Bacteriology Update 2017

1. Bacteriology Review
Update 2017

2. Beginning 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 aerobic and anaerobic conditions,
most so-named “aerobic” bacteria are actually facultative
• Microaerophilic – Grow better with reduced oxygen and elevated
carbon dioxide %
• Aerotolerant anaerobes– anaerobe is not killed by prolonged
exposure to oxygen – example: Clostridium tertium
• Lag Phase - >24 hrs old, growth is 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 collection using swab
1. Use swabs made of polyester or sponge like material, do not
use a cotton swab, traps bacteria /potentially toxic
2. After specimen collection, swab placed in Stuart’s or Amies
transport media (buffer solution with peptones)
3. These transport media preserve bacteria viability but do not
promote growth of bacteria
Urine collection – two methods suggested
1. Boric acid to place bacteria in stationary phase
2. Refrigerate within one hour
Purpose to maintain original colony count and maintain viability of
organisms
Tissues/Sterile body fluids – Sterile containers

4. Gram stain Procedure
1 minute
Rinse
Primary stain
Mordant
1 minute
Rinse
5-10 seconds
Rinse
Decolorization
Counter stain
1 minute
Rinse
Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color
Prepare thin film
of specimen on glass slide
Heat or methanol

5. Gram positive cocci
in pairs and chains
Streptococcus
Gram Positive cocci
in clusters
Staphylococcus
Gram positive rod
most like a
Bacillus species
Gram negative bacilli, enteric
Gram negative rod – Fusiform
Shaped – Fusobacterium
species
Visit the Gram stain tutorial
for more Gram stains

6. Assess quality of Sputum for
Bacterial culture with Gram stain
• Sputum specimen is rejected for bacterial culture, if sputum
judged to be “spit” not actually a deep cough specimen
• Perform Gram stain of (?) sputum specimen
• If <=25 epithelial cells – acceptable (right pix)
• If >= 25 epithelial cells /field (left pix)
• 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 Good
Sputum
10X objective 10X objective

7. Most used Agar Media -
BacteriologyBlood agar- 5% sheep’s blood agar
• Gauge hemolytic reaction of bacteria (alpha, beta, gamma)
• Culture a variety of Gram positive, Gram negative bacteria
and Yeast
Chocolate agar
• “Carmelized” blood agar – more enriched than blood agar
medium.
• Will support the growth of the same organisms as 5% Sheep’s
blood agar Plus more fastidious bacteria - like
Haemophilus influenzae and Neisseria gonorrhoeae

8. Most used agar media -
Bacteriology
• MacConkey agar – Supports growth of Gram negative rods (GNR),
and determines ability to ferment lactose, described as Selective
and Differential media
• Selective - crystal violet inhibits Gram positive organisms
• Differential agar - Lactose fermentation can be determined
• Lactose fermentation = pink (neutral red indicator)
• Non-lactose fermentation = no color
• After media is plated, incubate in aerobic incubator at 35˚ C for
24 – 72 hrs

9. MALDI-TOF Mass Spectrometry –
newest wide spread identification method
–Matrix-assisted laser desorption/ionization – time of flight
–Identification by analyzing proteins in bacteria
–Replaced many/most biochemical tests for bacterial ID

10. MALDI-TOF
• Laser is fired at the matrix/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 create spectrograph.
• Each bacterial 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
due to bound coagulase or “clumping factor”
All Staphylococcus are Positive for Catalase Enzyme
Staphylococcus aureus Coagulase Negative
Staph
Coagulase Enzyme Positive Coagulase Enzyme
Negative
+/- Yellow colony
Beta hemolysis
White colony
Most not hemolytic

13. Catalase Enzyme Reaction
Negative Positive
Bacteria added to Hydrogen Peroxide
Blood has innate catalase activity and will
give false positive catalase reaction

14. 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 [clot at either 4 or 24 hrs]
Staph aureus
Tube Coagulase
Reaction

15. 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)
• Food poisoning / Enterotoxins – stable to
heating at 100*C for 30 minutes
• Bacteremia and endocarditis
• Septic arthritis – Primary cause
• Toxins act as super antigens, recruit host
defense cells that liberate cytokines with
systemic effects

16. Unique Susceptibility Issues
MRSA =Methicillin resistant Staphylococcus aureus
–Altered Penicillin binding protein (PBP2) produced by the mecA
gene codes for resistance to oxacillin/methicillin /nafcillin
resistance
–Cefoxitin susceptibility testing is considered a more
sensitive indicator of resistance to oxacillin and provides
better detection of MRSA
–All MRSA are considered (reported) as resistant to
semisythetic penicillins and cephalosporins
–Vancomycin becomes a drug of choice
KB disk resistant

