FDA Bacteriological Analytical Manual (FDA’s Bacteriological Analytical Manual (BAM) presents the agency’s preferred laboratory procedures for microbiological analyses of foods and cosmetics.)
Introduction:
Staphytlococcus aureus (S. aureus) is the most virulent Staphylococcus species and causes a variety of infections in humans, both localized and systemic. Most commonly, S. aureus causes a localized skin infection after a skin break or wound. Another common site of entry is the respiratory system, and Staphylococcal pneumonia is a frequent complication of influenza. More serious illnesses may result when S. aureus enters the blood stream including osteomyelitis, endocarditis, sepsis and meninitis, among others. S. aurues is a major cause of nosomial (hosptial aqcquired) infections.
S. aureus produces several potential virulence factors such as alpha, beta, gamma and delta toxins, toxic shock syndrome toxin, enterotoxins, leucocidin, proteases, coagulase and clumping factor. The emergence of antibiotic-resistant forms of S. aureus (e.g., MRSA) is a major problem.
The surface of most strains is coated with protein A that binds the Fc region of IgG. Teichoic acids are bound to both peptidoglycan layer and cytoplasmic membrane.
Methicillin-resistant Staphylococcus aureus (MRSA):
Community-associated (CA) methicillin-resistant S aureus (MRSA) infections in the United States, epidemiologically defined as those occurring in individuals with no recent healthcare exposures, have been most often reported in younger, healthy individuals. Healthcare-associated (HA) MRSA infections, in contrast, are more likely to be diagnosed in older patients with comorbid conditions and are often invasive infections, such as BSIs or pneumonia.
Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of skin lesions in non-hospitalized people. (the hospitalized population is more likely to acquire systemic, bloodstream infections from MRSA. In the past, most of the infections caused by MRSA were confined to elderly patients in healthcare facilities and they were described as healthcare-associated MRSA (HA-MRSA). However, the epidemiology of MRSA changed overtime with the emergence of strains in patients with no previous history of hospitalization which were known as community-associated MRSA (CA-MRSA). CA-MRSA strains are more genetically diverse and belong to different clones than HA-MRSA. (see Udo, “Methicillin-resistant Staphylococcus aureus: An update on the epidemiology, treatment options and infection control”).
Staphylococcal scalded skin syndrome (SSSS)
Staphylococcal scalded skin syndrome (SSSS), also known as Ritter disease, is a potentially life-threatening infection caused by certain strains of Staphylococcus aureus (S. aureus) that release exfoliative toxins. Clinically, it is characterized by denudation of the skin and presents as large superficial blisters. The overall incidence of SSSS in the general population is estimated to be between 0.09 and 0.56 cases per one million people. However, SSSS is most commonly seen in children under the age of six. A study in the Czech Republic reported an incidence as high as 250 per one million children less than one year old. The increased incidence in young children is due to a lack of protective antibodies against exfoliative toxins and decreased renal clearance of the toxins as a result of immature renal function. The mortality rate of SSSS is less than five percent in children and greater than sixty percent in adults, likely due to an underlying immunodeficiency or comorbidity. See Brazel
S. aureus is a Gram-positive bacterium that frequently colonizes the eyes, ears, nares, umbilicus, and groin. All strains of S. aureus produce toxins, but only five percent of them release the exfoliative toxins A and B (ETA, ETB) that cause SSSS.
Particular Strains:
To date, 45 staphylococcal species and 24 subspecies have been described, using molecular methods. But how can S. aureus be distinguished from other staphylococcal species? Due to its high pathogenic potential, pathogenicity factors have primarily been used for differentiation. As early as 1903, Loeb categorized the genus Staphylococcus into two groups: coagulase-positive and coagulase-negative (CoNS) staphylococci. The coagulase test is based on the ability to produce free and/or cell wall-bound coagulase (clumping factor). While this classification remains relevant today, it is not sufficient to clearly identify S. aureus, as other staphylococcal species can also produce coagulase, such as Staphylococcus intermedius, Staphylococcus schleiferi subsp. coagulans, Staphylococcus hyicus, Staphylococcus lutrae, Staphylococcus delphini, and Staphylococcus pseudintermedius. However, these species are primarily associated with animals. For unequivocal identification of S. aureus, 16S rDNA sequencing and coagulase tests are required. (Peschel, “Staphylococcus aureus: a model for bacterial cell biology and pathogenesis” Bacteriology, 207(8), 2025)
USA300: Since at least 2004, the most often isolated MRSA strains in the United States have belonged to clonal complex (CC) 8 and CC5. USA300, which belongs to multilocus (ML) sequence type (ST) 8 (included in CC8), was initially associated with CA-MRSA infections, but since 2005 it has increasingly been recognized also as the cause of nosocomial infections. USA300 has become the most common MRSA strain circulating in the United States. Reports from the last 15 years indicate that the majority of MRSA SSTIs in the United States have their onset in the community and are caused by USA300.
USA500 is a closely related CC8 strain type that is easily distinguished from USA300 by wholegenome sequencing.
USA100 (usually ST5, which belongs to CC5) is most often a HA-MRSA strain. However, it has also been uncommonly isolated from epidemiologically defined CA-MRSA infections and from nasal carriage in individuals with no prior healthcare exposures.
