BACTERIOLOGY INAO

STREPTOCOCCUS, ENTEROCOCCUS, AND SIMILAR Group C S. dysgalactiae

ORGANISMS Group D Enterococcus spp.
 streptococcaceae consists of a large family of medically Streptococcus bovis complex
important spp., including streptococcus spp., and
enterococcus spp.
GROUP A: STREPTOCOCCUS PYOGENES
 organisms under this group that’s commonly seen in clinical
specimens are:  “GAS”
 S. pyogenes  MOST CLINICALLY IMPORTANT
 S. agalactiae  belongs to the Lancefield group A
 S. pneumoniae  considered as one of the MOST AGGRESSIVE PATHOGENS
 E. faecalis encountered in the clinical microbiology lab
 E. faecium  susceptible to bacitracin; PYR positive (+)
 Viridans streptococci group  infections of this bacteria are very prone to progression with
GENERAL CHARACTERISTICS the involvement of deeper tissues and organs
GRAM MORPHOLOGY  “flesh-eating bacteria” or severe invasive infection
 catalase-negative  Scarlet fever
 facultative anaerobic  → manifests rashes in the face and upper trunk
 GRAM+ cocci in pairs or CHAINS  Streptococcal shock syndrome
 spherical ovoid or lancet-shaped cocci VIRULENCE FACTORS OF S. PYOGENES
 some strains require additional CO2 for their initial isolation Streptolysin S  oxygen-STABLE
CATALASE TEST—a test to differentiate streptococcus from  NON-IMMUNOGENIC hemolysin (does
micrococcaceae not trigger an immune response)
 capable of lysing erythrocytes,
leukocytes, and platelets at room
temperature (20C–22C)
Streptolysin O  oxygen-LABILE (will be broken down
in the presence of oxygen)
STAPH: CATALASE+; STREP: CATALASE-  IMMUNOGENIC hemolysin
BASIS OF IDENTIFICATION  lyses the same cells and culture cells
 cell wall structure in streptolysin S but only in the
 hemolytic patterns on sheep blood agar (beta, alpha, absence of room temperature
gamma)  inhibited by CHOLESTEROL in the skin
 reaction or antibodies to specific bacterial antigen lipids (resulting in the absence of the
 the Lancefield Classification scheme development of protective antibodies
 biochemical identification associated with skin infection)
Streptococcal  erythrogenic toxins produced by
 molecular level: 16S ribosomal ribonucleic acid (rRNA) Pyrogenic
sequences lysogenic strains
Exotoxins  heat-labile
EPIDEMIOLOGY (SPEs)  may be released and produce scarlet
 many of these organisms are commonly found as part of the fever w/c occurs in association with
normal microbiome of the pharynx, mouth, lower GIT, and streptococcal pharyngitis manifested
vagina by rashes in the face and upper trunk
 rarely found in groups C and G (more on
 bacteria can cause a disease when:
Group A)
 other normal microbiota are depleted
 also acts as SUPERANTIGENS
 the bacterial inoculum is increased
 activating the immune system,
 virulent factors are heightened
especially macrophages and T-
 adaptive immunity is impaired
helper cells
S. PYOGENES
 induce the release of mediators
 upper respiratory tract and skin lesions (IL-1, IL-2, IL-6), tumor necrosis
S. PNEUMONIAE factor (alpha, beta), interferons, and
 upper respiratory microbiota cytokines which induces shock and
 When these organisms gain access to normally sterile sites (i.e., organ failure
blood, CSF, body pleural fluid, peritoneal fluid, pericardial M protein  anti-phagocytic cell wall
fluid, joint fluid, vascular tissue, etc.), they can cause life-
threatening infections.  very immunogenic
PATHOGENESIS AND SPECTRUM OF DISEASE  antibodies against this protein confer
immunity
BETA-HEMOLYTIC STREP  >60 M protein exist
 causes beta-hemolysis on blood agar; completely hemolyzed  CROSS-REACTION can lead to chronic
red cells around colonies infection assoc, with strep pyogenes.
 further categorized into Lancefield groups based on Class 1M protein: assoc with RHEUMATIC
serologically reactive carbohydrates in the cell wall FEVER (fever with endocarditis [inflammation of
the heart muscle], subcutaneous nodules, and
Group A S. pyogenes “GAS” polyarthritis and believed to usually follow s.
