10 enterobacteriaceae

肠杆菌科ENTEROBACTERIACEAEENTEROBACTERIACEAE

※ G-, non-spore forming rods. Most of them have

peritrichous flagella ( 周身鞭毛 ).

※ Primarily normal flora of gastrointestinal tract.

※ Free living, also transient colonizers of skin.

※ Facultative anaerobes: mixed acid fermentation

※ All ferment glucose; all reduce nitrates ( 硝酸盐 ) to nitrites

（亚硝酸盐） ; all are oxidase （ - ） .

Morphology & Identification

※ Lactose fermentation: normal flora （ + ） and

pathogens

（ - ） .

※ Primary isolation media include eosin ['iːəsɪn]-methylene-

blue (EMB) and MacConkey agar.

※ Differential selective media for specific organisms

including dyes and bile salts. (Salmonella-Shigella (SS)

medium, bismuth ['bɪzməθ]sulfite media.)

Escherichia （埃希菌属） Shigella （志贺菌属） Edwardsiella （爱德华氏菌属） Salmonella （沙门菌属） Citrobacter （柠檬酸细菌属） Klebsiella （克雷伯氏菌属） Enterobacter （肠杆菌属） Hafnia （二氧化铪属） Serratia （沙雷氏菌属）

Proteus （变形杆菌属） Providencia

（普罗威登斯菌属） Morganella

（摩根菌属） Yersinia

（耶尔森氏菌属） Erwinia （欧文氏菌属） Pectinobacterium （果胶菌属）

ClassificationClassification29 genera, over 100 species29 genera, over 100 species

1 、 Similar appearance and dyeability

§1 Common biological characteristics

§1 Common biological characteristics

1 、 Similar appearance and dyeability

2 、 Simple cultural conditions

※ Aerobic or facultative anaerobic —— can grow in

the presence or absence of O2

1. Similar appearance and dyeability

2. Simple cultural conditions

3. Vivid in biochemical reaction

※ Typically oxidase （ - ）※ Most strains ferment lactose & glucose with

the

production of acid and/or gas

§1 Common biological characteristics ( )★

Preliminary identification of enteric pathogens and intestinal Preliminary identification of enteric pathogens and intestinal

opportunisticopportunistic pathogens pathogens

Enteropathogenic bacteria

Intestinal opportunistic pathogens

do not ferment lactose

ferment lactose

lactose-fermentation ( 乳糖发酵试验）

Reactions on TSI （ TRIPLE SUGAR IRON ） agar slants

※ （ + ） in Nitrate Reduction Test ( 硝酸盐还原试验 )

对氨基苯磺酸

萘胺百浪多息

※ IMViC reaction :

Distinguish!

4. Complicated Antigenic Structure4. Complicated Antigenic Structure

※ ※ 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).

※ ※ LPS (endotoxin) –LPS (endotoxin) – OO antigen. antigen.

※ ※ various outer membrane proteins.various outer membrane proteins.

※ ※ Pili - various antigen types, some encoded by plasmidsPili - various antigen types, some encoded by plasmids

Antigenic structure

Over 700 antigenic types (serotypes) are

recognized based on O, H, and K antigens in E. coli E. coli (K

antigens be also called capsular antigen).

5 、 Resistance

These organisms have no spores, so the resistance

are not strong. The resistance of pathogenic strains to bile sa

lts （胆盐） and brilliant green （煌绿） is much higher t

han that of opportunisticopportunistic strains, so these two substances are

usually used as the inhibitors in enterobacteriaceae selective

medium.

6 、 Liable to variation

drug resistance variation

Virulence variation

Biochemical reaction variation

Conjugation

Transduction

lysogenic conversion

7 ． Production of bacteriocins

8 ． Some of enteric bacilli can produce

exotoxins.

