Gram Negative Bacilli

Gram Stain

Gram Negative Bacilli

Introduction to Disease Processes
Core
Fall 2006

images.MD

S. James Booth

Gram Negative Bacilli

Gram Negative Bacilli

Facultative Anaerobic Rods

Others:
Enterobacteriaceae
(not Enterobacteriaceae)
Escherichia
Salmonella
Vibrio
Aeromonas
Shigella
Haemophilus
Klebsiella
Enterobacter
Pasteurella
Francisella
Yersinia
Proteus
Others: Serratia,
Hafnia, Citrobacter,
etc.

Aerobic Rods
Pseudomonas
Others: Stenotrophomonas, etc.
Burkholderia
Legionella
Brucella
Bordetella
Bartonella

S. James Booth

S. James Booth

Gram Negative Bacilli

Gram Negative Bacilli

Microaerophilic rods

Anaerobic rods
(Separate Lecture)

Campylobacter
Helicobacter

Bacteroides
Porphyromonas
Prevotella
Fusobacterium

S. James Booth

S. James Booth

Gram Stain
Escherichia coli

Enterobacteriaceae
Facultative Anaerobic Rods

Escherichia
Salmonella
Shigella
Klebsiella
Enterobacter
Yersinia
Proteus
Others: Serratia, Edwardsiella, Providencia, Morganella,
Citrobacter, ~40 more
http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Gram_Stain/Gram_stain_images/index_gram_stain_images.html

S. James Booth

Gram Negative Rod

Enterobacteriaceae: Characteristics
Antigens
Cell wall (O Ag)
Flagella (H Ag)
Capsule (K, Vi)

Habitat
Normal GI flora (humans/animal)

oxidase positive

Pseudomonas
aeruginosa
Salmonella
Shigella

Physiology

Facultative anaerobes
Ferment glucose
Oxidase negative
Reduce nitrate

Escherichia coli

glucose fermentation; nitrate reduction

Salmonella
Shigella
E. coli
others

- +

Pseudomonas
others

Shigella

H2 S

lactose fermentation

- +
others

E. coli
others

- indole +

E.coli

Salmonella

S. James Booth

Lactose-Positive Colonies
on MacConkey Agar
S. James Booth

Red/pink Colonies

http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Gram_Stain/Gram_stain_images/index_gram_stain_images.html

http://www.rmit.edu.au/browse/About%20RMIT%2FHelp%2FMedia%20Assets%2FImage%2Fby%20subtype%2FPhotograph%20of%20culture%2F;ID=smyfchkof1ej1.JPG;STATUS=A

Indole Test
E. coli

E. coli Infections
Urinary tract
Neonatal meningitis

Neg

Gastrointestinal

Pos
Red ring

Sepsis with all of above + others

S. James Booth

Urinary Tract Infections

UTI

Cystitis (bladder):
Dysuria, frequency of urination, urgency

Epidemiology

Pyelonephritis (kidney):

Virulence

Community acquired
Nosocomial
P (pyelonephritis-associated) pili

Fever, chills, flank pain, dysuria,

nausea, vomiting

Diagnosis
Culture
Quantitation
S. James Booth

Urine Culture:
Pour Plate for Quantitation
>100,000 colony
forming units/mL of
urine in asymptomatic
patient is diagnostic;
Pregnant patients should
be treated for UTI;
elderly should not

Urine Culture:
Volumetric Loop

Loop designed to pick up

1/1000 of a mL; thus
100 colonies on a plate
corresponds to 100,000 (105)
bacteria per mL of urine
S. James Booth

http://gold.aecom.yu.edu/id/micro/index.html

E. coli GI Infections
Enterotoxigenic E. coli

(All food/water/person-to-person [fecal/oral])

Enterotoxigenic E. coli (ETEC)

Travelers; infants

Travelers/Infant diarrhea
Enteroaggregative E. coli also common cause of
travelers diarrhea

Enterohemorrhagic E. coli (EHEC)

Hemorrhagic colitis or hemolytic uremic
syndrome (HUS)
Enteropathogenic E. coli (EPEC; childhood diarrhea)
Enteroinvasive E. coli (EIEC; bacillary dysentery)
Enteroaggregative E. coli (EAggEC; travelers diarrhea;
childhood diarrhea)
Enteroadherent E. coli (EAEC; travelers diarrhea;
childhood diarrhea)

ETEC Heat Labile Toxin (LT)

Mucosal Cell
Reg
protein

adenylate
cyclase

Gm1 ganglioside
receptor

Heat-labile (LT): cAMP (fluid excretion)

Heat-stable (ST): cGMP (prevents re-absorption)
S. James Booth

Enterohemorrhagic E. coli
(EHEC)
Shiga toxin (Verotoxin; lysogenic)

cAMP

ADP-ribose

A subunit (1)

