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 INFECTIOUS DISEASE

BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY

VIDEO LECTURE


BACTERIOLOGY -  CHAPTER  ELEVEN

ENTEROBACTERIACEAE, VIBRIO, CAMPYLOBACTER AND
HELICOBACTER  

Dr Alvin Fox

     

SHQIP-ALBANIAN

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Logo image © Jeffrey Nelson, Rush University, Chicago, Illinois  and The MicrobeLibrary

Reading: Murray 6th Edition: Chapters 30

 

KEY WORDS
Opportunistic Gastroenteritis
Diarrhea 
Dysentery
Urinary tract infections
Lactose positive/negative
API strip
Enteropathogenic E. coli
Enterotoxigenic E. coli
Heat stable toxin
Heat labile toxin
Enteroinvasive E. coli
Enterohemorrhagic E. coli
Vero toxin (Shiga-like)
Hemolysin
Adhesive pili
Shigella
Bacillary dysentery
Shiga toxin
Salmonella typhi
Typhoid
Vi antigen
Salmonella enteritidis   (salmonellosis)
Salmonella cholerae-suis
Vibrio cholerae
Cholera
Choleragen (cholera toxin)
Yersinia entercolitica 
Campylobacter jejuni
Helicobacter pylori

ENTEROBACTERIACEAE

General 

This group of organisms includes several that cause primary infections of the human gastrointestinal tract. Thus, they are referred to as enterics (regardless of whether they cause gut disorders). Bacteria that affect the gastrointestinal tract include certain strains of E. coli and Salmonella, all 4 species of Shigella, and Yersinia entercolitica. The rheumatic disease, Reiter's syndrome (associated with HLA-B27), can result from prior exposure to Salmonella, Shigella, or Yersinia. Other organisms that are not members of the Enterobacteriacae, including Campylobacter and Chlamydia, are also causative agents of Reiter's syndrome. Yersina pestis (the cause of "plague") will be considered separately with other zoonotic organisms.

Members of this family are major causes of opportunistic infection (including septicemia, pneumonia, meningitis and urinary tract infections). Examples of genera that cause opportunistic infections are: Citrobacter, Enterobacter, Escherichia, Hafnia, Morganella, Providencia and Serratia. Selection of antibiotic therapy is complex due to the diversity of organisms.

Some of the organisms additionally cause community-acquired disease in otherwise healthy people. Klebsiella pneumoniae is often involved in respiratory infections. The organism has a prominent capsule aiding pathogenicity . The commonest community acquired ("ascending") urinary tract infection is caused by E. coli. The vast majority of urinary tract infections are ascending, often from fecal contamination. Proteus is another common cause of urinary tract infection; the organism produces a urease that degrades urea producing an alkaline urine.

 

Isolation and identification of Enterobacteriaceae

These are Gram-negative facultative anerobic rods. They lack cytochrome oxidase and are referred to as oxidase negative. They are often isolated from fecal matter on agar containing lactose and a pH indicator. Colonies that ferment lactose will produce sufficient acid to cause a color shift in the indicator (Figure 1). E. coli is a fermenter of lactose, while Shigella, Salmonella and Yersinia are non-fermenters. "Non-pathogenic" strains of E. coli (and other lactose-positive enterics) are often present in normal feces. Since they are difficult to differentiate from "pathogenic" E. coli, lactose-negative colonies are often the only ones identified in feces. All Enterobacteriaceae isolated from other sites (which contain low numbers of bacteria [e.g. urine] or are normally sterile [e.g. blood]) are identified biochemically, for example using the API 20E system. Important serotypes can be differentiated by their O (lipopolysaccharide), H (flagellar) and K (capsular) antigens. However, serotyping is generally not performed in the routine clinical laboratory.

agar-slant.jpg (114779 bytes) Figure 1A  Reactions in TSI agar slants.   For more information on this figure, please go here. © Neal R. Chamberlain, Kirksville College of Osteopathic Medicine, Kirksville, MO and The MicrobeLibrary
pathhek.jpg (16553 bytes)  Figure 1B Nonlactose fermenter on Hektoen agar which contains bile salts and acid indicators (bromthymol blue and acid fuchsin). The gram-positive bacteria are inhibited so the agar is selective for gram-negative bacteria. The lactose fermenters form orange colonies while the nonfermenters appear green to blue-green. This is especially helpful in distinguishing potential pathogens from normal flora in stool specimens. However, it is difficult to tell the non-fermenters from each other. The organism on this plate could be Salmonella, Proteus, or Shigella. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary

