We are searching data for your request:
Upon completion, a link will appear to access the found materials.
Does E. coli survive at any pH level? If I was to incubate it in agar of different pH, would it still form a bacterial lawn as it's called?
I assume you are asking if E. coli can survive extreme pH conditions and according to this study E. coli K-12 W3110 survives at pH 1.2 - pH 2.0 under low oxygen. This study cultured different strans overnight and exposed to pH 2.0 for 2 hours before diluting 1:80,000 and 1:400,000, under anoxic and aerated conditions. Dilutions where then plated allowing colonies to grow up overnight at 37 oC so this study measured survival as far as I can tell. E. coli can demonstrate robustness and E. coli survives and grows 'accustomed' at pH 7-8 based on the paper here. However it would be great if you clarified your agar pH and the E. coli genetic background you are interested in if you have a specific question about its particular limits (growth or survival).
Does E. coli survive at any pH level? - Biology
Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a high acid content (Figure 1). Recall that acidity is a function of the concentration of hydrogen ions [H + ] and is measured as pH. Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of all macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H + across the plasma membrane (see Cellular Respiration). If H + ions are neutralized by hydroxide ions, the concentration gradient collapses and impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity.
Figure 1. Lactic acid bacteria that ferment milk into yogurt or transform vegetables in pickles thrive at a pH close to 4.0. Sauerkraut and dishes such as pico de gallo owe their tangy flavor to their acidity. Acidic foods have been a mainstay of the human diet for centuries, partly because most microbes that cause food spoilage grow best at a near neutral pH and do not tolerate acidity well. (credit “yogurt”: modification of work by “nina.jsc”/Flickr credit “pickles”: modification of work by Noah Sussman credit “sauerkraut”: modification of work by Jesse LaBuff credit “pico de gallo”: modification of work by “regan76″/Flickr)
The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH. These values can cover a wide range, which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2.
Figure 2. The curves show the approximate pH ranges for the growth of the different classes of pH-specific prokaryotes. Each curve has an optimal pH and extreme pH values at which growth is much reduced. Most bacteria are neutrophiles and grow best at near-neutral pH (center curve). Acidophiles have optimal growth at pH values near 3 and alkaliphiles have optimal growth at pH values above 9.
Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see Figure 2). Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0.
Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Species of the archaean genus Ferroplasma live in acid mine drainage at pH values of 0–2.9. Lactobacillus bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are less tolerant of acidity. Acidophilic microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased negative surface charge that stabilizes them at low pH. Pumps actively eject H + ions out of the cells. The changes in the composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH.
At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5. Vibrio cholerae, the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0 it can survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH, the bright pink archaean Natronobacterium, found in the soda lakes of the African Rift Valley, may hold the record at a pH of 10.5 (Figure 3). Extreme alkaliphiles have adapted to their harsh environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton motive force in an alkaline environment. For example, the alkaliphile Bacillus firmus derives the energy for transport reactions and motility from a Na + ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point, due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles.
Figure 3. View from space of Lake Natron in Tanzania. The pink color is due to the pigmentation of the extreme alkaliphilic and halophilic microbes that colonize the lake. (credit: NASA)
Survival at the Low pH of the Stomach
Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori. This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005.
The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns out, this is not the case. In fact, H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH4 + and CO2. The ammonium ion raises the pH of the immediate environment.
This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a solution of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the urea, producing radioactive CO2 that can be detected in the patient’s breath. Because peptic ulcers may lead to gastric cancer, patients who are determined to have H. pylori infections are treated with antibiotics.
Think about It
- What effect do extremes of pH have on proteins?
- What pH-adaptive type of bacteria would most human pathogens be?
Key Concepts and Summary
- Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0.
- Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5. Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH.
- Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH.
Bacteria that grow in mine drainage at pH 1–2 are probably which of the following?
Bacteria isolated from Lake Natron, where the water pH is close to 10, are which of the following?
In which environment are you most likely to encounter an acidophile?
- human blood at pH 7.2
- a hot vent at pH 1.5
- human intestine at pH 8.5
- milk at pH 6.5
Fill in the Blank
A bacterium that thrives in a soda lake where the average pH is 10.5 can be classified as a(n) ________.
Lactobacillus acidophilus grows best at pH 4.5. It is considered a(n) ________.
Symptoms and Causes
What are the symptoms of an E. coli infection?
People who get infections with the STEC strain of E. coli can have the following symptoms:
- Stomach pains and cramps.
- Diarrhea that may range from watery to bloody.
- Loss of appetite or nausea.
- Low fever < 101 °F/ 38.5 °C (not all people have this symptom).
How soon do symptoms of E. coli infection develop?
You usually develop symptoms of a STEC infection within three to five days after drinking or eating foods contaminated with this E. coli bacteria. However, you could have symptoms as early as one day after exposure up to about 10 days later.
How long do symptoms of E. coli infection last? When will I feel better?
Your symptoms can last from five to seven days.
Other than diarrhea, are there serious illnesses caused by STEC strains of E. coli?
Most cases of E. coli infections are mild and do not cause a serious health risk. Cases resolve on their own with rest and drinking plenty of fluids. However, some strains can cause severe symptoms and even life-threatening complications, such as hemolytic uremic syndrome, which can lead to kidney failure and death.
What is hemolytic uremic syndrome?
Some people, especially children age five and under, who become infected with a STEC infection (the O157:H7 strain) develop a condition called hemolytic uremic syndrome (HUS). In this condition, toxins in your intestines from STEC cause diarrhea, travel into your bloodstream, destroy red blood cells and damage your kidneys. This potentially life-threatening illness develops in about 5% to 10% of people who are infected with STEC.
Early symptoms of HUS include:
As disease progresses, symptoms include:
- Decreased urination, blood in urine.
- Feeling tired.
- Pale-looking skin.
- Easy bruising.
- Fast heart rate.
- Sleepiness, confusion, seizures.
- Kidney failure.
If you develop severe diarrhea (lasting longer than three days or you can’t stay hydrated) or if you have bloody diarrhea, go to the hospital for emergency care. HUS, if it develops, occurs an average of 7 days after your first symptoms occur. It is treated with IV fluids, blood transfusions and dialysis (for a short period of time).
What causes an E. coli infection?
Technically, you develop an E. coli infection by ingesting (taking in by mouth) certain strains of E. coli bacteria. The bacteria travel down your digestive tract, releases a destructive toxin, called the Shiga toxin, which damages the lining of your small intestine. The growing infection causes your symptoms.
How did I get infected with E. coli?
You come into contact and swallow E. coli by eating contaminated food, drinking contaminated water or by touching your mouth with your hands that are contaminated with E. coli bacteria.
- Meats: Meats become contaminated with E. coli during the slaughtering process, when E. coli in animal intestines gets onto cuts of meat and especially when meat from more than one animal is ground together. If you eat undercooked meat (E. coli is killed when meat is thoroughly cooked), you can become infected with E. coli.
- Unpasteurized (raw) milk: E. coli on a cow’s udder and/or the milking equipment can get into the milk. Drinking contaminated raw milk can lead to an E. coli infection because it hasn’t been heated to kill the bacteria.
- Unpasteurized apple cider and other unpasteurized juices.
- Soft cheeses made from raw milk.
- Fruits and veggies: Crops growing near animal farms can become contaminated when E. coli-containing animal poop combines with rainwater and the runoff enters produce fields and lands on the produce. If you don’t thoroughly wash off the produce, E. coli enters your body when you eat these foods.
- E. coli in poop from both animals and humans can end up in all types of water sources including ponds, lakes, streams, rivers, wells, swimming pools/kiddie pools and even in local city water supplies that have not been disinfected. If you swallow contaminated water, you could get sick.
