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Bacteriology Update 2023
Margie Morgan, PhD, D(ABMM)
Objectives
• Bacteriology is a massive area of study, and it is impossible to
review everything in this slide set.
• This lecture reviews the most important organisms and
laboratory tests, and relevant antimicrobial therapy
• There has been many taxonomic updates over the last few
years. The updated name is noted in parenthesis, such as
Micrococcus (Kocuria) species.
Begin with Definitions
• Obligate Aerobe – require high level of oxygen (20%) to grow
• Obligate Anaerobe – >30 min exposure to oxygen can be deadly
• Facultative anaerobes – grow in both aerobic and anaerobic conditions,
most of the so-called “aerobic” bacteria are facultative (Escherichia coli)
• Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure
to oxygen, but grow best anaerobically, example: Clostridium tertium
• Microaerophilic – grow best with reduced oxygen and elevated % of
carbon dioxide (Streptococcus anginosus group)
• Lag Phase - >24 hrs old on agar plates, growth is slowing, not
appropriate for biochemical or susceptibility testing
• Stationary phase – Organisms alive but not replicating, suitable for
transportation of specimens to the laboratory
Specimen Collection - Aerobic
Throat / Wound / Abscess
1. Swabs should be either polyester fiber or flocked (prickly sponge)
2. Cotton fiber swabs should not be used, traps bacteria and potentially toxic
3. Specimen is collected with swab then placed in Stuart’s or Amie’s transport media
(buffered solution with peptones) for transport and storage
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
5. Transport media has stability limits / usually <= 72 hours
Urine (2 commonly used methods)
1. Cup with boric acid - organisms in stationary phase for transport
2. Refrigerate urine at 4*C within one hour after collection
3. Both methods maintain original colony count and viability of organisms
4. Plate urine onto agar plates within 24 hours of collection
Tissues/Sterile body fluid
1. Adequate volume transported in sterile container
Blood Culture Collection and Detection
• Two important collection issues
• Prevent contamination: Cleanse collection site using Chlorhexidine
• National Benchmark: Blood culture contamination rate should be <=3%
• Adequate volume of blood inoculated into blood culture bottles
• Adult blood culture (bottles shown) should approach 8-10 ml of blood per bottle
• One blood culture set consists of two bottles:
• One aerobic environment / one anaerobic environment
• Automated blood culture instruments to detect growth has
become the standard of practice
• Incubation for 5 days at 35*C
• Growth is detected by organisms causing an increase in the amount of C02 present
inside the bottle air space. This increase in CO2 triggers a fluorescent indicator to cause
instrument alarm indicating a positive bottle.
Gram Stain Procedure
1 minute
Rinse
Primary stain
Mordant 1 minute
Rinse
5-10 seconds
Rinse
Decolorization
Counter stain
1 minute
Rinse
Gram-positive organisms have a high amount of peptidoglycan in the cell wall. Peptidoglycan traps the
crystal violet in the cell wall which gives Gram-positive organisms a blue color.
Slide interpretation includes Gram-positive or Gram-negative staining (red/blue) designation and shape
of the stained organism.
Prepare thin film of specimen on
glass microscope slide
Heat or methanol
Gram Stain to Assess Quality of Sputum for
Performance of Bacterial Culture/ Is it spit or a
good expectorated deep cough?
• Expectorated sputum is examined for presence of
neutrophils (WBCs) and epithelial cells
• <10 epithelial cells/low power field (LPF) is observed
and >25 WBCs/LPF (exception for leukopenia)
• Sputum acceptable for performing bacterial culture
• If >10 epithelial cells / LPF
• Sputum is judged to be spit
• Bacterial culture should not be performed
• Request made for new “deep cough” specimen
Bad Sputum
Good Sputum
10X objective
Most common agar plating media
5% sheep’s blood agar (blood agar)
• Supports growth of numerous non-fastidious bacteria
and yeast
• Used to gauge hemolytic reactions of bacteria (alpha,
beta, gamma hemolysis)
Chocolate agar
• “Caramelized” blood agar – with added growth
enrichment
• Supports the growth of the same organisms as 5%
sheep’s blood agar plus fastidious bacteria – such as
Haemophilus influenzae and Neisseria gonorrhoeae
• Incubation of plates in a 5-8% CO2 incubator to nurture
for fastidious organimsms
Commonly used agars
• MacConkey agar – Selective and differential
medium for Gram negative bacilli
• Selective for Gram negative bacilli (GNRs)
• Inhibits growth of Gram-positive organisms due
to addition of crystal violet in the medium
• Differential for lactose fermentation
• Fermenters produce a pink colony due to
neutral red indicator turning colony pink
from acid production
• Lactose fermentation positive = pink
• No lactose fermentation = no color
• Lactose fermentation is a major
branchpoint in enteric Gram negative rod
identification
Lactose pos Lactose neg
MALDI-TOF Mass Spectrometry / Advancement in
the Identification of Bacteria
MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization – Time of flight)
– Identification by analyzing protein fingerprints of organisms
– Replaced many/most biochemical tests for bacteria identification
MALDI-TOF Theory
• Laser is fired at target plate containing matrix solution 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 tube by an
applied electrical field
• The time taken for the proteins to travel through the vacuum tube and
reach the detector depends on their mass/charge ratio (m/z) and
creates a spectrograph. This happens in @ 30 seconds.
• 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.
• **MALDI identifies 100’s of organisms, but there are still a few issues to
remember most notably, some organisms are closely related and not
well separated, such as Escherichia coli and Shigella species are too
similar to separate, so biochemicals must be performed to assist.
Safety in a Bacteriology Laboratory
• Risk assessment should be performed to establish safety precautions
for a safe working environment.
• Bacteriology laboratories handling routine cultures are considered
Biosafety Level 2.
• Biosafety Level 2 (BSL-2) is appropriate for handling moderate-risk
agents that cause human disease of varying severity by ingestion or
through percutaneous or mucous membrane exposure.
• Clinical specimens and procedures with possible aerosolization or
splashing performed in Biosafety Level 2 Cabinets with HEPA filtration
• Gloves and impermeable lab coats worn when working with patient
specimens
• Waste management program to properly dispose of biohazards
Gram Positive
Cocci
Most common
genera:
Staphylococcus &
Streptococcus
Staphylococcus
Gram stain
Gram positive cocci in random clusters – clusters
formed due to bound coagulase or “clumping factor”
All are Catalase enzyme test = Positive
Staphylococcus aureus Coagulase Negative Staph
Coagulase Enzyme Positive Coagulase Enzyme Negative
Sheep’s blood agar plate
+/- Yellow colony
Beta hemolysis
Sheep’s blood agar plate
White colony
Most non-hemolytic
Catalase Enzyme Reaction
Negative Positive
Bacteria placed in Hydrogen
Peroxide/ bubbles=positive reaction
Slide Coagulase reaction
Staphylococcus organism emulsified in rabbit plasma/
mixed well. Positive reaction = agglutination
Positive Coagulase enzyme = Staphylococcus aureus
Tube Coagulase Reaction
Rabbit plasma inoculated with organism and
incubated at 35˚C and observe for clot at 4 hours. If
no clot at 4 hours, read again at 24 hours
Negative tube coagulase
No clot formed/liquid =
Coagulase negative Staph
Positive Tube Coagulase
Clot formed at either 4 or
24 hours = Staph aureus
Slide Coagulase Reaction
Staphylococcus aureus
• Virulence mechanisms:
• Protein A – Primary virulence factor, surface protein, ability to
bind immunoglobulin and combat the immune response
• Toxins – many produced, act as super antigens, recruit host
defense cells that liberate cytokines with systemic effects
• Infections:
• Toxic shock syndrome (TSST-1 toxin)
• Scalded skin syndrome (Exfoliatin (SSS) exotoxin)
• Soft tissue infection (Panton valentine leukocidin toxin – PVL)
• Botrymycotic granules can be produced
• Food poisoning (vomiting) – Produce endotoxins stable to
heating at 100*C for 30 minutes
• Bacteremia
• Endocarditis
• Staph aureus is the most common cause of right-sided infective
endocarditis associated with IV drug abuse (@70 percent of cases)
• Primary cause of adult septic arthritis
Exfoliatin exotoxin
Onion skin peeling
PVL – soft tissue abscess
Botryomycotic granule
Methicillin Resistant Staph aureus (MRSA)
• Methicillin resistance develops due to the presence of altered
penicillin binding proteins (PBP2a) produced by the mecA gene.
• PBP2a codes for resistance to oxacillin/ methicillin/ nafcillin
resistance (the semisynthetic penicillin antibiotic class)
• Cephalosporin antibiotics are ineffective and reported as
resistant
• Vancomycin becomes an antibiotic of choice.
• Methods to detect MRSA:
• Resistant to oxacillin by susceptibility testing (old way)
• Resistant to cefoxitin by susceptibility test (better way)
• Molecular tests to detect PBP2a (mecA gene, mecC gene)
Of note: Emergence of mecC producing MRSA, these are not detected using
a mecA based test and best detected using cefoxitin susceptibility testing
The “D” Test for Inducible Clindamycin Resistance
• Question: Is S. aureus susceptible to Clindamycin?
• S. aureus isolates resistant to Erythromycin possess enzymes capable of
inducing Clindamycin resistance to emerge in the S. aureus organism
• Not detected by routine susceptibility testing
• D test – the inhibitory zone around clindamycin KB disk will be blunted to form
a “D” shape, meaning clindamycin was induced by erythromycin to become
resistant, this is termed “Inducible Resistance.”
D test positive
Inducible resistance
to clindamycin
Do not use for therapy
D test negative
Susceptible to clindamycin
Clindamycin can be used for
therapy
Nares is primary colonization site used for surveillance
Methods available for surveillance:
Culture on Chromogenic media
Differential media with chromogenic substrates that turn a specific
color to identify Staph aureus.
Selects for MRSA due to the addition of cefoxitin in the medium
Molecular assays can also be used to screen nares for presence
of MRSA. Increases the sensitivity of detection over culture
methods by 5-10% but greatly increases laboratory cost.
Mupirocin therapy for short term elimination of MRSA carriage in
nares and Chlorhexidine bathing to decolonize skin
Methicillin Resistant Staphylococcus aureus
(MRSA) Surveillance cultures to assist with
Hospital Epidemiology
Chromogenic Agar for MRSA
Coagulase Negative Staph (CNS)
@ 15 species human colonization/infection
• Staph epidermidis – most common species in humans
– Normal skin flora
– Pathogen of opportunity
– Common cause of catheter related bacteremia, endocarditis,
and prosthetic joint infection
– Pathogenicity from cell adhesion factors that form
biofilm on biologics and plastics, difficult to remove or treat
with antibiotics
• Staph saprophyticus –
– Urinary tract infection in the child-bearing age female
– This CNS adheres efficiently to epithelial cells
– The only CNS resistant to antibiotic Novobiocin (KB disk test)
White non-hemolytic colony
Resistant to
Novobiocin
• Staphylococcus lugdunensis
• Normal skin flora in humans and an emerging pathogen
• Pathogen in variety of infections particularly skin and soft
tissue infections
• Biochemical test of note: PYR spot test = pink = positive
• PYR = pyrolidonyl arylamidase
• Micrococcus (Kocuria) species/ close relative of Staph
• Gram positive cocci in tetrads
• Environmental contaminate/ seldom if ever a pathogen
• Mustard yellow colony
• Catalase enzyme = positive
• Coagulase enzyme = negative
Neg Pos
PYR Test
Coagulase Negative Staphylococcus (CNS)
Streptococcus
• Gram positive cocci in chains and pairs
• Catalase enzyme = negative
• Three groups based on hemolytic reaction produced
when grown on 5% sheep’s blood agar
• Alpha – greening of agar, partial hemolysis of RBCs
• Viridans Streptococcus, Streptococcus
pneumoniae, Granulicatella and Abiotrophia spp
• Beta – clearing of agar, complete hemolysis of RBCs
• Beta hemolytic Streptococcus, Streptococcus
pyogenes (A) and Streptococcus agalactiae (B)
• Gamma – no clearing of agar, intact RBCs
• Streptococcus bovis (gallolyticus)
Beta Hemolytic Streptococcus Typing
• Lancefield typing system: Beta hemolytic Streptococcus
are grouped (typed) by identifying the “C” carbohydrate
(CHO) and lipoteichoic acids present in the bacterial cell
wall.
• Classifies Beta Streptococcus into separate groups
(A, B, C, F, and G) identifying the ones commonly
associated with human infection
• The “C” CHO is detected in the Lancefield slide
agglutination test. CHO binds with specific monoclonal
antibody for each individual Streptococcus group.
