Listeria Monocytogenes: Understanding the enemy and plotting the defenses

Listeria Monocytogenes:  Understanding the enemy and plotting the defenses
By Eben van Tonder
6 January 2018 (Update:  4/2018)

For a brief overview of the identification of the organism, see Listeria Monocytogenes: Its discovery and naming.

Index

Summary
Background
Know Your Enemy
Gastroenteritis
Invasive Listeriosis
Link between Invasive Listeriosis and Gastrointestinal Symptoms
Distribution
Evaluating Control Measures
Focus areas for control measures
Cutting Plant and Chiller Rooms
Processing Plant Contamination
Factory Cleaning
Effective Equipment Cleaning
Cleaning as part of Plant Design
Potential Reservoirs of L. monocytogenes in Small Processing Plants
Other Areas for Potential L. monocytogenes Contamination
Other cleaning related steps
Compressed Air Must be Contaminant-Free
Control Points in Processing Steps and Validation
Thermal Inactivation
Acid Tolerance Response
Combined effect of pH and NaCl
Routine monitoring
Techniques for Sampling the Environment
Finished Product/In-Line Sampling
Sample testing and compositing
General Procedures for Sampling Listeria
Data Management
Critical Limits(CLs)and Records
Verification/Corrective Actions
Pork Anatomy and the Prevalence of Listeria
Conclusion

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Summary

In light of the unprecedented listeria outbreak in South Africa, we examine processes and procedures at the cutting plant, factory cleaning, thermal inactivation and acid tolerance response of Listeria monocytogenes.  We question everything we do and examine the nature of the bacteria in order to ensure that we have the right cleaning program and processing hurdles in place.

Key conclusions of the study are the importance of sanitising stations in the cutting department or plant, deboning and slicing departments.  Testing for ATP at plant start-up and three hours later, for listeria.  An aggressive use of QAC’s around drains and areas of heavy traffic with forklifts, pallet jacks, and human traffic.  Foam spraying access points into the offloading area and from the freezers into the factory.  The elimination of water during shift and where this is not possible, poisoning the water for bacteria, in particular, L. monocytogenes.  Regular hand sanitation at intervals throughout the shift.  The use of gloves.  Proper issuing of ingredients.  Bin liners.  Alkali detergent -> an acid-based sanitising sequence in cleaning and not acid first and then alkali.  Strict procedure must be enforced to prevent cross-contamination including maintenance tools and hosepipes.  Stripping all pumps once a month for thorough cleaning.  Fitting spacers to eliminate steel that is sandwiched together.

Proper utilisation of the smoking cycle to thermally inactivate any L. monocytogenes that may have come into production.  In order to achieve thermal inactivation in bacon production, at least three hours smoking/ thermal treatment is suggested at a core temperature of > 55 deg C.  A case will be made for maintaining our current NaCl levels (at least 4% NaCl to the meat) and processing it within 24 hours, while maintaining a meat temperature of < 7 deg C as an effective hurdle to prevent L. monocytogenes growth.

A maximum processing time before thermal treatment along with an ideal temperature range and salt % will be given which will restrict the multiplication of L. Monocytogenes.

The pork carcass is presented as a major root cause for the organism being present in pork factories.  Slaughtering and cutting departments must be aware of this and take special precautions in light of it.

The approach is to set up multiple hurdles for eradicating and retarding the growth of bacteria, including L. monocytogenes.

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Background

The current listeria outbreak in South Africa is the largest to date in recorded history with over 60 deaths as a result of listeriosis.  By 19 December there were 647 confirmed cases (550 on 5 December) i.e. almost 100 more in a question of 14 days.  It is reported that neonates are the most affected. The numbers indicate that it is one of the worst listeriosis cases in global history. “A large percentage (74%) of all the clinical isolates belonging to the same sequence type i.e. ST6 – this means that these isolates originate from a single source, most likely a food product on the market.  By the end of December, statistics indicated that the outbreak has not yet peaked. (Anelich Consulting)

In the army, one of the first lectures we got was “know your enemy” after which we started with fieldcraft and combat training.  I am reminded of the military approach in dealing with food safety.  Another important group of lectures dealt with Comm Ops Own Forces – Communication Operations Own Forces where a concerted effort is made to communicate key messages to own forces.  It will be important to have a similar approach to ensure that every member of staff buys into the overall approach, from the company management to the most recent temporary employee performing the simplest function on the factory floor, and everybody in between.

So, before we look at practical steps, lets first understand who our enemy is.

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Know Your Enemy

L. monocytogenes is a very dangerous and complex organism.  Understanding it and the risks it poses is a very important first step to focuses collective resolve and making it a priority.  Just how close we live to it, and how many of us over the past year or two had health issues as a result of ingesting it is an eye opener and shocking/ scary reality.  It is, in many regards, our biggest single threat from a bacterial perspective.

L. monocytogenes is a Gram-positive bacterium, in the division Firmicutes (Latin: firmus, strong, and cutis, skin, referring to the cell wall).  “This facultative intracellular bacterium can cause listeriosis, a severe invasive illness in humans, which may result in death. The risk of contracting listeriosis is high in immuno-compromised persons, the elderly, pregnant women, neonates, “people with weakened immune systems, such as persons immunocompromised by corticosteroids, anticancer drugs, graft-suppression therapy, and AIDS. Other conditions that may increase susceptibility to listeriosis are diabetes, cirrhosis, asthma, and ulcerative colitis. Healthy people are generally at a low risk of contracting L. monocytogenes-related illnesses; however, when heavily contaminated food is consumed, any person can be susceptible. Some research suggests that use of antacids also may increase the risk of contracting listeriosis.”  (Cutter, et al)

“Although listeriosis is relatively uncommon, it is a potentially fatal disease and frequently results in spontaneous abortions in pregnant women. Even though the symptoms may be relatively mild in the mother, the illness may be transferred to the fetus, causing serious illness or fetal death. Symptoms of L. monocytogenes may include meningitis, encephalitis, septicemia, spontaneous abortion, stillbirth, and influenza-like symptoms. The onset of the disease can occur anywhere from a few days up to 6 weeks after ingestion of L. monocytogenes bacteria, with the symptoms lasting from a few days to several weeks.”  (Cutter, et al)

Thirteen serotypes of L. monocytogenes have been identified, but only three serotypes (1/2a, 1/2b and 4b) are associated with the majority of sporadic cases of listeriosis; serotype 4b is linked to almost all recent outbreaks.”  (Thévenot, et al., 2006)  “Serotype 4b has been the type most commonly responsible for invasive listeriosis, whereas serotypes 1/2a and 1/2b have been the dominant isolates in outbreaks of gastroenteritis.”  (Say Tat Ooi, et al, 2005)

The fact that it is Gram-positive, gives us a clue to its dangerous toxin formation.  The classification as Gram-positive has nothing to do with gram, as in a measure of weight as I first thought when I heard the word many years ago.  It refers to the Danish scientist Hans Christian Gram (1853 – 1938) who “devised a method to differentiate two types of bacteria based on the structural differences in their cell walls. In his test, bacteria that retain the crystal violet dye do so because of a thick layer of peptidoglycan (a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall) and are called Gram-positive bacteria. In contrast, Gram-negative bacteria do not retain the violet dye and are coloured red or pink.”  (Diffen)

Generally, Gram-positive pathogens produce exocellular substances that typically account for most, if not all, of its ability to cause disease (i.e., virulence factors).  An exception to this is L. monocytogenes which is a Gram-positive intracellular pathogen.  It is a  facultative intracellular parasite which after invading the cell, directly spread to neighboring cells. It disseminates in host tissues sheltered from the immune system Virulent strains, such as L. monocytogenes, are known to secrete a number of exotoxic factors.  Exotoxic factors are molecules produced by bacteria, viruses, fungi, and protozoa that add to their effectiveness and enable them to achieve a number of results such as obtaining nutrition from the host and colonization.  In the case of L. monoctytogenes, the exotoxin factor produced enables the organism to spread from cell to cell with the aid of listeriolysin O (LLO) which it secretes.  LLO is thiol-activated and pore-forming but is not the cause of foodborne gastroenteritis syndrome.

