, at 20°C.

4 Discussion

Ammonia is naturally generated by indigenous microorganisms in moist chicken manure at appropriate temperature [7] and can cause a significant reduction of non-spore forming pathogens in stacked manure [2, 7]. However, it is practice in commercial poultry production to let the manure dry in order to reduce the detrimental effect ammonia on the birds [8]; under these circumstances the destruction of pathogens becomes less predictable. The present study indicates that treatment of the dry manure with ammonia results in a significant reduction of common pathogens. The amount of added ammonia in these experiments corresponds to 10 kg, or 13 l liquid ammonia, per ton manure as compared to a natural content of total nitrogen in chicken manure of 2–60 kg per ton [9]. By extending the exposure time it might be possible to decontaminate manure with concentrations of ammonia smaller than 10 kg per ton manure.

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