- You can swallow E. coli when it transfers from your hands directly to your mouth or onto the food you are eating. E. coli gets on your hands from touching poop (an invisible amount can be on your hands). You can get poop on your hands after changing your baby’s diapers, after having a bowel movement and not washing your hands completely, petting zoo or farm animals (many animals roll in or otherwise get E. coli from poop on their fur) or from poop on the hands of other people infected with disease-causing E. coli.
Is E. coli contagious?
When you hear the word “contagious,” you might immediately think of a cold or the flu – illnesses you can get from breathing in bacteria or viruses lingering in the air of a sick person’s cough or sneeze.
E. coli isn’t an airborne illness. It’s usually spread by eating contaminated food or drinking contaminated water that contains illness-producing strains of E. coli. (Remember not all strains of E. coli are harmful.)
E. coli can, however, be contagious and spread from person to person by the “oral-fecal route.” This means that harmful strains of E. coli are spread when people don’t wash their hands thoroughly with soap and water after they use the bathroom or otherwise touch poop (after changing baby diapers or older person’s incontinence undergarments, or petting zoo or farm animals that may have soiled fur) and they touch other people. People then get the invisible E. coli on their hands and swallow it when it is transferred from their hands to the food they eat or from putting their fingers in their mouth. E. coli spreads from person to person this way in settings such as day care centers and nursing homes.
Aging and Death in E. coli
Copyright: © 2005 Public Library of Science. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
As human beings, aging and death are an inevitable part of our lives. As we pass through each decade, the concrete signs of aging—greying hair, aches and pains, the gradual failure of one organ system after another—and the realization that we are mortal increasingly occupies our thoughts.
All other multicellular animals and plants also show clear signs of aging, as do some single-celled organisms. In the yeast Saccharomyces cerevisiae (baker's yeast), for example, the function of individual cells gradually declines with time, and each yeast cell has a finite life span. In organisms like this, it has been proposed that reproduction by asymmetric division is a prerequisite for aging. In other words, for a unicellular organism to age, when it divides, it must give rise to a “parent” cell and a smaller offspring cell (as in yeast), which then has to go through a juvenile phase of growth or differentiation before it divides. At each cell division, the parent cell becomes older until it reaches its natural life span and dies.
But what about organisms that produce two apparently identical cells when they divide? Do such organisms age? The assumption has been for some years that cells that divide symmetrically do not age and are functionally immortal. Eric Stewart and colleagues have now tested this idea by analyzing repeated cycles of reproduction in Escherichia coli , a bacteria that reproduces without a juvenile phase and with an apparently symmetric division.
E. coli is a rod-shaped organism that reproduces by dividing in the middle. Each resultant cell inherits an old end or pole and a new pole, which is made during the division. The new and the old pole contain slightly different components, so although they look the same, they are physiologically asymmetrical. At the next division, one cell inherits the old pole again (plus a brand new pole), while the other cell inherits, a not-quite-so-old pole and a new pole. Thus, Stewart and co-workers reasoned, an age in divisions can be assigned to each pole and hence to each cell.
The researchers used automated time-lapse microscopy to follow all the cell divisions in 94 colonies, each grown from a single fluorescently labeled E. coli cell. In all, the researchers built up a lineage for 35,049 cells in terms of which pole—old or new—each cell had inherited at each division during its history. They found that the cells inheriting old poles had a reduced growth rate, decreased rate of offspring formation, and increased risk of dying compared with the cells inheriting new poles. Thus, although the cells produced when E. coli divide look identical, they are functionally asymmetric, and the “old pole” cell is effectively an aging parent repeatedly producing rejuvenated offspring.
Stewart and his colleagues conclude that no life strategy is immune to the effects of aging and suggest that this may be because immortality is too costly or is mechanistically impossible. This may be bad news for people who had hoped that advances in science might eventually lead to human immortality. Nevertheless, E. coli should now provide an excellent genetic platform for the study of the fundamental mechanisms of cellular aging and so could provide information that might ameliorate some of the unpleasantness of the human aging process.
Questions and Answers
Escherichia coli (E. coli) bacteria normally live in the intestines of people and animals. Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. The types of E. coli that can cause diarrhea can be transmitted through contaminated water or food, or through contact with animals or persons.
E. coli consists of a diverse group of bacteria. Pathogenic E. coli strains are categorized into pathotypes. Six pathotypes are associated with diarrhea and collectively are referred to as diarrheagenic E. coli.
- Shiga toxin-producing E. coli (STEC)&mdashSTEC may also be referred to as Verocytotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC). This pathotype is the one most commonly heard about in the news in association with foodborne outbreaks.
- Enteropathogenic E. coli (EPEC)
- Enteroaggregative E. coli (EAEC)
- Enteroinvasive E. coli (EIEC)
- Diffusely adherent E. coli (DAEC)
Shiga toxin-producing E. coli (STEC)
Escherichia coli (abbreviated as E. coli) are a large and diverse group of bacteria. Although most strains of E. coli are harmless, others can make you sick. Some kinds of E. coli can cause diarrhea, while others cause urinary tract infections, respiratory illness and pneumonia, and other illnesses. Still other kinds of E. coli are used as markers for water contamination&mdashso you might hear about E. coli being found in drinking water, which are not themselves harmful, but indicate the water is contaminated. It does get a bit confusing&mdasheven to microbiologists.
Some kinds of E. coli cause disease by making a toxin called Shiga toxin. The bacteria that make these toxins are called &ldquoShiga toxin-producing&rdquo E. coli, or STEC for short. You might hear these bacteria called verocytotoxic E. coli (VTEC) or enterohemorrhagic E. coli (EHEC) these all refer generally to the same group of bacteria. The strain of Shiga toxin-producing E. coli O104:H4 that caused a large outbreak in Europe in 2011 was frequently referred to as EHEC. The most commonly identified STEC in North America is E. coli O157:H7 (often shortened to E. coli O157 or even just &ldquoO157&rdquo). When you hear news reports about outbreaks of &ldquoE. coli&rdquo infections, they are usually talking about E. coli O157.
In addition to E. coli O157, many other kinds (called serogroups) of STEC cause disease. Other E. coli serogroups in the STEC group, including E. coli O145, are sometimes called &ldquonon-O157 STECs.&rdquo Currently, there are limited public health surveillance data on the occurrence of non-O157 STECs, including STEC O145 many STEC O145 infections may go undiagnosed or unreported.
Compared with STEC O157 infections, identification of non-O157 STEC infections is more complex. First, clinical laboratories must test stool samples for the presence of Shiga toxins. Then, the positive samples must be sent to public health laboratories to look for non-O157 STEC. Clinical laboratories typically cannot identify non-O157 STEC. Other non-O157 STEC serogroups that often cause illness in people in the United States include O26, O111, and O103. Some types of STEC frequently cause severe disease, including bloody diarrhea and hemolytic uremic syndrome (HUS), which is a type of kidney failure.
Most of what we know about STEC comes from studies of E. coli O157 infection, which was first identified as a pathogen in 1982. Less is known about the non-O157 STEC, partly because older laboratory practices did not identify non-O157 infections. As a whole, the non-O157 serogroups are less likely to cause severe illness than E. coli O157, though sometimes they can. For example, E. coli O26 produces the same type of toxins that E. coli O157 produces, and causes a similar illness, though it is typically less likely to lead to kidney problems (called hemolytic uremic syndrome, or HUS).
People of any age can become infected. Very young children and the elderly are more likely to develop severe illness and hemolytic uremic syndrome (HUS) than others, but even healthy older children and young adults can become seriously ill.
The symptoms of STEC infections vary for each person but often include severe stomach cramps, diarrhea (often bloody), and vomiting. If there is fever, it usually is not very high (less than 101˚F/less than 38.5˚C). Most people get better within 5&ndash7 days. Some infections are very mild, but others are severe or even life-threatening.
Around 5&ndash10% of those who are diagnosed with STEC infection develop a potentially life-threatening complication known as hemolytic uremic syndrome (HUS). Clues that a person is developing HUS include decreased frequency of urination, feeling very tired, and losing pink color in cheeks and inside the lower eyelids. Persons with HUS should be hospitalized because their kidneys may stop working and they may develop other serious problems. Most persons with HUS recover within a few weeks, but some suffer permanent damage or die.