“Listeriosis is clinically defined when the bacterium is isolated from blood, cerebrospinal fluid, or an otherwise normally sterile site (such as the placenta).”  (Cutter, et al)

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Gastroenteritis

The fact that L. monocytogenes is responsible for gastroenteritis was first identified by Riedo et al. in 1989.  “Convincing evidence that L. monocytogenes could cause gastrointestinal illness came from an outbreak of febrile (a fewer) gastroenteritis that was associated with the consumption of contaminated chocolate milk. Symptoms developed in 75% of persons (45 of 60) who drank chocolate milk that had been served at a picnic. Indistinguishable strains of L. monocytogenes were isolated from unopened cartons of chocolate milk, from environmental specimens from the dairy that supplied the milk, and from the stool samples of 14 symptomatic persons.”  Commonly reported symptoms from outbreaks of gastroenteritis due to L. monocytogenes are listed below.  (Say Tat Ooi, et al, 2005)

symptoms l monocytogenes

“Bloody diarrhea was rare and was noted in 3% of cases. Although chills and sore throat were not reported in every outbreak, they were present in >65% of patients in some reports. It is also interesting to note that sleepiness (an unusual complaint in febrile gastroenteritis) was reported in 63% of cases described by Aureli et al., and fatigue was noted in 74% and 83% of cases reported by Dalton et al. and Frye et al., respectively.”  “The incubation period from the time of food ingestion to the onset of symptoms is usually 24 h or less, but it has ranged from 6 h to 10 days.”  (Say Tat Ooi, et al, 2005)

“Listerial gastroenteritis is typically self-limited without serious complications in healthy individuals. The usual duration of symptoms is 1–3 days but may be as long as 1 week. In most instances, only a small number of affected individuals required hospitalization because of illness—reportedly 2%  and 6.9% in 2 studies; however, in the outbreak described by Aureli et al., 19% of symptomatic persons were hospitalized. Almost all of those hospitalized have been children or elderly persons. In another outbreak, 4 young adults aged 17–27 years were reported to require inpatient care because of their illness. In 2 of these patients, L. monocytogenes was isolated from cultures of blood samples. The prevalence of bacteremia (the presence of bacteria in the blood) in patients with listerial gastroenteritis is unknown.”  (Say Tat Ooi, et al, 2005)

“Contaminated food appears to be the source of listerial infection in sporadic cases as well as in outbreaks.”  “There is no evidence of waterborne infection.”  (Say Tat Ooi, et al, 2005)

L. monocytogenes is found commonly in food, and recovery rates ranging from 2.2% to 92% have been reported in dairy and meat products during routine sampling [22]. The ingestion of listeriae must be a common event; however, clinical disease due to listeriae is rare, especially in healthy individuals. In reported gastroenteritis outbreaks, and in contradistinction to invasive listerial infection, the vast majority of people affected have been healthy, without obvious underlying disease.

It must also be noted that in the case of L. monocytogenes, unlike other syndromes caused by Gram-positive bacteria (with the exception of Clostridium prefingens), “the ingestion of viable cells is necessary for listeric infection to occur” (Jay, et al.; 2005:  532, 533)

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Invasive Listeriosis

The other illness caused by L. monocytogenes is invasive listeriosis which is severe and manifests itself usually clinically  in abortion, sepsis, and meningoencephalitis, a  medical condition that simultaneously resembles both meningitis, which is an infection or inflammation of the meninges, the three membranes that envelop the brain and spinal cord and encephalitis, which is an infection or inflammation of the brain itself.

“Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeria actively multiplies until the infection is controlled by a cell-mediated immune response.  This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal individuals, the continual exposure to listerial antigens, the toxin which induces an immune response in the body, probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level presence of the bacteria in the blood, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease.  (Vázquez-Boland, et al., J. A., 2001)

“Fetomaternal and neonatal listeriosis results from invasion of the fetus via the placenta.  Its consequence is abortion, usually from 5 months of gestation onwards, or the birth of a baby or stillborn fetus with generalized infection,. . . characterized by the presence of pyogranulomatous microabscesses disseminated over the body and a high mortality.”  “The infection is usually asymptomatic in the mother or may present as a mild flu-like syndrome with chills, fatigue, headache, and muscular and joint pain about 2 to 14 days before miscarriage. Less frequently (10 to 15% of perinatal cases), late neonatal listeriosis is observed.”  (Vázquez-Boland, et al., J. A., 2001)

The listerial infection most frequently reported in nonpregnant adults is that affecting the central nervous system (the brain and the spinal cord) (55 to 70% of cases).  Pure meningeal forms are observed in some cases, but infection normally develops as a meningoencephalitis accompanied by severe changes in consciousness, movement disorders, and, in some cases, paralysis of the cranial nerves.  The encephalitic form, in which Listeria organisms are isolated with difficulty from the cerebrospinal fluid (CSF), is common in animals but rare in humans (see below). Its course is usually biphasic, with an initial subfebrile phase lasting 3 to 10 days in which there may be headache, vomiting, visual disorders, and general malaise, followed in a second phase by the onset of severe signs of rhombencephalitis. The mortality rate for CNS infection is around 20% but may be as high as 40 to 60% if associated with concurrent, underlying debilitating disease. It has been estimated that L. monocytogenes accounts for 10% of community-acquired bacterial meningitis. Due to effective vaccination against Haemophilus influenzae, L. monocytogenes is now the fourth most common cause of meningeal infection in adults after Streptococcus pneumoniae, Neisseria meningitidis, and group B streptococci. However, in certain high-risk groups, such as cancer patients, L. monocytogenes is the most common cause of bacterial meningitis. Another frequent form of listeriosis (in some series of patients reported, even more, frequent than CNS infection) is bacteremia or septicemia (15 to 50% of cases), with a high mortality rate (up to 70%) if it is associated with severe underlying debilitating conditions. There are other atypical clinical forms (5 to 10% of cases), such as endocarditis (the third most frequent form), myocarditis, arteritis, pneumonia, pleuritis, hepatitis, colecystitis, peritonitis, localized abscesses (e.g., brain abscess, which accounts for about 10% of CNS infections by Listeria spp.), arthritis, osteomyelitis, sinusitis, otitis, conjunctivitis, ophthalmitis, and, in cows, mastitis.  (Vázquez-Boland, et al., J. A., 2001)

“The incubation period for the invasive illness is generally much longer, around 20 to 30 days.”  (Vázquez-Boland, et al., J. A., 2001)

“Host susceptibility plays a major role in the presentation of clinical disease upon exposure to L. monocytogenes. Thus, most listeriosis patients have a physiological or pathological defect that affects T-cell-mediated immunity. This justifies the classification of L. monocytogenes as an opportunistic pathogen. The groups at risk for listeriosis are pregnant women and neonates, the elderly (55 to 60 years and older), and immunocompromised or debilitated adults with underlying diseases. Listeriosis in nonpregnant adults is associated in most cases (>75%) with at least one of the following conditions: malignancies (leukemia, lymphoma, or sarcoma) and antineoplastic chemotherapy, immunosuppressants therapy (organ transplantation or corticosteroid use), chronic liver disease (cirrhosis or alcoholism), kidney disease, diabetes, and collagen disease (lupus).”  (Vázquez-Boland, et al., J. A., 2001)

“Human immunodeficiency virus (HIV) infection is also a significant risk factor for listeriosis. AIDS is the underlying predisposing condition in 5 to 20% of listeriosis cases in nonpregnant adults. It has been estimated that the risk of contracting listeriosis is 300 to 1,000 times higher for AIDS patients than for the general population. Nevertheless, listeriosis remains a relatively rare AIDS-associated infection, probably due to the preventive dietary measures taken by HIV-infected patients (avoidance of high-risk foods), the antimicrobial treatments that they receive regularly to treat or prevent opportunistic infections, and the fact that HIV infection does not significantly reduce the activity of the major effectors of immunity of Listeria spp. (innate immune mechanisms and the CD8+ T-cell subset.”  (Vázquez-Boland, et al., J. A., 2001)

“The health status of the patient greatly influences the outcome of listeriosis. Immunocompetent patients usually survive listeriosis, whereas those with underlying debilitating diseases often succumb to the infection (mean mortality rate for this group, >30 to 40%). Although most listeriosis cases are associated with underlying risk factors, there are also a few adult patients for whom no obvious predisposing condition can be identified.”  (Vázquez-Boland, et al., J. A., 2001)

“The following hypothetical scenario for the pathogenesis of listeriosis can be proposed. Clinical outcome of Listeria infection depends on three major variables: (i) the number of bacteria ingested with food, (ii) the pathogenic properties of the strain, and (iii) the immunological status of the host. In immunocompetent individuals with no predisposing conditions, ingestion of low doses of L. monocytogenes will probably have no effect other than the development or boosting of antilisterial protective immunity. In contrast, oral exposure to large doses is likely to result in an episode of gastroenteritis and fever and, depending on the virulence of the strain, possible invasive disease. Immunocompromised and debilitated individuals, however, cannot mount an immune response strong enough to control bacterial proliferation in the liver, the primary target organ of L. monocytogenes, and are therefore susceptible to invasive disease following the ingestion of a lower inoculum. Inefficiently restricted growth of L. monocytogenes in the hepatocytes in these individuals is likely to result in an increase in the critical mass of bacteria and their release into the bloodstream. The ensuing prolonged bacteremia will result in local infections in secondary target organs (particularly the brain and placenta) or in septicemic disease in severely immunocompromised hosts.”  (Vázquez-Boland, et al., J. A., 2001)

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Link between Invasive Listeriosis and Gastrointestinal Symptoms

An association between clinical episodes of invasive listeriosis and a history of gastrointestinal symptoms, including diarrhea, vomiting, and fever, was noticed some time ago. Investigations of recent food-borne outbreaks have provided compelling evidence that a febrile gastroenteritis syndrome (symptoms of fever and a runny tummy) may indeed be the main clinical manifestation of L. monocytogenes infection. The lesson to be learned from these epidemics is that L. monocytogenes should be sought as a possible disease-causing agent if you suffer from diarrhea and fewer.

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Distribution

Listeria monocytogenes is widely distributed in nature.  It is found in soil, animal and human feces, sewage, silage (fodder), and in water.  The natural habitat of these bacteria is thought to be decomposing plant matter where it lives on decaying organic matter.  Its association with dairy products is well known.  (Jay, et al.; 2005:  598)  Every factory has listeria.  It is that common and is carried into the factory through any one of the many access points.  From people walking it in under their boots, forklifts carrying it in through receiving doors from trucks or parking areas or through pallets that are not properly sanitised and cleaned to the cloths of factory workers and ineffective hand washing techniques.  Water is its transport medium through which it is distributed.

