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Gram Negative Stains and Infections
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PRESENTED BY: BRYANNA GRAY
CONTACT INFORMATION:
240-441-9227
[email protected]
Bacterial Cell Wall
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Bacterial cells are covered by a cell
envelope that is composed of a cell
membrane and a cell wall. The cell
membrane is a phospholipid bilayer that
regulates the transport of molecules into and
out of the cell. This is the weak structure
that would burst from the osmotic pressure
without reinforcement.
3 Primary Functions:
 Provide Semi-Permeable Barrier
Prevent loss of water
Desirable substance passage
 Protect against changes in osmotic
pressure
Main homeostasis and prevent bursting
 Prevent Digestion by the host enzyme.
FUN FACT
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• The chemical composition of the bacterial cell wall
is dramatically different from the mammalian cell
wall and this provides a number of potentially
attractive targets for the selective chemotherapy of
the bacterial infections.
GRAM STAINING
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CLASSIFYING BACTERIUM
Gram Staining
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Definition: a staining technique for the preliminary
identification of bacteria, in which a violet dye(crystal
violet) is applied, followed by a decolorizing
agent(grams alcohol or ethanol/acetone) and then a
red dye(safranin). The cell walls of certain bacteria
(denoted Gram-positive ) retain the first dye and
appear violet, while those that lose it (denoted Gramnegative ) appear red. Also called Gram's method.
Inventor: Hans Christian Gram
Purpose of Conducting a Gram Stain:
Helps determine the identity of bacterial sample
4 Steps to Gram Staining
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① Pour on crystal violet stain (a blue dye) and wait
60 seconds.
② Wash off with water and flood with iodine
solution.Wait 60 seconds.
③ Wash off with water and then "decolorize" with
95% alcohol.
④ Finally, counter-stain with safranin (a red dye).
Wait 30 seconds and wash off with water.
The Result of Gram Staining
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Gram Negative Stain Explanation
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 Crystal violet attaches to lipopolysaccharide layer
Lipopolysaccharide: (LPS), also known as lipoglycans and
endotoxins, are large molecules consisting of a lipid and a polysaccharide
composed of O-antigen, outer core and inner core joined by a covalent
bond they are found in the outer membrane of Gram-negative bacteria
and elicit strong immune responses in animals.
 Iodine acts as mordant
 Grams alcohol washes away LPS layer & with it the stain
 Safranin stains peptidoglycan
Outermost layer
lipopolysaccharide layer
peptidoglycan layer
Phospholipid bilayer
cytoplasm
Gram Negative Slide
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Review of Lab
Test/Classifications
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OXIDASE TEST
 OXYGEN CLASSIFICATIONS
 CATALASE TEST