The time between ingesting the STEC bacteria and feeling sick is called the &ldquoincubation period.&rdquo The incubation period is usually 3-4 days after the exposure, but may be as short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. HUS, if it occurs, develops an average 7 days after the first symptoms, when the diarrhea is improving.
STEC live in the guts of ruminant animals, including cattle, goats, sheep, deer, and elk. The major source for human illnesses is cattle. STEC that cause human illness generally do not make animals sick. Other kinds of animals, including pigs and birds, sometimes pick up STEC from the environment and may spread it.
Infections start when you swallow STEC&mdashin other words, when you get tiny (usually invisible) amounts of human or animal feces in your mouth. Unfortunately, this happens more often than we would like to think about. Exposures that result in illness include consumption of contaminated food, consumption of unpasteurized (raw) milk, consumption of water that has not been disinfected, contact with cattle, or contact with the feces of infected people. Some foods are considered to carry such a high risk of infection with E. coli O157 or another germ that health officials recommend that people avoid them completely. These foods include unpasteurized (raw) milk, unpasteurized apple cider, and soft cheeses made from raw milk. Sometimes the contact is pretty obvious (working with cows at a dairy or changing diapers, for example), but sometimes it is not (like eating an undercooked hamburger or a contaminated piece of lettuce). People have gotten infected by swallowing lake water while swimming, touching the environment in petting zoos and other animal exhibits, and by eating food prepared by people who did not wash their hands well after using the toilet. Almost everyone has some risk of infection.
Because there are so many possible sources, for most people we can only guess. If your infection happens to be part of the about 20% of cases that are part of a recognized outbreak, the health department might identify the source.
An estimated 265,000 STEC infections occur each year in the United States. STEC O157 causes about 36% of these infections, and non-O157 STEC cause the rest. Public health experts rely on estimates rather than actual numbers of infections because not all STEC infections are diagnosed, for several reasons. Many infected people do not seek medical care many of those who do seek care do not provide a stool specimen for testing, and many labs do not test for non-O157 STEC. However, this situation is changing as more labs have begun using newer, simpler tests that can help detect non-O157 STEC.
STEC infections are usually diagnosed through laboratory testing of stool specimens (feces). Identifying the specific strain of STEC is essential for public health purposes, such as finding outbreaks. Many labs can determine if STEC are present, and most can identify E. coli O157. Labs that test for the presence of Shiga toxins in stool can detect non-O157 STEC infections. However, for the O group (serogroup) and other characteristics of non-O157 STEC to be identified, Shiga toxin-positive specimens must be sent to a state public health laboratory.
Contact your healthcare provider if you have diarrhea that lasts for more than 3 days, or it is accompanied by high fever, blood in the stool, or so much vomiting that you cannot keep liquids down and you pass very little urine.
Non-specific supportive therapy, including hydration, is important. Antibiotics should not be used to treat this infection. There is no evidence that treatment with antibiotics is helpful, and taking antibiotics may increase the risk of HUS. Antidiarrheal agents like Imodium® may also increase that risk.
School and work exclusion policies differ by local jurisdiction. Check with your local or state health department to learn more about the laws where you live. In any case, good hand-washing after changing diapers, after using the toilet, and before preparing food is essential to prevent the spread of these and many other infections.
Colibacillosis (E. coli diarrhea)
Diarrhea associated with Escherichia coli can occur in young piglets within a few days of birth through well after weaning. Occasional cases of septicemia are attributable to E. coli. Edema disease, a unique form of colibacillosis, is presented separately (see Edema disease).
Colibacillosis affects pigs in all major swine-raising countries. There are many different types of E. coli, each of which may possess several of many virulence factors. Many outbreaks occur within the first week after birth. Others occur later during the nursing period. Still others occur about 1-2 weeks after weaning or following abrupt changes in environment or nutrition.
Colibacillosis has been recognized as an important diarrheal disease of pigs for over fifty years. For many years treatment and control were largely empirical. Recurring losses, as well as the importance of E. coli in other species including man, stimulated research on the disease. Research has largely paralleled development of confinement rearing. Advances in molecular biology have simplified specific identification of pathogenic coliforms and their genes related to virulence.
The coliform responsible for several recently reported food poisonings of people, O157:H7, does not appear to cause disease in swine nor is this serotype common in swine.
Pathogenic strains of E. coli are easily isolated, Gram-negative, flagellated bacilli. Most pathogenic strains form smooth to mucoid colonies some are beta-hemolytic. Virulence factors include fimbria (pili), enterotoxins (exotoxins), endotoxins, and capsules. Fimbria are the small hair-like processes on the bacterial surface that allow attachment to specific receptors on the surface of mucosal enterocytes of the small intestine (colonization). Pathogenic strains also produce one or more enterotoxins, which are exotoxins elaborated locally in the small intestine that can have either local or systemic effects. These strains are termed enterotoxigenic E. coli (ETEC).
There are five common, antigenically distinct pilus types found in pigs: F4 (K88), F5 (K99), F41, F6 (987P) and F18. The first four pilus types mediate adhesion in neonates. F18 is not associated with neonatal colibacillosis but is common in postweaning colibacillosis as is F4. Some strains have the ability to erode epithelium and are termed attaching and effacing E. coli (AEEC).
The toxins elaborated by pathogenic E. coli in swine (ETEC) are labile toxin (LT), stable toxin A (StA), stable toxin B (StB), and verotoxin (shiga-like toxin, SLT). The first three act locally causing hypersecretion of fluid from the intestine while verotoxin is responsible for the systemic vascular effects of edema disease (discussed separately).
In summary, there are many strains and types of E. coli present in swine and their environment. Enteropathogenic E. coli require the presence of both fimbria-mediated adhesion to enterocytes as well as elaboration of one or more toxins.
Potentially pathogenic E. coli are present in the intestinal tract and feces of many normal swine. Dams often act as immune carriers. Continuous farrowing, accompanied by poor sanitation and chilling, can increase the risk of colibacillosis. E. coli organisms contaminate the skin and mammary glands of dams and are ingested by nursing piglets. Piglets with little colostral or inherent immunity (enterocyte receptors for certain pilus types are not present in all genetic lines of swine) sicken first. Pathogenic coliforms are magnified by fecal shedding to further increase exposure of littermates. Disease occurrence and severity is related to dose ingested and the level of immunity derived from colostral immunity.
Pathogenic coliforms survive in contaminated buildings and can infect successive litters of pigs. Once present, E. coli tend to persist unless vigorous efforts are given to maintaining sanitation, husbandry, and environment.
Ingested pathogenic E. coli adhere to receptors on microvilli of enterocytes via pili. There they colonize, proliferate, and elaborate enterotoxins that cause excessive secretion of fluid and electrolytes by crypt epithelial cells which markedly exceeds absorptive capacity resulting in a net flow of tissue fluids into the lumen. Up to 40% of a piglet’s weight may be lost as fluid passed into the intestine. The enterotoxins, endotoxin, and/or adhesins may damage the microvilli and enterocytes as well. This reduces the absorption of electrolytes, water and endogenous secretions from the lumen. The large intestine, sometimes also affected, is unable to absorb the excess fluid and diarrhea results. Damage to epithelial cells sometimes leads to septicemia. Diarrhea usually continues until death results from dehydration and metabolic acidosis or from terminal septicemia.
Post-weaning colibacillosis is similar but with one additional consideration. Verotoxigenic strains elaborate verotoxin (shiga-like toxin) which has systemic effects on endothelium of blood vessels (edema disease). These strains are sometimes hemolytic on blood agar.