It is not only prevalent in nature and consequently in factories, but in our food also.  As we have already indicated, “L. monocytogenes is found commonly in food, and recovery rates ranging from 2.2% to 92% have been reported in dairy and meat products during routine sampling. The ingestion of listeriae must be a common event; however, clinical disease due to listeriae is rare, especially in healthy individuals. In reported gastroenteritis outbreaks, and in contradistinction to invasive listerial infection, the vast majority of people affected have been healthy, without obvious underlying disease.”  (Say Tat Ooi, et al, 2005)    It is therefore not surprising that the prevalence of asymptomatic stool carriage of L. monocytogenes in healthy adults occurs is said to be 1%–5%.  “A markedly increased prevalence of L. monocytogenes has been found in the stool specimens of patients receiving long-term treatment with gastric acid–suppressive medications, compared with patients who have normal gastric acid secretion.  (Say Tat Ooi, et al, 2005)

To show the widespread ingestion of l. monocytogenes, “Grif et al. studied the incidence of fecal carriage by examining 868 stool specimens obtained from 3 healthy volunteers during a 1-year period. Using culture and PCR, the researchers showed an incidence of 5–9 exposures to L. monocytogenes per person per year and an average of 2 episodes of asymptomatic fecal carriage (at least 2 positive results of cultures performed on consecutive days) per person per year; no episode of fecal shedding lasted >4 days, and shedding was not associated with any symptoms. In rare instances, promoting events may turn colonizing listeriae into invasive pathogens, as is suggested by instances in which the development of listeriosis followed shigella infection or colonoscopy.  Shigella infection is often associated by bloody diarrhea, but not always.”  (Say Tat Ooi, et al, 2005)

If it is that common, what factors cause people to get ill from it?  “Ingestion of a large inoculum of L. monocytogenes has been postulated as one of the factors in the pathogenesis of clinical illness. Farber studied healthy nonhuman primates that received doses of various concentrations of L. monocytogenes suspended in sterile whole milk. Only animals that received 109cells became noticeably ill; those that received 105 or 107 cells of L. monocytogenes did not become ill, which suggests that there is a dose-dependent response to L. monocytogenes. It is unclear what the minimum infecting dose is for either healthy or high-risk individuals.”  (Say Tat Ooi, et al, 2005)

“The concentration of L. monocytogenes found in food during microbiologic surveillance has been as high as 107 cfu/g (ten million) of food but is most often <104 cfu/g  (ten thousand) of food; on the other hand, in outbreaks of invasive listeriosis, counts have been >104 cfu/g (ten thousand) of food in most cases, even though colony counts from implicated food sources have ranged from 102 cfu/g (one hundred) to 109 cfu/g (one billion) of food. Similarly, in outbreaks of gastroenteritis, the degree of bacterial contamination in the implicated food source has varied from 3 ×101 cfu/g (ten) of food to 1.6 ×109 cfu/g (one billion) of food, but is most typically >105 cfu/g (one hundred thousand) of food. It should be remembered that, because a considerable amount of time may pass from the initial recognition of human disease to the sampling of potential food sources, the number of organisms found in food samples at the time of the outbreak investigation may not necessarily reflect the infecting dose. A retrospective calculation taking into consideration the growth rate of L.monocytogenes has estimated high-grade contamination with ∼1.0 ×107 cfu/g (one million) of food during an outbreak of acute febrile gastroenteritis that involved 16 of 44 healthy attendees of a catered party. Carrique-Mas et al. demonstrated a dose-dependent response in an outbreak of gastroenteritis related to consumption of contaminated cheese; illness developed in 36.3% of persons who had eaten 1–2 servings of cheese, 46.1% of those who had eaten 3–6 servings, and 77.8% of those who had eaten >6 servings.”  (Say Tat Ooi, et al, 2005)

This is however not the only factor determining if someone will become ill.  “In the outbreak that was associated with chocolate milk, there was no apparent difference in the amount of milk consumed by the 42 persons who became ill and the amount consumed by 15 persons who consumed milk but did not become ill, thereby raising the question of the role of the host in clinical illness.”  (Say Tat Ooi, et al, 2005)

As was already stated, suppression of gastric acidity possibly plays a role.  Generally speaking “gastric acidity is an important protective mechanism against many foodborne infections”, but its role in listerial infection is unclear. “Cobb et al. showed a markedly increased prevalence of Listeria in stool samples from patients who were receiving long-term treatment with H2 antagonists, compared with patients with normal gastric secretion. In animal models, rats that were pretreated with cimetidine could be infected with a significantly lower dose of virulent L. monocytogenes than could untreated rats. However, in a nonhuman primate model, there was no substantial difference in infecting dose between animals that were treated with antacids and control animals.  (Say Tat Ooi, et al., 2005)

Exactly how L. monocytogenes causes diarrhea is unknown, “but diarrhea is likely the result of direct invasion.”  The organism secretes listeriolysin O (LLO).  LLO is thiol-activated and pore-forming but is not the cause of foodborne gastroenteritis syndrome. “The organism is not known to produce any enterotoxins which affect the intestines and cause food poisoning, and invasion is suggested by fever, as well as by occasional bloody diarrhea and bacteremia (presence of the bacteria in the blood).  (Say Tat Ooi, et al, 2005)

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Evaluating Control Measures

I examine factory cleaning protocols, thermal inactivation and acid tolerance response.   Nesbakken et al. (1996), as reported by Thévenot, et al. (2006), “found L. monocytogenes at every stage of the fresh pork meat industry, with increasing prevalence from the slaughterhouse to the cutting room.”  The widespread occurrence of the bacteria, however, makes it likely that contamination can occur at any stage in the processing process with an increased risk at every stage of processing.

In evaluating the real risk of listeriosis from meat products, Thévenot reports that “the incidence of L. monocytogenes in meat products is generally low, even if the pathogen is present at low or moderate levels (Encinas et al. 1999; AFSSA 2000; FICT 2002). Even if a single bacterial cell has the potential to cause disease, epidemiological data indicate that foods involved in listeriosis outbreaks are those in which the organism has multiplied and in general, have reached levels significantly >1000 CFU g−1 (Ross et al.2002; Risk Assessment Drafting Group 2004).”  (Thévenot, et al.; 2006)

The approach is to set up multiple hurdles for eradicating and retarding the growth of bacteria, including L. monocytogenes.  Bacteria are able to resist small changes in an environment, “but severe or multiple changes stimulate complex stress responses that are generally directed to survival instead of growth (Booth 1998)”.  (Thévenot, et al.; 2006)

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Focus areas for control measures

Catherine N. Cutter, assistant professor, Department of Food Science, and William R. Henning, professor, Department of Animal Science from Penn State wrote a very handy article, Control of Listeria monocytogenes in Meat and PoultryAccording to them, “plants that have a regulatory requirement for HACCP are already operating under sanitation programs called Sanitation Standard Operating Procedures (SSOPs). In many cases, all or part of an SSOP may be transferred to the actual HACCP plan of a plant as sanitation becomes recognized as critical to the production of a safe meat product. There is no step later in the process that will control the hazard. The details of this transfer will vary from plant to plant and even product to product, based on the analysis and control methods chosen to address each potential hazard. Examples of CCPs developed from SSOPs might include:

  • personal hygiene
  • using color-coded clothing, such as blue frocks for RTE areas and white for raw product areas
  • recording the application and concentration of chemical sanitizers
  • use of microbiological or ATP bioluminescence tests to determine effectiveness of cleaning and sanitizing programs”

These measures are evaluated in light of the way that l. monocytogenes are most often transferred to food in a food factory environment, namely:

  1. employees, through their clothing, gloves, boots, or skin coming into direct contact with the product
  2. improperly cleaned and sanitized equipment
  3. the environment, through airborne bacteria or aerosol moisture droplets generated in other work areas

(Cutter, et al.)

“L. monocytogenes can grow in cool, damp environments such as those found in any processing area, in coolers, or on slaughter floors. Improper sanitation and/or incomplete removal of meat and fat from processing equipment can allow biofilms to develop. These biofilms provide nutrients and a place of attachment for growing bacteria, including L. monocytogenes.

Products that have been fully cooked and will be consumed as packaged, without further heat treatment, present the highest risk to consumers if contaminated with L. monocytogenes. In order to control L. monocytogenes contamination, plants must assess their product flow and identify the most likely sites of contamination. A pre-processing checklist has been developed to help processors evaluate areas of high risk.”  (Cutter, et al.)

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Cutting Plant and Chiller Rooms

Thévenot, et al. (2006) reports that “extensive study of meat contamination levels in the meat processing industry indicated that chilling and cutting significantly increased the contamination of pork meat (Nesbakken et al. 1996) while Van der Elzen and Snijders (1993) found that the environmental prevalence of the pathogen in chilling–cutting areas to be as high as 71–100%. These findings strongly suggest that post-slaughter processing is a significant cause of meat contamination and that contamination is amplified in the chilling and cutting room environment (Nesbakken et al. 1996).”  (Thévenot, et al.; 2006)  This means that the first major area of focus is the cutting areas and chilling rooms where several measures can be implemented depending on the plant design.  These include meat decontamination wash with acetic or lactic acid and redesigning the continues cleaning, end of shift and weekly deep clean cleaning regimes to deal with L. monocytogenes effectively.

