Section III: Gram Positive Cocci

Classification, Metabolic & Microscopic

Features of Gram Positive Cocci (GPC)
Algorithm for identification of Gram positive cocci

Metabolic and Microscopic Features of Gram Positive Cocci

Organism Growth Requirements Cellular Morphology and Staining Characteristics

O2 Requ. Optimal CO2 Incubation Period Shape Gram Arrangement Capsule Motility Spore Special
Staining/microscopy
/Spe-cial Features
Temp. Requ. Days Weeks Months

Staphylococcus Facultative anaerobic 37°C – 1 – – Cocci +ve In clusters + (many) – – –

aureus (Fig. 3.1.1)

Micrococcus sps Strictly aerobic 37°C – 1 – – Cocci +ve In small groups of – – – –

four/eights

Streptococcus Facultative anaerobic 37°C + 1 – – Cocci +ve In short/long chains + – – –

pyogenes
(group A)

Peptoccoccus niger Strictly anaerobic 37°C – Few – – Cocci +ve In singles/pars/clumps – – – –

Peptostreptococcus Strictly anae- 37°C – 5 – – Cocci +ve – – – – –

sps. robic/aero-tolerant
(some strains

Sarcinia sps. Strictly anaerobic 37°C – Few – – Cocci +ve In groups of 8 or more. – – –∆ –

Streptococcus Facultative anaerobe 37°C – 1 – – Cocci +ve In short chains + *– – –

agalactiae (group B
Streptococci)

Enterococcus sps. Facultative anaerobe 37°C – 1 – – Cocci +ve Oval cocci in pairs and ? –* – –
(group D strept) short chains

Streptococcus Facultative anaerobe 37°C + 1 – – Cocci +ve In short chains – – – –

‘viridans’ (some microaerophilic)

Streptococcus Facultative anaerobic 37°C + 1/typcal – – Cocci (lanceolate +ve In pairs + – – –

pneumoniae mophology shaped in pairs
may take few with broad end
days) opposite)
(Fig. 3.1.2a,b)

* Enterococci are non-motile except E. gallinarum and E. casseliflavus

∆ In Sarccinia spores reported, but not usually seen.
An Overview of the Media Requirements, Colonial
Characters and Diagnostic Characteristics of
Key Gram Positive Cocci
Basal Enriched Selective / indicator Characterization & confirmation of isolate

S. aureus Nutrient agar: Blood agar: - Mannitol-salt agar - Smear & staining characteristic
large, golden large, . - Salt milk agar & broth - Catalase test (Fig 1.9.10) (+)
yellow circular haemolytic containing 8-10% NaCl - Coagulase test (Fig. 3.2.2) (+)
colonies colonies - Ludlam’s medium containing - Mannitol fermentation (+)
(Fig. 3.2.1) LiCl, tellurite & polymyxin - DNase production (+)
Above three media used for - Phosphatase production (+)
isolating bacteria from
 specimens; as faeces - Glucose metabolism
- MacConkey: Tiny lactose - Presence of teichoic acid (+)
positive colonies - Phage typing

Basal media; as Enriched media Selective/ others Characterization and confirmation of isolation
Nutrient agar

S. No Blood agar: small Crystal violet blood agar: growth Smear & staining characteristics
growth (poor) pin-point, b +ve (medium inhibits other Catalase: negative
pyogenes haemolytic gram positive cocci) Ferments sugars producing acid only (no gas)
colonies (Fig. MacConkey: No growth (NG) Bacitracin sensitive
[Group A 3.2.3)
Presence of group A antigen
In serum/glucose
Streptococ broth, growth Hydrolysis of pyrrolidonyl napthalamide (PYR test)
appears as M-typing (about 80 Protein types)
cus]
granular turbidity T. typing
Not ferment ribose (helps to differentiate
S.pyogenes from other streptococci)

S. Poor growth Blood agar: b - Hippurate hydrolysis +ve

haemolytic CAMP test +ve (Fig. 3.2.4)
agalactiae colonies

[Group B

Streptococ

cus]

Enterococc Grow easily Blood agar: On MacConkey: tiny pink Bile aesculin hydrolysis test +ve, PYR test +ve
Usually non- colonies Heat resistant (60°C – 30 mins)
us spp. haemolytic Ability to gow at 45°C and in presence of 6.5%
NaCl
Resistant to trimethoprim – sulfamethoxazole

S. Easily grow Good growth - Catalase: negative

Facultative anaerobe
‘viridians’
Alpha haemolysis (often)

S. NG (scanty Blood agar: Macconkey- NG Catalase: negative

growth) haemolytic, Mice intraperitoneal inoculation Optochin sensitive
pneumonia dome shaped (rarely performed, if organism Bile soluble
e colonies, which expected to be scanty in Ferments inulin
later become flat specimen, animal dies in 1-3
with central Pathogenic to animal (animal pathogenicity test
day) (isolate organism from
umbonation +ve)
various sites)
Choclate agar; Ferments several sugars with acid but no gas
+ve (growth) (Hiss’s serum water /agar slopes)
Typing of isolate with appropriate antisera
(routinely not possible, nor required)

Clinical (Pathogenicity) Profile of Infection

Caused by Gram Positive Cocci
ORGANISM

Staphylococcus Invasive • Superficial As pustules, folliculitis, boils, carbuncles, styes,

aureus impetigo, abscesses, furuncle (Fig. 3.3.1) cellulitis
(Fig. 3.3.2) • Case: pgs 179-182

• Deep Osteomyelitis, pneumonia, empyema and others

Toxinoses • Food poisoning

• Toxic shock syndrome
• Scalded skin syndrome

S. epidermis Opportunistic pathogen in prosthetic devices; as urinary catheters, prosthetic valves

S.saprophyticus Urinary tract infection in sexually active females and an opportunistic pathogen

Micrococcus sps rarely opportunistic infections

Streptococcus Suppurative Local Sore throat (pharyngitis), pyoderma (localized skin

pyogenes (group A infection with vesicles/pustules)
Streptococci) Case: pgs 188-193
(Fig. 3.3.3)

Spreading Impetigo, Erysipela (Fig. 3.3.3), Scarlet fever,

necrotizing fascitis, cellutitis

Others Puerperal infection sepsis, wound infection,

abscesses

Non suppurative • Acute Rheumatic fever

(post streptococcal • Acute glomeulonephritis
sequelae)

Peptococcus niger Skin, soft tissue, respiratory tract and female genita tract infections

Streptococcus New born (infants) • Early birth onset-sepsis, pneumonia and meningitis (few days to a week of
agalactiae (group B birth)
Streptococci)

• Late onset syndrome (occurs 3-8 weeks after birth)

Often nosocomial, has decreased complications

 Adults • Puerperal infection and infections associated with gyanecologic surgery and
abortion

Streptococcus • Respiratory infection , skin infections, endocarditis, meningitis

groups C,G. and
S. dysgalactiae

Enterococcus sps • Urinary tract & biliary tract infections, Septicaemia, endocarditis &
(group D intra-abdominal infections • Case: pg 193
streptococcus) • Case: pg 193

Streptococcus • Abscesses
group F,G
(S. anginosus
group)

Streptococcus • Subacute bacterial endocarditis (associated ‘salivarius with ‘mitis), Dental

‘viridans’ caries (Associated with ‘mutans’), Brain and liver abscess
(S. mutans, S. • Case: pg 195
salivarius, S. mitis)

Streptococcus • Pneumonia Labar & bronchopneumonia, meningitis (all ages), sinusitis, otitis
pneumoniae media, arthritis, septicaemia and others • Case: pgs 196-198

Integrated Clinical Based Study of

Staphylococcus/Abscess
A 40 year man, Sohan Lal presented with an abscess on the right upper gluteal region He gave a history of having received an
intramuscular infection at the site, 10 days back Fig. 3.4.1. The aspirated pus was gram stained, revealed gram positive cocci.
The growth of the organism on blood agar medium is depicted in Fig. 3.2.1. (p. 175)

What is the provisional diagnosis, as to the most likely organism causing the infection in this case?
A.1 (a) The infection is most likely caused by S.aureus.
Which are the important tests that can be performed on the isolate to confirm it’s identity?
A.1 (b) Catalase and coagulase tests can help in confirming the identity of this isolate. DNAase test can further help (if the test is available) in
assessing the virulence of the isolate.
Who gave the nomenclature of Staphylococcus?
A.1 (c) (i) The name Staphylococcus was given by Sir Alexander Ogston (1880), who also studied its role in suppurative lesions.
To which family does Staphylococcus belong?
A.1 (c) (ii) Staphylococcus belongs to the gamily Micrococcaceae.
What are the other genera that belong to this family?
A.1 (c) (iii) The other three genera in this family are Micrococcus, Planococcus and Stomatococcus.
Is it possible for this infection to be endogenous in origin? If so, how would this individual have acquired
infection with this organism?
A.2 Yes. About 10-20% of adults can be nasal carriers of S.aureus and about 10% of a population can be carriers of this organism on perineum.
The case could have acquired the organism from any of these sites. The probability of acquiring such organisms, would be increased, if the
case had infections; as follcutitis etc.
Enumerate the virulence factors of S. aureus?
A.3 (a) These can be categorized into antigenic structure (of the organism). toxins and enzymes of S aureus. The key components of the
antigenic structure include capsule (in some strains), peptidoglycan, teichoic acid and protein A. The key toxins are haemolysins
(aipha, beta, gamma and delta), leucocidins, enterotoxins and exfoliative (epidermolytic) toxin. The important enzymes include
coagulase, deoxyribonucleases and phosphatase. (described at A3, Pg 184-185, Case 5)
Compare and contrast the virulent factors of S.aureus, S.pyogenes and S.pneumoniae.

A.3 (b)
S.aureus S.pyogenes S.pneumoniae

Cell associated Capsule (polysaccharide) Capsule (hyaluronic acid) Capsule (polysaccharide) Key virulent factor,
(antigenic structure) about 90 serotypes (refer A.4c, Case 9,
pg 197)

Teichoic acid (facilitate adhesion, are Fimbriae

protective)

Protein A M, T&R protein

Enzymes Coagulase Streptokinase

Deoxyribonuclease Deoxyribonuclease

Phospatase Nicotinamide adenine

dinucleotidase (NADase)
Hyaluronidase
Others

Toxins Haemolysins (alpha, beta, gamma and delta) Streptolysins (O&S) Toxins have little role

Leucocidins Pyrogenic toxin (respon- Hemolysins

sible for scarlet fever)

Toxic shock syndrome toxin Leucocidin

Exfoliative toxin (epidermolytic) Pneumolysin

Descibe the epidemiology of S.aureus infections.

