Catalase-Positive, Gram-Positive Cocci

Catalase-Positive, Gram-Positive Cocci

Learning Outcomes

The learning outcomes for this lecture include the ability to:

• Differentiate Staphylococcus from Micrococcus.

• List and describe media for Staphylococcus isolation.

• Differentiate Staphylococcus from Streptococcus using Gram stain, colonial morphology, and catalase reaction.

• State the principle and purpose of the catalase reaction.

• State the principle and purpose of the coagulase test.

• Differentiate bound coagulase from free coagulase and describe their detection.

• Describe the principle and purpose of mannitol salt agar (MSA).

• Differentiate Staphylococcus aureus from coagulase-negative staphylococci (CoNS).

• Identify virulence factors of Staphylococcus aureus and associated infections.

• Describe the Zone Edge test and its reaction mechanism.

• Describe inducible clindamycin resistance and its detection.

• List medically significant CoNS species and attributed infections.

• Describe the identification of Staphylococcus epidermidis and Staphylococcus saprophyticus.

• Discuss the clinical relevance of Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis, and other CoNS.

• State the purpose and principle of the novobiocin test.

Epidemology

Gram-Positive Cocci – Catalase Positive: General Characteristics

Microscopic Morphology

• Gram-positive cocci (GPC) – appear "perfectly round."

• Arrangement: Single cells, pairs, or clusters resembling "bunches of grapes."

Macroscopic Morphology

• Non-fastidious: Grow well on Blood Agar Plates (BAP) and Chocolate Agar (CHOC); no growth on MacConkey Agar (MAC).

• Colonies: Small to medium size after $18-24$ hours of incubation.

• Color: Beige, white, yellow, or light gold.

• Texture: Creamy, buttery, smooth.

• Hemolysis on BAP: Gamma ($\gamma$) or Beta ($\beta$) hemolysis.

Important Characteristics

• Catalase positive.

• Non-motile.

• Non-spore forming.

• Produce polysaccharide capsules and slime layers, leading to biofilm formation.

• Non-fastidious nutritional requirements.

• Aerobic or facultative anaerobes (except for Staphylococcus saccharolyticus, which is an obligate anaerobe).

• Common inhabitants of the skin and mucosal membranes of humans and warm-blooded mammals.

What is Catalase?

• Catalase is an enzyme commonly found in organisms exposed to oxygen.

• It catalyzes the decomposition of hydrogen peroxide ($2H<em>2O</em>2$) into water ($2H<em>2O$) and oxygen ($O</em>2$), as shown in the reaction: $2H<em>2O</em>2 \rightarrow 2H<em>2O + O</em>2$.

• Purpose: Protects organisms from oxidative damage.

• Catalase Test Caveat: When performing the test, avoid mixing bacterial inoculum with red blood cells (RBCs) from BAP, as this can cause weak bubbling (false positive) due to RBC catalase, which should not be reported as a positive result.

• A positive catalase test differentiates Gram-positive cocci genera: Staphylococcus species are positive, while Streptococcus species are negative.

Commonly Encountered Species

• Micrococcus spp. (M. luteus)

• Staphylococcus aureus

• Staphylococcus epidermidis

• Staphylococcus saprophyticus

• Staphylococcus lugdunensis

• Other Staphylococcus spp.: S. haemolyticus, S. warneri, S. capitis, S. simulans, S. hominis, S. intermedius, S. pseudintermedius, S. schleiferi.

Specific Organisms

Micrococcus spp.

• Found in the environment and part of normal skin flora.

• Often recovered alongside Staphylococcus spp. on the skin.

• Usually considered non-pathogenic, but can act as an opportunist.

• Produces a distinct yellow pigment on BAP.

Staphylococcus spp. Differentiation

• Staphylococcus species are initially differentiated by the coagulase test.

• Coagulase: An enzyme that clots plasma by converting fibrinogen to fibrin.

• Types of Coagulase: Bound coagulase and free coagulase.

• Differentiation categorizes staphylococci into coagulase-positive and coagulase-negative (CoNS) groups.

Coagulase-Positive Staphylococci

• Human Pathogens: Staphylococcus aureus (MSSA and MRSA), S. delphini, S. intermedius, S. lutrae, S. hyicus, S. pseudintermedius.

• S. pseudintermedius shows increasing incidence in human disease.

Coagulase-Negative Staphylococci (CoNS)

• Healthcare-Associated Infections (HAIs): S. epidermidis, S. lugdunensis, S. haemolyticus.