• Shown in picture is a positive test, with monoclonal
antibody coated latex beads for group A (Strep pyogenes)
agglutinating with the organism -
A
Streptococcus pyogenes
• Group A Streptococcus [GAS] produces an intense ring of beta
hemolysis around the small colony on 5% Sheep’s blood agar
• Biochemical tests used for identification:
• Bacitracin KB sensitivity test – GAS is inhibited by antibiotic
Bacitracin (A) producing a small zone of inhibition
• Not specific for GAS, inhibition also occurs with Beta hemolytic
Streptococcus group C
• PYR (pyrrolidonyl arylmidase) reaction
• Organism spotted onto moist PYR disk
• Wait 2 min – room temperature incubation
• Add cinnamaldehyde reagent
• Pink = positive = Streptococcus pyogenes
• **This test is not exclusive for Strep pyogenes –
Enterococcus and Staph lugdunensis are also positive
• Therapy : Penicillin, Amoxicillin or Cephalosporin antibiotics
No resistance reported to these agents
PYR reaction
+ --
• Streptolysin O and Streptolysin S toxins
• Comprise the ASO titer assay used to diagnosis Strep
pyogenes sequelae (rheumatic fever and GAS
glomerulonephritis)
• These toxins help GAS evade the immune system
• Toxin activity can be visually demonstrated on 5% Sheep’s
blood agar
• Streptolysin O toxin is oxygen labile
• Streptolysin S toxin is oxygen stable
• When both toxins are present, the stabbed area of the
media will demonstrate increased beta hemolysis
Streptococcus pyogenes (GAS) virulence factors
• M Protein prevents phagocytosis
• Hyaluronic capsule prevents phagocytosis
Streptococcus pyogenes / most common
Infections
• Pharyngitis (1)
• Impetigo (2)
• Erysipelas (3)
• Cellulitis (4)
• Necrotizing fasciitis (5)
• Puerperal sepsis
• Toxic Shock
• Scarlet fever (6)
3
4
5
6
1 2
Sequelae of Strep pyogenes Infection
Rheumatic fever
• Inadequate treatment of GAS skin or pharyngitis infection
• Family history, strain of GAS and multiple exposures can more
likely evolve into sequelae, occurs 10-30 days post infection
• Usually occurs in children 5 – 15 years
• Pathogenicity due to molecular mimicry: similarity between the proteins of
Strep A and human muscle tissue causes an autoimmune mechanism that
leads to confusion. The immune system is then armed to attack heart
(heart valves, muscle), joint, and bones not just GAS
• Usually leads to need for valve replacement surgery
Glomerulonephritis
• Post infection with Nephritogenic strain of GAS
• Leads to immune mediated destruction of the renal glomeruli
• Usually resolves without therapy but can progress
Streptococcus agalactiae (GBS)
• Biochemical tests:
– Camp test – Staph aureus strain that contains Camp factor streaked
perpendicular to group B Strep on a 5% sheep’s blood agar plate, Incubate 24 hr.
and view for intensified arrow shaped hemolysis. Positive test = GBS (see pix)
– Hippurate hydrolysis – used to detect the ability of GBS to hydrolyze the
chemical hippurate into glycine and benzoic acid by action of the
hippuricase enzyme – 4 hour incubation. Positive test = purple
pos
Staph aureus
Strep group B
Camp Test
Hippurate Hydrolysis
Increased area of
hemolysis
Streptococcus agalactiae [GBS]
• Pathogen of elderly
• Bacteremia and urinary tract infection,
• Acquisition most likely from the intestine
• Pathogen of neonate
• Bacteremia or central nervous system infection
• 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 8 – 28 days after birth
• Treatment: Penicillin or Cephalosporin (3rd generation)
Streptococcus agalactiae (GBS)
• Most effective way to prevent GBS neonatal infection is detect
colonization in pregnant
• Pregnant colonized (>=25%) in the cervix and/or rectal area
• All pregnant should be screened at 35 – 37 weeks of pregnancy
(Regulation/standard of practice)
• Enrichment methods for GBS screening are mandatory
• Cervix and rectal swab incubated in an enrichment broth for
18 hours at 35 ˚C then cultured onto 5% sheep’s blood agar.
• Initial incubation in Enrichment broth is also used to increase
sensitivity in molecular detection assays
• Ampicillin drug of choice for prophylaxis of pregnant women
testing positive for GBS
• Susceptibility testing for alternative therapies for GBS must be
performed in the penicillin allergic patient (Clindamycin)
• Most common species
• E. faecium and E. faecalis
• No defined virulence factors
• Gamma hemolytic on 5% Sheep’s blood agar
• Gram positive cocci in pairs and short chains
• Biochemical tests:
• Bile esculin agar pos = grows in presence of bile &
reduces esculin to esculetin to produce black color
• 6.5% NaCl tolerance = grows in presence of NaCl
• PYR reaction = positive
• E. faecium = arabinose fermentation positive
• E. faecalis = arabinose fermentation negative
Enterococcus
+ -
PYR
Neg Pos
Enterococcus
• Pathogen of opportunity
• Normal human intestinal flora
• Infections include UTI, bacteremia, and abdominal abscess
• Antimicrobial therapy:
• Intrinsic resistance to cephalosporin antibiotics
• Ampicillin plus Aminoglycoside can be synergistic for therapy of
endocarditis
• Vancomycin is an antibiotic of choice
• Unique susceptibility issues
• Acquired resistance to vancomycin known as vancomycin resistant
enterococcus or VRE. Resistance is due to acquisition of genetic
resistance genes:
• Van A resistance gene for E. faecium
• Van B resistance gene for E. faecalis
Streptococcus bovis (gallolyticus)
• Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype 1)
Isolation from blood culture is associated with colon cancer (73%)
• Streptococcus gallolyticus ssp. pasteurianus (S. bovis biotype 2)
Isolation from CSF in neonatal meningitis
• Gamma hemolytic, Gram positive cocci in pairs and short chains
• 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
Streptococcus pneumoniae
• Alpha hemolytic on 5% Sheep’s blood agar
• Gram positive bullet (lancet) shaped cocci in pairs
• Polysaccharide capsule = virulence factor / antiphagocytic
• Mucoid colony due to large amount of capsule produced
• Identification:
• Bile soluble – colonies dissolve Inhibited by Optochin –
in sodium deoxycholate (bile) ethylhydrocupreine hydrochloride
Zone of inhibition
must be >=14 mm
Autolytic Changes
Looks like tire
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
• 13-20 valent pneumococcal conjugate vaccine aids in preventing
invasive infections – those at risk need vaccination, infants, HIV,
immune suppressed, and elderly
• Susceptibility issues:
• Acquired resistance to Penicillin due to Penicillin binding proteins
• If susceptible,1st line therapy includes Penicillin or 3rd generation
Cephalosporin (Ceftriaxone)
Viridans Streptococcus
• Several species of viridans group Streptococcus are NF in mouth
and upper respiratory tract. Most common species:
S. mutans S. salivarius S. sanguis S. mitis
• Bile esculin negative
• Bile solubility negative
• Optochin resistant (zone size <=13 mm)
• Cause 30 – 40% cases of sub acute endocarditis on native valve,
usually due to bad dentition
• Cause abscess and various infections throughout the body in the
immune suppressed host
• Variable susceptibility patterns can have elevated MICs to
Penicillin so usual therapy is 3rd generation cephalosporin.
Streptococcus
pneumoniae
Viridans
Streptococcus
Viridans Streptococcus
unique species
• Streptococcus anginosis group:
• S. anginosus S. constellatus S. intermedius
• Normal flora in human mouth
• More virulent than “routine” viridans Streptococcus, due to capsule
• Grow best when incubated in 5 – 10% CO² incubation (microaerophilic)
• Butterscotch odor to colony
• Cause deep tissue abscess, bacteremia, endocarditis, and intra abdominal
infection
• Variable susceptibilities – so best to do susceptibility testing, always
susceptible to vancomycin
Nutritionally Variant Streptococcus
• Vitamin B6 (pyridoxal) deficient –
• Will not grow on agar medium without B6 supplementation
• Chocolate agar with supplements will support growth
• Will grow in blood culture bottle due to patient’s blood
containing vitamin B6
• Will not grow on 5% Sheep’s blood agar plate
• Will grow around Staph aureus streak, SA supplies vitamin B6
• MALDI-TOF can definitively identify
• Two genera:
• Abiotrophia defectiva
• Granulicatella adiacens
• Bacteremia and Endocarditis –
• More destructive to valve than “regular” viridans Streptococcus
• Elevated MIC’s to Penicillin, susceptible to 3rd generation
Cephalosporins.
• Combination therapy: Penicillin and Gentamicin can be effective
Satellite
next to S. aureus streak
Opportunistic Gram positive cocci
• Aerococcus ureae – Gram positive cocci in pairs and clusters
• Alpha hemolytic on 5% Sheep’s blood agar , difficult to identify, often confused with
viridans Streptococcus, MALDI-TOF can assist
• Emerging 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
• Can cause 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)
Gram Negative Cocci
Neisseria species
Moraxella catarrhalis
Gram Negative Cocci
• Neisseria species and Moraxella catarrhalis
• Small kidney bean shaped cocci in pairs
• Oxidase enzyme positive
• CTA (Cysteine Trypticase Agar) carbohydrate fermentation
tests are an older method to identify these organisms
• N. gonorrhea Gluc + Mal - Lac - Suc -
• N. meningitidis Gluc + Mal + Lac - Suc -
• N. lactamica Gluc + Mal + Lac+ Suc-
• M. catarrhalis Gluc - Mal - Lac - Suc -
• N. gonorrhea will NOT grow on 5% Sheep’s blood agar
• N. meningitidis will grow on 5% Sheep’s blood agar
Carbohydrate Fermentation Reactions
Compare (+) yellow fermentation
reaction 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
Growth on Chocolate Agar
Oxidase Enzyme Positive
Glucose + Glucose+/Maltose +
N. gonorrhea N. meningitidis
No growth BAP Grows on BAP
Neisseria meningitidis
• Meningitis, usually occurring in children and young adults
• Hallmark - petechiae (organisms crowd into capillaries) leads to
tissue necrosis and disseminated intravascular coagulation(DIC)
caused by organism endotoxin
• Progressive infection can be rapidly fatal (<24hrs)
• Colonization can occur in nasopharynx (10-20%)
• African meningitis belt – highest prevalence of disease in world
• 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 therapy,
asplenia, and HIV predispose to infection
• Adrenal necrosis known as Waterhouse Friderichsen syndrome
• Immunization at ages 2m, 12 yr, 16 yr, and in HIV to prevent
Neisseria gonnorrhea
• Sexually transmitted infection: urethrae,
endocervix, ocular, rectal, oropharynx, septic arthritis
• 10-20 % female ascend to PID but only 0.5% disseminate into bloodstream
• Gram stain of urethral discharge useful for male diagnosis only
• Gram stain of cervix can be problematic due to normal flora look-a-like organisms
• Culture: collect specimen with charcoal containing swabs, do not refrigerate
• Media: Selective Thayer Martin or Martin Lewis agar, chocolate agars with
increased nutritional supplementation and antibiotic trimethoprim
• Resistance: Beta lactamase enzyme and Chromosomal resistance mechanisms
• Emerging resistance in some parts of the world has increased the need to do
susceptibility assessment for patients not responding well to first line therapies
• Therapy: Ceftriaxone + Azithromycin or Doxycycline, combination therapy to
prevent development of resistance
Molecular testing for Neisseria gonorrhea
• Molecular amplification methods are the standard of practice and combo
testing for Chlamydia trachomatis is the norm due to high % of co-infections
• Urine, cervix/vaginal, throat and rectal – sites most often tested
• Molecular testing sensitive (96%) and specific (99%)
• Female: best specimen for diagnosis is cervix
• Urine <=10–15% less sensitive than cervix
• Male: Urine has become the standard specimen for diagnosis
• The older way for diagnosing Chlamydia trachomatis infection:
C. trachomatis culture -
Iodine staining of inclusions
in McCoy cell culture
Fluorescent antibody stain of C.
trachomatis infected cell –
positive cell contains green
staining Elementary bodies
Moraxella catarrhalis
• Colonizes the upper respiratory tract in children
• Infections: Pneumonia (COPD), sinusitis, primary
cause of otitis media in young children
• Gram stain of sputum can be helpful in diagnosis of
pneumonia (PMNs with Gram negative diplo-cocci)
• Hockey puck colony – able to push colony across
the agar surface without disruption
• Biochemical Tests:
• Oxidase enzyme positive
• DNA’ase enzyme positive
• Resistance: beta lactamase enzyme production (90%)
• Therapy: Augmentin or 3rd generation Cephalosporin
Gram Positive Rods
Corynebacterium
Bacillus
Listeria
Erysipelothrix
Corynebacterium
• Over 20 species, most are saprophytic
• Human normal flora, skin and nares
• Most grow well on 5% Sheep’s blood agar
• Gamma hemolytic gray colonies
• Catalase enzyme = positive
• “Diphtheroid” morphology on staining, Gram
positive rods in Chinese letter forms
• No spores produced
Corynebacterium diphtheriae
• Agent of Diphtheria
• Forms tough oropharyngeal membrane (Diphtheroid is Greek for
leather), bleeds when attempt to remove from throat
• Phage mediated exotoxin is distributed from the membrane causing
respiratory paralysis, heart, nervous system, and kidney damage
• Non-toxin producing strains cause cutaneous ulcers and
lesions
• Diagnosis: culture, toxin detection (Elek plate), serology,
and PCR to detect organism and study toxin genes
• Grows well on 5% Sheep’s blood agar, gray gamma
hemolytic colonies, catalase enzyme positive
• Selective medium: Cysteine Tellurite agar/ black colonies
• Metachromatic storage granules formed when grown on
Loeffler’s media and stained with methylene blue stain
• Treatment: Anti-toxin and Erythromycin or Penicillin
Elek plate
Metachromatic granules
Other Corynebacterium spp.
• Corynebacterium jeikeium
• Normal human skin flora / thrives on lipid
• Infects patients with indwelling plastic catheters and
devices and can lead to bacteremia
• Biofilms are formed on the plastic, protecting the organisms
from antibiotic treatment
• 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
+
Rhodococcus species
• Gram positive cocco-bacilli
• Partially acid fast (PAF) with modified acid-fast staining
• Related to Nocardia and Mycobacterium species
• Environmental organisms found in the soil
• Rhodococcus equi is the most common pathogen
• Zoonotic pathogen
• Capable of causing human pneumonia, bacteremia and
tissue invasion, more common in immune suppressed,
particularly HIV
• Grows well at 30*C and 35*C. producing salmon-pink
colonies.
• Identification best using MALDI-TOF
Bacillus species
• Large Gram positive rods with square ends
• Can be easily over-decolorized and appear reddish on Gram stain
• Spores produced which cause clearing in the bacillus
• Catalase enzyme positive
Spores/ clear areas in
bacillus
Variable staining / not clearly
Gram positive
Bacillus anthracis
• Agent of anthrax in herbivores
• Category A agent – priority biothreat pathogen
• Virulence factors: anthrax toxin and capsular polypeptide
• Infections:
• Wool sorter’s disease – acquired from handling contaminated
cow hides, produces a unique black eschar skin lesion
• Systemic infections: Pneumonia, sepsis, and meningitis
• Irregular shape to colony border on 5% Sheep’s blood agar (BAP)
Medusa head colonies
Gamma (no) hemolysis on BAP
Non-motile
Susceptible to penicillin
Bacillus cereus
• Most common Bacillus species causing infection
• Saprophytic environmental organism
• Nosocomial and opportunistic infections, particularly in immunocompromised,
and patients with indwelling or implanted devices
• Can be a contaminate or pathogen in blood cultures
• Cause skin infections in Intravenous drug users
• Associated with food poisoning: vomiting due to emetic toxin production and
diarrhea from diarrheal toxin. Refried rice has always been suspect.