Whether piglets contract colibacillosis depends on a balance between the number and virulence of pathogenic E. coli in the intestine, the pigs’ resistance to the disease, and environmental factors (temperature, humidity, sanitation, etc.). Neonatal piglets have incompletely developed immune systems and limited innate resistance. They are largely dependent on antibodies supplied in colostrum and milk. Anything that prevents piglets from obtaining colostrum leaves them susceptible to colibacillosis.
If purchased gilts farrow before they have developed antibodies to endemically present pathogenic E. coli, their colostrum and milk may not contain enough antibodies to protect their piglets. Also, as the nursing period progresses, piglets get less milk and the milk contains fewer antibodies. Chilling of piglets impairs intestinal motility and lowers resistance to infection. Massive exposure can overwhelm resistance. In recently weaned pigs, absence of milk antibodies and the different type of feed may contribute to outbreaks of colibacillosis.
Colibacillosis usually is signaled by the appearance of diarrhea. Piglets from gilts may be more severely affected than piglets nursing sows. The severity of the diarrhea varies. The hypersecretory diarrhea usually has an alkaline pH but varies in color. It may be clear and watery, especially in neonates, but may be white or yellow, influenced by type of ingesta and duration of the disease. Sick pigs occasionally vomit but vomiting is not as prominent as with transmissible gastroenteritis (TGE).
As diarrhea continues, there is progressive dehydration and the hair coat becomes roughened. Body temperature often is subnormal. Shivering often is noted unless an adequate supplementary heat source, such as heat lamps, is available. Signs are similar in pigs of various ages but tend to be more severe in younger pigs. Death losses can be severe if husbandry and environmental conditions are poor. Diarrhea tends to persist until intervention is accomplished.
E. coli is one of the most common causes of neonatal septicemia and polyserositis. Often, strains associated with septicemia are not enteropathogenic.
Dehydration is the most obvious clinical sign. The small intestine and colon may contain excess watery fluid or may be distended and gas-filled. There may be mild reddening and congestion of the stomach. Lesions often are surprisingly mild, especially in very young piglets. However, in outbreaks caused by certain pathogenic strains, usually in older postweaned pigs, there can be marked congestion of the gastrointestinal tract.
Microscopy of the mucosa of the small intestine reveals many coliforms adhered to microvilli of intestinal epithelial cells. Villi usually are intact. With some strains of E. coli, there may be necrosis of some villi and microvascular thrombosis in the lamina propria.
E. coli is a common cause of septicemia in neonates. In those cases, there is fibrinous polyserositis and arthritis.
Typical signs and lesions are useful but not definitive. The isolation of a uniform and high population of smooth, mucoid, or hemolytic E. coli from the small intestine is suggestive of colibacillosis. Diagnostic labs often use one of the following methods to more specifically identify the pathogenic E. coli:
- A slide agglutination test can identify the serogroup but does not confirm pathogenicity.
- Adhesin(s) can be identified using monoclonal antibodies.
- A polymerase chain reaction (PCR) can identify the pathogen genetically.
Genotyping by multiplex PCR is widely practiced to determine specific pilus and toxin genes present in the isolate.
Diagnostic methods do not identify important contributing factors such as chilling, poor sanitation or starvation. These often must be corrected if prevention or treatment is to be successful.
Colibacillosis has to be differentiated from other diarrheal diseases of young pigs. These include transmissible gastroenteritis (TGE), rotaviral infection, coccidiosis and Strongyloides parasitism. Starvation is also a major differential diagnosis.
As E. coli are commonly found in the small intestine of both normal and diseased pigs, microscopic examination of small intestinal sections can be useful in distinguishing the significance of the infection. Dense colonization of the brush border with adherent E. colishould be expected in most cases of colibacillosis with additional erosive or congestive changes present depending on the toxins that have been elaborated.
Many experienced veterinarians believe that colibacillosis is related largely to problems in housing and management which cause the disease secondarily. More detailed information on housing and management is available elsewhere. A few general guidelines on prevention and treatment follow:
Insofar as is possible, breeding stock should be obtained from a single source with no problems of colibacillosis. Dams should be acclimatized together for 3-6 weeks prior to breeding and during gestation so they can develop immunity to endemically occurring pathogens. This allows for the production of an adequate amount of specific antibodies in colostrum and milk.
It often is worthwhile to try to enhance the immunity of sows by using vaccines made from bacterial pili or toxins or both. Pregnant dams often are vaccinated twice at 2-3 week intervals prior to farrowing. A recent analysis of the value of vaccination, based on data from a National Animal Health Monitoring System (NAHMS) national survey, predicts that E. coli vaccination of sows would be cost effective for producers.
Another method of increasing colostral antibodies is feeding some farrowing house waste to sows during late gestation. Waste should include any pathogens present in the farrowing house and will stimulate formation of antibodies against them. Oral vaccination of sows with virulent E. coli cultured in milk was quite useful prior to the development of commercial injectable vaccines.
Use of the all in/all out system of raising piglets is recommended. Farrowing should occur in a facility thoroughly cleaned, disinfected, and dried between farrowings. The build-up of pathogens can be minimized by a vigorous, ongoing sanitation program.
The farrowing facility should be designed to provide a dry, comfortable environment for both dams and piglets. This requires lower temperatures (
70° F) for sows and areas warmed (to
90° F) for small piglets. Stresses on piglets should be minimized.
When precautionary efforts fail, a system should be available to treat piglets immediately if signs of colibacillosis appear. Antimicrobials can be administered to neonates orally or by injection. Due to the contagious nature of the organism, when treating sick pigs before weaning, all pigs (including those not scouring) in the litter also need to be treated at the same time. Weanlings can be given antibiotics in water. Antimicrobial sensitivity data is quite useful in selection of an appropriate antimicrobial. Oral electrolyte replacement solutions sometimes are used to help control dehydration. Various products may aid in prevention of postweaning colibacillosis. Plasma proteins, zinc oxide, organic acids, and probiotics are commonly used.
Some swine are genetically resistant to certain fimbriae of pathogenic E. coli. Breeding for genetically related resistance eventually may help control some forms of colibacillosis.
The Effects of Temperature on E. Coli
E. coli is a type of bacteria commonly found in the intestinal tracts of large mammals. Humans naturally harbor certain strains of E. coli in their intestines, but other strains can cause serious gastrointestinal illness. Cases of food poisoning are often linked to E. coli contamination from cattle and can infect beef and unpasteurized milk. It can also be spread to vegetables and fruits through manure compost. One of the reasons E. coli is such a common source of human illness is its resistance to heat and cold.
Foster, J. W. Escherichia coli acid resistance: tales of an amateur acidophile. Nat. Rev. Microbiol. 2, 898–907 (2004).
Lin, J. et al. Mechanisms of acid resistance in enterohemorrhagic Escherichia coli. Appl. Environ. Microbiol. 62, 3094–3100 (1996).
Lund, P., Tramonti, A. & De Biase, D. Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiol. Rev. 38, 1091–1125 (2014).
Foster, J. W. Acid stress responses of Salmonella and E. coli: survival mechanisms, regulation, and implications for pathogenesis. J. Microbiol. 39, 89–94 (2001).
Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F. & Foster, J. W. Control of acid resistance in Escherichia coli. J. Bacteriol. 181, 3525–3535 (1999).
Lin, J., Lee, I. S., Frey, J., Slonczewski, J. L. & Foster, J. W. Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J. Bacteriol. 177, 4097–4104 (1995).
Zhao, B. & Houry, W. A. Acid stress response in enteropathogenic gammaproteobacteria: an aptitude for survival. Biochem. Cell Biol. 88, 301–314 (2010).
Sen, H. et al. Structural and functional analysis of the Escherichia coli acid-sensing histidine kinase EvgS. J. Bacteriol. 199, e00310–e00317 (2017).
Hersh, B. M., Farooq, F. T., Barstad, D. N., Blankenhorn, D. L. & Slonczewski, J. L. A glutamate-dependent acid resistance gene in Escherichia coli. J. Bacteriol. 178, 3978–3981 (1996).