17. The “D” Test
• Is Staph aureus really susceptible to ClindamycinIs Staph aureus really susceptible to Clindamycin
• Why?? During therapy, S aureus isolates resistant toWhy?? During therapy, S aureus isolates resistant to
Erythromycin possess enzymes capable of inducingErythromycin possess enzymes capable of inducing
Clindamycin resistanceClindamycin resistance
• Kirby Bauer zone around Clindamycin will be blunted to form aKirby Bauer zone around Clindamycin will be blunted to form a
“D” if Clindamycin can be induced by Erythromycin to be“D” if Clindamycin can be induced by Erythromycin to be
resistant – so calledresistant – so called INDUCIBLE RESISTANCEINDUCIBLE RESISTANCE..
• Clindamycin should be reported as resistant by clindamycinClindamycin should be reported as resistant by clindamycin
induction and not used for therapy.induction and not used for therapy.
D test
positive
Inducible
resistance
D test negative
Clindamycin can
be used for
therapy

18. Nares is primary colonization site for MRSA and
the most common surveillance site
Culture methods for surveillance:
ChromAgar is commonly used - It is a selective
and differential media containing chromogenic
substrates that turn a specific color to ID Staph
aureus. Cefoxitin is added to the medium to
select for only MRSA.
Molecular assays (MA) can also be used to
screen nares for MRSA. MAs increase the
sensitivity of detection (@5-10%) but increases
laboratory costs.
Mupiricin can be used to nares eradicate carriage
Staphylococcus aureus (MRSA)
Hospital Epidemiology Issue:
Nares surveillance for MRSA

19. Coagulase negative Staph (CNS)
@ 15 species infect humans
• Staph epidermidis – 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 plastics like 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
–Novobiocin resistant by KB disk test
White non-hemolytic
colony
resistant

20. CNS & Related Cocci
• Staphylococcus hemolyticus –
• Can cause line related sepsis
• Hemolytic on blood agar
• Do not confuse with Staph aureus
• Staphylococcus lugdunensis
• ? greater pathogenicity than other CNS species
• Abscesses and bacteremia
• PYR test positive = pink
• Micrococcus species
• Mustard yellow colony – environmental contaminate
• Catalase positive / Gram positive cocci in tetrads
• Tube coagulase negative
• Does not ferment glucose
• Staphylococcus ferment glucose
Po
s
NegPYR TEST

21. Streptococcus
Gram positive cocci in chains and pairs
Catalase enzyme negative
Three groups based on hemolytic pattern on sheep’s blood agar
• Alpha – greening of agar, partial hemolysis of RBCs
• Viridans Streptococcus, Streptococcus pneumoniae,
Granulicatella/Abiotrophia
• Beta – clearing of agar, complete hemolysis of RBCs
• Beta hemolytic Streptococcus groups A - G
• Gamma – no clearing of agar, intact RBCs
• Streptococcus bovis (Strep gallolyticus)

22. Beta
Hemolytic
Streptococc
us
• Beta Streptococcus are grouped using the “C” carbohydrate (CHO)
in the bacteria cell wall
• Identifies Beta Strep groups – A, B, C, F, and G that are the ones
most commonly seen in human infections
• Lancefield grouping system uses the “C” CHO in the cell wall of
Streptococcus as the antigen in a slide agglutination test with
monoclonal antibody (A<B<C<D<F<G) attached to latex beads
See below slide for a positive and negative latex reaction.
• Immunologic typing is more accurate than biochemical testing to
group Beta hemolytic Streptococcus
+
-

23. Streptococcus pyogenes
• Genus/species name for Group A beta Streptococcus [GAS]
Biochemical identification:
• 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 – stands for group “A” Strep
• 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 PYR positive
• Therapy : Penicillin or Cephalosporin antibiotics
No resistance reported to these agents
PYR

24. • Streptolysin O and Streptolysin S toxins
• Comprise the ASO titer determination
• 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 is apparent on 5% Sheep’s
blood agar
Primary virulence factors:
M Protein – prevents
phagocytosis
Capsule – hyaluronic capsule
protects from phagocytosis