• Gram positive rod with spores
• Dull gray colony producing beta hemolysis on BAP
• Catalase positive
• Motile
• Resistant to penicillin
Listeria monocytogenes
• Small Gram positive rod / no spores produced
• Catalase enzyme positive
• Grows well on BAP producing subtle beta hemolysis
• Motility provides identifying information
• More motile at 25˚C than 35˚C
• Tumbling motility on wet mount examination
• Umbrella motility when inoculated into tubed media
• Bacteremia and meningitis in immune suppressed, pregnancy,
neonates, and elderly
• Culture or molecular amplification methods for diagnosis
• Grows well at 4˚C / infection from ingesting non pasteurized
cheese and milk products, Deli case foods
• Ampicillin drug of choice
• Intrinsic resistance to Cephalosporin antibiotics
Erysipelothrix
rhusiopathiae
• Small pleomorphic Gram positive rod
• No spores produced
• Catalase enzyme negative
• Alpha hemolytic colony on 5% sheep’s blood agar plate
• Only Gram positive rod that produces hydrogen sulfide (H2S)
MALDI-TOF ID is useful for this difficult to identify bacteria
– Infections from nature and animals, mostly swine, is an
occupational disease of farmers and butchers
– Virulence factor: capsule
– Soft tissue infection and bacteremia +/- endocarditis
– Intrinsic resistance to Vancomycin
H2S production detected on
Triple sugar iron agar (TSI)
Erysipelas – lacy skin
lesions of swine
Gram Negative Bacilli
•Escherichia coli
• Normal flora in human intestine
• #1 cause of UTI [@80% of cases]
• Bacteremia, neonatal meningitis,
and abdominal infections
• Lactose fermentation on MacConkey’s agar
• Eosin methylene blue agar (EMB) green sheen produced
• Spot indole reaction = positive / turns robin’s egg blue
• Detects breakdown of tryptophan from growth on BAP
Green sheen on
EMB agar
Indole positive
Lactose
fermentor
Escherichia coli
• Pathogen of diarrhea: Molecular testing is standard practice
• Enterotoxigenic (ETEC) E. coli cause of traveler’s diarrhea
• Entero-hemorrhagic E. coli (EHEC) (such as 0157:H7)
• Bloody diarrhea from eating undercooked beef or contaminated
veggies
• Pathogenicity from Shiga toxin production (2 types)
• Hemolytic uremic syndrome (HUS) can result with hemolytic
anemia, thrombocytopenia, and renal failure] particularly in young
children
• Old school: Culture on sorbitol MacConkey agar / does NOT
ferment sorbitol/ most all E. coli except EHEC ferment sorbitol
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 production
• Currant jelly sputum produced in alcoholics due to blood mixed
with Klebsiella capsular polysaccharide in sputum
• Klebsiella (Enterobacter) aerogenes new taxonomic classification
Extended Spectrum Beta Lactamase [ESBL]
•Enzymes produced by select species of Enterobacterales
• Confer resistance to Cephalosporins, Penicillins and Monobactam
(Aztreonam) antibiotics by opening the beta lactam ring of the antibiotic
and inactivating the antibiotic
• ESBLs do not attack Cephamycin antibiotics (cefoxitin, cefotetan) or the
Carbapenem antibiotic classes
•Plasmid mediated CTX-M beta lactamases (bla gene)are the most common
ESBL enzymes in the US currently, but many ESBL types can be found
worldwide
•Therapy for ESBL producing gram negative rods:
• Carbapenems: Imipenem, Meropenem, Doripenem, and
Ertapenem
Carbapenemases – CRE and CRO
•CRE = Carbapenamase resistant Enterobacteriales/ CRO= Carbapenamase
resistant organism (Pseudomonas)
•Carbapenemase beta lactamases lead to resistance to carbapenam antibiotics
(meropenem, imipenem, doripenem, ertapenem).
•Carbapenem-hydrolyzing-beta-lactamases are incorporated into the genetic
elements in transposons of Gram negative bacillus. Transposons can insert into
diverse plasmids which can transfer resistance to a broad spectrum of Gram
negative bacillus.
•Two CREs are getting the most attention:
• KPC – “Klebsiella pneumoniae carbapenemase” most common in the US
• NDM-1 – New Delhi metallo-beta-lactamase. Resistance determinants are numerous and
great concern about its spread.
• Other CRE/CROs: IMP, OXA-48, and VIM
•Infections with CRE/CRO producing GNRs can have a 50% fatality rate
Gram Negative Rods that do NOT Ferment Lactose!
• Proteus species
Colonies swarm in concentric layers on agar surface
• Proteus vulgaris – spot indole positive
• Proteus mirabilis – spot indole negative
• Normal flora in intestine
• Common cause of UTI and abdominal infections
• Intrinsic resistance to antibiotic Colistin due to mcr-1 gene
• Serratia marcescens
• Produces red pigmentation which can intensify at room temp
• Environmental contaminate
• Causes infection most usually in
• Immune suppressed
• Ventilator associated pneumonia
• Bacteremia
1. Glu/lac/suc
fermented
with gas
2. Glucose
only
fermented
3. Glucose
fermented
with H2S
production
4. No CHO
fermentation
Non fermenter
Triple Sugar Iron Agar (TSI)– 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
H2S production =
black medium
1 2 3 4
• Salmonella species
• Diarrhea with +/- fever and PMNs in the stool
• Infection from eating contaminated food (raw eggs, poultry, ground
beef or dairy) or direct contact with a sick person or animal
– Must ingest large #’s of organisms to make you ill, normal levels
of stomach acid is protective
• MacConkey agar - does not ferment lactose
• Produces hydrogen sulfide on selective media
• 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
Salmonella typhi
• Typhoid fever – high fever and sepsis, no diarrhea
• Human pathogen with most cases in US (75%) from international travel
• Post typhoid fever there can be carriage in gallbladder with passage in
feces. Can transmit to others by bad hygiene and food preparation
• Ingested organisms enter the bowel, rapidly move into the blood
stream and eventually the bone marrow
• Diagnosis: Blood cultures in early infection and bone marrow
culture in late stages (>1 month) of infection
• Vi capsular antigen unique to S. typhi
• Moustache of Hydrogen sulfide (H2S) produced in TSI medium slant
• This is a unique finding for S. typhi
Shigella species
• Diarrhea, +/-vomiting, fluid loss, PMNs 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]
• No lactose fermentation on agar
• Non motile
• No Hydrogen sulfide (H2S) produced
• 4 species based on somatic (cell wall) antigen
• S. dysenteriae Group A
• S. flexneri Group B
• S. boydii Group C
• S. sonnei Group D
Salmonella Shigella Agar (SS agar)
Shigella are colorless due to lactose
not being fermented. H2S produced by
Salmonella spp turning the colony black
Hektoen agar –
Salmonella produces H2S [Hydrogen sulfide]
producing black colonies
Shigella – green colonies, no H2S produced
Normal flora – orange colored due to
fermentation of lactose (E. coli)
Non-Lactose fermenter
Shigella
Salmonella
Normal Flora
Lactose fermented
H2S
Salmonella
Shigella
Salmonella
Yersinia enterocolitica
• Major reservoir – swine
• Humans infected by eating raw or undercooked pork
• Infections:
• Diarrhea
• Septicemia in patients with iron overload syndromes
• Mesenteric adenitis – symptomatic for right lower abdominal
pain which can be confused with appendicitis
• Infected blood products from transfusion have been reported
• Oxidase = negative
• Indole = negative
• Urease = positive Grows well at 4 °C
• CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for
Y. enterocolitica
Yersinia pestis
• Plague, Category A biothreat 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 infection of the lymphatic system, forms painful buboes
(lymph node swelling) at site of the bite
• Hemorrhagic lymph nodes spread organism into blood stream
• Pneumonia develops from blood stream infection
• Fatality >=50%
• Endemic in SW USA
• Grows well on blood agar
• catalase +, oxidase -
• Bipolar staining looks like a “safety pin”
• Vibrio cholera
• Natural environment is 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 which
– Increases cAMP with hyper secretion of NaCl and H20
– Death from dehydration and metabolic acidosis
• Curved “C” shape Gram negative rods can be seen
• Selective medium thio citrate bile sucrose agar, V. cholera
(TCBS) yellow color from sucrose fermentation
• Oxidase = positive ,
• Grows well in 1% salt solution
TCBS Agar
• Vibrio parahaemolyticus
• Diarrhea from ingestion of contaminated raw oysters
• Self limited in normal host, but serious in immune suppressed
• TCBS medium sucrose fermentation negative so green colony
• Vibrio vulnificus
• Diarrhea from ingestion of contaminated oysters
• Skin infection from injury in contaminated water
• Bacteremia can occur in certain patient populations following the
ingestion of contaminated oysters
• Patients with liver disease (cirrhosis) and patients with
increased serum iron
• Formation of painful skin lesions on lower extremities
with muscle necrosis
• 50% fatality rate
Acinetobacter baumannii
• Environmental saprophyte and normal flora on human skin
• Gram negative coccoid-bacilli
• Does not ferment lactose / Oxidase enzyme negative
• Opportunistic nosocomial pathogen
• Glucose oxidizer
• Pulmonary infection in vented patients
• Can acquire resistance to many antibiotics from antibiotic exposure
Stenotrophomonas maltophilia
• Rapid maltose oxidizer
• Gram negative bacillus
• Gun metal gray pigment
• Intrinsically resistant to many antibiotics, including carbapenems
• Nosocomial pathogen: super-colonizer after long term carbapenem therapy due to
intrinsic resistance to the carbapenem antibiotics (Imipenem and Meropenem)
Pseudomonas aeruginosa
• Fluorescent blue-green pigment produced (pyocyanin)
• Oxidase enzyme = positive
• Grape-like odor
• Grows at 42˚C
• Ps fluorescens/putida (a related species group does not grow at
42°C
• Major pathogen of cystic fibrosis
• Mucoid strains produced in the lung from the production of
polysaccharide capsule
• Major lung damage results from co-infection with Burkholderia
cepacia (gram negative rod)
• Nosocomial pathogen associated with exposure to water and moist
environments
• Intrinsically resistant to many antibiotics and acquire resistance to
carbapenems
Burkholderia pseudomallei
• Soil-dwelling bacterium endemic in tropical and subtropical regions
worldwide, particularly in Thailand and northern Australia.
• Infects humans and livestock such as goats, pigs, and sheep and causes the
disease melioidosis which is primarily a pneumonia. Mortality is 20 – 50%
• Biosafety level 3 containment and PPE for working with the organism/
Category A select agent needs to be handled with BSL-3 safety precautions
• Grows on a large variety of culture media including blood
and MacConkey agar in 24 hours at 35* C
• Colonies are wrinkled, have a metallic appearance, and possess an earthy odor.
• Can display bipolar staining when stain from culture growth (like Y. pestis)
• Difficult to identify using biochemicals, best using MALDI-TOF with
proper identification library
• Intrinsically resistant to many antibiotics via efflux pump mechanism
• Chryseobacterium (Elizabethkingia) meningosepticum
• Infections:
• Newborns: fatal meningitis and septicemia in the newborn
• Elderly/immune suppressed: bacteremia
• Environmental organism found in water
• Yellow colony
• Oxidase and Indole positive
• Haemophilus ducreyi
• Cause of venereal disease: Chancroid
• Painful necrotizing genital ulcers/inguinal lymphadenopathy
• School of fish appearance on Gram stain from lesion
• Requires hemin (X factor) to grow on solid media
Haemophilus influenzae
• Transmission – close contact and 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 (contains X and V factor)
• Will not grow on 5% sheep’s blood agar
• Requires 5-8% C0₂ for growth
• Effective vaccine targets invasive infections with H. influenzae type B
(Hib) effectively eliminating most childhood invasive infections
• Ampicillin resistance from beta lactamase enzyme productions [25 %],
3rd generation Cephalosporin becomes the antibiotic of choice
(Ceftriaxone) for invasive infections
Satellite phenomenon
Staph aureus supplies
the X and V factors
required
HACEK organisms
• Oral flora organisms. Due to poor detention or invasive dental procedures
organisms are introduced into bloodstream and can infect heart valves
• Fastidious Gram negative coccobacilli / need >=48 hrs to grow in culture
• Cause of 5 -10% of community acquired native valve endocarditis not related to IV drug
use
• Haemophilus species oxidase (-) catalase (-)
• Aggregatibacter (Actinobacillus) oxidase (-) catalase (+)
• Cardiobacterium hominis oxidase (+) indole (+)
• Eikinella corrodens oxidase (+), pits agar, bleach odor
• Kingella kingii oxidase (+), hemolytic on blood agar
• Major cause of septic joint infection in small children
Bordetella pertussis
• Whooping cough – 3 disease stages
• Prodromal – flu like disease – most contagious stage
• Catarrhal - classic whoop cough in small children
Toxin adheres to bronchial epithelial cells and cough
continues until toxin wears off
• Paroxysmal - recovery phase
• Human pathogen, inhabits nasopharynx
• Peripheral blood smear - Lymphocytosis with atypical, large,
irregular and deeply basophilic lymphocytes
• Tiny Gram negative coccobacillus
• Selective media = Regan Lowe Charcoal, growth in 3-5 days
• Molecular detection is standard of practice / greater sensitivity
• Reservoir for infection is in young adults due to waning
immunity. Reason for booster shots in young adults
Pasteurella multocida and P. canis
• Normal flora in many animals (zoonotic)
• Common pathogen of bite wound infections from cats and dogs
• Human pneumonia cases from close contact with animals
• 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 sensitive to penicillin
Capnocytophaga species
• Fusiform shaped Gram negative rods
• Fingerlike projections from colonies “Gliding”
• Require incubation in C02 for growth
• Oxidase negative
• Catalase negative
• Normal mouth flora (NF) in humans and animals
• C. canimorsus – NF in dogs, cause infected dog bites – high % of bite
infections lead to bacteremia and endocarditis
• Many Capnocytophaga species as NF in human mouth
• Infected mouth ulcers can lead to bacteremia
Brucella species
• Brucellosis, FUO, significant joint pain, intracellular pathogen of the Reticuloendothelial system
• Specimens: Blood (early in disease) and Bone Marrow (after first month)
• Automated Blood culture systems detect at >=5 days of incubation
• Serology can assist with diagnosis of chronic disease
• Culture: Small Gram negative coccobacilli, non hemolytic gray colony,
• Requires 5 – 10% 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 – infected pig exposure
• B. canis - Infected dog exposure
*Granuloma in
bone marrow
Campylobacter spp.
