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Bacteriology Review
Beginning definitions
• Obligate Aerobe – require oxygen (20%) to grow
• Obligate Anaerobe –>30 min of oxygen exposure can be toxic
• Facultative anaerobes – grow in aerobic and anaerobic
conditions, most “aerobic” bacteria are actually facultative
• Microaerophilic – Grow better with reduced oxygen and
elevated Carbon dioxide %
• Aerotolerant anaerobes– anaerobe not killed by prolonged
exposure to oxygen – example: Clostridium tertium
• Lag Phase - >24 hrs old, growth slowing, not appropriate for
biochemical or susceptibility testing
• Log Phase – Exponential growth – appropriate for all testing
• Stationary phase – appropriate for transporting specimens
Specimen collection
for Aerobic Bacteriology
Throat / Wound collected using swab
1. Swab placed in Stuart’s or Aimes transport media buffer
solution with peptones –
2. Preserve viability but not promote growth of bacteria
3. Swabs made of polyester or sponge like material (not
cotton) – cotton traps organisms
Urine
1. Boric acid to induce stationary phase
2. Refrigerate within one hour, Specimen stable for 24 hours
•
•

Purpose to maintain original colony count
Maintain viability of organisms
Gram stain

Primary stain
Mordant

10 seconds1 minute

Rinse
10 seconds1 minute

Rinse
Decolorization
Counter stain
C
o
u
n
t

5-10 seconds
10 seconds1 minute
Gram positive cocci
in pairs and chains
Streptococcus

Some Gram stains to
Remember!!!
Gram negative rod – fusiform
Shaped - Fusobacterium

Gram negative
bacillus
Most likely an
Enteric

Gram positive rod
most likely a
Bacillus species

Gram positive cocci in clusters,
Staphylococcus
Gram stain is used to assess quality of
Sputum specimen for culture
• Expectorated sputum specimen is rejected for bacterial
culture if actually “spit”:
– Perform Gram stain of sputum specimen
– If >= 25 epithelial cells /field
– Sputum is judged to be spit / not a sputum specimen
– Bacterial culture is not performed / it is rejected
– Request made for a new “deep cough” specimen
Bad Sputum

10X objective

Good
Sputum

10X objective
Most used Agar Media in Bacteriology
Blood agar- 5% sheep’s blood agar
gauge hemolytic reaction of bacteria (alpha, beta, gamma)
Grow variety of Gram positive & Gram negative bacteria and
Yeast

Chocolate agar – “cooked” blood agar – more enriched than
blood agar
Everything blood agar can grow Plus more fastidious bacteria
like Haemophilus influenzae and Neisseria gonorrhoeae
Most used agar media in Bacteriology
• MacConkey agar – Supports the growth of Gram negative
bacilli only (crystal violet inhibits Gram positive organisms)
Selective (only GNR) and Differential (Lactose fermentation)
Lactose fermentation = pink (neutral red indicator)
non-lactose fermentation = no color

• After media is plated incubated in aerobic and/or CO2
incubators at 35˚ C for 24 – 72 hours
Gram Positive Cocci
Staphylococcus
Streptococcus
Enterococcus
Staphylococcus
Gram positive cocci
clusters

All Staphylococcus are Positive for Catalase Enzyme
Staphylococcus aureus
Coagulase Enzyme Positive
+/- Yellow colony
Beta hemolytic

Coagulase Negative Staph
Coagulase Enzyme Negative
White colony
Most not hemolytic
Catalase Enzyme Reaction
Negative
Positive

Hydrogen Peroxide plus bacteria
Avoid blood agar – blood has innate
catalase activity and will give false
positive reaction
Tube Coagulase Reaction
Rabbit plasma inoculated with organism
Incubate at 35˚C
Read at 4 hours and if negative read again at 24
hours
Negative tube coagulase =no clot
Coagulase negative Staph

Positive tube coagulase [at 4 or 24 hrs] =
Clot
Staph aureus
Slide Agglutination Test to identify
Staphylococcus aureus
clumping

No clumping

• Clumping factor and protein A is found on the S. aureus cell wall.
• Fibrinogen and IgG to protein A is on the surface of the Staphaurex latex beads
• S. aureus will clump in the Staphaurex latex bead solution and
• positively identifies Staphylococcus aureus.
• Coagulase negative Staphylococcus species remain milky and do not clump in
• the solution.
•Advantage over tube coagulase – reaction time is 30 seconds vs. 24 hours
Staphylococcus aureus
• Primary virulence factor:

– Protein A – surface protein, ability to bind immunoglobulin and
combat the immune response

• Diseases and associated toxin:
–
–
–
–
–
–

Toxic shock syndrome (TSST-1 toxin)
Scalded skin syndrome (Exfoliatin (SSS) toxin)
Soft tissue infection (Panton valentine leucocidin toxin – PVL)
Septic arthritis – primary cause
Food poisoning (Enterotoxins)
Bacteremia and endocarditis
Susceptibility issues –(1) MRSA
• Methicillin Resistance (MRSA =Methicillin resistant S. aureus)
– Penicillin binding protein (PBP) produced by the mec A gene codes
for resistance to oxacillin/methicillin/penicillin
– All MRSA are considered resistant to the cephalosporin antibiotics
– Cefoxitin susceptibility testing is a more sensitive indicator of
resistance and provides better detection of MRSA than oxacillin
testing. Shown is a Kirby Bauer (KB) disk test that is resistant to
cefoxitin.
(2) The “D” Test for Clindamycin
• To accurately determine if Staph aureus/MRSA is susceptible
to Clindamycin
• During therapy, S aureus isolates resistant to Erythromycin
possess enzymes capable of inducing Clindamycin to become
resistant and therefore not useful for therapy.
• Kirby Bauer zone around Clindamycin will be blunted to form
a D if Clindamycin can be induced by Erythromycin to be
resistant – so called INDUCIBLE RESISTANCE.
• Clindamycin should be reported as resistant by clindamycin
induction and not used for therapy.
D test positive
Inducible
resistance

D test negative
Clindamycin can
be used for
therapy
Staphylococcus aureus (MRSA)
Hospital Epidemiology Issue:
Nasal surveillance for MRSA
Nares is the colonization site for MRSA and the
most common surveillance site for cultures.
Hospitals have surveillance programs to prevent
nosocomial spread of MRSA.
ChromAgar can be used to detect MRSA. It is a
selective and differential medium with
chromogenic substrates that turn specific colors
for Staph aureus. Cefoxitin is added to the
medium to detect methicillin resistant Staph
aureus (MRSA)
Molecular assays (MA) can also be used to
screen nares for MRSA. MAs increas sensitivity
of detection (@5-10%) and increase laboratory
costs.
Coagulase negative Staph (CNS)
@ 15 species infect humans

• Staph epidermidis – is the most common

– Major aerobic component of normal skin flora
– Common cause of subacute bacterial endocarditis
– Pathogenicity from cell adhesion factors forming
biofilm on biologics and plastic

White non-hemolytic
colony

• Staph saprophyticus –

– Urinary tract infections in the child bearing age female,
– This species of CNS adheres in greater #’s to epithelial cells
– Novobiocin resistant by KB disk test can be used as test to identify
Staph saprophyticus
resistant
CNS & Related Cocci
• Staph hemolyticus –

– Can cause line related sepsis
– This CNS is hemolytic on blood agar
– Do not confuse with Staph aureus

• Staph lugdunensis

– The PYR test is positive and used to identify
Pos
– Perhaps greater pathogenicity than other species

• Micrococcus species
–
–
–
–

Mustard yellow colored colony
Catalase positive / Gram positive cocci tetrads
Tube coagulase negative
Does not ferment glucose
• Staphylococcus ferments glucose

– Environmental contaminate

Neg
Streptococcus
Gram positive cocci in chains and or pairs
Catalase enzyme negative
Grouped by hemolytic pattern on 5% sheep’s blood agar
– Alpha – greening of agar, partial hemolysis of rbc
• Viridans Strep, Strep pneumoniae,
Granulicatella/Abiotrophia
– Beta – clearing of agar, complete hemolysis of rbc
• Beta hemolytic Strep groups A - G
– Gamma – no clearing of agar
• Streptococcus bovis
Beta hemolytic
Streptococcus
• Grouping of the Beta Streptococcus use the “C”
carbohydrate in the cell wall of the bacteria
• Identifies the Beta Strep groups – A, B, C, F, and G that
cause infections in humans.
• The Lancefield grouping system uses the “C” CHO in the
cell wall (antigen) in a slide agglutination reaction with
purchased monoclonal antibody attached to latex beads
See below slide for a positive and negative reaction.
• Immunologic typing is more accurate than biochemical
testing to group Streptococcus
+
-
Streptococcus pyogenes
•

Strep pyogenes – genus/species for Group A beta Streptococcus [GAS]
– Bacitracin KB sensitivity test – inhibited, no growth @ disk
– This test is not specific for Group A , it x-reacts with group C
– “A” disk is the Bacitracin disk
– PYR (pyrrolidonyl arylmidase) reaction
• Organism spotted on moist disk
• 2 min – RT incubation
• Add Cinnamaldehyde reagent
• Look for color reaction
• Pink = positive = Strep pyogenes
• This test is not exclusive for Strep pyogenes – Enterococcus and
Staph lugdunensis are also positive for PYR
– No resistance to Penicillin or Cephalosporin antibiotics
Primary virulence factors :
M Protein – prevents
phagocytosis
Capsule – hyaluronic capsule
protects from
phagocytosis

– Streptolysin O and Streptolysin S toxins
• Comprise the ASO titer

• Cell toxins lead to evasion from the immune system
• O toxin is oxygen labile S toxin is oxygen stable
• When both are active - most hemolysis occurs in the 5% Sheep’s
blood agar
Strep pyogenes diseases
• Diseases –
–
–
–
–
–
–
–
–

1

Pharyngitis
Impetigo (1)
Erysipelas (2)
2
Cellulitis (3)
Necrotizing fascitis (4)
Puerperal sepsis
Toxic Shock
Scarlet fever (5)

3

4

5
Sequelae of Strep pyogenes
infections
•
–
–
–
–
–
–

•
–
–
–
–

Rheumatic fever

Caused by inadequately treated Strep throat
Rise in the 1990s due to EIA tests being used as the only diagnostic test for
pharyngitis – EIA tests are not very sensitive – and led to children not receiving
antimicrobial therapy
All negative EIA specimens on children should be confirmed by culture
Similarity between the proteins of the Strep A and muscle tissue cause immune
system confusion
Immune system attacks the heart, joint and bones and causing inflammation – can
lead to valve replacement surgery
Anti-streptolysin O measures the amount of antibodies against Streptococcus group
A in the serum

Glomerulonephritis

10-14 days following skin infection or pharyngitis
Renal disease with inflammation of the glomeruli
ASO titers will be positive
Usually resolves with therapy
Streptococcus agalactiae (Group B)

Lancefield agglutination test demonstrates “B” cell wall CHO

Biochemical tests for identification:
Camp test – performed using a Staph aureus strain with Camp
factor perpendicular to group B Strep, incubate 24 hr.
intensifies toxin and produces arrow shaped hemolysis
Rapid hippurate hydrolysis
4 hr test for identification of GBS
Hippurate disk incubated with Group B Strep
Add ninhydrin reagent for color formation
Positive/Purple
pos

Arrow-like

Staph aureus

Strep group B
Strep agalactiae
Group B Streptococcus [GBS]
•

•

Pathogen of neonate – associated with in utero or perinatal organism
acquisition during birthing process
– Early onset – within 7 days
– Late 7 – 28 days from birth process.