• Urinary Tract Infections (UTIs):

  • S. saprophyticus: #2 cause of uncomplicated UTIs, especially in young, sexually active females.

  • S. epidermidis: Catheter-associated UTIs.

• Skin, Wound, Joint Infections: S. lugdunensis, S. haemolyticus, S. intermedius.

Staphylococcus aureus

• The primary human pathogen of the genus Staphylococcus.

• Causes infections in both healthy and immunocompromised individuals.

• Habitat: Skin and mucosal membranes of persistent carriers, intermittent carriers, transient carriers, or non-carriers.

• Can be recovered from almost any clinical specimen.

• Infections: Ranges from superficial skin infections to severe systemic infections.

Host Defense Against S. aureus

• Intact skin barrier and the skin microbiome are primary defenses.

• When skin barrier is broken: Polymorphonuclear neutrophils (PMNs) and innate immunity respond.

• Increased release of natural antimicrobial substances (e.g., lysozyme).

• Immune response and "pus" production.

Pathogenicity: Virulence Factors

S. aureus produces a wide array of virulence factors; each strain may possess a unique combination.

Enterotoxins

• Damage the gut, causing diarrhea and vomiting.

• Heat and acid stable, meaning they survive gastric acid and boiling at $100^{\circ}\text{C}$ for $30$ minutes.

• Toxins can still be present and harmful even if the organism is dead.

• $8$ serologically distinct enterotoxins (A, B, C, D, E, G, H, I).

• Found in up to $50\%$ of S. aureus isolates.

• Staphylococcal Food Poisoning: Caused by the toxin (primarily A, B, & D), not bacterial growth. Associated with contaminated food or inadequate refrigeration, often "picnic foods." It is self-limiting.

Toxic Shock Syndrome Toxin-1 (TSST-1)

• Previously known as Enterotoxin F.

• Causes endothelial and epidermal cell death.

• Superantigen: Induces an aggressive immune response, leading to T-cell proliferation and cytokine overproduction.

• Causes nearly all cases of menstruation-associated Toxic Shock Syndrome (TSS).

• Antibody protection against this toxin provides immunity.

Exfoliative Toxin

• Epidermolytic toxin: A serine protease that splits intracellular bridges of the epidermis.

• Causes the epidermal layer of skin to "slough off."

• Also known as Scalded Skin Syndrome Toxin (SSST).

• Staphylococcal Scalded Skin Syndrome (SSSS):

  • Can be localized or systemic.

  • In babies, it can result from inoculation of the umbilical stump with toxin-producing S. aureus.

  • Causes profuse peeling of the epidermal layer due to lysis of epidermal cells, revealing moist, red middle epidermis.

  • Lasts $2-4$ days; spontaneous recovery typically occurs in children.

  • Adult cases can lead to mortality.

Cytolytic Toxins

• Alpha ($\alpha$) hemolysin: Destroys platelets and tissues.

• Beta ($\beta$) hemolysin: Shows enhanced activity by acting on the sphingomyelin of red blood cell (RBC) membranes, causing lysis. It is a "hot-cold lysin," working best at $37^{\circ}$C and very well when stored at $4^{\circ}$C.

• Delta ($\delta$) hemolysin: Causes injury to cells and leukocytes but is less lethal.

• Gamma ($\gamma$) hemolysin: Damages white blood cells.

Exoenzymes

These enzymes facilitate the spread of microorganisms, causing deeper infections.

• Coagulase: Coagulates blood, protecting the bacteria from phagocytosis.

• Hyaluronidase: Hydrolyzes hyaluronic acid in connective tissue (a "spreading factor").

• Lipase: Breaks down fats and oils secreted by sebaceous glands on the skin surface.

• Protease: Destroys tissue proteins.

• Panton-Valentine Leukocidin (PVL): Toxic to white blood cells (WBCs), preventing clearance of infection. Associated with more serious and invasive skin and soft tissue infections (SSTIs).

Protein A

• A surface protein located on the cell wall.

• Binds the Fc portion of antibodies, preventing opsonization and phagocytosis.

• Decreases immune clearance of organisms at the site of infection.

• Contributes to increased swelling and pus formation.

S. aureus Infections

Localized Skin and Soft Tissue Infections (SSTIs)

These occur when endogenous strains enter the skin through wounds, follicles, or glands.