lnfmac.jpg (17671 bytes) Figure 1B Growth of a nonlactose fermenter on MacConkey agar which contains bile salts and crystal violet which inhibit the growth of gram-positive bacteria. The agar also contains lactose and a red dye that differentiates the lactose fermenters from the non-fermenters. Colonies of lactose fermenting bacteria are pink to red while the nonfermenters are colorless or transparent. This agar does not distinguish between the non-lactose fermenters; this growth could indicate several organisms - Proteus, Salmonella or Shigella, for example. In a stool specimen, it would be enough evidence to continue with further identification. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary

lnfemb.jpg (14403 bytes) Figure 1C  Growth of gram-negative bacteria that cannot ferment lactose on eosin methylene blue (EMB) agar which contains bile salts and dyes which inhibit growth of gram-positive bacteria. Growth on EMB agar is a useful diagnostic tool to distinguish between lactose fermenters and non-fermenters which will appear colorless.  Salmonella and Shigella, both non-lactose fermenting pathogens, can be distinguished from the more common intestinal flora which ferment lactose.  © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
 
 

Figure 2.  Bacteria (rod), yeast (round), and fungal hyphae (filamentous) on a kitchen sponge
©
Dennis Kunkel Microscopy, Inc.  Used with permission

Figure 3 E. coli (0157:H7) hemorrhagic type. Gram-negative, enteric, facultatively anaerobic, rod prokaryote. Potentially fatal to humans, contracted when contaminated meat is cooked inadequately. © Dennis Kunkel Microscopy, Inc.  Used with permission

 

Gastroenteritis, diarrhea and dysentery

Escherichia coli (Figure 3)

At the species level, E. coli and Shigella are indistinguishable. For practical reasons (primarily to avoid confusion), they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two organisms.

1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of gene probes has confirmed these strains as different from other groups listed below. There is a characteristic morphological lesion with destruction of microvilli without invasion of the organism which suggests adhesion is important. Clinically, one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools.

2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. They also cause "travellers diarrhea". Two types of plasmid-encoded toxins are produced. 

  • Heat labile toxins which are similar to choleragen (see cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and ions. 
  • Heat stable toxins. Guanylate cyclase is activated which inhibits ionic uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases.

3) Enteroinvasive E. coli (EIEC ) produce a dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery below).

4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157:H7 (figure 4).

Other kinds of EHEC are sometimes called "non-O157 EHEC". E. coli serogroups O26, O111, and O103 are those that most often cause illness in people in the United States. Most non-O157 EHECs cause less severe disease than O157:H7 but a few can cause more severe symptoms. Very often, non-O157 EHECs are not identified and much less is known about them.
EcoliO157H7.jpg (22484 bytes) Figure 4A  Transmission electron micrograph of Escherichia coli O157:H7  CDC/Peggy S. Hayes  psh1@cdc.gov 

ecoli0157diag.jpg (85595 bytes) Figure 4B  Chronology of E. coli O157:H7 infections, an emerging type of foodborne illness. CDC

 

CASE REPORT

Multistate Outbreak of E. coli O157:H7 Infections Associated with Lebanon Bologna

These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). However, they are taking on increasing importance (figure 4) with the recognition of outbreaks caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin (biochemically similar to shiga toxin - thus also known as "shiga-like") is highly associated with this group of organisms. The toxin is encoded by a lysogenic phage. Hemolysins (plasmid-encoded) are also important in pathogenesis.

Since these bacteria make shiga-like toxins, they are often called “Shiga toxin-producing” E. coli, or STEC for short. They are also called verocytotoxic E. coli (VTEC); these all refer to the same group of bacteria.

As noted above, there are at least four etiologically distinct diseases. However, in the diagnostic laboratory,  the groups are not generally differentiated and treatment is based on symptomatology. Usually, fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome).

Two major classes of pili are produced by E. coli: mannose-sensitive and mannose-resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium, allowing attachment. This aids in colonization by E. coli.

How does a patient become infected by an EHEC?

How can EHEC infections be prevented?

 

Figure 5. Shigella dysenteriae - Gram-negative, enteric, facultatively anaerobic, rod prokaryote; causes bacterial dysentery. This species is most often found in water contaminated with human feces.  © Dennis Kunkel Microscopy, Inc.  Used with permission

Shigella

Shigella (4 species; S. flexneri, S. boydii, S. sonnei (figure 5), S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer but does not penetrate. Usually within 2-3 days, dysentery results from bacteria damaging the epithelial  layers lining the intestine, often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). However, watery diarrhea is frequently observed with no evidence of dysentery. Shiga toxin (chromosomally-encoded), which is neurotoxic, enterotoxic and cytotoxic, plays a role. Its enterotoxicity can make the disease clinically appear as a diarrhea. The toxin inhibits protein synthesis (acting on the 70S ribosome and lysing 28S rRNA). This is primarily a disease of young children occurring by fecal-oral contact. Adults can catch this disease from children, although it can be transmitted by infected adult food handlers who contaminate food. The source in each case is unwashed hands. Man is the only "reservoir".