Pregnant women carry organism in the cervix and/or rectal area. All
should be tested at 35 – 37 weeks of pregnancy.
– Enrichment methods for GBS is standard of practice and must be used

• Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto
agar media. This broth can also be used as an enrichment method followed by
molecular testing.
• Carrot enrichment broth turns orange with growth of group B Strep

•

GBS is also a pathogen of the elderly – causes bacteremia & UTI

•

Susceptible to Penicillins and Cephalosporins
Carrot Broth
It used to be Streptococcus bovis
Taxon update –
1) Streptococcus gallolyticus ssp. gallolyticus (formerly S. bovis
biotype 1) associated with colonic cancer and endocarditis
2) Strep gallolyticus ssp. pasteurianus (formerly S. bovis
biotype II) associated with neonatal meningitis
Identification for S. gallolyticus (both ssp)
B
6.5% Salt

Enterococcus
Neg Pos

• Two most common species E. faecium and E. faecalis
• No well defined virulence factors
• Possesses the Group D CHO in the cell wall

Neg Pos

• Biochemical tests:
– Bile esculin positive – growth and black precipitin
Bile esculin
– Growth in 6.5% salt
– PYR positive (Group A beta Strep and Staph
PYR
lugdunensis also positive)*
+
– E. faecium = arabinose fermentation positive
– E. faecalis = arabinose fermentation negative
Enterococcus
•
•
•
•

Cause a variety of infections and is a pathogen of opportunity
Normal flora in the intestine
Cause UTI, bacteremia, and abdominal infections
Antimicrobial therapy:
–
Natural resistance to cephalosporins
–
Ampicillin/Aminoglycoside can be synergistic for therapy in cases of
endocarditis

•

Unusual susceptibility issues
–
Acquired resistance to vancomycin known as vancomycin resistant
enterococcus [VRE]
• Van A (E. faecium) resistance genes
• Van B (E. faecalis) resistance genes
Alpha hemolytic
Streptococcus
Streptococcus pneumoniae

Gram stain = gram positive bullet shaped cocci with capsule
Polysaccharide capsule = virulence factor

Identification:
–
–

Bile soluble – colonies dissolve in sodium deoxycholate (bile salts)
Optochin sensitive – 14mm or greater zone of inhibition around the
6mm optochin disk
Colonies dissolved
Inhibition
>=14mm

NO
Inhibition
Streptococcus pneumoniae
• Upper and Lower respiratory tract, sepsis and meningitis
• Prevent invasive disease with multi-serotype vaccine
• Acquired Resistance to Penicillin due to Penicillin binding
proteins (PBP)
• Minimum inhibitory concentration (MIC) testing necessary to
detect resistance to penicillin – in broth or Etest
Etest for
• Testing of CSF isolate:
Cefotaxime
– Penicillin R = >2 mcg/ml

• Resistance to Penicillin
• Cefotaxime 2nd drug of choice

MIC
Viridans Streptococcus
Several species of alpha hemolytic Streptococcus found as NF in
mouth and upper respiratory tract
S. mutans S. salivarius S. sanguis S. mitis
• Bile esculin slant negative
• Not bile soluble
Viridans Streptococcus
• Optochin resistant - <=13 mm
• Cause 30 – 40% cases of sub acute endocarditis / native valve
• Can cause abscesses and various infections throughout the
body especially in the immune suppressed host
• Variable susceptibility patterns, some isolates with elevated
MICs to Penicillin
Viridans Streptococcus
• The Streptococcus anginosis group
– S. anginosus S. constellatus S. intermedius

• Normal flora in human mouth
• More virulent than “normal” viridans
Streptococcus, perhaps due to capsule
• Cause deep tissue abscesses, bacteremia,
endocarditis, intra abdominal infections…
• Variable susceptibilities – so need to test
Nutritionally Variant Streptococcus
•

Vitamin B6 (pyridoxal) deficient – so will not grow on BAP without B6

•

Will grow in a patient blood culture bottle due to patient’s vit B6 in blood
• but not on the subculture 5% Sheep’s blood agar plate ( no vit B6)
• Requires streak of Staph aureus that supplies vitamin B6
• Nutritionally variant Strep will satellite @ S. aureus (see pix)

•

There are 2 species
– Abiotrophia
– Granulicatella

•

Endocarditis –
– More destructive to valve than
than “normal” viridans Strep
– Higher MIC’s to Pen or R

Satellite streptococcus
Gram Negative Cocci
Neisseria
Moraxella
Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
– Small kidney bean shaped cocci in pairs
– Oxidase enzyme positive

• CTA (Cysteine Trypticase agar) carbohydrate fermentations
are used for identification – glucose, maltose, lactose, sucrose
– N. gonorrhoeae Gluc + Mal - Lac - Suc – N. meningitidis Gluc + Mal + Lac - Suc – N. lactamica
Gluc + Mal + Lac+ Suc– M. catarrhalis
all negative
Dna’ase +
Neisseria species
gram negative diplococcus – intra and
extra cellular

Acinetobacter species –
Cocci are more round and not
usually in pairs – normal flora in
female
genital tract
Miniature CHO fermentation reactions for Neisseria
and Moraxella catarrhalis
control

+

Compare (+) reactions to negative (red) control well
Oxidase enzyme spot test:
Detects presence of enzyme cytochrome
oxidase
Add reagent
N,N dimethyl-p-phylenediamine oxalate
to filter paper with organism
positive = purple/ blue

+

+

Full size tube CHO fermentation test
Neisseria meningitidis
• Meningitis primarily in children and young adults
• Hallmark is petechiae (organisms crowd capillaries) – can lead
to tissue necrosis and DIC – infection can be rapidly fatal
• Carriage in Nasopharynx/ Susceptible to Penicillin
• Capsular polysaccharide is the primary virulence factor
• Complement deficiencies in 7,8,and 9 can predispose to
chronic type disease
• Adrenal necrosis is referred to Waterhouse
Friederichsen syndrome
• Type C is the current endemic strain in the USA – 1*problem
in college Freshmen living in dorm
• Immunization available for all serotypes except serogoup B
Neisseria gonorrhoeae
–
–
–
–
–
–
–

Acute urethritis, endocervix, ocular, rectal, oropharynx
10 – 20 % female ascend to PID but only 0.5% disseminate
Gram stain of urethral discharge useful for male diagnosis
Gram stain of cervix not specific for females
Transport for culture – charcoal swabs, No refrigeration
Medium of choice: Thayer Martin or Martin Lewis
Amplification methods [PCR] increases sensitivity of
detection and superior to culture
– Produces a beta lactamase enzyme and also Chromosomal
resistance
–

–

Therapy: Ceftriaxone +
Azithromycin or Doxycycline to
prevent
resistance – currently an
Moraxella catarrhalis
• Pneumonia, ocular, sinusitis, otitis media
• Gram stain of sputum can be helpful in
diagnosis of pneumonia
– Polys with gram negative diplococci
• Hockey puck colony – able to push
around on agar surface
– oxidase enzyme positive
– DNA’ase enzyme positive
– Resistant to ampicillin by beta lactamase enzyme
production
Gram Positive Rods
Corynebacteria
Bacillus
Listeria
Erysipelothrix
Corynebacterium
• Corynebacterium species are:

– Catalase +,
– Diphtheroid morphology (Chinese letter forms)
– no spores are produced

• C. diphtheriae – agent of Diphtheria

Elek plate

– Diphtheritic pseudo membrane produced and adherent in throat
– Phage mediated toxin is distributed from the membrane causing
respiratory paralysis
– Toxin detected by Elek immunoprecipitation procedure
– Grows well on 5% Sheep’s BAP
– Selective media Cysteine tellurite agar
Produces black colonies with brown halos

– Grown on egg containing Loeffler medium then stain
with methylene blue to look for metachromatic granules
that are colorful granules characteristic of C. diphtheria
Other Corynebacterium

• Corynebacterium jeikeium –
– normal skin flora bacteria / thrives on lipid
– Infects patients with plastic catheters and indwelling
devices by tunneling into the device from the skin
– Biofilms are formed on the plastic, protecting the
organism from antibiotic therapy
– Very resistant to most antibiotics
– Susceptible to vancomycin and tetracycline only
Red is (+)
• Corynebacterium urealyticum –
Urease reaction
– Rapidly urease positive diphtheroid
– Urinary tract infection in post renal transplants
– Resistant to many antibiotics – vancomycin susceptible
Bacillus species
Large gram positive rod –
boxcar shaped with square ends
Can over-decolorize easily and appear reddish
Spores produced – causes clearing in rod
Catalase enzyme positive
Bacillus anthracis - Agent of Anthrax
Natural infection of herbivores
Skin, pulmonary, GI and CSF infections
Unique black eschar skin lesions
Bioterrorism - effective due to highly
resistant spores that
can be easily disseminated into
the environment
Bacillus
Bacillus anthracis culture:

medusa head colonies on BAP
non-hemolytic on 5% Sheep’s blood agar
non-motile
Penicillin susceptible
With these reactions – contact public health
department for assistance with identification

Bacillus cereus – food poisoning with rapid onset (1 – 6 hr) of vomiting
Preformed emetic toxin in food
Fried rice is one of the most common food sources
beta hemolytic colony on Sheep’s blood agar and motile
Listeria monocytogenes
Small gram positive rod
Catalase positive / No spores produced
Subtle beta hemolysis on Blood agar
Tumbling motility on wet mount
Umbrella motility in tube media
More motile at 25˚C than 35˚C
Cold loving – grows well at 4˚C and the reason it is
abundant in refrigerated foods
Found in dairy products and deli case meats
Infections: Bacteremia in pregnant women and can
induce still births
Bacteremia and CNS in immune suppressed
Ampicillin is drug of choice/ resistant to Cephalosporins
Erysipelothrix rhusiopathiae
Small gram positive rod
Catalase negative
Alpha hemolytic colony
Only G+R that produces hydrogen
sulfide (H2S) in a TSI agar

H2S production on
Triple sugar iron agar (TSI)

Human skin infection acquired from swine erysipelas or
poultry and is most common in butchers
Uncommon isolate in human blood cultures
most common in drug addicts
high % of endocarditis with extensive damage to valve
Intrinsically resistant to Vancomycin
Gram negative bacilli
Enterics
Non fermenters
Fastidious
Enterobacteriaceae
Enteric Gram Negative Bacilli
Ferment glucose
Almost all are oxidase negative
Nitrate reduced to nitrates

Enteric Gram
Negative Rod
Ferment Glucose
Ferment Lactose
Ex. Escherichia
coli

Gram negative cell
Wall
Rod shaped
Simple flow chart
Identification
Strategy

Enteric Gram
Negative Rod
Ferment Glucose
Do NOT ferment
Lactose
Ex. Proteus species

Non fermenters
Glucose is not fermented
Oxidase positive and
negative
species

Gram negative Bacilli
Do NOT ferment
Glucose
Do not ferment
Lactose
Positive Oxidase
Pseudomonas
Burkholderia