Group B S. agalactiae “GBS” pyogenes respiratory tract infection) strep throat
but naulian bc antigen cross reacts with human
 BACTERIOLOGY INAO
heart and would have similar structure with M ALPHA-HEMOLYTIC STREPTOCOCCI
protein and (even tho naulian nakas strep throat, STREPTOCOCCUS PNEUMONIAE
ang iattack sa anitbodies is human
heartrheumatic fever)  susceptible to optochin; positive for bile solubility and
Class 2M protein: assoc with ACUTE quellung test (+)
GLOMERULONEPHRITIS (involves the  colonies are mucoid or flattened with a depressed center;
deposition of antigens and antibodies in the “coin-like” colonies
glomeruli, damaging the organ and characterized  seen in normal flora of upper respiratory tract (carriers)
by edema, hypertension, hematuria, and
 25%–50% of preschool children
proteinuria. It usually follows respiratory and
cutaneous infection)  36% of primary school- aged children
Lipotechoic  nearly 20% of adults are carriers (the prevalence
 permits bacterial adherence to the
acid of invasive serotypes has decreased due to the
respiratory epithelium
availability of vaccine)
STREPTOCOCCAL TOXIC SHOCK SYNDROME—similar to staph
 PERSON-TO-PERSON INFECTIONS during epidemics
shock syndrome in which there’s a MULTI-SYSTEM INVOLVEMENT
occur usually by DROPLET AEROSOLS (enhanced by
including renal and respiratory failure. treptococcal shock
upper respiratory tract infection and crowding)
syndrome is from the streptococcal infection through the mouth.
 develops when hosts immune system is impaired, most
There is a presence of rash, diarrhea and it can induce shock which
cases are ENDOGENOUS following aspiration of oral
can lead to death. Thus, it is very potent.
secretion containing normal flora that includes S.
S. pyogenes also elaborates about 20 extracellular products, pneumonia
including  CDC recommends newborns are vaccinated starting at 2
months of age and adults especially >65yrs old
 VARIOUS ENZYMES  pneumonia, meningitis (especially in infants and
 streptolysins elderly), spontaneous bacteremia (in persons who do
 hyaluronidase—may enhance spread of the not have a spleen), otitis media, sinusitis, and
organism thru CT; “flesh eating” spontaneous peritonitis
 streptokinase—promotes fibrinolytic activity by VIRULENCE FACTORS
converting plasminogen to plasmin
ANTIPHAGOCYTIC POLYSACCHARIDE CAPSULE
 deoxyribonucleases [DNAses]
 EVADES PHAGOCYTOSIS
 nicotinamide adenine dinucleotidase (NADase)  capable of mobilizing inflammatory cells mediated by
 pyrogenic (erythrogenic toxins) its cell wall structures
 has zero types A, B, and C w/c are produced by PNEUMOLYSIN
the isolates of S. pyogenes that was infected with  activates the CLASSIC COMPLEMENT PATHWAY
a specific TEMPERATE BACTERIOPHAGE same  mediates the suppression of the oxidative burst in
with corynebacterium phagocytes (providing for effective evasion of immune
 their pyrogenicity is caused by the direct action clearance)
on the hypothalamus
GROUP B S. AGALACTIAE (GBS) PHOSPHORYCHOLINE
 within the cell wall
 hippurate hydrolysis positive (+); positive CAMP test (+)  binds receptors for platelet-activating factors in
 usually associated with neonates endothelial cells, leukocytes, platelets, and tissue
 in which they get infected as they pass through the vaginal cells of the lungs (allows entry and spread of the
canal organism)
 colonization of maternal genital tract is assoc with VIRIDANS STREPTOCOCCI
colonization of infants and rest of neonatal disease  optochin resistant; PYR negative
acquired before or during the birthing process  NO hemolysis (alpha or gamma); smell like butterscotch
 with early-onset infections occurring within the first few (assoc with milleri)
days after delivery & late-onset infections appearing after 1w  The gamma-hemolytic version of viridans is PYR-negative as well.