Watery, mild cramps

Plasmid-encoded virulence factors: Pili & Toxins
Two enterotoxins

Inhibition of protein synthesis

Secreted by quorum sensing regulated
type III secretion system

Cl2 + H2O

B subunits (5)
S. James Booth

S. James Booth

Enterohemorrhagic E. coli

EHEC Disease

Epidemiology

Hemorrhagic colitis

Food-borne & person-to-person

Classically: hamburger in fast-food
restaurants
Currently: hamburger, raw veggies,
unpasteurized juices/milk, etc
Serotype O157:H7, others

Abdominal cramps/watery to bloody

diarrhea
No fever or pus
Possible progression to HUS
Hemolytic anemia
Thrombotic thrombocytopenia (TTP)
Renal failure

>2005: U.S. ~ 3,500 cases; NE ~ 60

Category B agent

Up to 5% mortality
S. James Booth

S. James Booth

Update on Multi-State Outbreak of

E. coli O157:H7 Infections From
Fresh Spinach, September 19, 2006
131 persons infected from 21 states
Among ill persons:

50% were hospitalized

15% developed kidney failure (HUS)
One adult died
Ninety-six (73%) were female
5% were children under age 5

Be aware of:
Enteroinvasive E. coli (EIEC; bacillary dysentery)
Enteropathogenic E. coli (EPEC; childhood
diarrhea)
Enteroaggregative E. coli (EAggEC; travelers
diarrhea; childhood diarrhea)
Enteroadherent E. coli (EAEC; travelers diarrhea;
childhood diarrhea)

S. James Booth

S. James Booth

E. coli Gastroenteritis

E. coli Gastroenteritis
Therapy

Laboratory diagnosis

Travelers (ETEC): re-hydration (e.g., chicken soup)

Enterotoxigenic (ETEC): not done

Enterohemorrhagic (EHEC)

Mild/moderate: loperamide; bismuth subsalicylate (or

others)
[modified from handout] Try to avoid antibiotics, but

Culture: Sorbitol-MacConkey Agar

Colorless: O157 (cant ferment sorbitol)

Severe: A fluoroquinolone (azithromycin if resistant)

Rifaximin (rifampin derivative)

Enterohemorrhagic (EHEC):
Re-hydration
role of antibiotics uncertain
http://www.ukneqasmicro.org.uk/pics1661.html

S. James Booth

S. James Booth

Lactose-Positive Colonies
on MacConkey Agar

Neonatal Meningitis
Escherichia coli ~29%
Streptococcus agalactiae ~ 34%
Signs/Symptoms

Red colonies

Respiratory distress, fever, irritability, failure to eat,

lethargy

S. James Booth

S. James Booth

Gram Stain
Shigella

Review: E. coli Therapy

Except for UTI (TMP/SMX), do not need to

know antimicrobials
Most GI: fluids
HUS: Controversial
Severe sepsis: (dont memorize) a cephalosporin
(cefotaxime, ceftriaxone, cefepime, ceftazidime)
or a fluoroquinolone
S. James Booth

Lactose-Negative colonies on
MacConkey Agar

Nonmotile Enterobacteriaceae

Colorless
(non-red)
colonies

Shigella
Yersinia

SYK

Klebsiella
S. James Booth

Note: Most cocci are also nonmotile

S. James Booth

August 11, 2006

Shigella

S. dysenteriae: Least common in U.S. (<1%)

S. sonnei: Most common in U.S. (~70-75%)
S. flexneri: (20 25%)
S. boydii: (~2%)

Shigella happens
By MARK ANDERSEN / Lincoln Journal Star
More than 30 probable cases of shigella, the parasitic
infection associated with prolonged and often
severe diarrhea, have been identified in Lancaster
County over the past two weeks.

Note: 160 confirmed cases in Nebraska in 2005

>16,000 U.S. cases

S. James Booth

Shigellosis

Shigellosis

Person to person
Low infecting dose
Shiga toxin (lysogenic)
Watery bloody

Sweden

Fever, cramps
PMNs, blood, and mucus in stool
No bacteremia

Treatment
Self-limiting, but
Fluids
Antibiotics to decrease duration of illness & person-to-person spread

Journal of Infection Volume 51, Issue 3 , October 2005, Pages 222-229

Shigellosis risk per 100,000 travellers in different regions of world

S. James Booth

Gram Stain
Salmonella

Salmonella Taxonomy
Confusing!
CDC and WHO
2 species

S. James Booth

S. enterica: 7 subspecies; > 2400 serovars

subspecies enterica: ~99% of all human pathogens
S. enterica subsp. enterica serovar Typhimurium
Classic (historical): S. typhimurium
S. enterica subsp. enterica serovar Enteritidis
Classic: S. enteritidis
S. enterica subsp. enterica serovar Typhi, etc.
Classic: S. typhi
S. bongori
Disease in cold-blooded animals