Gram negative Bacilli
Do NOT ferment
Glucose
Do NOT ferment
Lactose
Negative Oxidase
Acinetobacter
Stenotrophomonas
Enterics that Ferment Glucose
• Escherichia coli
–
–
–
–

Major aerobic NF in intestine
#1 cause of UTI
Common in abdominal infections and bacteremia
Indole reaction is positive

• Enterobacter species

Indole positive =
Robin’s egg blue

Green sheen
on
EMB agar

– Enterobacter cloacae and E. aerogenes most common
– Environmental organism with low pathogenicity
– Enterobacter sakasaki associated with neonatal meningitis

• Klebsiella species – K. pneumoniae most common

Mucoid colony

– Mucoid colony
– Currant jelly sputum in alcoholics due to blood mixed with capsular
polysaccharide in sputum
Enterics that do NOT ferment lactose!
No lactose fermentation = No color produced on

Macconkey agar
• Proteus species
Swarming colonies in layers on agar surface
• Indole positive – Proteus vulgaris
• Indole negative – Proteus mirabilis
• Serratia marcescens
– Red pigment produced and intensifies at room temp
– Infections in immune suppressed
– Ventilator associated pneumoniae
– Bacteremia
Gram negative bacillus
MacConkey agar
Lactose fermenter
fermenter

Lactose non

Gram stain enteric
bacilli – plump

Lactose fermentation

No lactose
fermentation
Triple Sugar Iron Agar – detect fermentation of
glucose, lactose and/ or Sucrose and the
production of hydrogen sulfide [H2S]

Glu/lac/suc
fermented
with gas

Glucose Glucose
fermented fermented
only
with H2S

No CHO
fermentation
Non fermenter
Extended Spectrum Beta Lactamase
Enzymes - “ESBL”
• ESBL enzymes are produced by numerous gram negative rods
– Confer resistance to Cephalosporins and synthetic Penicillins

• Plasmid mediated Tem 1 beta lactamase is the most common
enzyme produced
– Possible plasmid dissemination makes this an infection control issue

• Detect presence of ESBL in a gram negative rod
– Double disk test
– MIC breakpoints for the cephalosporins are set at very low MIC values
to catch subtle resistance

• ESBL producing GNR are treated with:
– Imipenem [Carbapenemases]
– Piperacillin/Tazobactam which blocks beta lactamase enzyme function
Typical ESBL Susceptibility Pattern
•

Escherichia coli
– Ampicillin R
Double disk test – demonstrates how
– Cefazolin R
the beta lactam blockers [clavulanate]
can stop
– Gentamicin R
the beta lactamase activity
– Cefotetan S
(Cephamycins are not cephalosporins)**
– Amikacin S
Cephalosporin
– Ceftazidime R
– Cefpodoxime R
Cephalosporin +
Clavulanate
– Piperacillin R
– Pip/Tazobactam S
(Tazobactam is a Beta lactam blocker)
– Imipenem S
Carbapenemase Enzyme producing Enterics
[CRE] and NDM-1 Enzyme
Carbapenemase enzymes are most commonly produced by
Klebsiella pneumonia and known as KPC which stands for K.
pneumoniae carbapenemase however, they have been found in many
genera of enteric gram negative rods
Resistant to all carbapenem antibiotics - imipenem, meropenem,
ertapenem, doripenem
CRE have cross resistance to virtually all antibiotic classes
- Polymyxins [colistin, polymyxin B] used for therapy
- High fatality rates associated with infections
Other resistance mechanisms:

New Delhi metallo-beta lactamase [NDM-1] enzymes – resistant to all
drug classes including carbapenems
Salmonella species
• Salmonella spp.
– Diarrhea with +/- fever – polys in the stool
– Infection from food ingestion – must ingest large #’s of
organisms to make you ill (1,00,000 bacteria)
– Eggs, meats and contaminated uncooked vegetables
– Does not ferment lactose/ produces Hydrogen sulfide
– Selective agars: SS and Hektoen
– Identification based on biochemical reactions to get to
genus and serologic typing “Kaufman White typing
scheme” to get to species
–
O Somatic (cell wall) antigen – Salmonella group B
–
H flagellar antigens – 2 phases [h1 & h2] speciates the
Salmonella – ie Salmonella enteritidis
– Vi capsular antigen – Found in S. typhi only
Salmonella Shigella
Agar (SS agar)
Salmonella and
Shigella are colorless
because they do not
ferment lactose

Hektoen agar –
Salmonella produces
H2S [Hydrogen sulfide] turning the
colonies black
Normal flora – orange colored due to
fermentation of lactose
Salmonella typhi
• Typhoid fever – fever, sepsis, high fatality rate
• Vi capsular antigen surrounds the D cell wall antigen
• Boil solution of organism for 15 minutes to destroy the
Vi capsular antigen and expose the cell wall antigen D
• Serotyping can then be performed for the D antigen
• Moustache of H2S in the TSI tube

– Carrier state in gallbladder
– Ingest organism, clears the bowel
– In @ 1 week then enters
– Blood stream & Bone Marrow
Shigella
• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool
[10%], tenesmus
• Human to human transmission
• Low #’s of organisms to make you ill [10 – 100 bacteria]
• Non motile and No H2S produced (differ from
Salmonella)
• Does not ferment lactose
• 4 species based on somatic antigen
– S. boydii
Group C
– S. dysenteriae
Group A
– S. flexneri
Group B
– S. sonnei
Group D
Yersinia enterocolitica
•
•
•
•
•

Diarrhea
Major reservoir – swine
Human usually infected by non pasteurized milk
Causes septicemia in iron overload syndromes
Unique infection Mesenteric adenitis – RLQ pain
which mimics appendicitis
• Grows well at 4*C (like Listeria)
• CIN agar (Cefsulodin-irgasan-novobiocin)
• Has been associated banked blood infections related
to Transfusion of infected products
Yersinia pestis - Plague
• Possible organism of bioterrorism
• Obligate flea/ rodent/ flea cycle
• Infected Flea bite - leads to Bubonic form/
painful buboes (lymph node swelling)
• Pneumonic form- bacteremic spread or
patient to patient fatality >=50%
• Southwestern part of USA
• Grows on BAP,
• catalase +, oxidase • Bipolar staining “safety pin”
Enteric Glucose Fermenters
that are Oxidase +
• Vibrio cholerae

TCBS AGAR

– Rice water stool produced due to mucus flecks in diarrhea
– Virulence is due to Toxin – receptor on the epithelial cell –
activates adenylate cyclase – increases cAMP – hyper
secretion of NaCl and H20 – death from dehydration
– It is a Halophilic bacteria which means it is a salt loving
organisms, 1% salt actually enhances growth
– Selective media – TCBS = thio citrate bile sucrose agar,
colony turns yellow due to sucrose fermentation
– 01 most virulent due to high production of toxin
Classic Gram Negative Rod Gram
Stains
Vibrio species

Campylobacte
r

Sea gull wings

C shaped
Enteric gram negative rod

Plumb and rectangular
Other Vibrio species
Vibrio parahaemolyticus
• Colony on TCBS medium is green – sucrose negative
• Diarrhea from ingestion of raw fish and shellfish

• Vibrio vulnificus
• Very virulent Vibrio species – infection leads to formation of
painful skin lesions with muscle necrosis on lower extremities
• Most prevalent in patients with pre existing liver disease
• 50% fatality rate even if treated
• Ingestion of raw oysters and shellfish
from coastal waters
• Green or yellow colonies TCBS
Do not ferment Glucose
Oxidase negative
• Acinetobacter – hospital environment and human skin
–
–
–
–
–

Major nosocomial pathogen
Acquires antibiotic resistance with pressure
Gram negative cocco-baccilli – big cocci
Acinetobacter baumannii – glucose oxidizer
Acinetobacter lwoffi – glucose non oxidizer

• Stenotrophomonas maltophilia
–
–
–
–

Rapid maltose oxidizer
Long Gram negative bacillus
Naturally resistant to many antibiotics
Nosocomial pathogen – super-colonizer when on long term Imipenem
therapy because Stenotrophomonas is naturally resistant to Imipenem
Glucose non-fermenter
Oxidase Positive
Pseudomonas aeruginosa
• Fluorescent pigment and blue-green
pigment (pyocyanin)
• Grape like odor
• Growth at 42˚C
Ps fluorescens/putida – no growth at 42*

• Major pathogen in cystic fibrosis
– Mucoid strains
– in combination with Burkholderia cepacia
can cause major lung damage

• Nosocomial pathogen –
associated with water
Glucose Non Fermenters/oxidase +
• Burkholderia cepacia
– Dry , yellow colony
– oxidase negative
– Problem organism in cystic fibrosis
• Flavobacterium (Chryseobacterium) meningiosepticum
– Associated with fatal septicemia in the neonate
– Low virulence
– Environmental source
Haemophilus species
Haemophilus influenza

– Causes a variety of infections

• Pneumonia, meningitis, otitis media, ocular

– Two nutritional factors for growth

–
–
–
–

• X = hemin
• V= NAD (nicotinamide adenine dinucleotide)
• Demonstrate by satellite phenomenon or X/V strips (slide 74)

Grows on chocolate agar
Will not grow on 5% sheep’s blood agar
Requires high level C0₂ [5 – 8%] for growth
Resistance to Ampicillin by beta lactamase production [1520 %]
+
Disk test for Beta lactamase
Detection
• Add bacteria to filter paper impregnated with Nitrocefin or
Cefinase test (yellow colored/chromogenic cephalosporin)
• Incubate at room temp (30 sec - 60 min) depending on
bacteria being tested

• Positive result is color change from yellow to red - beta
lactamase enzyme breaks down beta lactam ring to form
hydrolyzed red end product
• Detects resistance to Amp/Pen/Cephalosporin in
Haemophilus, N. gonorrhoea , Moraxella catarrhalis, and
anaerobic gram negative rods
Haemophilus species
• H. parainfluenza –
– requires V factor only
– Normal flora in the upper respiratory tract
– One of the HACEK organisms of endocarditis
• H. (Aggregatibacter) aphrophilus –
– No factor requirements for growth
– Infections: abscesses (liver, lung, brain) & endocarditis
• H. ducreyi –
– Requires X factor
– Cause of Chancroid - venereal disease
– “school of fish” appearance on stains
Demonstrates need for X and V factor
Satellite phenomena
HACEK group
• Oral flora that can become pathogens of
endocarditis usually caused by dental procedures
• Require 2-4 days to grow in patient blood cultures
• Haemophilus species = oxidase neg, catalase neg
• Actinobacillus actinomycetom-comitans = oxidase
neg, catalase pos
• Cardiobacterium hominis = oxidase pos
• Eikinella corrodens = oxidase pos, colony pits BAP
• Kingella kingii = oxidase pos, hemolytic on BAP, also
cause infections in small children (septic joints)
Bordetella pertussis
•
•
•

Whooping cough – three stages of disease

(1)Prodromal – flu like disease – most contagious stage
(2) Catarrhal - cough with classic whoop in small children
(3) Paroxysmal - recovery phase