• Folliculitis: Superficial inflammation of hair follicle(s). Usually resolves without complications but can progress.

  • Microscopic: $1\text{ mm}$ perifollicular red papule or pustule.

  • Treatment: Tetracycline or erythromycin ($500\text{ mg}$ $2\text{x/day}$).

• Furuncle (Boil): Inflammation of a hair follicle or sebaceous gland that has progressed to an abscess or pustule.

  • Areas: Sweat and abrasion.

  • Microscopic: About $1\text{ cm}$ tender red papule or fluctuant nodule.

  • Treatment: $1$. Incise & curettage. $2$. Dicloxicillin ($250\text{ mg}$ $4\text{x/day}$ for $10$ days) or Augmentin ($500\text{ mg}$ $2\text{x/day}$ for $10+\text{ days}$).

• Carbuncle: A larger and deeper lesion created by an aggregation and interconnection of a cluster of furuncles.

  • Areas: Sweat and abrasion, nape of neck.

  • Microscopic: Several cm diameter red plaque.

  • Treatment: $1$. Incise and curettage or excise. $2$. Dicloxicillin ($250\text{ mg}$ $4\text{x/day}$ for $10+\text{ days}$) or Rifampin ($300\text{ mg}$ $2\text{x/day}$ for $10+\text{ days}$ - note: causes orange body fluids).

• Impetigo: A skin infection involving the epidermis.

  • Characterized by vesicles (fluid-filled papules) that rupture and crust over, known as "honey-crusted lesions."

  • Most common in children ages $2-5$ years.

Invasive Infections

Infections of the dermis and epidermis can progress rapidly depending on strain-specific virulence factors and individual immune status.

• Septic arthritis: Infection of the joint space.

• Osteomyelitis: Infection of the bone.

• Bacteremia: Presence of bacteria in the blood, which can lead to:

  • Sepsis / Endocarditis: Infection of heart valves, forming vegetations.

• Other sterile body site infections.

Toxin-Mediated Infections

• Staphylococcal Food Poisoning (discussed under Enterotoxins).

• Staphylococcal Scalded Skin Syndrome (SSSS) (discussed under Exfoliative Toxin).

• Staphylococcal Toxic Shock Syndrome (TSS) (discussed under TSST-1).

  • Associated with tampon use.

  • Symptoms: High fever, rash on trunk and extremities, watery diarrhea, vomiting, dehydration, hypotension.

  • Fatal in $2-5\%$ of cases due to multiple organ system failure.

Other Coagulase-Positive Staphylococci

S. aureus is the primary human coagulase-positive pathogen.

• Other coagulase-positive Staphylococcus spp. (S. delphini, S. intermedius, S. pseudintermedius, S. schleiferi) are normal microbiota in animals (e.g., domestic dogs and cats).

• These are increasingly causing human SSTIs (from endogenous human flora or bite/scratch wounds).

• Notable cause of invasive infections in immunocompromised individuals.

Coagulase-Negative Staphylococci (CoNS)

• Refers to species that are tube-coagulase negative (though some CoNS produce bound coagulase).

• Normal flora of skin and mucous membranes of warm-blooded mammals; ubiquitous in human environments.

• Significantly less virulent than S. aureus.

• Significant cause of healthcare-associated infections (HAIs) or nosocomial infections.

• Common contaminants of human cultures.

CoNS Pathogenicity and Disease

• Virulence Factors:

  • Biofilms: Extracellular polysaccharide layer (glycocalyx) enhances binding to prosthetic devices, acts as a molecular scaffold, and provides protection from the immune system and antibiotics. Biofilm fragments can break off and seed other areas.

  • Acquire resistance genes.

• Opportunistic infections: Primarily associated with immunocompromised and hospitalized patients.

• Almost always associated with indwelling prosthetic devices.

• Exception: S. saprophyticus is a common cause of community-acquired UTIs in young, sexually active females (ranked second).

CoNS Infections

• Medical Devices: Shunts, IVs, catheters, prosthetic joints.

• Immunocompromised patients: Infections at sterile sites.

• Urinary Tract Infections:

  • S. epidermidis (SEPI): Associated with indwelling catheters.

  • S. saprophyticus (SSAP).

• Native Valve Endocarditis: S. lugdunensis (SLUG).

Micrococcus spp. & Rothia spp.

Micrococcus spp.

• Normal resident of human skin and the environment.

• Low virulence.

• Obligate aerobe.

• Can be mistaken for CoNS.