I also suggest the creation of a dedicated sanitising station in this department where all bins, crates and other equipment are sanitized before they either enter or leave the area.  Likewise, equipment such as knives should be sanitised whenever a new batch is started and completed at an appropriate time interval (ex. 20, 40 or 60-minute intervals) during processing of any particular batch.

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Processing Plant Contamination

Contamination in the processing plant can occur at several stages:

  • People, forklifts, pallet jacks, bins, crates, pallets, containers and other items entering or brought into the factory probably brings L. monocytogenes into the plant.
  • Water is its main transport mechanism and any moisture can carry it.  Drains is a particular problem area.
  • Raw material brought into the plant can be contaminated and hurdles may be inefficient to deal with the contamination and detection.
  • The contaminated raw material can contaminate surfaces and people who spread it.  These are well-recognised routes of contamination.
  • Similarly, where a meat sterilizing step is used before injection, for example, handling of unsterilized meat should be restricted to a specific area and workers leaving this area should perform proper handwashing before entering the rest of the processing plant.
  • A lack of a clear distinction and separation between workers from production and slicing/ packing and equipment belonging to each department is a major source of cross-contamination, particularly in circumventing the various hurdles designed to prevent cross-contamination.
  •  A particular focus should be on knives and similar equipment which can be shared between different departments within a production or between production and slicing/ packing – the standard best practice should be enforced that no such transfer of equipment can happen unless it has not been sanitised.  Plant maintenance/ engineering should receive special attention.  Along with cleaning staff, they often have unrestricted access to all parts of the plant and their tools should be viewed as major contributors to cross contamination.
  • “Poor personal hygiene, including simple procedures such as hand washing, has been identified as a causative mode of transmission of the pathogens (AFSSA 2000).”  (Thévenot, et al.; 2006)
  • “The areas of greatest relative risk for contaminating food in the factory environment are where Listeria has grown to high numbers. These growth niches must be sought and eliminated when found. Many factors affect microbial growth in niches, including moisture, nutrients, pH, oxidation-reduction potential, temperature, presence or absence of inhibitors, interactions between microorganisms in a population, and time (Faust and Gabis, 1988). Areas, where water, food (for microbial growth), and time (e.g. areas not accessible for cleaning) combine at a suitable temperature, produce microbial growth niches. Nutrition need not be visible to the naked eye to be adequate for microscopic life to grow. Consequently, the best places to sample for Listeria are those high moisture environments where the organism has had opportunity to incubate.”  (Kornacki, J. L, Seminar_Detecting_Sources_of_LM)

Here is a list of unsanitary operating practices by Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM.

unsanitory practices 1

unsanitory practices 2

unsanitory practices 3.png

unsanitory practices 4

unsanitory practices 5

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Factory Cleaning **

Post and during-shift cleaning

In general, the following applies to factory cleaning:

Step 1:  Remove gross, loose material such as meat.  “The cleaning step is important because it removes organic matter from processing surfaces (Chasseignaux et al. 2001; Thevenot et al.2005b). Besides harbouring bacterium, organic matter can reduce the efficiency of disinfectants [such as quaternary ammonium compounds (QACs)] or prevent disinfectants from reaching bacterium at the recommended concentrations (Gibson et al. 1999). Furthermore, Taormina and Beuchat (2002) showed that L. monocytogenes strains that were exposed to cleaning solutions which did not affect cell viability were more sensitive to subsequent treatment with sanitizing chemicals.”  (Thévenot, et al.; 2006)

In measuring results from last year June in the poultry industry Warnick Biersteker from Lionels Veterinary Supplies have demonstrated the superiority of a process where this step is performed without water.

Step 2:  Apply alkaline detergent foam and leave on for 10 to 15 minutes.  Mechanically remove stubborn dirt and residual substances and rinse the detergent off.

Step 3:  Ensure that residue water has been removed and the area is as dry as possible.  Now foam with an antimicrobial chemical in order to sanitize surfaces.

Thévenot, et al.’s 2006 article refers to work by Holah et al. (2002) who “observed an increasing tendency for disinfectants to be left on surfaces and not rinsed off prior to recommencing production.”  They comment on this that despite the claims of manufacturers that residual disinfectants will prevent subsequent surface microbial development, although low, sublethal concentrations may, in fact, enhance resistant.”

After discussing this particular point at length, it was decided not to rinse it off due to poor water quality results reported over the previous few years.  In an environment where water quality is high, Holah’s observations will be valid, but in South African context, the possibility for re-contamination through water outways the arguments.

I have found the creation of a sanitising station in the deboning, production and slicing departments useful where dedicated staff is responsible for sanitising crates, bins, knives and any other equipment before they either enter or leave the processing or slicing departments.  In larger processing plants, these steps are hard-wired into the plant design.

Thévenot, et al., (2006) reports that an “acid treatment (pH = 5·4) followed by an alkaline treatment (pH = 10·5) was not very effective against L. monocytogenes, whereas the opposite combination led to a three-log reduction of the bacterial population.”  Generally, they report that “the most effective treatments were combinations of alkaline, osmotic and biocide shocks.”

Related to the formation of biofilm, Thévenot, et al. (2006) refers to work of  Norwood and Gilmour (2000) who concludes that “in most food-processing environments and more precisely in pork meat industries, the daily use of sanitizers at correct concentrations preceded by detergent-aided cleaning, will remove all adhering organisms or biofilms.

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Effective Equipment Cleaning

“The following steps apply:

  1. dry cleaning
  2. prerinsing
  3. foaming and scrubbing
  4. rinsing
  5. application of chemical sanitizers
  6. visual inspection of equipment
  7. drying or removal of standing water (drying is important because it reduces the opportunity for Listeria to grow on floors–this–organism needs moisture to grow.)

Processors can establish the effectiveness of plant sanitation and learn the location of potential sources of contamination by conducting baseline microbial testing of both environmental and contact surfaces. These tests include microbiological analyses, including Aerobic Plate Counts (APC), generic Listeria or Listeria species (spp.), or ATP bioluminescence assays (see Figure 14). These can all be used to gain information about cleaning and sanitation procedures.”  (Cutter, et al.)

“Frequency of sanitation will be determined, to some extent, by the type of products and the risk involved. Equipment and tools that are only used to process RTE products should be sanitized before and after use. Do not place equipment parts on the floor to clean them. When cleaning product and equipment storage rooms, personnel must be careful not to splash water from the floor onto the product, thus possibly contaminating it with bacteria. Pay close attention to difficult-to-clean places where bacteria may easily hide.”  (Cutter, et al)

“Sanitizers that have proven most effective against L. monocytogenes are quaternary ammonia compounds (quats), chlorine solutions, and newer products containing peracetic acid. Some plants rotate sanitizers periodically (every month or two) to prevent bacterial resistance against any one sanitizer. Choose appropriate acid-based detergents to avoid “soapstone” or hard-water buildup that can lead to biofilms. Some plants alternate detergents, which changes the pH and may keep bacteria from adapting to a particular environment. (Care must be taken not to use chlorine and acid-based detergents simultaneously, due to potential chemical hazards to employees.) Processors should work with suppliers of these products and/or with sanitation professionals to develop specific usage plans for each particular operation.”  (Cutter, et al.)

Cutter and Henning provide the following useful recommendation for frequency of cleaning and sanitizing.

Recommended Frequency of Cleaning and Sanitizing
Area Frequency
All processing equipment Daily
Floors and drains Daily
Waste containers Daily
Storage areas Daily
Walls Weekly
Condensate drip Weekly/monthly
Coolers Weekly/monthly
Freezers Semi-annually

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Cleaning as part of Plant Design

“The following are some suggested design improvements for optimal contamination prevention.

  1. The storage of products, product flow, and the movement of people between raw and RTE areas are all very important. One of the first things that must be done is to eliminate traffic flow between RTE and raw areas–RTE products–must not come into contact with or be in proximity to raw products.
  2. RTE areas should be equipped with dehumidifying cooling units and drip pans for handling condensation. These units should be directed away from products in these areas and sanitized regularly. Make every effort to eliminate condensation in RTE work areas and coolers.
  3. Ceilings, floors, and walls should be smooth, sealed, and moisture-free.
  4. Air supply ducts should be filtered to prevent contaminants from entering the building or the room. RTE product storage rooms should be under positive air pressure so that air is not received from unfiltered or raw-product areas.
  5. Light fixtures should be designed so as not to collect dirt or moisture. Remove any difficult-to-clean overhead light fixtures from areas where RTE products are exposed.”

(Cutter, et al.)

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Potential Reservoirs of L. monocytogenes in Small Processing Plants

  • Floors and drains
  • Standing water
  • Ceilings and overhead pipes
  • Refrigeration/condensation units
  • Wet insulation (exposed to processing area)
  • Cleaning tools (sponges, brushes, squeegees)
  • Overhead rails and trolleys
  • Maintenance tools (wrenches, screwdrivers)
  • Wooden pallets
  • Forklifts and pallet jacks

(Cutter, at al.)

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Other Areas for Potential L. monocytogenes Contamination

  • Any recess or hollow object (rollers, switch boxes, box cutters, motor housings)
  • Rusted materials (equipment frames, pipes, shelving)
  • Cracked or pitted rubber hoses, door seals, walls
  • Icemakers
  • Air filters
  • Open bearings
  • Wheels Light switches (Cutter, et al.)

(Cutter, et al.)