• Gram = Tiny gram negative coccobacillus
• Inhabits Nasopharynx
• Harsh cough caused by toxin adhering to bronchial epithelial
cells, can last for months, especially deadly to young children
• PCR for diagnosis – most sensitive and specific
• Charcoal containing media for transport and culture – Regan
Lowe Charcoal agar
• Reservoir for infection – adults due to waning immunity
• Outbreaks due to lack of vaccination of children and adults
Pasteurella multocida
Primary cause of Cat and Dog bite wound infections
Can also cause pneumonia from close animal contact
Infections can disseminate to blood stream
Found as normal flora animal’s mouth
Small gram negative coccobacilli
Growth on 5% Sheep’s blood agar
Will not grow MacConkey agar (big clue)
Oxidase Positive
One of very few Gram negative rods
sensitive to penicillin
Capnocytophaga
• Fusiform shaped gram negative rods –
very pleomorphic
Gliding motility, fingerlike projections from colonies
• Oxidase negative, Catalase negative
• C. canimorsus – dog bites with bacteremia – high% of
infections lead to endocarditis

• Other Capnocytophaga species normal flora in the human
mouth and
• Can infect mouth ulcers induced by chemotherapy – this
becomes route of entry into bloodstream
Granuloma in bone
marrow

•
•

Brucella species

Disease – Fever of unknown origin, significant joint pain
Small gram negative coccobacilli – slow and difficult to grow in blood
cultures
– Castaneda biphasic blood culture held for 21 days [old school]

•
•
•

– Current: Automated Blood culture systems grow within 5 days
Specimens: blood and bone marrow most profitable
– Granuloma formation in bone marrow
Serology can be used for genera/species diagnosis
Brucella species in clinical specimen related to animal
species the patient was exposed:
– B. abortus – ingestion of raw cow milk
– B. melitensis – ingestion raw goat milk, feta cheese
– B. suis – contact with pigs B. canis - contact with dogs
Campylobacter
• Small curved bacilli –
shaped like sea gull wings
• C. jejuni – agent of diarrhea
–
–
–
–
–

Related to undercooked poultry ingestion
Requires selective media containing antibiotics -Skirrow’s blood agar
Thrives at 42˚C - can use to selectively culture Campylobacter
Requires microaerophilic atmosphere (high CO₂, low O₂)
Significant % Guillain-Barre syndrome as sequelae

C fetus – Bacteremia in the immune suppressed host / does
not cause diarrheal disease
C. jejuni – grows at 37˚C and 42˚, hippurate positive
C. fetus - grows at 37˚C and 25˚C hippurate negative
Francisella tularensis
Reservoir – rabbits, rodents, ticks and flies. Humans infected by
bug bites or directly from exposure to animal blood
Strongly associated with skinning rabbits with bare hands
Bacteria can penetrate small breaks in skin:
cause painful skin lesions –
enlarged lymph nodes –
leading to bacteremia (ulcerogladular tularemia)
Pneumonia
Great hazard to lab workers
Fastidious small gram negative cocco-bacillus
Culture media requires cysteine*
Helicobacter pylori
• Small curved bacilli
• Cause of acute gastritis
• Rapidly!! And strongly urease positive – used for detection
directly from antral biopsy tissue
• Difficult to grow in culture
• Stool antigen – diagnose and
test of cure
• Serum antibody can aid in
diagnosis and avoid biopsy
Legionella
Legionella pneumophila most common [6 serotypes]
• Requires cysteine in media for growth
• Media of choice Buffered Charcoal Yeast Extract agar
with colonies forming in 3-5 days
• Does not stain with gram stain in direct specimens
• Use silver impregnation stains in tissue
• Pulmonary disease associated with water,
cooling towers, shower heads, stagnant water
• Urinary antigen test to detect L. pneumophila type I infection
• Treatment: Erythromycin (macrolide)
Bacteria without cell walls
•
•
•

Mycoplasma and Ureaplasma – have cell membranes only! Media and
transport must contain sterols to protect the membrane to promote
survival and growth
Do not form discreet colonies on agar plates – must read plates under a
microscope to visualize organisms
PCR is becoming the detection method of choice since culture is
cumbersome and slow (3 – 14 days)

• M. pneumoniae –

Ureaplasma

– community acquired pneumonia
– Serology and PCR for diagnosis

• Genital mycoplasmas

Mycoplasma hominis

– M. hominis – fried egg colony, vaginitis, cervicitis, postpartum sepsis,
neonatal infections , pre rupture of membranes
– Ureaplasma urealyticum – rapid urea hydrolysis in broth, NGU &
upper genital tract infection, spontaneous abortion, neonatal
infections
Difficult to grow
• Bartonella henselae – Diagnose be PCR and/or serology
– Cat scratch disease – exposure to cat and cat boxes (excrement)
– Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV

• Bartonella quintana – cause of trench fever/ vector is the
body louse/Diagnose by Serology
• C. trachomatis Serovars L1,L2,& L3 cause of
Lymphogranuloma venereum
– Serology / clinical findings – lymphatics and lymph nodes involved

• C. pneumoniae (TWAR agent)- Pneumonia, Diagnose by PCR
• C. psittaci- psittacosis, pneumonia, exotic parrot exposure/
Serology and epidemiology
• Chlamydiae trachomatis
– trachoma - eye infection
Molecular Testing for Neisseria gonorrhoeae
and Chlamydia trachomatis

• Amplification of DNA by Polymerase Chain Reaction (PCR)
– Urine, Cervix, and urethral most often tested
– More sensitive than any culture based system
• Sensitivity/Specificity @ 96%/99%
• Females most sensitive specimen = cervix
• Males good sensitivity with urine / equal to urethral
• Has replaced culture methods as “Gold” standard of
Elementary bodies from
detection
infected cells using
Iodine staining
of inclusions from
Culture method

Fluorescent antibody
stain
More difficult to grow
• Ehrlichiosis –
–
–
–
–
–
–
–

Obligate intracellular pathogen
Anaplasma, inclusion in the PMN
Ehrlichia inclusion in the Monocytes
Vector = tick bite
fever, leukopenia, thrombocytopenia,
elevated serum aminotransferases,
no rash (differs Rocky Mountain Spotted Fever)

• Found in the south central, southeast , midwest USA
• PCR and serology for diagnosis
Spirochetes
• Borrelia burgdorferi - Lyme’s disease
– Primarily found in NE part of USA
– Vector = bite of the Ixodes tick
• Borrelia recurrentis - Relapsing fever
Vector =bite of the human body louse
Blood smear observe spirochete
Spirochetes
• Treponema pallidum
– Agent of Syphilis
– RPR and VDRL for antibody detection
– Molecular methods

Darkfield from chancre lesion

• Brachyspira – intestinal spirochete found
on the brush border of the intestine,
?? Role in disease
• Leptospira interrogans – Leptospirosis

– Fever with rash and renal involvement
– Urine from rats and other animals contaminate water supplies

Spirochete – Shepherd’s crook
Unusual Bacterial diseases
• Granuloma inguinale
– Klebsiella (Calymmatobacterium) granulomatis
– Rare STD – causes ulcerative genital lesions

• Streptobacillus moniliformis
–
–
–
–

Rat bite fever or Haverhill fever
L form or cell wall deficient bacteria
Inhibited by SPS in blood culture media
Needs serum supplementation to grow, will not grow in a
bacteriology culture
– Infection obtained from a rat bite
Anaerobes
• Anaerobic infections can occur in virtually
every organ and region of the body
• Polymicrobial – both aerobic and anaerobic species in the
infections
• Endogenous organisms (commensal flora) cause most
anaerobic infections
– Due to Trauma, vascular or tissue necrosis cutting off the oxygen
supply to the involved tissue

• Surgery plus antibiotics often necessary
• Anaerobic culture collection – must be oxygen free
– Gel contaning swabs
– Eswab,
– Evacuated vials (port o cult)/ oxygen free collection

– Do not refrigerate specimens – absorb oxygen and kill anaerobes
Anaerobes
•

PRAS media – pre reduced anaerobically sterile
– Media packaged in oxygen free environment
• Most common media used
– CDC anaerobic blood agar
– Kanamycin-vancomycin blood agar
– Bile Esculin agar
– Thioglycollate broth
– Chopped meat glucose broth
• Anaerobic gas pack jars – incubate cultures in anaerobic conditions
• Wet pack – add 10 ml water to hydrogen and CO2 generating
envelope/ requires palladium coated catalysts – generate heat
• Dry pack – (Anaeropack) Absorbs O2 and generates CO2
• Anaerobic bacteria can be poorly staining and bizarre or irregularly
shaped
Anaerobic Gram Negative Rods
Gram negative Anaerobic Bacillus
• Bacteroides fragilis group –
–
–
–
–
–
–
–
–
–
–

Pleomorphic gram negative rod
Growth in the presence of bile
Esculin positive – turning media black
Resistant to Penicillin and Kanamycin
Most common organisms in human bowel
B. fragilis
B. ovatus
B. thetaiotamicron – indole positive
B. uniformis
B. vulgatus

Growth on bile
Black pigment

• Resistant to Penicillin by beta lactamase enzyme –
• Metronidazole is antibiotic of choice
Gram Negative Anaerobic Bacillus
• Prevotella and Porphyromonas species
–
–
–
–
–

Will not grow in the presence of bile
Will not turn black on esculin media
Brick red fluorescence or black pigment
Normal commensal flora of the upper respiratory tract
Cause respiratory tract infection
Fusobacterium
Fusiform gram negative bacilli –
spindle shaped with pointed ends
• Normal flora in upper respiratory tract
• Associated with mouth and respiratory tract abscess
formation and liver abscess
• Vincent’s angina – necrotizing oral infection caused
by Fusobacterium species plus spirochetes
• Lemierre’s syndrome - oropharyngeal infection that
leads to thrombosis in jugular vein, septicemia, high
fatality rate, caused by Fusobacterium necrophorum
Anaerobic Gram Positive Rods
Clostridium species
• Gram positive bacilli (boxcar shaped ) with spores
– Some rods may over-decolorize and appear red

• Clostridium perfringens – most common species
–
–
–
–

Predominate anaerobic G+R in intestine
Double zone of beta hemolysis on BAP
Lecithinase produced on egg yolk agar
reverse camp test +

Lecithinase
Reverse Camp Test
Clostridium
•

Clostridium botulinum –
– Adult disease – Preformed heat labile toxin
ingested in mass produced or home-canned foods
– Infant disease - spore ingested from nature,
neurotoxin produced in gut,
• Begins with constipation and difficult sucking bottle
• Honey, soil, household dust
– Life threatening neuroparalytic disease
• Clostridium tetani
– Disease = Tetanus
– On Gram stain the cells appear like Tennis racket
– Toxin enters due to penetrating skin injury
– Disease in Non immunized
– Spastic contractions of voluntary muscles, hyperreflexia, lock jaw
(trismus)
Clostridium
• C. septicum –

– Bacteremia or Gas Gangrene in patient with underlying
malignancy
– Normal flora in the GI tract

• C. difficile –

– Disease = Antibiotic associated colitis
– Transmission can also occur in the hospital environment
due to the viability of the spores
– Diagnosis of infection:

EIA methods are not sensitive and are discouraged
PCR methods are more sensitive and specific for disease detection

– Two toxins produced:

