Phenotypic Identification of

Staphylococcus spp.

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 A. Background and Updates
B. Clinical Significance
C. Common Diseases
D. Laboratory Identification
E. Antimicrobial Susceptibility Test

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 At the end of the session, the participants must:
• Understand the techniques and principles in
identifying Staphylococcus spp.
• Understand the antimicrobial susceptibility results
of Staphylococcus spp.
• Form a diagram that will aid in the identification of
Staphylococcus spp.

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  In the Philippines, Methicillin-resistant Staphylococcus aureus (MRSA)
rate for 2017 is at 57% (n= 5,267). This was a statistically significant
decrease from the reported MRSA rate of 62% in 2016. (ARSP, 2018)
 As a result of the combination of increased use of intravascular devices
and an increase in the number of hospitalized immunocompromised
patients, Coagulase-Negative Staphylococcus spp. have emerged as a
major cause of nosocomial bloodstream infections. (PHE, 2014)
 People with MRSA are estimated to be 64% more likely to die than
people with a non-resistant form of the infection. (World Health
Organization, 2014)

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 Frequently colonize the skin and upper respiratory tract
 16 out of 52 Species can be found in human
 Commonly isolated species are:
 Staphylococcus aureus
 Staphylococcus epidermidis
 Staphylococcus saprophyticus
 Staphylococcus lugdunensis
 Staphylococcus haemolyticus
 Staphylococcus hominis

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  Gram-positive bacteria
 round/cocci
 grape-like clusters (asexual reproduction)
 Non-spore-forming bacteria
 Facultative anaerobes
 Heat-Resistant organism
 Can tolerate High Salt Content Media

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  HOSPITAL ACQUIRED (Nosocomial)
- hospital or health care facilities
- immunocompromised host
- a major cause of morbidity and mortality
 COMMUNITY ACQUIRED
-infection acquired outside health care setting
-infection present on admission
-close contact to a carrier host

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  Skin and soft Tissue Infections
- furuncles or boils “Pigsa” - Impetigo
- Post-operative wound infections - Cellulitis
 Deep Site infections:
- Bacteremia/Septicemia - Visceral abscesses
- Pericarditis - Endocarditis - Osteomyelitis

OSTEOMYELITIS FURUNCLE IMPETIGO

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 Toxin-Mediated Diseases
- Staphylococcal Gastroenteritis (Food Poisoning)
- Toxic Shock Syndrome
- Staphylococcal Scalded Skin Syndrome (SSSS)
 Pneumonia
- can be very lethal (infants and children)

STAPHYLOCOCCAL SCALDED SKIN TOXIC SHOCK SYNDROME

SYNDROME
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 CONTAMINANTS Normal Flora

• Isolation in pure culture from

infected siteCoNS
or body fluid
• Repeated isolation of same strain over
the course of infection
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  S. epidermidis – the most prevalent and persistent
Staphylococcus species in human skin

Infections :
- Bacteremia in immunocompromised hosts
- Surgical wound infections
- infections of indwelling foreign devices

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 S. haemolyticus - the 2nd most frequently encountered CoNS

Infections:
- Endocarditis
- Peritonitis

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 S. saprophyticus - part of the normal vaginal flora
-important opportunistic uropathogen
-common cause of community-acquired
UTI
Infections:
- Acute and Chronic UTI in females
- NGU (non-gonococcal urethritis) in males

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 www.google.com

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 No Special Precautions
Practice proper aseptic technique

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 Coagulase-neg Staph. sp
1. Describe the colony
morphology on BAP
 Size
 Color
 Hemolysis (Beta or
Gamma) Staphylococcus
aureus

Coagulase-neg
Staph. sp

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 2. Do Gram Staining
 Gram-positive cocci
 form clusters “grape like”
 occur singly, in pairs, tetrads, short chains

www.google.com

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 3. Catalase Test
-used to differentiate
Staphylococcus (+)
from Streptococcus (-)
-Reagent: Hydrogen
peroxide (H202)

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4. Coagulase Test
a. Slide Coagulase Test – screening
- detects clumping factor (bound
coagulase)
- rapid, but 15% of S. aureus are
negative
- read results in 10 seconds
***Colonies for testing must not
come from Mannitol salt agar (MSA)

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 b. Tube Coagulase Test - definitive
- detects free coagulase
- Reagent: commercially prepared Rabbit’s plasma/
dehydrated plasma containing citrate or
EDTA
- examine tube after: 2 hrs, 4 hrs and
24 hrs incubation
-Positive: Clot/Coagulate
Negative: no clot formation

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Gram-positive cocci in cluster

Rabbit's Plasma

Streptococcus spp (-)

Gram Stain

CoNS (-)

Staphylococcus spp (+)

Catalase Test

Coagulase Test

S. aureus (+)

3% H2O2

Colony morphology on BAP

Training Course on the Laboratory Diagnosis of Medically Important Bacterial

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 Coagulase Test

S. aureus (+) CoNS (-)

• VP test, urease test

• Sugar Fermentation test
• Novobiocin and
Polymixin B 300 disk
• Etc.

