ENTEROBACTERIACEAEENTEROBACTERIACEAE

Morphology & IdentificationMorphology & Identification Gram-negative non-spore forming rods. When motile, by

peritrichous flagella. Primarily normal flora of gastrointestinal tract. E.

coli>Klebsiella>Proteus>Enterobacter Free living, also transient colonizers of skin. Facultative anaerobes: mixed acid fermentation All ferment glucose; all reduce nitrates to nitrites; all oxidase

negative. Lactose fermentation: normal flora positive and pathogens

negative. Primary isolation media include eosin-methylene-blue (EMB)

and MacConkey agar. Differential selective media for specific organisms including

dyes and bile salts. (Salmonella-Shigella (SS) medium, bismuth sulfite media.)

ClassificationClassification~29 genera, over 100 species. ~29 genera, over 100 species.

– Escherichia– Shigella– Edwardsiella– Salmonella– Citrobacter– Klebsiella– Enterobacter– Hafnia– Serratia

– Proteus– Providencia– Morganella– Yersinia– Erwinia– Pectinobacterium

Antigenic Antigenic StructureStructure

– Most are motile by peritrichous flagella --Most are motile by peritrichous flagella --HH antigens. antigens.

– Capsule – Capsule – KK antigen (antigen ( ViVi for Salmonella).for Salmonella).– Cell envelope (wall)Cell envelope (wall)– LPS (endotoxin) –LPS (endotoxin) – OO antigen. antigen. – various outer membrane proteins.various outer membrane proteins.– Pili - various antigen types, some encoded by Pili - various antigen types, some encoded by

plasmids plasmids

Flagellar (H )Ag

Cell wall (O) Ag

Capsular (K)Ag

– septicemia, septicemia, – pneumonia, pneumonia, – meningitismeningitis– urinary tract infectionsurinary tract infections

Citrobacter EnterobacterEscherichiaHafniaMorganellaProvidenciaSerratia

Opportunistic diseases Opportunistic diseases --EnterobacteriaceaeEnterobacteriaceae

EnterobacteriaceaeEnterobacteriaceae:: gastrointestinal diseasesgastrointestinal diseases

– Escherichia coliEscherichia coli – SalmonellaSalmonella– ShigellaShigella – Yersinia entercoliticaYersinia entercolitica

• Histocompatibility antigen (HLA) B27Histocompatibility antigen (HLA) B27

– Enterobacteriaceae*Salmonella*Shigella*Yersinia

– NotNot EnterobacteriaceaeEnterobacteriaceae*CampylobacterCampylobacter*ChlamydiaChlamydia

Reiter's syndromeReiter's syndrome

• community acquired • otherwise healthy people

– Klebsiella pneumoniae * respiratory diseases* prominent capsule

–urinary tract infection–fecal contamination *E. coli*Proteus

– urease (degrades urea)urease (degrades urea)– alkaline urinealkaline urine

EnterobacteriaceaeEnterobacteriaceae

EnterobacteriaceaeEnterobacteriaceae

• gram negative facultative anaerobic rodsgram negative facultative anaerobic rods– – oxidase negative (no cytochrome oxidase)oxidase negative (no cytochrome oxidase)

• E. coli – lactose positive – not usually identified– lactose positive sp. common, healthy intestine

• Shigella, Salmonella,Yersinia– lactose negative– identified

FecesFeces

• other sitesother sites– identified biochemicallyidentified biochemically

EnterobacteriaceaeEnterobacteriaceae

SerotypesSerotypesreference laboratoryreference laboratory

– antigens antigens O (lipopolysaccharide) O (lipopolysaccharide) H (flagellar) H (flagellar) K (capsular) K (capsular)

Escherichia coliEscherichia coli

Escherichia coliEscherichia coli Toxins: two types of enterotoxin; Shiga-type

toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin

Type III secretion system Adhesions –colonization factors ; both pili or

fimbriae ;non-fimbrial factors involved in attachment. There are at least 21 different types of adhesions.

Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin)

Outer membrane proteins

E. coliE. coli fimbriae fimbriae

mannosemannose

Type 1 Type 1

• galactose galactose – glycolipids glycolipids – glycoproteins glycoproteins

PP

E.coli-urinary tract infection E.coli-urinary tract infection Is the leading cause of urinary tract infections which can lead toIs the leading cause of urinary tract infections which can lead to

acute cystitis (bladder infection) and pyelonephritis (kidney infection). acute cystitis (bladder infection) and pyelonephritis (kidney infection).

E.coli-Meningitis and SepsisE.coli-Meningitis and SepsisNeonatal meningitis – is the leading cause

of neonatal meningitis and septicemia with a high mortality rate. Usually caused by strains with the K1 capsular antigen.

Enteropathogenic Enteropathogenic E. coliE. coli fever infant diarrhea vomiting nausea non-bloody stools Destruction of surface microvilli1. loose attachment mediated by bundle

forming pili (Bfp); 2. Stimulation of intracellular calcium

level; 3. rearrangement of intracellular actin,

Enterotoxigenic Enterotoxigenic E. coliE. coli

AA watery diarrhea, nausea, abdominal watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. cramps and low-grade fever for 1-5 days.

TTravellers diarrhearavellers diarrhea and and diarrhea in diarrhea in children in developing countrieschildren in developing countries

Transmission is via contaminated food or Transmission is via contaminated food or water.water.

Enterotoxigenic Enterotoxigenic E. coliE. colidiarrhea like cholera diarrhea like cholera milder milder nursery travellers diarrhea nursery travellers diarrhea caused by LT, ST, or LT/ST.caused by LT, ST, or LT/ST.

Enterotoxigenic Enterotoxigenic E. coliE. coli Heat labile toxinHeat labile toxin

– like choleragenlike choleragen– Adenyl cyclase activated Adenyl cyclase activated – cyclic AMP cyclic AMP – secretion water/ionssecretion water/ions

Heat stable toxinHeat stable toxin– Guanylate cyclase activated Guanylate cyclase activated – cyclic GMPcyclic GMP– uptake water/ionsuptake water/ions

LT vs ST activityLT vs ST activity

E.coli-E.coli-Enteroinvasive (EIEC)Enteroinvasive (EIEC) The organism attaches to the intestinal mucosa

via pili 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.

resembles shigellosis

Enteroinvasive Enteroinvasive E. coliE. coli (EIEC(EIEC))Dysentery

- resembles shigellosis- elder children and adult

diarrhea

E.coli-E.coli-c. Enteropathogenic (EPEC)c. Enteropathogenic (EPEC)

Malaise and low grade fever diarrhea, vomiting, nausea, non-bloody stools

Bundle forming pili are involved in attachment to the intestinal mucosa.

This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin.

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

E.coli-E.coli-d. Enterohemorrhagic (EHEC) d. Enterohemorrhagic (EHEC)

Hemorrhagic – bloody, copious diarrhea– few leukocytes – afebrile

hemolytic-uremic syndrome – hemolytic anemia– thrombocytopenia (low platelets)– kidney failure

Enterohemorrhagic Enterohemorrhagic E. coliE. coli• Usually O157:H7

Transmission electron micrograph

Enterohemorrhagic Enterohemorrhagic E. coliE. coli

Vero toxin Vero toxin – “ “shiga-like”shiga-like”

Hemolysins Hemolysins

younger than 5 years old,causing hemorrhagic colitis

Enteroaggregative Enteroaggregative E. E. coli coli 肠集聚型大肠杆菌肠集聚型大肠杆菌

a cause of persistent, watery diarrhea with vomiting and dehydration in infants.

That is autoagglutination in a ‘stacked brick’ arrangement.

the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST).

The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

E.coli-E.coli-Enteroaggregative Enteroaggregative (EAggEC)(EAggEC)

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 incluse watery diarrhea, vomiting, dehydration and occasional abdominal pain.

Various Types of E. coliVarious Types of E. coli

Summary of Summary of E.coliE.coli strains that cause strains that cause

gastroenteritis.gastroenteritis.

