UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY

DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI

GENERA AND SPECIES TO BE CONSIDERED

֍ Beta-hemolytic streptococci • Enterococcus faecium

• Streptococcus pyogenes (group A • Other Enterococcus spp. isolated
streptococci) from humans
• Streptococcus agalactiae (group B  Enterococcus durans
streptococci)  Enterococcus mundtii
֍ Groups C, F, and G beta-hemolytic  Enterococcus dispar
streptococci  Enterococcus gallinarum
֍ Streptococcus pneumoniae  Enterococcus avium
֍ Viridans streptococci (alpha-hemolytic)  Enterococcus hirae
• Streptococcus mutans group  Enterococcus raffinosus
• Streptococcus salivarius group  Enterococcus casseliflavus
• Streptococcus mitis group ֍ Leuconostoc spp.
• Streptococcus bovis group ֍ Lactococcus spp.
• Streptococcus urinalis ֍ Globicatella sp.
• Streptococcus anginosus group ֍ Pediococcus spp.
(also called Streptococcus milleri ֍ Aerococcus spp.
group) ֍ Gemella spp.
֍ Nutritionally variant streptococci ֍ Helcococcus sp.
• Abiotrophia defectiva ֍ Alloiococcous otitidis
֍ Dolosicoccus paucivorans
• Granulicatella adiacens
֍ Facklamia spp.
• Granulicatella balaenopterae
֍ Dolosigranulum pigrum
• Granulicatella elegans
֍ Ignavigranum ruoffiae
֍ Enterococci (most commonly isolated)
֍ Tetragenococcus spp.
• Enterococcus faecalis
STREPTOCOCCI : GENERAL CHARACTERISTICS

1. Catalase negative gram positive cocci arranged in chains.

2. Non-motile, Nonspore-formers
3. Most are fastidious
4. Behave like facultative anearobes but are truly aerotolerant anaerobes. Some are capnophiles.
5. Grow on Enriched Media (Blood Agar, Chocolate Agar, Todd-Hewitt Broth) with small (0.5 to 2.0 µm)
pinpoint and translucent colonies.
6. More elongated than spherical
7. Ferments carbohydrates to lactic acid without gas production

CLASSIFICATION
SPECIES ANTIGEN HEMOLYSIS COMMON NAME DISEASES
S. pyogenes A Beta Group A Strep Rheumatic Fever, Scarlet
Fever, Pharyngitis,
glomerulonephritis,
pyogenic infection
S. agalactiae B Beta Group B Strep Neonatal sepsis,
meningitis, puerperal
fever
S. dysagalactiae C Beta Group C Strep Pharyngitis, impetigo
S. equi
S. bovis D Alpha, Gamma Nonenterococcus Endocarditis, UTIs
viridans strep
E. faecalis, E. D Alpha, Beta, Gamma Enterococcus UTIs
faecium Alpha
S. pneumoniae Pneumococcus Pneumonia, meningitis
Anginosus, mutans, A, C, F, G, N, or - Beta, Alpha, Gamma Viridans Endocarditis, dental
mitis, salivarius caries, tissue abscess

CLINICALLY SIGNIFICANT ISOLATES

֍ Streptococcus pyogenes
• Antigenic Structure and Virulence Factor
 Group A antigen
 M protein – peptidoglycan of the cell wall and extends to cell surface
 Most imporant virulence factor
 anti-phagocytic ; adherence to mucosal cells
 encoded by gene emm
 M1 serotype – most common serotype in pharyngitis
 Protein F / Fibronectin-binding protein
 Adhesion molecule that mediate attachment to host epithelial cells
 Lipoteichoic acids – secures attachment to oral mucosal cells
 Hyaluronic Acid Capsule – prevents phagocytosis ; masks antigens
 Streptolysins

