This document provides definitions and information about common aerobic bacteria: - It defines different types of bacteria based on their oxygen requirements including obligate aerobes and anaerobes. - It describes appropriate specimen collection and transport methods for different specimen types. - Key identification tests for common gram positive cocci like Staphylococcus, Streptococcus, and Enterococcus are summarized. - Antibiotic resistance in Staphylococcus aureus including MRSA is discussed. - Identification of clinically relevant beta hemolytic streptococci is covered.

1. Bacteriology Update 2020
Margie Morgan, PhD, D(ABMM)

2. Definitions
• Obligate Aerobe – require high level of oxygen (20%) to grow
• Obligate Anaerobe – >30 min of oxygen exposure can be deadly
• Facultative anaerobes – grow in both aerobic and anaerobic conditions,
most so-named “aerobic” bacteria are actually facultative (ie. E. coli)
• Microaerophilic – grow better with reduced oxygen and elevated
carbon dioxide %
• Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to
oxygen, but grow best anaerobically, example: Clostridium tertium
• Lag Phase - >24 hrs old on agar plates, growth is slowing, not appropriate
for biochemical or susceptibility testing
• Log Phase – <=24 hr growth that is appropriate for all testing
• Stationary phase – Organisms alive but not replicating, appropriate for
transporting specimens

3. Specimen Collection
for Aerobic Bacteriology
Throat / Wound collection
1. Collection swabs should be of polyester fiber or flocked (prickly sponge)
2. Cotton swab fibers should not be used for they trap bacteria and are potentially toxic
3. Specimen is collected on swab then placed in Stuart’s or Amie’s transport media (buffered
solution with peptones) for transport and storage up to 72 hrs.
4. Transport media preserves viability of the bacteria but does not promote growth of
bacteria, provides stasis of numbers prior to plating onto solid media
Urine collection – two methods most often used
1. Container with boric acid used to induce organisms into stationary phase
2. Refrigerate urine within one hour after collect
Both methods maintain original colony count and viability of organisms
Must plate urine within 24 hours of collection
Tissues/Sterile body fluid collection – collect in sterile container

4. Blood Cultures
• Two most important blood culture collection issues
• Preventing contamination: Cleanse collection site using Chlorhexidine
• National Benchmark: Blood culture contamination rate should be <=3%
• Adequate volume of blood drawn per blood culture bottle
• Adult blood culture should approach 8-10 ml of blood per bottle
• One Blood culture consists of two bottles:
• One aerobe and one anaerobe bottle
• Incubated at 35*C for 5 days
• Automated detection instruments for positivity is a
the standard of practice
• Growth is detected by increasing number of bacteria causing an increase in
the amount of C02 in the bottle air space. This triggers a fluorescent
indicator to produce a positive result.

5. Gram stain Procedure
1 minute
Rinse
Primary stain
Mordant 1 minute
Rinse
5-10 seconds
RinseDecolorization
Counter stain
1 minute
Rinse
Peptidoglycan in high amount in Gram positive cell wall traps the Crystal violet, hence blue color for
Gram positive organisms.
Document the color (red/blue) and shape of the stained organism.
Prepare thin film
of specimen on glass slide
Heat or methanol

6. Gram Stain to Assess Quality of Sputum for
Performing Bacterial Culture
• Expectorated sputum is examined for presence of
epithelial cells and neutrophils (WBCs)
• If <10 epithelial cells/Low Power Field (LPF) is
observed – acceptable for bacterial culture
Should also have >25 WBCs/LPF (except in
leukopenia)
• If >10 epithelial cells /LPF
• Sputum is judged to be spit
• Bacterial culture is not performed
• Request made to nursing staff for a “deep
cough” specimen
Bad Sputum
Good Sputum
10X objective

7. Most commonly used agar plated media
Blood agar- 5% sheep’s blood agar
• Used to gauge hemolytic reaction of bacteria (alpha, beta, gamma)
• Culture Gram positive & Gram negative bacteria, and Yeast
Chocolate agar
• “Caramelized” blood agar – more enriched than blood agar medium
• Supports the growth of the same organisms as 5% Sheep’s blood agar
plus fastidious bacteria – such as Haemophilus influenzae and
Neisseria gonorrhoeae

8. Commonly used agar media
• MacConkey agar – Selective and differential medium
• Selective - Supports growth of Gram negative rods but crystal violet
inhibits growth of Gram positive organisms
• Differential - Lactose fermentation can be determined by pink color
produced by colony, neutral red indicator turns pink from acid production
• Lactose fermentation positive = pink
• Non-lactose fermentation negative = no color
• Major branchpoint in enteric Gram negative rod identification

9. MALDI-TOF Mass Spectrometry
Matrix-Assisted Laser Desorption/Ionization – Time of flight
– Identification by analyzing protein fingerprints of bacteria
– Replaced many/most biochemical tests for identification of bacteria

10. MALDI-TOF Theory
• Laser is fired at target containing matrix and sample
• Laser energy is absorbed by the matrix and converted to
heat energy and ionizes the sample.
• Positive ions (proteins) are accelerated through a vacuum
by an applied electrical field
• The time taken for the proteins to reach the detector
depends on their mass/charge ratio (m/z) and creates
spectrograph.
• Each organism species has a different protein composition,
thus giving rise to a specific mass spectrograph.
• The mass spectrograph produced by a sample is then
compared with many thousands stored in a spectrograph
database to see which one it most closely matches. Thus
an identification is achieved.

11. Gram Positive
Cocci
Staphylococcus
Streptococcus
Enterococcus

12. Staphylococcus
Gram positive cocci in clusters – clusters formed
due to bound coagulase or “clumping factor”
Catalase enzyme test = Positive
Staphylococcus aureus Coagulase Negative Staph
Coagulase Enzyme Positive Coagulase Enzyme Negative
+/- Yellow colony
Beta hemolysis on
Sheep’s blood agar plate
White colony
Most not hemolytic
on Sheep’s blood agar
plate

13. Catalase Enzyme Reaction
Negative Positive
Bacteria placed in Hydrogen
Peroxide/ bubbles=positive reaction
Slide Coagulase reaction
Staphylococcus organism emulsified in rabbit
plasma/ mix well/ agglutination is positive rxn.
Positive Coagulase rxn = Staph aureus
Tube Coagulase Reaction
Rabbit plasma inoculated with organism /
Incubate at 35˚C / observe for clot at 4 hours
and if negative read again at 24 hours
Negative tube coagulase
No clot formed/liquid
Coagulase negative Staph
Positive Tube Coagulase
Clot formed
Staph aureus
Slide Coagulase Reaction