Managing of dehydration is of primary concern. Indeed, mild diarrhea is often not recognized as shigellosis. Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.

Long term consequences of a Shigella infection

Incidence of Shigella infections

 

 

  Figure 6. Salmonella - rod prokaryote (dividing); note the flagella. Causes salmonellosis (food poisoning). (x 20,800)  © Dennis Kunkel Microscopy, Inc.  Used with permission

salmonella1.jpg (116943 bytes) Figure 7a. Isolation Rate for Salmonella enteritidis by region, United States, 1974-1994 CDC 

Salmonella  (figure 6)

Based on genetic studies, there is a single species of Salmonella (Salmonella enterica). At the other extreme using appropriate antibodies, more than 2000 antigenic "types" have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi).

Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (most commonly S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs) (figure 7a). It does not have a human reservoir and usually presents as a gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2 - 5 days). Like Shigella, these organisms invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. cholerae-suis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required.

The severest form of salmonella infections, "typhoid" (enteric fever), caused by Salmonella typhi, is rarely seen in the US, although it is one of the historical causes of widespread epidemics and still is in the third world. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and, during this acute phase, gastrointestinal symptoms are noted. The organisms penetrates (usually within the first week) and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time, the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person (e.g. a food handler) can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used (see comments on cholera).

More on typhoid

 

Figure 7b
Yersinia enterocolitica - Gram-negative, facultatively anaerobic, rod prokaryote (dividing). This bacterium releases a toxin that causes enteritis with pain resembling appendicitis.
© Dennis Kunkel Microscopy, Inc.  Used with permission

Yersinia

Yersinia entercolitica (figure 7b) is a major cause of gastroenteritis (the main clinical symptom) in Scandinavia and elsewhere and is seen in the US. The organisms are invasive (usually without systemic spread). Typically the infection is characterized by diarrhea, fever and abdominal pain. However, systemic symptoms, after bacteremia, are seen. This organism can be transmitted by fecal contamination of water or milk by domestic animals or from eating meat products. It is best isolated by "cold" enrichment:  when refrigerated this organism survives while others do not. A similar, but less severe, disease is caused by Y. pseudotuberculosis. Antibiotic therapy is recommended.

 

 

 


Vibrio species

These are Gram-negative rods. They are comma shaped, facultative anaerobes which are oxidase positive. The most important vibrio, Vibrio cholerae (figure 8 and 9), is the causative agent of cholera. It has simple nutritional requirements and is readily cultivated. V. cholerae is found in the feces of an infected individual and ends up in the water supply if sewage is untreated. The organism is thus transmitted by drinking contaminated water. The organism survives in fresh water and, like other vibrios, in salt water. Food, after water contamination, is another means of transmission. Thus, it is primarily a disease of the third world. In the US, it is observed in the occasional international traveler, although it is sometimes seen after ingestion of sea-food. Once in the gut, the organism adheres to the epithelium of the intestine without penetration. Adhesion to the microvilli is thus important in pathogenesis. Cholera toxin is then secreted.

Choleragen (cholera toxin) is chromosomally encoded and contains two types of subunit (A and B). The B subunit binds to gangliosides on epithelial cell surfaces allowing internalization of the A subunit. B subunits may provide a hydrophobic channel through which A penetrates. The A subunit catalyses ADP-ribosylation of a regulator complex which in turn activates adenylate cyclase present in the cell membrane of the epithelium of the gut. The overproduction of cyclic AMP in turn stimulates massive secretion of ions and water into the lumen. Dehydration and death (without treatment) result. Thus, fluid replacement is the major component of treatment. Antibiotic therapy (including tetracycline) is additionally used. Vaccination is only partially effective and not generally recommended. It is most commonly used by international travelers.

