Enterobacteriaceae

Enterobacteriaceae

 Classification – more than15 different genera 

Escherichia

 

Shigella Edwardsiella



Salmonella

 

Citrobacter Klebsiella

 

Enterobacter Hafnia



Serratia

Enterobacteriaceae

 Proteus  Providencia 

Morganella



Yersinia



Erwinia



Pectinobacterium

Enterobacteriaceae

 Morphology and General Characteristics   Gram-negative, non-sporing, rod shaped bacteria Oxidase –  Ferment glucose and may or may not produce gas in the process (aerogenic vs anaerogenic)  Reduce nitrate to nitrite (there are a few exceptions)

Enterobacteriaceae

 Are facultative anaerobes  If motile, motility is by peritrichous flagella  Many are normal inhabitants of the intestinal tract of man and other animals  Some are enteric pathogens and others are urinary or respiratory tract pathogens  Differentiation is based on biochemical reactions and differences in antigenic structure

Enterobacteriaceae

 Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the the selective isolation of enteric pathogens.  Most of this media is selective by incorporation of dyes and bile salts that inhibit G+ organisms and may suppress the growth of nonpathogenic species of

Enterobacteriaceae.

 Many are differential on the basis of whether or not the organisms ferment lactose and/or produce H 2 S.

Enterobacteriaceae

 On CBA they all produce similar colonies that are relatively large and dull gray. They may or may not be hemolytic.

 The three most useful media for screening stool cultures for potential pathogens are TSI, LIA, and urea or phenylalanine agar.

 The antigenic structure is used to differentiate organisms within a genus or species.  Three major classes of antigens are found:

Enterobacteriaceae

 Somatic O antigens – these are the heat stable polysaccharide part of the LPS.    Variation from smooth to rough colonial forms is accompanied by progressive loss of smooth O Antigen.

Flagellar H antigens – are heat labile Envelope or capsule K antigens – overlay the surface O antigen and may block agglutination by O specific antisera.   Boiling for 15 minutes will destroy the K antigen and unmask O antigens. The K antigen is called the

Vi

Salmonella typhi

.

(virulence) antigen in

Antigenic Structure of Enterobacteriaceae

Enterobacteriaceae



Escherichia coli

 Normal inhabitant of the G.I. tract.   Some strains cause various forms of gastroenteritis.

Is a major cause of urinary tract infection and neonatal meningitis and septicemia.

  May have a capsule.

Biochemistry  Most are motile.

E. coli

   May be hemolytic on CBA – more common in pathogenic strains KEY tests for the normal strain:    TSI is A/A + gas LIA K/K Urea –   Indole + Citrate –  Motility + There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.

E. coli

 Antigenic structure - has O, H, and K antigens. K1 has a strong association with virulence, particularly meningitis in neonates.

 Virulence factors  Toxins  Enterotoxins – produced by enterotoxigenic strains of

E. coli

(ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin:  LT – is heat labile and binds to specific Gm 1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP.  Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.

E. coli

toxins

 ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT.

LT vs ST activity

E. coli

toxins

 Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).

Composition of subunits of enterotoxins

E. coli

toxins

 Shiga-type toxin – also called the verotoxin -produced by enterohemorrhagic strains of E. coli (EHEC) – is cytotoxic, enterotoxic, neurotoxic, and may cause diarrhea and ulceration of the G.I. tract.   There are two types shiga-like toxin 1 and shiga like toxin 2.

Inhibit protein synthesis by cleaving a 28S rRNA that’s part of the 60S subunit

E. coli

toxins

   Enteroaggregative ST-like toxin – produced by enteroaggregative strains of E. coli (EAEC) – causes watery diarrhea.

Hemolysins – two different types may be found: cell bound and secreted.  They lyse RBCs and leukocytes and may help to inhibit phagocytosis when cell bound.

Endotoxin  Type III secretion system to deliver effector molecules directly into the host cells.   Involved in inducing uptake of EIEC into intestinal cells.

Involved in development of an attachment and effacing lesion in EPEC characterized by microvilli destruction and pedestal formation.

Type III secretion system

Pedestal formation

E. coli

 Adhesions – are also called colonization factors and include both pili or fimbriae and non-fimbrial factors involved in attachment (e.g. intimin).   There are at least 21 different types of adhesions.

Antibodies to these may protect one from colonization.

 Virulence factors that protect the bacteria from host defenses   Capsule Iron capturing ability (enterochelin)  Outer membrane proteins - are involved in helping the organism to invade by helping in attachment (acting as adhesion) and in initiating endocytosis.

Types of adhesions

(intimin)

E. coli

 Clinical significance  Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).