• C. jejuni – Common cause of diarrhea in US, can progress to bacteremia in
HIV and immune suppressed
• Ingestion of undercooked poultry or juice contaminating raw food
• Sea gull shaped, poorly staining Gram negative rod
• Culture requires selective blood agar containing antibiotics to suppress
the overgrowth of normal bowel flora organisms
• Campy-BAP or Skirrow’s BAP
• Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂)
• Sequelae of diarrhea infection: Significant % Guillain-Barre syndrome
• C. fetus – Bacteremia in the immune suppressed host
• Source cattle and sheep
• Temperature tolerance aids in identification / but MALDI-TOF works best
C. jejuni – grows at 37˚C and 42˚ C, hippurate hydrolysis positive
C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative
• 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 and progress to:
• Painful skin lesions
• Enlarged lymph nodes
• Bacteremia (triad known as ulceroglandular tularemia)
• Pneumonia
• Small Gram negative rod
• Oxidase enzyme negative
• Requires cysteine in culture medium for growth
Francisella tularensis
Helicobacter pylori
• Acute gastritis with small % progressing to gastric
adenocarcinoma
• Human to human transmission due to poor hygiene
• Rapid and strong urease enzyme produced by this organism
• Can be used to detect organism in gastric antrum biopsy tissue
• Small curved Gram negative bacilli
• Difficult to grow, making culture not useful
• Stool antigen for diagnosis and test of cure useful
• Serum antibody detection obsolete – most adults IgG detected
• Organism stained by silver stains in gastric biopsies
• Treatment – Antibiotics and stomach acid suppression
Immunohistochemical
stain of gastric biopsy
Chronic active gastritis with plasma
cells and neutrophils in the lamina
propria. (H&E)
Legionella
pneumophila
• Pulmonary disease from exposure to aerosolized water
• Hyponatremia common in during infection
• Requires cysteine in culture medium for growth
• Selective medium: Buffered Charcoal Yeast Extract agar with
growth 3-5 days at 35*C in CO2 incubator
• Will not stain with usual Gram stain methods
• Must use carbol-fuchsin as counterstain to detect organism
• Silver impregnation stains used in fixed tissue
• Diagnosis: Urinary antigen test (EIA) detects Legionella
pneumophila type I in pulmonary infection
• Antibody response is delayed and seldom used for diagnosis
• Treatment: Erythromycin (macrolide)
BCYE
Blood agar
No growth
Bacteria without cell walls
• Mycoplasma and Ureaplasma species have no cell wall only cell membranes. Media for culture
must contain protective sterols. Molecular amplification is diagnostic method of choice.
• Lack of peptidoglycan cell wall, so will not Gram stain, no clear colonies produced on agar, and
cannot be treated with antibiotics that act by inhibiting cell wall formation
• M. pneumoniae
• Community acquired pneumonia
• High titer cold agglutinins with infection
• Genital mycoplasmas
• M. hominis form fried egg appearance on sterol containing agar
• Can cause vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of
membranes
• Ureaplasma urealyticum form dark metal-like appearance on sterol containing media
• Rapid hydrolysis of urea
• Non-gonococcal urethritis, upper genital tract infection, spontaneous abortion, and
neonatal infections
Ureaplasma
Mycoplasma hominis
Unusual and Difficult to Grow Bacteria
• Bartonella henselae
• Cat scratch disease from exposure to cat and cat excrement
• Bacillary angiomatosis – vascular skin lesions +/- invasion, associated with
HIV
• Bartonella quintana – cause of trench fever/ vector is the body louse
• Coxiella burnetii – cause of Q fever / zoonotic infection that can infect humans
• 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
Serologic and molecular assays for diagnosis
Unusual and Difficult to Grow Bacteria
• Klebsiella (Calymmatobacterium) granulomatis
• Agent of STD granuloma inguinale
• Infection leads to ulcerative genital lesions
• Streptobacillus moniliformis
• Rat bite fever or Haverhill fever
• Infection from untreated rat bite
• Cell wall deficient bacteria known as L form
• Difficult to culture; inhibited by SPS in blood culture media and requires
serum supplementation to grow
• Patient history is helpful, sequencing if necessary
• Tropheryma whipplei –
• Whipple disease
• Gram positive rod (Actinomycete) distant relative of
Mycobacterium avium and M. paratuberculosis
• Found in soil and farm animals
• Causes a diarrhea which can lead to malabsorption syndrome and
cardiac disease
• Characteristic findings in fixed tissue
Foamy macrophages in the
lamina propria
Unusual and Difficult to Grow Bacteria
• Ehrlichiosis – infection caused by Rickettsia spp
• Zoonotic infection
• Vector is the Ixodes tick (hard tick)
• Two genera cause infection / intracellular pathogen
• Anaplasma spp, inclusion (morula) in the PMN
• Ehrlichia spp inclusion in the monocyte
• Fever, leukopenia, thrombocytopenia,
• Elevated serum aminotransferases
• Ehrlichiosis has no rash, which differs from Rocky Mountain Spotted Fever
caused by Rickettsia rickettsia which is known for presence of rash
• Found in south central, southeast , and mid-west US
• PCR, serology, and examination of blood smear for diagnosis
Spirochetes
• Borrelia burgdorferi - Lyme’s disease
• Primarily found in Northeastern US
• Vector = Ixodes tick (hard tick)
• Acute disease: Fatigue, headache, fever, rash,
and erythema migrans lesion after tick bite
• Can progress to a chronic disease
• Diagnosis: Serology and molecular methods
• Borrelia species – Tick borne relapsing fever
• Western United States
• High fever (relapsing) with thrombocytopenia
• Muscle and joint aches
• Vector: Soft tick (Ornithorodos hermsi)
• Diagnosis: Blood smear observe spirochete
Spirochetes
• Treponema pallidum
• Agent of Syphilis
• Traditional Algorithm involves serologic screening for Syphilis using a
nontreponemal test (e.g., VDRL and RPR) first and followed by a
treponemal specific test such as EIA, TPPA, or FTA-ABS. This traditional
method has a high positive predictive value, but often misses early
primary and treated infections.
• New Reverse Algorithm for syphilis diagnosis consists of a first
performing a treponemal antibody screening immunoassay followed by
confirmatory nontreponemal antibody testing (RPR).This algorithm was
created to increase sensitivity of detection.
• Years past: Darkfield examination was used to view spirochetes from
lesions
Spirochete Infections
• Brachyspira
• Intestinal spirochetes are anaerobic bacteria
• Transmission is most likely contaminated water sources
• Most common in developing nations and HIV infected
• Intestinal spirochete found on the brush border of the
intestine
• Clinical significance is controversial
• Leptospira interrogans / Leptospirosis
• Fever with rash and renal involvement
• Urine from rats and other animals contaminate water supplies
• Microscopic agglutination test (CDC) is best diagnostic test
(1) Leptospirosis – Shepherd’s crook shaped spirochete
(2) Presence of spirochete in a renal tubule
(2)
1
Bacterial vaginosis
• Mixed anaerobic/aerobic bacterial infection
• Rather benign infection except in pregnancy
• Pregnant women are at increased risk due to hormone changes that happen during pregnancy.
Increased risk for premature birth and low birthweight.
• Discharge: Fish-like odor and alkaline pH >=4.5
• Usual NF organism of Lactobacillus is overgrown by:
• Gardnerella vaginalis (aerobic Gram variable rod) primary bacterial
marker for infection
• Mobiluncus (anaerobic Gram negative rod)
• Clue Cells are diagnostic for vaginosis and provides a more specific
indication of infection than growth in culture
• Molecular probe assays and amplification assays available as part of
“women’s health” screening panels
Clue Cell
Anaerobic Bacteria
• Anaerobic infections can occur in virtually any organ or region of
the body
• Most are polymicrobial –with both aerobic and anaerobic species
• Endogenous normal flora organisms cause most infections
• Due to trauma, vascular or tissue necrosis cutting there is a lowered
oxygen supply to the involved tissue that can lead to infection
• Treatment: Surgery to restore oxygen to tissue, remove necrotic
tissue and antimicrobial therapy
• Specimen collection for culture
• Gel containing swab
• ESwab
• Evacuated vials (port-o-cult)/ oxygen free vials for fluid
• Do not refrigerate specimen prior to culture, it will cause greater
absorption of oxygen and increase the death of anaerobes
Anaerobic culture
• PRAS media – pre reduced anaerobically sterile
• Media packaged in oxygen free environment
• Provides best opportunity for growth of anaerobes
• Most common anaerobic culture media include:
• CDC anaerobic enriched blood agar
• Kanamycin-vancomycin blood agar
• Bile esculin agar
• Thioglycollate broth
• Chopped meat glucose broth
• Anaerobic chambers – perform culture work in an oxygen free closed
cabinet so specimens and organisms are never exposed to oxygen
• Anaerobic gas packs and jars for anaerobic incubation of culture plates
• Wet pack – add 10 ml water to hydrogen and CO2 generating
envelope/ requires palladium coated catalysts to generate heat
and create oxygen free environment.
• Dry pack – (Anaeropack) absorbs O2 and generates CO2
PRAS
Bacteroides fragilis group
Pleomorphic irregular staining Gram negative rod
• Normal flora in the GI tract, most common anaerobe
• Grow in the presence of bile
• Growth on bile esculin media and turns media black
• Resistant to Penicillin and Kanamycin
• Infections: Related to the bowel such as GI abscess
• B. fragilis group organisms
• 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 Metronidazole resistance is suggesting a need to do
susceptibility testing to confirm susceptibility
Growth on bile/esculin media
Black pigment from Esculin
production (left) and growth on
Anaerobic Blood agar (right)
Prevotella and Prophyromonas
• Pleomorphic Gram negative rods
• Normal flora in the upper respiratory tract
• Infections: respiratory tract abscesses
• Will not grow in the presence of bile
• Will not turn black on bile esculin media
• Unique brick red fluorescence when placed under
UV light
• Colony forms natural black pigment after one week
of incubation
Fusobacterium spp.
• F. nucleatum Long thin gram negative
bacilli – spindle shaped with pointed ends
• Normal flora upper respiratory tract
• Infections: mouth and respiratory tract and liver
abscess
• Vincent’s angina – necrotizing oral co-infection
caused by Fusobacterium species and spirochetes
• F. necrophorum – pleomorphic gram negative bacilli in
filaments or chains
• Lemierre’s syndrome - oropharyngeal infection,
leads to thrombosis in jugular vein, septicemia,
with high fatality rate
Clostridium species
• Gram positive bacilli (boxcar shaped ), form spores which appear as
empty space in the bacilli
• May easily over-decolorize and appear gram negative
• Clostridium perfringens
• Infections: Food poisoning, necrotic tissue abscess (Clostridial
myonecrosis), bacteremia, cholecystitis
• Most common anaerobic Gram positive rod in intestine
• Important reactions:
• Double zone of beta hemolysis on BAP produced
• Lecithinase produced on egg yolk agar (clouds the agar)
• Reverse camp test positive
Lecithinase
Reverse Camp Test
Double zone of
hemolysis
• Clostridium botulinum – Botulism
• Adult disease – ingest preformed heat labile neurotoxin,
usually mass produced or in home-canned foods
• Infant disease - spore ingested from nature or product
of nature such as honey or household dust, spore
germinates in gut producing neurotoxin
• Begins with constipation and difficult sucking bottle
• Both forms are life threatening neuroparalytic diseases
• Clostridium tetani - Tetanus
• Gram stained cells appear like Tennis racket from
terminal spore
• Infection begins with penetrating skin injury with
introduction of the Tetanospasmin toxin
• Spastic contractions of voluntary muscles, hyper-reflexia,
lock jaw (trismus)
• Immunization to prevent
• Clostridium septicum –
• Bacteremia or gas gangrene in patient with underlying malignancy
• Hematogenous spread from GI tract leads to bacteremia – no trauma necessary
• Clostridioides (Clostridium) difficile –
• Disease: antibiotic associated colitis, pseudomembranes produced from toxin
production
• Toxin A – enterotoxin causes fluid accumulation
• Toxin B – potent cell cytotoxin, primary virulence factor (TcdB gene)
• Binary toxin – Nap1 strain, this strain has increased toxin production
• Diagnosis of infection:
• Standard of practice has become a two-step algorithm:
• Molecular amplification to detect TcdB gene indicating presence of toxin gene in the stool
• Enzyme immunoassay to detect active toxin in the stool
• If TcdB gene and toxin detected, the patient has C. difficile disease and requires therapy
• Alternate method: GDH (Glutamate dehydrogenase) antigen test uses antibodies to test for the
presence of the GDH, a protein present in all C. difficile isolates. Screening test with good sensitivity,
rapid turnaround time, and low cost.