De Biase, D., Tramonti, A., Bossa, F. & Visca, P. The response to stationary-phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system. Mol. Microbiol. 32, 1198–1211 (1999).
Iyer, R., Williams, C. & Miller, C. Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli. J. Bacteriol. 185, 6556–6561 (2003).
Soksawatmaekhin, W., Kuraishi, A., Sakata, K., Kashiwagi, K. & Igarashi, K. Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli. Mol. Microbiol. 51, 1401–1412 (2004).
Richard, H. & Foster, J. W. Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential. J. Bacteriol. 186, 6032–6041 (2004).
Zhang, M. et al. A genetically incorporated crosslinker reveals chaperone cooperation in acid resistance. Nat. Chem. Biol. 7, 671–677 (2011).
Mujacic, M. & Baneyx, F. Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli. Appl. Environ. Microbiol. 73, 1014–1018 (2007).
Tramonti, A., De Canio, M. & De Biase, D. GadX/GadW-dependent regulation of the Escherichia coli acid fitness island: transcriptional control at the gadY-gadW divergent promoters and identification of four novel 42 bp GadX/GadW-specific binding sites. Mol. Microbiol. 70, 965–982 (2008).
Seo, S. W., Kim, D., O'Brien, E. J., Szubin, R. & Palsson, B. O. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli. Nat. Commun. 6, 7970 (2015).
Harden, M. M. et al. Acid-adapted strains of Escherichia coli K-12 obtained by experimental evolution. Appl. Environ. Microbiol. 81, 1932–1941 (2015).
Krulwich, T. A., Sachs, G. & Padan, E. Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Microbiol. 9, 330–343 (2011).
Pienaar, J. A., Singh, A. & Barnard, T. G. Acid-happy: survival and recovery of enteropathogenic Escherichia coli (EPEC) in simulated gastric fluid. Micro. Pathog. 128, 396–404 (2019).
Kaur, P. & Asea, A. Loss of biofilm formation in an emerging foodborne pathogen Enteroaggregative Escherichia coli (EAEC) under acid stress. J. Cell Sci. Ther. 8, 260 (2017).
Small, P., Blankenhorn, D., Welty, D., Zinser, E. & Slonczewski, J. L. Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH. J. Bacteriol. 176, 1729–1737 (1994).
Wang, H. & Cronan, J. E. Only one of the two annotated Lactococcus lactis fabG genes encodes a functional β-ketoacyl-acyl carrier protein reductase. Biochemistry 43, 11782–11789 (2004).
Brown, J. L., Ross, T., McMeekin, T. A. & Nichols, P. D. Acid habituation of Escherichia coli and the potential role of cyclopropane fatty acids in low pH tolerance. Int. J. Food Microbiol. 37, 163–173 (1997).
Keweloh, H., Diefenbach, R. & Rehm, H. Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Arch. Microbiol. 157, 49–53 (1991).
Fozo, E. M. & Quivey, R. G. Jr Shifts in the membrane fatty acid profile of Streptococcus mutans enhance survival in acidic environments. Appl. Environ. Microbiol. 70, 929–936 (2004).
Quivey, R. G. Jr, Faustoferri, R., Monahan, K. & Marquis, R. Shifts in membrane fatty acid profiles associated with acid adaptation of Streptococcus mutans. FEMS Microbiol. Lett. 189, 89–92 (2000).
Cao, Y. J., Yang, J. M., Xu, X., Liu, W. & Xian, M. Increasing unsaturated fatty acid contents in Escherichia coli by coexpression of three different genes. Appl. Microbiol. Biotechnol. 87, 271–280 (2010).
De Wulf, P., McGuire, A. M., Liu, X. & Lin, E. C. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli. J. Biol. Chem. 277, 26652–26661 (2002).
Snyder, W. B. & Silhavy, T. J. Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli. J. Bacteriol. 177, 953–963 (1995).
DiGiuseppe, P. A. & Silhavy, T. J. Signal detection and target gene induction by the CpxRA two-component system. J. Bacteriol. 185, 2432–2440 (2003).
Henry, M. F. & Cronan, J. E. Jr. Escherichia coli transcription factor that both activates fatty acid synthesis and represses fatty acid degradation. J. Mol. Biol. 222, 843–849 (1991).
Feng, Y. & Cronan, J. E. Escherichia coli unsaturated fatty acid synthesis: complex transcription of the fabA gene and in vivo identification of the essential reaction catalyzed by FabB. J. Biol. Chem. 284, 29526–29535 (2009).
Campbell, J. W. & Cronan, J. E. Jr. Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene. J. Bacteriol. 183, 5982–5990 (2001).
Vogt, S. L. & Raivio, T. L. Just scratching the surface: an expanding view of the Cpx envelope stress response. FEMS Microbiol. Lett. 326, 2–11 (2012).
Raivio, T. L. Everything old is new again: an update on current research on the Cpx envelope stress response. Biochim. Biophys. Acta 1843, 1529–1541 (2013).
Fleischer, R., Heermann, R., Jung, K. & Hunke, S. Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli. J. Biol. Chem. 282, 8583–8593 (2007).
Perez, J. C. & Groisman, E. A. Acid pH activation of the PmrA/PmrB two-component regulatory system of Salmonella enterica. Mol. Microbiol. 63, 283–293 (2007).
Perez, J. C. et al. Evolution of a bacterial regulon controlling virulence and Mg 2+ homeostasis. PLoS Genet 5, e1000428 (2009).
Lioliou, E. E. et al. Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli. Biochim. Biophys. Acta 1725, 257–268 (2005).
Fritz, R., Stiasny, K. & Heinz, F. X. Identification of specific histidines as pH sensors in flavivirus membrane fusion. J. Cell Biol. 183, 353–361 (2008).
Lee, D. et al. RAP uses a histidine switch to regulate its interaction with LRP in the ER and Golgi. Mol. Cell 22, 423–430 (2006).
Thompson, A. N., Posson, D. J., Parsa, P. V. & Nimigean, C. M. Molecular mechanism of pH sensing in KcsA potassium channels. Proc. Natl Acad. Sci. USA 105, 6900–6905 (2008).
Laroche, C., Beney, L., Marechal, P. A. & Gervais, P. The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures. Appl. Microbiol. Biotechnol. 56, 249–254 (2001).
Sturr, M. G. & Marquis, R. E. Comparative acid tolerances and inhibitor sensitivities of isolated F-ATPases of oral lactic acid bacteria. Appl. Environ. Microbiol. 58, 2287–2291 (1992).
Mahon, M. J. pHluorin2: an enhanced, ratiometric, pH-sensitive green florescent protein. Adv Biosci. Adv. Biosci. Biotechnol. 2, 132–137 (2011).
Hickey, E. W. & Hirshfield, I. N. Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium. Appl. Environ. Microbiol. 56, 1038–1045 (1990).
Slonczewski, J. L., Rosen, B. P., Alger, J. R. & Macnab, R. M. pH homeostasis in Escherichia coli: measurement by 31 P nuclear magnetic resonance of methylphosphonate and phosphate. Proc. Natl Acad. Sci. USA 78, 6271–6275 (1981).
Alvarez-Ordonez, A. et al. Acid stress management by Cronobacter sakazakii. Int. J. Food Microbiol. 178, 21–28 (2014).
Ragheb, M. N. et al. Inhibiting the evolution of antibiotic resistance. Mol. Cell 73, 157–165 e155 (2019).
Gorden, J. & Small, P. L. Acid resistance in enteric bacteria. Infect. Immun. 61, 364–367 (1993).
Gilbert, R. J. & Roberts, D. Food hygiene aspects and laboratory methods. PHLS Micorbiol. Dig. 3, 32–34 (1986).
Tong, W. et al. Biosynthetic pathway for acrylic acid from glycerol in recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 100, 4901–4907 (2016).
