Gram-Negative Rods

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Transcript Gram-Negative Rods

Gram-Negative Rods

General Classification

Based on source or site of infection

1. Enteric tract 2. Respiratory tract 3. Animal sources

Source or site of infection

Enteric tract Primarily within:

Shigella, vibrio, Campylobacter

Both within and outside:

Escherichia, Salmonella

Enteric but outside only:

Klebsiella, Enterobacter, Serratia, Proteus, Providencia, Morganella, Pseudomonas, Bacteroides

Source or site of infection (continued)

Respiratory tract:

Haemophilus, Legionella, Bordetella

Animal sources:

Brucella, Francisella, Pasteurella, Yersinia

Classification Based on morphology, biochemical traits and genetic (phylogenetic) relationship Cocobacilli Vibrionaceae Pseudomonaseae Enterobacteriaceae

Enterobacteriaceae

A heterogenous family

Mostly found in colon of human and other animals

Different pathogenetic mechanisms

Facultative anaerobic

Glucose fermentation

None have cytochrome oxidase Reduce nitrates to nitrites

Most important genuses in Enterobacteriaceae family

Escherichia Shigella Salmonella Klebsiella Enterobacter Serratia Proteus Yersinia

In contrast to Enterobacteriaceae

Pseudomonaceae

Gram negative rods:

 Non-fermenting (Strict aerobic)  Not reduce nitrate  Oxidase-positive

General structure of cells in Enterobacteriaceae

All have

Endotoxin

Some have

Exotoxins

, mostly called

enterotoxins Three surface antigens: O antigen:

outer polysaccharide portion of the lipopolysaccharide (

repeating 3-4 oligosaccharide sugars 15-20 times

). A basis for the serologic typing (about 2000 types of

Salmonella

and 150 types of

E. coli

H antigen:

on the flagellar protein (in

E.coli

and

Salmonella

and not in

Klebsiella

and

shigella

Unusual H antigens in Salmonella

called

phase 1 and phase 2

. The organism can reversibly change in antigenicity to evade the immune response.

K polysaccharide antigen:

In encapsulated organisms such as

Klebsiella. Identified by quellung

(capsular swelling

) reaction

in the presence of

specific antisera

used for epidemiologic purposes. In

S. typhi

, it is called Vi (or virulence) antigen.

Quellung (capsular swelling) reaction

Coliforms

That part of this family which are

normal inhabitants of the colon

E. coli Enterobacter Klebsiella Citrobacter

So,

E.coli

is the indicator for fecal contamination of water supply:

Coliforms

Lactose fermentation, Acid and gas production, growth at 44.5 C and typical colony on EMB. 4 colony count per dL in drinking water is indicative of unacceptable fecal contamination.

Antibiotic therapy

Must be individually tailored to the antibiotic

sensitivity test

(Antibiogram).

Penicillin

and

cephalosporin

families.

Aminoglycosides

(Gentamicin, amikacin, kanamycin, streptomycin …),

Chloramphenicol, tetracyclines, quinolones

and

sulfonamides

Laboratory diagnosis

Culture for isolation

Suspected specimens are inoculated onto 2 media:

1. Blood Agar 2. A selective differential medium

(MacConkey’s agar or Eosin-methylene blue, EMB agar. The differential ability is based on lactose fermentation as the most important criterion in identification of these organism.

Non lactose fermenters form colorless colonies.

Selective effect is exerted by bile salts or bacteriostatic dyes .

Laboratory diagnosis (continued)

Culture for identification Screening biochemical tests

for a final definitive identification: Array of 20 or more biochemical tests to identify the species.

Serology

Usually in

Salmonella, Shigella

and

E.coli the

final detection is by serotyping using agglutination Ag+Ab test.

Triple Sugar Iron Agar

Almost enough to identify the genus

Indicator :

Phenol red

Components:

 Iron or Ferrous sulfate FeSO4 (ferrous sulfate) + Solfate reductase 

SH 2

 Sodium tiosufate Ferrous sulphide (FeS) Black

FeS

indicates the production of

SH 2

 3 sugars: glucose (0.1%), lactose (1%) , and sucrose (1%)  Pepton and yeast extract

Different observations for TSI

Reactions

Slant Acid Alkaline Alkalin Alkalin Button Acid Acid Acid Alkali n Ga s + + H 2 S + Representative genera Escherichia, Enterobacter, Klebsiella Shigella, Serratia Salmonella, Proteus Pseudomonas

Kligler Iron Agar

can be used as alternative with two sugers:

glucose & lactose

Urea Agar

Indicator:

Phenol red

Component :

Urea

If the bacterium Produces urease:

Urea ( NH 2 ) 2 CO NH 3 and CO 2 is hydrolyzed to light orange changes to reddish purple (in Proteus and K pneumoniae

Amonium citrate (Simmons Citrate)

Indicator: Bromothymol blue

If the bacterium can utilizes ammonium dihydrogen phosphate

(a salt of ammonium)

and

sodium citrate

as sole source of nitrogen and carbon, the indicator turns to blue at alkalin pH due to releasing ammonia.