• Selective medium Cycloserine, Cefoxitin, Fructose Agar [CCFA] – culture used for research
purposes, not for diagnosis of disease
Actinomyces
• Branching gram positive bacilli –
• No spores produced
• Aerotolerant, but grows best in anaerobic conditions
• Normal flora oral, GI, vagina, skin
• Infections: oral/facial (lumpy jaw), respiratory and GI
• Can form sulfur granules in tissue
• Actinomyces israelii – associated with oral, thoracic,
and abdominal infections, IUD infections
Bread crumb colonies produced in broth
Penicillin susceptible
Branching Gram positive rods
of Actinomyces – antler like
Molar tooth colony on surface
of blood agar plate
Sulfur granule
Aggregates of Actinomyces
Cervicofacial actinomycoses
–lumpy jaw
Propionibacterium acnes
(Cutibacterium)
• Pleomorphic, sometimes branching Gram positive rod
• Catalase positive
• Indole positive
• Normal flora - skin, oral, GU, and GI
• Can be seen as a pathogen in blood cultures or contamination due to not properly
cleaning the blood draw site
• Pathogen of acne vulgaris
• Opportunistic pathogen: cerebral shunt infections and other endovascular and
neurosurgical infections from organism gaining entrance from skin
• Firmly established as significant cause of prosthetic joint infection – particularly shoulder
joints
• Cultures should be held up to 7 days
• Therapy - Ampicillin
Sarcinia species
• The sarcina was the marching pack carried
by Roman legionaries, the heavy infantry of
the Roman legions. Due to the similar appearance
the organism was named Sarcinia.
• Gram-positive anaerobic coccus that has been
reported in the upper gastrointestinal tract
biopsies of patients with evidence of gastric stasis.
• The pathogenic role in humans is not entirely
known.

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Web Bacteriology 2023

  • 1. Bacteriology Update 2023 Margie Morgan, PhD, D(ABMM)
  • 2. Objectives • Bacteriology is a massive area of study, and it is impossible to review everything in this slide set. • This lecture reviews the most important organisms and laboratory tests, and relevant antimicrobial therapy • There has been many taxonomic updates over the last few years. The updated name is noted in parenthesis, such as Micrococcus (Kocuria) species.
  • 3. Begin with Definitions • Obligate Aerobe – require high level of oxygen (20%) to grow • Obligate Anaerobe – >30 min exposure to oxygen can be deadly • Facultative anaerobes – grow in both aerobic and anaerobic conditions, most of the so-called “aerobic” bacteria are facultative (Escherichia coli) • Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to oxygen, but grow best anaerobically, example: Clostridium tertium • Microaerophilic – grow best with reduced oxygen and elevated % of carbon dioxide (Streptococcus anginosus group) • Lag Phase - >24 hrs old on agar plates, growth is slowing, not appropriate for biochemical or susceptibility testing • Stationary phase – Organisms alive but not replicating, suitable for transportation of specimens to the laboratory
  • 4. Specimen Collection - Aerobic Throat / Wound / Abscess 1. Swabs should be either polyester fiber or flocked (prickly sponge) 2. Cotton fiber swabs should not be used, traps bacteria and potentially toxic 3. Specimen is collected with swab then placed in Stuart’s or Amie’s transport media (buffered solution with peptones) for transport and storage 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 5. Transport media has stability limits / usually <= 72 hours Urine (2 commonly used methods) 1. Cup with boric acid - organisms in stationary phase for transport 2. Refrigerate urine at 4*C within one hour after collection 3. Both methods maintain original colony count and viability of organisms 4. Plate urine onto agar plates within 24 hours of collection Tissues/Sterile body fluid 1. Adequate volume transported in sterile container
  • 5. Blood Culture Collection and Detection • Two important collection issues • Prevent contamination: Cleanse collection site using Chlorhexidine • National Benchmark: Blood culture contamination rate should be <=3% • Adequate volume of blood inoculated into blood culture bottles • Adult blood culture (bottles shown) should approach 8-10 ml of blood per bottle • One blood culture set consists of two bottles: • One aerobic environment / one anaerobic environment • Automated blood culture instruments to detect growth has become the standard of practice • Incubation for 5 days at 35*C • Growth is detected by organisms causing an increase in the amount of C02 present inside the bottle air space. This increase in CO2 triggers a fluorescent indicator to cause instrument alarm indicating a positive bottle.
  • 6. Gram Stain Procedure 1 minute Rinse Primary stain Mordant 1 minute Rinse 5-10 seconds Rinse Decolorization Counter stain 1 minute Rinse Gram-positive organisms have a high amount of peptidoglycan in the cell wall. Peptidoglycan traps the crystal violet in the cell wall which gives Gram-positive organisms a blue color. Slide interpretation includes Gram-positive or Gram-negative staining (red/blue) designation and shape of the stained organism. Prepare thin film of specimen on glass microscope slide Heat or methanol
  • 7. Gram Stain to Assess Quality of Sputum for Performance of Bacterial Culture/ Is it spit or a good expectorated deep cough? • Expectorated sputum is examined for presence of neutrophils (WBCs) and epithelial cells • <10 epithelial cells/low power field (LPF) is observed and >25 WBCs/LPF (exception for leukopenia) • Sputum acceptable for performing bacterial culture • If >10 epithelial cells / LPF • Sputum is judged to be spit • Bacterial culture should not be performed • Request made for new “deep cough” specimen Bad Sputum Good Sputum 10X objective
  • 8. Most common agar plating media 5% sheep’s blood agar (blood agar) • Supports growth of numerous non-fastidious bacteria and yeast • Used to gauge hemolytic reactions of bacteria (alpha, beta, gamma hemolysis) Chocolate agar • “Caramelized” blood agar – with added growth enrichment • Supports the growth of the same organisms as 5% sheep’s blood agar plus fastidious bacteria – such as Haemophilus influenzae and Neisseria gonorrhoeae • Incubation of plates in a 5-8% CO2 incubator to nurture for fastidious organimsms
  • 9. Commonly used agars • MacConkey agar – Selective and differential medium for Gram negative bacilli • Selective for Gram negative bacilli (GNRs) • Inhibits growth of Gram-positive organisms due to addition of crystal violet in the medium • Differential for lactose fermentation • Fermenters produce a pink colony due to neutral red indicator turning colony pink from acid production • Lactose fermentation positive = pink • No lactose fermentation = no color • Lactose fermentation is a major branchpoint in enteric Gram negative rod identification Lactose pos Lactose neg
  • 10. MALDI-TOF Mass Spectrometry / Advancement in the Identification of Bacteria MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization – Time of flight) – Identification by analyzing protein fingerprints of organisms – Replaced many/most biochemical tests for bacteria identification
  • 11. MALDI-TOF Theory • Laser is fired at target plate containing matrix solution 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 tube by an applied electrical field • The time taken for the proteins to travel through the vacuum tube and reach the detector depends on their mass/charge ratio (m/z) and creates a spectrograph. This happens in @ 30 seconds. • 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. • **MALDI identifies 100’s of organisms, but there are still a few issues to remember most notably, some organisms are closely related and not well separated, such as Escherichia coli and Shigella species are too similar to separate, so biochemicals must be performed to assist.
  • 12. Safety in a Bacteriology Laboratory • Risk assessment should be performed to establish safety precautions for a safe working environment. • Bacteriology laboratories handling routine cultures are considered Biosafety Level 2. • Biosafety Level 2 (BSL-2) is appropriate for handling moderate-risk agents that cause human disease of varying severity by ingestion or through percutaneous or mucous membrane exposure. • Clinical specimens and procedures with possible aerosolization or splashing performed in Biosafety Level 2 Cabinets with HEPA filtration • Gloves and impermeable lab coats worn when working with patient specimens • Waste management program to properly dispose of biohazards
  • 14. Staphylococcus Gram stain Gram positive cocci in random clusters – clusters formed due to bound coagulase or “clumping factor” All are Catalase enzyme test = Positive Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative Sheep’s blood agar plate +/- Yellow colony Beta hemolysis Sheep’s blood agar plate White colony Most non-hemolytic
  • 15. Catalase Enzyme Reaction Negative Positive Bacteria placed in Hydrogen Peroxide/ bubbles=positive reaction Slide Coagulase reaction Staphylococcus organism emulsified in rabbit plasma/ mixed well. Positive reaction = agglutination Positive Coagulase enzyme = Staphylococcus aureus Tube Coagulase Reaction Rabbit plasma inoculated with organism and incubated at 35˚C and observe for clot at 4 hours. If no clot at 4 hours, read again at 24 hours Negative tube coagulase No clot formed/liquid = Coagulase negative Staph Positive Tube Coagulase Clot formed at either 4 or 24 hours = Staph aureus Slide Coagulase Reaction
  • 16. Staphylococcus aureus • Virulence mechanisms: • Protein A – Primary virulence factor, surface protein, ability to bind immunoglobulin and combat the immune response • Toxins – many produced, act as super antigens, recruit host defense cells that liberate cytokines with systemic effects • Infections: • Toxic shock syndrome (TSST-1 toxin) • Scalded skin syndrome (Exfoliatin (SSS) exotoxin) • Soft tissue infection (Panton valentine leukocidin toxin – PVL) • Botrymycotic granules can be produced • Food poisoning (vomiting) – Produce endotoxins stable to heating at 100*C for 30 minutes • Bacteremia • Endocarditis • Staph aureus is the most common cause of right-sided infective endocarditis associated with IV drug abuse (@70 percent of cases) • Primary cause of adult septic arthritis Exfoliatin exotoxin Onion skin peeling PVL – soft tissue abscess Botryomycotic granule
  • 17. Methicillin Resistant Staph aureus (MRSA) • Methicillin resistance develops due to the presence of altered penicillin binding proteins (PBP2a) produced by the mecA gene. • PBP2a codes for resistance to oxacillin/ methicillin/ nafcillin resistance (the semisynthetic penicillin antibiotic class) • Cephalosporin antibiotics are ineffective and reported as resistant • Vancomycin becomes an antibiotic of choice. • Methods to detect MRSA: • Resistant to oxacillin by susceptibility testing (old way) • Resistant to cefoxitin by susceptibility test (better way) • Molecular tests to detect PBP2a (mecA gene, mecC gene) Of note: Emergence of mecC producing MRSA, these are not detected using a mecA based test and best detected using cefoxitin susceptibility testing
  • 18. The “D” Test for Inducible Clindamycin Resistance • Question: Is S. aureus susceptible to Clindamycin? • S. aureus isolates resistant to Erythromycin possess enzymes capable of inducing Clindamycin resistance to emerge in the S. aureus organism • Not detected by routine susceptibility testing • D test – the inhibitory zone around clindamycin KB disk will be blunted to form a “D” shape, meaning clindamycin was induced by erythromycin to become resistant, this is termed “Inducible Resistance.” D test positive Inducible resistance to clindamycin Do not use for therapy D test negative Susceptible to clindamycin Clindamycin can be used for therapy
  • 19. Nares is primary colonization site used for surveillance Methods available for surveillance: Culture on Chromogenic media Differential media with chromogenic substrates that turn a specific color to identify Staph aureus. Selects for MRSA due to the addition of cefoxitin in the medium Molecular assays can also be used to screen nares for presence of MRSA. Increases the sensitivity of detection over culture methods by 5-10% but greatly increases laboratory cost. Mupirocin therapy for short term elimination of MRSA carriage in nares and Chlorhexidine bathing to decolonize skin Methicillin Resistant Staphylococcus aureus (MRSA) Surveillance cultures to assist with Hospital Epidemiology Chromogenic Agar for MRSA
  • 20. Coagulase Negative Staph (CNS) @ 15 species human colonization/infection • Staph epidermidis – most common species in humans – Normal skin flora – Pathogen of opportunity – Common cause of catheter related bacteremia, endocarditis, and prosthetic joint infection – Pathogenicity from cell adhesion factors that form biofilm on biologics and plastics, difficult to remove or treat with antibiotics • Staph saprophyticus – – Urinary tract infection in the child-bearing age female – This CNS adheres efficiently to epithelial cells – The only CNS resistant to antibiotic Novobiocin (KB disk test) White non-hemolytic colony Resistant to Novobiocin
  • 21. • Staphylococcus lugdunensis • Normal skin flora in humans and an emerging pathogen • Pathogen in variety of infections particularly skin and soft tissue infections • Biochemical test of note: PYR spot test = pink = positive • PYR = pyrolidonyl arylamidase • Micrococcus (Kocuria) species/ close relative of Staph • Gram positive cocci in tetrads • Environmental contaminate/ seldom if ever a pathogen • Mustard yellow colony • Catalase enzyme = positive • Coagulase enzyme = negative Neg Pos PYR Test Coagulase Negative Staphylococcus (CNS)
  • 22. Streptococcus • Gram positive cocci in chains and pairs • Catalase enzyme = negative • Three groups based on hemolytic reaction produced when grown on 5% sheep’s blood agar • Alpha – greening of agar, partial hemolysis of RBCs • Viridans Streptococcus, Streptococcus pneumoniae, Granulicatella and Abiotrophia spp • Beta – clearing of agar, complete hemolysis of RBCs • Beta hemolytic Streptococcus, Streptococcus pyogenes (A) and Streptococcus agalactiae (B) • Gamma – no clearing of agar, intact RBCs • Streptococcus bovis (gallolyticus)
  • 23. Beta Hemolytic Streptococcus Typing • Lancefield typing system: Beta hemolytic Streptococcus are grouped (typed) by identifying the “C” carbohydrate (CHO) and lipoteichoic acids present in the bacterial cell wall. • Classifies Beta Streptococcus into separate groups (A, B, C, F, and G) identifying the ones commonly associated with human infection • The “C” CHO is detected in the Lancefield slide agglutination test. CHO binds with specific monoclonal antibody for each individual Streptococcus group. • Shown in picture is a positive test, with monoclonal antibody coated latex beads for group A (Strep pyogenes) agglutinating with the organism - A
