Bacteriology Update 2020

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

Definitions
• Obligate Aerobe – require high level of oxygen (20%) to grow
• Obligate Anaerobe – >30 min of oxygen exposur...

Specimen Collection
for Aerobic Bacteriology
Throat / Wound collection
1. Collection swabs should be of polyester fiber or...

Blood Cultures
• Two most important blood culture collection issues
• Preventing contamination: Cleanse collection site us...

Gram stain Procedure
1 minute
Rinse
Primary stain
Mordant 1 minute
Rinse
5-10 seconds
RinseDecolorization
Counter stain
1 ...

Gram Stain to Assess Quality of Sputum for
Performing Bacterial Culture
• Expectorated sputum is examined for presence of
...

Most commonly used agar plated media
Blood agar- 5% sheep’s blood agar
• Used to gauge hemolytic reaction of bacteria (alp...

Commonly used agar media
• MacConkey agar – Selective and differential medium
• Selective - Supports growth of Gram negati...

MALDI-TOF Mass Spectrometry
Matrix-Assisted Laser Desorption/Ionization – Time of flight
– Identification by analyzing pro...

MALDI-TOF Theory
• Laser is fired at target containing matrix and sample
• Laser energy is absorbed by the matrix and conv...

Gram Positive
Cocci
Staphylococcus
Streptococcus
Enterococcus

Staphylococcus
Gram positive cocci in clusters – clusters formed
due to bound coagulase or “clumping factor”
Catalase enzy...

Catalase Enzyme Reaction
Negative Positive
Bacteria placed in Hydrogen
Peroxide/ bubbles=positive reaction
Slide Coagulase...

Staphylococcus aureus
• Virulence mechanisms:
• Protein A – Primary virulence factor, surface protein, ability
to bind imm...

Methicillin Resistant Staphylococcus aureus
– Due to altered Penicillin binding proteins (PBP2a) produced
by the mecA gene...

The “D” Test for Inducible Clindamycin Resistance
• Is Staph aureus really susceptible to Clindamycin?
• During therapy, S...

Nares is primary colonization site and primary site used for
surveillance cultures
Culture methods used for nares surveill...

Coagulase negative Staph (CNS)
@ 15 species infect humans
• Staph epidermidis – most common in humans
– Major aerobic comp...

• Staphylococcus hemolyticus
• Cause catheter line related sepsis
• Beta hemolytic on 5% sheep’s blood agar
• Do not confu...

Streptococcus
Gram positive cocci in chains and pairs
Catalase enzyme = negative
Three groups based on hemolytic reaction
...

Beta Hemolytic
Streptococcus typing
• Beta hemolytic Streptococcus are grouped by the “C” carbohydrate
(CHO) present in th...

Streptococcus pyogenes
• Group A Streptococcus [GAS] – very beta hemolytic on blood agar
• Biochemical tests used for iden...

• Streptolysin O and Streptolysin S toxins
• Comprise the ASO titer assay that assists in the diagnosis of GAS
sequelae
• ...

Streptococcus pyogenes Infections
• Diseases –
• Pharyngitis
• Impetigo (1)
• Erysipelas (2)
• Cellulitis (3)
• Necrotizin...

Sequelae of Strep pyogenes Infection
Can occur 10 -30 days following impetigo or pharyngitis infection
Rheumatic fever
• D...

Streptococcus agalactiae (GBS)
• Biochemical tests:
– Camp test – performed using a Staph aureus strain that contains Camp...

Strep agalactiae [GBS]
• Pathogen of the elderly – Causes bacteremia and urinary tract infection, acquisition most likely
...

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

Enterococcus
• Pathogen of opportunity
• Usual intestinal normal flora
• Infections include UTI, bacteremia, and abdominal...

Streptococcus bovis (gallolyticus)
(Taxonomy is once again in the process of change)
• Streptococcus gallolyticus ssp. gal...

Streptococcus pneumoniae
Alpha hemolytic
Gram positive bullet (lancet) shaped cocci in pairs
Polysaccharide capsule = viru...

Streptococcus pneumoniae
• Normal inhabitant of the upper respiratory tract
• Infections: Upper and Lower respiratory trac...

Viridans Streptococcus
Several species of viridans group Streptococcus are NF in mouth and upper
respiratory tract.
Most c...