of age Moreover, they do not grow in the presence of 6.5% NaCl.
 newborns are affected but instead we assess the mothers bc  Most of them enter thru dental or surgical procedures which leads
to tooth abscesses, abdominal infections, bacteremia or valve
they are carrier endocarditis and late onsent prosthetic valve endocarditis
 All pregnant women at 36–37 weeks of gestation should have S. MUTANS—etiliogic agent of dental carries
vaginal or rectal specimens collected and processed for
S. MITIS—commonly assoc with endocarditis
detection of GBS. This specimen can be cultured or have a
S. BOVIS—bacteremia has been associated with
nucleic acid amplification test performed on them after
malignancies of the GIT; unique
enrichment in a selective culture broth.
 divided into 7 subtypes
 intrapartum antibiotic prophylaxis is given to the
 among them is S. gallolyticus
carriers (mothers) subsp. gallolyticus isolated from
 ADULTS: can cause postpartum endometritis, UTI, blood cultures of patients with
bacteremia, and skin & soft tissue infections, pneumonia and colonic cancer more often than others
osteomyelitis, endocarditis, meningitis, arthritis S.SALIVARIUS
 septicemia, pneumonia, and meningitis in newborns  isolated from the oral cavity and blood
S. ANGINOSUS
 formerly milleri group
 most common viridant streptococci
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responsible for liver, spleen, and brain  if the first swab yields negative,
abscesses inoculate the second swab
 very large and very complex (thus, are not into BA plate or selective
groupable by Lancefield serology) streptococcal BA plate
 can produce beta hemolysis; strains group  a TWO-PLATE CULTURE METHOD
A,C, F and G antigens  to increase recovery for
diagnosis of streptococcal
pharyngitis
GAMMA-HEMOLYTIC STREPTOCOCCI
ENTEROCOCCUS  both sheep BA and
 PYR (+); bile esculin hydrolysis (+) trimethoprimsulfamethoxazole
 grows in the presence of 6.5% NaCl (SXT) BA are inoculated.
 divided into E. faecium and E. faecalis Serologic test to detect streptolysin O
 the common cause of UTI in hospitalized persons and DNAs B antibodies in an acute and
 healthcare-associated infections (due to the development convalescent serum samples are used
of a particular resistance to multiple antibiotics that primarily to diagnose acute rheumatic
allows them to survive and proliferate esp in px receiving fever and acute glomerulonephritis
multiple antimicrobials causing SUPERINFECTIONS) following infection with gas.
 vancomycin-resistant enterococcus (VRE)
 resistance to vancomycin is possible because of van
genes MEDIA OF CHOICE
RESISTANT to ALL cephalosphorins and aminoglycosides BLOOD AGAR OR CHOCOLATE AGAR
 Streptococci grow well on BA or CA
VAN GENES  BA is preferred bc this is a differential media that can
 these are plasmid-borne genes (can create infection present the hemolytic factors of the organism (CA does not
control problems involving transmission of this have hemolytic pattern)
resistance)
 divided into three types: vanA, vanB, vanC ABIOTROPHIA & GRANULICATELLA
 vanA, vanB  special group of streptococci
→ confers high-level resistance  Both will not grow on BA or CA unless pyridoxal
→ predominantly found in E. faecium (vitamin B6) is supplied either by the placement of a
 vanC are differentiated from the intrinsic & lower- pyridoxal disc or by cross-streaking with a
level in resistance in yellow and motile Enterococcus staphylococcus (bc staphylococcus will break down
spp. the blood (because it’s beta-hemolytic) and release
the B6 that’s in the blood, which allows the
UNIDENTIFIED ENTEROCOCCI
Abiotrophia and Granulicatella to use the pyridoxal
 vagococcus fluvialis inside the lysed RBC.