Oxidase Test
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 The oxidase test is used to identify bacteria that
produce cytochrome c oxidase, an enzyme of the
bacterial electron transport chain. Bacteria that are
oxidase positive are aerobic, and can use oxygen as a
terminal electron acceptor in respiration.
Oxygen Classifications
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Catalase Test
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 The catalase test is used to differentiate
staphylococci (catalase-positive) from streptococci
(catalase-negative). The presence of catalase
enzyme in the test isolate is detected using hydrogen
peroxide. If the bacteria possess catalase
(catalase-positive), when hydrogen peroxide is
added bubbles of oxygen are observed.
Gram Negative Infections
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ENTEROBACTERIACEAE
GENUS
Enterobacteriaceae Highlighted
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Characteristics of Enterobacteriacea
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 Gram Negative Bacilli
 Facultative anaerobes
 Ubiquitous
-Except for few, most are present in the intestinal tract
of animals and humans as commensal flora; therefore,
they are sometimes called “fecal coliforms”
-Some live in water, soil and sewage
Biochemical Properties include:
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① Oxidase Negative
② Catalase Positive
③ Ferment glucose (dextrose)
④ Reduces nitrates to nitrites
Entry of Enterobacteriaceae
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 Contaminated medical devices
 Surgical site infections
 Intravenous (IV)
 Urinary Catheters
 Colonization of colon, perineum, urethra
 Pneumonias
 Contaminated food
**Large numbers usually needed**
Evasion
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 Capsule
 Anti-phagocytic
 Antigenic phase variation
 Altered expression of capsule flagella
 Plasmids
 Anti-microbial resistance
Enterobacteriaceae Infections:
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① E.Coli: Gastroenteritis
② Salmonella:a bacterium
Escherichia
coli (E.
coli)
that occurs mainly in the
intestine, especially a
serotype causing food
poisoning
Enterics
Salmonella
③ Shigella:a bacterium that
Shigella
is an intestinal pathogen
of humans and other
primates, some kinds of
which cause dysentery
Escherichia coli
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 Most significant species in
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the genus
Natural inhabitant of the
GI tract
Common isolate from
colon flora
Ferments glucose(+) and
lactose (+)
Positive indole and methyl
red tests
Usually motile
Green Metallic Sheen on
EMB agar
E.Coli Extraintestinal Infections
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 Urinary Tract Infection
 Pulmonary infection
 Bacteremia
 Meningitis
E.Coli Intestinal Infections
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•
•
•
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Diarrhea
Hemolytic-uremic syndrome
Dysentery
Groups –based on how they cause illness
ETEC -Enterotoxigenic
EPEC -Enteropathogenic
EHEC -Enterohemorrhagic
EIEC -Enteroinvasive
Enterotoxigenic E.coli (ETEC)
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 Enterotoxigenic (ETEC) – “traveler’s diarrhea”; watery diarrhea
without blood, nausea and vomiting; self-limiting; usually not
identified, other than patient history and lactose-positive organisms
cultured on differential media
 Colonization Factor Antigens CFA/I,CFA/II
-Facilitates bacteria adhesion
 Entertoxins
-Heat Labile
-Heat Stable
Heat Labile
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 In medicine, the term "labile" means susceptible to
alteration or destruction. For example, a heatlabile protein is one that can be changed or
destroyed at high temperatures. The opposite of
labile in this context is "stable.
 Heat Labile A-B Toxin
Heat Stable
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 Heat-stable enterotoxins (STs) are secretory
peptides produced by some bacterial strains, such as
enterotoxigenic Escherichia coli which are in general
toxic to animals. These peptides keep their 3D
structure and remain active at temperatures as high
as 100 °C.
Enteropathogenic E.Coli (EPEC)
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 Strain of Escherichia coli in which organisms adhere to
small bowel mucosa and produce characteristic changes in
the microvilli.
 This strain produces symptomatic, sometimes serious,
gastrointestinal illnesses, especially severe in neonates and
young children;
 Responsible for Pediatric Diarrhea (symptoms: N/V, fever,
watery diarrhea)
 Entertoxins:
-Heat Labile
-Heat Stable
EPEC in small intestines
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Pathogenic Mechanism
① Attaching and effacing
②
③
④
⑤
lesions
Microbes bind to the
epithelial cells via
bundle-forming pilli
(BFP)
Loose, then tight
binding
Effacement lesion
Associated pedestal
formation
Enteroinvasive E.Coli (EIEC)
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EIEC characteristics:
 Dysentery with bowel penetration
 Invasion and destruction of intestinal mucosa
 Watery diarrhea with blood
 Does NOT ferment lactose
 Identified via DNA probes
Enteroinvasive E.Coli (EIEC)
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Pathogenic Mechanism
① Invades intestinal
epithelial cells
② Lyses the phagosomal
vacuole
③ Moves through the
cytoplasm
④ Spreads to adjacent
cells
Enterohemorrhagic E.Coli (EHEC)
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 Enterohemorrhagic (EHEC serotype 0157:H7)
 Associated with dysentery, hemorrhagic colitis, hemorrhagic diarrhea
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
and hemolytic-uremic syndrome (HUS), which includes low platelet
count, hemolytic anemia, and acute renal failure
Symptoms: Diarrhea and Bloody Diarrhea
Potentially fatal, especially in young children
Undercooked hamburger, unpasteurized milk and apple cider have
spread the infection
Does NOT ferment sucrose
Identified by serotyping
Enterohemorrhagic E.Coli (EHEC)
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Pathogenic Mechanism
 Vero Toxin/ Shiga-like
toxin
 AB Toxin-inhibits
protein synthesis
(similar to Shiga toxin)
 Acid Resistant
 Low infectious dose
Escherichia coli TREATMENT
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GI Infections
 Mainly Rehydration Therapy
 Antibiotics & Antidiarrheals are not recommended
 Most people recover within 5 - 10 days w/o medicine
UTI and Systemic Infections- use antibiotics
 Sulfamethoxazole-Trimethoprim(Bactrim)
 Nitrofurantoin (Macrobid, Furadantin,
Macrodantin)
Salmonella sp.
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 Rod-shaped bacilli
 Lactose (-)
 H2S (+)
 Two Species of interest:
S. typhi and S. typhosa
 May enter the digestive
tract of humans/other
mammals in contaminated
food and cause abdominal
pains and violent diarrhea
 Appears grayish on EMB
agar.
Salmonella sp, Effects
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Diseases
Gastroenteritis
Septicemia
Enteric Fever
Carrier State
Symptoms
Fever
Abdominal cramps
Dysentery (blood diarrhea)
Salmonella sp. Pathogenic Effects
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 Gastroenteritis
Adhesion to microvilli
2.
Cellular invasion  Survival in vesicle
3.
Multiplication  ESCAPE
 Septicemia (all of the above +)
4.
Silent Septicemia  Uptake via Macrophages
5.
Survival in Macrophages vesicle  SYSTEMIC
 Enteric Fever (all of the above +)
6. Muliplication in lymph nodes, spleen, liver macrophages
7. Bacteremia release from macrophages to blood
8. High Fever (Endotoxin)
9. Reinvasion of intestine via gall bladder
 Carrier State
10. Established gall bladder infection
1.
Salmonella sp. Treatment
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 Self-limiting
 Hydration
 Usually resolves in 5-7 days
 Systemic Infection
 Ampicillin
 Sulfamethoxazole-Trimethoprim (Bactrim)
 Ciprofloxacin
Bactrim MOA
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Shigella sp.
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 Lactose (-)
 H2S (-)
 Non-motile
 Three Species of interest:
S. dysenteriae, S. sonnei &
S. fexneri
 Symptoms: Fever,
abdominal cramps,
dysentery.
 Presentation is similar to
Salmonella…WHY?
Shigella Entry
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 Fecal-oral transmission
 Acid Resistant- small numbers needed
Shigella Evasion
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 Cellular invasion-escape from endocytic vesicle,
multiplication in the cytoplasm  Spread to adjacent
cell
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Shigella Treatment
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Self-limiting
 Usually resolves within 5-7 days
 Hydration
 Ampicillin, Bactrim and Ciprofloxacin can be used
Gram Negative Infections
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NEISSERIA
GENUS
Neisseria Highlighted
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Neisseria Gonorrhoeae
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 Gram – diplococci
 Aerobic
 Cytochrome oxidase (+)
 Maltose (-)
 N.Gonorrhoeae:
Gonorrhea oldest STI,
venereal disease
involving inflammatory
discharge from the
urethra or vagina
Entry
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 Infects mucosal surfaces
 Ex. cervix, urethra, rectum, oropharynx,
nasopharynx, conjunctiva
Evasion
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 Capsule (antiphagocytic)
N. Gonorrhea Pathological Effects
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Gonorrhea
Symptomatic or asymptomatic
Transmission
Sexual Contact
Newborns
N. Gonorrhea Pathological Effects (continued)
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Men
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Asymptomatic infection
Acute urethritis
Purulent exudate w/
dysuria, polyuria, HA, fever
Anterior to posterior
urethral infection
Glands, ducts, and vesicles
may become infected
Chronic infection
Prostate, seminal vesicles,
and epididymitis may become
infected
Women
 Asymptomatic carriers
 Acute gonorrhea infection (lower
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tract)
Endocervix (traditional site of
infection)
Acute symptoms
Ab/pelvic pain, vaginal
discharge/dysuria
Chronic Gonorrhea infection
Tenderness in lower abdomen,
inflammation of the urinary tract
Pelvic Inflammatory Disease
(PID)
N. gonorrhoeae Extragenital Infections
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

