Staphylococcus: Gram-Positive Cocci

• Gram-positive cocci include the family Micrococcaceae.
• Genus examples comprise Staphylococcus, Micrococcus, Stomatococcus, and Planococcus.

Staphylococci Species

• Staphylococci include more than 50 different species
• Only a few can be associated with human disease
• Species associated with human disease include S. aureus, S. intermedius, S. epidermidis, S. saprophyticus, S. haemolyticus, and S. lugdenensis.
• Staphylococcus aureus is the most common isolated species.

Commensal Bacteria

• Some staphylococci species, such as S. aureus, S. epidermidis, and S. saprophyticus, are commensals of the skin, oropharynx, GIT, and urogenital tract
• Commensal bacteria can also be opportunistic pathogens waiting for ideal infection conditions to arise

Staphylococcus aureus

• Staphylococcus aureus is a significant human pathogen
• It can directly invade and destroy tissues
• It can give rise to superficial & deep-seated infections
• It produces various toxins
• It attaches to prosthetic valves
• It can cause bacteraemia
• It is responsible for nosocomial infections

S. aureus Infections

• S. aureus can cause pyogenic infections: folliculitis and impetigo which is a superficial skin infection in children
• It can cause clinical implications such as food poisoning (due to enterotoxins) and toxic shock syndrome (due to toxin-TSST1)
• Scalded Skin Syndrome (SSS), also known as Staphylococcal scalded skin syndrome (SSSS) or Ritter's disease, is a serious skin infection causing blistering and peeling via exfoliative toxins ETA & ETB

S. aureus Further Infections

• It can cause secondary infections such as endocarditis and bacteraemia
• Empyema can be a complication of pneumonia or post-surgery
• Some further clinical infections caused by S. aureus are osteomyelitis and septic arthritis
• It can also cause nosocomial infections because of catheters, intubation and poor hygiene by hospital staff

Other Staphylococci

• S. saprophyticus is known to cause UTI in young women, pyelonephritis, and urethritis and catheter-associated UTI
• S. epidermidis, along with S. aureus, can be associated with prosthetic devices such as joint prostheses, cardiovascular devices but can also cause Meningitis

Characteristics of staphylococci

• It can be gram-positive spherical-shaped cocci
• Growth in clusters of grapes is observed on agar
• Clinical specimens are seen as single cells or short chains
• Size is approximately 0.5 to 1µm

Lab Diagnosis of staphylococci Culture

• It can grow rapidly on non-selective media like Blood agar and MacConkey without crystal violet
• It can can grow under both aerobic and anaerobic conditions
• Colonies are typically smooth, low convex, and glistening
• S. aureus colonies can be golden pink on MacConkey
• Older colonies of S. aureus might appear translucent and sticky
• It gives a zone of β-hemolysis on Blood Agar
• S. aureus can grow in high salt concentrations

Catalase Test

• Catalase is an enzyme that converts hydrogen peroxide into water and oxygen
• Bacteria that contain the enzyme are usually aerobic (need oxygen) or facultative anaerobes
• The catalase test can be used to differentiate between staphylococcus and streptococci

Coagulase Positive

• Staphylococci are classified into two main coagulase groups
• Staphylococcus aureus and Staphylococcus intermedius are the only two staphylococci species that are coagulase positive.
• Coagulase is a protein emzyme produced by microorganisms that converts soluble fibrinogen to fibrin

Coagulase Negative

• Two forms of the coagulase test can be performed, bound (slide) to detect bound coagulase or clumping factor, and free (tube) to detect free coagulase
• The remaining staphylococci species (CNS Group) are coagulase-negative

Identification Plates

• Some identification plates include DNase, where S. aureus produces DNase, an enzyme which denatures DNA
• S. epidermidis and S. saprophyticus do not produce DNase
• The Mannitol Salt Agar helps in gram positive identification of cocci. It's comprised of NaCl, Mannitol and phenol red as pH indicator
• MSA plate is selective and differential
• Selective since it allows only high salt or saline-loving organisms to grow
• Differential since manitol fermenter organisms change the red pH indicator color (neutral) to yellow (acidic)
• Non-mannitol fermenters do not change the color of the media
• MSA allows Staphylococci growth especially for differentiating Staphylococcus aureus from others

Serology Test

• Rapid tests are available for testing purposes on S. aureus
• Virulence factors are the capsule, cell wall and invasins like hyaluronidase, staphylysin, leukocidin, leukotoxin, Coagulase and Staphylokinase
• Cell-bound proteins are also virulence factors
• Toxins include Toxic shock, exfoliation and emesis