Remember!
The use of latex agglutination kits, semi-automated kits (e.g. API) or
automated machines (Vitek, Phoenix, Maldi-Tof) has the potential to
accelerate and ease the routine identification of staphylococci and
other catalase-positive cocci.

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  Biochemical Test - Urease Test, Acetoin production
(Vogues Proskauer/VP Test)
 Sugar/Carbohydrate Fermentation Test
 Novobiocin Resistance (R=<16mm)
 Polymixin B 300 (R=<10mm)
 Latex agglutination test kits (e.g. Pastorex-Staphplus)
 Dnase test and Heat-stable nuclease test
 Automated machines (e.g. Vitek, Phoenix, Maldi-Tof)
 Semi-automated test kits (e.g. API)
http://www.biography.com/people/louis-
pasteur-9434402#synopsis
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 Vogues Proskauer/VP Test
- ability of the organism to
produce ACETOIN
- Reagent: (3:1)VP1 - α-naphthol
VP2 - 40% KOH
(+) = Red
(-) = Yellow/no color change

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 Urease Test
-ability of the organism to
split urea forming 2
molecules of ammonia by the
action of the enzyme UREASE
with resulting to alkalinity
(+) = Pink ;
(-) = Yellow/no color change

Training Course on the Laboratory Diagnosis of Medically Important Bacterial

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 Sugar/Carbohydrate
Fermentation Test

(+) = Yellow ;
(-) = orange/no color change

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 PB
Staphylococcus spp. CAT COAG CF VP TNase M MN LAC TRE NAG β-Gal PYR Urease 300-U Hemolysins
<10mm

S. aureus subsp. aureus + + + + + + + + + + - - d R +

S. aureus subsp. anaerobius - + - - + + - - - - - ND ND ND +

S. schleiferi subsp. coagulans + + - + + - + d - ND ND ND + ND +

S. schleiferi subsp. schleiferi + - + + + - + - d (+) (+) + - S +

S. lutre + + - + (±) + + + + ND + ND + ND +

S. intermedius + + d - + (±) + + (±) + + + + S D

S. delphini + + - - - + + + - ND ND ND + ND +

S. hyicus + d - - + - + + + + - - - R -

S. pseudintermedius + + - + + + + + + + + + + ND +

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 Colony PB
Staphylococcus spp. Pigment COAG CF VP TNase ODC BGAL 300-U Urease Hemolysins
/ Size <10mm

S. epidermidis -/- - - + - (d) - R + (d)

S. haemolyticus d/+ - - + - - - S - (+)

S. lugdunensis d/d - (+) + - + d d d (+)

S. schleiferi subsp. schleiferi - - + + + - + - - (+)

S. hyicus - d - - + - - R d -
S. capitis subsp. capitis -/- - - d - - - S - (d)
S. capitis subsp. urealyticus d/- - - d - - - ND + (d)
S. saccharolyticus -/- - - ND - ND ND ND ND -
S. warneri d/d - - + - - - S + (d)
S. pasteuri d/d - - d - - - ND + (d)
S. hominis subsp. hominis d/+ - - d - - - S + -

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 + : ≥ 90% positive

Staphylococcus spp. TRE M MN MNT SUC LAC

(trehalose) (maltose) (mannose) (mannitol) (sucrose) (lactose) (±) : ≥ 90% weakly positive

S. epidermidis - + (+) - + d d : 11-89 % positive

S. haemolyticus + + - d + d (d) : ≥ 90% negative

S. lugdunensis + + + - + +
- : ≥ 90% negative

S. schleiferi subsp. schleiferi d - + - - -

( ) : delayed reaction

S. hyicus + - + - + +
ND : Not Determined
S. capitis subsp. capitis - - + + (+) -
S. capitis subsp. urealyticus - + + + + (d)
S. saccharolyticus - - (+) - - -
S. warneri + (+) - d + d
S. pasteuri + (d) - d + d
S. hominis subsp. hominis d + - - (+) d

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 Novobiocin Resistant CoNS

Large Hemo- PYR TRE NAG

Staphylococcus spp. Col. lysins Urease NO3 ARAB XYL MN LAC SUC
(pyrro- (treha- (N-acetyl-
(arabinose) (xylose) (mannose) (lactose) (sucrose)
lidonyl) lose) glutamase)

S. saprophyticus subsp.
+ - + - - - - - d + + d
saprophyticus

S. saprophyticus subsp. bovis - - + + + - - - - + + +

S. cohnii subsp. urealyticus d d + d - - - + + - + d

S. hominis subsp. novobiosepticus - - + - - - - - d + - -

Training Course on the Laboratory Diagnosis of Medically Important Bacterial

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 LAT

API
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 VITEK 2 MALDI-TOF
Compact