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
 Steptolysin O | responsible for hemolysis in SBA incubated anaerobically
o Subsurface hemolysis
o Oxygen labile
o Lyses leukocytes, platelets, RBCs
o Highly immunogenic  high antibody production
 Streptolysin S | lyses leukocytes
o Surface Hemolysis
o Nonimmunogenic
o Oxygen Stable
o Hemolysis in aerobic conditions
 Streptokinase
 Acts on plasminogen to cause fibrinolysis
 Hyaluronidase
 Spreading factor
 Streptococcal Pyrogenic Exotoxins / Erythrogenic Toxins – superantigens
 Associated with Scarlet Fever
 SpeA
 SpeB
 SpeC
 SpeF
• Diseases and Epidemiology
 Bacterial Pharyngitis
 Strep thorat
 Often seen in children age 5 – 15
 Pyodermal Infections
 Impetigo – localized ; progress to weeping lesions ; inoculation on abrasions/ via
insect bites ; more on children aged 2 to 5
 Erysipelas – rare; also affects the subcutaneous tissue ; seen in elderly patients
o Acute spreading skin lesion; intensely erythematous with a plainly
demarcated irregular edge
 Cellulitis – deeper infection that may lead to gangrene in patients with
peripheral vesicular disease or diabetes mellitus; may lead to bacteremia/sepsis
 Scarlet Fever
 Caused by strains infected with the temperate bacteriophage T12  causes
production of Spe.
 Diffuse red rash on upper chest spreading to the trunk and extremities
 After rash disappears (5-7 days) desquamation occurs
 Type 2 Necrotizing Fascitis
 Rapid progressiong inflammation and necrosis of skin, subcutaneous fat, and
fascia
 NF : Flesh-eating disease, suppurative fasciitis, hospital gangrene, necrotizing
erysipelas
 Type 1 NF is caused by polymicrobial infection of aerobic and anaerobic
bacteria
 Type 3 NF – gas gangrene or clostridial myonecrosis
 Type 1 Saltwater NF variant – Vibrio spp. ; when skin wound is contaminated
with the bacteria in saltwater
 Streptococcal Toxic Shock Syndrome
 Entire organ system collapses leading to death
 Associated with SpeA
 M1-M3 isolates
 Greatest Risk : children with chikenpox, elderly patients
 Poststreptococcal Sequelae – may also be caused by GrpC S.equi subsp. zooepidemicus
 Rheumatic Fever
o Follows pharyngitis after 1 month
o Chronic : Rheumatic Heart Disease
o Mechanism : Antigenic Similarity between streptococcal antigens and
heart tissue causing cross-reactivity to streptococcal antibodies
 Acute Glomerulonephritis / Post-streptococcal Glomerulonephritis
o After cutaneous or pharyngeal infection
o More common in children than adults
o Antigen-antibody complexes deposit in glomerulus. When complement
acts it causes an inflammatory response causing glomerular damage
• Treatment : Drug of Choice is penicillin; Alternative : Erythromycin
• Features
 Beta Hemolytic
 Susceptible to Bacitracin - best to differentiate GAS from other B-hemolytic organisms
 PYR positive

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
 Enterococci are also positive with PYR; differentiate using Bacitracin
Susceptibility
֍ Streptococcus agalactiae
• Antigenic Structure and virulence factors
 Acid-stable polysaccharide in the cell wall
 Only speecies expressing B antigen
 Three capsular serotypes – each contain a terminal residue of sialic acid which amy
inhibiit activity of the alternative complement pathway
 Ia
 Ib
 II
 Important Virulence Factor : Polysaccharide Capsule
 Sialic Acid – most significant component
 Hemolysin
 CAMP Factor
 Neuraminidase
 DNAse
 Hyaluronidase
 Protease
• Diseases and Epidemiology
 Mastitis in cattles
 Most important cause of neonatal meningitis and leading cause of infantile death
 May cause premature birth
 Early Onset Infection of the Newborns
 Pneumonia and Sepsis
 Late-Onset Infection
 Meningitis and Sepsis
 GBS in the vagina – most important determining factor
 Pregnant women must be screened for GBS at 35th – 37th week of gestation
• Treatment
 Drug of choice is penicillin but may need higher doses compared to GAS
• Features
 Beta Hemolytic
 Resistant to Bacitracin
 CAMP and Hippurate Hydrolsis Positive
 differentiates GBS from Grp D/Enterococcus/Other B-hemolytic streptococci
 Some strains are able to grow in 6.5% NaCl.
 In NaCl broths with indicators, Streptococcus agalactiae (group B) will grow in
the media, however it does not produce an acid reaction. Turbidity, but no color
change will be present.
֍ Streptococcus pneumoniae
• Antigenic Structure and Virulence Factors
 Contains C-polysacchatide which is unrelated to the Lancefield C antigen
 C-substance reacts with CRP via chemical reaction
 Capsular Polysaccharide
 Antibody against it is protective
 Detected by Neufeld test
o + | Quellung Reaction / swelling
o Does not simply identify S. pneumoniae but serotypes it as well
 IgA protease
 Hemolysins – Pneumolysin
 Neuraminidase
 Hyaluronidase
• Diseases and Epidemiology
 primary cause of bacterial pneumonia, meningitis, and otitis media
 causes Lobar Pneumonia
 pneumonia is not usually a primary infection but is rather a result of normal defense
barrier disturbances.
 Prevention : Vaccination
 PPV – pneumococcal vaccine – protects against 13 serotypes that commonly
infects man
 PPSV23 – 23-valent pneumococcal polysaccharide vaccine
• Features
 Lancet shaped; Diplococci; Alpha Hemolytic (wide zone)
 Fastidious – require BHI, TSA with 5% Sheep RBC, Chocolate Agar
 Capnophile
 Colonies : round, glistening wet dome shaped
 Old Colonies undergo autolysis – coin with a raised rim appearance
 Resembles viridans