Motility

SIM medium: SH2, Indole, Motility Proteus: Swarms Differentiation between Enterobacter cloacae (motile) from Klebsiella pneumoniae (Non motile)

Indole (in SIM)

Tryptophan +

Tryptophanase

Intermediate products: Indole  Deamination  + ….

Detection: 5 drops Kovac’s reagent (contains para dimethyl-amino-benzaldehyde = PDAB) is added  Benzyl pyrol  Red ring

MR-VP 1. Methyl Red

MR-VP medium Components: Glucose, Phosphate After 48 hours  Methyl-red (1 droplet to 1 ml of medium) : I.

If pH < 4.4  Mixed acidic fermentation (Red color).

II. If pH ≥ 5  Butylen glycol fermentation (Yellow color).

MR-VP 2. Voges Proskauer

Butylen glycol fermentation

MR-VP medium: After 24-48 hours  Alcoholic

alpha naftool

(15 droplets to 1 ml medium) +

KOH

40% (10 droplets) (may come along

cratin

)  15-30 min: Red (If Acetoin “Acetyl methyl carbynol” exists.) II. No color change (If no Acetoin exist)

API 20E system

A plastic strips consist of 20 small wells containing dehydrated media components 1. The bacterium is suspended in sterile saline and added to each well.

2. The strip is incubated for 16-24 hours.

3. The colour reactions are noted as either positive or negative.

4. The test results can be entered into a computer programme to identify the bacterium.

API 20E system

Escherichia coli

Diseases 1. Diarrhea or dysentery

UTI

Sepsis

(The most common cause among negative rods) 4.

Neonatal meningititis

(One of the 2 important agents. The other is the

group B streptococci

due to colonization of vagina by these organisms in about 25% of pregnant women ). 5.

Nosocomial infection

UTI Sepsis Neonatal meningititis

Virolence factors:

Pili Capsule Endotoxin Two exotoxins (enterotoxins).

Pathogenesis

E. coli

attaches to the surface of

jejunum and ileum by Pili

 Bacteria synthesize

enterotoxins

(exotoxins determined by plasmids) 

Diarrhea

The

toxins

are strikingly

cell-specific

. Cells of

colon are lack of receptors

for the

toxins

E. coli pili (fimbriae)

mannose galactose glycolipids glycoproteins 43

Most important sub species of pathogenic E. coli

Enteropathogenic

E. coli

(EPEC) Enterotoxigenic

E. coli

(ETEC) Enteroinvasive

E. coli

(EIEC) Enterohemorrhagic

E. coli

(EHEC)

**Enteropathogenic E. coli (EPEC)**

destruction of surface microvilli Gut lumen fever diarrhea vomiting nausea non-bloody stools Diarrhea is self-limited and short duration (1-3 days) 45

Enterotoxigenic E. coli

Travellers diarrhea Diarrhea like cholera but milder

Diarrhea is self-limited and short duration (1-3 days) 46

**Enterotoxigenic E. coli (ETEC)**

Heat labile toxin (LT)



like choleragen



Activation of Adenylate cyclase



Cyclic AMP concentration



Secretion water/ions (potassium and chloride) Heat stable toxin (ST)



Activation of Guanylate cyclase



Cyclic GMP concentration



Uptake water/ions (Sodium and Chloride)

**Enterohemorrhagic E. coli (EHEC)**

Produce

verotoxin

which works like

Shiga toxin Hemorrhagic



bloody, copious diarrhea



few leukocytes Hemolytic-uremic syndrome



thrombocytopenia (low platelets)



hemolytic anemia



kidney failure

Enterohemorrhagic E. coli

Usually O157:H7 Flagella Transmission electron micrograph 49

**Enteroinvasive E. coli (EIEC)**

Very similar to shigella species (in biochemical and morphological traits) Invades to epithelial mucosal cells Cause enteric inflammation Non lactose fermentative Non motile

E. coli Transmission

By: Meat products or sewage-contaminated vegetables

UTI

The most common agent for UTI and nosocomial UTI. (Cystitis, pyelonephritis): fever, chills, flank pain Occurs primarily in women

Systemic infection

Capsule and endotoxin

play a prominent role Capsular polysaccharide interferes with phagocytosis (Serotype having

causes

neonatal meningitis

LPS

during sepsis causes fever, hypotension and disseminated intravascular coagulation.