Biochemical, Morphological and Structural Characteristics
At the species level, the genetic diversity of S. aureus is relatively high. Evolution within the host is driven by genetic variation within its small genome (2.8–3.2 Mbp), which encodes 2,500–3,000 proteins and is carried on a single chromosome associated with one or more plasmids (24–27). The stable component (core genome) is complemented by a set of accessory genes (accessory genome) that can mediate antibiotic resistance, virulence, or immune evasion mechanisms and are often carried on mobile genetic elements. Antibiotic resistance genes are generally found on plasmids, transposons, or the staphylococcal cassette chromosome, whereas phages and pathogenicity islands carry virulence and immune evasion determinants. The genetic diversity of S. aureus is also important in the laboratory setting, as different strains can yield varying results in the same assay or different phenotypes for the same mutation. This variability can lead to a lack of reproducibility between laboratories using different strains. Table 1 summarizes the most relevant S. aureus strains used in research, including their origins and main characteristics. (Peschel, “Staphylococcus aureus: a model for bacterial cell biology and pathogenesis” Bacteriology, 207(8), 2025)
S. Aureus are alpha hemolytic and destroy red blood cells. They are facultative anaerobic, coccal bacteria. They appear as grape-like clusters (because they reproduce asexually by binary fission, and the two daughter cells remain attached) and have large, golden-yellow colonies, often with hemolysis when grown on blood agar plates.
Protein A of Staphylococcus aureus (“SpA): refers to a 42 kda multi-domain protein isolated from S. aureus. SpA is bound to the bacterial cell wall via its carboxy-terminal cell wall binding region, referred to as the X domain. At the amino-terminal region, it includes 5 immunoglobulin binding domains, referred to as E, D,A,B and C. Each of these domains contains about 58 amino acid residues and they share 675-90% amino acid sequence identity. SpA is widely used for antibody purification due to its high affinity for immunoglobulins. For more on SpA as an affinity ligand for purification of antibodies see “Protein Purification”.
Pathogenesis
Adhesion Molecules:
The surface of Staphylococcus aureus is ‘decorated’ with proteins that are covalently anchored
to the cell wall peptidoglycan. Structural and functional analysis has identified four distinct classes of surface proteins, of which microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) are the largest class. These surface proteins have numerous functions, including adhesion to and invasion of host cells and tissues, evasion of immune responses and biofilm formation. Thus, cell wall-anchored proteins are essential virulence factors for the survival of S. aureus in the commensal state and during invasive infections, and targeting them with vaccines could combat S. aureus infections. See Hook
S. aureus owes much of its pathogenic process to proteins such as Surface protein A (SpA) anchored to its cell well. These adhesins facilitate evasion of the host immune response and host colonisation by binding to plasma proteins and host endothelial cells. SpA is anchored to the bacterial cell surface via an LPXTG motif, which is typical of microbial surface components that recognize adhsive matrix molecules. The binding activity of SpA acts to cloak the bacerial cell with IgG, thus blocking any interaction with Fc receptors on nuetrophils and hingering phagocytosis (Atkins, Molecular Immunology 45, 2008, 1600-1611).
S. aureus causes disease by either production of toxin or direct invasion and destruction of tissue. Some toxins produced include exfoliative toxin which causes staphylococcal scalded skin syndrome (SSSS), toxic shock syndrome (TSS) and enterotoxins which are heat resistant toxins that cause food poisoning.
Coagulases also contribute to S. aureus virulence by assisting in colonisation and evasion of the immune system rather than by targeting host cells
Staphylococcal food poisoning (SFP), caused by staphylococcal enterotoxins, is a global common foodborne illness among various types of foodborne diseases (FBD). In the United States alone, nearly 0.24 million cases of SFP are reported every year. (See Mothadica “Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance”)
Inhibition of Neutrophil Migration: S. aureus has evolved an arsenal of factors to evade innate immunity. Neutrophils represent the most abundant polymorphonuclear leukocyte population in blood and are a crucial defense against S. aureus. The pathogen, in turn, targets all stages of neutrophil recruitment as well as neutrophil effector functions. Staphylococci keep neutrophils from migrating toward the site of infection by impeding chemotaxis via the secretion of staphylococcal superantigen-like 5 and 10 (SSL5 and SSL10); chemotaxis inhibitory protein of S. aureus (CHIPS); and its homologs, formyl peptide receptor-like inhibitory proteins (FLIPr and FLIPr-like). Further, SSL5 prevents neutrophils from rolling on endothelial cells, a crucial step in their recruitment process. This interference is aided by staphylococcal secretion of extracellular adherence protein, which blocks adherence of neutrophil ligands to cognate endothelial adhesion receptors. Interestingly, S. aureus can also stimulate neutrophil chemotaxis via the secretion of formylated PSMs. See Missiakas