 lactococcus garvieae  Cultures that appear positive and show chaining, and
 lactotoccus lactis gram-positive cocci on gram stain but do not grow
on subculture should be subcultured again with a
 ‘sometimes misidentified as enterococci’. Vagococcus pyridoxal disc to consider the possibility of nutrient
spp., are motile while Lactococcus spp. are not. variable pyridoxal dependent streptococcus such as
 Both are susceptible to vancomycin and fail to form gas S. meteor, abiotrophia & granulicatella bacteremia
in Mann, Rogosa, and Sharpe (MRS) broth.
5% SHEEP BA with SXT
 However, they have the same presentation as
enterococcus in terms of PYR, LAP, and 6.5% NaCl  for isolating group A streptococci from throat swabs
broth positive.  a bacitracin disc is placed on the initial inoculum streak
 Vancomycin suscetibility is a distinguishing charac to aid in the identification
from entercoccus GENITAL CARRIAGE OF GBS
 TODD HEWITT BROTH
 LIM BROTH
LABORATORY DIAGNOSIS To detect genital carriage of group B streptococci
during pregnancy, a vaginal or rectal swab is inoculated
DIRECT DETECTION METHODS into the Todd Hewitt broth which contains antimicrobials
ANTIGEN DETECTION MOLECULAR
such as gentamycin, nalidixic acid, and colistin and
METHODS nalidixic acid again which suppresses the growth of
normal vaginal microbiota and allows the growth of GBS
 latex agglutination  PCR for: only. After 24hrs of incubation, the Lim both should be
 enzyme-linked immunosorbent  Christie-Atkins- subcultured into the sheep blood agar or it can also be
assay (ELISA) technologies Munch-Petersen subcultured into the CHROMagar strep B where it will
readily available commercial kits; very (CAMP) factor for produce a mauve color. Aside from vaginal rectal swabs,
specific but false negative results may GBS (cfb gene) we can also do urine for group b streptococci infection.
occur if the specimen contain low  C5a peptidase However, it must have >104 CFU/mL in a pregnant
numbers of S. pyogenes. gene (scpB) to female to be used as a marker for a possibility of GBS by
 TWO THROAT swabs detect group B vaginal carriage.
 recommended streptococci ENTEROCOCCOSEL AGAR
 if the first swab yields  Selective differential medium based on esculin
positive, the second swab hydrolysis
can be discarded  Selective by incorporation of inhibitory oxgall
 BACTERIOLOGY INAO
bile salts to inhibit growth of other GRAM+ beta-lysin of staphylococcus aureus. GBS
organisms (with exception of enterococcus or are streaked perpendicular to a streak of S.
GBS) aureus on sheep blood agar
RESULT
Sodium azide for the inhibition of gram-negative POSITIVE: enhanced hemolysis is
organisms. However, occasionally other bacteria may indicated by an arrowhead-shaped zone of
display the dark brown precipitate. Hence, bile esculin beta-hemolysis at the juncture of the two
agar and enterococcosel agar with vancomycin are used organisms
for primary screening to detect VRE. NEGATIVE: no enhancement of hemolysis
HIPPURATE to detect the ability of the bacteria to
GRANADA AGAR HYDROLYSIS hydrolyze substrate hippurate into glycine
 Chromogenic broth including carrot broth media can TEST
be used as enrichment broth. and benzoic acidby the action of the
 Colonies of beta-hemolytic group b strep will convert hippuricase enzyme
the color of the tube from clear to YELLOW OR PRINCIPLE:
ORANGE. However, non-hemolytic GBS will not  hippurate is the glycine conjugate of
change the tube color and when used, a negative benzoic acid
broth would still need to be planted into solid media  when Hippurate is hydrolysed by an
for recovery of these strains organism, glycine and benzoic acid
 Carrot broth for GBS
are formed
 Selective non-chromogenic enrichment broth can be
subcultured into the Granada agar. Colonies of GBS  glycine is deaminated by the oxidizing