Viridans Streptococcus
unique species
• Streptococcus anginosis group: includes
• S. anginosus S. constellatus S. intermed...

Nutritionally Variant Streptococcus
• Vitamin B6 (pyridoxal) deficient –
• Will not grow on agar medium without B6 supplem...

Rare Opportunistic Gram pos cocci
• Aerococcus ureae – Gram positive cocci in pairs and clusters
• Alpha hemolytic on bloo...

Gram Negative Cocci
Neisseria spp
Moraxella catarrhalis

Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
• Small kidney bean shaped cocci in pairs within WBC
• O...

CHO Fermentation Reactions
Compare (+) yellow reactions to
negative (red) control well
+
+ +
Oxidase enzyme spot test:
Det...

Neisseria meningitidis
• Colonization occurs in nasopharynx (10-20%)
• African meningitis belt – highest prevalence in wor...

Neisseria gonorrhoeae
• STD – sites of infection include urethrae, endocervix, ocular, rectal,
oropharynx, septic arthriti...

Amplification Testing for Neisseria gonorrhoeae
and Chlamydia trachomatis
• Amplification methods (Polymerase Chain Reacti...

Moraxella catarrhalis
• Infections: Pneumonia, ocular, sinusitis, otitis media
• Gram stain of sputum can be helpful in di...

Gram Positive Rods
Corynebacteria
Bacillus
Listeria
Erysipelothrix

Corynebacterium
• Over 20 species, most are saprophytic
• Human normal flora, especially skin and nares
• Most gamma hemol...

Corynebacterium diphtheriae
• Agent of Diphtheria
• Diphtheritic adherent pseudo membrane produced in throat
• Non-toxin p...

Other Corynebacterium
• Corynebacterium jeikeium –
• Normal skin flora bacteria / thrives on lipid
• Infects patients with...

Bacillus species
• Large Gram positive rods with square ends – boxcar shape
• Can over-decolorize easily and appear reddis...

Bacillus anthracis
Anthrax
• Category A agent – highest threat to public health and bioterrorism – spores
are viable for d...

• Bacillus anthracis
• Irregular shape to colony border
Medusa head colonies on BAP
No hemolysis on 5% Sheep’s blood agar
...

Listeria monocytogenes
• Small Gram positive rod/ no spores
• Catalase = positive
• Subtle beta hemolysis on 5% blood agar...

Erysipelothrix
rhusiopathiae
• Small Gram positive rod
• Catalase enzyme = negative
• Alpha hemolysis produced on blood ag...

Gram negative bacilli

• Escherichia coli
• Normal flora in human intestine
• #1 cause of UTI [@80% of cases]
• Also, bacteremia, neonatal mening...

• Enterobacter species
• Enterobacter cloacae complex, most common species
• Environmental GNRs with low pathogenicity
• U...

Enterics that do NOT ferment lactose!
• Proteus species
Colonies swarm in layers on agar surface
• Proteus vulgaris – spot...

1. Glu/lac/suc
fermented
with gas
2. Glucose
fermented
3. Glucose
fermented
with H2S
4. No CHO
fermentation
Non fermenter
...

• Salmonella species
• Diarrhea with +/- fever – polys in the stool
• Infection from eating contaminated food (such as raw...

Salmonella typhi
• Typhoid fever – fever, sepsis, treatable with antibiotics
• Human pathogen - most cases in US (75%) fro...

Shigella
• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool
• Infection : Human to human transmission /control ...

Salmonella Shigella Agar (SS agar)
Salmonella and Shigella are colorless
due to lactose not being fermented –
H2S produced...

Yersinia enterocolitica
• Major reservoir – swine
• Humans infected by eating raw or undercooked pork
• Infections:
• Diar...

Yersinia pestis - Plague
• Category A agent – call public health!
• Obligate flea/ rodent/ flea cycle in nature
• Human in...

• Vibrio cholera
• Natural environment saltwater
• Halophilic (salt loving) – salt enhances growth
• Rice water diarrheal ...

• Vibrio parahaemolyticus
• Diarrhea from ingestion of raw oysters
• Usually self limited but serious in immune suppressed...

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

Acinetobacter species –
• Environmental saprophyte and normal flora on human skin
• Gram negative coccoid-bacilli
• Lactos...

