2.2 Human pathogens that can infect plants

Our human-centric view of disease often leads to sweeping assumptions that human pathogenic bacteria are devoted to human hosts. Species of Salmonella, Serratia, Enterobacter, and Enterococcus are commonly considered problematic human pathogens that are frequently found in the nosocomial environment, and which cause food poisoning, general infections, and septicaemia. Actually, other bacterial species that cause skin, wound, and urinary tract infections (e.g. Bacillus cereus, Proteus vulgaris) can also be found in rhizosphere environments.

Relatively recent studies, however, have begun to uncover that these human-pathogenic bacterial species are also capable of colonizing and causing disease in a wide variety of plant hosts. Various studies have indicated that human pathogenic bacteria enter the root tissue at sites of lateral root emergence. Notably, many of these studies have been conducted under laboratory conditions, providing evidence for the phytopathogenic potential of these bacterial species. However, the incidence of plant disease caused by many of these human pathogens in the natural environment remains unknown.

he occurrence of human pathogenic bacteria in the rhizosphere has been ascribed to several factors, including the high nutritional content, protection from ultraviolet (UV) radiation, and the availability of water films for dispersal and for preventing desiccation. Others have argued that the abundant and highly diverse indigenous rhizosphere microbial communities provide a strong barrier against the invasion of human pathogens. For example, the growth of S. enterica and E. coli O157:H7 on roots of Arabidopsis thaliana was strongly inhibited by a plant-associated strain of Enterobacter asburiae. Nevertheless, many human pathogenic bacteria can be highly competitive for nutrients and produce various antimicrobial metabolites allowing them to colonize and proliferate on plant surfaces in the presence of the indigenous microbial communities. Interestingly, the mechanisms involved in rhizosphere colonization and antimicrobial activity of human pathogenic bacteria appear to be similar to those involved in virulence and colonization of human tissues.

Next, we will discuss the biology and pathogenesis of some of the most common causative agents of infection associated with the consumption of plant foods.

 2.2.1. Salmonella

This genus is composed of two species; Salmonella enterica and Salmonella bongori. S. enterica, subdivided into hundreds of serovars, is the causal agent of many human, animal, and bird diseases worldwide, including gastroenteritis and typhoid fever. It is the pathogen most frequently linked to consumption of fruit and vegetables. Despite its clear adaptation to survival in human hosts, Salmonella has also been detected in the rhizosphere of several crop plants. For a long time, Salmonella was assumed to survive on plants after a more or less accidental infection. However, this notion has recently been challenged. Salmonella bacteria are able to persist in soil. Studies on native Conzattia multiflora, a legume tree indigenous to Mexico, revealed the existence of Salmonella plant-borne lines isolated from nodule-like structures. S. enterica ssp. enterica serovar Typhimurium (S. typhimurium) has been found in the rhizosphere of several crop plants including: wheat (Triticum sativum), oilseed rape (Brassica napus), and strawberry (Fragaria ananassa). S. enterica levels on wild tomato (Solanum pimpinellifolium) were lower than on domesticated tomato cultivars (Solanum lycopersicum). An additional infection route could be water, which was in contact with human or animal waste. Contaminated water is a well-known means of dissemination of numerous pathogens, including Salmonella. In this way, bacteria can move through the water supply system and subsequently reach crop fields where the contaminated water is used for irrigation. S. enterica populations were bigger in the phyllosphere of tomato plants irrigated with contaminated water than in plants grown from seeds in pre-infested soil. Infection of plants with Salmonella may also occur with the help of other organisms. Co-inoculation with the nematode Caenorhabditis elegans and Salmonella Newport of lettuce, strawberries or carrots placed on top of animal or composted manure resulted in plant infection with these bacteria; conversely, the plant tissues were not infected with the pathogen in the absence of the nematode. Salmonella can also be transported via insects, e.g. by the pharaoh ant (Monomorium pharaonis), moving from indoor facilities to the environment, and therefore coming in contact with a range of possible hosts, including plants. The interaction between Salmonella and arbuscular mycorrhzal fungi (AMF) has demonstrated a higher persistence of Salmonella in plants colonized by AMF, which indicates another layer of interactions in the rhizosphere.