Sanitary significanceSanitary significanceTotoal bacterial number: number of Totoal bacterial number: number of

bacteria contained per ml or gm of the bacteria contained per ml or gm of the sample; the standard of drinking water is sample; the standard of drinking water is less than 100.less than 100.

Coliform bacteria index: the number of Coliform bacteria index: the number of coliform bacteria detected out per 1000 coliform bacteria detected out per 1000 ml sample; the standard of drinking ml sample; the standard of drinking water is less than 3water is less than 3

Escherichia coli Genetically E. coli and Shigella are genetically highly closely related. 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 that 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. Also cause "traveler’s 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 and water uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases.

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

4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157: H7. These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). Outbreaks are often 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 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.

ShigellaShigella

ShigellaShigellaS. flexneri, S. boydii, S. sonnei, S. S. flexneri, S. boydii, S. sonnei, S.

dysenteriaedysenteriae– bacillary dysenterybacillary dysentery– shigellosisshigellosis

bloody fecesbloody fecesintestinal painintestinal painpuspus

Genral Genral featuresfeatures

Pili. Pili. Most strains can not ferment Most strains can not ferment

lactose; S. sonnei can slowly_ lactose; S. sonnei can slowly_ ferment lactose.ferment lactose.

According to O antigen, 4 groupsAccording to O antigen, 4 groupsEasily causing drug-resistence.Easily causing drug-resistence.

ShigellosisShigellosis

within 2-3 dayswithin 2-3 days– epithelial cell damageepithelial cell damage

Shiga toxinShiga toxinenterotoxicenterotoxiccytotoxiccytotoxicinhibits protein synthesisinhibits protein synthesis

– lysing 28S rRNA

ShigellaShigella attachment and penetration attachment and penetration

WWithin 2-3 daysithin 2-3 days EEpithelial cell pithelial cell

damagedamage

Clinical significanceClinical significanceman only "reservoir"man only "reservoir"mostly young children mostly young children

– fecal to oral contactfecal to oral contact– children to adultschildren to adults

transmitted by adult food handlerstransmitted by adult food handlers– unwashed handsunwashed hands

Clinical significanceClinical significance 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).

Is is rare for the organism to disseminate. The severity of the disease depends upon the

species one is infected with. S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.

ImmunityImmunity

SIgA.

Diagnosis of Shigella infectionDiagnosis of Shigella infection

Specimen: stool. Culture and Identification Quick immunological methods:1. Immunofluorescent “ball” test; 2. Coagglutination.

PreventionPrevention

streptomycin dependent (SD) dysentery vaccine.

Treating shigellosisTreating shigellosis

manage dehydrationpatients respond to antibiotics ,

Problem of drug-resistance– disease duration diminished

Shigella 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"). Shiga toxin (chromosomally encoded) is neurotoxic, enterotoxic and cytotoxic plays a role. The toxin inhibits protein synthesis (acting on the 80S 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. However it can be transmitted by infected adult food handlers, contaminating food. The source in each case is unwashed hands. Man is the only "reservoir".

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.

SalmonellaSalmonella

Salmonellosis may present as one of several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia.

The antigenic structures of salmonellae The antigenic structures of salmonellae used in serologic typingused in serologic typing

SalmonellaSalmonella2000 antigenic "types”2000 antigenic "types”disease categorydisease category

– S. enteritidisS. enteritidis– many serotypesmany serotypes

– S. cholerae-suisS. cholerae-suis– S. typhiS. typhi

Virulence factorsVirulence 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 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

Enteric or typhoid feverEnteric or typhoid fever Enteric or typhoid fever occurs when the bacteria Enteric or typhoid fever occurs when the bacteria

leave the intestine and multiply within cells of the leave the intestine and multiply within cells of the reticuloendothelial system. reticuloendothelial system.

The bacteria then re-enter the intestine, causing The bacteria then re-enter the intestine, causing gastrointestinal symptoms. gastrointestinal symptoms.

Typhoid fever has a 10-14 day incubation period Typhoid fever has a 10-14 day incubation period and may last for several weeks.and may last for several weeks.

