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 Bacterial identification 
  in the diagnostic 
  laboratory versus 
  taxonomy

  
Enterobacteriaceae,
  Vibrio, Campylobacter
  and Helicobacter

 
Enterobacteriaceae
  Vibrio
  Campylobacter and  
    Helicobacter


BACTERIOPHAGE  
EXCHANGE OF  
  GENETIC 
  INFORMATION

Streptococci  
Streptococcus  
  pneumoniae
and 
  Staphylococci  

SPIROCHETES AND 
  NEISSERIA  

ANAEROBES AND 
  PSEUDOMONAS - 
  OPPORTUNISTIC 
  INFECTIONS 

MYCOBACTERIA,  
  CORYNEBACTERIA
  AND LEGIONELLA  

BORDETELLA AND 
  HAEMOPHILUS  

RICKETTSIA, 
  EHRLICHIA, COXIELLA 
  AND BARTONELLA
  

ZOONOSES  
  LISTERIA, 
  FRANCISELLA,   
  BRUCELLA, BACILLUS  
  AND YERSINIA 





















 

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 ENTEROBACTERIACEAE, VIBRIO, CAMPYLOBACTER 
 AND HELICOBACTER  
     

Dr Alvin Fox
Medical Microbiology, MBIM 650/720 Lecture: 36 and 37

Reading: Murray Third Edition: Chapters 29-31

 cutting-dk.jpg (46206 bytes) Plastic kitchen cutting board surface - rod bacteria in a smooth matrix; filamentous bacteria -smooth oval cells; fungal hyphae with textured spores
 (x 2,70) © Dr Dennis Kunkel, University of Hawaii. Used with permission

 

ENTEROBACTERIACEAE

General 

This group of organisms include 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 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 which ferment lactose will produce sufficient acid to cause a color shift in the indicator. 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 e.g. 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.

Gastroenteritis, diarrhea and dysentery

(i) Escherichia coli

e coli-dk.jpg (22552 bytes) Rod-Shaped Bacterium, E. coli (division) (SEM x22,245)  E. coli (0157:H7) - a rod prokaryote. Hemorrhagic type  © Dr Dennis Kunkel, University of Hawaii. Used with permission

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. a) 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. b) 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. 

EcoliO157H7.jpg (22484 bytes) Transmission electron micrograph of Escherichia coli O157:H7  CDC/Peggy S. Hayes  psh1@cdc.gov  ecoli0157diag.jpg (85595 bytes) Chronology of E. coli O157:H7 infections, an emerging type of foodborne illness. CDC

 

These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). However, they are taking increasing importance 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; encoded by a lysogenic phage. Hemolysins (plasmid encoded) are also important in pathogenesis.

As noted above, there are at least 4 etiologically distinct diseases. However, in the diagnostic laboratory generally the groups are not differentiated and treatment would be on symptomatology. Generally 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.

 

(ii) Shigella

shig-dk.jpg (14696 bytes) Shigella sonnei - rod prokaryote; causes shigellosis (bacterial dysentery) (x 22,400) © Dr Dennis Kunkel, University of Hawaii. Used with permission

Shigella (4 species; S. flexneri, S. boydii, S. sonnei, 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 epithelium lining layers of 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, frequently watery diarrhea is 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 60S 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 contaminating 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.

(iii) Salmonella

salmon-dk.jpg (16097 bytes) Salmonella typhimurium - rod prokaryote (dividing); note the flagella. Causes salmonellosis (food poisoning). (x 20,800)  © Dr Dennis Kunkel, University of Hawaii. Used with permission

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).

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

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). 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, they 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).

 

(iv) Yersinia

Yersinia entercolitica 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.

 

VIBRIOS

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

vibchol-dk.jpg (18397 bytes) Vibrio cholerae - vibrio-shaped prokaryote that causes cholera (x 15,575) © Dr Dennis Kunkel, University of Hawaii. Used with permission

These are Gram negative rods, comma shaped, facultative anaerobe which are oxidase positive. The most important vibrio, Vibrio cholerae, 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 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. Not as severe as cholera but a non-bloody diarrhea is observed.

 

 

CAMPYLOBACTER AND HELICOBACTER

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

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

The most common of the Campylobacter 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 has been accepted in the last few years as the major cause of stomach ulcers where it colonizes. 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 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 the US 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. 


 

 


 






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