25. 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

26. Sequelae of Strep pyogenes
Infections
Rheumatic fever
• Cause: inadequately treated Strep throat
• Rise in the 1990s due to EIA tests being used as the only diagnostic test for
pharyngitis – EIA tests are only 60-70% sensitive – and led to children not
receiving antimicrobial therapy. Negative EIA specimens must be confirmed
by culture
• Similarity between the proteins of Strep A and muscle tissue cause immune
system confusion which leads to immune system to attack heart, joint, and
bones
• Anti-streptolysin O serum test measures the amount of antibodies
produced against Streptococcus group A
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

27. Streptococcus agalactiae
(Group B)
• Biochemical tests for identification:
–Camp test – performed using a Staph aureus strain with
Camp factor streaked perpendicular to group B Strep,
incubate 24 hr. / intensifies toxin production and
produces arrow shaped hemolysis
–Rapid hippurate hydrolysis -
4 hr identification of GBS –
Hippurate disk
Saline inoculated with Group B Strep
Ninhydrin reagent added for color formation
• Positive = Purple
pos
Arrow
Staph aureus
Strep group B

28. Strep agalactiae [GBS]
• Pathogen of the elderly – Bacteremia and urinary tract infection
• Pathogen of neonate – in utero or perinatal organism acquisition during
birthing process
• Early onset – within 7 days
• Late 7 – 28 days from birth process.
• Treatment: Penicillin or Cephalosporin (3rd
generation)
• Pregnant women carry organism in the cervix and/or rectal area.
• All pregnant must be tested at 35 – 37 weeks of pregnancy.
• Enrichment methods for GBS are standard of practice and must be used
• Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then
subculture onto Blood agar. This broth can also be used as an
enrichment method to enrich for molecular testing.
• Carrot enrichment broth turns orange with growth of group B Strep
Carrot Broth

29. Streptococcus bovis
(gallolyticus)
Streptococcus gallolyticus ssp. gallolyticus
formerly S. bovis biotype 1
associated with endocarditis and colon cancer (73%) when isolated
from blood cultures
Strep gallolyticus ssp. pasteurianus
formerly S. bovis biotype II- associated with neonatal meningitis
Biochemical reactions:
Both species have similar biochemical reactions
6.5% NaCL

30. • Two most common species
• E. faecium
• E. faecalis
• No well defined virulence factors
• Possesses the Group “D” CHO in the cell wall
• Biochemical tests:
• Bile esculin positive – growth and black precipitin
• Growth in 6.5% salt
• PYR positive (Group A beta Strep and Staph
lugdunensis also positive)*
• E. faecium = arabinose fermentation positive
• E. faecalis = arabinose fermentation negative
Enterococcus
Bile esculin
PYR
+ -
Neg Pos
PYR

31. Enterococcus
• Variety of infections / Pathogen of opportunity
• Intestinal normal flora
• Cause UTI, bacteremia, and abdominal infections
• Antimicrobial therapy:
• Natural resistance to cephalosporin antibiotics
• Ampicillin plus Aminoglycoside can be synergistic for therapy
in cases of endocarditis
• Unique susceptibility issues
• Acquired resistance to vancomycin known as vancomycin
resistant enterococcus or VRE
• Van A (E. faecium) resistance genes
• Van B (E. faecalis) resistance genes

32. Alpha hemolytic
Streptococcus
Streptococcus pneumoniae
Gram stain = Gram positive bullet (lancet) shaped cocci in pairs
Polysaccharide capsule = virulence factor, antiphagocytic
Identification:
• Bile soluble – colonies dissolve in sodium deoxycholate (bile)
• Optochin sensitive – 14mm or greater zone of inhibition around the
6mm optochin disk – “P” = pneumococcus
Inhibition
>=14mm
NO
Inhibition
Colonies dissolved

33. Streptococcus pneumoniae
• Upper and Lower respiratory tract infection, sepsis and
meningitis
• Multi-serotype vaccine aids preventing invasive infections
• Susceptibility issues:
• Acquired Resistance to Penicillin due to Penicillin binding
proteins (PBP)
• Minimum inhibitory concentration (MIC) testing necessary to
detect resistance to penicillin
• Testing of CSF isolate a result of
• Penicillin = >2 mcg/ml is resistant
• Therapy –
• If susceptible 1st
line therapy is either
Penicillin or 3rd
generation CephalosporinMIC
MIC

34. Viridans Streptococcus
Several species of alpha hemolytic Streptococcus are NF in
mouth and upper respiratory tract
S. mutans S. salivarius S. sanguis S. mitis
• Bile esculin slant = negative
• Not bile soluble
• Optochin resistant with zone size <=13 mm
• Ethylhydrocupreine hydrochloride
• Cause 30 – 40% cases of sub acute endocarditis/native valve
• Can cause abscess and various infections throughout the
body in the immune suppressed host
• Variable susceptibility patterns, some isolates with elevated
MICs to Penicillin
Viridans Streptococcus