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
 Optochin Susceptible
 Differentiates it from other alpha-hemolytic strep

֍ Viridans Streptococci
• constituents of the normal microbiota of the upper respiratory tract, the female genital tract, and
the gastrointestinal tract
• fastidious, with some strains requiring CO2 for growth
• Five groups
 S. mitis group
 S. mitis
 S. pneumoniae
 S. sanguis
 S. oralis
 S. mutans group
 S. mutans
 S. sobrinus
 S. salivarius group
 S. salivarius
 S. vestibularis
 S. bovis group
 S. equinus
 S. gallolyticus
 S. infantarius
 S. alactolyticus
 S. anginosus group
 S. anginosus
 S. constellatus
 S. intermedius
• Viridans streptococci are the most common cause of subacute bacterial endocarditis, a condition
associated with a transient bacteremia.
• The ability to ferment sugars, Voges-Proskauer (VP) reaction, β-D-glucuronidase activity, and
hippurate hydrolysis are used for the differentiation of species within the viridans group.
֍ Enterococcus
• Natural inhabitants of the intestinal tracts of humans and animals
• Group D antigen
• Most enterococci are nonhemolytic or α-hemolytic, although some strains show β-hemolysis.
 PYR + like GAS : differentiate via Bacitracin Susceptibility Test
 Enterococcus is resistant to Bacitracin
 PYR is useful in differentiating them from other Group D streptococci.
• Enterococci sometimes exhibit a pseudocatalase reaction—weak bubbling in the catalase test.
• In contrast to streptococci, enterococci have the ability to grow under extreme conditions—for
example, in the presence of bile or 6.5% NaCl or at 45° C or alkaline pH.
 Some strains of GBS are able to grow on 6.5% NaCl and rarely GAS
 Aerococci may also be bile-esculin-positive and may grow in 6.5% NaCl
• these organisms are capable of acquiring and exchanging genes encoding resistance to
antimicrobial agents
• This genus is the first clinically relevant group of gram-positive cocci to acquire and disseminate
resistance to vancomycin, thus the name vancomycin-resistant Enterococcus (VRE)
• Vagococcus fluvialis, Lactococcus garvieae, and Lactococcus lactis are sometimes misidentified
as enterococci, often called unidentified enterococci.
 Vagococcus spp. are motile differentiating them from the lactococci.
 Both lactococci and vagococci are susceptible to vancomycin and fail to form gas in
Mann, Rogosa, and Sharpe (MRS) broth;
 Both lactococci and vagococci are pyrrolidonyl arylamidase (PYR) and leucine
aminopeptidase (LAP) positive; and grow in 6.5% NaCl broth.
֍ Miscellaneous
• low virulence and are almost exclusively associated with infections involving compromised hosts
 A possible exception is the association of Alloiococcus otitidis with chronic otitis media
in children
• Certain intrinsic features, such as resistance to vancomycin among Leuconostoc spp. and
Pediococcus spp., may contribute to the ability of these organisms to survive in the hospital
environment.