Treatment

Antibiogram

for most infections A combination of

ampicillin

and

gentamicin in neonatal meningitis Rehydration

for diarrhea

Prevention

No passive or active immunization

Prompt withdraw of

catheters and intravenous lines Caution regarding uncooked food

and unpurified water while traveling.

Klebsiella

Klebsiella ozaenae

- Ozena (the atrophy of nose with bad smelling)

K. rhinoscleromatis -

Rhinoscleroma (A granulomatosis in nose and pharynx)

Klebsiella penomoniea

- 5% in upper respiratory and GI systems - Nosocomial infection

Klebsiella oxytoca

- Nosocomial infection

Lab detection

Large mocoidal colonies.

Lac pos.

Not motile

Proteus

P. mirabilis

 UTI (Alkalic environment in urinary tracts) due to urease 

kidney stone

P. vulgaris

 Nosocomial infection

Shigella

Species:

S. dysenteriae S. sonnei S. flexeneri S. bouedi

There are more than 40 shigella serotypes.

Important properties

- Non lactose fermenting - Distiguishable from Salmonella by: no gas, no H 2 S, nonmotile.

- Having an enterotoxin called

Shiga toxin

Shigellosis

Only a human disease Transmitted from person to person by asymptomatic carriers (oral-fecal transmission) 4 F’s –

fingers, flies, food, feces Food-born

outbreaks outnumber

water-born

outbreaks by

2 to 1

In mental hospitals

and

day-care nurseries Children <7

accounts for half of shigella positive stool culture

Shigella disentery type 1

(Shiga bacillus): - Labile Exotoxin (effective both on

intestine

and

CNS

works like

verotoxin

E.coli

)

Pathogenesis

Exclusively in

gastrointestinal tract Bloody diarrhea

(dysentery):

Invading

the mucosa of the

distal ileum

and

colon

Local inflammation

accompanied by

ulceration

occurs The organisms

rarely penetrate the wall

enter the bloodstream

unlike salmonellae.

Invasion next cells  microabcess colon and distal ileum   ulcer to epithelial cells (M cells)   bleeding  spreading to formation in the wall of necrosis of mocusal layer psudomemberane .

Clinical findings

Incubation

period:

1-2 days

Symptoms:

Fever, abdominal cramps,

followed by

diarrhea

(watery at first but later contains blood and mucus).

  M

ild or severe

disease depending: - The

The

species of Shigella age

of the patient

Most invasive species

S. dysenteriae

causes the most severe disease

S. sonnei

causes mild disease but more frequent

Shiga toxin neurotoxic effects

Shiga toxin CNS abnormalities can include

lethargy disorientation paralysis coma Neurotoxicity occurs most often in children and the elderly and is often fatal.

Clinical findings

Resolves in 2-3 days

but antibiotic can shorten the course.

Serum

antibodies appear after recovery

but are not protective.

Most patients spread bacteria only short time after recovery but

few people stay chronic carriers

Most shigellosis

cases are in

children less than 10 years

Treatment

50% cases self recovery

is in

2-5 days

The main treatment:

Fluid

and

electrolyte replacement.

No antibiotic in mild cases

Antibiogram test:

Trimethoprim - sulfamethoxazole

Ampiciln

Prevention

Interruption of fecal-oral transmission

by proper sewage disposal,

chlorination

of water and

personal hygiene

No vaccine Antibiotic prophylactic

not recommended

Lab diagnosis

Samples

: feces, rectal swabs

Culture

and

biochemical tests

: Non-lactose fermenter

Serology tests:

Slide agglutination to detect its species

Lab diagnosis

Invasive enteric infections:

Shigella, Salmonella or Campylobacter

Toxin-producing organism:

V. cholerae

E. coli, Clostridium perfringens

or certain viruses or

Entamoeba histolytica

Methylene blue

stain of a

fecal sample

to determine whether

PMNs

are present.

Salmonella

Important properties

Not lactose - fermentive Produce H 2 S, Gas, motile

Salmonella nomenclature

Is complicated.

Currently there are two recognized species:

S. enterica

and

S. bongori

S. enterica

has

5 main subspecies

S. enterica enterica

is relevant with human infections (specific or zoonotic).