14. Staphylococcus aureus
• Virulence mechanisms:
• Protein A – Primary virulence factor, surface protein, ability
to bind immunoglobulin and combat the immune response
• Toxins - act as super antigens, recruit host defense cells that
liberate cytokines with systemic effects
• Disease associated toxins:
• Toxic shock syndrome (TSST-1 toxin)
• Scalded skin syndrome (Exfoliatin (SSS) toxin)
• Soft tissue infection (Panton valentine leukocidin toxin – PVL)
• Food poisoning / Enterotoxins – Toxins stable to
heating at 100*C for 30 minutes
• Bacteremia
• Endocarditis
• Septic arthritis – Primary cause of adult septic arthritis
Exfoliatin toxin
Onion skin
peeling
PVL – soft tissue
abscess

15. Methicillin Resistant Staphylococcus aureus
– Due to altered Penicillin binding proteins (PBP2a) produced
by the mecA gene that codes for resistance to
oxacillin/methicillin /nafcillin resistance (semisynthetic penicillin
antibitotic)
– Ways to detect MRSA
1. Can use molecular PBP2a, produced by the mecA gene to
detect resistance
2. Cefoxitin susceptibility testing is considered a more
sensitive indicator of resistance to these antibiotics and
provides more sensitive detection of MRSA
– All MRSAs are reported resistant to oxacillin, nafcillin and
methicillin and all cephalosporin antibiotics. None of these
antibiotics should be used for therapy
– Vancomycin becomes antibiotic of choice
– Of note: Recently there has been an emergence of mecC
producing MRSA, these are not detected using a mecA test.
They are best detected by testing for resistance to cefoxitin.

16. The “D” Test for Inducible Clindamycin Resistance
• Is Staph aureus really susceptible to Clindamycin?
• During therapy, S aureus isolates resistant to Erythromycin possess
enzymes capable of inducing Clindamycin resistance
• This cannot be detected by regular susceptibility testing
• With the D test – the zone around Clindamycin KB disk will be blunted
to form a “D” shape, meaning Clindamycin was induced by
Erythromycin to be resistant – so called “INDUCIBLE RESISTANCE”
• Clindamycin should be reported as resistant by clindamycin induction
testing and clindamycin should not used for therapy.
D test positive
Inducible resistance
to clindamycin
Do not use for therapy
D test negative
Susceptible to Clindamycin
Clindamycin used for therapy

17. Nares is primary colonization site and primary site used for
surveillance cultures
Culture methods used for nares surveillance cultures:
Chromogenic media is commonly used – It is selective for MRSA
due to the addition of cefoxitin. It is differential due to
chromogenic substrates that turn a specific color to identify Staph
aureus
Molecular assays (MA) can also be used to screen nares for
MRSA colonization. MA increase the sensitivity of detection over
culture methods by 5-10% but greatly increase laboratory costs.
Mupirocin therapy can be placed in nares to eradicate carriage
Chlorhexidine bathing is used to decolonize skin
Methicillin Resistant Staphylococcus aureus
(MRSA) Surveillance cultures to assist with
Hospital Epidemiology

18. Coagulase negative Staph (CNS)
@ 15 species infect humans
• Staph epidermidis – most common in humans
– Major aerobic component of normal skin flora
– Common cause of subacute bacterial endocarditis
– Pathogenicity from cell adhesion factors form
biofilm on biologics and plastics such as heart valves,
Catheters and replacement joints, making treatment difficult
• Staph saprophyticus –
– Urinary tract infection in the child bearing age female,
– This CNS adheres in greater #’s to epithelial cells
– Only CNS resistant to Novobiocin (KB disk test)
White non-hemolytic colony
Resistant to
Novobiocin

19. • Staphylococcus hemolyticus
• Cause catheter line related sepsis
• Beta hemolytic on 5% sheep’s blood agar
• Do not confuse with Staph aureus!
• Staphylococcus lugdunensis
• ? greater pathogenicity than other CNS
• Infections mostly abscess and bacteremia
• Biochemical test of note: PYR test positive = pink
• Closely related organism:
• Micrococcus species
• Gram positive cocci in tetrads
• Mustard yellow colony – environmental contaminate
• Catalase = positive
• Coagulase enzyme = negative
• Does not ferment glucose
• Staphylococcus ferment glucose
Neg Pos
PYR Test
Coagulase Negative Staph (CNS)

20. Streptococcus
Gram positive cocci in chains and pairs
Catalase enzyme = negative
Three groups based on hemolytic reaction
produced with growth on 5% sheep’s blood agar
• Alpha – greening of agar, partial hemolysis of RBCs
• Viridans Streptococcus, Streptococcus pneumoniae,
Granulicatella and Abiotrophia
• Beta – clearing of agar, complete hemolysis of RBCs
• Beta hemolytic Streptococcus such as Streptococcus
pyogenes and Streptococcus agalactiae
• Gamma – no clearing of agar, intact RBCs
• Streptococcus bovis (gallolyticus)

21. Beta Hemolytic
Streptococcus typing
• Beta hemolytic Streptococcus are grouped by the “C” carbohydrate
(CHO) present in the bacteria cell wall. This typing system is known as
the Lancefield typing system.
• Classifies the Beta Strep groups – A, B, C, F, and G, the groups most
commonly isolated with human infections
• The “C” CHO in the cell wall is the antigen used in a slide agglutination
test and it bonds with specific monoclonal antibody of each individual
Streptococcus group. Shown in picture is Strep group A typed using
monoclonal antibody coated latex beads to group A
-
A

22. Streptococcus pyogenes
• Group A Streptococcus [GAS] – very beta hemolytic on blood agar
• Biochemical tests used for identification:
• Bacitracin KB sensitivity test – GAS are inhibited by antibiotic
Bacitracin
• This test is not specific for GAS, inhibition also occurs with
Beta hemolytic Streptococcus group C
• PYR (pyrrolidonyl arylmidase) reaction
• Organism spotted onto moist PYR disk
• 2 min – RT incubation
• Add Cinnamaldehyde reagent
• Pink = positive = Streptococcus pyogenes
• This test is not exclusive for Strep pyogenes –
Enterococcus and Staph lugdunensis also pink (+)
• Therapy : Penicillin, Amoxicillin or Cephalosporin
antibiotics
No resistance reported to these agents
PYR

23. • Streptolysin O and Streptolysin S toxins
• Comprise the ASO titer assay that assists in the diagnosis of GAS
sequelae
• Cell toxins lead to evasion from the immune system
• Toxin activity can be demonstrated on 5% Sheep’s blood agar media
• O toxin is oxygen labile S toxin is oxygen stable
• When both toxins are present, the stabbed area of the media will
demonstrate increased beta hemolysis.
Primary virulence factors:
M Protein – prevents
phagocytosis
Capsule – hyaluronic
capsule prevents
phagocytosis

24. Streptococcus pyogenes Infections
• Diseases –
• Pharyngitis
• Impetigo (1)
• Erysipelas (2)
• Cellulitis (3)
• Necrotizing fascitis (4)
• Puerperal sepsis
• Toxic Shock
• Scarlet fever (5)
1
2
3
4
5