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
LABORATORY DIAGNOSIS AND IDENTIFICATION TECHNIQUES

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
֍ Bacitracin Susceptibility
• Historically, bacitracin susceptibility has been used as an inexpensive test for presumptive
identification of S. pyogenes.
• This method is helpful in screening for GAS in throat cultures. The throat swab is used to
inoculate SBA containing SMZ, and a bacitracin disk is placed directly onto the agar. Growth of
most interfering respiratory microbiota is inhibited by SMZ, but S. pyogenes and S. agalactiae
grow.
• β-Hemolytic colonies that grow and are susceptible to bacitracin are presumptively identified as S.
pyogenes.
• S. pyogenes (group A) is susceptibile to bacitracin and resistant to SMZ
• S. agalactiae (group B) is resistant to both bacitracin and SMZ
֍ CAMP Test [Christie, Atkins, and Munch-Petersen]
• used for presumptive identification of GBS [Listeria monocytogenes is also CAMP +]
 Differentiate GBS and Listeria by Catalase and Gram stain
• can be performed in 3 ways
 use of a β-lysin–producing strain of S. aureus
 the use of a disk impregnated with the β-lysin
 rapid CAMP test (or spot CAMP test), involves placing a drop of extracted β-lysin on the
area of confluent growth of the suspected GBS. After incubation at 35° C for at least 20
minutes, enhanced hemolysis is observed
• The result is a characteristic arrowhead-shaped hemolytic pattern [only if with S. aureus]
• Some group A streptococci will be CAMP test positive if the test plate is incubated in a candle jar,
in a CO2 atmosphere, or under anaerobic conditions. Therefore, ambient air incubation should be
used
• L. ivanovii only shows a positive CAMP reaction when using an alternative CAMP test method, in
which Rhodococcus equi replaces S. aureus.
• Reverse CAMP + : Arcanobacterium haemolyticum, Clostridium perfringens
 Arcanobacterium is CAMP + if streaked against S. agalactiae
֍ Hippurate Hydrolysis
• A useful test for differentiating S. agalactiae from other β-hemolytic streptococci
 Enterococci can also be hippurate hydrolysis positive.
• S. agalactiae possesses the enzyme hippuricase (also called hippurate hydrolase), which
hydrolyzes sodium hippurate to form sodium benzoate and glycine.
• The glycine reacts with ninhydrin giving the purple color positive result
• Requires 30 min incubation
֍ Pyrrolidonyl-α-Naphthylamide Hydrolysis
• provides a high probability for the presumptive identification of the β-hemolytic GAS and the
nonhemolytic group D streptococci
• S. pyogenes is the only species of Streptococcus that is PYR positive.
• Other genera that are PYR positive include Enterococcus, Aerococcus, and Gemella
• detects the activity of L-pyrrolidonyl arylamidase, also called pyrrolidonyl aminopeptidase
֍ Leucine Aminopeptidase
• is a peptidase that hydrolyzes peptide bonds adjacent to a free amino group.
• leucine-β-naphthylamide, is hydrolyzed to β-naphthylamine. After the addition of p-
dimethylaminocinnamaldehyde reagent, a red color develops.
• most helpful in differentiating Aerococcus and Leuconostoc spp. from other gram-positive cocci.
• Streptococcus, Enterococcus, and Pediococcus spp. are LAP positive
• Aerococcus and Leuconostoc spp. are LAP negative.
֍ Voges-Proskauer Test
• distinguish the small-colony–forming β-hemolytic anginosus group containing group A or C
antigens from large-colony–forming pyogenic strains with the same antigens
• anginosus group are positive
• detects acetoin production from glucose
• a heavy suspension of bacteria is made in 2 mL of VP broth.
• After about 6 hours of incubation at 35° C, a few drops of 5% α-naphthol and 40% KOH are
added.
• The tube is shaken vigorously to increase the concentration of dissolved oxygen, and the broth is
incubated at room temperature for 30 minutes.
• the formation of a red or pink color is a positive reaction
֍ β-D-Glucuronidase
• detects the action of β-D-glucuronidase, an enzyme found in isolates of large-colony–forming β-
hemolytic group C and G streptococci but not in the small-colony–forming β-hemolytic S.
anginosus group.
• Several commercially prepared rapid assays are available.
• A fluorogenic assay using methylumbelliferyl-β-D-glucuronide has also been described.
֍ Bile Esculin and Salt Tolerance
• mainstays in identification schemes for nonhemolytic, catalase-negative, gram-positive cocci