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Other cleaning related steps *

  • QAC’s will be used to cover the floor area of high traffic areas and be applied to the floor around all drains to manage the eventuality of drains backing up.
  • Door foamers for entry points into the freezers and loading bays will be proposed to management and for the doors from the freezers leading into the factory.
  • Chemical rings will be put in all drain baskets and all evaporator water drip trays.
  • All water dripping in the factory will aggressively me eliminated.
  • The floor will be fixed to prevent puddles from forming.
  • Metal that is sandwiched will be separated by spacers to allow for cleaning.
  • Hot water will be made available for cleaning in all departments and in the wash bay in particular.
  • Hand disinfecting stations will be expanded and the number increased.
  • Maintenance team’s habits will be studied to determine risky behavior for cross contamination.  Tools will be included in daily cleaning schedules.  The feasibility of a maintenance room for each department will be evaluated.
  • All equipment will be evaluated in terms of the easy by which covers can be removed.
  • Pumps will be opened and cleaned once a month.
  • All equipment will be stripped completely for thorough cleaning once a month.
  • Trolly cleaning will be stepped up and product arrangement limited to no lower than 30cm off the ground to prevent contamination by splashing.
  • Trolly wheels will receive special attention.
  • Regular hand disinfection procedure during shift.
  • Proper treatment and cleaning of cleaning equipment.
  • A proper plant startup procedure will be applied whereby surfaces will be swabbed with ATP test strips before processing is allowed to start.  All equipment will be inspected before every new shift start.  Three hours after plant startup, tests will be performed for listeria.
  • Hand sanitation will be extended to include apron and cuffs sanitation every time when hand sanitation would have been performed.
  • A blend of sodium lactate, acetate, diacetate will be considered for preserving properties.
  • Proper raw materials issuing procedures and a correct use of the outer-inner packaging.
  • A map of the factory will be drawn up where all risks will be highlighted and a task list with priorities will be drawn up.  The general approach is that no stone will be left unturned to provide the safest food processing environment possible.

Thank to Dr. Joseph Yates for taking me through these points.*

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Compressed Air Must be Contaminant-Free

“Compressed air must be purified of contaminants before use in the food industry. The contaminants are water vapor and moisture, solid particulates (including spores) and oil aerosols and vapors.

The presence of moisture is the primary concern for the food industry because moisture creates the ideal habitat for microorganisms and fungus. Moisture may reside in the piping system near point-of-use applications where compressed air comes into contact with food products. Microorganisms and fungus can grow inside the piping system and then be blown into food products or food containers.

In order to inhibit the growth of microorganisms and fungi, pressure dewpoints must be below -15 F (-26 C). Drying the compressed air to a specified pressure dewpoint is the simple way to eliminate moisture in the compressed air system. The dewpoint specification will be of either +37 F (+3 C) or -40 F (-40 C). In some facilities, both of these specifications may be used to reduce energy costs associated with drying the compressed air – depending upon whether compressed air has any possibility of coming into contact with food products.

Solid particulates must be removed with filtration products from the compressed air system. When compressed air is dried below -15 F (-26 C), harmful microorganisms and fungi are converted into spores. These spores are now a “solid particulate” which must be filtered. Other sources of solid particulates are coatings on the air compressor rotors, pipe-scale from the compressed air piping system, and ambient dust and particulates which may be ingested by the air compressor. It is recommended, when selecting compressed air filtration products, that care is taken to request coalescing filters tested to the new ISO Standard 12500 Parts 1-3.

Oil aerosols and vapors are another significant concern. One myth in compressed air systems is that the use of an oil-free air compressor frees the system of any compressed air treatment requirements. This is not the case. Ambient air ingested by air compressors will carry water vapor, particulates, and hydrocarbons and compressed air dryers and filters are always therefore required.

compressed air
The U.K. Code of Practice for Food Grade Air

System #1: Contact

“Contact” is defined in the code as, “the process where compressed air is used as a part of the production and processing including packaging and transportation of safe food production.” Another way of defining this is simply if compressed air comes into direct contact with food products. If this is the case, the end user must know that the compressed air must be purified to the “Contact” purity-level as defined in the Code. We often hear the term “incidental contact” used in the U.S. This is an ambiguous term. It is recommended that engineers clearly define between “Contact” and “Non-Contact”.

Here is a application example of compressed air coming into “Contact” with food. Vegetable peeling machines utilize compressed air to prepare raw food stocks for packaging and consumption. The vegetable peelers use a jet nozzle of air to peel onions and other vegetables.⁴

In this type of “Contact System”, The U.K. Code of Practice recommends a -40 F (-40 C) pressure dewpoint which will ensure that no microorganisms can grow. This can be accomplished with desiccant (adsorption) type compressed air dryers located in the compressor room (centralized air treatment). Each facility will have to determine if further point-of-use air dryers (de-centralized) are required to ensure the dewpoint specifications. Point-of-use air dryers may be of either desiccant (adsorption) or membrane-type technology.

Coalescing filters are required to remove solid particulates and total oil (aerosol + vapor) to the specification levels. Please note that activated carbon filters will be required as well to remove oil vapors. As with the air dryers, each facility will have to determine if de-centralized filtration is required in addition the the centralized filtration.

System #2: Non-Contact High-Risk

Non-Contact is defined in the code as, “the process where compressed air is exhausted into the local atmosphere of the food preparation, production, processing, packaging or storage.” Within this section we have a High-Risk and Low-Risk distinction. A Non-Contact High-Risk situation may be where compressed air is used in a blow-molding process to create a package – and then product is introduced into the package later in the day. Many food processors and have their own in-house production lines to create their own packaging. Without proper air treatment, it is possible that oil, moisture, and particulates (notably bacteria) could be present on the packaging – waiting for the food product!

The U.K. Code of Practice clearly states that “Non-Contact High-Risk” compressed air systems should establish the same compressed air purity specifications as “Contact” systems.

System #3: Non-Contact Low-Risk

In “Non-Contact Low-Risk” systems, The U.K. Code of Practice recommends a +37 F (+3 C) pressure dewpoint. This can be accomplished with refrigerated type compressed air dryers located in the compressor room (centralized air treatment). Each facility will have to determine if further point-of-use air dryers (de-centralized) are required to ensure the dewpoint specification.

Defining a Non-Contact Low-Risk system is equally important to define because it is common to see food industry systems “over-protect” their compressed air systems. Most plants have significant portions (over 50%) of their compressed air going to “plant air” applications. These “plant air” applications will have absolutely no contact with food products or food-packaging machinery. It is important to understand this relationship and design your system accordingly. We often see desiccant air dryers used to dry all the compressed air in the facility to a -40 F (-40 C) dewpoint – when only 40% of the compressed air needs this dewpoint.

It is worth noting that refrigerated type compressed air dryers normally have significantly lower associated energy costs than desiccant air dryers. Desiccant air dryers will use a portion (can be 15%) of the compressed air to regenerate the desiccant bed and/or use electric heaters. Refrigerated dryers use relatively small refrigeration compressors and can be cycling or non-cycling.

Coalescing filters are required to remove solid particulates and total oil (aerosol + vapor) to the same specification levels as “Contact” systems. Please note that activated carbon filters will be required as well to remove oil vapors. As with the air dryers, each facility will have to determine if de-centralized filtration is required in addition the the centralized filtration.”  From article by Air Technology Group Hitachi America, Ltd., Industrial Components & Equipment Division, Three Types of Food-Industry Compressed Air Systems.

Three Types of Compressed Air Systems

The food industry, faced with the question, of how to specify a safe and efficient compressed air system, must first define how compressed air is used in their facility. The U.K. Code of Practice for Food Grade Air provides a comprehensive resource on compressed air systems in the food industry. The Code was jointly developed, in 2006, by the British Retail Consortium (BRC) and the British Compressed Air Society (BCAS). For more information on acquiring a copy of the Code, visit www.bcas.org.uk. The Code defines three specific types of compressed air systems in the food industry; systems with contact with food, non-contact high-risk, and non-contact low-risk.

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Control Points in Processing Steps and Validation

The processing steps must be evaluated and where possible adjusted to accommodate the reversal of any possible contamination that occurred.

Do you manufacture products that will support the growth of L. monocytogenes?

“Processors of RTE products should ask three questions when determining the risk of exposure of their products to Listeria monocytogenes:

  1. Do validation results support the efficacy of kill steps used in processing?
  2. If products are exposed to an environment not known to be free of L. monocytogenes, what post-processing steps, if any, are in place to prevent contamination in that environment?
  3. What does the finished product testing reveal about the status of the products with respect to L. monocytogenes contamination?

In order to be guaranteed incapable of supporting the growth of L. monocytogenes, a product must have at least one of the following characteristics:

  1. Water activity (AW) value of= 0.85 or less
  2. pH at 4.6 or below when measured at 24°C.
  3. Stored in an unopened, sealed container that is commercially sterile under non-refrigerated storage (retorted or aseptically filled)
  4. Laboratory evidence demonstrates that the growth of infectious or toxigenic organisms cannot occur
  5. Composition of product naturally does not support the growth of microorganisms

Since L. monocytogenes is relatively easy to kill by thermal processing, the chief concern in controlling L. monocytogenes is the prevention of recontamination after cooking. It is important to verify that all thermal processes and procedures meet the requirements for pathogen destruction.”

(Cutter, et al.)

In particular, the following must be considered.