25. Sequelae of Strep pyogenes Infection
Can occur 10 -30 days following impetigo or pharyngitis infection
Rheumatic fever
• Due to inadequate treatment of GAS skin or pharyngitis infection
• Family history (genes), strain of GAS and multiple exposures to GAS
can more likely evolve into sequelae
• Usually occurs in children 5 – 15 years
• Pathogenicity due to molecular mimicry: there is similarity between the proteins
of Strep A and human muscle tissue that cause an autoimmune mechanism that
lead to confusion. The immune system is armed to attack heart (heart valves,
muscle), joint, and bones
• ASO titers will be elevated
• Usually leads to need for valve replacement surgery
Glomerulonephritis
• Post infection with Nephritogenic strain of Strep pyogenes
• Leads to immune mediated destruction of the renal glomeruli
• ASO titers will be elevated
• Usually resolves without therapy

26. Streptococcus agalactiae (GBS)
• Biochemical tests:
– Camp test – performed using a Staph aureus strain that contains Camp factor
streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin production
that produces arrow shaped hemolysis (see pix below)
– Rapid hippurate hydrolysis –
– Hippurate hydrolysis test is used to detect the ability of bacteria to hydrolyze
hippurate into glycine and benzoic acid by action of hippuricase enzyme present in
GBS – 4 hour test. Positive test = purple
pos
Staph aureus
Strep group B
Camp TestHippurate Hydrolysis
Increased area of
hemolysis

27. Strep agalactiae [GBS]
• Pathogen of the elderly – Causes bacteremia and urinary tract infection, acquisition most likely
from the intestine
• Pathogen of neonate bacteremia or CNS – In utero or perinatal organism acquisition during
birthing process, infection in @ 1/2000 births
• Early onset infection – within 7 days of birth
• Late onset infection - within 8 – 28 days of birth
• Treatment: Penicillin or Cephalosporin (3rd generation)
• @ 25% of pregnant women colonized in the cervix and/or rectal area with GBS
• All pregnant must be screened at 35 – 37 weeks of pregnancy for GBS (Regulation)
• Enrichment methods for GBS screening are standard of practice
• Cervix and Rectal swab incubated in an enrichment broth for 18 hours at 35 ˚C then
cultured onto Blood agar. Enrichment broth can also be used to increase sensitivity in
molecular testing methods
• Ampicillin drug of choice to treat GBS positive pregnant woman during delivery
• Susceptibility testing for alternative therapies must be performed in PCN allergy
• Clindamycin testing with Inducible resistance testing confirmation must be done prior to
using Clindamycin

28. • Two most common species
• E. faecium and E. faecalis
• No defined virulence factors
• Group “D” CHO in the cell wall
• Biochemical tests:
• Bile esculin agar = growth/black
• 6.5% NaCl tolerance (turbidity/growth)
• PYR = positive
• E. faecium = arabinose fermentation positive
• E. faecalis = arabinose fermentation negative
Enterococcus
+ -
PYR
Neg Pos

29. Enterococcus
• Pathogen of opportunity
• Usual intestinal normal flora
• Infections include UTI, bacteremia, and abdominal abscess
• Antimicrobial therapy:
• Natural resistance to cephalosporin antibiotics
• Ampicillin plus Aminoglycoside can be synergistic for therapy in cases of
endocarditis
• Vancomycin is an antibiotic of choice
• Unique susceptibility issues
• Acquired resistance to vancomycin known as vancomycin resistant
enterococcus or VRE. Resistance due to acquisition of genetic material:
• Van A (E. faecium) resistance gene
• Van B (E. faecalis) resistance gene

30. Streptococcus bovis (gallolyticus)
(Taxonomy is once again in the process of change)
• Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype 1)
Isolation is associated with colon cancer (73%) when isolated from a
patients blood culture
• Streptococcus gallolyticus ssp. pasteurianus
formerly S. bovis biotype II- associated with neonatal meningitis
• Biochemical reactions:
Bile esculin slant positive
6.5% NaCl no growth
PYR reaction negative
Susceptible to Penicillin
Bile Esculin Positive
6.5% No Growth 6.5% Growth
PYR Negative PYR Positive
Strep gallolyticus Enterococcus

31. Streptococcus pneumoniae
Alpha hemolytic
Gram positive bullet (lancet) shaped cocci in pairs
Polysaccharide capsule = virulence factor and antiphagocytic
Identification:
Bile soluble – colonies dissolve Inhibited by Optochin – ethyl
in sodium deoxycholate (bile) hydrocupreine hydrochloride
Zone of inhibition
must be >=14 mm
Autolytic
Changes

32. Streptococcus pneumoniae
• Normal inhabitant of the upper respiratory tract
• Infections: Upper and Lower respiratory tract infection (Lobar
pneumonia), Sepsis, Meningitis, middle ear, ocular, sinus
• Asplenic and immune suppressed patients particularly at risk
• 13 valent pneumococcal conjugate vaccine aids in preventing invasive
infections – those at risk need vaccination
• Susceptibility issues:
• Acquired Resistance to Penicillin due to Penicillin binding proteins
(PBP)
• If test susceptible,1st line therapies include Penicillin or 3rd
generation Cephalosporin (Ceftriaxone)

33. Viridans Streptococcus
Several species of viridans group Streptococcus are NF in mouth and upper
respiratory tract.
Most commons species include:
S. mutans S. salivarius S. sanguis S. mitis
• Bile esculin = negative
• Bile solubility = negative
• Optochin resistant with a zone size <=13 mm
• Cause 30 – 40% cases of sub acute endocarditis on native valve usually due
to bad dentition
• Can cause abscess and various infections throughout the body in the
immune suppressed host
• Variable susceptibility patterns, some isolates with elevated MICs to
Penicillin. Usual therapy is 3rd generation cephalosporin.
Viridans
Streptococcus
Streptococcus
pneumoniae

34. Viridans Streptococcus
unique species
• Streptococcus anginosis group: includes
• S. anginosus S. constellatus S. intermedius
• Normal flora in human mouth
• More virulent than “normal” viridans Streptococcus, due to capsule
• Grows best when incubated in 5 – 10% CO² incubation = Microaerophilic
• Butterscotch odor to colony
• Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal
infections…
• Variable susceptibilities – so best to do susceptibility testing, always
susceptible to vancomycin

35. Nutritionally Variant Streptococcus
• Vitamin B6 (pyridoxal) deficient –
• Will not grow on agar medium without B6 supplementation
• Will grow in blood culture bottle due to vitamin B6 in patient’s blood
• Will not grow on 5% Sheep’s blood agar plate
• Will grow with Staph aureus streak that supplies vitamin B6
• Nutritionally variant Streptococcus will satellite @ S. aureus streak
• Two species:
• Abiotrophia defectiva
• Granulicatella adiacens
• Endocarditis –
• More destructive to valve than “regular” viridans Streptococci
• Higher MIC’s to Penicillin, susceptible to 3rd generation Cephalosporins.
• Combination therapy: PCN and Gentamicin
Satelliting streptococcus
next to S. aureus streak