Liu, C. et al. Functional balance between enzymes in malonyl-CoA pathway for 3-hydroxypropionate biosynthesis. Metab. Eng. 34, 104–111 (2016).
Ma, Y. & Marquis, R. E. Thermophysiology of Streptococcus mutans and related lactic-acid bacteria. Antonie van. Leeuwenhoek 72, 91–100 (1997).
Aboulwafa, M. & Saier, M. H. Jr Lipid dependencies, biogenesis and cytoplasmic micellar forms of integral membrane sugar transport proteins of the bacterial phosphotransferase system. Microbiology 159, 2213–2224 (2013).
Markevics, L. J. & Jacques, N. A. Enhanced secretion of glucosyltransferase by changes in potassium ion concentrations is accompanied by an altered pattern of membrane fatty acids in Streptococcus salivarius. J. Bacteriol. 161, 989–994 (1985).
Chang, Y. Y. & Cronan, J. E. Jr Membrane cyclopropane fatty acid content is a major factor in acid resistance of Escherichia coli. Mol. Microbiol. 33, 249–259 (1999).
Wang, A. Y., Grogan, D. W. & Cronan, J. E. Jr Cyclopropane fatty acid synthase of Escherichia coli: deduced amino acid sequence, purification, and studies of the enzyme active site. Biochemistry 31, 11020–11028 (1992).
Prost, L. R. et al. Activation of the bacterial sensor kinase PhoQ by acidic pH. Mol. Cell 26, 165–174 (2007).
Surmann, K., Cudic, E., Hammer, E. & Hunke, S. Molecular and proteome analyses highlight the importance of the Cpx envelope stress system for acid stress and cell wall stability in Escherichia coli. Microbiologyopen 5, 582–596 (2016).
Weatherspoon-Griffin, N. et al. The CpxR/CpxA two-component system up-regulates two Tat-dependent peptidoglycan amidases to confer bacterial resistance to antimicrobial peptide. J. Biol. Chem. 286, 5529–5539 (2011).
Bernal-Cabas, M., Ayala, J. A. & Raivio, T. L. The Cpx envelope stress response modifies peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein YgaU. J. Bacteriol. 197, 603–614 (2015).
Siryaporn, A. & Goulian, M. Cross-talk suppression between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli. Mol. Microbiol. 70, 494–506 (2008).
Stincone, A. et al. A systems biology approach sheds new light on Escherichia coli acid resistance. Nucleic Acids Res. 39, 7512–7528 (2011).
Chakraborty, S. & Kenney, L. J. A new role of OmpR in acid and osmotic stress in Salmonella and E. coli. Front. Microbiol. 9, 2656 (2018).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
Zhao, G., Weatherspoon, N., Kong, W., Curtiss, R. 3rd & Shi, Y. A dual-signal regulatory circuit activates transcription of a set of divergent operons in Salmonella typhimurium. Proc. Natl Acad. Sci. USA 105, 20924–20929 (2008).
Edwards, R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149–157 (1998).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCt Method. Methods 25, 402–408 (2001).
The Role of Pathogenic E. coli in Fresh Vegetables: Behavior, Contamination Factors, and Preventive Measures
2 Center for Research in Microbiological Sciences, Institute of Sciences, Benemerita Universidad Autonoma de Puebla, 14 South and San Claudio Avenue, University City, San Manuel, CP 72590, Puebla, Mexico
3 Food Safety & Processing, Mississippi State University, Starkville, MS 39762, USA
Many raw vegetables, such as tomato, chili, onion, lettuce, arugula, spinach, and cilantro, are incorporated into fresh dishes including ready-to-eat salads and sauces. The consumption of these foods confers a high nutritional value to the human diet. However, the number of foodborne outbreaks associated with fresh produce has been increasing, with Escherichia coli being the most common pathogen associated with them. In humans, pathogenic E. coli strains cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and other indications. Vegetables can be contaminated with E. coli at any point from pre- to postharvest. This bacterium is able to survive in many environmental conditions due to a variety of mechanisms, such as adhesion to surfaces and internalization in fresh products, thereby limiting the usefulness of conventional processing and chemical sanitizing methods used by the food industry. The aim of this review is to provide a general description of the behavior and importance of pathogenic E. coli in ready-to-eat vegetable dishes. This information can contribute to the development of effective control measures for enhancing food safety.
The consumption of fresh produce has increased notably in recent years due to multiple contributions of nutrients and functional properties [1, 2]. Over the last 30 years, there has been a 25% increase in the average amount of fresh produce consumed per person in the USA . A diet rich in fruit and vegetables has been shown to protect against various types of cancer and chronic illnesses, such as coronary heart disease . However, at the same time, consumption of fresh produce is associated with a growing number of foodborne outbreaks due to bacterial contamination of these products .
Leafy greens, such as lettuce, spinach, and fresh herbs, are some of the vegetables most frequently linked to bacterial infections . In the United States, from 1990 to 2005, the Food Safety Project reported that at least 713 produce-related outbreaks were associated with foodborne disease, of which 12% involved fresh fruits and vegetables [4, 7]. In 2011, the Advisory Committee on the Microbiological Safety of Food (ACMSF) reported that, in the UK, there were 531 cases of reported illness, including one death, related to the consumption of fruits and vegetables between 2008 and 2010 . In the same year, Germany reported an outbreak of Shiga toxin-producing E. coli (STEC) serotype O104 : H4 at the end of the outbreak, 3785 cases of illness were reported outside of Germany, identifying contaminated sprouted seeds as responsible for the foodborne outbreak .
It should be emphasized that the effect of foodborne diseases affects not only the sick person but also has considerable economic repercussions. On the one hand, there are costs related to the sick person, including medical care and absenteeism from work and school. On the other hand, there are the costs on society, including the decrease in worker productivity, expenses of research on the outbreak, the loss of income due to food companies closing, legal expenses for litigation related to diseases, and the expenses in public medical services .
It has been shown that how crops are harvested, processed, and distributed has enhanced both the supply and variety of products, which may also have increased the risk of more widespread outbreaks. The increase in illness associated with consumption of fresh produce reflects a documented increase in food contamination .
Foodborne illness may be the cause of fresh produce contamination by pathogenic bacteria, viruses, and protozoa [11–14]. This contamination may originate from manure, soil, sewage, surface water, or wildlife  it may also occur during washing, slicing, soaking, packing, and food preparation . Among the bacteria associated with foodborne illnesses are Listeria monocytogenes , E. coli , Shigella soney , Salmonella , and Staphylococcus aureus .
Survival and growth of these microorganisms depend on several factors, including the specific features of the microorganism, fruit ripeness, environmental conditions, plant development, bacterial resistance to the plant metabolic processes, plus harvest, and postharvest processes . Particularly, some pathogenic microorganisms can internalize and adhere to the plant surface . Unfortunately, current industrial sanitizing and washing treatments of fruits and vegetables (e.g., triple washing of prepackaged leafy greens) do not guarantee the total elimination of pathogens . Therefore, this review considers the main E. coli pathotypes associated with foodborne outbreaks due to fresh produce consumption.
Furthermore, some recently introduced processes, considered to prevent the contamination of raw vegetables, are also described. They range from the production stages to the hygienic conditions during food preparation, from “the field to the table.” Reij and Den Aantrekker  reported that important factors contributing to the presence of pathogens in prepared foods are insufficient hygiene (1.6%), cross contamination (3.6%), processing or storage in inadequate rooms (4.2%), contaminated equipment (5.7%), and contamination by personnel (9.2%).
2. Incidence of E. coli
The most common vegetables associated with E. coli STEC are sprouts and green leafy vegetables (Table 1). The possible source of the contamination of sprouts is the seed that is used (it was possible to see that there were many contaminated seed lots). In the case of leafy greens, it appears that contaminated water (drag water from cattle lots or water contaminated by other sources) is the most common source of contamination. Many outbreaks reported around 30 cases, with the ratio of hospitalizations to cases ranging from 18 to 67%.