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DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI

• bile esculin test is a two-step test detecting growth of bacteria in the presence of 40% bile and the
ability to hydrolyze esculin.
 Group D streptococci and Enterococcus spp. test positive
• Organisms positive for bile esculin are separated into group D streptococci or Enterococcus by the
salt tolerance test.
 Growth in 6.5% NaCl broth is used to identify Enterococcus and Aerococcus organisms.
 Some species of Pediococcus and Leuconostoc grow in 6.5% NaCl broth when incubated
for 24 hours.
 Group D streptococci do not grow in a 6.5% NaCl broth.
֍ Optochin Susceptibility
• a filter paper disk containing optochin (ethylhydrocuprein hydrochloride) is added to the surface
of an SBA plate that has just been inoculated with an α-hemolytic Streptococcus.
• Incubate overnight at at 35° C in a CO2 incubator.
• A zone of inhibition greater than 14 mm (6-mm disk) or 16 mm (10-mm disk) is considered
susceptible and a presumptive identification of S. pneumoniae.
֍ Bile Solubility
• takes advantage of the S. pneumoniae autocatalytic enzyme amidase
• suspension of S. pneumoniae in a solution of sodium deoxycholate lyses, and the solution becomes
clear

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI
SUMMARY ON IDENTIFICATION

• Except for Abiotrophia and Granulicatella (unless BAP/CAP is supplemented with pyridoxal/Vitamin B6),
all the organisms listed on the first page will grow on standard laboratory media such as 5% sheep blood
and chocolate agars.
 Most organisms will grow on agar media within 48 hours of inoculation.
• Most of the organisms within this group are facultative anaerobes, with some preferring a CO2-enriched
environment.
 Laboratories typically incubate blood or chocolate agar plates in 5% to 10% carbon dioxide. This
is the preferred atmosphere for S. pneumoniae and is acceptable for all other genera discussed.
• The cellular arrangement and the type of hemolysis are important considerations in identification.
 If the presence of hemolysis is uncertain, the colony should be moved aside with a loop and the
medium directly beneath the original colony should be examined by holding the plate in front of a
light source.
 Visualization of beta-hemolysis is enhanced by anaerobic conditions.
 blood agar plates should be inoculated by stabbing the inoculating loop into the agar
several times
• For isolating group A streptococci from throat swabs, the most common medium is 5% sheep blood agar
supplemented with trimethoprimsulfamethoxazole (SXT), to suppress the growth of normal microbiota.
 A bacitracin disc is placed on the initial inoculum streak to aid in identification. However, this
medium also inhibits growth of groups C, F, and G beta-hemolytic streptococci.
 Use of the 0.04-U bacitracin disk is no longer recommended for S. pyogenes, because groups C
and G streptococci are also susceptible to this agent.
• The PYR and hippurate or CAMP tests can be used to identify groups A and B streptococci, respectively.
 A brown halo around colonies on bile esculin agar and a positive PYR reaction is indicative of
Enterococcus sp.
• To detect genital carriage of group B streptococci during pregnancy, a vaginal or rectal swab is inoculated
into Todd-Hewitt broth, such as LIM (Todd-Hewith with CNA).
 After 24-hour incubation the LIM broth is subcultured to 5% sheep blood agar.
 LIM broth can also be subcultured to CHROMagar Strep B, designed specifically for
detection of GBS with sensitivity close to 100%.
⸋ Colonial appearance of GBS are mauve colored.
 Latex agglutination confirmation test can be performed directly from the plates on suspected
colonies.
 All plates negative for GBS should be incubated for an additional 24 hours.
 S. agalactiae is able to hydrolyze hippurate and is positive in the CAMP test.
 The CAMP test detects production of a diffusible, extracellular protein that enhances the
hemolysis of sheep erythrocytes by Staphylococcus aureus.
 A positive test is recognized by the appearance of an arrowhead shape at the juncture of
the S. agalactiae and S. aureus streaks
• Minute beta-hemolytic streptococci are all likely to be of the S. anginosus group
 a positive Voges-Proskauer test and negative PYR test identify a beta-hemolytic streptococcal
isolate as such.
• Screening test for vancomycin susceptibility is often useful for differentiating among many alpha-
hemolytic cocci.
 All streptococci, aerococci, gemellas, lactococci, and most enterococci are susceptible
 pediococci, leuconostocs, and many lactobacilli are typically resistant
• Colonies suspicious as S. pneumoniae (small, gray, moist, alpha-hemolytic; center may be depressed) must
be tested for either bile solubility or susceptibility to optochin (ethylhydrocupreine hydrochloride).
 The bile solubility test is confirmatory and is based on the ability of bile salts to induce lysis of S.
pneumoniae. Streptococcus pseudopneumoniae is partially soluble, and other alpha-hemolytic
streptococci are insoluble.
 In the optochin test, which is presumptive, a filter paper disk (“P” disk) impregnated with optochin
is placed on a blood agar plate previously streaked with a lawn of the suspect organism. The plate
is incubated at 35°C for 18 to 24 hours and read for inhibition.
 S. pneumoniae produce a zone of inhibition, whereas viridans streptococci grow up to the
disk and thus are resistant.
 Occasional strains of S. oralis, S. mitis, and S. pseudopneumoniae are also sensitive
• Once S. pneumoniae has been ruled out as a possibility for an alpha-hemolytic isolate, viridans streptococci
and enterococci must be considered.
 Aerococcus, Facklamia, Gemella, Globicatella, Halococcus, Lactococcus, Leuconostoc, and
Pediococcus can all resemble viridian streptococcus.
 Pediococcus can be confused with enterococci, because they are bile-esculin positive and cross-
react with group D antisera..
• Differentiation of enterococci, group D streptococci, and lactococci is traditionally based on the ability of
the organisms to hydrolyze esculin in the presence of 40% bile; other streptococci do not.
 The esculetin in bile esculin agar reacts with an iron salt to form a dark brown precipitate
surrounding the colonies.