The susceptibility to contamination by S. enterica via soil differs among agricultural crops. Members of the Brassicaceae family presented higher bacterial populations than tomato and lettuce. However, lettuce was shown to have a more highly contaminated phyllosphere, suggesting that other contamination routes, such as irrigation water, are also effective. Another noteworthy possibility is that the plant itself might contribute to infection. A study performed on alfalfa sprouts demonstrated that, during seed germination, the endosperm breakdown causes the release of reducing sugars and other organic molecules, which can be found in the irrigation water and, in consequence, produce a growth medium for S. enterica. Since bacteria metabolize these molecules as a source of nutrients, plant exudates seem to be a suitable nutrient source. The high multiplication rates of some Salmonella strains in effluxes of germinating seeds has prompted the suggestion of saprophytic growth. Furthermore, other plant pathogenic bacteria may contribute to the infection with Salmonella. Pectinolytic bacterial pathogens, which cause soft rot and in this way mobilize nutrients, are frequently associated with infection of fruits and vegetables by S. enterica. Salmonella is not only capable of colonizing the phyllosphere; it has been shown to infect the plant and cause death of plant organs. Inoculation of Salmonella in Arabidopsis via shoot or root tissues resulted in chlorosis, wilting, and eventually death of the infected tissues within seven days.

 2.2.2. Escherichia coli

Escherichia coli is a bacterium that is commonly found in the gastrointestinal tract of humans and warm-blooded animals. Due to its high prevalence in the gut, E. coli is used as the preferred indicator to detect and measure faecal contamination in the assessment of food and water safety. Considered as harmless commensals, E. coli strains constitute about 1 percent of the normal gut microbial population. While most of the strains within the gut are beneficial for human gastrointestinal function, others are harmful. Pathogenic E. coli strains are distinguished from others by their ability to cause serious illness as a result of their genetic elements for toxin production, adhesion to and invasion of host cells, interference with cellular metabolism and tissue destruction. To be transmitted from one host to another, these bacteria must leave their host and get into the surrounding environment. There is evidence that some E. coli can survive for several weeks outside the host, and even grow in water or soil. But it is on plant matter that E. coli colonization has become a concern, since although most types of E. coli are harmless, the presence of pathogenic strains on fruit and vegetables presents a food safety risk. Produce-associated outbreaks of E. coli O157 infections linked to consumption of leafy green vegetables have been increasingly recognized. However, several E. coli clones have acquired pathogenicity islands via horizontal gene transfer that enable them to cause urinary tract infections and diarrheal diseases. Diarrheal E. coli are divided into six categories that can cause illness ranging from moderate diarrhea to severe systemic diseases (e.g. hemolytic-uremic syndrome).

Shiga toxin-producing E. coli

Shiga toxin-producing E. coli (STEC) is a zoonotic pathogen colonizing mainly cattle and small ruminants. Although cattle products, principally beef, are the most commonly recognized sources of E. coli O157 infections, fruits and vegetables consumed raw are also an important source. Three leaf attachment mechanisms have been described in E. coli O157. First, contrary to nonpathogenic E. coli, the pathogenic strain STEC O157:H7 adheres strongly to tomato skin, spinach leaves, and roots of alfalfa sprouts. Adhesion to these surfaces is mediated by curli. The adhesion of E. coli O157, as well as the related enteropathogenic E. coli (EPEC), to a variety of salad leaves is mediated by the filamentous type secretion system (T3SS). The E. coli O157 secretion system is designed to translocate effector proteins into mammalian cells; protein translocation is mediated by an ATPase and is dependent on a translocation pore that is inserted into the plasma membrane. The flagella also play a role in E. coli O157 leaf attachment. Taken together, these data suggest that E. coli O157 employ multiple mechanisms to colonize plants and are well adapted to this biosphere. Like Salmonella, E. coli O157 can also reach the sub-stomatal cavity and the spongy mesophyll and survive in this environment.