A.3 (c) It is very important to study the epidemiology of S. aureus (including MRSA), as they are responsible worldwide for
causing nosocomial infections (including outbreaks), being a common cause of surgical wound infections. Currently; S.
aureus (including MRSA) has been reported to be an important pathogen, even in community acquired infections. So, it
is important to understand, how these infections may be controlled.
S. aureus is a part of the normal human flora. Shortly after birth, the neonate may be colonized, by organism from
neighbouring human surroundings. These sites include the umbilical stump, perineal area and skin. Later on in life, the
carriage rate of the organism in healthy person varies between 25-50%, depending on the area, season and local
epidemiological factors. The colonization may be transient or persistent. The commonest site for colonization is the
anterior nares (nasal vestibule), to be followed by skin, perineal area, oropharynx and vagina (the latter site was
evaluated, after reporting of the outbreak of toxic shock syndrome in adult premenopausal women).
Source of infection: Human cases and carriers.
Mode of transmission:
• Direct contact with infected patient/carrier (commonest mode)
• Indirect contact with environment; as fomites (bed linen, blankets, door knob) or airborne (droplets having
desquamated epithelial cells).
Type of infection:
• In a hospital setting, the infection are often exogenous, often acquired from the hospital care giver.
 • In a community setting, infections are often endogenous, i.e., acquired from self colonized strain present on the
desquamated cells, from the skin or nose.
Some groups of individuals have been reported to have higher rates of colonization with S. aureus than the general
population, as the doctors, nurses and hospital ward attendants. The patients with increased rate of colonization include
insulin dependent diabetics, HIV infected patients, injection drug users and individuals (patients) with various
dermatologic conditions.
In an hospital setting, infection often occurs by transient colonization of hands of hospital personnel, who transfer strains
from one patient to another.
The reasons for S. aureus to be an important nosocomial agent include:
(i) Ability to survive harsh environment, as with high salt concentration
(ii) Presence of numerous virulent factors in the organism
(iii) Ability to persist intracellularly in certain phagocytes
(iv) Potential to acquire resistance to antimicrobials.
The nosocomial strains in an hospital usually belong to certain phage types (e.g., phage type 80/81) and are usually
multidrug resistant. This makes them have the potential to cause outbreaks.
What is the primary modality of management of this case (pyogenic abscess)?
A.4 (a) The primary modality of management of such of a case is surgical, i.e., incision and drainage of the lesion, to be
followed by antimicrobial therapy.
Is the administration of antimicrobial sufficient to resolve the infection in this case?
A.4 (b) Administration of antimicrobial alone won’t be able to resolve this infection, as this agent won’t be able to reach the
interior of the lesion, in an adequate concentration level to be effective
This case was started on oral cephalexin, but the treatment was changed after the availability of the antibiotic
susceptibility report, which categorized the isolate to be a methicillin resistant S. aureus.
What is this process (of changing antimicrobials) called?
A.5 (a) ‘De-escalation’
What is the likely resistance mechanism in the incriminated pathogen, that could be responsible for this
scenario?
A.5 (b) The presence of mec A gene in a S.aureus isolate, results in acquistion of new penicillin  binding protein (PBP), which
makes it as an methicillin resistant S.aureus (MRSA). These isolates are resistant to penicillinase stable penincillins. The
latter antibiotics were developed, when this organism started developing resistance to penicillin, due to production of β-
lactamase enzyme, that degrades the beta-lactam ring of penicillin, making this antibiotic ineffective. Currently; around
30% S.aureus of isolates produce this enzyme. The MRSA isolates have a modified cell wall protein termed penicillin
binding protein 2 (PBP-2), which has significantly reduced affinity for all beta lactam antimicrobial agents, making these
isolates resistant to all penicillins, cephalosporins, monobactams (as aztreonam) and carbapenems (as impenem). This
makes the elimination of these isolates difficult.
 Penicillin resistance in streptococcus pneumoniae is by mod focation of existing PBP.
What does GISA stand for? Explain.
A.6 ‘GISA’ stands for Glycopeptides intermediate S.aureus. These isolates have reduced susceptibility to vancomycin.
Descibe genome of S.aureus. Mention its role in pathogenicity especially antibiotic resistance.
A.7 Size: 2800 kbp, ‘circular’, with prophage, plasmid and transposon
They are: • Lysogenized
• Have unique ‘pathogenicity’ or ‘genomic islands’, which are mobile genetic elements, containing clusters for enterotoxins and
antibiotic resistance.
• Have Staphylococcal cassette chromosome mec (SCC mec), are islands, containing methicillin resistance (mec A gene).
The expression of virulence determinant (both toxin and non-toxin mediated) is dependent on series of regulatory genes (e.g., accessory gene
regulator). Also see A.5b.
What toxin mediated syndromes, can a case infected with S.aureus infection develop?
A.8 (a) Food poisoning,Toxic shock syndrome and Staphylococcal scalded skin syndrome.
Describe the pathogenesis of toxic shock syndrome.
A.8 (b) Toxic shock syndrome develops, when the S.aureus starts producing an exotoxin called toxin shock syndrome toxin 1
(TSST-1). About 20% of S.aureus isolates causing bacteremia contain the TSST-1 gene, but only a small fraction express
it in a clinical setting.
Descibe the role of Staphylococcal enterotoxin, Toxic shock syndrome toxin (TSST) and exfolative toxin in S.
aureus pathogenicity.
A.8 (c) Enterotoxin: About one-third of S. aureus strains, produce one of the atleast six (A-F) serologically distinct
enterotoxins, responsible for staphylococcal food poisoning. Most of these strains belong to bacteriophage
group III.
The gene for this toxin appears to be chromosomally mediated but is regulated by a plasmid borne protein. The
molecular weight of these toxins vary from 26-30 kDa. The toxins are heat-stable, so the heating of the food may destroy
the bacteria, but not the toxin. So; laboratory processing of such food may not yield the organism.
The toxins are superantigens and act by stimulating a subset of T lymphocytes, producing large amounts of interleukins.
These can increase the intestinal persistalsis and can have an emetic effect.
The foods often implicated are milk products (as milk, sweets), potato salad and meats. These foods promote the growth
of S. aureus. A tiny amount of enterotoxin; as small as few microgram can cause food poisoning.
The common symptoms of the illness include nausea, vomiting, diarrhea and at times dehydration and hypotension may
occur. The absence of fever, rapidity of onset and epidemic nature of the illness can be a pointer to the illness, to be due
to this entity.
The diagnosis of this entity is by demonstration of the enterotoxin and/or bacteria in the implicated food and/or
vomiting/stool. The toxin can be detected by serological tests; as latex agglutination and ELISA.
The treatment is essentially supportive and symptomatic. The illness can be prevented by screening and isolating a food
handler, who may be carrier of toxigenic S. aureus.
TSST (Toxic shock syndrome toxin):
This is a toxin produced by S. aureus, which is distinct from the enterotoxins, previously described in S. aureus food
poisoning. This toxin; which also acts as a superantigen, may produce enteritis but does not produce food poisoning and
instead produces a distinct syndrome called toxic shock syndrome. The strains often belong to phage group I. The toxin
has a M.W. of 22 kDa and resembles enterotoxin F. The toxin induces production of interleukin-1, TNF  and other
cytokines; which produce leakage and cellular damage of endothelial cells resulting in widespread organ damage.
This syndrome gained recognition in the early 1980s, when an national outbreak in USA, occurred in young, apparently
healthy, menstruating women. Epidemiological investigations revealed a strong association between menstruation and
use of a recently introduced high absorbent tampoon in the market. The withdrawal of the tampon; resulted in marked
decline of these cases. Apparently, the TSST producing S. aureus isolates had contaminated the tampoon, which had
resulted in the absorption of the toxin from the female genital tract.
This syndrome is currently seen in both sexes. The syndrome begins with non-specific ‘flu’ like symptoms. Evidence of a
clinical S. aureus infection is not a prerequisite for development of this illness. The multisystem illness is characterized
by high fever, headache, subcutaneous oedema, vomiting, diarrhea and erythroderma. Severe cases can go into acute
renal failures, D.I.C., brain encephalopathy, shock and death.
The diagnosis of this entity is essentially clinical. The diagnosis is based on a constellation of findings rather than on one
specific finding. The case should be negative serologically for diseases; as measles, leptospirosis and Rickettsial
diseases. Culture of blood and/or cerebrospinal fluid should be negative for organisms other than S. aureus. TSST can be
demonstrated by latex agglutination test/ELISA. TSST genes can be demonstrated by PCR based tests.
Exfoliative (Epidermolytic) toxin:
This toxin is a group of two antigenically distinct antigens, namely toxin A (M.W.-30 kDa) and toxin B (M.W.-29 kDa).
These toxins act as superantigens. Toxin A is of chromosomal origin, heat stable (100°C – 30 min) whereas toxin B is of
plasmid origin and heat labile. These toxins are responsible for staphylococcal scalded skin syndrome (SSSS), which
affects mainly newborns and children. The pathogenic role of the exfoliative toxin in this syndrome is demonstrated, by
the presence of specific antibodies; protective in nature in both man and mice. The toxin causes separation and loss of
most superficial layers of the epidermis. The disease can vary from the localized form; in which blister formation is seen
to a generalized form of the staphylococcal scalded skin syndrome.
Enumerate the principles and describe the treatment of cases infected with S. aureus.
A.9 CASES:
Gen-principles:
• Surgical incision and drainage is essential for suppurative lesions
• Device removal may be necessary, in infected ones
• No conclusion on duration of treatment but invasive lesions may require 4-8 weeks of parenteral (i/v) treatment.
Antimicrobials have adjunctive role, if lesions are abscesses.
I: If isolate is penicillin sensitive PnG (however many reports have found > 80% isolates resistant to this antibiotic.
II: If isolate is penicillin resistant, but methicillin sensitive, i.e., MSSA  Naficillin, oxacillin–semisynthetic penicillinase
resistant penicillins (SPRPs).
(Methicillin* was withdrawn after about 1 yr of usage)
III: If isolate is *Methicillin resistant S.aureus (MRSA), Vancomycin is drug of choice (MRSA).
Alternatives
Quinupristin/Dalfopristin
Linezolid, Tigecycline, Levofloxacin, Doxycycline, Daptomycin, Teicoplanin, Ceftobiprole
Some groups have advocated combination of ciprofloxacin and Rifampicin to deal with the possibility of emergence of drug
resistance.
IV: If isolate is Vancomycin Intermediate susceptible S. aureus/Vancomycin resistant (VISA/VRSA)∆-----------limited options,
as Linezolid, daptomycin and quinupristin/dalfopristin.
* The cause of MRSA is mec A novel gene (acquired likely from S. sciuri) which alters penicillin-binding protein (PBP) on S. aureus cell membrane. The attered
PBP2a of these strains have decreased affinity for  lactam antibiotics. Such isolates are often resistant to other groups of antimicrobials as quinolones,
aminoglycosides and macrolide.
∆ The cause of VRSA is VAn A gene, acquired from a Vancomycin resistant strain of Enterococcus faecalis by conjugation (VISA is due to increase of cell wall
thickness of S. aureus)
Approach and treatment of S. aureus carriers, see A.7, Pg 187, Case 5.