35. 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,
perhaps due to capsule
• Cause deep tissue abscess, bacteremia, endocarditis, intra
abdominal infections…
• Variable susceptibilities – so best to do susceptibility testing

36. Nutritionally Variant
Streptococcus
• Vitamin B6 (pyridoxal) deficient –
• will not grow on media without B6 supplementation
• Will grow in a patient blood culture bottle due to
vitamin B6 in patient’s blood
• Will not subculture onto 5% Sheep’s blood agar plate (no vit B6)
• Will grow with Staph aureus streak (supplies vitamin B6)
• Nutritionally variant Strep will satellite @ S. aureus streak
• Two species:
• Abiotrophia spp
• Granulicatella spp
• Endocarditis –
• More destructive to valve than
than “regular” viridans Strep
• Higher MIC’s to Penicillin
Satellite streptococcus
Next to S. aureus streak

37. Rare Opportunistic Gram pos
cocci• Aerococcus ureae – Gram positive cocci in pairs and clusters
• Alpha hemolysis , difficult to identify
• 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
• 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 – Gram positive cocci in chains
• Intrinsic resistance to vancomycin – bacteremia in immune suppressed
• Watch out! Do not confuse with VRE

38. Gram Negative Cocci
Neisseria
Moraxella

39. Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
• Small kidney bean shaped cocci in pairs
• 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 +
• N. gonorrhoae will NOT grow on 5% Sheep’s blood agar
• N. meningitids will grow on 5% Sheep’s blood agar

40. Miniature CHO fermentation reactions for
Neisseria and Moraxella catarrhalis
Compare (+) yellow 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
control
Full size tube CHO fermentation test

41. Neisseria meningitidis
• Meningitis in children and young adults
• Hallmark is petichiae (organisms crowd into capillaries) tissue
necrosis and DIC (endotoxin) /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
Friderichsen syndrome
• Type C is the current endemic strain in the
USA – 1*problem in college Freshmen living in dorm
Type B is the classic epidemic strain
• Immunization available for all serotypes

42. Neisseria gonorrhoeae
• Acute urethritis, endocervix, ocular, rectal, oropharynx,
septic arthritis
• 10 – 20 % female ascend to PID but only 0.5% disseminate
• Gram stain of urethral discharge useful for male diagnosis,
however, cervix Gram stain not specific for females
• Transport – charcoal swabs, Eswab, No refrigeration
• Media: Selective Thayer Martin or Martin Lewis agar
• Amplification methods [PCR] increase sensitivity of
detection and superior to culture – standard of practice
• Produces a beta lactamase enzyme and also Chromosomal
resistance : Therapy: Ceftriaxone + Azithromycin or
Doxycycline to prevent resistance

43. Neisseria gonorrhoeae
gram negative
diplococcus – intra and
extra cellular
Acinetobacter species –
Cocci are larger and more round than
Neisseria, not usually in pairs –
normal flora in female genital tract
Primary reason why female Gram stains
Cannot be trusted.

44. Molecular Testing for NeisseriaMolecular Testing for Neisseria
gonorrhea and Chlamydiagonorrhea and Chlamydia
trachomatistrachomatis
• Amplification of DNA by Polymerase Chain ReactionAmplification of DNA by Polymerase Chain Reaction
(PCR) is the Standard of practice(PCR) is the Standard of practice
• Urine, Cervix, and Urethral most often testedUrine, Cervix, and Urethral most often tested
• More sensitive than culture based systemsMore sensitive than culture based systems
• Sensitivity/Specificity @ 96%/99%Sensitivity/Specificity @ 96%/99%
• Female: most sensitive specimen is cervixFemale: most sensitive specimen is cervix
• Female urine @ 10 – 15% less sensitiveFemale urine @ 10 – 15% less sensitive
• Males virtual equal sensitivity with urine and urethralMales virtual equal sensitivity with urine and urethral
C. trachomatis culture -
Iodine staining of inclusions
in McCoy cell culture –
previous method of choice
Elementary
bodies in
fluorescent
antibody stain of
C. trachomatis
infected cell

45. Moraxella catarrhalis
• Pneumonia, ocular, sinusitis, otitis media
• Gram stain of sputum can be helpful in
diagnosis of Moraxella pneumonia
• Polys with gram negative diplo-cocci
• 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 – Cefinase test can be used
• Therapy: Augmentin or 2nd
or 3rd
generation Cephalosporin