Enteroaggregative E. coli and enterotoxigenic E. coli

Enteroaggregative E. coli (EAEC) may be an important cause of bacterial gastroenteritis. However, the reservoir of EAEC is unknown. There have been observed the following two patterns of bacterial distribution following 1 h incubation of EAEC with lettuce leaves: (i) diffuse adherence to the epidermis and (ii) localized adhesion to the guard cell of the stomata. The binding to the epidermis is mediated by the pilus, which is known to play a role in colonization of the human gut, whereas aggregation around the stomata is mediated by flagellae.

Enterotoxigenic E. coli (ETEC) is an important cause of infantile and travelers’ diarrhea and causes severe watery diarrhea in calves and piglets. The flagellum is the main adhesin mediating the attachment of ETEC to the epidermis of lettuce leaves.

 2.2.3. Serratia marcescens

Members of the genus Serratia cause important infections in humans, animals, and insects. Taxonomically, the genus Serratia is confusing, and currently there are 14 recognized species, with 2 subspecies. The type species, Serratia marcescens, is an opportunistic human pathogen commonly found in the respiratory and urinary tracts of humans, and is responsible for approximately 1.4% of nosocomial infections that can be life-threatening.

While environmental S. marcescens strains are often red due to the production of prodigiosin, the strains associated with hospital outbreaks are mostly non-pigmented. As many S. marcescens strains are also resistant to multiple antibiotics, it represents a growing problem for public health. However, relatively little is known about the factors that contribute to S. marcescens pathogenesis within its host.

Yet, despite its animal virulence factors, Serratia has also been found to be a common phytopathogen. S. marcescens is recognized as a phloem-resident pathogen that causes cucurbit yellow vine disease of pumpkin (Cucurbita moschata L.), watermelon, and yellow squash (Cucurbita pepo L.), which is marked by wilting, phloem discoloration, and yellowing of foliage. S. marcescens produces a biofilm along the sides of the phloem vessels, blocking the transport of nutrients and eventually causing the plant to wilt and die.

 2.2.4. Enterobacter cloacae

Another bacterium that has also shown cross-kingdom pathogenesis is the Gram-negative bacterium Enterobacter cloacae. It is an important nosocomial pathogen responsible for bacteremia, lower respiratory tract infections, skin, and soft-tissue infections, as well as urinary tract infections. E. cloacae synthesizes a Shiga-like toxin II-related cytotoxin, which has been implicated in an infant case of haemolytic-uremic syndrome. Although there is evidence that E. cloacae has evolved to colonize the human host, it has also been identified as the causal agent of grey kernel disease of macadamia (Macadamia integrifolia). The onset of grey kernel disease not only affects the quality of the kernels produced by the tree, but also results in grey discoloration and a foul odor. E. cloacae also causes bacterial soft rot disease in dragon fruit (Hylocereus spp.), bacterial leaf rot in Odontioda orchids, and is also responsible for internal yellowing disease in papaya and onions.

Enterobacter bulb decay develops after onions are harvested, cured, and stored. The decay usually occurs in a few scales of the onion bulb and the tissue develops a brown color giving the bulb a dirty ring appearance when cut in half. If storage lots of onions have a high enough incidence of Enterobacter bulb decay (>2–5%), the whole lot cannot be sold and results in a significant loss to the grower. The mechanism of how E. cloacae causes bulb decay, as well as the specificity of the virulence factors employed in each of these hosts are unknown and, as a result, the development of disease control methods for bulb decay are limited.