Toxin A – enterotoxin causing fluid accumulation
Toxin B – potent cell cytotoxin – detected in PCR assays*
Actinomyces
• Branching gram positive bacilli - do not form spores
• Normal flora in the mouth
• Capable of forming sulfur granules
in tissue – often found on normal tonsil
• Actinomyces israelii – associated with oral, thoracic,
and abdominal infections, IUD infections
B
Branching Gram positive rods
of Actinomyces – antler like

Molar tooth colony
Sulfur granule
Propionibacterium acnes
•
•
•
•
•
•
•

Pleomorphic gram positive rod
Catalase positive
Spot indole positive
Normal flora skin, oral, GU and GI
Potential contaminate in blood cultures
Can be pathogen in cerebral shunt infections
Firmly established as significant cause of prosthetic joint
infection – particularly shoulder joints
• Cultures should be held 7- 14 days to detect
• Therapy - Ampicillin
Bacterial vaginosis
• Clue cells are diagnostic
• Mixed anaerobic/aerobic bacterial infection are the two most
common organisms in vaginosis:
– Gardnerella vaginalis (aerobic gram variable rod) and Mobiluncus
(anaerobic curved gram negative rod)
– Human blood agar for the culture of Gardnerella,
it is beta hemolytic and distinctive

• Nugent score Gram (for BV diagnosis)
– Healthy = Lactobacillus Gram positive rods
– Intermediate = mixed bacterial types
– BV = Gardnerella and Mobiluncus on smear

