Intestinal Pathogens
I. Agents
II. General patterns of disease
III. Epidemiology
IV. Bacterial intestinal pathogens - types of diseases
A. Cholera - prototypic toxigenic disease
B. Shigellosis - prototypic invasive disease
C. Other intestinal pathogens
Intestinal Pathogens
A large number of pathogens can cause intestinal disease. These include bacteria, viruses, protozoans and fungi
Intestinal Pathogens
Viruses - rotavirus
Norwalk agent
Vibrio cholerae
Shigella
E. coli
Campylobacter jejuni
Salmonella spp.
Protozoans
In industrialized nations, the leading causes of intestinal infections are:
Viruses - rotavirus, which causes winter epidemics in
children, and Norwalk agent, which is associated
with summer outbreaks in children and adults
E. coli
Campylobacter jejuni
Salmonella spp.
Worldwide: cholera and dysentery are major causes of morbidity and mortality.
General patterns of disease:
Diarrhea may result from toxicity or invasion, symptoms result from the multiplication of the organisms and invasion of intestinal epithelium and/or the production of toxins (Figure 1, Figure 2). Disease may range from a mild diarrhea (which even though mild can be a severe problem for an infant or malnourished individual) to a systemic, fatal septicemia or toxemia
Epidemiology
These are spread by fecal-oral route. The pathogens are excreted in the feces and then spread by the famous 4 Fs: feces, fingers, food and flies. If the pathogen or its toxin is primarily food borne, the disease will be referred to as food poisoning. These diseases usually reflect fecal contamination of food and water and thus are associated with poor sanitation, the exception is the toxin type food poisonings such as botulism and S. aureus in which the ingested toxin or organisms may come from environmental rather than fecal contamination. Most cases are associated with contaminated food or water or the presence of healthy carriers in the community.
These diseases may be epidemic or endemic.
Reservoir: For many enteric pathogens, humans are the reservoir, but for others such as the Salmonella species, there may be many animal hosts.
We will discuss only a few of these diseases, in particular, those caused by the enteric bacteria. These bacteria produce 5 basic disease patterns:
1. Enterotoxigenic - (prototype is V. cholerae)
Bacteria attach to small intestine - secrete toxin - watery
diarrhea
2. Dysentery - (Shigella)
Mucosal invasive and multiplication within epithelial cells
terminal ileum and large intestine
3. Destruction of brush border without further invasion
4. Invasion to mesenteric lymph nodes or,
5. Septicemia
Some species such as E. coli and Salmonella will fall into several of these groups depending on the individual strains:
E. coli 1,2,3,5
S. typhi 5, but S. enteriditis 1, 4
Campylobacter 1,2, 4
Difficult to identify these pathogens in many instances - how do you distinguish between a normal flora E. coli and one which is the cause of disease. It is often necessary to determine the presence of specific virulence factors rather than simply identifying the bacterium
Dysentery
Bacillary dysentery (mention amebic) results from infection with any Shigella
4 species: all pathogenic for man
Shigella dysenteriae**
S. flexneri
S. boydii
s. sonnei*
Listed in descending order of severity of illness
*most prevalent in the U.S.
**least prevalent in the U.S.
Epidemiology: narrow host range-man is the reservoir,
Infecting dose: 10-1000 organisms
extremely infectious/very easily spread
can be spread by fomites since the required dose is so small
hand washing rarely effective in eradicating organisms
High morbidity except in endemic areas where there is some build-up of immunity.
Very hard to control spread of disease, especially any place where sanitation is "so-so" (daycare centers, institutions, armies, large gatherings Rainbow people)
Incubation period: 1-4 days
Symptoms: Sudden onset
severe abdominal cramps
fever
diarrhea-blood and mucous often present
small volume, frequent
usually not a watery diarrhea like cholera, although a watery diarrhea is sometimes seen due to Shiga toxin acting on the small intestine (Shiga toxin released in transit or an atypical colonization of the small intestine)
neurologic sxs: lethargy, confusion
Pathology: Colonizes the colonic epithelium
Ulceration of ileal and colonic epithelium affecting the large intestine (absorptive function)
thus, see the small volume, bloody, mucoid stools
watery, voluminous diarrheas caused when small
intestine involved (secretory function)
Distinctive lesions: small colonic ulcers (little craters)
ulcers / abscesses covered by pseudomembrane
which consists of bacilli, dead epithelial cells,
debris, fibrin, and polymorphonuclear leukocytes.