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-> Thermal Inactivation

Temperature is the most important hurdle against L. monocytogenes.  “It grows in a temperature range of +0·4 to 45°C, while the optimum growth temperature lies between 30 and 37°C (AFSSA 2000). Listeria monocytogenes grows in refrigerated foods (Augustin 1999), and this is of major importance for risk assessment of foodstuffs: even when initial contamination is low, the organism can multiply during refrigeration and reach levels up to 100 CFU g−1 (AFSSA 2000). Besides, the temperature home refrigerators are often closer to 9°C than 4°C (Sergelidis et al. 1997), which also favours L. monocytogenes growth.”  (Thévenot, et al.; 2006)

Several factors necessitate a careful evaluation of the heating step in processing due to the “organism’s high level of heat tolerance relative to those of other non-spore-forming foodborne pathogens.”  Not only is its heat tolerance high, but several factors which it is likely to encounter in the processing environment increases its heat tolerance.  For example, “previous studies involving L. monocytogenes have demonstrated that the composition of the heating medium affected the thermal inactivation of this organism. The heat resistance of L. monocytogenes was stronger in ground pork than in pork slurry, and its heat resistance was stronger in meat slurry than in phosphate buffer.”

Adding curing agents also increase its hear tolerance.  “In comminuted meats, the addition of phosphates, salt, or curing-salt mixes has been found to increase the thermotolerance of L. monocytogenes. The thermotolerance of L. monocytogenes is further increased when this organism has endured an environmental stress such as sublethal heat shock, osmotic stress, starvation, exposure to acid or alkali, exposure to ethanol, or exposure to hydrogen peroxide.  Therefore, heat-processing procedures for foods should be designed to destroy L. monocytogenes in its most heat resistant state and thus to provide an adequate margin of safety against this pathogen.” (Lihono, et al, 2003).

Lihono et al from Iowa State University used a quadratic linear response model in 2003 to “describe the combined effects and interactions of different parameters on the thermal inactivation of starved L.monocytogenes” which involved D-values (times [reported in minutes] required for a 90% reduction in viable cells), “modelled as a function of heating temperature, SPP concentration, and NaCl concentration”.  The model is based on starved L.monocytogenes since “water used for cleaning and rinsing food contact surfaces generally provides a low-nutrient environment for microorganisms.  Certainly, the stress from prolonged deprivation of nutrients can induce increased microbial resistance to subsequent chemical and physical challenges.”  (Lihono, et al, 2003)

In their study, they reported that “the addition of 3 and 6% NaCl to pork slurry significantly (P, 0.05) increased the D-values for the organism.”  Let’s look at their findings more carefully.  (Lihono, et al, 2003)

At a NaCl and SPP T concentration of 0, and temperature of 57.5 deg C, the observed D-value was 2.79 minutes (2.93, calculated).  When the salt % was increased to 3%, the D-value increased by 177% to 7.75 (observed) and 6.4 (predicted).  At 6% NaCl, it rose by another 88% to an astounding 14.59 observed minutes and 13.95 predicted.   Adding the phosphates (SPPT), at a 6% salt level and a phosphate concentration of 0.5%, reduce the D-value to 8.2 observed and 8.91 predicted.  At a salt concentration of 3%, and SPPT at 0.5%, the D-value is reduced to 4.03 observed and 4.16 predicted.

In their report, they site Juneja and Eblen who “demonstrated that the addition of NaCl (at 1.5 to 6%) to beef gravy protected L. monocytogenes against thermal inactivation at all temperatures (55 to 65 deg C) tested in their study.  Similar findings were reported by Yen et al., who demonstrated that the heat resistance of L.monocytogenes increased when the organism was heated at 60 deg C in ground pork with added NaCl.”  They commented on their own work and said that generally, their results on “the thermal inactivation of starved L. monocytogenes in pork slurry with added NaCl are consistent with those of other studies on the thermal inactivation of this pathogen in broth, pork, and beef. The investigators who conducted those studies reported an increase in the heat resistance of L. monocytogenes in various meat blends containing 3 to 4% NaCl. Therefore, the results of the present study can be used to predict the thermal inactivation of L. monocytogenes as affected by added NaCl in meat products.”  (Lihono, et al, 2003)

Faber, as reported by Jay, et al., found the same results in sausage-type meat.  The D-value ar 62 deg C was 61 seconds, “but when cure ingredients were added, the D value increased to 7.1 minutes, indicating heat-protective effects of the cured components, which consisted of nitrite, dextrose, lactose, corn syrup and 3% (w/v) NaCl.  (Jay, et al.; 2005:  602)

Thévenot, et al., (2006) reports that “products which are cooked at fairly low temperatures (50–60°C) may not be L. monocytogenes free even after if cooking times are long (FICT 2002).”  (Thévenot, et al.; 2006)  This leaves us with the question of an optimum cooking temperature for bacon.  It is my experience that bacon starts changing to a pale cooked colour as opposed to the pinkish-reddish cooked cured colour at a temperature of 48 deg C.  Faber’s 62 deg C is therefore out of the question.  Jay, et al states that cooking meat to an internal temperature of 71 deg C for 2 minutes will destroy L. monocytogenes, (Jay, et al.; 2005:  602) which in the case of bacon production is even more unworkable.  I am pitching the core temperature for bacon at 55 deg C for at least three hours (our standard smoking/ thermal processing time) based on the work of Lihono, et al. (2003) and will continue to search the literature for more precise guidance and submit this suggestion to academics for comment.

Uninoculated minced beef was placed in vacutainers and allowed to equilibrate in water-baths pre-adjusted to 50, 55 or 60 deg C (McMahon 1997), reported by Bolton, et al (2000).  They reported the D-values for Listeria monocytogenes in minced beef using a laboratory water-bath.  At 50 deg C, the D-value was 32.7, 32.7 and 36.1 minutes respectively and at 55 deg C, 3.4 and 3.2 minutes.  It is interesting that they report that at 48 deg C, the solid beef sample showed a D-value of 88.6 minutes and 92.6 minutes respectively, heated in a commercial retort.  This, I take to predict the ineffectiveness of a smoker setting of 48 deg C for core temperature requirement as far as inactivating L. monocitogenes.  It must also be noted that due to the variable factors enhancing the heat resistance of this bacteria in a factory setting, one must opt for an as high temperature as can be tolerated. (Bolton, et al.; 2000)

Frankfurters that was processed to an internal temperature of 71.1 deg C has been shown to effect at least a 3-log cycle reduction of strain Scott A..  (Jay, et al.; 2005:  602)

Why does the presence of salt increase the D-value?

Lihono, et al. says “the influence of salts on the thermal inactivation of microorganisms is largely due to reduced water activity and increased osmotic pressure of the heating medium.”  (Lihono, et al, 2003)

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->  Acid Tolerance Response

It is reported that the pH minimum for the growth of two strains was 3.5 and 4.0 in a chemically defined medium using HCl.  On beef treated with 2% lactic and acetic acid, acid adaption did not protect L. monocytogenes strain Scott A.   The importance for us is that we have implemented a carcass wash procedure with lactic acid sprayed with very low pressure as one of many control measures.  Another consequence of its “acid adaption is that this characteristic provides protection not only the ability to develop acid resistance but also resistance against HHP” as well as freezing.  (Jay, et al., 2003:  530) This is of concern since HPP is currently being considered for several new product formulations.  This warrants further investigation.

“In general, the minimum growth pH of any bacterium is a function of temperature of incubation, general nutrient composition of growth substance, water activity, and the presence and quality of NaCl and other salt inhibitors.”  (Jay, et al.; 2005:  595)

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->  Combined effect of pH and NaCl

The interaction between pH, NaCl and incubation periods have been studied extensively.  Time to reach visible growth (expressed in days) is, in one study, representing the number of days required to achieve a 100-fold increase in the number of L. monocytogenes. The findings from this study are instructive.  “At pH 4.66, time to visible growth was 5 days at 30 deg C with no NaCl added. This changes to 8 days with 4.0% NaCl at 30 deg C and 13 days with 6% NaCl.  At a temperature of 5 deg C, and a pH of 7.0, growth occurs only after 9 days with no NaCl added, 15 days at 4% NaCl and 28 days for 6.0% NaCl.  (Jay, et al.; 2005:  597)

This would indicate that adding 4% NaCl to the meat and processing it within 24 hours, maintaining a meat temperature of < 7 deg C is an effective hurdle to prevent L. monocytogenes growth.  In light of this, I will evaluate our freezer room and track the effective storage temperature of meat during the resting phase.

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Routine monitoring

I am completely re-designing our approach to monitoring by following the methods and philosophy as outlined by Dr. Kornacki and explained to me by Dr. Yates. *

Zones

Dr. Kornacki makes the following very good suggestion which will be discussed company management and with Food safety department to incorporate this approach into our monthly monitoring plan.  He writes, “a number of food processors have found it helpful to beak down their routine environmental sampling program into four zones (Hall, 2004; ICMSF, 2002, Kornacki and Gurtler, 2007). Zone 1 samples are those taken from direct and indirect (e.g. overhead pipes) product contact areas. Zone 2 samples are surfaces adjacent to Zone 1 and include areas like equipment framework and guards. Zone 3 includes surfaces in RTE product zones such as floors, drains, walls, equipment. Zone 4 areas are more remote from the ready-to-eat product zones such as warehouses, loading docks, employee break rooms, and locker rooms. Rotating sampling sites at an appropriate frequency will result in covering a wider region of the factory environment.”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

An Approach to In-factory risk assessment

“The probability of RTE product contamination is affected by a number of variables
including but not limited to

a.) proximity of microbial growth niches to the product stream,

b.) number of growth niches,

c.) spatial relationship of niches to product stream,

d.) microbial populations in niches,

e.) extent of niche disruption, and

f.) exposure of product stream to the environment (Faust and Gabis, 1988).