Aspects related to case theme/examination assessment

Enumerate coagulase negative staphylococci (CONS). Descibe the epidemiology, pathogenesis, laboratory
diagnosis and treatment of infections caused by CONS.
A.10 These are staphylococci, which are other than S. aureus and are coagulase negative, hence designated as CoNS in short
• Co-coagulase • N – Negative • S – Staphylococci
There are about 32 species which come in this category. Out of it, half have been associated with human infection.
The common species isolated from pathogenic lesions are:
• S. epidermis – most common CoNS isolate, therefore many laboratories designate all CoNS as S. epidermis, a practice
that needs to be discouraged.
S. saprophyticus, S. hominis (causes bacteremia in malignancy cases), S. saccharolyticus (causes endocarditis)
S. lugdunensis, S. schleiferi (cause native valve endocarditis and osteomyelitis)
Note: Original valve is term used in comparison to implanted/artificial valve
• Epidemiology: Of all the CoNS, S. epidermis is most frequently isolated. It is a normally found on the skin. Carriage
rate of it is exceedingly high, for this reason, it is commonly isolated; as an contaminant from clinical cultures. Similarly,
this organism has also been found to contaminant other specimens; as CSF, respiratory samples, hence their isolation
should be interpreted carefully.
In the past they were rare cause of significant infections, but now they are increasingly linked to human infections. This
is likely to be due to increased population that is immunocompromised/debilitated and has artificial devices; as
implanted catheters (i/v catheters, dialysis catheters), prosthetic devices (as artificial joint) and cardiac related devices
(heart valves, stents, pacemakers). The bacterium is transmitted by contact with infected persons and hospital personnel.
For this reason, it is an important nosocomial pathogen.
S. saprophyticus is a commensal of the skin and genital mucosa. It causes urinary tract infection in young women.
• Pathogenesis:
 CoNS are opportunistic bacteria. Cell envelope factors that help attachment to plastic (artificial) surfaces act as virulent
factor. Following initial adherence of the organism to the surfaces, further adherence is provided by the polysaccharide
glycocalyx (slime), produced in significant quantities by this organism. Biofilm appears to act as barrier protecting the
organism. The resistance of many CoNS to multiple antimicrobial agents, contributes further to their persistence in the
body. The capacity of S. saprophyticus to cause UTI in young women, appears to be related to its enhanced capacity, to
adhere to uroepithelial cells (related to 160 kDa haemagglutinin/adhesin).
• Pathogenicity:
Commonly cause infections in immunocompromised/debilitated patients as:
Bacteremia (as in premature infants, diabetic patients, patients on steroids, transplant recipients),
UTI and Osteomyelitis
Infections also in patients with indwelling devices (see epidemiology above).
• Laboratory diagnosis:
The diagnosis is not difficult using standard bacteriological media, however their interpretation is difficult, i.e., giving
significance to their isolation. Following aspects need to be given importance, while reporting this.
(i) Presence of clinical relevant features and laboratory parameters, increases the significance of the isolation.
(ii) Repeated isolation of the organism adds significance to the isolation
The isolate repeatedly cultured should have same antibiogram and/or a closely related DNA fingerprint.
S. saprophyticus is novobiocin resistant.
• Treatment:
Frequently, the removal of the infected indwelling device, if possible is helpful. This is easily possible in i/v catheters etc. but is
difficult to perform in implants; as artificial hip joint. However; sometimes chronically infected devices don’t respond to
antimicrobials and there is no choice than to remove them.
Vancomycin is the drug of choice for infections caused by S. epidermis. These isolates often can be multidrug resistant.
Trimethoprim – Sulfamethoxazole or norfloxacin (quinolone) is effective in treatment of S. saprophyticus infections.
Describe Micrococcus.
A.11 This is another genus in the family Micrococcaceae. These are commensals, free living, coagulase negative, but catalase
positive. Their pathogenic significance is doubtful.
Egs: Micrococcus ^roseus, Micrococcus luteus and Micrococcus varians
On culture, they often produce beautiful colonies with red, yellow or orange pigments. Their identification is important, as their
isolation from a clinical specimen would indicate the isolate to be of minimal importance, as it would mostly indicate specimen
contamination. Micrococci appear as gram positive cocci, staine darker and non uniformly (than staphylococci), as per in
groups of four (tetral) or eight and size of coccus is larger than that of staphylococcus. They break down carbohydrates
oxidatively in comparison to fermentative breakdown by staphylococci.
Named so, because of red pigment it produces.

Integrated Clinical Case Based Study of

Staphylococcus/Cellulitis
A 40 year old man presented with acute pain in right arm and shoulder. Examination of the affected part revealed swelling with
glistening erythema and purplish discoloration of the skin with pus discharge.

What is the clinical differential diagnosis of the above case?

A.1 (i) Abscess (burst) (ii) Cellulitis of the affected part
(iii) Erysipelas (iv) Necrotizing fascitis
What are the likely pathogens that can be implicated in this case?
A.2 (i) S.aureus (ii) Streptococci
(iii) Anaerobes; as Bacteroides (iv) Coliforms
(v) Pseudomonas species (vi) Mixed infection
What is the typical pathological finding in a case of S.aureus infection? Desribe the pathogenesis of pyogenic
S.aureus infections.
A.3 It is presence of abscess. Leucocytes constitute the primary host defense mechanism against S. aureus. Migration initiated of
leucocytes to site of infection, resulting from orchestrated expression of molecules on endothelium; initiated by
TNF , IL-1 and IL-6. The initial intense polymorphonuclear response (PMN), is followed by infiltration of fibroblasts and
macrophages. The host cellular response (which includes deposition of fibrin and collagen) usually contains infection but if not
controlled, infection spreads to neigbouring tissue or blood.
The degree of virulence of S. aureus is considered to be modest. It would be interesting to compare the pathogeniciy of this to
coagulase negative staphylococci, which is believed to be low but with increased use of implanted catheters, and prosthesis
have emerged as important iatrogenic pathogens.
So the significance of diminution of host defense, when it occurs is of importance. The following are the key factors
(i) breech skin/mucosa (ii) neutropenia (acquired defect) and (iii) presence of foreign body.
Pathogenesis
The virulence factors of S. aureus can be categorized into
Cell wall associated factors
• Capsule: A few strains of S. aureus contain polysaccharide capsule, which is antiphagocytic (inhibits phagocytosis) and protects the
organism.
• Protein A: It is present in cell wall of 90% of strains (especially Cowan 1). It binds to Fc moiety of IgG, exerting an antiphagocytic
effect. It is used in coagglutination category of antigen-antibody reactions, as it can bind to the Fc portion of IgG leaving the Fab
portion of the antibody to bind to antigen.
• Teichoic acid: It is a polymer of ribitol phosphate and forms major antigenic determinant of cell wall of S. aureus. It mediates
adhesion of the organism to host tissue and is anti-complementary (protects organism from complement). It is absent in coagulase
negative staphylococci and micrococci.
• Peptidoglycan: It is a polysaccharide polymer which activates complement and may participate in inflammatory reactions.
Toxins:
They are categorized as toxins, as they affect host cell function and/or morphology. Their action may be mediated by enzymes or by inducers,
by acting as superantigens
(1) Cytolytic exotoxins (haemolysins): As the name indicate; these cause lysis of red blood cells of several species. These cytolytic
exotoxins, also act on a wide variety of other cell types; as platelets, leucocytes and cells of the skin (dermonecrotic). Four types of
these namely, alpha, beta, gamma and delta have been characterized.
Of these, alpha () toxin is the most important in pathogenicity. It is chromosomally mediated. Chemically; it is a protein with MW
of 33 kDa. It is inactivated at 60°C, however its activity is regained paradoxically, if it is heated to between 80-100°C, due to the heat
labile inhibitor getting inactive at higher temperature. The alpha toxin can be toxoided but neither toxoid nor anti- -haemolysin has
been shown to be of any value in the treatment or prevention of chronic staphylococcal infection.
Beta-haemolysin is also active on a variety of cells. It has a molecular weight of 35 kDa and exhibits hot-cold phenomenon, i.e.,
haemolytic properties of toxin on RBC’s become evident at 37°C, after the RBC’s have been exposed to cold temperature.
(2) Leucocidins:
These damage leucocytes and macrophages, as the name of toxin indicates. This toxin is also named ‘Panton-Valentine’ leucocidin,
after its discoverers.
(3) Exfoliative toxin, enterotoxin and toxic shock syndrome toxin (TSST-1) also have superantigen activity. (See A 8c, pg. 181-182)
Enzymes:
• Coagulase: It is an enzyme, which can convert fibrinogen of the plasma (not serum) into fibrin. The fibrin can coat the
staphylococci and make it resistant to opsonization and phagocytosis. However; it’s deposition around the pathogenic
lesion, may result in it’s localization. There are two forms of this enzyme, whose differences are given in the table 3.5.1.
For the methods for their performance, refer, page ....., chapter 9, Section 1. This enzyme is present in almost all isolates
of S. aureus and it’s presence helps to differentiate S. aureus from the less virulent staphylococci, often designated as
coagulase negative staphylococci (CONS).
 Table 3.5.1: Differences between free and bound coagulase