Multiplication of bacterial cells results in the
release of endotoxin and metabolic byproducts
Shigella elicits a strong inflammatory response
stimulation of chemotactic factors (via endotoxin, etc.)
influx of PMNs lysis of host cells-->release of interleukins, etc. additional PMNs coming to area
-->inflammatory response, cramping, fever....
Bacteria are not found beyond the lamina propria
have only limited invasive potential
do not cause septicemias
i.e. highly invasive in terms of gaining entry to the intestinal epithelial cells, but do not invade systemically--limited localized invasion
Self-limiting disease
life-threatening in infants/small children due to dehydration or chronic malnutrition
Pathogenicity: once ingested, organisms must
Survive transit through the stomach (acid resistant)
Attach to colonic epithelial cells
Invade the cells
Multiply within the epithelial cells
Spread intracellularly and intercellularly
Note: Intracellular parasite ( vs. intracellular pathogen)
Virulence Factors:
(1.) Adherence
2. Ability to penetrate epithelial cells
3. Ability to multiply in epithelial cells
4. Ability to spread to adjacent cells
(5.) Toxins
() not well defined as far as contribution to virulence
Toxins:
In 1953, Van Heynigan reported the isolation and purification of a toxin from culture supernatants of S. shigae (S. dysenteriae). This was classified as a neurotoxin, Shiga toxin , since it caused paralysis and death in mice after injection. In natural infections, it behaves more as an enterotoxin and enterotoxic activity was noted in rabbit ligated ileal loops. (re: cholera assay)
However, Shigella are found in the large intestine, there is a relatively small effect on the ileum.
The toxin also has cytotoxin activity in tissue culture cells. If applied to a cell monolayer, it lyses the cells
Shiga toxin: inhibits protein synthesis in mammalian cell extracts
inhibition does not require NAD
distinct from activity of diphtheria toxin
(target: ribosome- cleaves adenine from 3' end of 28S
rRNA component of the ribosome thus, blocks
aminoacyl tRNA binding and inhibits peptide
elongation)
Shiga-like Toxin (SLT)
Originally thought Shiga toxin only found in S. dysenteriae, but more recent studies have demonstrated the presence of a closely related toxin called Shiga-like toxin in cultures of S. flexneri and S. sonnei:
O'Brien et al. isolated and partially purified SLT from S.
Genetics of Shiga Toxin Production:
Chromosomally encoded in Shigella: stx locus stx A stx B
1 A subunit - enzymatic activity
+ 5 B subunits - binding activity
Presence of the toxin is a contributing factor, but not essential to virulence
Regulation:
Synthesis of Shiga toxin repressed by Fe like diphtheria and botulinum toxins
Life cycle of the shigellae in host cells (Figure 3)
Each of these steps/stages is absolutely necessary in order for the production of disease
Adherence: clearly involved in the early steps
Shigella are probably taken in through M cells and then enter through the subepithelial spaces
Penetration=Invasion=Entry:
Appears to be one of the most crucial steps in
producing disease
Mechanism: directed phagocytosis by non-
professional phagocyte
Lysis of the Vesicle
Once inside the cell, the microbe must be able to
disrupt the phagosomal vesicle and gain entry into
the cytosol
Mediated by a contact hemolysin
Intracellular Multiplication:
Growing in an intestinal epithelial cell which is not equipped to kill
Protected from humoral immune response components
Protected from macrophages
Exposed to different environmental cues
Spread to Adjacent Cells:
This ability is required for production of disease
Involves actin polymerization induced by a Shigella
surface protein
See bacteria associated with host cell protrusions
which extrude into adjacent host cells
Genetics of Virulence
Large (220 kB) plasmid required for virulence
Virulence Plasmid:
Family of large plasmids in Shigella spp
Encodes a number of proteins expressed on the surface of the Shigella which are involved in: attachment, induction of phagocytosis, lysis of the phagocytic vacuole (contact hemolysin), and spread to adjacent cells
Temperature Regulation of plasmid-encoded virulence factors
Expressed at 37ºC (temp within the host)
Not expressed at 30ºC
Use T D as environmental signal to alert microbe to location
Several chromosomal loci are also required for virulence
ETEC strains produce a cholera-like syndrome
Virulence of these strains is associated with the production of toxins but they must be able to adhere and colonize the small intestine - adhere through specific pili or fimbrae
LT - This toxin is related to cholera toxin
86K protein composed of 1 a subunit and 5 b subunits
antigenic cross reactivity between the two
functionally the same - bind to GM1, stimulate adenylate cyclase. A second LT has been isolated from porcine strains related to CT but distinct from human LT
The E. coli LT is usually plasmid encoded while CT is chromosomal
Another class of E. coli toxins are the heat stable toxins:
STA - active in a variety of animals and
STB - only affects pigs
These are small polypeptides - ~18 amino acids - show some variation at the amino terminus but the carboxyterminus does not appear to vary
ST binds rapidly to cells and activates intestinal guanylate cyclase
Increased levels of cGMP causes membrane alterations which lead to net secretion of fluid and electrolytes
ST genes also are plasmid encoded
STA gene is a transposon - flanked by inverted repeats of IS1
ST and LT usually found on the same plasmid - LT+, ST+ strains produce the most severe disease
LT+/ST- and LT-/ST+ strains have also been isolated and are pathogenic
Enteropathogenic E. coli (EPEC)
1940's and 1950's, it was noted that E. coli isolated from cases of infant diarrhea fell into certain O:H serotypes. These particular serogroups were only rarely found in healthy children. Coined the term "enteropathogenic" E. coli
These strains cause diarrhea in volunteers, but lack LT and ST and are not associated with dysentery-like invasion. These strains colonize and cause destruction of brush border.