3. Contamination Factors in Fresh Vegetables
There are three types of factors that affect microbiota present in fresh products: physical, chemical, and biological. Physical factors, such as pH, temperature, and moisture, affect the growth and some metabolic activities of microbiota. Chemical factors include the availability and nutrients in vegetables that may be used by microorganisms. Finally, biological factors include the presence of competitive microbiota and bacterial-plant interactions . Fresh produce may be contaminated at any point in the production chain between farm and table. It has been shown that produce contamination is high during three periods: in the field, during initial processing, and in the kitchen . Table 2 lists agricultural factors (organic fertilizer, irrigation water, soil, and spraying of pesticides and insecticides) and postharvest practices (handling, collection, washing, processing, transportation, and packaging) that can cause the contamination of raw vegetables by various pathogenic microorganisms, including E. coli [12, 26–28].
3.1. Preharvest Factors
Soil and improperly composted animal manure are considered to be the main preharvest contamination factors. Soil is a natural reservoir for a large variety of human pathogens, including pathogenic E. coli, due to the addition of animal waste . E. coli O157 : H7 may survive in the soil from 7 to 25 weeks depending on soil types, humidity level, and temperature. This bacterium can also survive during crop storage or distribution . According to Launders et al. , the presence of STEC O157 in potatoes represents a risk because it may cause cross contamination with other foods that are consumed raw. Furthermore, in organic food production, the use of animal manure is a common practice several reports relate this type of crop system to the presence of fecal contamination, particularly during the leafy vegetable harvest . According to the Centers for Disease Control and Prevention (CDC) , several US states were affected by the consumption of organic spinach contaminated with STEC O157.
Domestic animals and wildlife also represent a potential source of pathogenic bacteria, particularly for lettuce and leafy greens at preharvest stages along the coast of California and in Yuma, AZ . Berger et al.  showed that the feces of wildlife are involved in vegetable contamination and may cause E. coli O157 : H7 outbreaks. Jay-Russell et al.  studied a potential reservoir for pathogenic E. coli in feces from coyotes and dogs. Insects could also be a source of plant contamination. Contaminated flies have been shown to transfer E. coli to plant leaves or fruits . In addition, Lynch et al.  found that intensive agricultural practices have forced crop fields to be too close to animal production areas. The ecological consequences of this proximity have increased the likelihood of contamination by E. coli O157 : H7 in wildlife: the percentages tested positive in unspecified duck was 5% (1/20 total samples) in Washington, USA in large mammals including deer, such as the black-tailed deer (Odocoileus hemionus columbianus), it was 11.1% (1/9 total samples) in California, USA, in unspecified deer, it was 25% (1/4 total samples) in Ireland, in feral pig (Sus scrofa), it was 14.9% (13/87 total samples) in California and in small mammals in England, such as the rabbit (Oryctolagus cuniculus), it was 48.8% (20/41 total samples). All sample types were feces, anal and cloacal swabs, or gastrointestinal contents from individual animals, unless otherwise noted .
Seasons are another important environmental condition that affects the prevalence of E. coli in vegetables. For example, E. coli contamination in cilantro and parsley significantly increased in fall compared to that found in spring and winter . The finding of E. coli in irrigation water has been associated with the presence of feces from cattle and other animals, especially during heavy rainfall.
There are current reports on outbreaks caused by the consumption of lettuce irrigated with water contaminated with E. coli O157 : H7 . However, the risk associated with the use of contaminated water for irrigation depends on the irrigation system used. There is a lower probability risk of spreading pathogens from contaminated water through drip irrigation versus overhead sprinkler systems . Another study shows that well water used for irrigation may be contaminated with E. coli O157 : H7 from feces of cattle or other animals, which can be observed especially during heavy rainfall. Also, karst formations occur when acidic water begins to break down bedrock surfaces, allowing surface water to enter fractures in limestone, contaminating the groundwater, which then favors the survival of E. coli in karst streams for long periods .
An additional factor during the handling and harvesting of crops are the workers’ hands. They can become a vehicle for contamination during preharvest due to the lack of access to latrines or handwashing stations .
3.2. Postharvest Contamination
In some cases, the presence of E. coli in vegetables, such as alfalfa sprouts, fresh spinach, and raw clover sprouts, is significantly higher at final postharvest stages compared to early stages of handling . This may be due to subsequent direct contamination or by pathogen multiplication during postharvest procedures in raw vegetables.
The confirmation of E. coli in postharvest packing steps indicates possible fecal contamination and the potential presence of enteric pathogens of fecal origin. According to Zhang et al. , when E. coli O157 : H7 was isolated from certain types of fresh vegetables, the prevalence was relatively low, but this microorganism can cause illness in consumers.
Water is employed in many steps, such as washing, chill tanks, sprays, and shipping ice during the postharvest process. The washing procedure is required to remove soil and debris from vegetables and some microorganisms. In spite of this, if the water used is contaminated, washing, slicing, soaking, packaging, and preparation may be the original source of E. coli transmission to vegetables. The use of contaminated water in hydrocoolers in which fresh products are stored may generate vegetable contamination . Other sources of potential contamination with E. coli during the preparation of green leafy vegetables (salads) include the water baths or dump tanks used by packers and the lack of cooling during storage . In addition, food contamination may occur if the vegetables are prepared with unclean implements in restaurants or home kitchens. Lynch et al.  mentioned that the establishment of pathogens, such as E. coli, in vegetables may occur through cross contamination by the food handler’s hands due to poor hygiene when raw meat or poultry are also being prepared.
Some outbreaks have been associated with the cutting of vegetables during salad preparation. The fresh-cut produce used in the salads has been linked to the bacterial growth. Estrada-Garcia et al.  discussed the risk of acquiring an ETEC infection when chopped vegetables are used in the preparation and consumption of Mexican salsa. Due to the great diversity of possible sources of contamination in fresh vegetables, more studies are required to learn how to prevent and correct contamination during pre- and postharvest processing.
3.3. Preharvest and Postharvest Preventive Measures for Fresh Produce
During preharvest, some pathogens may be transferred to the environment by application of inadequately composted animal manure . Therefore, it is essential to use fertilizers that are properly “stabilized.” One way to stabilize them is through the use of composting, in which the organic matter is decomposed by the action of microorganisms for a certain period of time (e.g., 3 or 15 days) at a designated temperature (131°F), followed by a stage of curing under colder conditions. These conditions reduce the levels of pathogenic microorganisms, promote the decomposition of cellulose and lignin, and stabilize their composition [42, 43]. Untreated human sewage should not be used to fertilize vegetables and crops for human consumption , unless it complies with the specifications for the use of biosolids according to regulation .
There is a risk of microbial contamination from water associated with irrigation systems due to the relationship between the volume of water retained on the crop’s surface, the amount of food consumed, and time harvest [45, 46]. Likewise, there is a recognized need to establish GAPs (Good Agricultural Practices) based on produce safety standard protocols for the irrigation of fresh produce . During postharvest, wash water can be a transmission vehicle for pathogens, especially when this water is reused . In addition, E. coli can survive for relatively long times in tap water, which can have serious consequences for the health of consumers. This point was revealed in incidents occurring in the water supply system of Walkerton, Canada, which was contaminated with E. coli O157 : H7 seven people died, and more than 2,300 people became ill .
In addition, the risk of reclaimed water may be reduced through treatment and disinfection systems, such as activated charcoal, reverse osmosis, membrane filtration, chlorination, ozonation, and UV irradiation however, some systems are often expensive, particularly in developing countries .
Postharvest treatment of fruit and vegetables is also involved in food contamination these treatments include handling, storage, transportation, and cleaning. Various studies reveal that food workers were frequently engaged in unsafe food handling, promoting microbial contamination of ready-to-eat foods. This typically occurs because food handlers are asymptomatic carriers of pathogenic microorganisms or have poor personal hygiene. Measures to diminish the risk of contamination by food workers include implementing proper handwashing and improving personal hygiene .