46. Gram Positive Rods
Corynebacteria
Bacillus
Listeria
Erysipelothrix

47. Corynebacterium
•Over 20 species, most not pathogenic
•Gamma hemolytic gray colonies
•Catalase +
•Diphtheroid morphology – Gram positive Chinese letter forms
•No spores produced

48. Corynebacterium
diphtheriae• Agent of Diphtheria
• Diphtheritic adherent pseudo membrane produced in throat
• Phage mediated exotoxin is distributed from the membrane
causing respiratory paralysis (virulence factor)
• Toxin detected by Elek immuno-diffusion test
• Grows well on 5% Sheep’s BAP
• Selective medium Cysteine Tellurite agar
• Produces black colonies with brown halos
• Grow on egg containing Loeffler medium then stain
with methylene blue to observe metachromatic granules -
colorful storage granules characteristic of C. diphtheria
Elek plate
Tellurite agar
Metachromatic granules

49. 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
• Corynebacterium urealyticum –
• Rapidly urease positive diphtheroid
• Urinary tract infection in post renal transplants
• Resistant to many antibiotics – vancomycin susceptible
Red is (+) for
Urease reaction

50. Bacillus species
•Large gram positive rods with square ends - boxcar
•Can over-decolorize easily and appear reddish
•Spores produced – causes clearing in rod
•Catalase enzyme positive
•Most are motile
spores

51. Bacillus anthracis
• Anthrax – Category A agent – highest threat to public health
and bioterrorism
• Natural infection of herbivores – infected animals can have
fatal infection and contaminate the environment for years
• Virulence factors: anthrax toxin and capsular polypeptide
• Skin is the most common presentation -Woolsorter’s disease
– acquired from handling imported hides
• Unique black eschar skin lesions
• Pneumonia, sepsis, and meningitis
uncommon

52. Bacillus
• Bacillus anthracis culture:
• Medusa head colonies on BAP
No hemolysis on 5% Sheep’s blood agar
Non-motile
Penicillin susceptible
With these reactions – Alert public health
department for assistance
• Bacillus cereus –
• Food poisoning with rapid onset (1-6 hr) vomiting
• Preformed emetic toxin in food
• Fried rice is one of the most common food sources
• Traumatic wound infection from contaminated soil
• Beta hemolytic colony on Sheep’s blood agar
• Motile

53. Listeria monocytogenes
• Small gram positive rod/ no spores
• Catalase positive*
• 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 reason why it is
abundant in refrigerated foods like dairy and
deli case meats
• Infections: Bacteremia pregnant & still births
• Infections: Bacteremia and CNS in immune suppressed
• Ampicillin / intrinsic resistance to Cephalosporins

54. Erysipelothrix rhusiopathiae
• Small gram positive rod
• Catalase enzyme negative
• Alpha hemolysis on blood agar
• Only G+R that produces hydrogen
sulfide (H2S) in a TSI agar slant
–Human infections acquired from
swine with erysipelas or poultry
Infections most common in butchers
–Bacteremia uncommon
• most common in drug addict
• High % endocarditis /extensive damage
–Intrinsic resistance to Vancomycin
H2S production on
Triple sugar iron agar (TSI)
Erysipelas – lacy skin
lesions of swine

55. Gram negative bacilli
Enterics – ferment Glucose
Non fermenters – do not ferment Glucose
Fastidious

56. Gram negative bacillus
MacConkey agar
Lactose fermenter
Lactose non
fermenter
Gram stain enteric
bacilli – plump
No lactose
fermentation
Lactose fermentation

57. Enteric GNRs / ferment lactose
• Escherichia coli
• Major aerobic NF in intestine
• #1 cause of UTI [@80%]
• Bacteremia, neonatal meningitis
• Abdominal infections
Biochemicals:
• Spot indole reaction = positive
• Green sheen produced on EMB agar (Eosin methylene blue)
• Pathogen of diarrhea
• Enterotoxigenic E. coli – Traveler’s diarrhea
• Enterohemorrhagic E. coli [0157:H7]
• Bloody diarrhea usually acquired from
eating undercooked cow meat from an infected cow
• HUS – hemolytic uremic syndrome [hemolytic anemia,
thrombocytopenia, and renal failure] particularly in children
• Does not ferment sorbitol – most other E. coli types ferment
Sorbitol
Green sheen on
EMB agar
Mucoid colony
Indole positive
Robin’s egg blue