• Colony morphology: Lemon yellow colony, $\gamma$-hemolytic.

• Microscopic morphology: Large GPC in tetrads.

• Tests: Catalase positive, Bacitracin sensitive, Modified Oxidase positive.

• Metabolism: Oxidative, not fermentative.

Rothia spp.

• Normal resident of the oropharynx.

• Low virulence; implicated in underlying malignancy or neutropenic patients.

• Formerly named Stomatococcus mucilaginosus.

• Can be mistaken for CoNS.

• Colony morphology: Small, white, sticky, $\gamma$-hemolytic. Strongly adherent to agar.

• Microscopic morphology: Large GPC.

• Tests: Catalase positive (some variable), Modified oxidase negative.

Laboratory Diagnosis of Staphylococcus spp.

General Growth Characteristics

• Understanding basic growth requirements is critical for isolation and identification.

• Determines specimen collection, transport procedures, and primary media selection.

• Most Staphylococcus spp. are non-fastidious and facultative organisms.

• Do not require special collection or transport media.

• Grow readily on enriched non-selective primary media (BAP, CHOC, Thioglycollate).

• Facultative (grow with $O<em>2$, $CO</em>2$, or anaerobically).

• Visible colonies appear after > $18$ hours of incubation.

Primary Media

• Grows readily on BAP, CHOC, and thioglycollate.

• Selective Media for Staphylococcus spp. isolation:

  • Mannitol Salt Agar (MSA): May include oxacillin.

    • Principle: Selective and differential agar.

    • Selective component: High salt concentration ($7.5\%\ NaCl$) selects for halophilic (salt-tolerant) organisms like Staphylococcus spp.

    • Differential component: Contains mannitol sugar and phenol red pH indicator. Fermentation of mannitol produces acid, causing a pH drop and a color change of the medium from red to yellow.

  • Columbia Colistin-Nalidixic Acid Agar (CNA):

    • Principle: Enriched and selective for Gram-positive organisms.

    • Enriched: Contains $5\%$ sheep blood.

    • Selective: Colistin and nalidixic acid antibiotics inhibit Gram-negative bacteria.

  • Phenylethyl Alcohol Agar (PEA):

    • Principle: Enriched and selective for Gram-positive organisms.

    • Enriched: Contains $5\%$ sheep blood.

    • Selective: PEA inhibits most Gram-negative bacteria.

  • CHROMagar Staph. aureus.

Microscopic Morphology: Gram Stains

• Performed as a direct smear from a colony.

• Observe for pus (purulence), WBCs, and bacteria.

• Caution: Organisms older than $48$ hours can easily over-decolorize, leading to false Gram-negative results.

Colony Morphology (BAP)

• Staphylococcus aureus: Small-medium white, grey, yellow, or gold colonies. Most exhibit a large zone of $\beta$-hemolysis.

• Coagulase Negative Staphylococci (CoNS): Small, white colonies; most are $\gamma$-hemolytic. S. haemolyticus and some S. lugdunensis can be $\beta$-hemolytic.

• Micrococcus spp.: Small, lemon-yellow, $\gamma$-hemolytic colonies. Often smaller than CoNS at $24$ hours incubation. Microscopically, appear as Gram-positive cocci in tetrads.

• Rothia spp.: Small, white, sticky, $\gamma$-hemolytic colonies.

Key Laboratory Identification Tests

Catalase Test

• Principle: Detects the presence of the enzyme catalase.

• Procedure: Place $1$ drop of $H<em>2O</em>2$ on a clean glass slide, transfer $1$ colony with a wooden stick or loop, and observe for immediate bubble formation.

• Staphylococcus and Micrococcus spp. are positive.

Identification Tests for S. aureus

• Slide Coagulase Test:

  • Principle: Detects bound coagulase (clumping factor) which directly converts fibrinogen to fibrin.

  • Procedure: Combine rabbit plasma with a suspect colony on a slide, rotate for $10$ seconds.

  • Confirmation: Negative tests should be confirmed with the tube method, especially if colony morphology is consistent with S. aureus ($\beta$-hemolytic).

• Latex Agglutination Kits:

  • Principle: Detects clumping factor, free coagulase, and/or Protein A.

  • Used more commonly than slide coagulase due to sensitivity and longer shelf-life.

  • Always read package inserts, consider patient immune status and sample source, and correlate results with colony morphology.