36. Rare Opportunistic Gram pos cocci
• Aerococcus ureae – Gram positive cocci in pairs and clusters
• Alpha hemolytic on blood agar , difficult to identify, often confused with viridans
Streptococcus
• Urinary tract pathogen
• Rothia mucilaginosa – Gram positive cocco-baccilli
• Neutropenia and gut problems predispose to infection
• Normal flora in the oral cavity and upper respiratory tract
• Pathogen in dental caries and periodontal disease
• Bad teeth can lead to bacteremia with endocarditis
• Gemella morbillorum–
• Easily over decolorized, Gram positive in pairs – requires CO2 to grow
• Normal flora in oral cavity
• Bacteremia with endocarditis
• Leuconostoc mesenteroides– Gram positive cocci in chains
• Intrinsic resistance to vancomycin
• Bile esculin = negative
• Bacteremia in immune suppressed
• Watch out! Do not confuse with vancomycin resistant enterococcus (VRE)

37. Gram Negative Cocci
Neisseria spp
Moraxella catarrhalis

38. Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
• Small kidney bean shaped cocci in pairs within WBC
• Oxidase enzyme = positive
• CTA (Cysteine Trypticase Agar) carbohydrate fermentations 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 enzyme positive
• N. gonorrhoeae will NOT grow on 5% Sheep’s blood agar
• N. meningitidis will grow on 5% Sheep’s blood agar

39. CHO Fermentation Reactions
Compare (+) yellow reactions to
negative (red) control well
+
+ +
Oxidase enzyme spot test:
Detects production of enzyme cytochrome
oxidase
Add reagent N,N trimethyl-p-
phenylenediamine dihydrochloride to filter
paper with organism smear
positive = blue to purple color
control
Full size CHO fermentation tubes
Growth on Chocolate Agar
Oxidase Enzyme Positive
Glucose + Glucose+/Maltose +
N. gonorrhoeae N. meningitidis
No growth BAP Grows on BAP

40. Neisseria meningitidis
• Colonization occurs in nasopharynx (10-20%)
• African meningitis belt – highest prevalence in world
• Meningitis, usually occurs in children and young adults
• Hallmark - petechiae (organisms crowd into capillaries) leads
to tissue necrosis and DIC (endotoxin)
• Infection can be rapidly fatal (<24hrs)
• Capsular polysaccharide is primary virulence factor
• N. meningitidis serotypes A,B,C Y and W, most common
• Complement deficiencies in factors 7,8,and 9, Eculizumab,
asplenia, and HIV predispose to infection
• Adrenal necrosis in this infection referred to as
Waterhouse Friderichsen syndrome
• Immunization at ages 2m, 12 yr, 16 yr, and in HIV

41. Neisseria gonorrhoeae
• STD – sites of infection include urethrae, endocervix, ocular, rectal,
oropharynx, septic arthritis
• 10-20 % female ascend to PID but only 0.5% disseminate throughout body
• Gram stain of urethral discharge useful for male diagnosis only
• Gram stain of cervix can be problematic due to NF look alike organisms,
such as Acinetobacter species in the female
• Transport for culture – charcoal swabs at room temperature
• Media: Selective Thayer Martin or Martin Lewis agar, chocolate type agar
with increased nutrition and antibiotic trimethoprim
• Amplification methods [PCR] have become standard of practice, improved
sensitivity comparted to culture methods
• Has both beta lactamase enzyme and Chromosomal resistance
mechanisms
• Therapy: Ceftriaxone + Azithromycin or Doxycycline, combination therapy
to prevent development of resistance

42. Amplification Testing for Neisseria gonorrhoeae
and Chlamydia trachomatis
• Amplification methods (Polymerase Chain Reaction (PCR) have become the
standard of practice, combo testing due to possible co-infection
• Urine, cervix/vaginal, throat and rectal – sites most often tested
• More sensitive than culture
• Sensitivity/Specificity @ 96%/99%
• Female: most sensitive specimen is cervix
• Urine @ 10 – 15% less sensitive
• Males: Equal sensitivity with urine and urethral, urine specimen of
choice
• The ancient ways: C. trachomatis culture -
Iodine staining of inclusions
in McCoy cell culture –
previous method of choice
Fluorescent antibody stain of
C. trachomatis infected cell –
positive cell contains green
staining Elementary bodies

43. Moraxella catarrhalis
• Infections: Pneumonia, ocular, sinusitis, otitis media
• Gram stain of sputum can be helpful in diagnosis of pneumonia
(Polys with gram negative diplo-cocci)
• Hockey puck colony – able to push colony across the agar surface
• Biochemical Tests:
• Oxidase enzyme = positive
• DNA’ase enzyme = positive
• Produces a beta lactamase enzyme
• Therapy: Augmentin or 2nd or 3rd generation Cephalosporin

44. Gram Positive Rods
Corynebacteria
Bacillus
Listeria
Erysipelothrix

45. Corynebacterium
• Over 20 species, most are saprophytic
• Human normal flora, especially skin and nares
• Most gamma hemolytic gray colonies on 5% Sheep’s blood agar
• Catalase = positive
• “Diphtheroid” morphology on Gram stain – Gram positive rods in Chinese
letter forms
• No spores produced

46. Corynebacterium diphtheriae
• Agent of Diphtheria
• Diphtheritic adherent pseudo membrane produced in throat
• Non-toxin producing strains can cause wound infection
• Phage mediated exotoxin is distributed from the membrane causing
respiratory paralysis
• Exotoxin detected by Elek immuno-diffusion test
• Grows well on 5% Sheep’s BAP
• (1) Selective medium Cysteine Tellurite agar
• Produces black colonies with brown halos
• (2) Metachromatic granules produced from growth on egg containing
Loeffler medium -stain with methylene blue to observe colorful storage
granules
Elek plate
Tellurite
agar
Metachromatic
granules

47. Other Corynebacterium
• Corynebacterium jeikeium –
• Normal skin flora bacteria / thrives on lipid
• Infects patients with indwelling plastic catheters and devices
leading to bacteremia
• Biofilms are formed on the plastic surface, layers are formed
protecting the organism from antibiotic therapy
• Resistant to most antibiotics
• Susceptible to vancomycin and tetracycline
• Corynebacterium urealyticum –
• Urease = positive
• Cause of urinary tract infection in post renal transplants
• Resistant to many antibiotics but vancomycin susceptible
Red is (+) for
Urease reaction +

48. Bacillus species
• Large Gram positive rods with square ends – boxcar shape
• Can over-decolorize easily and appear reddish on Gram
• Spores produced – clearing in bacillus on Gram
• Catalase enzyme = positive
spores

49. Bacillus anthracis
Anthrax
• Category A agent – highest threat to public health and bioterrorism – spores
are viable for decades in soil
• Natural infection of herbivores – infected animals can have fatal infection
and contaminate the environment for years
• Virulence factors: anthrax toxin and capsular polypeptide
• Infections:
• Wool sorter’s disease – skin infection acquired from handling infected imported
hides produces a unique black eschar skin lesions
• Pneumonia, sepsis, and meningitis