58. Enteric GNRs that ferment
Lactose•Enterobacter species
• Enterobacter cloacae and E. aerogenes most common
• Environmental organisms with low pathogenicity
• Enterobacter (Cronobacter) sakazakii associated with
neonatal meningitis
•Klebsiella species –
• K. pneumoniae most common
• Mucoid colony
• Currant jelly sputum in alcoholics due to blood mixed with
capsular polysaccharide in sputum

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

60. Glu/lac/suc
fermented
with gas
Glucose
fermented
only
Glucose
fermented
with H2S
No CHO
fermentation
Non fermenter
Triple Sugar Iron Agar – Media for GNR to detect
fermentation of glucose, lactose and/or Sucrose and
production of hydrogen sulfide [H2S]
Fermentation= yellow
Gas = Disruption of the agar
H2S
No fermentation =
red

61. Salmonella species
• Salmonella species
• Diarrhea with +/- fever – polys in the stool
• Infection from food
–Eggs, meats and contaminated uncooked vegetables,
–must ingest large #’s of organisms to make you ill (1,00,000
bacteria), stomach acid is protective
• Does not ferment lactose/ produces Hydrogen sulfide
• Selective agars for growth: SS and Hektoen
• 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 – S. typhi only

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

63. Salmonella typhi
• Typhoid fever – fever, sepsis, high fatality rate
• Vi capsular antigen - surrounds the D cell wall antigen
• Boil solution of S. typhi for 15 minutes to destroy the Vi
capsular antigen and expose the cell wall D antigen
• Serotyping can then be performed for the D antigen
• Moustache of H2S produced in TSI tube
• Carrier state post typhus in gallbladder
• Ingested organism enters the bowel
then in @ 1 week moves on to the
Blood stream & Bone Marrow –

64. Shigella
• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool ,
tenesmus, usually treated with Quinolones
• Infection : Human to human transmission /control with
good hygiene
• Low #’s of organisms to make you ill [10 – 100 bacteria]
• Non motile and No H2S produced ( – Green colonies on
Hektoen agar
• 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

65. Yersinia enterocolitica
•Major reservoir – swine
•Humans infected by drinking non pasteurized milk
•Infections:
• Diarrhea
• Septicemia in iron overload syndromes
• Mesenteric adenitis – RLQ pain which mimics appendicitis
• Infected blood products from transfusion
•Grows well at 4 °C (like Listeria)
•CIN agar (Cefsulodin-irgasan-novobiocin) is
selective agar to grow Y. enterocolitica

66. Yersinia pestis - Plague
• Category A agent
• Obligate flea/ rodent/ flea cycle in nature
• In human, a rat flea bite - leads to Bubonic plague,
infection of the lymphatic system, painful buboes
(lymph node swelling) at site of the bite
• Hemorrhagic lymph nodes spread to blood
• Pneumonic form from blood stream infection
• Fatality >=50%
• Endemic in Southwestern USA
• Grows on blood agar
• catalase +, oxidase -
• Bipolar staining “safety pin”

67. Glucose Fermenters
Oxidase Positive
•Vibrio cholera
• Natural environment is saltwater
• Halophilic (salt loving) – enhances growth
• Rice water diarrheal stool from mucus flecks
• 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 media – thio citrate bile sucrose agar,
yellow color due to sucrose fermentation
TCBS Agar

68. Other Vibrio species
•Vibrio parahaemolyticus
• Diarrhea from ingestion of raw /oysters
• Usually self limited, worse in immune suppressed
• TCBS medium = green = sucrose fermentation negative
•Vibrio vulnificus
• Ingestion of shellfish raised in coastal waters
• Infections:
• Diarrhea
• Skin infection from injury exposed to coastal waters
• Bacteremia - leads to formation of painful skin lesions on lower
extremities with muscle necrosis / This form in patients with pre liver
disease – increased serum iron - 50% fatality rate

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

70. GNRs - Do not ferment Glucose
Oxidase negative
• Acinetobacter species – isolated from hospital environment
and human skin, large gram negative cocco-bacilli
• Ac. baumannii a major nosocomial pathogen
• Glucose oxidizer
• Acquires antibiotic resistance with pressure
• Ac. lwoffi –
• glucose non oxidizer
• Stenotrophomonas maltophilia
• Rapid maltose oxidizer
• Long Gram negative bacillus
• Naturally resistant to many antibiotics
• Gun metal gray pigment
• Nosocomial pathogen – super-colonizer when on long term
Imipenem therapy due to natural resistance to Imipenem