Naming the salmonella

S. enterica enterica: 1. S. typhi 2. S. paratyphi (A, B, C…) 3. S. typhimurium 4. S. choleraesuis

5. S. enteritidis (1500 serotypes)

Diseases

Enterocolitis

(

S. typhimurium & S. enteritidis

)

Enteric fever (typhoid fever)

(

S. typhi and S. paratyphi

)

Septicemia with metastatic abscesses

(

S. choleraesuis)

Diseases

Enterocolitis

(

S. typhimurium & S. enteritidis

) An

invasion

epithelial

and

subepithelial tissue

of small and large intestines .

Penetration both through and between the mucosal cells

: Inflammation and diarrhea .

PMN response limits the infection to the gut

and the adjacent

mesenteric lymph nodes

The

dose of Salmonella

required: at least

10 5 - 10 8

while for

Shigella: 10 3

organisms.

Clinical findings of Enterocolitis

Incubation period:

6-48 hours Symptoms:

Nausea, Vomiting, Abdominal pain and diarrhea with or without blood The disease is self-limited. Treatment only in very young and very old.

S. Typhimurium & S. enteritidis

: the

most common cause of enterocolitis.

S. typhimurium colonies

Typhoid or Enteric fever

Typhoid

Enteric fever

caused by

S. typhi

and

paratyphi (

A, B and C) .  The

illness is slow

, with fever and constipation rather than vomiting and diarrhea.

 After the first week,

bacteremia

becomes sustained.

High fever

tender abdomen

, and

enlarged spleen

occur.

Typhoid (Enteric fevers)

Infection

begins in small intestine

but

few gastrointestinal symptoms

occur.

The organisms multiply in the

mononuclear phagocytes

peyer’s patches

, then spread to the

phagocytes of the liver, gallbladder

and

spleen

leading to

bacteremia

and then

fever

Typhoid (continued)

Rose spot

(rose-coloured papules) on the abdomen are associated with typhoid fever but occur only rarely.

The disease begins to

resolve by the third week

but

intestinal hemorrage

perforation

can occur.

3% of typhoid fever patients become chronic carriers

. The

carrier rate is higher among women.

Septicemia

S. choleraesuis

: most often cause septicemia.

Symptoms: 

Fever



Little or no enterocolitis



Focal symptoms

: bone, lung, or meninges.

Septicemia

(

S. choleraesuis)

Accounts for only about

5-10% of Salmonella infections

and occurs: More common in patients with

chronic disease

children with enterocolitis

. It leads to

seeding of many organs

commonly:

osteomyelitis

pneumonia

, and

meningitis

Typhoid vaccine

Two types:  Live vaccine  Subunit vaccine (a vi capsular polysaccharide vaccine)

Transmission of salmonella

Ingestion of

food

and

water

contaminated by

human

and

animal wastes

S. typhi

, transmitted only by

humans

, but other species have a significant animal

reservoir

Human sources:



Temporarily

excrete

the organism during or shortly after enterocolitis 

2. Chronic carriers

Transmission

The most frequent

animal source

poultry and eggs

, but

meat products

that are

inadequately cooked

have been implicated as well.

Dogs and other pets

including

turtles

are additional

sources

Lab. diagnosis

Enterocolitis

: isolated from stool

Enteric fever

: blood culture during first 2 weeks of illness.

Septicemia:

Blood culture

Lab. diagnosis

MacConkey, EMB, Hekton Enteric agar, XLD, Endo agar Lactose -, other biochemical tests Gas and H 2 S (

S. type

: no gas)

Serological tests

by their O, H and Vi

antigens Serological detection

for Ab if culture is negative:

Vidal test

Treatment

Enterocolitis

: 

Self-limited.



Fluid and electrolyte replacement.



Antimicrobial agents

are indicated only for

neonates

or persons with

chronic disease

who are

at risk of septicemia

and

disseminated abscesses

Treatment

Enteric fever and Septicemia: Ampicillin

chloramphenicol



Ampicillin

: in patients who are

chronic carriers

S. typhi



Cholecystectomy

may be necessary to

abolish the chronic carrier state



Focal absesses

should be

drained

Prevention

Public health

and

personal hygiene

measures.

Proper

sewage treatment

chlorinated water

supply

Cultures of stool samples from food handlers

Two

vaccine

confer protection against

S. typhi

but no common

Proper cooking

of poultry and meat

Pasteurization of milk

Genus: Yersinia Species: Yersinia pestis and Yersinia enterocolitica Small

gram negative bacillus,

bipolar

staining (like a safety pin)

Capsule

in freshly isolated organism but lost with passage Plague or black death

None motile

Disease

Virulence factors

Capsule

antigen (F-1) which protects against phagocytosis.