Consequently, our investigational approach has been to break down our factory observations and sampling into regions of “high”, “medium” and “indirect” relative risk of product contamination.

Areas of high risk are somewhat analogous to Zone 1. These exist where moist, entrapped (or standing) residues are located in close proximity to the product stream.  Such an area might include the back plate of a poorly sealed positive displacement pump used to remove product from a heat exchanger (e.g. pasteurizer), or residues entrapped in poorly designed valves located subsequent to a validated Critical Control Point (CCP) in the process stream.

Indirect risk samples would include those from Zones 2-4 that do not produce an obvious direct risk of product contamination. However, the microbial ecology of food processing environments is so dynamic that one does not always readily observe the connection, say between a forklift with Listeria-contaminated wheels observed in a raw processing area, but later charged in a common area with forklifts dedicated for use in ready-to-eat product production (RTE) areas (thereby cross-contaminating the wheels of the RTE forklift(s) from the floor). Cross-contaminated rotating RTE area forklift wheels may later splash (or aerosolize) Listeria onto exposed product.

We defined “medium risk” areas as those places similar to high-risk areas, but before some process or procedure with likely potential to reduce the microbial load by an undetermined amount. These usually require a challenge study or process validation to determine the lethality and risk. Medium risks may also be areas or practices which might result in contamination of the product infrequently. One example of a medium risk site may be exposed, and cooling, 135 deg F (57 deg C) molten cheese product in an area with potential for contamination (e.g. ceiling watermarks over the product or near employee cross traffic through wet floor areas). This is clearly not a desirable situation and in the limited context of a risk assessment walk-through, would be ranked as a high risk, if the product were not heated. Immediate corrective action to eliminate the contamination potential would still be recommended. In this example, it is not clear if the process temperature and time are adequate to sufficiently destroy a population of Listeria monocytogenes. The product matrix plays a significant part in bacterial heat resistance (Stumbo, 1965). Therefore, knowledge of  Listeria monocytogenes heat resistance over a range of temperatures in this product matrix is also needed. If this is not known, a laboratory-based thermal challenge study with a multistrain cocktail of Listeria monocytogenes or perhaps a pilot plant based study with appropriate surrogate microorganisms could be done (Kornacki, 2002; Eblen, 2005).

These types of studies may result in discovery of a previously unknown CCP. Criteria for selection of surrogates have been described by the FDA (Anonymous, 2000). USDA has also emphasized the importance of process validation studies (Engeljohn, 2004).”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

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Techniques for Sampling the Environment

“A number of techniques exist for sampling the factory environment (Evancho, et al,2001). More commonly used approaches include the use of pre-sterilized, inhibitor-free sponges, traditional swabs, and contact plates. A comparison of the advantages and disadvantages of these approaches is represented in the table below. In more recent times the use of 1-Ply composite tissue have shown to provide another effective alternative that may provide a lower cost alternative to the above (Vorst, et al, 2004). Sponges and swabs must be pre-hydrated with sterile neutralizing buffer before sampling. Hydration of the swab or sponge facilitates greater microbial recovery from a surface and neutralizing buffer is used to prevent residual sanitizer in the sample from destroying the target organism prior to testing.”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

sampling approaches

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Finished Product/In-Line Sampling

Finished Product Testing

“Finished product testing cannot be relied upon as the sole determinant of a Listeria-free product. No amount of finished product sampling and testing short of assaying the entire product with a perfect method can guarantee that the product is Listeria-free. Finding a problem through finished product testing is likely in situations where the incidence of product contamination is high (see table below). However, this is rarely the case. For example, L. monocytogenes was recovered from 1.6% of 32,800 packages of frankfurters using a method six times more sensitive than the standard USDA/FSIS product composite enrichment method (Wallace, et al, 2003a). Tompkin (2002) stated that “…it should be possible in most food processes that include a validated listericidal step (e.g. cooking) to keep the prevalence of product contamination <0.5%.” It is impractical to test enough samples to gain high confidence of detecting contaminated lots with such low contamination incidences. Consider a product contaminated at the 1% level. In theory, 299 randomly selected samples per lot are required to gain a 95% chance that at least one sample would test positive (Table 5; Midura and Bryant, 2001).

If the true incidence is 0.1% it would take 2996 samples per lot and so forth. Therefore any finished product testing should be viewed as part of a comprehensive Listeria-control program including Good Manufacturing Practices, HACCP, and its other prerequisite programs. Knowing where to look and taking appropriate environmental samples and appropriate corrective action is far more effective than extensive product testing.” (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

In-line sampling

Sometimes it is impractical to sample product contact surfaces of some processing equipment. We have found rigorous application of statistical sampling techniques at selected locations before and after an inaccessible area has been effective in isolating areas of product contamination.

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(Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

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Sample testing and compositing

Test methods

“Numerous conventional and rapid assays exist for the recovery of Listeria.”  I am in contact with suppliers such as 3M who work across the world on sampling and will get guidance from them by an Official Method of Analysis published by the AOAC (http://www.aoac.org/ILM/july_aug_05/oma.htm.). We will rapidly incorporate such methods and I will ensure that these have been scientifically validated for our particular sample matrix, especially in instances when we need to use a non-approved method.  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

Compositing product samples

At Woody’s we have already changed to a composite sampling approach, but my fear is that it is still not being implemented in the most advantages manner.  “The ability to combine multiple randomly collected samples into one will clearly save testing costs.”  What we so far have failed to do and will correct immediately, is to ensure that our compositing schemes have been validated.  Dr. Kornacki states that compositing schemes should also be validated. “Inappropriate sampling schemes can lead to misleading test results, as described above. The same is true for inappropriate compositing schemes. Some RTE meat sample compositing schemes yielded inconsistent results depending upon the type of meat product sampled and the Listeria assay performed (Curiale, 2000).  Other approaches to sampling may also afford enhanced recovery of Listeria or reduced labor intensiveness, such as product or package rinses (Wallace, et al, 2003b; Luchansky, et al, 2002).”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)  I will study the matter and amend the approach once clarity emerge.

Molecular subtyping

“Once the samples are collected, tested, and isolates recovered a variety of molecular subtyping techniques may be applied such as PFGE, RAPD, RepPCR, and 16s rDNA sequencing. Manufacturers tend to use specialized laboratories for this work, but some have developed in-house techniques for this purpose. In-factory Listeria testing is not recommended. These approaches have been useful in revealing specific patterns of Listeria transmission that would otherwise not have been understood (Pruett, 2005).”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

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General Procedures for Sampling Listeria

  • “A responsible employee with proper training should be chosen to conduct this testing. It is important to have the same employee conduct the testing on a regular basis to ensure the consistency of the procedures.
  • Samples must always be taken in the same manner and be of the same size area sampled.
  • For large, flat surfaces such as tables, floors, drip pans, etc., swab an area of 100 square centimeters by rubbing a moistened sponge back and forth across it; then flip the sponge over and swab the same area perpendicular to the original sponge strokes.
  • Remove drain covers and swab the interior surfaces and throats of the drains.
  • For small or confined spaces (chain conveyor links, machine interiors, knife holders, etc.), swab several spaces or as large a total surface area as possible.
  • Make sure the sponge bag is clearly marked with the sample date, sample location, and company name.
  • Keep good records of exactly where each sample was taken.
  • If the sampled area is a food-contact surface, it is advisable to sanitize the swabbed area immediately after sampling. By doing this, any questions are eliminated about the disposition of the product that touched that surface if the tests are positive for Listeria.
  • If Listeria is found, clean-up and sanitation efforts should be intensified in that area to eliminate the source and keep it under control. After several weeks of intensified cleaning, the same area(s) should be resampled to verify that the contamination has been eliminated.”

(Cutter, et al)

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Data Management

“Some companies have diligently tracked this microbe and amassed a lot of information.  Unless one manages this data properly important trends can be missed. For example, assume Listeria monocytogenes was recovered from a site during post-operational sampling. Corrective action is taken and it tests negative at the next sampling. The company might assume effective corrective action occurred. Analysis of trend data throughout the year may tell a different story. Perhaps, the site was positive approximately once per month for 12 consecutive months. Clearly, a better corrective action would need to be applied. Eifert (2002) has shown how Pivot tables can be used for precisely this type of analysis.”  (Dr. Kornacki from his  Seminar_Detecting_Sources_of_LM)

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Critical Limits (CLs) and Records

“All CCPs must have a critical limit (CL) that is both observable and measurable. “Measurable,” in this case, means that careful records must be kept of a wide range of variable factors, from the number of violations of a clothing policy to the amount of chemical sanitizer used in an area over a given period of time.”  (Cutter, et al.)