Ascending urinary tract infection

Urinary tract infections (UTI)

 New evidence in women who suffer from recurrent UTIs suggests that this is due to the formation of pod-like

E. coli

biofilms inside bladder epithelial cells.

 Bacteria living on the edges of the biofilms nay break off leading to a round of infection.

Pod-like biofilm

E. coli

infections

 Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate.

  Usually caused by strains with the K1 capsular antigen .

Gastroenteritis – there are several distinct types of

E. coli

that are involved in different types of gastroenteritis:      enterotoxigenic

E. coli

(ETEC), enteroinvasive

E. coli

(EIEC), enteropathogenic

E. coli

(EPEC) , enteroaggregative

E. coli

(EAEC), and enterohemorrhagic

E. coli

(EHEC).

Various types of

E. coli

E. coli gastroenteritis

  ETEC – is a common cause of traveler’s diarrhea and diarrhea in children in developing countries.

 The organism attaches to the intestinal mucosa via colonization factors and then liberates enterotoxin.

 The disease is characterized by a watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days.  Transmission is via contaminated food or water.

EPEC – Bundle forming pili are involved in attachment to the intestinal mucosa.      The type III secretion system inserts the tir (translocated intimin receptor) into target cells, and intimate attachment of the non-fimbrial adhesion called intimin to tir occurs. Host cell kinases activated to phosphorylate tir which then causes a reorganization of host cytoskeletal elements resulting in pedestal formation and development of an attaching and effacing lesion The exact mode of pathogenesis is unclear, but it is probably due to the attachment and effacement.

Diarrhea with large amounts of mucous without blood or pus occurs along with vomiting, malaise and low grade fever.

This is a problem mainly in hospitalized infants and in day care centers.

BFP

EPEC EPEC EPEC

Pedestal formation

EPEC

Tir injected

E. coli gastroenteritis

  EIEC – The organism attaches to the intestinal mucosa via pili and outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa.  There is lateral movement of the organism from one cell to adjacent cells.  Symptoms include fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous, and pus .

EAEC – Mucous associated autoagglutinins cause aggregation of the bacteria at the cell surface and result in the formation of a mucous biofilm .   The organisms attach via pili and liberate a cytotoxin distinct from, but similar to the ST and LT enterotoxins liberated by ETEC. Symptoms include watery diarrhea, vomiting, dehydration and occasional abdominal pain.

E. coli

gastroenteritis

 EHEC – The organism attaches via pili to the intestinal mucosa and liberates the shiga-like toxin .  The symptoms start with a watery diarrhea that progresses to bloody diarrhea without pus and crampy abdominal pain with no fever or a low-grade fever.

   This may progress to characterized by low platlet count, hemolytic anemia, and kidney failure. hemolytic-uremic syndrome that is This is most often caused by serotypes O157:H7.

This strain of

E. coli

can be differentiated from other strains of

E. coli

by the fact that it does not ferment sorbitol in 48 hours (other strains do).    A sorbitol-Mac (SMAC) plate (contains sorbitol instead of lactose) is used to selectively isolate this organism.

One must confirm that the isolate is toxin before reporting out results.

E. coli

O1547:H7 using serological testing and confirm production of the shiga-like Serotypes of E. coli other than O157H7 have now been found to cause this disease

Summary of

E.coli

strains that cause gastroenteritis.

E.coli

 Antimicrobic therapy- E. coli is usually susceptible to a variety of chemotherapeutic agents, though drug resistant strains are increasingly prevalent.  It is essential to do susceptibility testing.

 Treatment of patients with EHEC infections is not recommended because it can increase the release of shiga-like toxins and actually trigger HUS

Shigella species

 Shigella   Contains four species that differ antigenically and, to a lesser extent, biochemically.



S. dysenteriae

(Group A)   

S. flexneri

(Group B)

S. boydii

(Group C)

S. sonnei

(Group D) Biochemistry       TSI K/A with NO gas LIA K/A Urea – Motility All ferment mannitol except

S. dysenteriae S. sonnei

may show delayed lactose fermentation

Shigella species

 Antigenic structure  Differentiation into groups (A, B, C, and D) is based on O antigen serotyping; K antigens may interfere with serotyping, but are heat labile.

 O antigen is similar to

E. coli

, so it is important to ID as

Shigella

before doing serotyping.

 Virulence factors  Shiga toxin – is produced by

S. dysenteriae

and in smaller amounts by

S. flexneri

and

S. sonnei.

  Acts to inhibit protein synthesis by inactivating the 60S ribosomal subunit by cleaving a glycosidic bond in the 28S rRNA constituents.