Salmonellosis
Two gastrointestinal forms
Gastroenteritis
Localized
S. enterica: S. typhimurium, S. enteritidis
> 2400 other serotypes (O, H, Vi antigens)
Animal hosts

Enteric (typhoid/paratyphoid) fever

Bacteremic (invasive)
Salmonella typhi (S. paratyphi; others)
Human host

Salmonella Gastroenteritis
(salmonellosis; enterocolitis; enteritis)

Ingestion of contaminated food or contact

w/infected animals
Poultry, swine, cattle, rodents, and infected
humans
Raw milk/raw milk products, undercooked or raw
eggs/ egg products, meat and meat products,
contaminated water

Pets: turtles, tortoises, iguanas, chicks, dogs, cats

Salmonella Gastroenteritis
Most Common Serotypes

Enterocolitis Pathophysiology
Infectious dose > 100,000
Localized invasion of epithelium of small/large
intestines
Infection limited to lamina propria/adjacent mesenteric
lymph nodes
Results in inflammation and watery diarrhea
Bacteremia rare (infants, immunosuppressed)

EID: Volume 12, Number 3, March 2006

Salmonella Enterocolitis
Incubation period: 8-48 hours (1-2 days)
Nausea, vomiting, progressing to abdominal
cramping and diarrhea

Enterocolitis
Diagnosis/Therapy/Prevention
Stool Culture
Lactose neg, H2S +
Serogrouping: In U.S., serogroups B, C, D responsible for most
infections
S. enteritidis and S. typhi are serogroup D

Self-limiting

may be bloody

Fluids/electrolytes
Antimicrobials for immunosuppressed, young children, elderly

Usually self-limiting (5-7 days)

Sanitation
Epidemiology: 2005 U.S. ~ 45,000 cases
NE ~ 219 cases

MacConkey Agar
Salmonella

Triple Sugar Iron (TSI)

Black = H2S
positive

S. James Booth

Typhoid Fever
(enteric fever)

Human-to-human
Contaminated food/beverages handled by persons
shedding S typhi from stools (less commonly
urine) or water from sewage contaminated with
S typhi
Shellfish from water polluted by raw sewage
Canned meat produced using faulty canning
processes may cause outbreaks

Typhoid Fever: Pathophysiology

Small Intestine
Infection of M cells of Peyer patches
Replication, spread to other phagocytic cells
Systemic spread
Organs, esp. reticuloendothelial tissues of liver,
spleen, bone marrow, gallbladder, Peyers patches (reinfection)

Carrier state ~3%

http://sprojects.mmi.mcgill.ca/tropmed/disease/typhoid/geo.htm

Peyer Patches

Typhoid Fever
Incubation period: 1-2 weeks
Fever usually begins 1 week postexposure

http://www.bu.edu/histology/p/12006loa.htm

Malaise, anorexia, myalgias, arthralgia, cough,

sore throat, and headache
Mental confusion
Relative bradycardia
Not as tachycardic as expected for degree of fever

Rose spots
may appear
typically on anterior chest wall
http://www.rcai.riken.go.jp/eng/group/epi/image/01.jpg

Rose Spots

Typhoid Fever
Diagnosis
Culture: blood or bone marrow
Serology: Widal test (agglutination)
DNA probes/PCR

Therapy
A fluoroquinolone or ceftriaxone
Use of antimicrobials mortality from ~20% to near 0

Prevention
http://www.medicalhealthcareinfo.com/content/Symptoms_of_typhoid_fever_may_1.php
http://www.mmc.org.au/image.php?id=000031

Sanitation
Vaccination (high risk travel)
Control of carriers

Epidemiology: U.S. 2005 ~ 320 cases

Gram Stain
Klebsiella pneumoniae

Mucoid Colonies
Klebsiella pneumoniae

S. James Booth
http://www.austin.cc.tx.us/microbugz/03morphology.html

Source unknown

Pneumonia

Diseases: K. pneumoniae

Lobar pneumonia

Lobar pneumonia & bronchopneumonia (next

slide)

Affects large area or most of lobe

Often Klebsiella or pneumococcus

Bronchopneumonia

Urinary tract infections

Other opportunistic: burns, wound, etc.