9. Opportunistic pathogen -9. Opportunistic pathogen -EnterobacteriaceaeEnterobacteriaceae

— — septicemia(septicemia( 败血症）败血症）

— — pneumoniapneumonia （肺炎）（肺炎）

— — meningitismeningitis （脑炎）（脑炎）

— — urinary tract infectionsurinary tract infections

（泌尿系统感染）（泌尿系统感染）

Citrobacter EnterobacterEscherichiaHafniaMorganellaProvidenciaSerratia

EnterobacteriaceaeEnterobacteriaceae:: gastrointestinal diseasesgastrointestinal diseases

— — some serological type of Escherichia colisome serological type of Escherichia coli

— — SalmonellaSalmonella

— — ShigellaShigella

— — Yersinia entercoliticaYersinia entercolitica

§2 Escherichia coliEscherichia coli （（ E. coliE. coli ））

Coliform enterobacteria which ferment lactose

Characteristics

Morphology

※ usually motile, produce peritrichous

flagella

※ some produce polysaccharide capsules

※ smooth, translucent （半透明） , colourless

colonies, 2-3 mm in diameter in 18h on nutrient

agar

※ temperature (15-45℃)

Characteristics

Antigenic structureAntigenic structure

Over 700 antigenic types (serotypes) are recognized

based on O, H, and K antigens.

▲ Lipopolysaccharide LPS (heat-stable somatic

antigens ) — O antigens

▲ flagellar antigens — H antigens

▲ "capsular" antigens — K antigens

Diseases caused by Diseases caused by E. coliE. coli

E. coli is responsible for three types of infections

in humans:

※ urinary tract infections (UTI),

※ neonatal meningitis,

※ intestinal diseases (gastroenteritis ，肠胃炎 ).

1. pathogenic material

Pathogenicity

⑴ Adhesins

Include:

— Colonization Factor Antigen Ⅰ 、Ⅱ、Ⅲ

— aggregative adherence fimbriae Ⅰ 、 II

— bundle-forming pili

—Intimin

— P pili

— invasive plasmid antigen proteins (Ipa),

— Dr pili

(2) Exotoxins

△ Shiga-type toxin:Ⅰ 、Ⅱ

△ Heat labile enterotoxin:Ⅰ 、Ⅱ

△ Heat stable enterotoxin: a 、 b

△ Hemolysin A(HlyA)



△ Heat stable enterotoxin:a 、 b


Shiga-type toxins( 志贺毒素 )

Shiga-type toxins – also called verotoxin -produced by

enterohemorrhagic strains of E. coli (EHEC) – is cytotoxic,

enterotoxic, neurotoxic, and may cause diarrhea and ulceration

of the G.I. tract.

Two types: shiga-like toxin 1 and 2.

Inhibit protein synthesis by

cleaving a 28S rRNA that’s part of the

60S subunit

Stxlysogenic phage

intestinal villi( ['vɪliː] 绒毛）kindey epithelial cells

Gb3(glycolipid receptor)

Split 28S rRNA in 60S subunit

Termination of host protein synthesis

Upregulation gene expression of inflammatory factor

The pathogenic mechanisms of shiga(-type) toxins

Cell death

villus

The chemcial constitution and pathogenesis of heat labile enterotoxin （不耐热肠毒素 )

Share 70% homology with Cholera toxin

Subunit ASubunit B

cell membrane

Adenyl Cyclase

ATP cAMP 腹泻腹泻diarrhea

GM1 receptor

H2O CI- Na+ are Secreted greatly



△ Heat stable enterotoxin: a 、 b


The chemcial constitution and pathogenesis of and pathogenesis of Heat stable enterotoxin (耐热肠毒素 )

ST

STa

STb

guanylate cyclase

cGMP↑ 腹泻腹泻diarrhea

Disturbance in Water-Electrolyte Balance

⊕

No homology with Cholera toxin

ST – is heat stable and binds to specific receptors to stimulate

the production of cGMP with the same results as with LT.

Both enterotoxins are composed of five beta subunits (for

binding) and 1 alpha subunit (has the toxic enzymatic

activity).

LT vs ST activityLT vs ST activity

Hemolysin A( 溶血素 A)

This toxin is considered play an important role in the

pathogenesis of diseases caused by uropathogenic E.coli,

but the mechanisms are not known nowadays.

Clinical finding

(1) Extraintestinal infection: Opportunities pathogenic

① urinary tract infections ： infected by UPEC

(uropathogenic E.coli ） ② septicaemia, bacteremia

③ neonatal meningitis

Results of ectopic parasitism ['pærəsaɪtˌɪzəm]( 异常寄生）

(2) intestinal diseases (Gastroenteritis)

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)

enterohemorrhagic E. coli (EHEC)

caused by Pathogenic E. coli

Enterotoxigenic Enterotoxigenic E. coliE. coli (ETEC): (ETEC): cause diarrhea in infants and cause diarrhea in infants and travelers travelers

Pathogenic E. coli — ETEC

Involves two steps: Intestinal colonization, Diarrheagenic

enterotoxin(s)

Transmission is via contaminated food or water.