  • 24. Streptococcus pyogenes • Group A Streptococcus [GAS] produces an intense ring of beta hemolysis around the small colony on 5% Sheep’s blood agar • Biochemical tests used for identification: • Bacitracin KB sensitivity test – GAS is inhibited by antibiotic Bacitracin (A) producing a small zone of inhibition • Not specific for GAS, inhibition also occurs with Beta hemolytic Streptococcus group C • PYR (pyrrolidonyl arylmidase) reaction • Organism spotted onto moist PYR disk • Wait 2 min – room temperature incubation • Add cinnamaldehyde reagent • Pink = positive = Streptococcus pyogenes • **This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis are also positive • Therapy : Penicillin, Amoxicillin or Cephalosporin antibiotics No resistance reported to these agents PYR reaction + --
  • 25. • Streptolysin O and Streptolysin S toxins • Comprise the ASO titer assay used to diagnosis Strep pyogenes sequelae (rheumatic fever and GAS glomerulonephritis) • These toxins help GAS evade the immune system • Toxin activity can be visually demonstrated on 5% Sheep’s blood agar • Streptolysin O toxin is oxygen labile • Streptolysin S toxin is oxygen stable • When both toxins are present, the stabbed area of the media will demonstrate increased beta hemolysis Streptococcus pyogenes (GAS) virulence factors • M Protein prevents phagocytosis • Hyaluronic capsule prevents phagocytosis
  • 26. Streptococcus pyogenes / most common Infections • Pharyngitis (1) • Impetigo (2) • Erysipelas (3) • Cellulitis (4) • Necrotizing fasciitis (5) • Puerperal sepsis • Toxic Shock • Scarlet fever (6) 3 4 5 6 1 2
  • 27. Sequelae of Strep pyogenes Infection Rheumatic fever • Inadequate treatment of GAS skin or pharyngitis infection • Family history, strain of GAS and multiple exposures can more likely evolve into sequelae, occurs 10-30 days post infection • Usually occurs in children 5 – 15 years • Pathogenicity due to molecular mimicry: similarity between the proteins of Strep A and human muscle tissue causes an autoimmune mechanism that leads to confusion. The immune system is then armed to attack heart (heart valves, muscle), joint, and bones not just GAS • Usually leads to need for valve replacement surgery Glomerulonephritis • Post infection with Nephritogenic strain of GAS • Leads to immune mediated destruction of the renal glomeruli • Usually resolves without therapy but can progress
  • 28. Streptococcus agalactiae (GBS) • Biochemical tests: – Camp test – Staph aureus strain that contains Camp factor streaked perpendicular to group B Strep on a 5% sheep’s blood agar plate, Incubate 24 hr. and view for intensified arrow shaped hemolysis. Positive test = GBS (see pix) – Hippurate hydrolysis – used to detect the ability of GBS to hydrolyze the chemical hippurate into glycine and benzoic acid by action of the hippuricase enzyme – 4 hour incubation. Positive test = purple pos Staph aureus Strep group B Camp Test Hippurate Hydrolysis Increased area of hemolysis
  • 29. Streptococcus agalactiae [GBS] • Pathogen of elderly • Bacteremia and urinary tract infection, • Acquisition most likely from the intestine • Pathogen of neonate • Bacteremia or central nervous system infection • 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 8 – 28 days after birth • Treatment: Penicillin or Cephalosporin (3rd generation)
  • 30. Streptococcus agalactiae (GBS) • Most effective way to prevent GBS neonatal infection is detect colonization in pregnant • Pregnant colonized (>=25%) in the cervix and/or rectal area • All pregnant should be screened at 35 – 37 weeks of pregnancy (Regulation/standard of practice) • Enrichment methods for GBS screening are mandatory • Cervix and rectal swab incubated in an enrichment broth for 18 hours at 35 ˚C then cultured onto 5% sheep’s blood agar. • Initial incubation in Enrichment broth is also used to increase sensitivity in molecular detection assays • Ampicillin drug of choice for prophylaxis of pregnant women testing positive for GBS • Susceptibility testing for alternative therapies for GBS must be performed in the penicillin allergic patient (Clindamycin)
  • 31. • Most common species • E. faecium and E. faecalis • No defined virulence factors • Gamma hemolytic on 5% Sheep’s blood agar • Gram positive cocci in pairs and short chains • Biochemical tests: • Bile esculin agar pos = grows in presence of bile & reduces esculin to esculetin to produce black color • 6.5% NaCl tolerance = grows in presence of NaCl • PYR reaction = positive • E. faecium = arabinose fermentation positive • E. faecalis = arabinose fermentation negative Enterococcus + - PYR Neg Pos
  • 32. Enterococcus • Pathogen of opportunity • Normal human intestinal flora • Infections include UTI, bacteremia, and abdominal abscess • Antimicrobial therapy: • Intrinsic resistance to cephalosporin antibiotics • Ampicillin plus Aminoglycoside can be synergistic for therapy of endocarditis • Vancomycin is an antibiotic of choice • Unique susceptibility issues • Acquired resistance to vancomycin known as vancomycin resistant enterococcus or VRE. Resistance is due to acquisition of genetic resistance genes: • Van A resistance gene for E. faecium • Van B resistance gene for E. faecalis
  • 33. Streptococcus bovis (gallolyticus) • Streptococcus gallolyticus ssp. gallolyticus (S. bovis biotype 1) Isolation from blood culture is associated with colon cancer (73%) • Streptococcus gallolyticus ssp. pasteurianus (S. bovis biotype 2) Isolation from CSF in neonatal meningitis • Gamma hemolytic, Gram positive cocci in pairs and short chains • 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
  • 34. Streptococcus pneumoniae • Alpha hemolytic on 5% Sheep’s blood agar • Gram positive bullet (lancet) shaped cocci in pairs • Polysaccharide capsule = virulence factor / antiphagocytic • Mucoid colony due to large amount of capsule produced • Identification: • Bile soluble – colonies dissolve Inhibited by Optochin – in sodium deoxycholate (bile) ethylhydrocupreine hydrochloride Zone of inhibition must be >=14 mm Autolytic Changes Looks like tire
  • 35. 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 • 13-20 valent pneumococcal conjugate vaccine aids in preventing invasive infections – those at risk need vaccination, infants, HIV, immune suppressed, and elderly • Susceptibility issues: • Acquired resistance to Penicillin due to Penicillin binding proteins • If susceptible,1st line therapy includes Penicillin or 3rd generation Cephalosporin (Ceftriaxone)
  • 36. Viridans Streptococcus • Several species of viridans group Streptococcus are NF in mouth and upper respiratory tract. Most common species: S. mutans S. salivarius S. sanguis S. mitis • Bile esculin negative • Bile solubility negative • Optochin resistant (zone size <=13 mm) • Cause 30 – 40% cases of sub acute endocarditis on native valve, usually due to bad dentition • Cause abscess and various infections throughout the body in the immune suppressed host • Variable susceptibility patterns can have elevated MICs to Penicillin so usual therapy is 3rd generation cephalosporin. Streptococcus pneumoniae Viridans Streptococcus
  • 37. Viridans Streptococcus unique species • Streptococcus anginosis group: • S. anginosus S. constellatus S. intermedius • Normal flora in human mouth • More virulent than “routine” viridans Streptococcus, due to capsule • Grow best when incubated in 5 – 10% CO² incubation (microaerophilic) • Butterscotch odor to colony • Cause deep tissue abscess, bacteremia, endocarditis, and intra abdominal infection • Variable susceptibilities – so best to do susceptibility testing, always susceptible to vancomycin
  • 38. Nutritionally Variant Streptococcus • Vitamin B6 (pyridoxal) deficient – • Will not grow on agar medium without B6 supplementation • Chocolate agar with supplements will support growth • Will grow in blood culture bottle due to patient’s blood containing vitamin B6 • Will not grow on 5% Sheep’s blood agar plate • Will grow around Staph aureus streak, SA supplies vitamin B6 • MALDI-TOF can definitively identify • Two genera: • Abiotrophia defectiva • Granulicatella adiacens • Bacteremia and Endocarditis – • More destructive to valve than “regular” viridans Streptococcus • Elevated MIC’s to Penicillin, susceptible to 3rd generation Cephalosporins. • Combination therapy: Penicillin and Gentamicin can be effective Satellite next to S. aureus streak
  • 39. Opportunistic Gram positive cocci • Aerococcus ureae – Gram positive cocci in pairs and clusters • Alpha hemolytic on 5% Sheep’s blood agar , difficult to identify, often confused with viridans Streptococcus, MALDI-TOF can assist • Emerging 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 • Can cause 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)
  • 40. Gram Negative Cocci Neisseria species Moraxella catarrhalis
  • 41. Gram Negative Cocci • Neisseria species and Moraxella catarrhalis • Small kidney bean shaped cocci in pairs • Oxidase enzyme positive • CTA (Cysteine Trypticase Agar) carbohydrate fermentation tests are an older method to identify these organisms • N. gonorrhea Gluc + Mal - Lac - Suc - • N. meningitidis Gluc + Mal + Lac - Suc - • N. lactamica Gluc + Mal + Lac+ Suc- • M. catarrhalis Gluc - Mal - Lac - Suc - • N. gonorrhea will NOT grow on 5% Sheep’s blood agar • N. meningitidis will grow on 5% Sheep’s blood agar
  • 42. Carbohydrate Fermentation Reactions Compare (+) yellow fermentation reaction 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 Growth on Chocolate Agar Oxidase Enzyme Positive Glucose + Glucose+/Maltose + N. gonorrhea N. meningitidis No growth BAP Grows on BAP
  • 43. Neisseria meningitidis • Meningitis, usually occurring in children and young adults • Hallmark - petechiae (organisms crowd into capillaries) leads to tissue necrosis and disseminated intravascular coagulation(DIC) caused by organism endotoxin • Progressive infection can be rapidly fatal (<24hrs) • Colonization can occur in nasopharynx (10-20%) • African meningitis belt – highest prevalence of disease in world • 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 therapy, asplenia, and HIV predispose to infection • Adrenal necrosis known as Waterhouse Friderichsen syndrome • Immunization at ages 2m, 12 yr, 16 yr, and in HIV to prevent
  • 44. Neisseria gonnorrhea • Sexually transmitted infection: urethrae, endocervix, ocular, rectal, oropharynx, septic arthritis • 10-20 % female ascend to PID but only 0.5% disseminate into bloodstream • Gram stain of urethral discharge useful for male diagnosis only • Gram stain of cervix can be problematic due to normal flora look-a-like organisms • Culture: collect specimen with charcoal containing swabs, do not refrigerate • Media: Selective Thayer Martin or Martin Lewis agar, chocolate agars with increased nutritional supplementation and antibiotic trimethoprim • Resistance: Beta lactamase enzyme and Chromosomal resistance mechanisms • Emerging resistance in some parts of the world has increased the need to do susceptibility assessment for patients not responding well to first line therapies • Therapy: Ceftriaxone + Azithromycin or Doxycycline, combination therapy to prevent development of resistance
  • 45. Molecular testing for Neisseria gonorrhea • Molecular amplification methods are the standard of practice and combo testing for Chlamydia trachomatis is the norm due to high % of co-infections • Urine, cervix/vaginal, throat and rectal – sites most often tested • Molecular testing sensitive (96%) and specific (99%) • Female: best specimen for diagnosis is cervix • Urine <=10–15% less sensitive than cervix • Male: Urine has become the standard specimen for diagnosis • The older way for diagnosing Chlamydia trachomatis infection: C. trachomatis culture - Iodine staining of inclusions in McCoy cell culture Fluorescent antibody stain of C. trachomatis infected cell – positive cell contains green staining Elementary bodies
  • 46. Moraxella catarrhalis • Colonizes the upper respiratory tract in children • Infections: Pneumonia (COPD), sinusitis, primary cause of otitis media in young children • Gram stain of sputum can be helpful in diagnosis of pneumonia (PMNs with Gram negative diplo-cocci) • Hockey puck colony – able to push colony across the agar surface without disruption • Biochemical Tests: • Oxidase enzyme positive • DNA’ase enzyme positive • Resistance: beta lactamase enzyme production (90%) • Therapy: Augmentin or 3rd generation Cephalosporin
  • 48. Corynebacterium • Over 20 species, most are saprophytic • Human normal flora, skin and nares • Most grow well on 5% Sheep’s blood agar • Gamma hemolytic gray colonies • Catalase enzyme = positive • “Diphtheroid” morphology on staining, Gram positive rods in Chinese letter forms • No spores produced
  • 49. Corynebacterium diphtheriae • Agent of Diphtheria • Forms tough oropharyngeal membrane (Diphtheroid is Greek for leather), bleeds when attempt to remove from throat • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis, heart, nervous system, and kidney damage • Non-toxin producing strains cause cutaneous ulcers and lesions • Diagnosis: culture, toxin detection (Elek plate), serology, and PCR to detect organism and study toxin genes • Grows well on 5% Sheep’s blood agar, gray gamma hemolytic colonies, catalase enzyme positive • Selective medium: Cysteine Tellurite agar/ black colonies • Metachromatic storage granules formed when grown on Loeffler’s media and stained with methylene blue stain • Treatment: Anti-toxin and Erythromycin or Penicillin Elek plate Metachromatic granules
  • 50. Other Corynebacterium spp. • Corynebacterium jeikeium • Normal human skin flora / thrives on lipid • Infects patients with indwelling plastic catheters and devices and can lead to bacteremia • Biofilms are formed on the plastic, protecting the organisms from antibiotic treatment • 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 +
  • 51. Rhodococcus species • Gram positive cocco-bacilli • Partially acid fast (PAF) with modified acid-fast staining • Related to Nocardia and Mycobacterium species • Environmental organisms found in the soil • Rhodococcus equi is the most common pathogen • Zoonotic pathogen • Capable of causing human pneumonia, bacteremia and tissue invasion, more common in immune suppressed, particularly HIV • Grows well at 30*C and 35*C. producing salmon-pink colonies. • Identification best using MALDI-TOF
  • 52. Bacillus species • Large Gram positive rods with square ends • Can be easily over-decolorized and appear reddish on Gram stain • Spores produced which cause clearing in the bacillus • Catalase enzyme positive Spores/ clear areas in bacillus Variable staining / not clearly Gram positive
  • 53. Bacillus anthracis • Agent of anthrax in herbivores • Category A agent – priority biothreat pathogen • Virulence factors: anthrax toxin and capsular polypeptide • Infections: • Wool sorter’s disease – acquired from handling contaminated cow hides, produces a unique black eschar skin lesion • Systemic infections: Pneumonia, sepsis, and meningitis • Irregular shape to colony border on 5% Sheep’s blood agar (BAP) Medusa head colonies Gamma (no) hemolysis on BAP Non-motile Susceptible to penicillin