will appear yellow to orange for ease of detection. agent ninhydrin (which is reduced
 Nucleic acid amplification test (NAAT) can be used to during the process)
detect GBS directly in vaginal rectal specimen or can  the end product of the ninhydrin
be used to detect GBS in Lim or carrot broth culture. oxidation reacts to form a purple-
INCUBATION CONDITIONS AND DURATION colored product
RESULT
 STREP are facultative anaerobes POSITIVE: deep blue/violet color in 30mins
 some prefer a CO2-enriched environment (false positive if it exceeds 30 mins)
 5% to 10% CO2 (beta-hemolysis is enhanced by anaerobic NEGATIVE: colorless or slightly yellow-pink
condition) color
 Because of STREPTOLYSIN O, BA should be inoculated by Medium must contain only the Hippurate bc
stabbing the inoculating loop into the agar several times for ninhydrin might react with any free amino acids
observation of subsurface hemolysis present in the growth media or other broths
TESTS TO DIFFERENTIATE STREP AND ENTEROCOCCUS OPTOCHIN TAXO P
TEST PRINCIPLE: optochin
BACITRACIN Principle: a disk (Taxo A) impregnated with
TEST a small amount of bacitracin (0.04 units) is (ethylhydroxycupreine hydrochloride/P
placed on an agar plate disk), is placed on a lawn of organism on
RESULT a sheep blood agar plate
POSITIVE: any zone of inhibition >10mm; RESULT
susceptible (S. pyogenes) POSITIVE: zone of inhibition ≥ 14mm in
NEGATIVE: No zone of inhibition; diameter; with 6mm disk (S. pneumoniae
resistant (S. agalactiae) only)
L- a test for the presumptive identification of NEGATIVE: no zone of inhibition
PYRROLIDONYL BILE
ARYLAMIDASE GAS and enterococciby the presence of differentiates S. pneumoniae from other
(PYR) TEST the enzyme I-pyrrolidonyl arylamidase SOLUBILITY alpha-hemolytic streptococci as the
PRINCIPLE: the enzyme I-pyrrolidonyl TEST former is bile soluble
arylamidase hydrolyzes the I- pyrrolidonyl-
b-naphthylamide substrate to produce a b- PRINCIPLE: bile or a solution of a bile salt
naphthylamine. The b-naphthylamine can (e.g., sodium deoxycholate) rapidly lyses
be detected in the presence of N, N- pneumococcal colonies
methylamino-cinnamaldehyde reagent by  lysis depends on the presence of an
the production of a bright red precipitate. intracellular autolytic enzyme
RESULT (amidase) (this enzyme is
POSITIVE: bright red (PINK) color within demonstrated by allowing the bile
5mins culture to age in the incubator)
NEGATIVE: no color change or an orange  bile salt lowers the surface tension
color between the bacterial cell membrane
CAMP TEST Christie-Atkins-Munch-Peterson (CAMP) test, and the medium (thus, accelerating the
to differentiate group streptococci from other organism’s natural autolytic process)
streptococcus spp.Listeria monocytogenes  bile salts activate the autolytic enzyme
also produces a positive result with this test
which induces clearing of the culture
PRINCIPLE: certain organisms (including
RESULT
group B streptococci) produce a diffusible
POSITIVE: suspension clears
extracellular hemolytic protein (CAMP
factor) that acts synergistically with the NEGATIVE: suspension remains turbid
 BACTERIOLOGY INAO
NEGATIVE: growth and no blackening of
QUELLUNG  also called the Neufeld reaction medium
TEST  the gold standard technique for
serotyping S. pneumoniae GROUP A (S. PYOGENES)
 this is a microscopic precipitin test  susceptible to bacitracin
 used to identify pneumococci or  B-hemolytic
 PYR+
determine the capsular stereotype of
GROUP B (S. AGALACTIAE)
individual pneumococcal isolates (to
 B-hemolytic
detect the capsule of pneumococcus)  Hippurate hydrolysis+
PRINCIPLE: anti-capsular bodies that are  CAMP TEST+
present in the serum would react with a STREPTOCOCCUS PNEUMONIAE
carbohydrate material of the  Optochin SUSCEPTIBLE
pneumococcal capsule (causing a micro-  Bile solubility+