Vibrio parahemolyticus is usually transmitted by ingestion of raw sea-food and thus is not commonly seen in the US. The organism grows best in high concentrations of salt.  A non-bloody diarrhea is observed but it is not as severe as cholera

 

ANIMATION
Pathology of Cholera
© Alan House and Mike Hyman, Department of Microbiology, North Carolina State University, Raleigh, N.C. and The MicrobeLibrary

 
Figure 8a
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater.  © Dennis Kunkel Microscopy, Inc.  Used with permission

Figure 8b
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater.  © Dennis Kunkel Microscopy, Inc.  Used with permission

vibrio-chol.jpg (78573 bytes) Figure 8c Vibrio cholerae. Leifson flagella stain (digitally colorized).  CDC/Dr. William A. Clark 

Figure 9. Vibrio cholerae - Gram-negative, facultatively anaerobic, curved (vibrio-shaped), rod prokaryote; causes Asiatic cholera.  © Dennis Kunkel Microscopy, Inc.  Used with permission

 

campylo-cdc.jpg (50711 bytes) Figure 10a. Campylobacter fetus. Leifson flagella stain (digitally colorized).  CDC/Dr. William A. Clark

Figure 10b
Campylobacter jejuni is an enteric, curved-rod prokaryote (bacterium). It is the bacterium that causes campylobacteriosis, one of the most common bacterial causes of diarrheal illness in the United States. It is a relatively fragile bacterium that is easily killed by cold or hot temperatures. Birds are carriers due to their body temperature being just right to host the bacteria. Improper handling of raw poultry or undercooked fowl is usually the source of infection in humans.
 © Dennis Kunkel Microscopy, Inc.  Used with permission


Campylobacter and Helicobacter

These two groups of Gram-negative organisms are both curved or spiral shaped and are genetically related.

The most common of the Campylobacter (figure 10) causing human disease are C. jejuni. The organism infects the intestinal tract of several animal species (including cattle and sheep) and is a major cause of cause of abortions. The organism is transmitted to man in milk and meat products. Watery diarrhea predominates but dysentery is common. The organism is invasive but generally less so than Shigella. Malaise, fever and abdominal pain are other disease features. Bacteremia is observed in a small minority of cases. The organism is microaerophilic and grows best at 42oC. It is frequently isolated under these conditions  using selective media . It can be treated with antibiotics but is usually a self-limiting disease.

Helicobacter pylori (figure 11) has been accepted in the last few years as the major cause of stomach ulcers. The organism chronically lives in and on the stomach mucosa of man. Culture is the preferred method of diagnosis but may miss a number of cases. The organism characteristically produces a urease which generates ammonia and carbon dioxide. This aids in detecting and identifying the isolated organism. Urease is produced in such large amounts that it can be directly detected in mucosa sampled after endoscopy. Alternatively, 13C or 14C labeled CO2 is detected in the breath after feeding labeled urea. Production of ammonia is a factor in pathogenesis (in locally neutralizing stomach acid). Antibiotic therapy eliminates the organism, peptic ulcers heal and relapses are generally avoided.

 

Conclusion
Sanitary measures protect the water supply, avoiding contamination with sewage. This is the primary reason that epidemics with life-threatening pathogens (e.g cholera and typhoid) are rarely seen in western countries but are commonly seen in the third world. Other less severe diseases (e.g. salmonellosis, EHEC) are still common from eating contaminated animal products, which has been less well controlled. Shigella, which has a human host, would be even more difficult to eradicate. Vaccination is rarely used and, indeed, is an expensive way to go compared to sewage treatment. In severe diarrhea, fluid replacement is essential. Antibiotic therapy is used in severe local infection and always in systemic disease.

 

 

Figure 10c
Campylobacter jejuni - Gram-negative, enteric, curved (vibrio-shaped), rod prokaryote. Found in the gastrointestinal tract of humans and animals, it can travel to the oral cavity and genitourinary tract. Causes gastroenteritis, especially in infants.
 © Dennis Kunkel Microscopy, Inc.  Used with permission

 

 

helico.jpg (42726 bytes)  helico2.jpg (12000 bytes) Figure 11a
Helicobacter pylori electron micrographs; fastidious microaerophile; typical helical shape shown in EM; causative agent of chronic gastritis, peptic ulcers and gastric cancer. Image can be used to describe the helical morphology of the organism. Average size: 1micron by 2-5 microns. Organism is
in log phase of growth.  © Cindy R. DeLoney, Loyola University of Chicago, Chicago, Illinois and
The MicrobeLibrary

Figure 11b
Helicobacter pylori
- Gram-negative, spiral to pleomorphic, spiral rod prokaryote. It can move by means of tiny flagella at the end of the cell. There are many strains of H. pylori which are distinguished by the human disease with which they cause. H. pylori infection is the main cause of chronic superficial gastritis and it is associated with both gastric and duodenal ulcers. It lives in the interface between the surface of gastric epithelial cells (the lining of the stomach). It often clusters at the junctions of epithelial cells.
© Dennis Kunkel Microscopy, Inc.  Used with permission

 Figure 11c
Helicobacter pylori - Gram-negative, spiral to pleomorphic, spiral rod prokaryote.
© Dennis Kunkel Microscopy, Inc.  Used with permission
 

 

 

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