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Bacteriology

  • 2. Beginning definitions • Obligate Aerobe – require oxygen (20%) to grow • Obligate Anaerobe –>30 min of oxygen exposure can be toxic • Facultative anaerobes – grow in aerobic and anaerobic conditions, most “aerobic” bacteria are actually facultative • Microaerophilic – Grow better with reduced oxygen and elevated Carbon dioxide % • Aerotolerant anaerobes– anaerobe not killed by prolonged exposure to oxygen – example: Clostridium tertium • Lag Phase - >24 hrs old, growth slowing, not appropriate for biochemical or susceptibility testing • Log Phase – Exponential growth – appropriate for all testing • Stationary phase – appropriate for transporting specimens
  • 3. Specimen collection for Aerobic Bacteriology Throat / Wound collected using swab 1. Swab placed in Stuart’s or Aimes transport media buffer solution with peptones – 2. Preserve viability but not promote growth of bacteria 3. Swabs made of polyester or sponge like material (not cotton) – cotton traps organisms Urine 1. Boric acid to induce stationary phase 2. Refrigerate within one hour, Specimen stable for 24 hours • • Purpose to maintain original colony count Maintain viability of organisms
  • 4. Gram stain Primary stain Mordant 10 seconds1 minute Rinse 10 seconds1 minute Rinse Decolorization Counter stain C o u n t 5-10 seconds 10 seconds1 minute
  • 5. Gram positive cocci in pairs and chains Streptococcus Some Gram stains to Remember!!! Gram negative rod – fusiform Shaped - Fusobacterium Gram negative bacillus Most likely an Enteric Gram positive rod most likely a Bacillus species Gram positive cocci in clusters, Staphylococcus
  • 6. Gram stain is used to assess quality of Sputum specimen for culture • Expectorated sputum specimen is rejected for bacterial culture if actually “spit”: – Perform Gram stain of sputum specimen – If >= 25 epithelial cells /field – Sputum is judged to be spit / not a sputum specimen – Bacterial culture is not performed / it is rejected – Request made for a new “deep cough” specimen Bad Sputum 10X objective Good Sputum 10X objective
  • 7. Most used Agar Media in Bacteriology Blood agar- 5% sheep’s blood agar gauge hemolytic reaction of bacteria (alpha, beta, gamma) Grow variety of Gram positive & Gram negative bacteria and Yeast Chocolate agar – “cooked” blood agar – more enriched than blood agar Everything blood agar can grow Plus more fastidious bacteria like Haemophilus influenzae and Neisseria gonorrhoeae
  • 8. Most used agar media in Bacteriology • MacConkey agar – Supports the growth of Gram negative bacilli only (crystal violet inhibits Gram positive organisms) Selective (only GNR) and Differential (Lactose fermentation) Lactose fermentation = pink (neutral red indicator) non-lactose fermentation = no color • After media is plated incubated in aerobic and/or CO2 incubators at 35˚ C for 24 – 72 hours
  • 10. Staphylococcus Gram positive cocci clusters All Staphylococcus are Positive for Catalase Enzyme Staphylococcus aureus Coagulase Enzyme Positive +/- Yellow colony Beta hemolytic Coagulase Negative Staph Coagulase Enzyme Negative White colony Most not hemolytic
  • 11. Catalase Enzyme Reaction Negative Positive Hydrogen Peroxide plus bacteria Avoid blood agar – blood has innate catalase activity and will give false positive reaction
  • 12. Tube Coagulase Reaction Rabbit plasma inoculated with organism Incubate at 35˚C Read at 4 hours and if negative read again at 24 hours Negative tube coagulase =no clot Coagulase negative Staph Positive tube coagulase [at 4 or 24 hrs] = Clot Staph aureus
  • 13. Slide Agglutination Test to identify Staphylococcus aureus clumping No clumping • Clumping factor and protein A is found on the S. aureus cell wall. • Fibrinogen and IgG to protein A is on the surface of the Staphaurex latex beads • S. aureus will clump in the Staphaurex latex bead solution and • positively identifies Staphylococcus aureus. • Coagulase negative Staphylococcus species remain milky and do not clump in • the solution. •Advantage over tube coagulase – reaction time is 30 seconds vs. 24 hours
  • 14. Staphylococcus aureus • Primary virulence factor: – Protein A – surface protein, ability to bind immunoglobulin and combat the immune response • Diseases and associated toxin: – – – – – – Toxic shock syndrome (TSST-1 toxin) Scalded skin syndrome (Exfoliatin (SSS) toxin) Soft tissue infection (Panton valentine leucocidin toxin – PVL) Septic arthritis – primary cause Food poisoning (Enterotoxins) Bacteremia and endocarditis
  • 15. Susceptibility issues –(1) MRSA • Methicillin Resistance (MRSA =Methicillin resistant S. aureus) – Penicillin binding protein (PBP) produced by the mec A gene codes for resistance to oxacillin/methicillin/penicillin – All MRSA are considered resistant to the cephalosporin antibiotics – Cefoxitin susceptibility testing is a more sensitive indicator of resistance and provides better detection of MRSA than oxacillin testing. Shown is a Kirby Bauer (KB) disk test that is resistant to cefoxitin.
  • 16. (2) The “D” Test for Clindamycin • To accurately determine if Staph aureus/MRSA is susceptible to Clindamycin • During therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducing Clindamycin to become resistant and therefore not useful for therapy. • Kirby Bauer zone around Clindamycin will be blunted to form a D if Clindamycin can be induced by Erythromycin to be resistant – so called INDUCIBLE RESISTANCE. • Clindamycin should be reported as resistant by clindamycin induction and not used for therapy. D test positive Inducible resistance D test negative Clindamycin can be used for therapy
  • 17. Staphylococcus aureus (MRSA) Hospital Epidemiology Issue: Nasal surveillance for MRSA Nares is the colonization site for MRSA and the most common surveillance site for cultures. Hospitals have surveillance programs to prevent nosocomial spread of MRSA. ChromAgar can be used to detect MRSA. It is a selective and differential medium with chromogenic substrates that turn specific colors for Staph aureus. Cefoxitin is added to the medium to detect methicillin resistant Staph aureus (MRSA) Molecular assays (MA) can also be used to screen nares for MRSA. MAs increas sensitivity of detection (@5-10%) and increase laboratory costs.
  • 18. Coagulase negative Staph (CNS) @ 15 species infect humans • Staph epidermidis – is the most common – Major aerobic component of normal skin flora – Common cause of subacute bacterial endocarditis – Pathogenicity from cell adhesion factors forming biofilm on biologics and plastic White non-hemolytic colony • Staph saprophyticus – – Urinary tract infections in the child bearing age female, – This species of CNS adheres in greater #’s to epithelial cells – Novobiocin resistant by KB disk test can be used as test to identify Staph saprophyticus resistant
  • 19. CNS & Related Cocci • Staph hemolyticus – – Can cause line related sepsis – This CNS is hemolytic on blood agar – Do not confuse with Staph aureus • Staph lugdunensis – The PYR test is positive and used to identify Pos – Perhaps greater pathogenicity than other species • Micrococcus species – – – – Mustard yellow colored colony Catalase positive / Gram positive cocci tetrads Tube coagulase negative Does not ferment glucose • Staphylococcus ferments glucose – Environmental contaminate Neg
  • 20. Streptococcus Gram positive cocci in chains and or pairs Catalase enzyme negative Grouped by hemolytic pattern on 5% sheep’s blood agar – Alpha – greening of agar, partial hemolysis of rbc • Viridans Strep, Strep pneumoniae, Granulicatella/Abiotrophia – Beta – clearing of agar, complete hemolysis of rbc • Beta hemolytic Strep groups A - G – Gamma – no clearing of agar • Streptococcus bovis
  • 21. Beta hemolytic Streptococcus • Grouping of the Beta Streptococcus use the “C” carbohydrate in the cell wall of the bacteria • Identifies the Beta Strep groups – A, B, C, F, and G that cause infections in humans. • The Lancefield grouping system uses the “C” CHO in the cell wall (antigen) in a slide agglutination reaction with purchased monoclonal antibody attached to latex beads See below slide for a positive and negative reaction. • Immunologic typing is more accurate than biochemical testing to group Streptococcus + -
  • 22. Streptococcus pyogenes • Strep pyogenes – genus/species for Group A beta Streptococcus [GAS] – Bacitracin KB sensitivity test – inhibited, no growth @ disk – This test is not specific for Group A , it x-reacts with group C – “A” disk is the Bacitracin disk – PYR (pyrrolidonyl arylmidase) reaction • Organism spotted on moist disk • 2 min – RT incubation • Add Cinnamaldehyde reagent • Look for color reaction • Pink = positive = Strep pyogenes • This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis are also positive for PYR – No resistance to Penicillin or Cephalosporin antibiotics
  • 23. Primary virulence factors : M Protein – prevents phagocytosis Capsule – hyaluronic capsule protects from phagocytosis – Streptolysin O and Streptolysin S toxins • Comprise the ASO titer • Cell toxins lead to evasion from the immune system • O toxin is oxygen labile S toxin is oxygen stable • When both are active - most hemolysis occurs in the 5% Sheep’s blood agar
  • 24. Strep pyogenes diseases • Diseases – – – – – – – – – 1 Pharyngitis Impetigo (1) Erysipelas (2) 2 Cellulitis (3) Necrotizing fascitis (4) Puerperal sepsis Toxic Shock Scarlet fever (5) 3 4 5
  • 25. Sequelae of Strep pyogenes infections • – – – – – – • – – – – Rheumatic fever Caused by inadequately treated Strep throat Rise in the 1990s due to EIA tests being used as the only diagnostic test for pharyngitis – EIA tests are not very sensitive – and led to children not receiving antimicrobial therapy All negative EIA specimens on children should be confirmed by culture Similarity between the proteins of the Strep A and muscle tissue cause immune system confusion Immune system attacks the heart, joint and bones and causing inflammation – can lead to valve replacement surgery Anti-streptolysin O measures the amount of antibodies against Streptococcus group A in the serum Glomerulonephritis 10-14 days following skin infection or pharyngitis Renal disease with inflammation of the glomeruli ASO titers will be positive Usually resolves with therapy
  • 26. Streptococcus agalactiae (Group B) Lancefield agglutination test demonstrates “B” cell wall CHO Biochemical tests for identification: Camp test – performed using a Staph aureus strain with Camp factor perpendicular to group B Strep, incubate 24 hr. intensifies toxin and produces arrow shaped hemolysis Rapid hippurate hydrolysis 4 hr test for identification of GBS Hippurate disk incubated with Group B Strep Add ninhydrin reagent for color formation Positive/Purple pos Arrow-like Staph aureus Strep group B
  • 27. Strep agalactiae Group B Streptococcus [GBS] • • Pathogen of neonate – associated with in utero or perinatal organism acquisition during birthing process – Early onset – within 7 days – Late 7 – 28 days from birth process. Pregnant women carry organism in the cervix and/or rectal area. All should be tested at 35 – 37 weeks of pregnancy. – Enrichment methods for GBS is standard of practice and must be used • Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto agar media. This broth can also be used as an enrichment method followed by molecular testing. • Carrot enrichment broth turns orange with growth of group B Strep • GBS is also a pathogen of the elderly – causes bacteremia & UTI • Susceptible to Penicillins and Cephalosporins Carrot Broth
  • 28. It used to be Streptococcus bovis Taxon update – 1) Streptococcus gallolyticus ssp. gallolyticus (formerly S. bovis biotype 1) associated with colonic cancer and endocarditis 2) Strep gallolyticus ssp. pasteurianus (formerly S. bovis biotype II) associated with neonatal meningitis Identification for S. gallolyticus (both ssp) B
  • 29. 6.5% Salt Enterococcus Neg Pos • Two most common species E. faecium and E. faecalis • No well defined virulence factors • Possesses the Group D CHO in the cell wall Neg Pos • Biochemical tests: – Bile esculin positive – growth and black precipitin Bile esculin – Growth in 6.5% salt – PYR positive (Group A beta Strep and Staph PYR lugdunensis also positive)* + – E. faecium = arabinose fermentation positive – E. faecalis = arabinose fermentation negative
  • 30. Enterococcus • • • • Cause a variety of infections and is a pathogen of opportunity Normal flora in the intestine Cause UTI, bacteremia, and abdominal infections Antimicrobial therapy: – Natural resistance to cephalosporins – Ampicillin/Aminoglycoside can be synergistic for therapy in cases of endocarditis • Unusual susceptibility issues – Acquired resistance to vancomycin known as vancomycin resistant enterococcus [VRE] • Van A (E. faecium) resistance genes • Van B (E. faecalis) resistance genes
  • 31. Alpha hemolytic Streptococcus Streptococcus pneumoniae Gram stain = gram positive bullet shaped cocci with capsule Polysaccharide capsule = virulence factor Identification: – – Bile soluble – colonies dissolve in sodium deoxycholate (bile salts) Optochin sensitive – 14mm or greater zone of inhibition around the 6mm optochin disk Colonies dissolved Inhibition >=14mm NO Inhibition
  • 32. Streptococcus pneumoniae • Upper and Lower respiratory tract, sepsis and meningitis • Prevent invasive disease with multi-serotype vaccine • Acquired Resistance to Penicillin due to Penicillin binding proteins (PBP) • Minimum inhibitory concentration (MIC) testing necessary to detect resistance to penicillin – in broth or Etest Etest for • Testing of CSF isolate: Cefotaxime – Penicillin R = >2 mcg/ml • Resistance to Penicillin • Cefotaxime 2nd drug of choice MIC
  • 33. Viridans Streptococcus Several species of alpha hemolytic Streptococcus found as NF in mouth and upper respiratory tract S. mutans S. salivarius S. sanguis S. mitis • Bile esculin slant negative • Not bile soluble Viridans Streptococcus • Optochin resistant - <=13 mm • Cause 30 – 40% cases of sub acute endocarditis / native valve • Can cause abscesses and various infections throughout the body especially in the immune suppressed host • Variable susceptibility patterns, some isolates with elevated MICs to Penicillin
  • 34. Viridans Streptococcus • The Streptococcus anginosis group – S. anginosus S. constellatus S. intermedius • Normal flora in human mouth • More virulent than “normal” viridans Streptococcus, perhaps due to capsule • Cause deep tissue abscesses, bacteremia, endocarditis, intra abdominal infections… • Variable susceptibilities – so need to test