Although these strains do not produce LT or ST, they do make at least one toxin which may play a role in disease:
Shiga-like toxin (SLT)
Enteroinvasive E. coli - dysentery, clinically looks like shigellosis
These strains are capable of invasion and multiplication within colonic epithelial cells.
A 220 kb plasmid with considerable sequence homology to the S. flexneri virulence plasmid is found in these strains
Considering the promiscuity of the enteric in nature - not surprising that many common plasmids and common pathogenic mechanisms are encountered
Cloned genes are being used as probes to detect these pathogenic E. coli in cultures or clinical specimens
Salmonella species also a heterogeneous group:
Enterotoxigenic strains produce a labile toxin similar in function and antigenicity to cholera toxin
S. typhi and paratyphi - represent the most invasive of the enterics cause typhoid or paratyphoid fever, respectively
S. typhi and paratyphi A are strictly human pathogens (paratyphi B has other animal reservoirs)
Typhoid (Figure 4)
begins with the ingestion of S. typhi from contaminated food or water, the organisms penetrate the intestinal mucosa but do not produce intestinal symptoms at this stage. Gain access to the regional lymphatic and spread systemically.
In the lamina propria of a non-immune host - elicit an influx of macrophages. Those which are not ingested locally, drain into mesenteric lymph node and may be phagocytosed or gain access to blood - filtered out by the reticulendothelial cells throughout the body - dissemination. Bacilli continue to multiply and are not killed by macrophages. These are facultative intracellular pathogens.
Incubation period of 10 - 14 days following ingestion -
appearance of symptoms:
Onset of symptoms associated with a second, sustained bacteremia
Fever, rose spots - probably both manifestations of endotoxemia, splenemegaly, abdominal pain and diarrhea
Like many intracellular pathogens, S. typhi produces a more chronic disease
Carriers are not uncommon and ~3% become chronic carriers - may continue to carry and shed the organism for life - e.g. Typhoid Mary
S. typhi grows very well in bile, can persist in gall bladder and biliary tract, and cells are continuously shed into intestinal tract. These are the best transmitters of the disease.
Immunity to S. typhi relatively complex - both humoral and cellular immunity involved
Bacteria have a capsule, Vi antigen, not antiphagocytic but Vi+ cells are more virulent. May help protect the bug by shielding the O antigens from antibody which may be present in the blood
Antibodies against Vi are more often found in chronic carriers
Anti O or H antigens associated with acute cases, H ab persist, O Ab may cross react with other enterics
Immunity may require development of cellular immunity, activated macrophages and T cells which can prevent bacteremia in previously infected or chronically infected individuals.
The most common Salmonella infections now are S. enteriditis (type 4 on chart)
Short incubation period 8 - 48 h. Usually follows consumption of contaminated food but can be acquired from animals:
pet turtles and other reptiles often carry Salmonella
domestic fowl, ducks, turkeys - 40% of healthly turkeys are infected
direct contact with the animals may results in infection - more commonly - food is contaminated during processing - eggs from infected hens, improperly butchered turkeys etc.
Organisms penetrate the epithelium and multiply within the lamina propria, drain into mesenteric lymph nodes but rarely become septicemic except in compromised hosts.