71. Glucose non-fermenter
Oxidase Positive
Pseudomonas aeruginosa
• Fluorescent & blue-green pigment (pyocyanin)
• Grape-like odor
• Growth at 42˚C
• Ps fluorescens/putida group– no growth at 42°C
• Major pathogen of cystic fibrosis
• Mucoid strains [polysaccharide capsule]
• In combination with Burkholderia cepacia
can cause major lung damage
• Nosocomial pathogen –
associated with water & moisture
• Intrinsically resistant to many antibiotics

72. Glucose Non
Fermenters/oxidase +
• Burkholderia cepacia
• Low virulence – found in the environment
• Problem organism in cystic fibrosis:
infection causes extensive lung damage
• Dry , yellow colony
• Oxidase positive
• Esculin 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

73. Haemophilus influenzae
• Variety of infections
• Transmission – close contact/secretions
• Virulence factor – capsular polysaccharide
• Small pleomorphic Gram negative rod
• Requires 2 nutritional factors for growth:
• X = hemin
• V= NAD (nicotinamide adenine dinucleotide)
• Grows on chocolate agar (has X and V factor)
• Will not grow on 5% sheep’s blood agar
• Requires high level C0₂ [5 – 8%] for growth
• Effective vaccine targets the H. influenza type B (HIB) –
effectively eliminated most childhood invasive infections
• Resistance to Ampicillin due to beta lactamase enzyme [20 %],
3rd
gen Cephalosporin becomes the antibiotic of choice

74. Test for Beta lactamaseTest for Beta lactamase
DetectionDetection
• Add bacteria to filter paper impregnated with Nitrocefin orAdd bacteria to filter paper impregnated with Nitrocefin or
Cefinase (yellow colored/chromogenic cephalosporin)Cefinase (yellow colored/chromogenic cephalosporin)
• Incubate at room temp for @ 1 minuteIncubate at room temp for @ 1 minute
• Positive result is a color change fromPositive result is a color change from yellow to redyellow to red ––
• Beta lactamase enzyme breaks down beta lactam ring ofBeta lactamase enzyme breaks down beta lactam ring of
Nitrocefin to a hydrolyzed red end productNitrocefin to a hydrolyzed red end product
• Detects resistance to Amp/Pen/1Detects resistance to Amp/Pen/1StSt
gen Cephalosporin ingen Cephalosporin in
Haemophilus species, N. gonorrhea , Moraxella catarrhalis,Haemophilus species, N. gonorrhea , Moraxella catarrhalis,
and anaerobic Gram negative rodsand anaerobic Gram negative rods
• Does NOT detect the ESBL enzyme of enteric GNRsDoes NOT detect the ESBL enzyme of enteric GNRs

75. Satellite phenomena
Small pleomorphic GNR
On Gram stain
Growth on Chocolate agar
Growth between X and
V strips
Staph aureus supplies the
X and V factors required

76. More Haemophilus species
• H. parainfluenza –
• requires V (NAD) factor only
• Usually normal flora in the upper respiratory tract
• However, one of the HACEK organisms of endocarditis
• H. (Aggregatibacter) aphrophilus – (new taxonomy)
• No X and V 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

77. HACEK group
• Oral flora organism that due to poor detention or
dental procedures become agents of endocarditis
• Fastidious Gram negative coccobacilli
• 5 -10% of community acquired native valve endocarditis
unrelated to IV drug use
• Require 2-4 days to grow in patient blood cultures
• Haemophilus species = oxidase (-) catalase (-)
• Aggregatibacter (Actinobacillus) oxidase (-) catalase (+)
• Cardiobacterium hominis = oxidase (+)
• Eikinella corrodens = oxidase (+), colony pits BAP
• Kingella kingii = oxidase (+), hemolytic on BAP, also
cause of septic joint infection in small children

78. 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
– Pertussis Toxin adheres to bronchial epithelial cells and cough
continues until toxin wears off – can be months
– (3) Paroxysmal - recovery phase
• Tiny Gram negative coccobacillus
• Human pathogen - Inhabits Nasopharynx
• Peripheral blood - Lymphocytosis with atypical
large, irregular and deeply basophilic lymphocytes
• Charcoal media for transport and culture –
Regan Lowe Charcoal agar / growth in 3-5days
• PCR more sensitive than culture for diagnosis
• Reservoir for infection – young adults due to waning immunity.
Reason behind initiative for booster shots in young adults