Salmonella typhi is the most common species Salmonella typhi is the most common species isolated from this salmonellosis. isolated from this salmonellosis.

HHuman reservoiruman reservoir::carrier state common CContaminated foodontaminated food::water supply water supply PPoor sanitary conditions oor sanitary conditions

TyphoidTyphoid

•acute phase, gastroenteritis acute phase, gastroenteritis

gall bladdergall bladder–shedding, weeksshedding, weeks

SepticemiaSepticemia-occurs 10-14 days-occurs 10-14 days– lasts 7 dayslasts 7 days

gastrointenteritisgastrointenteritis

胆囊 --- 肠道 --- 粪排菌 /肠 壁淋巴组织肾 ----- 尿肝脾 ----- 肿大骨髓 ------ 受抑制皮肤 ---- 血栓出血 -- 玫瑰疹

伤寒和付伤寒的致病过程伤寒和付伤寒沙门菌小肠上部粘膜

肠系膜淋巴结

固有层淋巴结

进入血液 再次进入血液第一次菌血症 第二次菌血症

Typhoid -TherapyTyphoid -TherapyAntibioticsAntibiotics

– essentialessentialVaccinesVaccines Vi (capsular) antigen Vi (capsular) antigen ::protectiveprotective

SalmonellaSalmonella gastroenteritisgastroenteritis

Salmonella gastroenteritis is the most common Salmonella gastroenteritis is the most common form of salmonellosis and generally requires an form of salmonellosis and generally requires an 8-48 hour incubation period and may last from 8-48 hour incubation period and may last from 2-5 days. 2-5 days.

Symptoms include nausea, vomiting and Symptoms include nausea, vomiting and diarrhea diarrhea ((non-bloody stoolnon-bloody stool)). Salmonella . Salmonella enteritidis is the most common isolate.enteritidis is the most common isolate.

poultrypoultry 家禽 , eggs, eggs. . no human reservoirno human reservoir self-limiting (2 - 5 days)self-limiting (2 - 5 days)

SalmonellaSalmonella septicemia septicemia

Salmonella septicemia (bacteremia) may be caused by any species but S. cholerae-suis is common. This disease resembles other Gram-negative septicemias and is characterized by a high, remittent fever with little gastrointestinal involvement.

Immunity (Immunity (S. typhiS. typhi))Vi (capsular) antigen Vi (capsular) antigen

– protectiveprotective

DDiagnosisiagnosisA. SpecimensA. Specimens a) Enteric fever: blood, bone marrow, a) Enteric fever: blood, bone marrow,

stool, urine.stool, urine. b) Food poisoning: stool, vomitus, b) Food poisoning: stool, vomitus,

suspected food.suspected food. c) Septicemia: blood. c) Septicemia: blood. B. Culture and identificationB. Culture and identificationC. Widal testC. Widal test

Salmonella 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 (S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs). It does not have a human reservoir and usually presents as 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. 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 organism 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

KlebsiellaKlebsiella– NF of GI tract, but potential pathogen in other areas – 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

KlebsiellaKlebsiella K. pneumoniae (Friedlander bacilli): may K. pneumoniae (Friedlander bacilli): may

cause primary pneumonia, urinary tract and cause primary pneumonia, urinary tract and wound infections, bacteremia, meningitis, etc.wound infections, bacteremia, meningitis, etc.

K. rhinoscleromatis: pathogen of K. rhinoscleromatis: pathogen of granumatous destruction of nose and granumatous destruction of nose and pharynx.pharynx.

K. ozaenae: causes chronic atrophic rhinitis.K. ozaenae: causes chronic atrophic rhinitis.

Proteus Proteus General characteristics: “swarming” phenomenon General characteristics: “swarming” phenomenon

on nonselective agaron nonselective agar (P.vulgaris; P.mirabilis and (P.vulgaris; P.mirabilis and P.myxofaciens) P.myxofaciens)

P.vulgaris strains (OX-19, OX-K, OX-2)have P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). common antigen with Rickettsia (Weil-Felix test).

urinary tract infections; food poisoningurinary tract infections; food poisoning..