This plays a role in the ulceration of the intestinal mucosa.

Shigella species

 Outer membrane and secreted proteins  These proteins are expressed at body temperature and upon contact with M cells in the intestinal mucosa they induce phagocytosis of the bacteria into vacuoles. 

Shigella

destroy the vacuoles to escape into the cytoplasm.  From there they spread laterally (Polymerization of actin filaments propels them through the cytoplasm.) to epithelial cells where they multiply but do not usually disseminate beyond the epithelium.

Shigella

attachment and penetration

Shigella

attachment

Shigella

penetration

Shigella

invasion continued

Shigella

 Clinical significance  Causes shigellosis or bacillary dysentery.       Transmission is via the fecal-oral route.

The infective dose required to cause infection is very low (10-200 organisms).

There is an incubation of 1-7 days followed by fever, cramping, abdominal pain, and watery diarrhea (due to the toxin)for 1-3 days. This may be followed by frequent, scant stools with blood, mucous, and pus (due to invasion of intestinal mucosa). It is rare for the organism to disseminate. The severity of the disease depends upon the species one is infected with. 

S. dysenteria sonnei

and is the most pathogenic followed by

S. boydii

.

S. flexneri

,

S.

Shigella

 Antimicrobial therapy  Sulfonamides are commonly used as are streptomycin, tetracycline, ampicillin, and chloramphenicol.

 Resistant strains are becoming increasingly common, so sensitivity testing is required.

Salmonella

 Salmonella  Classification has been changing in the last few years.

 There is now 1 species:

S. enteritica

, and 7 subspecies: 1, 2 ,3a ,3b ,4 ,5, and 6.

  Subgroup 1 causes most human infections Clinically Salmonella isolates are often still reported out as serogroups or serotypes based on the Kauffman-White scheme of classification.

 Based on O and H (flagella) antigens    The H antigens occur in two phases; 1 and 2 and only 1 phase is expressed at a given time.

Polyvalent antisera is used followed by group specific antisera (A, B, C1, C2, D, and E) Salmonella typhi also has a Vi antigen which is a capsular antigen.

Phase variation of

Salmonella

Salmonella

  Biochemistry     TSI K/A + gas and H 2 S:

S. typhi

produces only a small amount of H 2 S and no gas , and

S. paratyphi A

produces no H 2 S LIA K/K with H 2 S with

S. paratyphi

Urea – A giving K/A results Motility +   Citrate +/ Indole Virulence factors  Endotoxin – may play a role in intracellular survival   Capsule (for

S. typhi

and some strains of S

. paratyphi

) Adhesions – both fimbrial and non-fimbrial

Salmonella virulence factors

 Type III secretion systems and effector molecules – 2 different systems may be found:   One type is involved in promoting entry into intestinal epithelial cells The other type is involved in the ability of

Salmonella

to survive inside macrophages   Outer membrane proteins - involved in the ability of

Salmonella

to survive inside macrophages Flagella – help bacteria to move through intestinal mucous  Enterotoxin - may be involved in gastroenteritis  Iron capturing ability

Salmonella

 Clinical Significance – causes two different kinds of disease: enteric fevers and gastroenteritis.  Both types of disease begin in the same way, but with the gastroenteritis the bacteria remains restricted to the intestine and with the enteric fevers, the organism spreads  Transmission is via a fecal-oral route, i.e., via ingestion of contaminated food or water.

Salmonella

 The organism moves through the intestinal mucosa and adheres to intestinal epithelium.

 Effector proteins of the type III secretion system mediate invasion of enterocytes and M cells via an induced endocytic mechanism.  Salmonella multiplies within the endosome .

Salmonella invasion of epithelial cells

Salmonella

 The endosome moves to the basal side of the cell and

Salmonella

are released and may be phagocytosed by macrophages.

  For gastroenteritis the

Salmonella

multiply and their presence induces a strong inflammatory response which causes most of the symptoms seen in gastroenteritis (mild to moderate fever with diarrhea and abdominal cramps).

 The inflammatory response prevents the spread beyond the GI tract and eventually kills the bacteria.

In enteric fevers (typhoid and paratyphoid) the Salmonella disseminate before they multiply to high enough levels to stimulate a strong inflammatory response so the initial symptoms are only a low-grade fever and constipation.

Salmonella

   The bacteria move via the lymphatics and bloodstream to the liver and spleen where phagocytosis and multiplication occurs.

The bacteria re-enter the bloodstream to disseminate throughout the body to all organs causing fever, headaches, myalgia, and GI problems. Rose spots (erythematous, muculopapular lesions) are seen on the abdomen. Osteomyelitis, cystitis, and gall bladder infections may occur.