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 Enterococcosel agar is a selective differential medium based on the esculin hydrolysis and is also
selective by incorporation of inhibitory oxgall (bile salts) to inhibit growth of other gram-positive
organisms, with the exception of group D streptococci, and sodium azide to inhibit growth of
gram-negative organisms.
 However, occasionally other bacteria may display the dark brown precipitate.
 Bile esculin agar and enterococcosel agar with vancomycin are used for a primary screening to
detect vancomycin-resistant enterococci
• Except for species not usually isolated from humans (Enterococcus saccharolyticus, E. cecorum, E.
columbae, and E. pallens), all enterococci hydrolyze PYR and possess group D antigen
 Most clinical laboratories identify Enterococcus spp. presumptively by demonstrating that the
isolate is PYR and LAP positive and that it grows at 45°C and in 6.5% NaCl.
 However, the recent discovery of S. urinalis presents a problem in this regard.
 S. urinalis and the commonly isolated Enterococcus spp. exhibit identical reactions in the
four tests listed above and they only differ in the ability to grow at 10°C (S. urinalis
cannot grow at this temperature).
• Leuconostoc produces gas from glucose in MRS broth; this distinguishes it from all other genera, except
the lactobacilli.
 However, unlike Leuconostoc spp., lactobacilli appear as elongated bacilli when Gram stained
from thioglycollate broth.
• Several organisms (e.g., Leuconostoc, Pediococcus, Lactococcus, Helcococcus, Globicatella,
Tetragenococcus, Streptococcus urinalis, and Aerococcus viridans) will show growth on bile esculin agar
and in 6.5% salt broth; therefore these two tests no longer solely can be used to identify enterococci.
 Aerococcus, Gemella, and Pediococcus grow as large, spherical cocci arranged in tetrads or pairs
or as individual cells.
 Leuconostoc may elongate to form coccobacilli, although cocci are the primary morphology.