The World Health Organization  suggests 5 basic steps to prevent contamination of food by E. coli and other enteropathogens: (1) separating raw and cooked foods, (2) keeping the work area clean, (3) cook (the food thoroughly), (4) keeping food at safe temperatures, and (5) using safe water and raw materials.
Other actions to decrease food contamination are the use of better disinfectants. Recently, studies examined different and novel disinfectants for produce disinfection, such as chlorine dioxide, ozonized water, and electrolyzed oxidizing water. However, all these methods have their own limitations, making them unsuitable for an extensive application . For example, ozonized water has been approved as GRAS (generally recognized as safe) by the FDA (Food and Drug Administration) as an effective disinfectant against bacteria, fungi, protozoa, and microbial spores however, ozone is very unstable and may be toxic, causing eye and respiratory system irritation . Other alternatives are those proposed by Qi et al. , using sodium persulfate activated by ferrous sulfate and sodium hydroxide, which effectively inactivate E. coli O157 : H7. According to the Food Safety Modernization Act (FMSA) for fresh products, food handlers should receive education on the appropriate use of sanitizing agents and on the principles of food hygiene and safety . Another important measure is for managers to be well trained in microbiology, so they can properly supervise preparation of the agents. These training measures could contribute to the reduction of foodborne disease outbreaks associated with the consumption of raw vegetables.
While the most commonly used sanitizer is chlorine at 100 to 200 ppm, other alternative sanitizers, including ozone, peroxyacetic acid, and chlorine dioxide, are actively being evaluated for efficacy against pathogenic and spoilage microorganisms. Peracetic acid (80 ppm), chlorine (100 and 200 ppm), chlorine dioxide (3 and 5 ppm), and ozone (3 ppm) reduce populations >5 log of E. coli O157 : H7 inoculated on apples, lettuce, strawberries, and cantaloupe. Sensory panels only detected the use of 80 ppm peracetic acid on chopped lettuce and 200 ppm sodium hypochlorite on whole apples, with the other treatments being acceptable for consumers . Angeles-Núñez  showed that poor hygiene in containers and transportation may be sources of contamination and may be counteracted with an adequate system of washing, disinfection, and the application of good agricultural practices. The main risk factors of contamination during transportation include following improper production practices, temperature abuse, unsanitary cargo areas, improper loading or unloading procedures, damaged packaging, shipping containers in inadequate condition, poor employee habits, and road conditions .
Another approach, the use of Modified Atmosphere Packaging (MAP) of fresh fruits and vegetables results in an extended shelf life. MAP systems generally utilize an internal package atmosphere other than air in a hermetically sealed package of suitable permeability. O2, CO2, and N2 are the most commonly employed. The effect of MAP in inhibiting the growth of pathogens is more influenced by the type of vegetables than by the particular gas used. According to the study conducted by Abadias et al. , the population of E. coli O157 : H7 was higher in fresh-cut carrots (7.0–8.4 log cfu·g −1 ) at 25°C after 3 days of storage, while in fresh-cut melon, the bacterium reached populations of 8.5 and 8.9 log cfu·g −1 after 1 day of storage in modified atmosphere packaging, no growth was observed in the fresh-cut pineapple.
As mentioned in the previous section, the food processing industry has been using chemical decontamination (hypochlorite, peroxyacetic acid, organic acid, hydrogen peroxide, trisodium phosphate, and ozone) and physical decontamination (gamma irradiation) of ready-to-eat fresh produce. However, it has been recently reported that the nonthermal method of pulsed ultraviolet (PUV) light is a more effective method for reducing EHEC biofilm on fresh produce and packaging materials. A different strategy is focused on the use of plant commensal microbiota to compete with pathogens for diffusible factors or carbon sources in vegetal leaves and roots [4, 10, 56–59].
Recent studies are focusing on improving the efficacy of antimicrobial agents by increasing the lethal activity on pathogenic microorganisms such as E. coli, specifically focusing on the toxicity of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radical. These agents usually accumulate after exposing the bacteria to a stressor agent, such as an antimicrobial. According to Hong et al. , the blocking of ROS accumulation by exogenous mitigating agents slowed or inhibited the E. coli poststressor death, and they concluded that the lethal action of the agents depends in part of an amplifying accumulation of ROS that exceeds primary damage repair.
4. Survival Conditions and Persistence Mechanisms
Escherichia coli is an innocuous member of the human and warm-blooded animal gut microbiota however, pathogenic strains may cause intestinal and extraintestinal infections. These primary hosts may acquire E. coli from water and food contaminated with feces therefore, the presence of E. coli is used as an indicator of fecal contamination.
Some E. coli strains have been isolated from various plants used for human consumption, and these plants, such as spinach, lettuce, alfalfa, cress, bean, arugula, tomato, and radish, are considered a secondary host [61–64]. These plants have physical barriers such as wax, cuticle, cell wall, trichomes, and stomata (natural pores). It has been shown that some bacteria use stomata as entrance points to the leaf interior. Several human pathogenic bacteria can survive on and penetrate the plant interior in the apoplast they can remain in this environment with low metabolic activity, and they are able to survive drastic changes in temperature, pH, osmolality, and nutrient deprivation [65, 66].
Pathogenic E. coli possess adherence factors for human epithelial colonization, and it has been shown that several of these factors are also used for adherence to raw vegetables . On the contrary, the plant offers E. coli a harsh environment with aerobic conditions, lower temperature, low pH, a high level of UV (ultraviolet) energy, and aerial surfaces (phyllosphere), which are poor in nutrients and contain antimicrobial secondary metabolites . However, diarrheagenic E. coli have evolved mechanisms for vegetal attachment that vary according to the strain and plant involved (Table 3). Contamination of raw vegetables with E. coli is important since vegetables are used for fresh food preparations and since low doses of infection are sufficient to cause intestinal disease (E. coli O157 : H7 <100 or even <10) .
Materials and Methods
Empirical Data on E. coli Concentration and Decay Rate
The die-off patterns of E. coli in dairy slurry, cattle dung, and biosolids were analyzed from the published peer-reviewed literature to develop an overview of the E. coli concentration and decay rate (k) as presented in Table 1. In this case, 12 relevant papers were utilized to generate the data under five categories—(1) un-amended soil, (2) E. coli spiked soil, (3) biosolids, (4) dairy slurry, and (5) cattle dung. These studies were deemed relevant based on the availability or possibility of derivation of initial E. coli concentration and k value. The heterogeneous nature of the above five categorized materials and their diverse treatment conditions like moisture level, seasonality, application dose, and condition were also considered to cover the wide range of data set. Data were obtained from tables or log-linear regression equations where available (Himathongkham et al., 1999 Oliver et al., 2006 Lang and Smith, 2007 Martinez et al., 2013 Hodgson et al., 2016 Roberts et al., 2016) otherwise, data were extracted from digitized figures to derive log-linear regression equation by plotting Log10 CFU g 𢄡 dw vs. Time (days) (Avery et al., 2004, 2005 Oliver et al., 2010 Schwarz et al., 2014 Biswas et al., 2018 Ellis et al., 2018). The die-off pattern of pathogens can be described by the first-order kinetics Equation (1), which upon integration gives the linear Equation (2) (Mubiru et al., 2000 Martinez et al., 2013). This natural logarithm based linear Equation (2) was converted to the base 10 logarithm (i.e., Log10) based Equation (3) and compared with a straight line equation (y = mx+c) to get the slope (m) and subsequently, the die-off or decay rate (k) values were obtained using Equation (4) (Table 1). The linear Equation (2) can be transformed to an exponential model (Equation 5) to assess the risk of E. coli content in soil after application of different organic residues like dairy slurry, sewage sludge, and cattle dung (Vinten et al., 2004).
where C is the E. coli concentration per unit of mass or volume and k is the die-off or decay rate.