Symptoms of paratyphoid fevers (due to

S. paratyphi

B, or C) are similar to but less severe than those that occur with typhoid fever (due to

S. typhi

) A,

Salmonella

 Diagnosis of typhoid fever   Blood cultures are positive during the first week and after the second week Stool cultures and sometimes urine cultures are positive after the second week   The Widal test is a serological test for antibodies against

Salmonella typhi.

One looks for a 4-fold rise in titer between acute and convalescent stages.

10% of those infected become short term carriers and a smaller % become long-term carriers due to persistence of the bacteria in the gallbladder or urinary bladder.

Salmonella

 Antimicrobial therapy  Enteric fevers – use chloramphenicol usually. Resistant strains have emerged making antimicrobial susceptibility testing essential.

 Gastroenteritis – usually doesn’t require antimicrobic therapy.  Replace lost fluids and electrolytes.

Comparison of

Shigella

versus

Salmonella

invasion

Shigella Salmonella

Enterobacteriaceae



Citrobacter

   TSI K/A or A/A both + gas and H 2 S LIA K/A + H 2 S Urea usually +  Motility +  Are opportunistic pathogens causing urinary tract or respiratory tract infections and occasionally wound infections, osteomyelitis, endocarditis, and meningitis.

Enterobacteriaceae



Edwardsiella tarda

    TSI K/A + gas and H 2 S LIA K/K +H 2 S Urea – Citrate –   Indole + Clinical significance – causes GI disease in tropical and subtropical countries

Enterobacteriaceae

 Klebsiella  NF of GI tract, but potential pathogen in other areas   TSI A/A + gas LIA K/K  Urea +   Citrate + MR-, VP+   Motility Has both O and K antigens

Klebsiella

 Virulence factors  Capsule  Adhesions  Iron capturing ability  Clinical significance  Causes pneumonia, mostly in immunocompromised hosts.  Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia)  A major cause of nosocomial infections such as septicemia and meningitis

Enterobacteriaceae



Enterobacter

 NF of GI tract  TSI, LIA, and urea give variable results depending upon species  Citrate +  Clinical significance  Nosocomial infections  Bacteremia in burn patients

Enterobacteriaceae



Serratia

 A free-living saprophyte   TSI A/A or K/A; +/- gas (does not ferment lactose) LIA usually K/K  Citrate +   Motility + Urea +/   Has been found in RT and UT infections Is resistant to many antimicrobics

Enterobacteriaceae



Proteus, Providencia, and Morganella

 Are all part of the NF of the GI tract (except

Providencia).

  All motile, with

Proteus

swarming PA +   Lysine deamination + (LIA R/A) Urea + for most, strongly + for

Proteus

  TSI variable (know the reactions for each in the lab!) Indole – only

P. mirabilis

is -

Proteus, Providencia, and Morganella

  Virulence factors  Urease – the ammonia produced may damage the epithelial cells of the UT Clinical Significance  UT infections, as well as pneumonia, septicemia, and wound infections 

Yersinia

 Three species are important pathogens in man   

Yersinia pestis

– causes plague

Yersinis enterocolitica

– enteropathogenic

Yersinia pseudotuberculosis

– enteropathogenic

Yersinia species

 Identification 

Y. pestis

can be separated from

pseudotuberculosis Y. enterocolitica

and

Y.

by the fact that it is non-motile.

Y. enterocolitica

motile at 37 0 and

Y. pseudotuberculosis

C, and motile at 22 0 C.

are both non 

Y. pestis

is identified based on the following:  Non-motile   Bipolar staining Slow growth of small colonies on ordinary culture media – it grows better at lower temperature (25-30 0 C)

Yersinia pestis

bipolar staining

Yersinia species

    TSI K/A no gas LIA K/A Urea –   Guinea pig or mouse pathogenicity studies: LD 50 <10 Direct fluorescent antibody test  New DNA probe test

Yersinia pestis

– virulence characteristics   Endotoxin – is responsible for many of the symptoms Murine toxin – causes edema and necrosis in mice and rats, but has not been shown to play a role in human disease

Y. pestis

      Fraction 1 – a protein component of the antiphagocytic protein capsule. Also blocks flea digestion.

V antigen virulence – a secreted protein that controls expression of many of the virulence genes plus it appears to have another unknown function that is essential for Pla – a protease that activates plasminogen activator (acts as a fibrinolysin) and degrades C3b (prevents formation of complement membrane attack complex) and C5a (prevents attraction of phagocytes) Psa – a pilus adhesion for attachment Iron acquisition and sequestering system Type III secretion system  YopB and YopD – disrupt actin cytoskeleton in phagocytic cells to evade phagocytosis

Y. pestis



Y. pestis

– clinical significance  In man plague occurs in two forms; bubonic and pneumonic  Bubonic plague – transmitted by fleas from an infected rodent (is endemic in our local mountains).