• Tube Coagulase Test:

  • Principle: Detects free (extracellular) coagulase, also called "Staphylocoagulase." Extracellular coagulase activates coagulase-reacting factor (CRF) to form a complex resembling thrombin, which then converts fibrinogen to fibrin (a delayed reaction).

  • Procedure: Check for coagulation in $4-6$ hours (clots can autolyse after >$20-24$ hours, leading to false negatives).

  • Considered the gold standard test for S. aureus.

Differentiating Micrococcus spp. from Staphylococcus spp.

• Modified Oxidase (Microdase disk):

  • Principle: Detects the enzyme oxidase, which transfers hydrogens to an oxygen molecule during aerobic respiration.

  • Micrococcus spp. will be positive.

  • Most Staphylococcus spp. will be negative.

• Bacitracin Susceptibility ($0.04\text{ U}$ disk):

  • Micrococcus spp. are susceptible (>10\text{ mm} zone).

  • Staphylococcus spp. are resistant (<10\text{ mm} zone).

  • Note: Many Micrococcus strains are now resistant to bacitracin; use colony morphology and modified oxidase to confirm.

Table 9-1: Differentiation of Staphylococcus from Micrococcus

Further Testing to ID CoNS

The extent of identification (presumptive ID = CoNS vs. full ID = species-level with sensitivities) depends on:

• Laboratory policies.

• Sample source.

• Patient scenario / immune status.

• Novobiocin Susceptibility (for urine cultures):

  • Purpose: Test for S. saprophyticus.

  • Principle: Utilizes a $5\text{ }\mu \text{g}$ novobiocin disk.

  • S. saprophyticus is resistant (<16\text{ mm} zone).

  • S. epidermidis and other CoNS are susceptible (>16\text{ mm} zone).

• PYR Test:

  • Principle: Detects pyrrolidonyl peptidase enzyme. Disks impregnated with L-pyrrolidonyl-$\beta$-naphthylamide (substrate). Hydrolysis of the substrate yields free $\beta$-naphthylamine, which produces a red color after reagent addition.

  • Purpose: Important for differentiating S. aureus from S. lugdunensis, both of which may be Staph latex positive.

  • S. aureus: Negative.

  • S. lugdunensis: Positive.

  • S. haemolyticus: Positive.

Other Identification Methods

• Molecular methods:

  • PCR (Polymerase Chain Reaction): Specifically to differentiate between resistant strains (MSSA and MRSA).

  • rRNA sequencing: For speciation in positive blood cultures.

  • Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight MS (MALDI-TOF MS).

  • MSSA: Methicillin-Susceptible Staphylococcus aureus.

  • MRSA: Methicillin-Resistant Staphylococcus aureus.

Laboratory Identification Flowchart Summary

Based on Gram-positive cocci that are catalase positive:

1. Gram Stain & Catalase Test: GPC, catalase positive.

2. Coagulase Test: Positive leads to Staphylococcus aureus. Negative leads to Coagulase-negative Staphylococcus spp. or Micrococcus spp.

3. For Coagulase Negative results: Further differentiate.

   • Oxidase/Bacitracin susceptibility: Positive oxidase with susceptible bacitracin indicates Micrococcus spp. Negative oxidase with resistant bacitracin indicates Staphylococcus spp. (CoNS).

   • For CoNS:

     • If from urine source, perform Novobiocin susceptibility: Susceptible (>16\text{ mm}) implies S. epidermidis or other $\gamma$-hemolytic CoNS. Resistant (<16\text{ mm}) implies S. saprophyticus.

     • For other CoNS or specific differentiation, a PYR test can help differentiate S. lugdunensis (PYR positive) from S. aureus (PYR negative) which can have similar latex results.

Comparison Table of CoNS, S. aureus, Micrococcus, Rothia

Antimicrobial Susceptibility Testing

Antimicrobial Therapy

• Vital for the management and treatment of staphylococcal infections.

• A broad spectrum of agents may be used.

Beta-Lactam Antibiotics

• Mechanism: Inhibit cell wall synthesis by binding to and irreversibly inactivating penicillin-binding proteins (PBPs), which are responsible for crosslinking peptidoglycan chains.

• This inhibits the formation of a structurally stable cell wall, leading to bacterial lysis (bactericidal effect).

• Effective against many Gram-positive and Gram-negative organisms.

Mechanisms of Resistance: $\beta$-Lactamase

• Mechanism: $\beta$-lactamase is an enzyme produced by microorganisms that hydrolyzes the $\beta$-lactam ring, preventing the antibiotic from binding to PBPs.