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Verification/Corrective Actions

“Verification of sanitation procedures should be conducted periodically in small plants to reassure plant operators and inspectors that a sanitation program is effective in preventing the presence of L. monocytogenes on equipment surface areas and in the final product. Microbial swab tests on equipment and/or product should be used to verify the program’s effectiveness (see discussion of testing procedures above).”  (Cutter, et al)

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Pork Anatomy and the Prevalence of Listeria

One of the ways that listeria enters the processing facility is through the pork carcass.  Contamination of pork carcasses during slaughter occurs in one of two ways: “previously infected live animal or cross-contamination of the carcass from the slaughter environment.” (Baer, et al.; 2013)  Hellsrom, et al. (2010) found that from 1,962 samples taken from farms, 119 (6%) were L. monocytogenes positive.”  (Hellstrom, et al., 2010)

->  Digestive Tract

The digestive tract of the animal is not as much of a source for L. monocytogenes infection as one may expect.  “L. monocytogenes is ubiquitous and persists in the environment (Ryser and Marth 2007), which allows for infection of swine at the farm. Unlike Salmonella and Campylobacter, which exist at high levels in the feces of pigs, L. monocytogenes does not flourish in the intestines of swine perhaps due to the competitive microflora (Bunˇci´c 1991). In a study of finishing swine, only 1.7% of conventionally raised swine were positive for shedding L. monocytogenes, which is much lower than Salmonella and Campylobacter prevalence (Fosse and others 2009).

However, production stage and management practices can significantly affect the prevalence of L. monocytogenes in swine. In feces, sows have a higher prevalence of L. monocytogenes than piglets (Fenlon and others 1996). However, cull sows had a lower prevalence of L. monocytogenes than market pigs when tested during slaughter, which could be attributed to lower pig densities of sows at the farm compared to market hogs (Wesley and others 2008). Moreover, fewer animals being processed at sow harvesting facilities allows for less cross-contamination to occur and reduces water use, contributing to a drier slaughter environment and reduced likelihood of contamination with Listeria spp. (Wesley and others 2008).”  (Baer, et al.; 2013)

->  Tonsils

“L. monocytogenes is present in higher levels in the tonsils with a prevalence of 12% to 45% (Bunˇci´c 1991; Autio and others 2000; Hellstrom and others 2010).”  (Baer, et al.; 2013)

A study in The Netherlands has shown that of 44 samples taken inside the cutting room, 36.4% of the pork neck and shoulder were positive, 27.3% legs,  11.4% belly.  Only 1 carcass, when swabbed on the inside, was positive.  (Baer, et al.; 2013)

Of 29 environmental samples taken inside the cutting room, a massive 86.2% were positive highlighting the importance of cutting room sanitation and the correct procedures.  (Baer, et al.; 2013)

anatomy of the pig

pork tonsils
Anatomical localization and histological characteristics of the porcine tonsils. (a) Median section through a porcine head. The pharyngeal tonsil (large oval area) and tubal tonsil (small oval area) in the nasopharynx, the tonsil of the soft palate at the oral side of the soft palate (larger rectangular area, detail shown in upper right insert), and the lingual tonsil (smaller rectangular area) and the paraepiglottic tonsil at the lateral base of the epiglottis (encircled area, detail shown in the lower right insert) are indicated. (b–f) Histological sections through parts of the lingual tonsil (b), the tonsil of the soft palate (c), the pharyngeal tonsil (d), the tubal tonsil (e), and the paraepiglottic tonsil (f), respectively. The encircled areas in (c), (d), and (f) delineate a tonsillar follicle with a central crypt.

->  Farming method

“Organic production led to increased prevalence of L. monocytogenes in tonsil (47%) and pluck (13%) samples compared to conventional production (12% and 1%, respectively) (Hellstrom and others 2010).”  (Baer, et al.; 2013)  Hellstrom, et al. found that “Prevalence in all sample types was higher in organic pork production than in conventional production.”

The following risk factors have been identified on farms that increase the likelihood for L. monocytogenes infestation, “large group size, contact with pet and pest animals, manure treatment, use of coarse feed, access to outdoor area, hygiene practices, and drinking from the trough.”  Much more study is needed in this area.”  “Large numbers of pigs in one pen facilitate contact with more pigs, thus spreading the bacterium. Moreover, because L. monocytogenes is common in the environment, outdoor areas may be a source of contamination.  Coarse feed is frequently contaminated with L. monocytogenes. Although these practices appear to be associated with the prevalence of L. monocytogenes, they may otherwise be advantageous with regard to pig welfare, which is one aim in organic production, and some of these practices are also required by law.”  In addition to these, “controlling pest animals and restricting the entrance of pets and birds into piggeries reduces the prevalence of L. monocytogenes. Drinking water can easily be contaminated when pigs drink from a common trough; using nipple drinkers may be prudent. In addition, liquid manure compared with solid manure as well as mechanical removal of manure reduces the prevalence of L. monocytogenes.  Some earlier studies have reported similar results, showing that farm management practices, such as specific pathogen–free herds and type of feed, influence the prevalence of L. monocytogenes.”  (Hellstrom, et al., 2010)

It should be noted that Hellstrom, et al. (2010) also found that in their study, “farms with the highest prevalence of L. monocytogenes had no contaminated carcasses. This shows that a high prevalence of L. monocytogenes in pigs does not inevitably lead to highly contaminated meats. Several preventive actions can be utilized in the slaughtering process to reduce contamination of pathogenic bacteria, including proper cleaning and disinfection of equipment and good operating protocols. With good manufacturing practices, contamination from pigs to the food chain may be substantially reduced, and thus, solid hygienic practices are of the utmost importance during slaughter.” (Hellstrom, et al., 2010)

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Conclusion

This article is a “work in progress” and a discussion document.  Work on it will continue and updates will be made available after every update for comment.

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*  Particular thanks to Dr. Josehp Yates from Red Arrow for intensively working through this with me and offering numerous bits of advice, provided overall direction and graciously made this list of references available.  I am looking forward to working with him over the next year when he will do an in-person plant inspection to evaluate our application of what we work on and to identify areas that we are missing.  Dr Yates can be contacted at j.yates@redarrowusa.com.

**  I want to thank Warnich Biersteker from Lionels Veterinary Supplies in Cape Town for the evaluation of the section on cleaning protocols.  Thank you for your tireless input and your forensic approach.  Warnick can be contacted at warnich7@gmail.com.

References

Bolton, D. J., McMahon, C.M., Doherty, A.M.,  Sheridan, J.J., McDowell, D.A.,  Blair, I.S. and Harrington, D.. 2000. Thermal inactivation of Listeria monocytogenes and Yersinia enterocolitica in minced beef under laboratory conditions and in sous-vide prepared minced and solid beef cooked in a commercial retort.  Journal of Applied Microbiology 2000, 88, 626-632

Baer, A. A., Miller, M. J., and Dilger, A. C..  2013.  Pathogens of Interest to the Pork Industry: A Review of Research on Interventions to Assure Food Safety (Baer_et_al-2013-Comprehensive_Reviews_in_Food_Science_and_Food_Safety).  Institute of Food Technologists; Vol. 12, 2013; Comprehensive Reviews in Food Science and Food Safety: p  183 – 287.

Cutter, C. N., Henning, W. R..  Control of Listeria monocytogenes in Meat and PoultryPenn State.

Hellstrom, S.,  Laukkanen, R., Siekkinen, K-M, Ranta, J., Maijala, R., and Korkeala, H..  Listeria monocytogenes Contamination in Pork Can Originate from Farms.  Journal of Food Protection, Vol. 73, No. 4, 2010, Pages 641–648, International Association for Food Protection.  (L monocytogenes in Pork Can Originate from Farms)

Jay, J. M., Loessner, M. J., Golden, D. A..  2005.  Modern Microbiology, Seventh Edition.  Springer.

Kornacki, J. L..  Detecting Sources of Listeria monocytogenes in the Ready-To-Eat Food Processing; EnvironmentKornacki Microbiology Solutions, Inc., Listeria Detection RTE Plants

Lihono, M. A., Mendonca, A.F., Dickson, J.S., and Dixon, P. M..  2003.  Predictive Model To Determine the Effects of Temperature, Sodium Pyrophosphate, and Sodium Chloride on Thermal Inactivation of Starved Listeria monocytogenes in Pork Slurry.  Journal of Food Protection, Vol.66, No.7, 2003, Pages 1216–1221 Copyright (c) International Association for Food Protection.

Riedo, F. X., Pinner, R. W., Tosca, M. L., et al. A point-source foodborne listeriosis outbreak: documented incubation period and possible mild illness, J Infect Dis, 1994, vol.
170  (pg. 693 – 6)

Say Tat Ooi, Bennett Lorber; Gastroenteritis Due to Listeria monocytogenesClinical Infectious Diseases, Volume 40, Issue 9, 1 May 2005, Pages 1327–1332, https://doi.org/10.1086/429324

Thévenot, D., Dernburg, A., Vernozy‐Rozand, C. 2006.  An updated review of Listeria monocytogenes in the pork meat industry and its products.  Journal of Applied Microbiology, Volume 101, Issue 1 July 2006, Pages 7–17.

Three Types of Food-Industry Compressed Air Systems, by Air Technology Group Hitachi America, Ltd., Industrial Components & Equipment Division.

Vázquez-Boland, J. A., Kuhn, M., Berche, P., Chakraborty, T., Domínguez-Bernal, G., Goebel, W., … Kreft, J. (2001). Listeria Pathogenesis and Molecular Virulence Determinants. Clinical Microbiology Reviews14(3), 584–640. http://doi.org/10.1128/CMR.14.3.584-640.2001

http://anelichconsulting.co.za/index.php/industry-news

https://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria

Photo credit:

Anatomy of the pig from:  https://www.minipiginfo.com/pig-anatomy-and-terminology.html

Detailed breakdown of the tonsils:  https://www.hindawi.com/journals/jir/2011/472460/