THE STREPTOCOCCUS-LIKE ORGANISMS

֍ Abiotrophia and Granulicatella

• Formely known as the nutrionally variant streptococci
• Grow as satellite colonies around other bacteria
• require sulfhydryl compounds for growth
• significant cause of bacteremia, endocarditis, and otitis media
• linked to a few cases of osteomyelitis, endophthalmitis after cataract extraction, brain abscess,
chronic sinusitis, septic arthritis, meningitis, and breast implant– associated infections
• Granulicatella adiacens, Granulicatella elegans, and Granulicatella balaenopterae have been
isolated from blood cultures and tissue samples.
 Endocarditis resulting from these organisms is difficult to treat because of the increased
tolerance of the organisms to antimicrobial agents.
 Surgery is usually required to achieve a cure.
• should be suspected when gram-positive cocci resembling streptococci are observed in positive
blood cultures that subsequently fail to grow on subculture
 use an SBA plate with a S. aureus streak and examine it for satellitism.
 Alternatively, media can be supplemented with 10 mg/L pyridoxal hydrochloride.
• Granulicatella spp. are often misdiagnosed and require a combination of approaches for
identification
֍ Aerococcus
• A common airborne organism
• widespread, opportunistic pathogen causing an increasing number of infections associated with
bacteremia, endocarditis, and UTI in immunocompromised patients
• Aerococcus viridans has been linked to cases of bacteremia and endocarditis.
• Aerococcus urinae and Aerococcus sanguinicola
 have been associated with invasive diseases, such as sepsis, endocarditis, lymphadenitis,
and peritonitis, often originating from the urinary tract.
 UTIs are seen more often in elderly women while invasive infections occur most
frequently in elderly men, but the outcome is generally favorable.
 Able to aggregate platelets ; form biofilms
• Diagnosis / Isolation | Identification Techniques
 Aerococci resemble viridans streptococci on culture but are microscopically similar to
staphylococci in that they occur as tetrads or clusters
 sometimes show a weak catalase or pseudocatalase reaction
 grow in the presence of 6.5% NaCl and can easily be confused with enterococci
 Some strains of A. viridans are bile esculin positive and PYR positive.
 A. urinae is bile esculin negative and PYR negative.
 16S ribosomal ribonucleic acid (rRNA) sequencing and MALDITOF testing seem to
accurately identify A. urinae, A. sanguinicola, and A. viridans.
֍ Gemella
• are similar in colony morphology and habitat to viridans streptococci

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 UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | CATALASE NEGATIVE GRAM-POSITIVE COCCI

• easily decolorize on Gram staining and often appear as gram-negative cocci in pairs, tetrads,
clusters, or short chains.
• isolated from cases of endocarditis, wounds, and abscesses.
• The most significant species is Gemella haemolysans
֍ Lactococcus
• gram-positive cocci that occur singly, in pairs, or in chains and are physiologically similar to
enterococci.
• previously classified as group N streptococci
• isolated in cases of UTI and endocarditis
• Production of acid from carbohydrates is useful in distinguishing it from enterococci
֍ Leuconstoc
• Irregular cocoid morphology
• share several phenotypic and biochemical characteristics with Lactobacillus spp., viridans
streptococci, Pediococcus spp., and Enterococcus spp. and are sometimes misidentified.
• intrinsically resistant to vancomycin
• frequently found on plant surfaces and vegetables, and in milk products
• isolated from cases of meningitis, bacteremia, UTIs, and pulmonary infections
• Species associated with infection include
 Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc
lactis, Leuconostoc mesenteroides, and Leuconostoc pseudomesenteroides.
• Biochemical identification is based on the
 absence of catalase, PYR, and LAP activities;
 hydrolysis of esculin in the presence of bile;
 growth in the presence of 6.5% NaCl;
 production of gas from glucose
֍ Pediococcus
• facultatively anaerobic, gram-positive cocci (arranged in pairs, tetrads, and clusters) that can grow
at 45° C.
• intrinsically resistant to vancomycin
• associated with infections in patients who have underlying gastrointestinal abnormalities or who
have previously undergone abdominal surgery
• have also been linked to bacteremia, abscess formation, and meningitis
• Biochemical characteristics used to identify them include
 a positive bile esculin test
 the presence of LAP activity
 absence of PYR activity
 do not produce gas from glucose
 some grows in 6.5% NaCl

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