50. • Bacillus anthracis
• Irregular shape to colony border
Medusa head colonies on BAP
No hemolysis on 5% Sheep’s blood agar
Non-motile
Susceptible to penicillin
Alert public health department – possible B. anthracis
• Bacillus cereus –
• Two diseases most common:
• (1) Food poisoning
• Rapid onset of vomiting within 1 – 6 hours
• Preformed emetic toxin produced in food and ingested
• Fried rice is one of the most common food sources
• (2) Traumatic wound infection from contaminated soil
• Beta hemolytic colony on 5% Sheep’s blood agar
• Motile
• Resistant to Penicillin

51. Listeria monocytogenes
• Small Gram positive rod/ no spores
• Catalase = positive
• Subtle beta hemolysis on 5% blood agar
• Similar to Streptococcus agalactiae
• More motile at 25˚C than 35˚C
• Tumbling motility on wet mount
• Umbrella motility in tubed media
• Cold loving – Grows well at 4˚C
• Infection from refrigerated foods
• Cheese – non pasteurized
• Deli case foods
• Milk products approaching expiration date
• Ampicillin drug of choice –
• Intrinsic resistance to Cephalosporins

52. Erysipelothrix
rhusiopathiae
• Small Gram positive rod
• Catalase enzyme = negative
• Alpha hemolysis produced on blood agar plate
• Only G+R that produces hydrogen
sulfide (H2S) - can detect on TSI agar slant
– Human infections acquired from swine with Erysipelas infection
– Isolated rarely from infections post cat bite
– Bacteremia in humans uncommon
• Occurs in IV drug addict
• High % endocarditis /extensive valve damage
– Intrinsic resistance to Vancomycin
H2S production on
Triple sugar iron agar (TSI)
Erysipelas – lacy skin
lesions of swine

53. Gram negative bacilli

54. • Escherichia coli
• Normal flora in human intestine
• #1 cause of UTI [@80% of cases]
• Also, bacteremia, neonatal meningitis,
and abdominal infections
• Ferments lactose when grown on MacConkey agar
• Spot indole reaction = positive / robin’s egg blue color
• Detects breakdown of tryptophan from growth on blood agar
• Green sheen produced when grown on Eosin methylene blue agar (EMB)
• Pathogen of diarrhea:
• Enterotoxigenic (ETEC) E. coli is the cause of traveler’s diarrhea
• Enterohemorrhagic E. coli (EHEC) (such as 0157:H7)
• Bloody diarrhea acquired from eating undercooked meat from an infected
cow – pathogenicity from Shiga toxin production
• Hemolytic uremic syndrome (HUS) can result [hemolytic anemia,
thrombocytopenia, and renal failure] particularly in young children
• When grown on MAC agar with sorbitol / does NOT ferment sorbitol/ most
all E. coli except EHEC ferment sorbitol
Green sheen on
EMB agar
Indole positive
Lactose
fermentor

55. • Enterobacter species
• Enterobacter cloacae complex, most common species
• Environmental GNRs with low pathogenicity
• Usually infects a compromised host
• Enterobacter (Cronobacter) sakazakii associated with neonatal
meningitis
• Klebsiella species
• K. pneumoniae most common species
• Mucoid colony due to capsule
• Currant jelly sputum in alcoholics due to blood mixed with
capsular polysaccharide in sputum

56. Enterics that do NOT ferment lactose!
• Proteus species
Colonies swarm in layers on agar surface
• Proteus vulgaris – spot indole positive
• Proteus mirabilis – spot indole negative
• Normal flora in intestine
- Common in UTI, abdominal infections
• Serratia marcescens
• Produces red pigmentation - intensifies at room temp
• Environmental contaminate
• Causes infection in
• Immune suppressed
• Ventilator associated pneumonia
• Bacteremia

57. 1. Glu/lac/suc
fermented
with gas
2. Glucose
fermented
3. Glucose
fermented
with H2S
4. No CHO
fermentation
Non fermenter
Triple Sugar Iron Agar (TSI)– Used to detect fermentation of glucose,
lactose and/or sucrose and production of hydrogen sulfide [H2S] in GNRs
CHO Fermentation= yellow medium
Gas production= Disruption of the agar
H2S
No CHO
fermentation =
Red medium
1 2 3 4

58. • Salmonella species
• Diarrhea with +/- fever – polys in the stool
• Infection from eating contaminated food (such as raw eggs) or direct
contact with a sick animal
– must ingest large #’s of organisms to make you ill (1,00,000 bacteria),
normal levels of stomach acid is protective
• Does not ferment lactose
• Produces hydrogen sulfide on TSI slant and selective agars
• Motile
• Identification based on biochemical reactions and serologic typing
• Kaufman White serologic typing for speciation of Salmonella
• O Somatic (cell wall) antigen – Salmonella group “B”
• H flagellar antigens – 2 phases [h1 & h2]
• Vi capsular antigen – Salmonella typhi only

59. Salmonella typhi
• Typhoid fever – fever, sepsis, treatable with antibiotics
• Human pathogen - most cases in US (75%) from international travel
• Post typhi infection there can be carriage in gallbladder (passed in feces),
spread by bad hygiene and food handled by person shedding S. typhi
• Ingested organisms enter the bowel, then move into the blood stream and eventually
the bone marrow
• Diagnosis best made with blood cultures and in late stages (>1 month) bone marrow
culture
• Vi capsular antigen can aid in serologic identification
• Moustache of H2S produced in TSI medium slant

60. Shigella
• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool
• Infection : Human to human transmission /control with good hand
hygiene
• Ingestion of low #’s of organisms make you ill [10 – 100 bacteria]
• Does not ferment lactose
• Non motile
• No H2S produced
• 4 species based on somatic antigen
• S. boydii Group C
• S. dysenteriae Group A
• S. flexneri Group B
• S. sonnei Group D

61. Salmonella Shigella Agar (SS agar)
Salmonella and Shigella are colorless
due to lactose not being fermented –
H2S produced by Salmonella turning
colony black
Hektoen agar –
Salmonella produces H2S [Hydrogen sulfide]
producing black colonies
Shigella – green colonies
Normal flora – orange colored due to
fermentation of lactose (E. coli)
Non-Lactose fermenter
Shigella
Salmonella
Normal Flora
Lactose fermented
H2S

62. Yersinia enterocolitica
• Major reservoir – swine
• Humans infected by eating raw or undercooked pork
• Infections:
• Diarrhea
• Septicemia in patients with iron overload syndromes
• Mesenteric adenitis – Infection symptomatic for Right Lower Abdominal
pain which mimics appendicitis
• Infected blood products from transfusion have been reported
• Oxidase = neg, Indole = neg, Urease = pos, Grows well at 4 °C
• CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for Y.
enterocolitica