Endotoxin Exotoxins

(block

beta adrenergic receptors

)

Coagulase Fibrinolysin Pesticin I

(a bacteriocin)

antigen protein

Pathogenesis Urban cycle

Transmission of the bacteria among

urban rats

with the

rat flea

as vector to human.

This cycle predominates during times of

poor sanitation

. Wartime, when rats proliferate and come in contact with the fleas in the sylvatic cycle.

Sylvatic cycle Humans are accidental hosts

and cases of plague occur as a result of being bitten by a flea that is a part of the sylvatic cycle.

Event within the flea

The flea ingests the bacteria while taking a blood meal from a bacteremic rodent.  The blood clots in the flea’s stomach owing to the action of the enzyme coagulase made by the bacteria  The bacteria are trapped in the fibrin and proliferate to large numbers.  The mass of organisms and fibrin block the proventriculus of the flea’s intestinal tract. During its next blood meal the flea regurgitates the organisms into the next animal  Because the proventriculus is blocked, the flea gets no nutrition. 

becomes hungrier



loses its natural host selectivity for rodents human

 

more readily bites a

The bacteria inoculated by bite spread to the

regional lymph nodes

 become

swollen and tender called buboes

and this plague is called

bubonic plague

.  The organisms can reach high

concentrations in the blood

and disseminate to form

abscesses in many organs including lungs

.  The endotoxin -related symptoms, including

disseminated intravascular coagulation

and

cutaneous hemorrhages

, probably were the genesis of the term ‘ black death ’.

Respiratory droplet transmission

Respiratory droplet transmission of the organism from patients with pneumonic plague can occur.

Clinical findings

Bubonic plague

, is the most frequent form, begins with

pain and swelling of the lymph nodes draining

the site of the flea bite and systemic symptoms such as high

fever

myalgia

, and

prostration

The

buboes

are an

early characteristic

finding.

Septic shock

and

pneumonia

are the main life threatening subsequent events.

Epidemiology

Endemic in the wild rodents

of Europe and Asia for thousands of years.

99%

of cases of plague occur in

Southeast Asia

Enzootic (sylvatic)

cycle consists of transmission among

wild rodent by fleas

Rodents are relatively resistant

to disease.

Humans are accidental hosts

and cases of plague occur as a result of being bitten by a flea that is a part of the sylvatic cycle.

Pneumonic plague

can arise either from

inhalation of an aerosol

or from

septic emboli

that

reach the lung

Untreated bubonic

plague is

fatal

in approximately

half of the cases

Untreated pneumonic

plague is

invariably fatal

Lab. diagnosis

The best procedure:

Smear and culture of blood or pus from the bubo.

Giemsa’s or Wayson’s stain

reveals the typical safety-pin appearance of the organism better than does Gram’s stain.

Fluorescent-antibody staining

can be used to identify the organism in tissues.

rise in antibody titer

to the capsule antigen can be useful.

Treatment

The treatment of choice is a

combination

streptomycin and tetracycline

Due to the rapid progression of the disease,

treatment should not wait for the results of the bacteriologic culture

Incision and drainage

of the buboes are

not usually necessary.

Prevention

Prevention Controlling the spread of rats

in urban areas.

Preventing rats from entering the country

by ship or airplane.

Avoiding flea bites Avoiding contact with dead wild rodents

A patient with plague must be placed in strict isolation

(quarantine) for

72 hours after antibiotic therapy

is started.

Only

close contacts need to receive prophylactic tetracycline

. There is

no vaccine

for citizens normally. But a

killed organism vaccine protecting bubonic but not pneumonic plague was used by USA forces

during Vietnam wars.

Yersinia enterocolitica

Motile: flagella are present at 22 c.

No capsule Primarily a zoonotic disease (cattle, deer, pigs, and birds)

Yersiniosis : Incubation 1-2 days

. Acute

Y. enterocolitica

infections produce severe diarrhea in humans, along with Peyer's patch necrosis, chronic lymphadenopathy , and hepatic or splenic abscesses. Fever and right-sided abdominal pain.

Lab diagnosis

Culture: Cold enrichment Culture on blood Sample: Feces

Y. pseudotuberculosis

Motile: flagella are present at 22 c., No capsule. Culture on blood.

Primarily a zoonotic disease (cattle, deer, pigs, and birds).

In animals, can cause tuberculosis -like symptoms , including localized tissue necrosis and granulomas in the spleen , liver , and lymph node .

In humans, symptoms of Pseudotuberculosis (Yersinia) are similar to those of infection with

Yersinia enterocolitica

(fever and right-sided abdominal pain), except that the diarrhea is often absent.