  • 35. Nutritionally Variant Streptococcus • Vitamin B6 (pyridoxal) deficient – so will not grow on BAP without B6 • Will grow in a patient blood culture bottle due to patient’s vit B6 in blood • but not on the subculture 5% Sheep’s blood agar plate ( no vit B6) • Requires streak of Staph aureus that supplies vitamin B6 • Nutritionally variant Strep will satellite @ S. aureus (see pix) • There are 2 species – Abiotrophia – Granulicatella • Endocarditis – – More destructive to valve than than “normal” viridans Strep – Higher MIC’s to Pen or R Satellite streptococcus
  • 37. Gram Negative Cocci • Neisseria species and Moraxella catarrhalis – Small kidney bean shaped cocci in pairs – Oxidase enzyme positive • CTA (Cysteine Trypticase agar) carbohydrate fermentations are used for identification – glucose, maltose, lactose, sucrose – N. gonorrhoeae Gluc + Mal - Lac - Suc – N. meningitidis Gluc + Mal + Lac - Suc – N. lactamica Gluc + Mal + Lac+ Suc– M. catarrhalis all negative Dna’ase +
  • 38. Neisseria species gram negative diplococcus – intra and extra cellular Acinetobacter species – Cocci are more round and not usually in pairs – normal flora in female genital tract
  • 39. Miniature CHO fermentation reactions for Neisseria and Moraxella catarrhalis control + Compare (+) reactions to negative (red) control well Oxidase enzyme spot test: Detects presence of enzyme cytochrome oxidase Add reagent N,N dimethyl-p-phylenediamine oxalate to filter paper with organism positive = purple/ blue + + Full size tube CHO fermentation test
  • 40. Neisseria meningitidis • Meningitis primarily in children and young adults • Hallmark is petechiae (organisms crowd capillaries) – can lead to tissue necrosis and DIC – infection can be rapidly fatal • Carriage in Nasopharynx/ Susceptible to Penicillin • Capsular polysaccharide is the primary virulence factor • Complement deficiencies in 7,8,and 9 can predispose to chronic type disease • Adrenal necrosis is referred to Waterhouse Friederichsen syndrome • Type C is the current endemic strain in the USA – 1*problem in college Freshmen living in dorm • Immunization available for all serotypes except serogoup B
  • 41. Neisseria gonorrhoeae – – – – – – – Acute urethritis, endocervix, ocular, rectal, oropharynx 10 – 20 % female ascend to PID but only 0.5% disseminate Gram stain of urethral discharge useful for male diagnosis Gram stain of cervix not specific for females Transport for culture – charcoal swabs, No refrigeration Medium of choice: Thayer Martin or Martin Lewis Amplification methods [PCR] increases sensitivity of detection and superior to culture – Produces a beta lactamase enzyme and also Chromosomal resistance – – Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent resistance – currently an
  • 42. Moraxella catarrhalis • Pneumonia, ocular, sinusitis, otitis media • Gram stain of sputum can be helpful in diagnosis of pneumonia – Polys with gram negative diplococci • Hockey puck colony – able to push around on agar surface – oxidase enzyme positive – DNA’ase enzyme positive – Resistant to ampicillin by beta lactamase enzyme production
  • 44. Corynebacterium • Corynebacterium species are: – Catalase +, – Diphtheroid morphology (Chinese letter forms) – no spores are produced • C. diphtheriae – agent of Diphtheria Elek plate – Diphtheritic pseudo membrane produced and adherent in throat – Phage mediated toxin is distributed from the membrane causing respiratory paralysis – Toxin detected by Elek immunoprecipitation procedure – Grows well on 5% Sheep’s BAP – Selective media Cysteine tellurite agar Produces black colonies with brown halos – Grown on egg containing Loeffler medium then stain with methylene blue to look for metachromatic granules that are colorful granules characteristic of C. diphtheria
  • 45. Other Corynebacterium • Corynebacterium jeikeium – – normal skin flora bacteria / thrives on lipid – Infects patients with plastic catheters and indwelling devices by tunneling into the device from the skin – Biofilms are formed on the plastic, protecting the organism from antibiotic therapy – Very resistant to most antibiotics – Susceptible to vancomycin and tetracycline only Red is (+) • Corynebacterium urealyticum – Urease reaction – Rapidly urease positive diphtheroid – Urinary tract infection in post renal transplants – Resistant to many antibiotics – vancomycin susceptible
  • 46. Bacillus species Large gram positive rod – boxcar shaped with square ends Can over-decolorize easily and appear reddish Spores produced – causes clearing in rod Catalase enzyme positive Bacillus anthracis - Agent of Anthrax Natural infection of herbivores Skin, pulmonary, GI and CSF infections Unique black eschar skin lesions Bioterrorism - effective due to highly resistant spores that can be easily disseminated into the environment
  • 47. Bacillus Bacillus anthracis culture: medusa head colonies on BAP non-hemolytic on 5% Sheep’s blood agar non-motile Penicillin susceptible With these reactions – contact public health department for assistance with identification Bacillus cereus – food poisoning with rapid onset (1 – 6 hr) of vomiting Preformed emetic toxin in food Fried rice is one of the most common food sources beta hemolytic colony on Sheep’s blood agar and motile
  • 48. Listeria monocytogenes Small gram positive rod Catalase positive / No spores produced Subtle beta hemolysis on Blood agar Tumbling motility on wet mount Umbrella motility in tube media More motile at 25˚C than 35˚C Cold loving – grows well at 4˚C and the reason it is abundant in refrigerated foods Found in dairy products and deli case meats Infections: Bacteremia in pregnant women and can induce still births Bacteremia and CNS in immune suppressed Ampicillin is drug of choice/ resistant to Cephalosporins
  • 49. Erysipelothrix rhusiopathiae Small gram positive rod Catalase negative Alpha hemolytic colony Only G+R that produces hydrogen sulfide (H2S) in a TSI agar H2S production on Triple sugar iron agar (TSI) Human skin infection acquired from swine erysipelas or poultry and is most common in butchers Uncommon isolate in human blood cultures most common in drug addicts high % of endocarditis with extensive damage to valve Intrinsically resistant to Vancomycin
  • 50. Gram negative bacilli Enterics Non fermenters Fastidious
  • 51. Enterobacteriaceae Enteric Gram Negative Bacilli Ferment glucose Almost all are oxidase negative Nitrate reduced to nitrates Enteric Gram Negative Rod Ferment Glucose Ferment Lactose Ex. Escherichia coli Gram negative cell Wall Rod shaped Simple flow chart Identification Strategy Enteric Gram Negative Rod Ferment Glucose Do NOT ferment Lactose Ex. Proteus species Non fermenters Glucose is not fermented Oxidase positive and negative species Gram negative Bacilli Do NOT ferment Glucose Do not ferment Lactose Positive Oxidase Pseudomonas Burkholderia Gram negative Bacilli Do NOT ferment Glucose Do NOT ferment Lactose Negative Oxidase Acinetobacter Stenotrophomonas
  • 52. Enterics that Ferment Glucose • Escherichia coli – – – – Major aerobic NF in intestine #1 cause of UTI Common in abdominal infections and bacteremia Indole reaction is positive • Enterobacter species Indole positive = Robin’s egg blue Green sheen on EMB agar – Enterobacter cloacae and E. aerogenes most common – Environmental organism with low pathogenicity – Enterobacter sakasaki associated with neonatal meningitis • Klebsiella species – K. pneumoniae most common Mucoid colony – Mucoid colony – Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum
  • 53. Enterics that do NOT ferment lactose! No lactose fermentation = No color produced on Macconkey agar • Proteus species Swarming colonies in layers on agar surface • Indole positive – Proteus vulgaris • Indole negative – Proteus mirabilis • Serratia marcescens – Red pigment produced and intensifies at room temp – Infections in immune suppressed – Ventilator associated pneumoniae – Bacteremia
  • 54. Gram negative bacillus MacConkey agar Lactose fermenter fermenter Lactose non Gram stain enteric bacilli – plump Lactose fermentation No lactose fermentation
  • 55. Triple Sugar Iron Agar – detect fermentation of glucose, lactose and/ or Sucrose and the production of hydrogen sulfide [H2S] Glu/lac/suc fermented with gas Glucose Glucose fermented fermented only with H2S No CHO fermentation Non fermenter
  • 56. Extended Spectrum Beta Lactamase Enzymes - “ESBL” • ESBL enzymes are produced by numerous gram negative rods – Confer resistance to Cephalosporins and synthetic Penicillins • Plasmid mediated Tem 1 beta lactamase is the most common enzyme produced – Possible plasmid dissemination makes this an infection control issue • Detect presence of ESBL in a gram negative rod – Double disk test – MIC breakpoints for the cephalosporins are set at very low MIC values to catch subtle resistance • ESBL producing GNR are treated with: – Imipenem [Carbapenemases] – Piperacillin/Tazobactam which blocks beta lactamase enzyme function
  • 57. Typical ESBL Susceptibility Pattern • Escherichia coli – Ampicillin R Double disk test – demonstrates how – Cefazolin R the beta lactam blockers [clavulanate] can stop – Gentamicin R the beta lactamase activity – Cefotetan S (Cephamycins are not cephalosporins)** – Amikacin S Cephalosporin – Ceftazidime R – Cefpodoxime R Cephalosporin + Clavulanate – Piperacillin R – Pip/Tazobactam S (Tazobactam is a Beta lactam blocker) – Imipenem S
  • 58. Carbapenemase Enzyme producing Enterics [CRE] and NDM-1 Enzyme Carbapenemase enzymes are most commonly produced by Klebsiella pneumonia and known as KPC which stands for K. pneumoniae carbapenemase however, they have been found in many genera of enteric gram negative rods Resistant to all carbapenem antibiotics - imipenem, meropenem, ertapenem, doripenem CRE have cross resistance to virtually all antibiotic classes - Polymyxins [colistin, polymyxin B] used for therapy - High fatality rates associated with infections Other resistance mechanisms: New Delhi metallo-beta lactamase [NDM-1] enzymes – resistant to all drug classes including carbapenems
  • 59. Salmonella species • Salmonella spp. – Diarrhea with +/- fever – polys in the stool – Infection from food ingestion – must ingest large #’s of organisms to make you ill (1,00,000 bacteria) – Eggs, meats and contaminated uncooked vegetables – Does not ferment lactose/ produces Hydrogen sulfide – Selective agars: SS and Hektoen – Identification based on biochemical reactions to get to genus and serologic typing “Kaufman White typing scheme” to get to species – O Somatic (cell wall) antigen – Salmonella group B – H flagellar antigens – 2 phases [h1 & h2] speciates the Salmonella – ie Salmonella enteritidis – Vi capsular antigen – Found in S. typhi only
  • 60. Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless because they do not ferment lactose Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] turning the colonies black Normal flora – orange colored due to fermentation of lactose
  • 61. Salmonella typhi • Typhoid fever – fever, sepsis, high fatality rate • Vi capsular antigen surrounds the D cell wall antigen • Boil solution of organism for 15 minutes to destroy the Vi capsular antigen and expose the cell wall antigen D • Serotyping can then be performed for the D antigen • Moustache of H2S in the TSI tube – Carrier state in gallbladder – Ingest organism, clears the bowel – In @ 1 week then enters – Blood stream & Bone Marrow
  • 62. Shigella • Diarrhea, +/-vomiting, fluid loss, polys and blood in stool [10%], tenesmus • Human to human transmission • Low #’s of organisms to make you ill [10 – 100 bacteria] • Non motile and No H2S produced (differ from Salmonella) • Does not ferment lactose • 4 species based on somatic antigen – S. boydii Group C – S. dysenteriae Group A – S. flexneri Group B – S. sonnei Group D
  • 63. Yersinia enterocolitica • • • • • Diarrhea Major reservoir – swine Human usually infected by non pasteurized milk Causes septicemia in iron overload syndromes Unique infection Mesenteric adenitis – RLQ pain which mimics appendicitis • Grows well at 4*C (like Listeria) • CIN agar (Cefsulodin-irgasan-novobiocin) • Has been associated banked blood infections related to Transfusion of infected products
  • 64. Yersinia pestis - Plague • Possible organism of bioterrorism • Obligate flea/ rodent/ flea cycle • Infected Flea bite - leads to Bubonic form/ painful buboes (lymph node swelling) • Pneumonic form- bacteremic spread or patient to patient fatality >=50% • Southwestern part of USA • Grows on BAP, • catalase +, oxidase • Bipolar staining “safety pin”
  • 65. Enteric Glucose Fermenters that are Oxidase + • Vibrio cholerae TCBS AGAR – Rice water stool produced due to mucus flecks in diarrhea – Virulence is due to Toxin – receptor on the epithelial cell – activates adenylate cyclase – increases cAMP – hyper secretion of NaCl and H20 – death from dehydration – It is a Halophilic bacteria which means it is a salt loving organisms, 1% salt actually enhances growth – Selective media – TCBS = thio citrate bile sucrose agar, colony turns yellow due to sucrose fermentation – 01 most virulent due to high production of toxin
  • 66. Classic Gram Negative Rod Gram Stains Vibrio species Campylobacte r Sea gull wings C shaped Enteric gram negative rod Plumb and rectangular
  • 67. Other Vibrio species Vibrio parahaemolyticus • Colony on TCBS medium is green – sucrose negative • Diarrhea from ingestion of raw fish and shellfish • Vibrio vulnificus • Very virulent Vibrio species – infection leads to formation of painful skin lesions with muscle necrosis on lower extremities • Most prevalent in patients with pre existing liver disease • 50% fatality rate even if treated • Ingestion of raw oysters and shellfish from coastal waters • Green or yellow colonies TCBS
  • 68. Do not ferment Glucose Oxidase negative • Acinetobacter – hospital environment and human skin – – – – – Major nosocomial pathogen Acquires antibiotic resistance with pressure Gram negative cocco-baccilli – big cocci Acinetobacter baumannii – glucose oxidizer Acinetobacter lwoffi – glucose non oxidizer • Stenotrophomonas maltophilia – – – – Rapid maltose oxidizer Long Gram negative bacillus Naturally resistant to many antibiotics Nosocomial pathogen – super-colonizer when on long term Imipenem therapy because Stenotrophomonas is naturally resistant to Imipenem
  • 69. Glucose non-fermenter Oxidase Positive Pseudomonas aeruginosa • Fluorescent pigment and blue-green pigment (pyocyanin) • Grape like odor • Growth at 42˚C Ps fluorescens/putida – no growth at 42* • Major pathogen in cystic fibrosis – Mucoid strains – in combination with Burkholderia cepacia can cause major lung damage • Nosocomial pathogen – associated with water