Affects patches throughout one or (frequently) both lungs

CAP: pneumococcus; Immunosuppressed: Klebsiella, others
Right lobar pneumonia

S. James Booth

Common Signs and Symptoms

of Acute Bacterial Pneumonia
In Children and Adults

Bronchopneumonia

MedPix
http://www.studentbmj.com/issues/00/02/education/13.php

http://rad.usuhs.mil/medpix/medpix_home.html?#top

Common Causes of Pneumonia

in Alcoholics
Klebsiella pneumoniae
Anaerobes
Occasionally Haemophilus influenzae

Shaking chills
Fever
Chest pain
Cough (often productive)
Shortness of breath (dyspnea)

S. James Booth

10

Urease

K. pneumoniae
Epidemiology: NF
Virulence factors: Capsule
Signs/Symptoms

O
H2N C NH2

Pneumonia: bloody sputum (currant jelly)

urease

NH4+ + CO2

UTI: typical +/- stones (urease +)

Bacteremia
# 2 behind E. coli in nosocomial bacteremia

http://www.foodsubs.com/Fruitpre.html

Therapy: dont need to know (same as E. coli)

S. James Booth

Urease Test

Enterobacter
Nosocomial UTIs
Immunocompromised
Multiple antimicrobial resistance common
Dont learn therapy

Negative

Positive

S. James Booth

S. James Booth

Gram Stain
Proteus

Proteus

Swarming
Urease +
P. mirabilis (most common)
UTI (#2 most common; usually nosocomial)
Therapy: dont need to know (ampicillin)

S. James Booth
S. James Booth

11

Swarming: Proteus

Swarming: Proteus
Note rings

Note rings

Swarming is due to bacterial differentiation into

hyper-flagellated swarmer cells followed by
de-differentiation into dividing non-swarming
vegetative cells. Rings appear after several cycles.
S. James Booth
S. James Booth

Swarming: Proteus

Yersinia

Note rings

Y. enterocolitica
Enterocolitis (especially pediatric)
Watery diarrhea

Enteric fever (mild)

Y. pestis
Plague
Black Board: See handout for learning objectives
S. James Booth
S. James Booth

Gram Stain
Vibrio cholerae

Vibrio cholerae
Electron Micrograph

Note
curved
rods

Single polar
flagellum
Curved
rod
S. James Booth

Source unknown

12

Vibrio cholerae
Human to human
Enterotoxin: similar m.o.a. as LT (E. coli)
8 in cAMP 8 fluid/electrolyte secretion

Watery diarrhea (abdominal cramps, vomiting, nausea)

Severity is variable
Rice-water stools

Vibrio parahaemolyticus
Sea food associated gastroenteritis
Occasionally V. vulnificus

Vibrio vulnificus
Sea water associated wounds

(also with rotavirus & arsenic poisoning)

Dehydration (severe can lead to rapid death)

Fluids/electrolytes (primary tx)

Vibrio: Necrotizing Fasciitis

Occasionally V. parahaemolyticus

Note: Both are halophiles

http://www.visualdx.com/vibrio/

Aeromonas
Gram-negative facultative anaerobe
A. caviae, A. hydrophila, A. schubertii, A.
veronii, others

Fresh/brackish water; soil

Gastroenteritis
Usually mild diarrhea
Acute enteritis in AIDS

Wound infections
doi:10.1016/j.injury.2005.05.016

Quad Plate Haemophilus influenzae

Haemophilus
H. influenzae
Encapsulated: meningitis
Nonencapsulated: conjunctivitis, otitis media,
sinusitis

H. ducreyi
Chancroid

http://www.who.int/csr/resources/publications/drugresist/en/IAMRmanual.pdf

13

Haemophilus influenzae Meningitis

Chocolate Agar
Haemophilus influenzae
Supplemented
with X and V

Rapid onset: Fever, headache, stiff neck,

irritability, vomiting
Neurologic sequelae
Hearing loss, mental retardation, convulsions, etc.

S. James Booth

Quad Plate
Haemophilus influenzae

H. Influenzae
will grow only
in this quadrant

X+V

Haemophilus influenzae

S. James Booth

Haemophilus influenzae
Other diseases: Hib (encapsulated)
Acute epiglottitis
Septic arthritis (< 2 y.o.)
Cellulitis (<2 y.o.)

Nonencapsulated (non-typable)

http://education.med.nyu.edu/courses/microbiology/courseware/infect-disease/Gram_Neg_Bacilli5.html

Haemophilus ducreyi
Chancroid
A sexually transmitted disease characterized by
painful genital ulceration & inflammatory inguinal
adenopathy
2005: U.S. 17 cases

Conjunctivitis
Otitis media: #2-3; St. pneumo. #1; Staph. aureus #2-3)
Sinusitis: H. influ & St. pneumo.

14

Pasteurella multocida

Cat Bite: Pasteurella multocida

Cellulitis assoc. with animal bites/scratches

esp. cats

http://knowledge.emedicine.com/splash/shared/pub/cotw/0014answer.html

Francisella tularensis
Tularemia
Black Board: See handout for learning
objectives

15