Free coagulase Bound coagulase

- Secreted free into culture medium - Is constituent of cell wall

- Heat labile - Heat stable

- Detected by tube test - Detected by slide test

- One antigenic type identified - 8 antigenic type (A-H) identified

- Considered more diagnostic of S. aureus - Less diagnostic

- Requires coagulase-reacting factor for it’s action - Does not require CRF

• Catalase: Staphylococci produce this enzyme, which converts hydrogen peroxide into nontoxic H2O and oxygen.
*Because staphylococcal phagocyte killing is mediated by toxic oxygen radicals, produced by PMN, it has been
postulated that catalase production by counteracting host defense mechanisms, correlates with pathogenicity.
• Fibrinolysin (staphylokinase)-role not clear
• Nucleases, lipases and protease-role not clear
• Penicillinase: As many as 80% of Staphylococcus aureus strains are resistant to penicillin. This is because of the
presence of enzyme, penicillinase (beta-lactamse), which is often plasmid mediated in these strains (gene also present on
chromosome). The enzyme inactivates penicillin and cephalosporins, by splitting the beta lactam ring. Staphylococci
produces four types (A-D) of penicillinases. These plasmids are transmitted amongst the staphylococci strains both by
transduction and conjugation. These plasmids may also carry resistance to some heavy metals; as mercury, arsenic and
some antibiotics; as erythromycin.
• Penicillin binding protein (PBP-2a): Is a membrane bound enzyme (contrast with beta lactamase, which is
extracellular). Some multiresistant S. aureus strains widely referred to as methicillin resistant S. aureus (MRSA) are
resistant to penicillinase resistant penicillins, cephalosporins and some other groups of drugs. There were designated as
MSRA, as the strains were resistant to methicillin, which was used to treat the penicillinase resistant staphylococci. The
resistance is mediated by mec A gene, which is a part of the mobile genetic element termed staphylococcal cassette
chromosome mec (SCC mec). This chromosomal mediated resistance gene is hypothesized to have been acquired by
horizontal transfer from a related staphylococcal species, S. sciur. This gene is responsible for production of low affinity
penicillin binding proteins on the cell wall (instead of normal PBP), which have less binding to many antibiotics, making
them ineffective.
The steps involved in the pathogenesis are:
(1) Inoculation and asymptomatic colonization of tissue surface site. The anterior nare is the principal site of staphylococcal colonization
in man. The biology of the colonization process is poorly understood. Staphylococci are opportunists. For the initiation of the
infection, a breech in the cutaneous or the mucosal barrier is necessary. The presence of microcapsule (in some strains), protein A and
the ability to internalize, helps the organism to evade host defense mechanisms and is critical in invasion.
Once the infection is initiated, the pathogenesis of Staphylococcal infection is complex and obscure.
(2) Invasion of tissue because of organism enzymes; as cytotoxins, lipase, proteases, hyaluronideses and thermonucleases. The enzymes
facilitate the spread of infection across tissue surfaces but their precise role is not clear.
(3) This can lead to bacteremia, which may be asymptomatic or symptomatic.
(4) This can lead to metastatis of infection to sites; as bones, lungs.
(5) Evasion and host defense.
The immunity is not strong or long testing, as is evident by the continuous susceptibility of the individual to infections
throughout life. The reasons for this are not understood.
What relevant investigations would you like to perform in this case?
A.4 (a) (i) Pus culture with antimicrobial susceptibility testing
(ii) Blood culture
(iii) Appropriate radiologic investigation to delineate the extent of infection.
Tabulate the characteristics of S. aureus, S. epidermis and S. saprophyticus.
A.4 (b)
Table 3.5.2: Characteristics of Three Species of Staphylococcus

S. aureus S. epidermidis S. saprophyticus

Coagulase test + – –

Mannitol fermentation + – –

Production of
   DNAase + – –
   Phosphatase + –/+ –

Protein A in the cell wall + – –

Lysostaphin senstivity + – –

Novobiocin resistance – – +

What is the role of bacteriophage in typing of S.aureus? Describe the technique.

A.5 Purpose: Strain typing
Principle: Strains of S. aureus are lysed by more than one specific phage, the pattern of lysis serves as marker for the strain. An
international set of 23 standard phages of S. aureus are used. Susceptibility of S. aureus strains to various temperate phages,
provides the basis for a phage typing system.
Most strains of S. aureus are lysogenic, but they are immune to them. These can lyse some other strains. The bacteriophages are
propagated on special strains of S. aureus, which also serve as controls for the specificity of bacteriophages.
Benefit:
• Provides epidemiologic information
• No help in treatment
Limitation of technique:
• Is an phenotypic typing system, so less valuable than genotypic typing systems; as DNA finger printing and ribotyping
• Technique is
– Complex
– Time consuming
– Requires trained personnel
• Non specific results can be due to
– Spontaneous lysis by phages
– Activation of latent phages
Procedure:
• Lawn culture of S. aureus strain to be typed is prepared
• One RTD of 23 standard typing phages applied on the lawn culture
• Plate incubated overnight and read subsequently
The National reference center for staphylococcal phage typing in India is located at department of Microbiology, Maulana Azad
Medical College, New Delhi.
Interpretation:
Phage type of strain is expressed by the designation of the phages that lyse it.
The commonest phage type prevalent in most parts of India is 52/52A/80/81 (which means that it is lysed by these phages). If
two S. aureus strains have same phage type, one cannot conclude that they are identical or same. However if two S. aureus
 strains have two different phage types, one can be sure that the two strains are ‘different’. So the typing help to distinguish S.
aureus strains.
How should this case be managed?
A.6 The lesion should be surgically explored and managed; appropriately. Appropriate antimicrobials should be started empirically
and then ‘de-escalated’ [changed, if indicated]; if necessary.
Classify S.aureus carriers. How are they treated?
A.7 CARRIERS
• Skin
• ^Nasal
• Others
^often accompanied with skin carriage
Indication of treatment: Surgeons, nurses, healthcare workers; especially if carry MRSA
Skin: Local cream with antimicrobials
Nasal carrier:
• Locally: neomycin and bacitracin
• Intranasally: mupirocin
• Systemic: rifampicin can be considered in chronic nasal carriers
Complication (of treatment): Some consider such regimes useless, as these encourage replacement of susceptible S.
aureus strains with multidrug resistance.
How are S.aureus infections controlled?
A.8 (i) Screening of hospital staff; as surgeons, nurses and health care workers for S. aureus (especially MRSA). If found
positive, may be given non-clinical duties, until negative for S. aureus spontaneously or with treatment. Intranasal
mupirocin may be helpful in carriers.
(ii) Monitoring of the nosocomial S. aureus strains in the hospital (by antibiotic susceptibility pattern, phage type etc.).
(iii) Isolation and effective treatment of infected cases. Topical application of antimicrobial agent can prevent dissemination
of infection to others.
(iv) Reinforcement of health-care associated policies in the hospital; as mandatory hand hygiene.
(v) Monitoring of sterilization of instruments etc. in the hospital.

Integrated Clinical Based Study of

S.pyogenes/Sore Throat
A 6 year old girl, Ashima presented to a medical practioner with sore throat and fever. He did not order for any microbiological
investigation and prescribed drugs for providing symptomatic relief, only.

Which is the essential investigation that the practioner should have in the ordered for above scenario? Justify
it.
A.1 (a) It is essential to get a throat swab culture performed on every case (of child) of sore throat, to rule out group A
streptococcal infection (identifying the cause of pharyngitis is not possible clinically). The practioner should have
requisitioned this investigation. In case culture test is not feasible, antigen detection for group A streptococcus, in throat
swab specimen may be performed. Definitive treatment for group A streptococcal infection is necessary and penincillin
prophylaxis is also recommended to prevent long term post-streptcoccal sequelae; as rheumatic heart disease.
Enumerate the key non-suppurative complications of S. pyogenes infection. Compare and contrast them in a
tabular fashion.
A.1 (b)
Table 3.6.1: Comparison of Acute rheumatic fever and acute post-streptococcal glomerulonephritis

Acute rheumatic fever Acute post-streptococcal

Glomerulonephritis

Preceding infection Sore throat Skin infection/sore throat

Prior sensitization Essential Not necessary

Latent period Longer 2-4 weeks Shorter (1-3 weeks)

S. pyogenes (serotypes Any, but some are more associated Pyoderma types and throat infection types
implicated)

Hereditary tendency Present Not known

Pathology Characteristic lesion is ‘Aschoff nodule’ Antigen-antibody complexes on glomerular basement

membrane and increased PMN infiltrate

Pathogenesis Most likely due to cross-reactivity between antigens of May be due cross reactivity between nephritogenic
organism and myocardium/heart valves streptococci and glomerular antigen or due to immune
complexes deposition, leading to activation of C3 and
C5, leading to tissue destruction

Complement level Uaffected Lowered

Serological response Elevated ASO (titer > 200) and anti DNAase titres ASO titres may not increase
Anti DNAase levels increased (t.ter>300)

Clinical profile Often dependent on inflammation of joints and heart Albuminuria, haematuria, oedema

Course Progressive or static Usually spontaneous resolution

Prognosis Variable Good (in 80-90%)

Antimicrobial prophylaxis Indicated (beneficial) Not indicated

Describe the procedure for taking throat swab?

A.2 (a) The case is made to sit on a stool and asked to lift the head back and preferably close the eyes. The throat is well
illuminated and the tongue is depressed with a tongue depressor and the patient is asked to say ‘Ah’. The throat
(including the tonsillar and the inflamed area) is swabbed vigorously with the swab and placed in the tube to be
transported to the lab (procedure done with gloved hands)
It is important to collect it properly, so that the specimen does not get contaminated with the commensal flora of oral
cavity, while collecting sample, which could result in a fallacious report
What medium can be used to transport the throat swab, if delay in the transport of the clinical sample to the
laboratory is expected?
A.2 (b) Pike’s medium

The growth that has occurred on the blood agar plate inoculated with the throat swab (pin point size colonies with beta
hemolysis). Fig. 3.2.3.

Enumerate the likely pathogens based on the mentioned colonial characteristics.

A.3 (a) Beta haemotytic colonies on blood agar in context with the current clinical picture could be S.pyogenes, Haemophilus
haemolyticus and Listeria monocytogenes.
Describe the classification of beta hemolytic streptococci.
A.3 (b) Rebecca Lancefield in 1933 classified -haemolytic streptococci into distinct serogroups, a study that helped
tremendously in understanding the epidemiology of streptococcal infections. The streptococci can be classified on the
basis of oxygen requirements into aerobes, facultative anaerobes and obligate anaerobes Fig. 3.6.1. Most streptococci
which cause human pathogenicity are facultative anaerobes, although some are obligate anaerobes.
 The streptococci can be further classified on the basis of their haemolysis on blood agar into ,  and  streptococci. The
diferentiating features of haemolysis are given in table 3.6.2. Most streptococci that are human pathogens are 
haemolytic.