63. Yersinia pestis - Plague
• Category A agent – call public health!
• Obligate flea/ rodent/ flea cycle in nature
• Human infection usually from a rat flea bite - leads to Bubonic plague
that is the infection of the lymphatic system, forms painful buboes
(lymph node swelling) at site of the bite
• Hemorrhagic lymph nodes spread Y. pestis into blood stream
• Pneumonia develops from blood stream infection
• Fatality >=50%
• Endemic in SW USA
• Grows well on blood agar
• catalase +, oxidase -
• Bipolar staining “safety pin”

64. • Vibrio cholera
• Natural environment saltwater
• Halophilic (salt loving) – salt enhances growth
• Rice water diarrheal stool from mucus flecks (classic)
• Virulence due to enterotoxin production –
– Receptor on epithelial cell in small bowel –
– Activates adenyl cyclase –
– Increases cAMP with hyper secretion of NaCl and H20
– Death from dehydration and metabolic acidosis
• Curved “C” shape Gram negative rod
• Selective medium – thio citrate bile sucrose agar,
(TCBS) yellow color from sucrose fermentation
• Oxidase = positive , grows in 1% salt solution
TCBS Agar

65. • Vibrio parahaemolyticus
• Diarrhea from ingestion of raw oysters
• Usually self limited but serious in immune suppressed
• TCBS medium = green = sucrose fermentation negative
• Vibrio vulnificus
• Infection by ingestion of raw shellfish
• Infections:
• Diarrhea
• Skin infection from injury in water
• Bacteremia - leads to formation of painful skin lesions on
lower extremities with muscle necrosis / This form seen in
patients with liver disease (cirrhosis) – patients have increased
serum iron - 50% fatality rate

66. Classic Gram Negative Rod Gram Stains
Campylobacter Vibrio species
Enteric gram negative rod
Sea gull wings
C shaped
Plumb and rectangular

67. Acinetobacter species –
• Environmental saprophyte and normal flora on human skin
• Gram negative coccoid-bacilli
• Lactose non fermenter
• Oxidase enzyme negative
• Ac. baumannii – nosocomial/common opportunistic pathogen particularly endo-trachs
• Glucose oxidizer
• Can acquire resistance to many antibiotics from antibiotic exposure
• Ac. lwoffi – less commonly isolated
• Glucose non oxidizer
• Stenotrophomonas maltophilia
• Rapid maltose oxidizer
• Gram negative bacillus
• Gun metal gray pigment
• Intrinsically resistant to many antibiotics
• Nosocomial pathogen – super-colonizer after long term carbapenem therapy due to
intrinsic resistance to the carbapenems (Imipenem and Meropenem)

68. Pseudomonas aeruginosa
• Fluorescent & blue-green pigment (pyocyanin)
• Oxidase enzyme positive
• Grape-like odor
• Grows at 42˚C
• Ps fluorescens/putida group– no growth at 42°C
• Major pathogen of cystic fibrosis
• Mucoid strains produced due to polysaccharide capsule
• In combination with Burkholderia cepacia
can cause major lung damage
• Nosocomial pathogen
associated with exposure to water and moist environments
• Intrinsically resistant to many antibiotics

69. • Burkholderia cepacia
• Gram negative tod
• Low virulence – Environmental saprophyte
• Problem organism in cystic fibrosis, particularly in
co-infection of lung with Pseudomonas aeruginosa
• Dry , yellow colony
• Oxidase positive
• Esculin hydrolysis positive
• Chryseobacterium (Elizabethkingia) meningosepticum
• Infections:
• Newborns: fatal meningitis and septicemia in the newborn
• Elderly/immune suppressed: bacteremia
• Low virulence
• Environmental source – water
• Yellow colony,
• Oxidase and Indole positive

70. Haemophilus influenzae
• Transmission – close contact/secretions
• Virulence factor – capsular polysaccharide
• Small pleomorphic Gram negative rod
• Requires 2 nutritional factors for growth:
• X factor = hemin
• V factor = NAD (nicotinamide adenine dinucleotide)
• Grows on chocolate agar (has X and V factor)
• Will not grow on 5% sheep’s blood agar
• Requires C0₂ [5 – 8%] for growth
• Effective vaccine targets invasive infections with H. influenzae type B (HIB)
– effectively eliminating most childhood invasive infections
• Resistance to Ampicillin due to beta lactamase enzyme productions [25 %],
3rd generation Cephalosporin becomes the antibiotic of choice
(Ceftriaxone) for invasive infections

71. Haemophilus influenza
Growth only between X
and V strips
Staph aureus supplies the
X and V factors required
Factor X and V test:
Demonstrates the need for both
X and V factor
Demonstration of the satellite
phenomenon

72. Other Haemophilus species
• H. parainfluenza –
• Requires V (NAD) factor only
• Normal flora in the upper respiratory tract
• Member of HACEK organism of endocarditis
• H. (Aggregatibacter) aphrophilus
• No X and V factor requirements for growth
• Infections: abscess (liver, lung, brain) & endocarditis
• H. ducreyi –
• Requires X factor
• Cause of Chancroid - venereal disease
• Painful necrotizing genital ulcers/inguinal lymphadenopathy
• “School of fish” appearance on stains

73. HACEK group
• Oral flora organisms - Due to poor detention or invasive dental procedures
organisms introduced into bloodstream and infect the heart valves
• Fastidious Gram negative coccobacilli / need 2-4 days to grow in culture
• 5 -10% of community acquired native valve endocarditis (not IV drug
use)
• Haemophilus species oxidase (-) catalase (-)
• Aggregatibacter (Actinobacillus) oxidase (-) catalase (+)
• Cardiobacterium hominis oxidase (+)
• Eikinella corrodens oxidase (+), pits the agar, smells like
bleach
• Kingella kingii oxidase (+), hemolytic on blood agar
• Major cause of septic joint infection in small children

74. Bordetella pertussis
• Whooping cough – 3 stages of disease
(1) Prodromal – flu like disease – most contagious stage
(2) Catarrhal - cough - with classic whoop in small children
toxin adheres to bronchial epithelial cells and cough
continues until toxin wears off – can be months
(3) Paroxysmal - recovery phase
• Human pathogen - inhabits Nasopharynx
• With disease, peripheral blood - Lymphocytosis with atypical
large, irregular and deeply basophilic lymphocytes
• Tiny Gram negative coccobacillus
• Regan Lowe Charcoal agar / growth in 3-5 days
• PCR is now standard of practice / more sensitive
• Reservoir for infection – young adults due to waning immunity.
Reason behind initiative for booster shots in young adults

75. Pasteurella multocida/canis
• Normal flora in many animals (zoonotic)
• Bite wound infections from cats and dogs
• Human pneumonia from close cat and dog contact
• Small Gram negative coccobacilli
• Growth on 5% Sheep’s blood agar
• Non hemolytic grey colony
• No growth on MacConkey agar
• Oxidase positive
• One of very few GNRs that is
sensitive to penicillin