• Direct $\beta$-lactamase tests: Performed only on certain bacteria to predict resistance to $\beta$-lactam antibiotics, eliminating the need for direct AST.

• Bacteria tested and detected resistance:

  • H. influenzae: Ampicillin, Amoxicillin

  • N. gonorrhoeae: Ampicillin, Amoxicillin, Penicillin

  • M. catarrhalis: Ampicillin, Amoxicillin, Penicillin

  • S. aureus: Ampicillin, Amoxicillin, Penicillin

• Nitrocefin (Cefinase) test: Uses chromogenic cephalosporin (nitrocefin), which is yellow when the $\beta$-lactam ring is intact and pink when hydrolyzed. A rapid and sensitive direct detection method, confirmed with KB disk diffusion.

• Zone Edge Test: Also known as the "beach and cliff effect," this phenomenon confirms the presence of $\beta$-lactamase enzymes in bacterial isolates. Most Staphylococcus produce $\beta$-lactamase.

Altered Penicillin Binding Protein: PBP 2a

• Primary mechanism of MRSA resistance: An altered penicillin binding protein (PBP 2a) renders all currently available $\beta$-lactams ineffective.

• Genetic basis: Resistance is conferred by the mecA gene, which produces the altered PBP 2a.

• The mecA gene is carried on a mobile DNA element (SCCmec), which can be readily transferred between species (horizontal gene transfer).

• This mechanism is responsible for widespread antibiotic resistance.

Community-Acquired MRSA (CA-MRSA)

• Typically acquired in community settings (schools, gyms, households).

• Often associated with skin and soft tissue infections (abscesses, cellulitis, folliculitis).

• Strains often carry genes encoding toxins such as Panton-Valentine Leukocidin (PVL).

• Typically harbor the SCCmec type IV element, a small mobile genetic element containing the mecA gene.

Healthcare-Associated MRSA (HA-MRSA)

• Typically acquired in healthcare settings (hospitals, nursing homes, long-term care facilities).

• Often associated with invasive infections (bloodstream infections, surgical site infections (SSI), pneumonia).

• Typically carry larger SCCmec elements (types I-III), which may contain additional antibiotic resistance genes, making them more resistant to multiple classes of antibiotics.

Screening for MRSA

1. MRSA screening agar: MSA + oxacillin, or CHROMagar.

2. Cefoxitin (FOX) $30\text{ }\mu \text{g}$ disk diffusion:

   • <19\text{ mm} zone: Resistant.

   • >20\text{ mm} zone: Susceptible.

MRSA Confirmation

• PBP 2a Latex Agglutination Kit.

• Detection of mecA gene via PCR is the gold standard.

• CoNS are also increasingly resistant to $\beta$-lactams through similar mechanisms.

Macrolide Resistance

• The increase in MRSA isolation has led to increased use of macrolide antibiotics for treatment.

• Lincomycin macrolide antibiotics are hydrophobic and diffuse well into tissues.

• Clindamycin is the macrolide of choice for deep tissue MRSA infections.

• Macrolide resistance expression:

  • Constitutive mechanism: Constant expression.

  • Inducible mechanism: Activated by the presence of erythromycin, typically identified in erythromycin-resistant S. aureus strains.

Macrolide Resistance: D-Test

• Purpose: Performed when an organism appears susceptible to clindamycin but resistant to erythromycin by automated AST systems. Confirms the clindamycin result is correct.

• Clinical Relevance: Patients taking erythromycin long-term risk their Staph and Strep turning on the erm gene.

• The erm gene codes for production of an enzyme for erythromycin resistance, and this gene can also trigger the bacteria to produce another enzyme for resistance to clindamycin.

• When to set up D-Zone: When in vitro presentation shows erythromycin resistant and clindamycin susceptible.

• Procedure:

  • Inoculate a Mueller Hinton plate with a $0.5$ McFarland lawn of S. aureus.

  • Add clindamycin ($2\text{ }\mu \text{g}$) and erythromycin ($5\text{ }\mu \text{g}$) disks, $15\text{ mm}$ apart.

• Inducible clindamycin resistance (positive result):

  • Erythromycin induces the production of a resistance enzyme to clindamycin.

  • Bacteria near the erythromycin disk will become resistant to clindamycin.

  • This results in a flattened D-shaped zone of inhibition around the clindamycin disk.