Pathogenesis of ETECPathogenesis of ETEC

ETEC – is a common cause of traveler’s diarrhea and

diarrhea in children in developing countries.

The disease is characterized by a watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days.


EnterotoxinsEnterotoxins

LT (heat-labile) toxin

ST (heat-stable) toxin


固有层

Pathogenic E. coli — EIEC (enteroinvasive E. coli )

▲ Shigella-like E coli

▲ invasive (penetrate and multiply within epithelial

cells)

▲ does not produce shiga toxin

▲ EIEC – The organism attaches to the intestinal

mucosa via pili and outer embrane 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.

Pathogenic E. coli — EIEC (enteroinvasive E. coli )

▲ Symptoms include fever, severe abdominal cramps, malaise

（不适） , and watery diarrhea followed by scanty stools

containing blood, mucous, and pus.

Pathogenic E. coli — EPEC (enteropathogenic E. coli )

▲ bundle forming pili (intimin)

▲ moderately invasive "attaching and effacing"

▲ does not produce LT or ST; some reports of shiga-like toxin

▲ usually infantile diarrhea; watery diarrhea similar to

ETEC, some inflammation, no fever; symptoms probably

result mainly from invasion rather than toxigenesis

Pathogenic E. coli — EAEC (enteroaggregative E. coli )

EAEC –aggregation of the bacteria at the cell surface and result

in the formation of a mucous biofilm.

▲ The organisms attach via AAFI/II and Bfp and produce a

cytotoxin .

▲ Symptoms include watery diarrhea, vomiting, dehydration

and occasional abdominal pain.

Pathogenic E. coli — EHEC

Enterohemorrhagic Enterohemorrhagic E. coliE. coli (EHEC) (EHEC)

▲ represented by a single strain (serotype O157:H7)

▲ adhesins (probably mediated by fimbriae)

▲ moderately invasive

▲ produces shiga toxin but not LT or ST

▲ copious bloody discharge intense inflammatory response, may

be complicated by hemolytic uremia ( 出血性尿毒症）

▲ pediatric diarrhea caused by this strain can be fatal due to

acute kidney failure.

Pathogenic E. coli

TreatmentTreatment

▲ Treatment is based on symptomatology （症候学） .

▲ Fluid replacement is the primary treatment

▲ Antibiotics are generally not used except in severe diseases or

diseases that has progressed to a systemic stage

(e.g.hemolytic-uremia syndrome).

§2 ShigellaShigella （（志贺菌属）

agents of bacillary dysentery

Shigella

Morphology and StructureMorphology and Structure

※ G-,

※ PiliPili, nonmotile,

※ facultatively anaerobic,

※ non-spore-forming rods

※ Most strains Most strains failure to ferment lactose; S. sonneiferment lactose; S. sonnei (( 宋内志宋内志

贺菌贺菌） can can slowly ferment lactose.slowly ferment lactose.

※ closely related with Escherichia coli

※ Easily causing drug-resistence.Easily causing drug-resistence.

Antigenic structure and classification

speciesgrou

ptype subtype

S. dysenteriae

A 1 ～ 10 8a,8b,8c

S.flexneriB 1 ～ 6 ， X ， Y

Variant

1a,1b,2a,2b,3a,3b,3c ，

4a,4b

S.boydii C 1 ～ 18

S.sonnei D 1

According to the specific O antigen, Shigella are classified

into 4 groups, more than 40 serotypes(include subtypes).

O antigen

K antigen: non-specificity

Group-specific

Type-specificCan be use for typing

Shigella

里急后重结肠炎

[tɪ'nezməs]

ShigellaVirulenceVirulence

1. invasin

▲ encoded by plasmids — Invasion Plasmid Antigens (Ipa) A-D.

▲ induces the endocytic uptake of shigellae by M cells, epithelial cells,

and macrophages.

▲ deform the plasma membrane of contiguous cells.