Table 3.6.2: Differentiating features of alpha, beta and *gamma haemolysis

Alpha-haemolysis Beta-haemolysis

- Zone of partial haemolysis seen around the colony (unlysed RBCs - Zone of complete haemolysis seen around the colony (all RBCs in the
seen in the zone) zone are lysed)
- Zone of lysis is narrow (1-2 mm in width) - Zone of lysis is wide (2-4 mm in width)
- Zone has indefinite margin - Zone has definite margin
- Zone is greenish in color (as RBCs are incompletely digested) - Zone is colorless (as complete haemolysis occurs)

*Gamma haemolysis implies no haemolysis (NH)

The -haemolytic streptococci are classified by the Lancefield system. The serologic grouping based on reactions of
specific antisera with cell wall carbohydrate antigens of the organism, identities 20 groups, A to V (without I and J). Out
of these groups, group A streptococci are of most human importance. The group A streptococci (GAS) are further
divided; on the basis of M protein precipitin reaction, T protein agglutination method and R antigens on the cell surface.
Streptococci based on oxygen requirement

Aerobes/facultative anaerobes Obligate anaerobe e.g. Peptostreptococci

(details, see ..... chapter)
Based on haemolysis

Alpha haemolytic *Beta haemolytic e.g. Gamma haemolytic

e.g. S. viridans S. pyogenes (most e.g. enterococci
(commensal / are pathogenic) (most are non-
opportunistic haemolytic)
pathogens
20 Lancefield group
(A-V) without I and J
(some streptococci in
these group may be
alpha or non-
haemolytic)

*Based on M protein, further divided into more than 100 Griffith types

Fig. 3.6.1: Classification of Streptococci

What is the reservoir of group A streptococcus (GAS)?

A.4 (a) The upper respiratory tract of man including the throat, nasopharynx and nose is the primary reservoir for GAS.
Descibe the epidemiology of group A streptococcal pharyngitis.
A.4 (b) GAS pharyngitis:
Agent: S. pyogenes
Any serotype can cause acute rheumatic fever, but M type 5 is more associated with this entity. Acute glomerulonephritis
is associated with certain pyodermal types; as 49, 53-55 and pharyngitis strains; as 1 and 12.
Source of infection:
Human upper respiratory tract (including throat, nasopharynx and nose) of cases and carriers.
• Occasionally even anal carriers.
• Food, also source (less common) of outbreaks.
• Non human source of infection include bovine mastitis, through milk
 • Serotyping and molecular typing is important in epidemiological study.
• Control measures include early detection of carriers, cases and early antibiotic therapy.
Mode of transmission:
• It is spread by respiratory secretions, which can be acquired by direct contact with the mucosa, through
contaminated dust/fomites or through large droplets produced by coughing, sneezing or even conversation.
Droplet transmission is effective at short distances of 2-5 feet. Fomites is not important in spread through GAS
survive for short period in dried secretions.
Host:
The infections occur worldwide. The respiratory infections are commonest in children amongst 5-8 year age group. For
this reason, rheumatic fever is common in 5-15 year age group. Immunity occurs following infection is type specific and
is associated with antibody to the M protein. However; reinfections are common due to multiplicity of serotypes.
• Asymptomatic carrier rate is usually low (sometimes carriage rates of greater than 10% is documented in children)
• Throat carriers outnumber nasal carriers, but nasal carriers have greater infectivity than throat carriers.
• Acute cases are most important in spread of infection (much more than carriers).
Environment:
Infection is common in closed places. Outbreaks of infection are common in boarding schools and military camps.
Describe erysipela.
A.4 (c) It is an acute and diffuse infection of the skin, involving the superficial lymphatics (subcutaneous tissue). It affects all
age groups, including adults and often involves the face. It is characterized, by lesion having fiery red advancing
erythema with swollen, red and indurated skin.
Describe scarlet fever.
A.5 (a) It is a complication of streptococcal pharyngitis caused by some strains of S. pyogenes that produce certain pyrogenic
exotoxins (erythrogenic toxin). The disease is uncommon in the tropics and does not occur in India. The disease has
become less common in recent years for unknown reasons. The cases have fever, pharyngitis and characteristic rash that
may occur due to hypersensitivity reaction and requires prior exposure to toxin.
Is scarlet fever prevalent in India? What are the possible reasons for the prevalent scenario?
A.5 (b) Scarlet fever has not been reported from India, despite common occurrence of GAS infections in the country. The reason
for this could be the absence of strains in this area, having the temperate bacteriophage, responsible for the erythrogenic
toxin.
Describe the pathogenesis of group A streptococcal pharyngitis.
A.6 (a) The attachment (pharyngitis) of GAS to epithelial cells of the host is the first step. The organism may come in an inhaled
droplet. The attachment of the organism occurs by the M protein, lipoterchoic acid and F protein of the organism with the
fibronectin on epithelial cells of the host. This leads to bacterial colonization stage.
In some cases, the bacteria proliferates, secretes various toxins, causing damage to surrounding cells, invading the
mucosa and eliciting inflammatory response. The enzymes; as streptokinase, deoxyribonucleases and hyaluronidase lead
to spread of the infection. The organism may result in spread of the infection causing cellulitis, fascitis and other
manifestations.
The virulenic is determined by the ability of the organism to adhere, avoid phapocytosis (imediated by capsule, M and M
like proteins) and production toxins.
Descibe the role of S. pyogenes antigens, enzymes and toxins in it’s pathogenicity.
A.6 (b) The schematic representation of the organism is depicted in Fig. 3.6.1.
Capsule on the outer side
• Present in some strains
• Composed of hyaluronic acid, production of excessive amount of capsule gives a mucoid appearance to the colonies.
• Is weakly antigenic/non-immunogenic, antibodies to it have not been shown to be protective. The possible reason for this is the
resemblance of the hyaluronic acid of human connective tissue to that of the capsule.
• The capsule can prevent the organism from ingestion and killing by phagocytes (also prevents complement action by
preventing binding of complement components).
Fimbriae:
• They extend from the cell membrane (where they are anchored) passing through the cell-wall and capsule to be extending
outside it (so may be the outermost structure).
M. Protein:
• It is a major protein of cell wall of group A streptococci.
• It is a fibrillar protein, which extends from the peptidoglycan to the outside cell surface.
• More than 100 M types are known and it is the basis of Griffith typing
• M. protein is a major virulence factor (bacterium is non-infectious, in the absence of M. protein).
• It is antiphagocytic and facilitates the attachment of the organism to the epithelial cells.
• Antibodies to M protein (overcome resistance to phagocytosis) are opsonic/protective. Thus, persons with antibodies to a
specific M type acquired due to prior infection are protected against subsequent infection with Group A streptococci of the
same type but not against different M types. So; individuals may have numerous GAS infections in their life-time, as they
encounter new M protein types, for which they have no antibodies.
• The carboxy terminal of the M protein is attached to the peptidoglycan of the cell wall and the amino terminal extends towards
the surface.
• The antigenic specificity of the M protein lies in the amino-terminal portion, which is the most variable portion of the molecule
and is available for immune surveillance.
• The resistance of the organism (with M protein) to phagocytosis may partly be due to the binding of the fibrinogen to M
protein molecules on streptococci surface, which could interfere with the deposition of opsonic and complement fragments and
it’s activation on the organism surface.
• Typing of strains on the basis of M protein is done conventionally in a few reference labs by antisera.
• Typing for assignment of M protein type is also done by PCR, which is based on amplification of variable region of M protein
gene.
Lipoteichoic acid:
It is also associated with the M protein and the complex play a role in the adhesion of the organism to the (host) oral epithelial cells.
T and R protein:
These are the two other major proteins of the cell-wall of the organism. T typing of the strains has value in the epidemiological
surveillance of infection.
Group specific carbohydrate (polysaccharide):
The major component of the cell wall of the organism is the peptidoglycan, which provides rigidity to the organism. Within this
matrix, lies the group specific antigen (polysaccharide), which is composed of a polymer of rhamnose and N-acetyl glucosamine. On
the basis of these antigens, S. pyogenes is divided into 20 groups (A to V) without I and J (Lancefield groups). The specific ‘C’ antigen
can be extracted by numerous techniques. One of the commonest extraction method is done with hydrochloric acid method
(Lancefield’s acid extraction method) and typing done with type specific antisera.
Protein F (fibronectin-binding protein):
It mediates attachment to fibronectin in the pharyngeal epithelium.
Extracellular Products/Toxins/Other virulence factors:
Streptolysin O:
• As name indicates, it causes lysis of cells, and is antedated ‘O’, as it is oxygen labile (is active only in its reduced
form).
• Is a protein with haemolytic property.
• Causes lysis of host cells (as cardiac tissue), neutrophils and platelets.
• Haemolysis not seen around surface colonies (as is oxygen labile), but causes haemolysis in pour plates
• Is immunogenic/antigenic.
• Antibodies to streptolysin ‘O’, i.e., antistreptolysin O (ASLO) antibodies appears in sera following streptococcal
infection. An ASO titre exceeding 200 todd units indicates recent or recurrent streptococcal infection.
• The streptolysin ‘O’ cross-reacts with similar haemolysins of streptococcus groups C and G, pneumolysin of S
pneumoniae, tetanolysin of C. tetani and similar toxins produced by C. perfringens, B cereus and L.
monocytogenes.
Streptolysin ‘S’:
• ‘S’ in the name indicates oxygen stability.
• Is a protein.
• Causes lysis of leucocytes.
• Is responsible for haemolysis seen around surface colonies.
• Is not antigenic
Streptococcal pyrogenic exotoxins A, B and C (previously known as erythrogenic toxin):
• The toxin got the current name, as induction of fever is the primary effect of the toxin.
• Produced only by some strains of S. pyogenes.
• Three antigenically distinct forms of exotoxins, A, B and C are described.
• Type A and Type B toxins are coded by bacteriophages that lysozenize certain strains of S. pyogenes.
• Type C toxin is a cysteine protease that is chromosomally mediated.
• Lack of prevalence of strains having temperate phages coding exotoxins A and B in India, may be responsible for
absence of scarlet fever, despite high frequency of GAS infections.
• Scarlet fever uncommon now
• Toxin responsible for rash of scarlet fever
Some strains are associated with severe invasive streptococcal infections as; necrotizing fascitis and streptococcal
toxic shock syndrome.
• Exotoxin A acts as a superantigen causes massive release of cytokines (from helper T cells and macrophages).
Exotoxin B is a protease that causes rapid tissue destruction.
• This toxin has been used to develop susceptibility and diagnostic tests as Dick test and Schultz Charlton reaction
(test) for Scarlet fever. These tests are of historical importance, as Scarlet fever is uncommon and not a serious
infection.
Streptokinase (Fibrinolysin):
Two types of streptokinase, namely streptokinase A and B exist. Antibodies produced against them can be used in
retrospective diagnosis of disease, as the enzyme is antigenic
• It is an enzyme which activates (catalyzes) conversion of plasminogen into plasmin, which can cause fibrin
digestion.
• It breaks down the fibrin laid around the infected site, thus facilitating the spread of infection.
• The fibrinolytic property is used therapeutically in drugs that cause break down of thrombus and embolus, thus
can be life-saving in management of myocardial infarction, if used initially.
• It is also produced by streptococcal groups C and G.
Deoxyribonucleases (Streptodornase):
• As the name indicates, are enzymes that depolymerize/liquefy DNA.
• Four types of deoxyribonuceases, namely A, B, C and D are known.
• These enzymes liquefy the viscous DNA that collects in thick pus, making serous the character of the exudate.
• This enzyme helps in the spread of infection.
• Enzyme is antigenic. Demonstration of antideoxyribonuclease B antibody in serum of a suspected case is
diagnostic of GAS infections, particularly of skin.
• Enzyme prepartions containing this enzyme along with streptokinase are used clinically in liquefying thick
exudate; as present in empyemas.
Hyaluronidase:
• As the name indicates, is an enzyme that breaks down hyaluronic acid.
• By splitting hyaluronic acid of the host tissue, it can help in the spread of infection.
• A dilemma with this enzyme is that this enzyme, would also cause self destruction of it’s capsule (if present). It is
seen that strains that produce large amounts of this enzyme are non-capsulate.
Other products:
• Some strains produce enzymes as amylases, lipases, phosphatases etc. Their role in the pathogenesis is not clear.
Some M types of GAS produce a lipoproteinase called ‘serum opacity factor’. It is so named, as the enzyme when
applied of an agar gel containing horse serum, produces opacity (because of lipoproteinase activity).
Most of the pathology of GAS infections is due to direct invasion/toxins of this organism. Mention two
pathological entities attributed to GAS that are antibody mediated.
A.6 (c) Acute Rheumatic fever (ARF) and Acute Glomerulonephritis (AGN) are antibody mediated.
Describe Impetigo (pyoderma) and mention it’s importance.
A.6 (d) Impetigo (Pyoderma): It is a localized infection of the skin, usually on exposed parts of the skin, as legs and face. The
infection usually starts after the organisms invades the skin, after minor trauma, as skin abrasions. It is common in
children and is caused by limited serotypes of S. pyogenes. The infection is acquired by direct contact with an infected
person or fomites. This skin lesion is a major cause for initiation of acute glomerulonephritis in tropics.
Can this case (under discussion) be treated without performing the antibiotic susceptibility test?
A.7 (a) Yes. Once group A streptococcus is isolated, it is not nececessary to perform the antimicrobial susceptibility test. This is
so, as no resistance has been reported to penicillin, despite usage for over 50 years for this organism.
Descibe the treatment guidelines of Group A streptococcal infection.
A.7 (b) Penicillin G is the DOC. In cases allergic to it, drugs; as erythromycin, clarithromycin, Azithromycin, clindamycin or
oral cephalosporin; as cephalexin can be used.
• S. pyogenes has not acquired resistance to PnG.
• In pharyngitis, the duration should be 10 days to ensure complete eradication of the organism. It prevents
development of post-streptococcal sequelae; as ARF, but does not prevent development of AGN (though can
eradicate GAS from skin).
• It does not have any effect in an established case of ARF or AGN but chemoprophylaxis as with oral penicillin
must be given to prevent streptococcal reinfection (with different M type) to prevent recurrence of ARF.
Chemoprophylaxis (with long acting penincillin) for different periods is recommended. In cases with history of
ARF, prophylaxis upto 21 years of page is recommended, whereas in cases of RHD, lifelong prophylaxis is
recommended (having one episode of ARF is a risk factor for subsequent episodes, if patient is reinfected with S.
pyogenes)
• No definite guideline for management of asymptomatic carriers exist.
• Recently, strains resistant to erythromycin have been reported. Infection with different M types of GAS is
possible. Vaccine containing multiple M protein epitopes is being evaluated in animal models.
After an attack of group A sore throat, for how long should antimicrobial prophylaxis be instituted?
A.8 The aim of the antimicrobial prophylaxis (mostly pencillin) is to prevent the recurrent episodes of rheumatic fever, which are
most common in the first five years, after the initial diagnosis is made.
The prophylaxis may be extended for individuals, who have had recurrent disease, have rheumatic disease or work in
environment that predispose them to exposure to GAS infection.
Is there any vaccine available for S. pyogenes?
A.9 Currently no vaccine is available. Considering the frequency and seriousness of GAS infections, development of a vaccine is
desirable. The scientist are targeting the M proteins and variety of virulence factors, as C5 peptidase. The challenge in a
potential vaccine against the M protein component, is to identify epitopes that would induce the production of protective
antibodies against maximum M types as possible, while ensuring that these antibodies do not react against human tissues.