  • 70. Glucose Non Fermenters/oxidase + • Burkholderia cepacia – Dry , yellow colony – oxidase negative – Problem organism in cystic fibrosis • Flavobacterium (Chryseobacterium) meningiosepticum – Associated with fatal septicemia in the neonate – Low virulence – Environmental source
  • 71. Haemophilus species Haemophilus influenza – Causes a variety of infections • Pneumonia, meningitis, otitis media, ocular – Two nutritional factors for growth – – – – • X = hemin • V= NAD (nicotinamide adenine dinucleotide) • Demonstrate by satellite phenomenon or X/V strips (slide 74) Grows on chocolate agar Will not grow on 5% sheep’s blood agar Requires high level C0₂ [5 – 8%] for growth Resistance to Ampicillin by beta lactamase production [1520 %] +
  • 72. Disk test for Beta lactamase Detection • Add bacteria to filter paper impregnated with Nitrocefin or Cefinase test (yellow colored/chromogenic cephalosporin) • Incubate at room temp (30 sec - 60 min) depending on bacteria being tested • Positive result is color change from yellow to red - beta lactamase enzyme breaks down beta lactam ring to form hydrolyzed red end product • Detects resistance to Amp/Pen/Cephalosporin in Haemophilus, N. gonorrhoea , Moraxella catarrhalis, and anaerobic gram negative rods
  • 73. Haemophilus species • H. parainfluenza – – requires V factor only – Normal flora in the upper respiratory tract – One of the HACEK organisms of endocarditis • H. (Aggregatibacter) aphrophilus – – No factor requirements for growth – Infections: abscesses (liver, lung, brain) & endocarditis • H. ducreyi – – Requires X factor – Cause of Chancroid - venereal disease – “school of fish” appearance on stains
  • 74. Demonstrates need for X and V factor Satellite phenomena
  • 75. HACEK group • Oral flora that can become pathogens of endocarditis usually caused by dental procedures • Require 2-4 days to grow in patient blood cultures • Haemophilus species = oxidase neg, catalase neg • Actinobacillus actinomycetom-comitans = oxidase neg, catalase pos • Cardiobacterium hominis = oxidase pos • Eikinella corrodens = oxidase pos, colony pits BAP • Kingella kingii = oxidase pos, hemolytic on BAP, also cause infections in small children (septic joints)
  • 76. Bordetella pertussis • • • Whooping cough – three stages of disease (1)Prodromal – flu like disease – most contagious stage (2) Catarrhal - cough with classic whoop in small children (3) Paroxysmal - recovery phase • Gram = Tiny gram negative coccobacillus • Inhabits Nasopharynx • Harsh cough caused by toxin adhering to bronchial epithelial cells, can last for months, especially deadly to young children • PCR for diagnosis – most sensitive and specific • Charcoal containing media for transport and culture – Regan Lowe Charcoal agar • Reservoir for infection – adults due to waning immunity • Outbreaks due to lack of vaccination of children and adults
  • 77. Pasteurella multocida Primary cause of Cat and Dog bite wound infections Can also cause pneumonia from close animal contact Infections can disseminate to blood stream Found as normal flora animal’s mouth Small gram negative coccobacilli Growth on 5% Sheep’s blood agar Will not grow MacConkey agar (big clue) Oxidase Positive One of very few Gram negative rods sensitive to penicillin
  • 78. Capnocytophaga • Fusiform shaped gram negative rods – very pleomorphic Gliding motility, fingerlike projections from colonies • Oxidase negative, Catalase negative • C. canimorsus – dog bites with bacteremia – high% of infections lead to endocarditis • Other Capnocytophaga species normal flora in the human mouth and • Can infect mouth ulcers induced by chemotherapy – this becomes route of entry into bloodstream
  • 79. Granuloma in bone marrow • • Brucella species Disease – Fever of unknown origin, significant joint pain Small gram negative coccobacilli – slow and difficult to grow in blood cultures – Castaneda biphasic blood culture held for 21 days [old school] • • • – Current: Automated Blood culture systems grow within 5 days Specimens: blood and bone marrow most profitable – Granuloma formation in bone marrow Serology can be used for genera/species diagnosis Brucella species in clinical specimen related to animal species the patient was exposed: – B. abortus – ingestion of raw cow milk – B. melitensis – ingestion raw goat milk, feta cheese – B. suis – contact with pigs B. canis - contact with dogs
  • 80. Campylobacter • Small curved bacilli – shaped like sea gull wings • C. jejuni – agent of diarrhea – – – – – Related to undercooked poultry ingestion Requires selective media containing antibiotics -Skirrow’s blood agar Thrives at 42˚C - can use to selectively culture Campylobacter Requires microaerophilic atmosphere (high CO₂, low O₂) Significant % Guillain-Barre syndrome as sequelae C fetus – Bacteremia in the immune suppressed host / does not cause diarrheal disease C. jejuni – grows at 37˚C and 42˚, hippurate positive C. fetus - grows at 37˚C and 25˚C hippurate negative
  • 81. Francisella tularensis Reservoir – rabbits, rodents, ticks and flies. Humans infected by bug bites or directly from exposure to animal blood Strongly associated with skinning rabbits with bare hands Bacteria can penetrate small breaks in skin: cause painful skin lesions – enlarged lymph nodes – leading to bacteremia (ulcerogladular tularemia) Pneumonia Great hazard to lab workers Fastidious small gram negative cocco-bacillus Culture media requires cysteine*
  • 82. Helicobacter pylori • Small curved bacilli • Cause of acute gastritis • Rapidly!! And strongly urease positive – used for detection directly from antral biopsy tissue • Difficult to grow in culture • Stool antigen – diagnose and test of cure • Serum antibody can aid in diagnosis and avoid biopsy
  • 83. Legionella Legionella pneumophila most common [6 serotypes] • Requires cysteine in media for growth • Media of choice Buffered Charcoal Yeast Extract agar with colonies forming in 3-5 days • Does not stain with gram stain in direct specimens • Use silver impregnation stains in tissue • Pulmonary disease associated with water, cooling towers, shower heads, stagnant water • Urinary antigen test to detect L. pneumophila type I infection • Treatment: Erythromycin (macrolide)
  • 84. Bacteria without cell walls • • • Mycoplasma and Ureaplasma – have cell membranes only! Media and transport must contain sterols to protect the membrane to promote survival and growth Do not form discreet colonies on agar plates – must read plates under a microscope to visualize organisms PCR is becoming the detection method of choice since culture is cumbersome and slow (3 – 14 days) • M. pneumoniae – Ureaplasma – community acquired pneumonia – Serology and PCR for diagnosis • Genital mycoplasmas Mycoplasma hominis – M. hominis – fried egg colony, vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes – Ureaplasma urealyticum – rapid urea hydrolysis in broth, NGU & upper genital tract infection, spontaneous abortion, neonatal infections
  • 85. Difficult to grow • Bartonella henselae – Diagnose be PCR and/or serology – Cat scratch disease – exposure to cat and cat boxes (excrement) – Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV • Bartonella quintana – cause of trench fever/ vector is the body louse/Diagnose by Serology • C. trachomatis Serovars L1,L2,& L3 cause of Lymphogranuloma venereum – Serology / clinical findings – lymphatics and lymph nodes involved • C. pneumoniae (TWAR agent)- Pneumonia, Diagnose by PCR • C. psittaci- psittacosis, pneumonia, exotic parrot exposure/ Serology and epidemiology • Chlamydiae trachomatis – trachoma - eye infection
  • 86. Molecular Testing for Neisseria gonorrhoeae and Chlamydia trachomatis • Amplification of DNA by Polymerase Chain Reaction (PCR) – Urine, Cervix, and urethral most often tested – More sensitive than any culture based system • Sensitivity/Specificity @ 96%/99% • Females most sensitive specimen = cervix • Males good sensitivity with urine / equal to urethral • Has replaced culture methods as “Gold” standard of Elementary bodies from detection infected cells using Iodine staining of inclusions from Culture method Fluorescent antibody stain
  • 87. More difficult to grow • Ehrlichiosis – – – – – – – – Obligate intracellular pathogen Anaplasma, inclusion in the PMN Ehrlichia inclusion in the Monocytes Vector = tick bite fever, leukopenia, thrombocytopenia, elevated serum aminotransferases, no rash (differs Rocky Mountain Spotted Fever) • Found in the south central, southeast , midwest USA • PCR and serology for diagnosis
  • 88. Spirochetes • Borrelia burgdorferi - Lyme’s disease – Primarily found in NE part of USA – Vector = bite of the Ixodes tick • Borrelia recurrentis - Relapsing fever Vector =bite of the human body louse Blood smear observe spirochete
  • 89. Spirochetes • Treponema pallidum – Agent of Syphilis – RPR and VDRL for antibody detection – Molecular methods Darkfield from chancre lesion • Brachyspira – intestinal spirochete found on the brush border of the intestine, ?? Role in disease • Leptospira interrogans – Leptospirosis – Fever with rash and renal involvement – Urine from rats and other animals contaminate water supplies Spirochete – Shepherd’s crook
  • 90. Unusual Bacterial diseases • Granuloma inguinale – Klebsiella (Calymmatobacterium) granulomatis – Rare STD – causes ulcerative genital lesions • Streptobacillus moniliformis – – – – Rat bite fever or Haverhill fever L form or cell wall deficient bacteria Inhibited by SPS in blood culture media Needs serum supplementation to grow, will not grow in a bacteriology culture – Infection obtained from a rat bite
  • 91. Anaerobes • Anaerobic infections can occur in virtually every organ and region of the body • Polymicrobial – both aerobic and anaerobic species in the infections • Endogenous organisms (commensal flora) cause most anaerobic infections – Due to Trauma, vascular or tissue necrosis cutting off the oxygen supply to the involved tissue • Surgery plus antibiotics often necessary • Anaerobic culture collection – must be oxygen free – Gel contaning swabs – Eswab, – Evacuated vials (port o cult)/ oxygen free collection – Do not refrigerate specimens – absorb oxygen and kill anaerobes
  • 92. Anaerobes • PRAS media – pre reduced anaerobically sterile – Media packaged in oxygen free environment • Most common media used – CDC anaerobic blood agar – Kanamycin-vancomycin blood agar – Bile Esculin agar – Thioglycollate broth – Chopped meat glucose broth • Anaerobic gas pack jars – incubate cultures in anaerobic conditions • Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts – generate heat • Dry pack – (Anaeropack) Absorbs O2 and generates CO2 • Anaerobic bacteria can be poorly staining and bizarre or irregularly shaped
  • 94. Gram negative Anaerobic Bacillus • Bacteroides fragilis group – – – – – – – – – – – Pleomorphic gram negative rod Growth in the presence of bile Esculin positive – turning media black Resistant to Penicillin and Kanamycin Most common organisms in human bowel B. fragilis B. ovatus B. thetaiotamicron – indole positive B. uniformis B. vulgatus Growth on bile Black pigment • Resistant to Penicillin by beta lactamase enzyme – • Metronidazole is antibiotic of choice
  • 95. Gram Negative Anaerobic Bacillus • Prevotella and Porphyromonas species – – – – – Will not grow in the presence of bile Will not turn black on esculin media Brick red fluorescence or black pigment Normal commensal flora of the upper respiratory tract Cause respiratory tract infection
  • 96. Fusobacterium Fusiform gram negative bacilli – spindle shaped with pointed ends • Normal flora in upper respiratory tract • Associated with mouth and respiratory tract abscess formation and liver abscess • Vincent’s angina – necrotizing oral infection caused by Fusobacterium species plus spirochetes • Lemierre’s syndrome - oropharyngeal infection that leads to thrombosis in jugular vein, septicemia, high fatality rate, caused by Fusobacterium necrophorum
  • 98. Clostridium species • Gram positive bacilli (boxcar shaped ) with spores – Some rods may over-decolorize and appear red • Clostridium perfringens – most common species – – – – Predominate anaerobic G+R in intestine Double zone of beta hemolysis on BAP Lecithinase produced on egg yolk agar reverse camp test + Lecithinase Reverse Camp Test
  • 99. Clostridium • Clostridium botulinum – – Adult disease – Preformed heat labile toxin ingested in mass produced or home-canned foods – Infant disease - spore ingested from nature, neurotoxin produced in gut, • Begins with constipation and difficult sucking bottle • Honey, soil, household dust – Life threatening neuroparalytic disease • Clostridium tetani – Disease = Tetanus – On Gram stain the cells appear like Tennis racket – Toxin enters due to penetrating skin injury – Disease in Non immunized – Spastic contractions of voluntary muscles, hyperreflexia, lock jaw (trismus)
  • 100. Clostridium • C. septicum – – Bacteremia or Gas Gangrene in patient with underlying malignancy – Normal flora in the GI tract • C. difficile – – Disease = Antibiotic associated colitis – Transmission can also occur in the hospital environment due to the viability of the spores – Diagnosis of infection: EIA methods are not sensitive and are discouraged PCR methods are more sensitive and specific for disease detection – Two toxins produced: Toxin A – enterotoxin causing fluid accumulation Toxin B – potent cell cytotoxin – detected in PCR assays*
  • 101. Actinomyces • Branching gram positive bacilli - do not form spores • Normal flora in the mouth • Capable of forming sulfur granules in tissue – often found on normal tonsil • Actinomyces israelii – associated with oral, thoracic, and abdominal infections, IUD infections B
  • 102. Branching Gram positive rods of Actinomyces – antler like Molar tooth colony Sulfur granule
  • 103. Propionibacterium acnes • • • • • • • Pleomorphic gram positive rod Catalase positive Spot indole positive Normal flora skin, oral, GU and GI Potential contaminate in blood cultures Can be pathogen in cerebral shunt infections Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints • Cultures should be held 7- 14 days to detect • Therapy - Ampicillin
  • 104. Bacterial vaginosis • Clue cells are diagnostic • Mixed anaerobic/aerobic bacterial infection are the two most common organisms in vaginosis: – Gardnerella vaginalis (aerobic gram variable rod) and Mobiluncus (anaerobic curved gram negative rod) – Human blood agar for the culture of Gardnerella, it is beta hemolytic and distinctive • Nugent score Gram (for BV diagnosis) – Healthy = Lactobacillus Gram positive rods – Intermediate = mixed bacterial types – BV = Gardnerella and Mobiluncus on smear