76. Capnocytophaga species
• Fusiform shaped Gram negative rods
• Fingerlike projections from colonies “Gliding”
• Dependence incubation in C02 for growth
• Oxidase negative
• Catalase negative
• Normal mouth flora in humans and animals
• C. canimorsus – Associated with dog bites – high % of bite infections lead
to bacteremia and endocarditis
• Capnocytophaga species (many) NF in humans
• Infect mouth ulcers induced by chemotherapy
• Can lead to bacteremia

77. Brucella species
• Brucellosis, FUO, significant joint pain, intracellular pathogen of the Reticuloendothelial system
• Specimens: Blood and Bone Marrow
• Old: Castaneda biphasic blood culture held for 21 days
• Current: Automated Blood culture systems with growth @ 5 -6 days
• Laboratory acquired Brucellosis is common* Careful
• Serology can assist with chronic disease
• Culture: Small Gram negative coccobacilli, non hemolytic gray colony, requires C02 to grow
• Oxidase positive, urease enzyme positive
• Zoonosis – Infection from ingestion of raw milk, animal exposure, inhalation
• B. abortus – raw cow milk
• B. melitensis – raw goat milk, feta cheese
• B. suis – pigs
• B. canis - dogs
Granuloma in bone
marrow

78. Campylobacter spp.
• C. jejuni – Diarrhea, common cause in US, bacteremia in HIV
and immune suppressed
• Ingestion of undercooked poultry / juice contaminating raw food
• Sea gull shaped, poorly staining Gram negative rod
• Culture requires selective blood agar with antibiotics / Campy-BAP, Skirrow’s BAP
• Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂)
• Sequelae - Significant % Guillain-Barre syndrome
• C fetus – Bacteremia in the immune suppressed host.
• Source cattle and sheep.
• Temperature tolerance aids in identification
C. jejuni – grows at 37˚C and 42˚ C, hippurate hydrolysis positive
C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative

79. • Reservoir – rabbits, rodents, ticks and flies.
• Humans infected by insect bites or from exposure to animal blood
(such as skinning rabbits with bare hands)
• Bacteria can penetrate small breaks in skin:
• cause painful skin lesions
• enlarged lymph nodes
• leading to bacteremia (ulceroglandular tularemia)
• Pneumonia
• Great hazard to lab workers by aerosolization
• Small Gram negative rod/ oxidase negative
• Requires cysteine in culture medium for growth
• Found in chocolate agar but not blood agar
Francisella tularensis

80. Helicobacter pylori
• Acute gastritis – % progress to gastric adenocarcinoma
• Human to human transmission/ fecal - oral route, poor hygiene
• Rapid and strong urease enzyme –
• Can be used for detection directly from gastric antrum biopsy tissue
• Small curved Gram negative bacilli
• Difficult to grow
• Stool antigen/diagnosis and test of cure
• Serum antibody detection obsolete/ not clinically useful
• Organism stained by silver stains in GI
• Treatment – Antibiotics and acidsuppression
Immunohistochemical
stain of gastric biopsy

81. Legionella
pneumophila
• Pulmonary disease – association with water
• Requires cysteine in culture medium for growth
• Buffered Charcoal Yeast Extract agar/ growth 3-5 days
• Will not show on Gram stains using safranin
• Carbol fuchsin counterstain will stain Legionella
• Use silver impregnation stains in tissue
• Urinary antigen test detects L. pneumophila type I infection – the
most common cause of infection
• Serology
• Treatment: Erythromycin (macrolide)
BCYEBlood agar
No growth

82. Bacteria without cell walls
• Mycoplasma spp and Ureaplasma spp – cell membranes only! Media contain sterols to
protect the membrane
• Lack of peptidoglycan cell wall – means unable to Gram stain, no colonies produced on agar,
cannot be treated by antibiotics that act by inhibiting cell wall formation
• M. pneumoniae –
• Community acquired pneumonia
• PCR for diagnosis
• Presence of cold agglutinins
• Genital mycoplasmas
• M. hominis – fried egg on agar surface, vaginitis, cervicitis, postpartum sepsis, neonatal
infections , pre rupture of membranes
• Ureaplasma urealyticum – Dark metal-type appearance, rapid urea hydrolysis in broth,
NGU & upper genital tract infection, spontaneous abortion, neonatal infections
• PCR current method for a more sensitive diagnosis
UreaplasmaMycoplasma hominis

83. Unusual and difficult to grow
• Bartonella henselae
• Cat scratch disease – exposure to cat and cat excrement
• Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV
• Bartonella quintana – cause of trench fever/ vector is the body louse
• Diagnose Bartonella by Serology/PCR
• Chlamydia trachomatis Serovars L1,L2,& L3
Lymphogranuloma venereum – STD that involves
lymphatics and lymph nodes
• Chlamydia pneumoniae (TWAR agent)- Pneumonia
• Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure
• Diagnosis: Serology and PCR assays

84. Unusual and difficult to grow
• Ehrlichiosis – Rickettsia bacteria
• Zoonotic intracellular pathogen
• Vector – Ixodes tick *(hard tick)
• 2 Genera cause Ehrlichiosis
• Anaplasma spp, inclusion (morula) in the PMN
• Ehrlichia spp inclusion in the Monocytes
• Fever, leukopenia, thrombocytopenia,
• Elevated serum aminotransferases,
• No rash (differs from Rocky Mountain Spotted Fever caused by
Rickettsia rickettsii)
• Found in south central, southeast , midwest US
• PCR, serology, and exam of blood smear for diagnosis

85. Spirochetes
• Borrelia burgdorferi - Lyme’s disease
• Primarily found in NE part of US
• Vector = Ixodes tick
• Acute disease: Fatigue, headache, fever, and rash
• Can have progression to chronic disease
• Diagnosis: Serology and PCR
• Borrelia species – Tick borne relapsing fever
• Western United States
• High fever (relapsing) with thrombocytopenia
• Muscle and joint aches
• Vector: tick (Ornithorodos hermsi)
• Diagnosis: Blood smear observe spirochete

86. Spirochetes
• Treponema pallidum
• Syphilis
• RPR and VDRL for antibody detection
• Molecular methods for diagnosis
• 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
Darkfield from chancre lesion
(1) Leptospirosis – Shepherd’s crook
(2) Presence of spirochete in a renal
tubule
(1) (2)

87. Unusual and difficult to grow
• Granuloma inguinale
• Klebsiella (Calymmatobacterium) granulomatis
• Rare STD – causes ulcerative genital lesions
• Streptobacillus moniliformis
• Rat bite fever or Haverhill fever
• Infection from rat bite
• L form - cell wall deficient bacteria
• Inhibited by SPS in blood culture media
• Needs serum supplementation to grow
• Tropheryma whipplei - Whipple’s disease
• Gram positive rod (Actinomycete) distant relative
Mycobacterium avium and M. paratuberculosis
• Found in soil and farm animals
• Diarrhea – can lead to malabsorption Foamy macrophages in the
lamina propria