Integrated Clinical Based Study of

Enterococcus/Septicaemia
A 6 year old girl, Sabeena admitted in a PICU (paediatic ICU) was on central venous line for 15 days. She received ceftazidime
and gentamicin. She remained stable, but subsequently presented with high spikes of fever Her blood culture yielded
Enterococcus faecalis. Without waiting for the antimicrobial susceptibility result, she was started on a presumptive treatment
with vancomycin. The case did not respond to the administered antimicrobial and repeat blood culture again yielded
Enterococus faecalis.
What infection has this child likely developed?
A.1 The case has most likely developed a central venous line induced nosocomial septicaemia.
What is your microbiological diagnosis?
A.2 Her clinical condition is most likely due to vancomycin resistant enterococci (VRE)
What are the risk factors for developing enterococal infections?
A.3 Old age, prolonged hospitalization, prolonged antimicrobial adminstration, debility or conditions in which mucosal and/or
epithelial barriers have been disrupted.
What are the characteristics of an isolate that suggest it to be an enterococcal one?
A.4  microscopically appear as gram positive cocci in pairs and small chains, (ii) growth on MacConkey appear; as tiny pink
colonies and as black colonies on tellurite blood agar, ability to grow in presence of 40% bile, 6.5% NaCl and pH 9.6,
Heat test positive (ability to withstand heat at 60°C for 30 mins)
What is the combination of drugs used for treating enterococcal infections?
A.5 Penicllin or ampicillin is often used in combination with an aminoglycoside. The combination of these drugs gives synergistic
activity, i.e., either of the above two classes of drugs used individually, can not achieve a therapeutic effect, as achieved by their
combination.
What does high level aminoglycoside resistance in enterococcus convey?
A.6 Common regime for treatment of serious enterococcal infections, as septicemia involve combination of cell wall inhibitors; as; penicillin,
ampicillin or vancomycin with aminoglycosides as; streptomycin or gentamicin.The addition of the cell wall inhibitor agent helps in the
penetration of the aminoglycoside into the bacterial cytoplasm, making the intrinsically resistant organism; aminoglycoside susceptible. The
presence of high level aminoglycoside resistance (HLAR) in enterococci makes the synergy of cell wall inhibitors and aminoglycosides
ineffective. HLAR in enterococci is defined, as the minimum inhibitory concentration of aminoglycoside for the isolate to be greater than
2000 g/ml. Among the many mechanisms for the resistance of these isolates, is the secretion of enzymes by these isolates, which inactivate
the aminoglycosides by different mechanisms; as adenylation or phosporylation
What antimicrobials are commonly used for treating vancomycin resistant enterococci (VRE)?
A.7 Quinupristin/dalfopristin, Linezolid OR Combination of ciprofloxacin and rifampicin.
Prolonged therapy is required for resolution of most infections, which may be 6-8 weeks.

Integrated Clinical Based Study of

S.viridans/Infective Endocarditis (I.E.)
A 60 year old man, Sohrahudin presented with a 2 month history of low grade fever and facial swelling. He is a known diabetic
and has long standing hypertension. Echocardiography has revealed vegetations on the implanted Valve.

What is your clinical differential diagnosis?

A.1 Infective endocarditis, connective tissue disorders and chronic infection (as brucellosis, tuberculosis, etc).
What are the common microbes that are implicated in Infective endocarditis?
A.2 Staphylococcus epidermis, Streptococcus ‘viridians’, S.aureus, Enterococcus spp., C. burnetti
Which criteria are commonly used for making a diagnosis of Infective endocarditits (I.E.)?
A.3 Modified Duke criteria are often used for diagnosis of endocarditis, which has major and minor criteria. The cases get mainly
catergorized into final I.E, possible I.E. and IE excluded.
Classify endocarditis and enumerate the common microbes associated with the different categories?
A.4 Subacute, Acute and *Prosthetic valve endocarditis are the common types of endocarditis. These are most commonly associated
with S.viridans, S.aureus and S.epidermis, respectively.
Which is the most important microbiological investigation that can be done to make a diagnosis of I.E.?
Describe the procedure.
A.5 Blood culture is the most important microbiological investigation. Briefly, a minimum of three three blood cultures should be
performed in the first twenty four hours. 5-10 ml of blood each should be inoculated to a set of two blood culture bottles, used
 for each of three blood cultures. Such procedure is employed, so that the intermittent release of organisms that may occur in this
disease can be detected.
Which is the most useful test to establish a diagnosis of I.E.?
A.6 Echocardiography is the most useful diagnostic tool for IE. Transesophageal echocardiography has been demonstrated to be
more sensitive and specific than transthoracic echocardiography to demonstrate valvular vegetations.
Mention the key principles that should be utilized in initiating antimicrobial therapy for I.E.?
A.7 The incriminated microbe should obviously be susceptible to the antimicrobial being instituted. Besides this the levels of
adminstered antimicrobials should exceed the MIC level for the incriminated microbe. Lastly, prolonged therapy is required for
resolution of most infections, which may be 6-8 weeks .
Mention one preventive strategy in relation to I.E.
A.8 Person with I.E. must have minor procedures; as dental extraction performed under antimicrobial cover.
If Streptococcus bovis had been isolated from this case, what system (organ) in the case was likely to be source
of this etiological agent?
A.9 Gastrointestinal lesions; as polyps or malignancies; as carcinoma are associated with S.bovis endocarditis.