▲ IcsB plasmid-encoded protein lyses the plasma membranes,

resulting in intercellular bacterial spread.

Shigella

2. Endotoxin

cause fever, shock, bloody, mucoid stools, and abdominal

pain (cramps and tenesmus) .

VirulenceVirulence

Shigella

3. exotoxin-- Shiga toxin(vero toxin)

▲ chromosomally-encoded

▲ neurotoxic, enterotoxic and cytotoxic

▲ The toxin inhibits protein synthesis (acting on the 60S

ribosome and lysing 28S rRNA). Its enterotoxicity can make

the disease clinically appear as a diarrhea.

VirulenceVirulence

ShigellaShigella attachment attachment and penetrationand penetration

ShigellaShigella

— Clinical significance

△ Causes shigellosis ( 志贺氏菌病 )or bacillary dysentery.

○ ○ bloody fecesbloody feces

○ ○ intestinal painintestinal pain

○ ○ puspus

△ Transmission is via the fecal-oral route.

△ The infective dose 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 .

— S. dysenteria is the most pathogenic followed by S. flexneri, S.

sonnei and S. boydii.

Shigella

▲ occurring by fecal-oral contact

▲ Man is the only “reservoir”, mostly young childrenmostly young children

children to adultschildren to adults

transmitted by adult food handler (unwashed hands)transmitted by adult food handler (unwashed hands)

Epidemiology(Epidemiology( 流行病学 )

ImmunityImmunity

SIgA

Shigella

Treatment and ControlTreatment and Control

▲ Managing of dehydration is of primary concern.

▲ Patients with severe dysentery are usually treated with

antibiotics (e.g. ampicillin).

▲ prevention: attenuated live vaccine

slide agglutination tests

Sample

Macc 、 SS plate

Biochemical reaction

Serological identification Sugar fermentation

※ procedure of laboratory diagnosis

DiagnosisDiagnosisShigella

Shigella

Diagnosis — SamplingDiagnosis — Sampling

▲ Positive cultures are most often obtained from blood-tinged

plugs of mucus in freshly passed stool specimens obtained

during the acute phase of disease

▲ Rectal swabs may also be used if the specimen is deposited in

a buffered glycerol saline holding solution

Shigella

Diagnosis — IsolationDiagnosis — Isolation

primary differential/selective media

SS agar

ShigellaDiagnosis — IsolationDiagnosis — Isolation

▲ colorless, non-lactose-fermenting colonies

▲ Secondly tubed slants ( 斜面 ) of Kligler's

Iron Agar or Triple Sugar Iron

Agar.

▲ Shigella species produce an

alkaline slant and an acid butt

with no bubbles of gas in the agar.

Shigella

Diagnosis — IdentificationDiagnosis — Identification

▲ slide agglutination tests with antisera

▲ polymerase chain reaction (PCR).

▲ Enzyme-linked immunosorbent assay (ELISA)

Salmonella (Salmonella (沙门菌属 )

Salmonella

Structure & Antigenic TypesStructure & Antigenic Types

▲ G-, flagellated, facultatively anaerobic bacilli

▲ three major antigens:

H or flagellar antigen;

O or somatic antigen;

Vi antigen (only a few serovars)

SalmonellaSalmonella

▲ 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 infection

△ Based on O and H (flagella) antigens

There are 58 O antigens in salmonella, put the

salmonella that have the same O antigen together and

make a group. There are 42 groups of salmonella (A,

B,C and so on).

△ The H antigens occur in two phases; 1 and 2 and only 1

phase is expressed at a given time.

phase 1: high specific, the basis of subtype

phase 2: non-specific.

△ Salmonella typhi also has a Vi antigen which is a capsular

antigen, it is also non-specific.

Salmonella Virulence FactorsVirulence Factors

▲ the ability to replicate intracellularly

▲ the ability to invade cells

invasin: Vi (capsular) antigen

▲ Adhesions – both fimbrial and non-fimbrial

▲ a complete lipopolysaccharide coat-LPS (endotoxin), also may play

a role in intracellular survival

▲ 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

Salmonella — Clinical Manifestations

1) Gastroenteritis—food poisoning

▲ Symptoms usually begin 6 to 48 hours after ingestion of

contaminated food or water

▲ the cardinal manifestation is diarrhea.