 2.2.5. Enterococcus

Cross-kingdom pathogenesis is not limited to Gram-negative bacteria. Enterococci are part of the normal intestinal flora of humans and animals, but are also important pathogens responsible for serious infections, especially in immunocompromised patients. With increasing antibiotic resistance, enterococci are recognized as nosocomial pathogens that can be challenging to treat. The genus Enterococcus includes more than 17 species, but only a few can cause local or systemic clinical infections, including urinary tract and abdominal infections, wound infections, bacteremia, and endocarditis. Clinical isolates of E. faecalis have been found to produce hemolysin—a virulence factor that leads to the lysis of red blood cells. Hemolytic strains exhibit multiple drug resistance more frequently than non-hemolytic strains, whereas strains isolated from fecal specimens of healthy individuals display a low (17%) incidence of hemolysin production. E. faecalis is not only capable of infecting mammalian and nematode hosts, but also the plant Arabidopsis thaliana, causing plant death seven days after inoculation under laboratory conditions. The manifestation of disease initially begins once the bacterium has successfully attached itself to the leaf surface, where upon entry into the leaf tissue through the stomata or wounds, E. faecalis multiplies and colonizes the intercellular spaces of the plant host and causes rotting and disruption of the plant cell wall and membrane structures. The phytopathogenicity of E. faecalis appears to involve some of the same genetic determinants involved in animal pathogenesis, including the quorum-sensing system gene and a serine protease. E. faecalis clearly uses a general disease strategy that allows it to use the same virulence factors for exploiting two different hosts.

 2.2.6. Listeria monocytogenes

L. monocytogenes is best known as a serious human pathogen that is transmitted to humans via consumption of contaminated food products. However, it is also a bacterium that occurs commonly in soils, associated with decaying plant matter. Thus, it can be naturally associated with any plant product grown in contact with soil. Unusually for human pathogens, it can use plant sugars for growth and hence, the organism is often a problem associated with minimally processed fruit or vegetables. Specifically, when vegetables or fruit are cut, sugars are released which the organism can utilize for growth.

Listeria in plant tissue. It is still unclear whether L. monocytogenes can become internalized in plant tissue.

The information currently available on the internalization of Listeria is from very limited studies and some of the results are contradictory. It has been shown that, like E. coli, Listeria can enter the stomata of leaves but whether this is an active process or a passive entrapment of bacteria within the structure (which is far larger than the bacterial cell) has not been determined. However, bacteria able to enter natural openings in the plant can be protected from various sanitizers following closure of the guard cells. Since the organism is commonly found in soil, the entry of Listeria into leaf tissue is more likely to occur post-harvest rather than during growth. The fruit and vegetable crops can be contaminated post-harvest when there is a big enough temperature differential between the crop and wash water to allow movement of water into the plant tissue. Following entry, it is likely that the bacteria are transported via the mass flow of water entering the plant and moving within it.

The exception to this is when Listeria-contaminated manure has been used as a fertilizer, as occurred in the Maritime coleslaw outbreak in 1981. This was the first outbreak of L. monocytogenes that could be definitely linked to food and was caused by commercially prepared coleslaw in Canada and resulted in at least 41 cases and 7 confirmed deaths. In this case, it was found that manure from Listeria-infected sheep had been used as an organic fertilizer when growing the cabbages used to prepare the salad, leading to heavy leaf contamination.

Growth of Listeria on fresh produce. Produce commodities can differ in their ability to support growth of L. monocytogenes. Some studies have been carried out to compare growth on intact and damaged or cut produce. Growth has been reported on intact tomatoes, but studies on carrots, cabbage, peppers, and cantaloupe melon reported growth only on cut, shredded or damaged produce. In general, Listeria grows well on shredded lettuce, with populations increasing during storage. Survival and growth patterns on vegetables have been found to vary with strain, product type, and packaging atmosphere. It has been suggested that packaging under modified atmosphere increases the ability of L. monocytogenes to grow by inhibiting competing microflora, but this remains to be proven, and the effect may differ by commodity, atmosphere composition, and storage conditions. However, modified atmosphere packaging (MAP) evidently does not inhibit the growth of L. monocytogenes, and the prolonged shelf life may in itself increase the listeriosis risk.

Significant differences have been observed in terms of the ability of Listeria to survive in heat and acidic conditions and on packaged vegetables. Cooked or pasteurized produce usually supports rapid growth and may represent a risk after post-processing contamination, especially if not refrigerated and consumed without heating. It is now accepted that most produce commodities support the growth of L. monocytogenes.