Integrated Clinical Based Study of

S.pneumoniae/Pneumonia
A 55 year old man, Ghanshyam Singh presented to the ICU with high grade fever and respiratory distress. He had a long
history of alcohol intake. Sputum sample was not available from this case, as he had only non-producive cough. Blood
culture was performed, which grew alpha haemolytic colonies whose, microscopy revealed gram positive cocci;
predominantly in pairs. His chest X-ray revealed infiltrates in upper and middle lobes of right lung.

What is the clinical diagnosis in the above case?

A.1 Community acquired pneumonia. The case under discussion has developed this pathology, while being in the community (and
not during stay in the hospital). The etiological agent is likely to be pneumococcus.
What screening test can be done on the isolated suspected pneumococal colonies to have its presumptive
diagnosis?
A.2 (a) Optochin sensitivity can be performed on the isolated colonies to arrive at a presumptive diagnosis of S.pneumoniae (this
isolate is susceptible to this chemical agent, which is ethyl dihydrocuprein). Because of typical growth of pathogen on
blood agar medium, fungal, viral or tubercular causes of pneumonia are unlikely.
What is Quellung reaction? Mention it’s historical role.
A.2 (b) Quellung reaction (latin ‘quellung’: swollen) was described by Neufeld in 1902. The test consists of the addition of a
drop of homologus antiserum to a suspension of capsulated bacteria; as pneumococci, resulting in apparent capsular
swelling; capsule becoming clearly delineated and refractile.
This test was used in the past, for identification when specific antisera were used, as part of the treatment protocol.
Can S.pneumoniae be present in an individual, without causing any morbidity?
A.3 (a) Yes. The normal colonization rate of this organism in the human throat (upper respiratory tract) is about 5-10%.
Who is credited with the discovery of S. pneumoniae?
A.3 (b) Louis Pasteur and Sternberg in 1881
Descibe the epidemiology of pneumococcal infections.
A.3 (c) S. pneumoniae colonies the nasopharynx. The organism colonizes the infant at about 6 month of age. Later on the
organism can be isolated from about 20-40% of healthy children and 5-10% of healthy adults. The infection has a
seasonal incidence, being more common in winter (pneumococcal pneumonia). No significant animal reservoir for this
pathogen exists.
 Pneumococcal infection are leading cause of deaths worldwide. Infections due to serotype 3 are most virulent. Cases are
seen more often in the two extreme age groups. The incidence of pneumococcal bacteremia is high in children upto two
years of age.
Infection with S. pneumoniae, usually result in carriage of the organism for few months. Disease results; when the host
resistance is lowered due to stress, malnutrition viral infection, alcoholism etc.
The organism spreads from one individual to another, as a result of direct contact and respiratory droplet transmission. It
is for this reason that closed and crowded spaces as military camps, day care centers, crowded wards are associated with
pneumococcal outbreaks.
There are two systems for the more than 90 serotypes (based on distinct capsular composition) of S. pneumoniae. In the
American system, serotypes are numbered in the sequence in which they were identified. The strains that frequently
cause disease were generally the earliest to be identified and therefore tend to have lower numbers. The Danish system
places serotypes into groups, based on antigenic similarities.
Serotyping was useful in the 1930s, when type specific antisera were administered as part of treatment protocol and
again in the 1990s, when used to track the source of outbreaks. Capsule switching which has reported to occur in
pneumococcus team limits the epidemiological reliability of strain serotyping. Currently; molecular techniques as pulsed
–field Gel electrophoresis and multilocus sequence are used to type these strains.
Which predisposing factor in this case likely led this case to the present condition?
A.4 (a) Consumption of alcohol of long standing duration.
What is the possible mechanism by which this factor (alcohol consumption) likely played a part in the
pathogenesis of the current case?
A.4 (b) Inhibition of the ability of polymorphonuclear neutrophils (PMNs) to ingest the organism.
What are the key virulent factors of S. pneumoniae?
A.4 (c) Polysaccharide capsule (the various vaccines are based on this) and cytolysin (pneumolysisn) which acts on both
alveolar epithelial cells and pulmonary endothelial cells.
Describe the pathogenesis of pneumococcal infections.
A.4 (d) The first essential step is the bacterial adherence (colonization) to human pharyngeal cells through the specific interaction of bacterial
surface adhesins; as pneumococcal surface antigen A and epithelial cell receptors. The latter are glycoconjugates containing specific
disaccharides.
Once colonization has occurred, infection occurs, if the organisms are carried into anatomically contiguous areas; such as the
eustachian tube or the nasal sinuses or the lungs and their clearance does not readily occur. The normal non-specific defense
mechanisms; as cough reflex, epiglottal reflex, mucociliary movement and patency of eustachian tube and sinuses help in the
organism clearance. So; conditions; as otitis media and pneumococcal pneumonia usually result, when the non-specific defense and
specific defense mechanisms are lacking.
It is important to study the conditions that predispose to pneumococcal infection because they not only help to understand the
pathogenesis of the disease but also help in deciding, which individuals are to be vaccinated. Following are the conditions that
frequently predispose to pneumococcal infections:
Increased likelihood of exposure:
• Day care centers
• Homes for the eldery
• Military training camps
• Prisons
Defective antibody formation:
• Primary deficiencies; as selective IgG deficiency.
• Secondary deficiencies due to diseases; as HIV infections, lymphoma, multiple myeloma etc.
• Complement deficiencies
Insufficient number of PMN:
• Drug induced neutropenia
• Aplastic anemia
Defective clearance of bacteremia:
 • Hypoplenia/asplenia
• Splenectomy
Respiratory infection:
• Air pollution
• Allergy
• Cigarette smoking
• Asthma
Multifactorial:
• Infancy and aging
• Stress
• Glucocorticosteroid treatment
• Malnutrition
• D.M.
• Alcoholism
• Cirrhosis
• Renal insufficiency
• Chronic disease hospitalization
S. pneumoniae produces few toxins unlike S. pyogenes and S. aureus, which produce a variety of tissue damaging substances. The
chief virulent factor of the pneumococcus is the capsule. It is made up of repeating oligosaccharides, that are synthesized within the
cytoplasm. Nearly; every clinical isolate of S. pneumoniae, contains an capsule and only rarely have uncapsulated isolates been
implicated in infections. Anticapsular antibody provides the best specific protection against pneumococcal infection. In the absence of
anticapsular antibody, the phagocytic cells have limited capacity to ingest and destroy pneumococci. The role played by pneumococcal
toxins; as haemolysin, leucocidin and pneumolysin is limited. However pneumolysin may be considered as a virulent factor, as it has
cytotoxicity potential.
The capacity of pneumococci to cause disease, depends on its capacity to replicate (extracellularly) in host tissue (escaping ingestion
and killing by host phagocytic cells) and ability to generate an intense inflammatory response damaging tissues. The cell wall
components of pneumococcus; as teichoic acid, peptidoglycan, phospharylcholine etc. may play a part in the pathogenesis process.
The principal site of pneumococcal clearance from the blood stream is believed to be the spleen. It is for this reason that
overwhelming pneumococcal infection occurs in children and adult after splenectomy. The incidence of pneumococcal infections also
occurs many fold in this population.
Numerous studies have shown that specific antipneumococcal antibodies can occur following infection with the
organism. These antibodies appear approximately a week after infection and are type specific, i.e., immunity is only
against the serotype with which infection has occurred.
After two days of hospitalization, the consciousness of the case deteriorated. The case also developed neck
stiffening. Lumbar puncture revealed CSF, which had cloudy appearance.
What is your clinical diagnosis?
A.5 Acute meningitis.
What could be the reasons for cloudy appearance of CSF?
A.6 The CSF can be cloudy either due to increased number of leucocytes and/or microbes.
What microbiological tests can be done to make a presumptive diagnosis in this case?
A.7 Gram stain of the CSF can throw light on the type of infection Emphasis during smear reporting should be given on the type of
leucocytes, bacteria/yeast present in the CSF. Empiric therapy can be started based on these findings.
Latex agglutination test on the CSF can also be done to detect the presence of antigen of common pathogens.Culture of the CSF
and/or blood is a time consuming investigation.
What are the differentiating features between S.pneumoniae and S.viridans?
A.8 Distinguishing features of key alpha haemolytic streptococci i.e., S. pneumoniae and ‘S. viridans’:
S. pneumoniae S. viridans
Microscopic features

Shape Lanceolate with broad ends facing each other Round/oval

Arrangement Pairs/sort chain Chains

Capsule Present (mostly) Absent

Quellung reaction Positive Negative

Growth (cultural) characteristics

On blood agar Alpha haemolytic, transparent, draughtsman colonies Alpha haemolytic

In liquid media Uniform turbidity Granular turbidity

Identification tests

Bile solubility + -

Insulin fermentation + -

Optochin sensitivity + -

Animal pathogenicity test

Intraperitoneal mouse inoculation Fatal Non fatal

What is the treatment protocol in managing pneumococcal infections with special reference to meningitis?
A.9 Pencillin had been the drug of choice till the 1970s, when increasing number of penicillin resistant strains started getting
isolated. Currently about 10-15% of pneumococcal strains are penicillin resistant (have altered bacterial penicillin binding
proteins with lowered affinity for penicillin) and some other beta lactams antibiotics also demonstrate resistance.
Strains with susceptibility (sensitivity) to penicillin are defined as those, which have MIC value of < 0.1 µg/ml, intermediate
susceptible with MIC 0.1-1 µg/ml and resistant with MIC > 2.0 µg/ml. These definitions are based on drug levels achievable in
CSF during treatment of meningitis. The drug concentration achievable in the blood, lung, sinuses and middle ear are actually
much higher than CSF, thus the MIC needs to be interpreted with reference to the infections being treated. The beta-lactam
antibiotics are successful in treatment of otitis media, sinusitis etc. but not meningitis, if the cause is penincillin resistant
pneumococci.
The appearance of resistance in pneumococcal isolates is worrisome and is likely to arise via horizontal transfer of genetic
material, as resistance to other antibiotics as chloramphenicol, erythromycin and clindamycin is often present. Resistance in
penicillin intermediate susceptible strains is likely due to spontaneous mutation, which necessitate higher concentration of
penicillin to saturate the penicillin binding proteins.
Penicillin–susceptible pneumococci are susceptible to all commonly used cephalosporins. Penicillin-intermediate susceptible
strains are likely to be susceptible to many third-generation cephalosporins including cefotaxime and ceftriaxone. Meningitis
due to a penicillin resistant strains is not likely to respond with third generation cephalosporins. Vancomycin is the drug of
choice, if the pneumococcus is ceftriaxone resistant.
Could this episode in this individual have been prevented?
A.10 Yes. If this individual had taken the pneumococcal vaccine (if the strain which caused this episode was included as one of the
antigens in the components of the vaccine). Details see page......