79. Pasteurella multocida/canis
• Bite wound infections from cats and dogs
• Human pneumonia from close cat and dog contact
• Infections can disseminate to blood stream
• Normal flora in many animals (zoonotic)
• Small Gram negative coccobacilli
• Growth on 5% Sheep’s blood agar
• Non hemolytic grey colony
• Will not grow on MacConkey agar (big clue)
• Oxidase Positive
• One of very few GNR genera that is
sensitive to penicillin

80. Capnocytophaga species
• Fusiform shaped Gram negative rods –
very pleomorphic
• Fingerlike projections from colonies “Gliding”
• Dependence on CO2 incubation for growth
• Oxidase negative, Catalase negative
• Normal mouth flora in humans and animals
• C. canimorsus – Associated with dog bites – high % of these
bite infections lead to bacteremia and endocarditis
• Capnocytophaga species (many) in human
• Infect mouth ulcers induced by chemotherapy
• Bacteremia

81. Brucella species
• Disease – Brucellosis, fever of unknown origin, significant joint pain,
intracellular pathogen of the RES system
• Specimens: Blood and bone marrow (granuloma formation)
• Serology
• Small Gram negative coccobacilli – difficult/slow to grow in culture
• Old: Castaneda biphasic blood culture held for 21 days
• Current: Automated Blood culture systems with growth @ 5 -6 days
• Laboratory acquired Brucellosis is common
• Zoonosis – ingestion of raw milk, animal exposure, inhalation
• Brucella species dependent upon animal species the patient was exposed:
• B. abortus – raw cow milk
• B. melitensis – raw goat milk, feta cheese
• B. suis – pigs
• B. canis - dogs
Granuloma in bone marrow

82. Campylobacter spp.
• “Curved” Gram negative rod
• C. jejuni – Diarrhea, one of most common causes in US,
bacteremia in HIV and immune suppressed
• Ingestion of undercooked poultry / juice contaminating raw food
• Culture requires selective blood agar with antibiotics / Campy-BAP,
Skirrows 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˚, hippurate hydrolysis positive
C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis
negative

83. • Reservoir – rabbits, rodents, ticks and flies. Humans
infected by insect bites or directly from exposure to animal
blood
• Associated with 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 aerolization
• Fastidious small gram negative cocco-bacillus
• Culture media requires cysteine*
Francisella tularensis

84. Helicobacter pylori
• Small curved Gram negative bacilli
• Acute gastritis – can progress to gastric adenocarcinoma
• Human to human transmission/ fecal - oral route
• Rapid and strong urease enzyme – used for detection directly
from gastric antrum biopsy tissue
• Difficult to grow in culture
• Stool antigen/diagnosis and test of cure
• Serum antibody for past exposure
• Organism stained by silver stains in GI
• Treatment – Antibiotics and acid
suppression
Histologic exam of gastric
biopsy
Hematoxylin-eosin stain or
Warthin-Starry Silver stain

85. Legionella
• Legionella pneumophila [6 serotypes]
• Pulmonary disease – strong association with water
• Requires cysteine in culture medium for growth
• Buffered Charcoal Yeast Extract agar/ 3-5 days
• Will not show on Gram stain of specimens
• Use silver impregnation stains in tissue
• Urinary antigen test detects L. pneumophila type I infection –
the most common cause of infection
• Treatment: Erythromycin (macrolide)
BCY
E

86. 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 diagnosis
UreaplasmaMycoplasma hominis

87. 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
lymphatics and lymph nodes involved
• Chlamydia pneumoniae (TWAR agent)- Pneumonia
• Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure
• Diagnosis: Serology and PCR assays

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

89. 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 recurrentis - Relapsing fever
• High fever (relapsing) with thrombocytopenia
• Vector: body louse
• Diagnosis: Blood smear observe spirochete

90. Spirochetes
• Treponema pallidum
• Syphilis
• RPR and VDRL for antibody detection
• Molecular methods
• 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)

91. Unusual Bacterial diseases
•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, will not grow on
routine bacteriology media
• Detailed history aids diagnosis

92. 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

93. • 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
Anaerobic Bacteria

94. Anaerobic culturing
• 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
environment
• 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

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

96. 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
Growth on
bile/esculin media
Black pigment from
Esculin production

97. 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

98. 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 form
filaments or chains of rods
• Lemierre’s syndrome - oropharyngeal infection,
leads to thrombosis in jugular vein, septicemia,
with high fatality rate

99. Anaerobic Gram Positive
Rods

100. 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

101. • 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

102. •C. septicum –
• Bacteremia or Gas Gangrene in patient with underlying
malignancy
• Hematogenous spread from GI tract – no trauma necessary
•C. 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

103. 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.

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

105. Propionibacterium 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