88. Bacterial vaginosis
• A mixed anaerobic/aerobic bacterial infection
• The normal balance of vaginal flora is disrupted
• Rather benign infection except in pregnancy
• Discharge: Fishy like odor of discharge, alkaline pH >=4.5
• Usual NF organism of Lactobacillus are overgrown by:
• Gardnerella vaginalis (aerobic Gram variable rod)
• Mobiluncus (anaerobic curved Gram negative rod)
• Increased #s Gardnerella vaginalis is the bacterial marker for infection
– Human blood agar media of choice, Beta hemolytic, Gram variable staining
• Clue Cells are diagnostic and more specific than growth in culture
• Molecular probe assays and amplification assays available as part of “women’s health”
screening panels
Clue Cell

89. Anaerobic Bacteria
• Anaerobic infections can occur in virtually every any organ or
region of the body
• Polymicrobial –with both aerobic and anaerobic species
• Endogenous organisms / usually normal flora
• Increase in numbers and cause infection due to trauma, vascular or
tissue necrosis cutting off the oxygen supply to the involved tissue
• Treatment: Usually surgery to restore oxygen and remove
necrotic tissue plus antibiotics
• Anaerobic culture collection
• Gel containing swabs
• ESwab
• Evacuated vials (port o cult)/ oxygen free
• Do not refrigerate specimens – absorb oxygen in cold temperatures
and can more rapidly kill anaerobes

90. Anaerobic culture
• PRAS media – pre reduced anaerobically sterile
• Media packaged in oxygen free environment
• Most common media used
• CDC anaerobic enriched blood agar
• Kanamycin-vancomycin blood agar
• Bile esculin agar
• Thioglycollate broth
• Chopped meat glucose broth
• Anaerobic chambers – perform all culture work in an oxygen free closed
cabinet
• Anaerobic gas pack jars – for anaerobic incubation of agar plates
• Wet pack – add 10 ml water to hydrogen and CO2 generating
envelope/ requires palladium coated catalysts – generates heat
• Dry pack – (Anaeropack) Absorbs O2 and generates CO2
PRAS

91. Anaerobic Gram
Negative Rods
Unusual shapes and sometimes poorly staining by Gram

92. Bacteroides fragilis group
Pleomorphic irregular staining Gram negative rod
• Grows in the presence of bile
• Growth on Bile esculin media is black
• Resistant to Penicillin and Kanamycin
• Normal flora in GI tract
• Infections: Related to the bowel/ GI abscess
• foul smelling/gas produced
• B. fragilis group
• B. fragilis – most common species
• B. ovatus
• B. thetaiotamicron ( indole reaction positive)
• B. uniformis
• B. vulgatus
• Resistant to Penicillin by beta lactamase enzyme / Metronidazole is antibiotic of choice,
however recent reports of resistance is suggesting a need to do susceptibility testing.
Growth on
bile/esculin media
Black pigment from
Esculin production

93. Prevotella and Prophyromonas
–Pleomorphic Gram negative rod
–Normal flora in the upper respiratory tract
–Infections: respiratory tract abscesses
–Will not grow in the presence of bile
–Will not turn black on esculin media
–Unique** Brick red fluorescence and black pigment
formed on Blood agar after one week of incubation

94. Fusobacterium spp.
• F. nucleatum Long thin gram negative
bacilli – spindle shaped, pointed ends
• Normal flora upper respiratory tract
• Infections: mouth and respiratory tract abscess formation and liver
abscess
• Vincent’s angina – necrotizing oral infection caused by
Fusobacterium species and spirochetes
• F. necrophorum – pleomorphic gram negative bacilli that
filaments or chains of rods
• Lemierre’s syndrome - oropharyngeal infection,
leads to thrombosis in jugular vein, septicemia,
with high fatality rate

95. Anaerobic Gram Positive
Rods

96. Clostridium species
• Gram positive bacilli (boxcar shaped ) form spores
• Bacilli may over-decolorize and appear red
• Clostridium perfringens
• Infections: Food poisoining, necrotic tissue abscesses (Clostridial
myonecrosis), bacteremia, cholecystitis
• Most common anaerobic G+R in intestine
• Double zone of beta hemolysis on BAP
• Lecithinase produced on egg yolk agar
• Reverse camp test positive
Lecithinase
Reverse Camp Test

97. • Clostridium botulinum – Botulism
– Adult disease – Preformed heat labile neurotoxin
ingested is mass produced or in home-canned foods
– Infant disease - spore ingested from nature or product
produced in nature such as honey or household dust,
neurotoxin produced in gut
• Begins with constipation and difficult sucking bottle
– Both forms are ife threatening neuroparalytic disease
• Clostridium tetani - Tetanus
• Gram stained cells appear like Tennis racket
• Tetanospasmin toxin enters by penetrating skin injury
• Spastic contractions of voluntary muscles,
hyper-reflexia, lock jaw (trismus)
• Immunization to prevent

98. • C. septicum –
• Bacteremia or Gas Gangrene in patient with underlying
malignancy
• Hematogenous spread from GI tract – no trauma necessary
• Clostridioides (Clostridium) difficile –
• Disease: antibiotic associated colitis, pseudomembranous
colitis from toxin production
• Toxin A – enterotoxin causing fluid accumulation
• Toxin B – potent cell cytotoxin – primary virulence
• Binary toxin – so called Nap1 strain is produces larger amount of
toxin
• Diagnosis of infection:
• EIA methods [toxin A/B] are insensitive but detect active toxin
• PCR methods [toxin A/B] are more sensitive, detect toxin genes
• Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA]
• Infants have @ 70% colonization rate – do not test

99. Actinomyces
• Branching gram positive bacilli –
• do not form spores
• Aerotolerant, but grow best anaerobic
• Normal flora oral, GI, vagina, skin
• Infections: usu. oral/facial (lumpy jaw)
• Form sulfur granules in tissue
• Can be found on normal tonsil
• Actinomyces israelii – associated with oral, thoracic, and
abdominal infections, IUD infections
Bread crumb colonies in broth
Molar tooth colony on agar plates
Penicillin susceptible.

100. Branching Gram positive rods
of Actinomyces – antler like
Molar tooth colony
Sulfur granule
Clumps of Actionomyces

101. Propionibacterium acnes
renamed: Cutibacterium acnes
• Pleomorphic Gram positive rod
• Catalase positive, Indole positive
• Normal flora - skin, oral, GU and GI
• Contaminate in blood cultures from skin NF
• Pathogen of acne vulgaris
• Opportunistic pathogen: cerebral shunt infections and other endovascular and
neurosurgical infections
• Firmly established as significant cause of prosthetic joint infection – particularly
shoulder joints
• Cultures should be held up 7- 14 days
• Therapy - Ampicillin