Laboratory Diagnosis and Treatment (Overview)

An Overview of the Comparative Approach in Laboratory Diagnosis of
Key Gram Positive Cocci (Aerobic) Organisms
Organism / Specimen Stain enhanced microscopy Detection of Serological Tests Culture of Differential Diagnosis Antimicrobial
Disease microbial antigen/metabolite/ Organisms in Susceptibility
Media/Characterizati
genome
on and
Confirmation of
isolate
Staphylococcus - exudates Gram staining; gram positive - - Antistaphylolysin titre may Nutrient - Staphylococcus saprophyticus - usual tests an
aureus - anterior nare swab (in cocci in clusters help in detecting deep deep agar: large, golden - other caogulase negative tested usually
carriers) infection in body as yellow circular staphylococci Vancomycin,
osteomyeltis (titre>2 units/m) colonies aminoglyco-
- food, vomitus, stool (in food - Anaerobic cocci
considered to be significant sides,erythrom
poisoning) Details see in - Micrococcus species chloramphen
- blood (in septicaemia) characterization and
- Streptococcus species trimethoprim-
confirmation of
- serum sulphamethox
isolate: pg 175
ciprofloxacin
For detecting m
resistance, can
cefoxotin disc s

Streptococcus - exudates Gram staining (cocci in chains) Latex agglutination, - Antistreptolysin ‘O’ titre (Acb, Nutrient agar - Other Streptococcal groups - Organism is u
pyogenes - throat swab coagglutination & enzymes see p 191-92) : NG namely B,C,&G sensitive to P
immunoassays techniques - levels greater than 200 todds - Streptococcus ‘viridans’ Erythromycin
- blood culture Details see in
available for detecting microbial units indicative of active susceptibility
- serum (for antistreptolyim O characterization and
antigen in sample (antigen is infection, Anti-Dnase B levels routinely put u
& antiDNase level in confirmation of
extracted from specimen) also estimated
Rheumatic heart heart isolate, p 176
disease & acute glomeru-
lonephritis, respectively
- sputum

Streptococcus - exudates - Gram staining (diplococci - Latex agglutination, - Nutrient agar: - Streptococcus ‘viridans’ - Special proto
pneumoniae - cerebrospinal fluid lanceolate shaped with broad coagglutination and counter NG (scanty growth) followed for p
ends facing each other immunoelectro phoresis antimicrobial
- blood Details see in
- India Ink preparation (capsule techniques available to detect characterization and
test on specia
demonstration) the antigen from various blood agar
confirmation if isolate
samples; as cerebrospinal
- Quellung test (addition of
fluid
antiserum makes capsule
swell apparently)

An Overview of the Antimicrobial options in the infections caused by Gram Positive Cocci
Cell Wall Inhibitors Cell-Membrane Amino Acid Synthesis Nucleic Acid Other
Inhibitors Inhibitors Synthesis Inhibitors

ORGANISM

Staphylococcus aureus • Penicillin G (DOC) • Erythomycin (DOC)

(non penicillinase • Cefazolin (if penicillin allergy) (if penicillin allergy)
producer)

Staphylococcus aureus • Cloxacillin (oral) • Clindamycin Fluoroquinolones

(penicillinase producer)
• Nafcillin (parentral) • Linezolid
(but methicillin susceptible)

• Cephalosporins

Pn + lactamase  inhibitors; as
amoxicillin + clavulanic acid
(DOC)

• Carbapenems; as Meropenem

• Daptomycin

Staphylococcus aureus • Vancomycin (DOC) • Linezolid Fluroquinolone • Vancomycin(DOC)

(methicillin resistant) • Daptomycin • Quinupristin- ± Gentamicin ±
Dalfopristin Rifampin

Staphylococcus aureus • Daptomycin • Linezolid

(VISA/VRSA) • Quinupristin-
Dalfopristin

Nasal Staphylococcal Mupirocin

colonization

S. epidermis (coagulase • Cloxacillin

negative Staphylococci) • Naficillin
• Vancomycin

Streptococcus pyogenes • PnG (DOC), PnV Erythromycin

(group A) • Cephalexin (if patient is allergic
(if penicillin allergy) to penicillin)
 Antimicrobial prevention is required for cases with history of rheumatic fever, before procedures; as dental, that may induie
bectermia Adequate therapeutic levels of drug must be maintained for 10 days to prevent development of acute rheumatic fever

Drugs have no role on acute rheumatic fever and acute glomerulonephritis (established cases).
But persons who have recovered from ARF, must be given prophylactic treatment to prevent recurrences. Acute GN does not recur,
so no use of prophylactic antimicrobials in it.

Streptococcus groups • PnG (DOC), PnV • Erythromycin

(hemolytic B, C & G) • Ampicllin • Azithromycin
• Cephalosporins • Clarithromycin
(3rd generation) • Clindamycin
• Daptomycin
• Vancomycin

*Streptococcus group D • PnG + Aminoglycosides

(General) (as gentamicin) (DOC)

*Enterococcus faecalis • Ampicillin + Gentamicin (DOC)

• Vancomycin + Gentamicin

*Enterococcus faecium • Vancomycin + Gentamicin (DOC, if isolate is vancomycin susceptible)

• Linezolid (DOC, if isolate is vancomycin resistant
• Quinupristin Dalfopristin (DOC, if isolate is vancomycin resistant)

Streptococcus • PnG + Gentamicin (DOC)

‘viridans’ • Cephalosporins
• Vancomycin

Streptococcus • PnG (DOC), PnV Erythromycin • Trimethoprim-

pneumoniae • Cephalosporin Azithromycin Sulfamethoxazole
• Carbapenem Clindamycin • Fluoro-quinolone
• Vancomycin

Anaerobic cocci; as • PnG • Clindamycin

Peptococcus • Metronidazole

- Lactamases are enzymes that hydrolyze the  - lectan ring of te -lactam category of antimicrobials, inactivating the drug. Such enzymes are specific for penicillins,
cephalosporins and carbopenems, designated as penicillinases, cephalosporinases and carbapenemases; respectively.
* Sumergostoc cp,nomatopms are essemtoal.

Assessment/Examination Questions
1. Classify the lesions caused by S. aureus. p. 177
2. Describe in detail the epidemiology of S. aureus infections. A 3c., p. 180
3. Classify the S. aureus carriers. Mention their importance and treatment. A7., p. 187
4. Enumerate the virulence factors of S.aureus. Compare and contrast the virulence factors of S. aureus, S. pyogenes and S. pneumoniae.
A3., p. 184-185, A3b., p. 179
5. Describe the pathogenesis of S. aureus infections. Mention the role played by the genome of S. aureus in antibiotic resistance. A 3., p.
184-86, A 7., p. 181
6. Tabulate the key differences between S. aureus, S. epidermis and S.saprophyticus. Describe coagulase test. A 4b., p. 186, p. 185, p. 67
7. Describe in detail the laboratory diagnosis of S. aureus infections. p. 173-175
8. Enumerate coagulase negative staphylococci (CONS). Describe the epidemiology, laboratory diagnosis and treatment of CONS
infections. A. 10., p. 182-183.
9. Describe Micrococcus. A 11., p. 183
10. Outline the morphology and cultural characteristics of S. aureus. p. 173-175
11. Describe the importance and treatment of infections caused by Methicillin resistant S. aureus (MRSA). A 9., p. 182, p. 200
12. Describe bacteriophage typing. A 5., p. 186-187
13. Enumerate toxin mediated syndromes, S. aureus can cause. Describe Staphylococcal food poisoning and Staphylococcal scalded skin
syndrome. A 8b, c., p 181-182
14. Outline the treatment of S. aureus infections. p. 200, A 9., p. 182
15. How are S. aureus infections controlled? A 8., p. 187
16. Classify the lesions caused by S. pyogenes. Compare and contrast the non-suppurative complications of S. pyogenes in a tabular fashion.
p. 177 and A 3b., 179
17. Describe the classification of beta hemolytic streptococci. A 3b., p. 189
18. What is the reservoir of group A streptococcus? Describe the epidemiology of group A streptococcal pharyngitis. A4a, b., p. 190
19. Describe the pathogenesis of group A streptococcal pharyngitis. A 6a., 190 and A 6b., p. 191-193
20. Describe the role of S. pyogenes antigens, enzymes and toxins in the pathogenicity. A 6b., p. 191-193
21. Outline the morphology and cultural characteristics of S. pyogenes. p. 174, 176
22. Describe the laboratory diagnosis of streptococcal sore throat. p. 199, p. 174, 176
23. Can a case of streptococcal sore throat be treated without performing the antibiotic susceptibility testing? Describe the treatment
guidelines of group A streptococcal infection including antimicrobial prophylaxis. A 7a., p. 193, p. 200
24. Describe antigenic structure of S. pyogenes. Diagramatically depict cell wall of S. pyogenes. Discuss the relevance of cell wall antigens
to virulence and classification. A 6b., p. 191
25. Describe the laborarory diagnosis of rheumatic fever. A 1b., p. 1888
26. Is Scarlet fever prevalent in India? What are the possible reasons for this scenario? A 5b., p. 190
27. Describe Lancefield grouping. A 3b., p. 179
28. Describe Group B streptococci, Group D streptococci, Enterococci, Streptococcus ‘viridians’. p. 176, cases 7, 8., p. 194-195
29. Describe Streptolysins, Streptokinase and Streptodornase. p. 191-192
30. Describe Heat test and CAMP test. p. 176
31. Outline the morphology and cultural characteristics of S. pneumoniae. p. 174, 176
32. What are the key virulent factors of S. pneumoniae? Describe the pathogenesis of pneumococcal infections. A 3a., p. 196
33. Can S. pneumoniae be present in an individual without causing any morbidity? Describe the epidemiology of pneumococcal infections.
A 3a., p. 196, A3c., p. 196
34. Describe the laboratory diagnosis of pneumococcal infections. p. 199, p. 173-176
35. Tabulate the differences between of S. pneumoniae and S. viridans in a tabulated fashion. A 8., p. 198
36. Describe quelling reaction, bile solubility test and optochin sensitivity test. A 2a,b., p. 196, A 8., p. 198
37. Describe Pneumococcal vaccine. p. 629