  • 54. Bacillus cereus • Most common Bacillus species causing infection • Saprophytic environmental organism • Nosocomial and opportunistic infections, particularly in immunocompromised, and patients with indwelling or implanted devices • Can be a contaminate or pathogen in blood cultures • Cause skin infections in Intravenous drug users • Associated with food poisoning: vomiting due to emetic toxin production and diarrhea from diarrheal toxin. Refried rice has always been suspect. • Gram positive rod with spores • Dull gray colony producing beta hemolysis on BAP • Catalase positive • Motile • Resistant to penicillin
  • 55. Listeria monocytogenes • Small Gram positive rod / no spores produced • Catalase enzyme positive • Grows well on BAP producing subtle beta hemolysis • Motility provides identifying information • More motile at 25˚C than 35˚C • Tumbling motility on wet mount examination • Umbrella motility when inoculated into tubed media • Bacteremia and meningitis in immune suppressed, pregnancy, neonates, and elderly • Culture or molecular amplification methods for diagnosis • Grows well at 4˚C / infection from ingesting non pasteurized cheese and milk products, Deli case foods • Ampicillin drug of choice • Intrinsic resistance to Cephalosporin antibiotics
  • 56. Erysipelothrix rhusiopathiae • Small pleomorphic Gram positive rod • No spores produced • Catalase enzyme negative • Alpha hemolytic colony on 5% sheep’s blood agar plate • Only Gram positive rod that produces hydrogen sulfide (H2S) MALDI-TOF ID is useful for this difficult to identify bacteria – Infections from nature and animals, mostly swine, is an occupational disease of farmers and butchers – Virulence factor: capsule – Soft tissue infection and bacteremia +/- endocarditis – Intrinsic resistance to Vancomycin H2S production detected on Triple sugar iron agar (TSI) Erysipelas – lacy skin lesions of swine
  • 58. •Escherichia coli • Normal flora in human intestine • #1 cause of UTI [@80% of cases] • Bacteremia, neonatal meningitis, and abdominal infections • Lactose fermentation on MacConkey’s agar • Eosin methylene blue agar (EMB) green sheen produced • Spot indole reaction = positive / turns robin’s egg blue • Detects breakdown of tryptophan from growth on BAP Green sheen on EMB agar Indole positive Lactose fermentor
  • 59. Escherichia coli • Pathogen of diarrhea: Molecular testing is standard practice • Enterotoxigenic (ETEC) E. coli cause of traveler’s diarrhea • Entero-hemorrhagic E. coli (EHEC) (such as 0157:H7) • Bloody diarrhea from eating undercooked beef or contaminated veggies • Pathogenicity from Shiga toxin production (2 types) • Hemolytic uremic syndrome (HUS) can result with hemolytic anemia, thrombocytopenia, and renal failure] particularly in young children • Old school: Culture on sorbitol MacConkey agar / does NOT ferment sorbitol/ most all E. coli except EHEC ferment sorbitol
  • 60. 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 production • Currant jelly sputum produced in alcoholics due to blood mixed with Klebsiella capsular polysaccharide in sputum • Klebsiella (Enterobacter) aerogenes new taxonomic classification
  • 61. Extended Spectrum Beta Lactamase [ESBL] •Enzymes produced by select species of Enterobacterales • Confer resistance to Cephalosporins, Penicillins and Monobactam (Aztreonam) antibiotics by opening the beta lactam ring of the antibiotic and inactivating the antibiotic • ESBLs do not attack Cephamycin antibiotics (cefoxitin, cefotetan) or the Carbapenem antibiotic classes •Plasmid mediated CTX-M beta lactamases (bla gene)are the most common ESBL enzymes in the US currently, but many ESBL types can be found worldwide •Therapy for ESBL producing gram negative rods: • Carbapenems: Imipenem, Meropenem, Doripenem, and Ertapenem
  • 62. Carbapenemases – CRE and CRO •CRE = Carbapenamase resistant Enterobacteriales/ CRO= Carbapenamase resistant organism (Pseudomonas) •Carbapenemase beta lactamases lead to resistance to carbapenam antibiotics (meropenem, imipenem, doripenem, ertapenem). •Carbapenem-hydrolyzing-beta-lactamases are incorporated into the genetic elements in transposons of Gram negative bacillus. Transposons can insert into diverse plasmids which can transfer resistance to a broad spectrum of Gram negative bacillus. •Two CREs are getting the most attention: • KPC – “Klebsiella pneumoniae carbapenemase” most common in the US • NDM-1 – New Delhi metallo-beta-lactamase. Resistance determinants are numerous and great concern about its spread. • Other CRE/CROs: IMP, OXA-48, and VIM •Infections with CRE/CRO producing GNRs can have a 50% fatality rate
  • 63. Gram Negative Rods that do NOT Ferment Lactose! • Proteus species Colonies swarm in concentric layers on agar surface • Proteus vulgaris – spot indole positive • Proteus mirabilis – spot indole negative • Normal flora in intestine • Common cause of UTI and abdominal infections • Intrinsic resistance to antibiotic Colistin due to mcr-1 gene • Serratia marcescens • Produces red pigmentation which can intensify at room temp • Environmental contaminate • Causes infection most usually in • Immune suppressed • Ventilator associated pneumonia • Bacteremia
  • 64. 1. Glu/lac/suc fermented with gas 2. Glucose only fermented 3. Glucose fermented with H2S production 4. No CHO fermentation Non fermenter Triple Sugar Iron Agar (TSI)– 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 H2S production = black medium 1 2 3 4
  • 65. • Salmonella species • Diarrhea with +/- fever and PMNs in the stool • Infection from eating contaminated food (raw eggs, poultry, ground beef or dairy) or direct contact with a sick person or animal – Must ingest large #’s of organisms to make you ill, normal levels of stomach acid is protective • MacConkey agar - does not ferment lactose • Produces hydrogen sulfide on selective media • 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
  • 66. Salmonella typhi • Typhoid fever – high fever and sepsis, no diarrhea • Human pathogen with most cases in US (75%) from international travel • Post typhoid fever there can be carriage in gallbladder with passage in feces. Can transmit to others by bad hygiene and food preparation • Ingested organisms enter the bowel, rapidly move into the blood stream and eventually the bone marrow • Diagnosis: Blood cultures in early infection and bone marrow culture in late stages (>1 month) of infection • Vi capsular antigen unique to S. typhi • Moustache of Hydrogen sulfide (H2S) produced in TSI medium slant • This is a unique finding for S. typhi
  • 67. Shigella species • Diarrhea, +/-vomiting, fluid loss, PMNs 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] • No lactose fermentation on agar • Non motile • No Hydrogen sulfide (H2S) produced • 4 species based on somatic (cell wall) antigen • S. dysenteriae Group A • S. flexneri Group B • S. boydii Group C • S. sonnei Group D
  • 68. Salmonella Shigella Agar (SS agar) Shigella are colorless due to lactose not being fermented. H2S produced by Salmonella spp turning the colony black Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black colonies Shigella – green colonies, no H2S produced Normal flora – orange colored due to fermentation of lactose (E. coli) Non-Lactose fermenter Shigella Salmonella Normal Flora Lactose fermented H2S Salmonella Shigella Salmonella
  • 69. Yersinia enterocolitica • Major reservoir – swine • Humans infected by eating raw or undercooked pork • Infections: • Diarrhea • Septicemia in patients with iron overload syndromes • Mesenteric adenitis – symptomatic for right lower abdominal pain which can be confused with appendicitis • Infected blood products from transfusion have been reported • Oxidase = negative • Indole = negative • Urease = positive Grows well at 4 °C • CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for Y. enterocolitica
  • 70. Yersinia pestis • Plague, Category A biothreat 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 infection of the lymphatic system, forms painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread organism into blood stream • Pneumonia develops from blood stream infection • Fatality >=50% • Endemic in SW USA • Grows well on blood agar • catalase +, oxidase - • Bipolar staining looks like a “safety pin”
  • 71. • Vibrio cholera • Natural environment is 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 which – Increases cAMP with hyper secretion of NaCl and H20 – Death from dehydration and metabolic acidosis • Curved “C” shape Gram negative rods can be seen • Selective medium thio citrate bile sucrose agar, V. cholera (TCBS) yellow color from sucrose fermentation • Oxidase = positive , • Grows well in 1% salt solution TCBS Agar
  • 72. • Vibrio parahaemolyticus • Diarrhea from ingestion of contaminated raw oysters • Self limited in normal host, but serious in immune suppressed • TCBS medium sucrose fermentation negative so green colony • Vibrio vulnificus • Diarrhea from ingestion of contaminated oysters • Skin infection from injury in contaminated water • Bacteremia can occur in certain patient populations following the ingestion of contaminated oysters • Patients with liver disease (cirrhosis) and patients with increased serum iron • Formation of painful skin lesions on lower extremities with muscle necrosis • 50% fatality rate
  • 73. Acinetobacter baumannii • Environmental saprophyte and normal flora on human skin • Gram negative coccoid-bacilli • Does not ferment lactose / Oxidase enzyme negative • Opportunistic nosocomial pathogen • Glucose oxidizer • Pulmonary infection in vented patients • Can acquire resistance to many antibiotics from antibiotic exposure Stenotrophomonas maltophilia • Rapid maltose oxidizer • Gram negative bacillus • Gun metal gray pigment • Intrinsically resistant to many antibiotics, including carbapenems • Nosocomial pathogen: super-colonizer after long term carbapenem therapy due to intrinsic resistance to the carbapenem antibiotics (Imipenem and Meropenem)
  • 74. Pseudomonas aeruginosa • Fluorescent blue-green pigment produced (pyocyanin) • Oxidase enzyme = positive • Grape-like odor • Grows at 42˚C • Ps fluorescens/putida (a related species group does not grow at 42°C • Major pathogen of cystic fibrosis • Mucoid strains produced in the lung from the production of polysaccharide capsule • Major lung damage results from co-infection with Burkholderia cepacia (gram negative rod) • Nosocomial pathogen associated with exposure to water and moist environments • Intrinsically resistant to many antibiotics and acquire resistance to carbapenems
  • 75. Burkholderia pseudomallei • Soil-dwelling bacterium endemic in tropical and subtropical regions worldwide, particularly in Thailand and northern Australia. • Infects humans and livestock such as goats, pigs, and sheep and causes the disease melioidosis which is primarily a pneumonia. Mortality is 20 – 50% • Biosafety level 3 containment and PPE for working with the organism/ Category A select agent needs to be handled with BSL-3 safety precautions • Grows on a large variety of culture media including blood and MacConkey agar in 24 hours at 35* C • Colonies are wrinkled, have a metallic appearance, and possess an earthy odor. • Can display bipolar staining when stain from culture growth (like Y. pestis) • Difficult to identify using biochemicals, best using MALDI-TOF with proper identification library • Intrinsically resistant to many antibiotics via efflux pump mechanism
  • 76. • Chryseobacterium (Elizabethkingia) meningosepticum • Infections: • Newborns: fatal meningitis and septicemia in the newborn • Elderly/immune suppressed: bacteremia • Environmental organism found in water • Yellow colony • Oxidase and Indole positive • Haemophilus ducreyi • Cause of venereal disease: Chancroid • Painful necrotizing genital ulcers/inguinal lymphadenopathy • School of fish appearance on Gram stain from lesion • Requires hemin (X factor) to grow on solid media
  • 77. Haemophilus influenzae • Transmission – close contact and 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 (contains X and V factor) • Will not grow on 5% sheep’s blood agar • Requires 5-8% C0₂ for growth • Effective vaccine targets invasive infections with H. influenzae type B (Hib) effectively eliminating most childhood invasive infections • Ampicillin resistance from beta lactamase enzyme productions [25 %], 3rd generation Cephalosporin becomes the antibiotic of choice (Ceftriaxone) for invasive infections Satellite phenomenon Staph aureus supplies the X and V factors required
  • 78. HACEK organisms • Oral flora organisms. Due to poor detention or invasive dental procedures organisms are introduced into bloodstream and can infect heart valves • Fastidious Gram negative coccobacilli / need >=48 hrs to grow in culture • Cause of 5 -10% of community acquired native valve endocarditis not related to IV drug use • Haemophilus species oxidase (-) catalase (-) • Aggregatibacter (Actinobacillus) oxidase (-) catalase (+) • Cardiobacterium hominis oxidase (+) indole (+) • Eikinella corrodens oxidase (+), pits agar, bleach odor • Kingella kingii oxidase (+), hemolytic on blood agar • Major cause of septic joint infection in small children
  • 79. Bordetella pertussis • Whooping cough – 3 disease stages • Prodromal – flu like disease – most contagious stage • Catarrhal - classic whoop cough in small children Toxin adheres to bronchial epithelial cells and cough continues until toxin wears off • Paroxysmal - recovery phase • Human pathogen, inhabits nasopharynx • Peripheral blood smear - Lymphocytosis with atypical, large, irregular and deeply basophilic lymphocytes • Tiny Gram negative coccobacillus • Selective media = Regan Lowe Charcoal, growth in 3-5 days • Molecular detection is standard of practice / greater sensitivity • Reservoir for infection is in young adults due to waning immunity. Reason for booster shots in young adults
  • 80. Pasteurella multocida and P. canis • Normal flora in many animals (zoonotic) • Common pathogen of bite wound infections from cats and dogs • Human pneumonia cases from close contact with animals • 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 sensitive to penicillin
  • 81. Capnocytophaga species • Fusiform shaped Gram negative rods • Fingerlike projections from colonies “Gliding” • Require incubation in C02 for growth • Oxidase negative • Catalase negative • Normal mouth flora (NF) in humans and animals • C. canimorsus – NF in dogs, cause infected dog bites – high % of bite infections lead to bacteremia and endocarditis • Many Capnocytophaga species as NF in human mouth • Infected mouth ulcers can lead to bacteremia
  • 82. Brucella species • Brucellosis, FUO, significant joint pain, intracellular pathogen of the Reticuloendothelial system • Specimens: Blood (early in disease) and Bone Marrow (after first month) • Automated Blood culture systems detect at >=5 days of incubation • Serology can assist with diagnosis of chronic disease • Culture: Small Gram negative coccobacilli, non hemolytic gray colony, • Requires 5 – 10% 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 – infected pig exposure • B. canis - Infected dog exposure *Granuloma in bone marrow