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 Chromagar

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Training Course on the Laboratory Diagnosis of Medically Important Bacterial
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Training Course on the Laboratory Diagnosis of Medically Important Bacterial
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Training Course on the Laboratory Diagnosis of Medically Important Bacterial
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 3. Observe for
color change.
2. Pick colonies Pink/Red =
1. Moisten the
surrounding the
disk with distilled
PEN disk and blot
Positive (+)
water
on the disk No Color
change =
Negative (-)
Note: If the nitrocefin test is (-), the
penicillin zone-edge test should be
performed before reporting the
isolate as PEN (S) in cases where
PEN may be used for therapy
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 POSITIVE = Sharp or
a “cliff” zone edge
(beta-lactamase
production)

NEGATIVE = Fuzzy or a
“beach” zone edge (no
beta-lactamase
production)

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 METHICILLIN RESISTANT Staphylococcus
aureus (MRSA)

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 In the Philippines, Methicillin-resistant Staphylococcus aureus
(MRSA) rate for 2017 is at 57% (n= 5,267). This was a
statistically significant decrease from the reported MRSA rate of
62% in 2016. (ARSP, 2018)
 They are transmissible, which an outbreak can occur when one
strain is transmitted to other patients
 MRSA is a major cause of hospital-acquired infections that are
becoming increasingly difficult to combat because of emerging
resistance to all current antibiotic classes.

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 Detection of MRSA
 MecA mediated Oxacillin Resistance using
Cefoxitin
- Cefoxitin is used as a surrogate for mecA-
mediated Oxacillin resistance.

 Report Oxacillin susceptible or resistant based

on the cefoxitin result.

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 Detection of MRSA
 Cefoxitin - screen disk to oxacillin

< 21 mm = Resistant – Methicilin

Resistant Staphylococcus aureus
(MRSA)
> 22 mm = Susceptible – Methicilin
Susceptible Staphylococcus aureus
Cefoxitin: 18 mm (Resistant)
(MSSA)

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 Detection of MRSA
Medium : Mueller Hinton Agar with 4% NaCl for oxacillin only
: Mueller Hinton Agar without NaCl for cefoxitin and
other antibiotics
Incubation : 33-35°C, full 24 hrs. for oxacillin/vancomycin, (above
35°C may not detect MRSA)
: 16-20 hrs. for cefoxitin and other antibiotics

Minimal Inhibitory
Concentration (MIC)
= > 4 ug/ml

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 Detection of MRSA
Detection of mecAgene by
Polymerase Chain Reaction (PCR)

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 Macrolide Resistant Staphylococcus spp.
(Inducible Clindamycin Resistance)

- Occurrence of induced resistance of

Clindamycin caused by the D-zone
presence of MLSB gene and erm gene
- Can be detected by disk
approximation test (D-zone Test)
using Clindamycin disc against
Erythromycin disc.

A distance of 15 – 26mm between

Erythromycin and Clindamycin
**see Supplementary info. V
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 Macrolide Resistant Staphylococcus spp.
(Inducible Clindamycin Resistance)
ICR (+) = Flattening of
clindamycin zone
adjacent to erythromycin
disk D-zone

ICR (-) = No flattening of

clindamycin zone
adjacent to erythromycin
disk

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 Macrolide Resistant Staphylococcus spp.
(Inducible Clindamycin Resistance)
INTERPRETATION
Erythromy Clindamy Report
cin (E) cin (CD)
S S Susceptible (S) to both and ICR (-)
R R Resistant (R) to both and ICR (-)
R S (ICR +) Resistant (R) to both and indicate
the presence of ICR
R S (ICR -) Erythromycin (R) and Clindamycin
(S) and ICR (-)

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 Factors that can cause false reactions:
 Colonies from BAP can cause false positive
catalase reaction
 Staphylokinase - enzyme that can lyse the clot after
prolonged incubation
 Contaminated media
 Mixed culture
 Contaminated reagents

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 CASE STUDY

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A patient suffering from skin blister (Ritter Disease) was
subjected for wound culture. Laboratory results show:
- Clear hemolysis, Gram-positive cocci in cluster
- Catalase (+), PYR (-)
- Coagulase Slide (+), Coag. Tube (-)
- Mannose, Lactose, Maltose are (+)
- DNASE, Urease and VP (+)
Question:
1. Identify the most probable organism causing the disease.
Why?
2. What test would you need to differentiate your answer in Q1
with Staph. intermedius and Staph. pseudintermedius?

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 References..
• Arsrl (2018). Arsp 2017 data summary report [online]. Accessed on December 21, 2017.
Available at http://arsp.com.ph/wpcontent/uploads/2017/06/2016_annual_
report_summary.pdf
• C.-J. Chen, Y.-C. Huang. New epidemiology of Staphylococcus aureus infection in Asia.
Clinical Microbiology and Infection, Volume 20, Issue 7, 2014, Pages 605-623,
ISSN 1198-743X. https://doi.org/10.1111/1469-0691.12705.
• Who (2014). WHO’s first global report on antibiotic resistance reveals serious,
worldwide threat to public health [online]. Accessed on Jan 22, 2018. Available
at http://www.who.int/mediacentre/news/releases/2014/amr-report/en/
• Public Health England (2014). UK Standards for Microbiology investigations [online].
Accessed on Jan 23, 2018. Available at http://www.sfam.org.uk

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