Local infection based on site of contact:
-Conjunctivitis
-Ophthalmia neonatorum (newborn)
Pharyngitis or Proctitis
- Either may be transmitted sexually
Symptoms
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 Fever, lower abdomen pain, increased risk of ectopic
pregnancy

Sites of infection

Endometrium (endometritis), fallopian tubes
(salpingitis)
N. gonorrhoeae Laboratory Diagnosis
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①
②
③
④
Smear of urethral pus with gram (-) diplococcus
Carbohydrate utilization (glucose fermenter)
Direct Fluorescence Antibody Test
ELISA
N.Gonorrhoeae Treatment and Resistance
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Treatment options:
① Cephalosporins(Ceftriaxone)
Preferred agent for most N. gonorrhoeae infections
②
Fluoroquinolones (Ciprofloxacin, levofloxacin)
Resistance:
Resistance to antibiotics exists and is rapidly emerging
Examples include:
①
Penicillinase Producing Neisseria Gonorrhoeae
(PPNG )
②
Increasing Fluoroquinolones resistance limits use
Neisseria meningitidis
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Characteristics
Gram (-) diplococci
Oxidase (+)
Aerobic
Glucose & Maltose (+)
Meningococcal disease is a
contagious infection spread
by close contact with an
infected person, such as
living together or kissing.
 Grows on chocolate agar
with Co2 production
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N. meningitidis Pathogenic Mechanism
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Evasion