precipitin reaction on the surface of the S.  A-hemolytic
pneumonia)  Colonies are mucoid or flattened with a depressed center
 this antigen-antibody reaction causes  “Coin like colonies”
 Quellung test
a change in the refractive index of the
ENTEROCOCCUS
capsule (for it to appear swollen and  PYRase+
more visible)  Grow in the presence of 6.5% NaCl
 after the addition of a counter stain  Bile esculin hydrolysis+
(methylene blue), the pneumococcal VIRIDANS
cells would stain dark blue and are  A-hemolytic (optochin resistant; PYRase-)
surrounded by a sharply demarcated  Y (Gamma)-hemolytic (PYRase- and do not grow in 6.5%
“halo” which represents the outer edge NaCl)
of the capsule
ANTIMICROBIAL SUSCEPTIBILITY TESTING
 the light transmitted through the
 groups A, B, C, and G streptococcus are all susceptible
capsule appears brighter than either
to penicillin
the pneumococcal cells or the
 testing for resistance to macrolides, clindamycin, and
background
the tetracyclines is performed
 single cells, pair chains, and clumped
 in the case of penicillin allergy
cells may have a positive quelling
 a D zone test
reaction
 if erythromycin resistant
 capsulated pneumococci are
 clindamycin and erythromycin disks are separated by
highly virulent compared to
12mm instead of the 15-16mm recommended for testing
unencapsulated pneumococcus
S. aureus
SALT PRINCIPLE: heart infusion broth
TOLERANCE containing 6.5% NaCl is used as the test Catalase separates micrococci spp. (+) & streptococci spp. (-).
TEST medium(this broth also contains a small Streptococci are then differentiated by their hemolytic pattern.
amount of glucose and bromcresol purple
For beta-hemolytics, S. pyogenes and S. agalactiae are
as the indicator for the acid production)
differentiated from each other through the
RESULT
POSITIVE: visible turbidity in the broth, (1) bacitracin test in which the former is sensitive/susceptible and
with or without a color change from purple to the latter is resistant
yellow
NEGATIVE:no turbidity and no color (2) CAMP test in which the former is negative and the latter is
positive and
change
BILE  used for the presumptive identification (3) PYR test in which the former is positive and the latter is negative.
ESCULIN of enterococci
TEST  differentiates enterococci and group D For alpha-hemolytics, they are differentiated from each through
streptococci from non- group d viridans Taxo P or optochin test. If the result is positive, it indicates S.
streptococci pneumoniae. Otherwise, it indicates enterococcus or other vridans
PRINCIPLE: gram-positive bacteria streptococcus spp. if that’s the case, a PYR test is performed to
other than some streptococci and differentiate enterococcus from the other viridans streptococcus
enterococci are inhibited by the bile salts spp. if result is positive, it indicates the former and if negative, it
in this medium. Organisms capable of indicates the latter. For further confirmatory, 6.5% is added.
growth in the presence of 4% bile and able Leuconostoc and pediococcus rarely cause disease. However,
to hydrolyze esculin to esculetin will identification of these spp. are important as they are intrinsically
demonstrate growth (esculin reacts with resistant to vancomycin. Meanwhile, aerococcus spp. are also
Fe3+ and forms a dark brown to black identified as they are morphologically similar to enterococcus. The
precipitate) key difference between the two (especially in a urine culture) is
RESULT when a catalase- negative colony consists of gram-positive cocci in
POSITIVE: growth and blackening of the tetrads and clusters, it indicates aerococcus spp., but if in pairs or
agar slant
 BACTERIOLOGY INAO
short chains, it indicates enterococcus. Moreover, take note of the
PYR and LAP results of the four spp. Indicated above