  • 83. Campylobacter spp. • C. jejuni – Common cause of diarrhea in US, can progress to bacteremia in HIV and immune suppressed • Ingestion of undercooked poultry or juice contaminating raw food • Sea gull shaped, poorly staining Gram negative rod • Culture requires selective blood agar containing antibiotics to suppress the overgrowth of normal bowel flora organisms • Campy-BAP or Skirrow’s BAP • Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂) • Sequelae of diarrhea infection: Significant % Guillain-Barre syndrome • C. fetus – Bacteremia in the immune suppressed host • Source cattle and sheep • Temperature tolerance aids in identification / but MALDI-TOF works best C. jejuni – grows at 37˚C and 42˚ C, hippurate hydrolysis positive C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative
  • 84. • 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 and progress to: • Painful skin lesions • Enlarged lymph nodes • Bacteremia (triad known as ulceroglandular tularemia) • Pneumonia • Small Gram negative rod • Oxidase enzyme negative • Requires cysteine in culture medium for growth Francisella tularensis
  • 85. Helicobacter pylori • Acute gastritis with small % progressing to gastric adenocarcinoma • Human to human transmission due to poor hygiene • Rapid and strong urease enzyme produced by this organism • Can be used to detect organism in gastric antrum biopsy tissue • Small curved Gram negative bacilli • Difficult to grow, making culture not useful • Stool antigen for diagnosis and test of cure useful • Serum antibody detection obsolete – most adults IgG detected • Organism stained by silver stains in gastric biopsies • Treatment – Antibiotics and stomach acid suppression Immunohistochemical stain of gastric biopsy Chronic active gastritis with plasma cells and neutrophils in the lamina propria. (H&E)
  • 86. Legionella pneumophila • Pulmonary disease from exposure to aerosolized water • Hyponatremia common in during infection • Requires cysteine in culture medium for growth • Selective medium: Buffered Charcoal Yeast Extract agar with growth 3-5 days at 35*C in CO2 incubator • Will not stain with usual Gram stain methods • Must use carbol-fuchsin as counterstain to detect organism • Silver impregnation stains used in fixed tissue • Diagnosis: Urinary antigen test (EIA) detects Legionella pneumophila type I in pulmonary infection • Antibody response is delayed and seldom used for diagnosis • Treatment: Erythromycin (macrolide) BCYE Blood agar No growth
  • 87. Bacteria without cell walls • Mycoplasma and Ureaplasma species have no cell wall only cell membranes. Media for culture must contain protective sterols. Molecular amplification is diagnostic method of choice. • Lack of peptidoglycan cell wall, so will not Gram stain, no clear colonies produced on agar, and cannot be treated with antibiotics that act by inhibiting cell wall formation • M. pneumoniae • Community acquired pneumonia • High titer cold agglutinins with infection • Genital mycoplasmas • M. hominis form fried egg appearance on sterol containing agar • Can cause vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes • Ureaplasma urealyticum form dark metal-like appearance on sterol containing media • Rapid hydrolysis of urea • Non-gonococcal urethritis, upper genital tract infection, spontaneous abortion, and neonatal infections Ureaplasma Mycoplasma hominis
  • 88. Unusual and Difficult to Grow Bacteria • Bartonella henselae • Cat scratch disease from exposure to cat and cat excrement • Bacillary angiomatosis – vascular skin lesions +/- invasion, associated with HIV • Bartonella quintana – cause of trench fever/ vector is the body louse • Coxiella burnetii – cause of Q fever / zoonotic infection that can infect humans • 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 Serologic and molecular assays for diagnosis
  • 89. Unusual and Difficult to Grow Bacteria • Klebsiella (Calymmatobacterium) granulomatis • Agent of STD granuloma inguinale • Infection leads to ulcerative genital lesions • Streptobacillus moniliformis • Rat bite fever or Haverhill fever • Infection from untreated rat bite • Cell wall deficient bacteria known as L form • Difficult to culture; inhibited by SPS in blood culture media and requires serum supplementation to grow • Patient history is helpful, sequencing if necessary • Tropheryma whipplei – • Whipple disease • Gram positive rod (Actinomycete) distant relative of Mycobacterium avium and M. paratuberculosis • Found in soil and farm animals • Causes a diarrhea which can lead to malabsorption syndrome and cardiac disease • Characteristic findings in fixed tissue Foamy macrophages in the lamina propria
  • 90. Unusual and Difficult to Grow Bacteria • Ehrlichiosis – infection caused by Rickettsia spp • Zoonotic infection • Vector is the Ixodes tick (hard tick) • Two genera cause infection / intracellular pathogen • Anaplasma spp, inclusion (morula) in the PMN • Ehrlichia spp inclusion in the monocyte • Fever, leukopenia, thrombocytopenia, • Elevated serum aminotransferases • Ehrlichiosis has no rash, which differs from Rocky Mountain Spotted Fever caused by Rickettsia rickettsia which is known for presence of rash • Found in south central, southeast , and mid-west US • PCR, serology, and examination of blood smear for diagnosis
  • 91. Spirochetes • Borrelia burgdorferi - Lyme’s disease • Primarily found in Northeastern US • Vector = Ixodes tick (hard tick) • Acute disease: Fatigue, headache, fever, rash, and erythema migrans lesion after tick bite • Can progress to a chronic disease • Diagnosis: Serology and molecular methods • Borrelia species – Tick borne relapsing fever • Western United States • High fever (relapsing) with thrombocytopenia • Muscle and joint aches • Vector: Soft tick (Ornithorodos hermsi) • Diagnosis: Blood smear observe spirochete
  • 92. Spirochetes • Treponema pallidum • Agent of Syphilis • Traditional Algorithm involves serologic screening for Syphilis using a nontreponemal test (e.g., VDRL and RPR) first and followed by a treponemal specific test such as EIA, TPPA, or FTA-ABS. This traditional method has a high positive predictive value, but often misses early primary and treated infections. • New Reverse Algorithm for syphilis diagnosis consists of a first performing a treponemal antibody screening immunoassay followed by confirmatory nontreponemal antibody testing (RPR).This algorithm was created to increase sensitivity of detection. • Years past: Darkfield examination was used to view spirochetes from lesions
  • 93. Spirochete Infections • Brachyspira • Intestinal spirochetes are anaerobic bacteria • Transmission is most likely contaminated water sources • Most common in developing nations and HIV infected • Intestinal spirochete found on the brush border of the intestine • Clinical significance is controversial • Leptospira interrogans / Leptospirosis • Fever with rash and renal involvement • Urine from rats and other animals contaminate water supplies • Microscopic agglutination test (CDC) is best diagnostic test (1) Leptospirosis – Shepherd’s crook shaped spirochete (2) Presence of spirochete in a renal tubule (2) 1
  • 94. Bacterial vaginosis • Mixed anaerobic/aerobic bacterial infection • Rather benign infection except in pregnancy • Pregnant women are at increased risk due to hormone changes that happen during pregnancy. Increased risk for premature birth and low birthweight. • Discharge: Fish-like odor and alkaline pH >=4.5 • Usual NF organism of Lactobacillus is overgrown by: • Gardnerella vaginalis (aerobic Gram variable rod) primary bacterial marker for infection • Mobiluncus (anaerobic Gram negative rod) • Clue Cells are diagnostic for vaginosis and provides a more specific indication of infection than growth in culture • Molecular probe assays and amplification assays available as part of “women’s health” screening panels Clue Cell
  • 95. Anaerobic Bacteria • Anaerobic infections can occur in virtually any organ or region of the body • Most are polymicrobial –with both aerobic and anaerobic species • Endogenous normal flora organisms cause most infections • Due to trauma, vascular or tissue necrosis cutting there is a lowered oxygen supply to the involved tissue that can lead to infection • Treatment: Surgery to restore oxygen to tissue, remove necrotic tissue and antimicrobial therapy • Specimen collection for culture • Gel containing swab • ESwab • Evacuated vials (port-o-cult)/ oxygen free vials for fluid • Do not refrigerate specimen prior to culture, it will cause greater absorption of oxygen and increase the death of anaerobes
  • 96. Anaerobic culture • PRAS media – pre reduced anaerobically sterile • Media packaged in oxygen free environment • Provides best opportunity for growth of anaerobes • Most common anaerobic culture media include: • CDC anaerobic enriched blood agar • Kanamycin-vancomycin blood agar • Bile esculin agar • Thioglycollate broth • Chopped meat glucose broth • Anaerobic chambers – perform culture work in an oxygen free closed cabinet so specimens and organisms are never exposed to oxygen • Anaerobic gas packs and jars for anaerobic incubation of culture plates • Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts to generate heat and create oxygen free environment. • Dry pack – (Anaeropack) absorbs O2 and generates CO2 PRAS
  • 97. Bacteroides fragilis group Pleomorphic irregular staining Gram negative rod • Normal flora in the GI tract, most common anaerobe • Grow in the presence of bile • Growth on bile esculin media and turns media black • Resistant to Penicillin and Kanamycin • Infections: Related to the bowel such as GI abscess • B. fragilis group organisms • 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 Metronidazole resistance is suggesting a need to do susceptibility testing to confirm susceptibility Growth on bile/esculin media Black pigment from Esculin production (left) and growth on Anaerobic Blood agar (right)
  • 98. Prevotella and Prophyromonas • Pleomorphic Gram negative rods • Normal flora in the upper respiratory tract • Infections: respiratory tract abscesses • Will not grow in the presence of bile • Will not turn black on bile esculin media • Unique brick red fluorescence when placed under UV light • Colony forms natural black pigment after one week of incubation
  • 99. Fusobacterium spp. • F. nucleatum Long thin gram negative bacilli – spindle shaped with pointed ends • Normal flora upper respiratory tract • Infections: mouth and respiratory tract and liver abscess • Vincent’s angina – necrotizing oral co-infection caused by Fusobacterium species and spirochetes • F. necrophorum – pleomorphic gram negative bacilli in filaments or chains • Lemierre’s syndrome - oropharyngeal infection, leads to thrombosis in jugular vein, septicemia, with high fatality rate
  • 100. Clostridium species • Gram positive bacilli (boxcar shaped ), form spores which appear as empty space in the bacilli • May easily over-decolorize and appear gram negative • Clostridium perfringens • Infections: Food poisoning, necrotic tissue abscess (Clostridial myonecrosis), bacteremia, cholecystitis • Most common anaerobic Gram positive rod in intestine • Important reactions: • Double zone of beta hemolysis on BAP produced • Lecithinase produced on egg yolk agar (clouds the agar) • Reverse camp test positive Lecithinase Reverse Camp Test Double zone of hemolysis
  • 101. • Clostridium botulinum – Botulism • Adult disease – ingest preformed heat labile neurotoxin, usually mass produced or in home-canned foods • Infant disease - spore ingested from nature or product of nature such as honey or household dust, spore germinates in gut producing neurotoxin • Begins with constipation and difficult sucking bottle • Both forms are life threatening neuroparalytic diseases • Clostridium tetani - Tetanus • Gram stained cells appear like Tennis racket from terminal spore • Infection begins with penetrating skin injury with introduction of the Tetanospasmin toxin • Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw (trismus) • Immunization to prevent
  • 102. • Clostridium septicum – • Bacteremia or gas gangrene in patient with underlying malignancy • Hematogenous spread from GI tract leads to bacteremia – no trauma necessary • Clostridioides (Clostridium) difficile – • Disease: antibiotic associated colitis, pseudomembranes produced from toxin production • Toxin A – enterotoxin causes fluid accumulation • Toxin B – potent cell cytotoxin, primary virulence factor (TcdB gene) • Binary toxin – Nap1 strain, this strain has increased toxin production • Diagnosis of infection: • Standard of practice has become a two-step algorithm: • Molecular amplification to detect TcdB gene indicating presence of toxin gene in the stool • Enzyme immunoassay to detect active toxin in the stool • If TcdB gene and toxin detected, the patient has C. difficile disease and requires therapy • Alternate method: GDH (Glutamate dehydrogenase) antigen test uses antibodies to test for the presence of the GDH, a protein present in all C. difficile isolates. Screening test with good sensitivity, rapid turnaround time, and low cost. • Selective medium Cycloserine, Cefoxitin, Fructose Agar [CCFA] – culture used for research purposes, not for diagnosis of disease
  • 103. Actinomyces • Branching gram positive bacilli – • No spores produced • Aerotolerant, but grows best in anaerobic conditions • Normal flora oral, GI, vagina, skin • Infections: oral/facial (lumpy jaw), respiratory and GI • Can form sulfur granules in tissue • Actinomyces israelii – associated with oral, thoracic, and abdominal infections, IUD infections Bread crumb colonies produced in broth Penicillin susceptible
  • 104. Branching Gram positive rods of Actinomyces – antler like Molar tooth colony on surface of blood agar plate Sulfur granule Aggregates of Actinomyces Cervicofacial actinomycoses –lumpy jaw
  • 105. Propionibacterium acnes (Cutibacterium) • Pleomorphic, sometimes branching Gram positive rod • Catalase positive • Indole positive • Normal flora - skin, oral, GU, and GI • Can be seen as a pathogen in blood cultures or contamination due to not properly cleaning the blood draw site • Pathogen of acne vulgaris • Opportunistic pathogen: cerebral shunt infections and other endovascular and neurosurgical infections from organism gaining entrance from skin • Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints • Cultures should be held up to 7 days • Therapy - Ampicillin
  • 106. Sarcinia species • The sarcina was the marching pack carried by Roman legionaries, the heavy infantry of the Roman legions. Due to the similar appearance the organism was named Sarcinia. • Gram-positive anaerobic coccus that has been reported in the upper gastrointestinal tract biopsies of patients with evidence of gastric stasis. • The pathogenic role in humans is not entirely known.