Pseudomonas aeruginosa
• Fluorescent & blue-green pigment (pyocyanin)
• Oxidase enzyme positive
• Grape-like odor
• Grows ...

• Burkholderia cepacia
• Gram negative tod
• Low virulence – Environmental saprophyte
• Problem organism in cystic fibrosi...

Haemophilus influenzae
• Transmission – close contact/secretions
• Virulence factor – capsular polysaccharide
• Small pleo...

Haemophilus influenza
Growth only between X
and V strips
Staph aureus supplies the
X and V factors required
Factor X and V...

Other Haemophilus species
• H. parainfluenza –
• Requires V (NAD) factor only
• Normal flora in the upper respiratory trac...

HACEK group
• Oral flora organisms - Due to poor detention or invasive dental procedures
organisms introduced into bloodst...

Bordetella pertussis
• Whooping cough – 3 stages of disease
(1) Prodromal – flu like disease – most contagious stage
(2) C...

Pasteurella multocida/canis
• Normal flora in many animals (zoonotic)
• Bite wound infections from cats and dogs
• Human p...

Capnocytophaga species
• Fusiform shaped Gram negative rods
• Fingerlike projections from colonies “Gliding”
• Dependence ...

Brucella species
• Brucellosis, FUO, significant joint pain, intracellular pathogen of the Reticuloendothelial system
• Sp...

Campylobacter spp.
• C. jejuni – Diarrhea, common cause in US, bacteremia in HIV
and immune suppressed
• Ingestion of unde...

• Reservoir – rabbits, rodents, ticks and flies.
• Humans infected by insect bites or from exposure to animal blood
(such ...

Helicobacter pylori
• Acute gastritis – % progress to gastric adenocarcinoma
• Human to human transmission/ fecal - oral r...

Legionella
pneumophila
• Pulmonary disease – association with water
• Requires cysteine in culture medium for growth
• Buf...

Bacteria without cell walls
• Mycoplasma spp and Ureaplasma spp – cell membranes only! Media contain sterols to
protect th...

Unusual and difficult to grow
• Bartonella henselae
• Cat scratch disease – exposure to cat and cat excrement
• Bacillary ...

Unusual and difficult to grow
• Ehrlichiosis – Rickettsia bacteria
• Zoonotic intracellular pathogen
• Vector – Ixodes tic...

Spirochetes
• Borrelia burgdorferi - Lyme’s disease
• Primarily found in NE part of US
• Vector = Ixodes tick
• Acute dise...

Spirochetes
• Treponema pallidum
• Syphilis
• RPR and VDRL for antibody detection
• Molecular methods for diagnosis
• Brac...

Unusual and difficult to grow
• Granuloma inguinale
• Klebsiella (Calymmatobacterium) granulomatis
• Rare STD – causes ulc...

Bacterial vaginosis
• A mixed anaerobic/aerobic bacterial infection
• The normal balance of vaginal flora is disrupted
• R...

Anaerobic Bacteria
• Anaerobic infections can occur in virtually every any organ or
region of the body
• Polymicrobial –wi...

Anaerobic culture
• PRAS media – pre reduced anaerobically sterile
• Media packaged in oxygen free environment
• Most comm...

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

Bacteroides fragilis group
Pleomorphic irregular staining Gram negative rod
• Grows in the presence of bile
• Growth on Bi...

Prevotella and Prophyromonas
–Pleomorphic Gram negative rod
–Normal flora in the upper respiratory tract
–Infections: resp...

Fusobacterium spp.
• F. nucleatum Long thin gram negative
bacilli – spindle shaped, pointed ends
• Normal flora upper resp...

Anaerobic Gram Positive
Rods

Clostridium species
• Gram positive bacilli (boxcar shaped ) form spores
• Bacilli may over-decolorize and appear red
• Cl...

• Clostridium botulinum – Botulism
– Adult disease – Preformed heat labile neurotoxin
ingested is mass produced or in home...

• C. septicum –
• Bacteremia or Gas Gangrene in patient with underlying
malignancy
• Hematogenous spread from GI tract – n...

Actinomyces
• Branching gram positive bacilli –
• do not form spores
• Aerotolerant, but grow best anaerobic
• Normal flor...

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

Propionibacterium acnes
renamed: Cutibacterium acnes
• Pleomorphic Gram positive rod
• Catalase positive, Indole positive
...

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