Invasion of epithelial cell  followed by invasion of blood
stream and systemic spread

Anti-phagocytic capsule

Facultative intracellular parasite
Adherence

Pilin–initial adhesion

Opa–firm adhesion
Pathological Effects

Lipooligosaccharide(LOS)  endotoxin

Sequestration of iron from host

IgA protease-Destroys soluble IgA

Beta-lactamase-Inactivates beta-lactam antibiotics
N. meningitidis Symptoms
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 Common symptoms: sudden fever, HA, and stiff neck.
 Other symptoms :n/v, increased sensitivity to light, and


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
confusion.
Children and infants may show inactivity, irritability,
vomiting, or poor reflexes.
Sequelae: deafness, mental retardation, behavior defects.
Meningococcal disease can also cause an infection of the
blood  tiredness, vomiting, cold hands and feet, chills,
severe aches and pain, fast breathing, diarrhea, and a
dark purple rash.
Meningococcal disease is very serious and can be
fatal. In fatal cases, deaths can occur in as little as a few
hours.
N. meningitidis Treatment
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Empiric

3rd generation cephalosporins(ex. Ceftriaxone,
Cefotaxime)
Targeted

Penicillin, Ampicillin, or Ceftriaxone
Prophylaxis

Ciprofloxacin
Immunization

Covers 4 serotypes of N. meningitidis

Does not protect against other serotypes
Gram Negative Infections
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VIBRIO
GENUS
Vibrio cholerae
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 Gram - curved rod
Etiological agent of cholera:
1. Toxin based, similar to
the heat labile toxin of E.
coli
2.
1A5B Toxin
3.
Cellular cAMP increase,
while cellular Chloride
ions and water decrease

Oral Fecal Transmission
V. Cholerae Symptoms
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 Severe diarrhea, rice water stools (thin mucus flexs)

Dehydration and shock --> death
V. Cholerae Treatment
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Treatment

Water and ion replacement

Most patients can be treated with oral fluid/
electrolyte replacement

IV fluids used for severe cases
Antibiotics usually unnecessary, but if need be:

Doxycycline

Azithromycin
Temperature Classifications
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Five Temperature Classifications
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① Psychrophile : 5 to 15 degrees Celsius
② Psychrotroph: Room temperature 20 to 30
degrees Celsius
③ Mesophile: between 25 to 45 degrees Celsius
(humans are mesophile) our normal flora is an
example of mesophile
④ Thermophile: between 45 to 70 degrees Celsius
think hot tubs, or springs
⑤ Hyperthermophile: 70 + degrees Celsius
Why is optimum temperature important?
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① Substrates fit into an enzymes active site like a key.
② Proteins change with temperature for instance
frying an egg the protein changes from a liquid into
a gel like solid.
③ Placing an enzyme at a particular temperature that
it is not normally in will change its shape.
Lock and Key
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 Substrates fit into an enzymes active site like a key.
How does temperature effect a protein?
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 Frying an egg the protein changes from an liquid into
a gel like solid. Placing an enzyme at a particular
temperature that it is not normally in will change its
shape.
Check Your Knowledge:
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① What is the mechanism of action of Bactrim?
② Why is optimum temperature important?
③ Define lipopolysaccharide
④ What is the purpose of gram staining?
⑤ What are the 3 families discussed?
⑥ What are the pathogenic mechanism of each
bacteria discussed?
⑦ What are the symptoms of each bacteria
discussed?
ANY QUESTIONS FOR ME?!?!?!?!?!
References:
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①
②
③
④
⑤
⑥
⑦
⑧
http://www.ppdictionary.com/gnbac.htm
Parija SC (Jan 1, 2009). Textbook of Microbiology & Immunology. India: Elsevier.
ISBN 8131221636.
Kulp A, Kuehn MJ (2010). "Biological functions and biogenesis of secreted bacterial
outer membrane vesicles". Annu. Rev. Microbiol. 64: 163–84.
doi:10.1146/annurev.micro.091208.073413. PMC 3525469. PMID 20825345.
http://study.com/academy/lesson/bacterial-cell-walls-and-the-gram-stain-test.html
Dr. Kulkarn “Antibiotics Lecture” September 18,2015
D’Amico, Salvino et al. “Psychrophilic Microorganisms: Challenges for Life.” EMBO
Reports 7.4 (2006): 385–389. PMC. Web. 18 May 2016.
– Clinical Microbiology; Cecile Sanders & Keri Brophy-Martinez Chapter 16
Paula Darsaw “Gram Negative Infections” August 15, 2015