▲ nausea, vomiting, abdominal cramps, myalgia, headache,

fever and chills are common

▲ The duration of fever and diarrhea is usually 2 to 7 days


2) Septicemia

▲ an intermediate stage of infection – no intestinal symptoms

and the bacteria cannot be isolated from fecal specimens.

▲ it may remain localized in the intestine or disseminate to the

bloodstream


▲ severe systemic form, may be fatal

▲ The best studied enteric fever is typhoid fever

▲ may be preceded by gastroenteritis

▲ an incubation period of 10 to 14 days

▲ symptoms of enteric fevers are nonspecific:fever,

anorexia[ˌænə'reksiə] ( 厌食症） , headache, myalgias, and

constipation （便秘）

3) Enteric fevers — typhoid3) Enteric fevers — typhoid



★ primary bacteraemic phase:

(7- 10 days of the incubation period )

▲ invade the epithelium

▲ spread to mesenteric [mesən'terɪk] （肠系膜的） lymph

nodes & throughout the body

▲ be taken up by the reticuloendothelial cells （网状内皮细胞）

△ infect the liver, spleen, gallbladder, bones, meninges

[mə'nɪndʒiːz]

▲ invade bloodstream via thoracic duct [θɔː'ræsɪk] （胸导管）



★ second and heavier bacteraemic phase(2-3 weeks)

▲ pass into the blood( the onset of the fever and other signs of

clinical illness)

▲ From the gallbladder a further invasion of the intestine

results.

▲ Peyer patches （淋巴集结） & other gut lymphoid tissues

become involved in an inflammatory reaction

▲ followed by necrosis, sloughing （蜕皮） and the

formation of typhoid ulcers



▲ Onset: 2 weeks/ insidious/early symptoms

▲ Progression :

the temperature shows a stepladder rise over the 1st week of the

illness, remains high for 7-10 days and then falls by lysis during

the 3rd or 4th week.

physical signs include a relative bradycardia [brædɪ'kɑːdɪə]

（心搏徐缓 ) , hepatomegaly, splenomegaly ( 肝脾肿大） and often a rash of rose spots.

▲ Relapse: shorter and milder.

▲ Complications: severe intestinal haemorrhage and perforation


4) The prolonged carrier state4) The prolonged carrier state

▲ continue to excrete the salmonellae for a year or

more

▲ The bacilli are most commonly present in the

gallbladder

DDiagnosisiagnosis

A. 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 identification

C. Widal testC. Widal test

Salmonella — laboratory diagnosis

Isolation & identificationIsolation & identification

▲ Blood culture

the definitive diagnosis of enteric fever

most commonly found during the first 7-10 days

and during relapses

▲ Stool and urine culture

In typhoid fever, positive form the 2nd week and

urine cultures from the 3rd week of the infection.

▲ Specimens should be plated on several EMB media.

▲ Laboratory identification of the genus Salmonella is done by

biochemical tests.

▲ Biochemical reactions of suspicious colonies are then determined

on triple sugar iron agar and lysine-iron agar.

▲ serologic type is confirmed by serologic testing.

▲ It can be confirmed by antigenic analysis of O and H antigens

using polyvalent and specific antisera.

Salmonella — laboratory diagnosis

Isolation & identificationIsolation & identification

Salmonella-laboratory diagnosis

Widal testWidal test

▲ a tube test for determining the quantity of agglutinating antibodies, or agglutinins, in the serum of a patient with typhoid fever

Salmonella-laboratory diagnosis-widal test

▲ Generally, in typhoid cases, it is valuable that the

titre of specific

O antibodies is ≥1:80 or the titre of specific H

antibodies is ≥1:160.

▲ In paratyphoid cases, if the titre of specific H

antibodies is ≥1:80, the result is （ + ） .

Salmonella

Control and TreatmentControl and Treatment

▲ Vaccines are available for typhoid fever and are

partially effective.

▲ Typhoid fever and enteric fevers should be treated

with antibiotics.

Exercises:

IMViC test ST LT ETEC EIEC EPEC EHEC EAEC Enteric fevers

1.Please describe the Common biological characteristics of enterobacteriaceae briefly.

2.Please describe the pathogenic mechanism of salmonella.

10 enterobacteriaceae

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Transcript of 10 enterobacteriaceae