 The bacteria travel in the blood to the nearest lymph node where they are engulfed by fixed macrophages.

     A high fever develops and the lymph nodes in the groin and armpit become enlarged (buboes) as the bacteria proliferate and stimulate an inflammatory response. The bacteria growing in the lymph node leak into the bloodstream. Lysis of the bacteria releases LPS, causing septic shock. Subcutaneous hemorrhages, probably due to LPS causing DIC gave the disease the name, the black death, in the middle ages. The untreated mortality rate is quite high.

Buboes and pneumonia

Y. pestis

 Eventually bacteria reach the lungs where they are ingested by lung macrophages to cause pneumonic plague.

 Pneumonic plague – this can be transmitted directly to others via aerosol. Direct inhalation of aerosols containing the organism produces a form of the disease that progresses much more rapidly and the mortality rate is close to 100%.

 Treatment for plague  Streptomycin or tetracycline are effective

Yersinia species



Yersinia enterocolitica

and

Yersinia pseudotuberculosis identification –

 Both are motile at 22-25 0 C, but non-motile at 37 0 C       Both exhibit bipolar staining Both grow better at lower temperatures and produce small colonies at 37 0 C TSI A/A (sucrose, not lactose fermentation) for

Y. enterocolitica

; K/A for

Y. pseudotuberculosis

LIA K/A for both Urea + for both ODC + for

Y. enterocolitica

only

Yersinia species

 Cefsulodin-irgasan-novobiocin (CIN) agar is a selective media developed specifically for the isolation of

Y. enterocolitica

from gastrointestinal specimens.   The media also contains mannitol and phenol red to differentiate mannitol from non-mannitol fermenting organisms.

The media is incubated at Yersinia are the only Enterobacteriaceae that will grow on the media. room temperature and  

Aeromonas

and

Pleisiomonas

, both members of the Vibrionaceae will also grow. After 48 hours at RT, Y. enterocolitica and Y. pseudotuberculosis both produce typical pink (from mannitol fermentation) colonies with a bulls-eye appearance.

Y. enterocolitica

growth on CIN

Yersinia species

 Y. enterocolotica – virulence factors   Enterotoxin similar to E. coli ST (increases cGMP leading to watery diarrhea) Adhesions – include both fimbrial and non-fimbrial adhesions.   At least four different adhesions have been identified thus far.

Antiphagocytic proteins – include both outer membrane and secreted proteins.  Some are actually injected directly into the host via a type III secretion mechanism.   Some interfere with signal transduction in host cells, thus interfering with the ability of PMNs to respond to signals leading them to the invading bacteria. Others disrupt the actin cytoskeleton and lead to death of the PMNs.

Yersinia species

    V antigen - a secreted protein that controls expression of many of the virulence genes plus it appears to have another unknown function that is essential for virulence Iron capturing ability Yad A – an outer membrane protein that interferes with C3b binding to bacteria thus preventing the formation of a membrane attack complex.

 Endotoxin Y. pseudotuberculosis – virulence factors  Has all of the same virulence factors as

Y. enterocolitica

except the enterotoxin.

Yersinia species



Yersinia enterocolitica

significance   and

Y. pseudotuberculosis

– clinical Both are acquired by ingestion of contaminated food or water.

Y. enterocolitica

is a common cause of human disease, whereas,

Y. pseudotuberculosis

is mainly a disease of other animals.

  Both cause a disease involving fever and abdominal pain. Y. enterocolitica also causes a watery diarrhea.

After ingestion, the bacteria invade the intestinal epithelium by invasion of M cells.  They are transcytosed through the M cells and released at the basal surface.  Once through the intestional epithelium, the bacteria penetrate into the underlying lymphoid tissue, where they multiply both inside and outside host cells.

Yersinia species

   Multiplication of the bacteria produces an inflammatory response that is responsible for the extreme pain associated with the infections (resembles acute appendicitis) Fever is due to the activity of the LPS endotoxin.

Sometimes they drain into adjacent mesenteric lymph nodes, causing mesenteric lymphadenitis.

 Reactive arthritis may occur in some people following

Y. enterocolitica

infection.  It is thought to be due to cross reacting T cells or antibodies that attack the joints.

Summary of

Yersinia

infections

Yersinia species

 Antimicrobic susceptibility - must do antimicrobial susceptibility testing.