Therapy

• All strains of staphylococci should be tested for antibiotic resistance
• Penicillin was the initial drug of choice
• Resistant strains developed which hydrolyse the β-lactam ring of the penicillin and other antibiotics, called Penicillinase (β-lactamases)

Semi-synthetic Penicillin

• Semi-synthetic penicillins, such as methicillin and oxacillin, were then developed and used
• Resistance developed to semi-synthetic drugs, rendering them ineffective
• Resistance genes can be found on a plasmid that can be transmitted to another bacteriu

Plasmids

• A plasmid is a small DNA molecule physically separate from, chromosomal DNA within a cell that can replicate indipendently

Resistance

• Methicillin resistance is indicative of multiple resistance
• Resistance has been reported to Methicillin due to acquisition of the chromosomal mecA gene
• MecA codes for an altered penicillin-binding protein PBP2', so the subunits of the peptidoglycan will bind to altered penicillin-binding proteins
• MRSA, resistant to methicillin, are also resistant to β-lactam antibiotics
• The drug of choice is Vancomycin
• Resistance to vancomycin in E. feacalis has also been reported
• S. aureus is becoming infected with the plasmid containing this vancomycin resistance

MRSA

• MRSA is methicillin-resistant Staphylococcus aureus, therefore resistant to synthetic penicillins
• It is resistant to a synthetic penicillin-methicillin
• It causes many lethal infections in humans
• It can easily transfer resistance to other species directly or indirectly
• It first arose in hospitals, but has since spread and is now referred to as Community-acquired MRSA
• Carriers tend to be health workers of this super bug

MRSA Testing

• Testing for MRSA resistance can be conducted in various ways, for example

1. Disk diffusion methods, testing for oxacillin (synthetic penicillin), cefoxatin and cloxacillin
2. VITEK (Automation)
3. PCR

Inducible Clindamycin Resistance

• Inducible clindamycin resistance can be detected via disk diffusion method using clindamycin and erythromycin disks
• A 15-µg Erythromycin disk is placed in proximity to a 2-µg Clindamycin disk on an agar plate inoculated by a staphylococcal isolate, spaced approx. 15 mm apart
• The plate is then incubated overnight.
• Indicated by a flattening of the zone of inhibition around the Clindamycin disk shaping in a "D"
• This means that the Erythromycin induced Clindamycin resistance
• Laboratories should provide tests to help results for Clindamycin to be reported as susceptable or resistant

Mechanism of Action

• Erythromycin (a macrolide) and clindamycin (a lincosamide) can inhibit protein synthesis by binding to bacterial cells' 50S ribosomal subunits
• In staphylococci, resistance to both antimicrobial agents can occur through methylation of their ribosomal target sit
• Such resistance is typically mediated by erm

Clinical Significance

• Three main erm (erythromycin ribosome methylation) genes, erm(A), erm(B) and erm(C), have been described in staphylococci
• The erm(A) gene is located on transposon Tn554, which has insertion sites in the Staphylococcus aureus chromosome
• This resistance can be constitutive, where rRNA methylase is always produced, determining S. aureus isolates, or erythromycin/clindamycin resistance when in vitro testing is used
• Can be be produced conditionally, where methylase is produced only with an inducing agent and routine invitro
• Isolates become resistant to Erythromycin but appear susceptible to Clindamycin

Inducible Clindamycin Testing Procedure

• Prepare 0.5 McFarland standard suspension of Erythromycin resistant S. aureus isolates
• Transfer this culture to Muller Hinton Agar (MHA) plates
• Place Clindamycin (2-µg) and Erythromycin (15-µg) disks approximately 15 mm apart (measured edge to edge)
• Lastly incubate the plate for 16 to 18 hours at 37C
• So a summary of lab testing to ID Staph Aureus involves identifying gram-positive cocci, doing a catalase test, identification plates and finally the MH sensitivities testing for MRSA or D-test

Staph saprophyticus

• Known to be an important cause of UTIs, such as pyelonephritis and cystitis, especially in sexually active young women
• Also causes catheter-associated urinary tract infections in men and women
• Virulent factors adhere to the epithelial cells in the urinary tract and to the urethral cells

Staph saprophyticus LAB ID

• Coagulase negative
• DNase negative
• Non-hemolytic on blood agar
• Fermentation of mannitol is variable
• Resistant to novobiocin
• Test to differentiate coagulase-negative staphylococci