Biofilm Formation: Biofilm production contributes to the persistence of MRSA infections, as strains exhibiting enhanced virulence thrive within these protective structures. Almost 60% of all infections in vivo conditions are believed to be casued by bacteria embedded in biofilms. Biofilm formation requires a suitable substrate. Coloniation occurs more frequently on rough surfaces becasue of the higher surface area and favorable physicochemical properites. This means that implanted medical device materials can be easy targets. Biofilm fomration takes place in multiple stages, including attachment, formation/maturation and dispersal. The initial attachment of a bacterium to biotic (endovasclar, bone or joint) or abiotic (prosthetic device/catheter) surfaces involves a number of proteins known as Microbial Surface Components Recognizing Adhsive Matric Molecuels (MSCRAMn) The main MSCRAM involved in S. aureus adhesion are cluping factor A (ClfA) and clumping factor B (ClfB). These clumping factor prtoeins guide intial attachment by binding to fibrinogen. Other MSCRAMs include bone sialo-binding protein (Bbp) which attaches to the extracellular matrix and collagen adhesin (Can) which binds collagen. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Biofilm-associated MRSA can cause a broad range of infections ranging from SSTIs to deep seated sinfections include blood stream infections, osteomyelitis, and infective endocarditis. Microorganisms in biofilms have the ability to enter a dormant state for extended periods, ranging from weeks to years. This latent condition can persist until conducive conditions arise, leading to the manifestation of localized or systemic indications and simptoms of infection. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Entry/Colonisation
Adhesion to tissues is required for bacterial colonisation to occur. For this purpose, S aureus express surface adhesins which interact with host matrix proteins such as fibronectin, collagen, etc. In addition, Staphylococci are able to bind several serum proteins, such as IgG, possibly masking the bacteria from the immune system of the host.
The most studied receptor in S aureus is protein A, a cell wall associated protein, which binds to the Fc and the Fab regions of IgG from several species. The fact that protein A binds so well has been taken advantage of for the purification of IgG (see “biotechnology”, “protein purification” and “affinity A” under “chromatography”).
Transmission and Symptoms:
Methicillin-resistant S. aureus is a common contaminant of all kinds of surfaces one touches such as gym equipment, airplane tray tables, electornic devices, razors.
MRSA infections of the skin tend to be raised, red, tender, localized lesions, often featuring pus and feeling hot to touch. They occur easily in breaks in the skin caused by injury, shaving or even just abrasion. They may localize around a hair follicle. Fever is a common feature.
Diagnosis:
PCR is routinely used to diagnose MRSA. Alternatively, cultivation on blood agar is a useful technique.
DNA Next Generation Sequencing: Isolates from the HUP Clinical Microbiology Laboratory were stored prospectively at −80°C. To prepare DNA for sequencing, frozen cultures were streaked on blood agar plates and incubated overnight at 37°C. Single isolates were passaged onto fresh blood agar plates before single colonies were isolated for sequencing. Isolates were sequenced at the Penn/ Children’s Hospital of Philadelphia (CHOP) Microbiome Center. Sequence libraries were prepared using the Illumina Nextera kit and sequenced by Illumina Hi-seq. See David
Selective media such as mannitol salt agar are used. The production of catalase, an enzyme that breaks down hydrogen peroxide accumulated during oxidative metabolism can be used to differentiate the staphylococci, which product it from the streptococci which do not.
One key technique for separating S. aureus from other species of Staphylococcus is the coagulase test. By definition, any staph isolate that coagulates plasma is S. aureus. All others are coagulase negative.
Keep in mind that an increasing number of dangerous skin infections are being caused by gram negative pathogen, Vibrio vulnificus. A gram stain of the would guide you to the right direction.
Case Study: A 20-year-old young male medical student presented to the dermatological outpatient department (OPD) with complaints of erythematous skin rash spread throughout the body. The rashes were observed on the hands, legs, and on the trunk. The patient gave a history of scuba diving three days before he started to notice these skin lesions. The patient was found to be very anxious but otherwise was clinically normal. The patient was referred to the department of clinical microbiology for further microbiological evaluation. The skin rash was isolated but present in many areas of the body including the hands and legs. Most skin lesions coincided with the cuts. The palms and soles showed no signs of rash. There were multiple areas of erythematous skin, with raised and white fluid filled bumps. The area surrounding the affected skin was thoroughly cleansed with 70% alcohol. A sterile scalpel blade was then used to scrape the affected area. The sample was then inoculated into a blood agar, and nutrient agar and incubated at 370 C. The sample was also smeared on a clean and grease free slide. On the grams stain, occasional gram-positive cocci in clusters were seen. Microscopy (KOH mount) for fungal elements was negative. After overnight incubation, a moderate growth of 2-3 mm small, round, moist, convex, and opaque beta-hemolytic colonies were observed. The colonies showed yellow coloured non-diffusible pigment on the nutrient agar. Grams stain from the culture confirmed the presence of gram-positive cocci in clusters. The cultured bacterium was confirmed as Staphylococcus aureus by a positive coagulase test, and fermentation of mannitol. See Kandi
Treatment
S aureus are usually treatment with penicillins or cephalosporins. Methicillin resistant S. aureus are resistant to all beta-lactams, however. Only vancomycin is effective.(a characteristic that can be used to distinguish them from S. epidermidis which are gamma hemolytic).
When penicillin, a β-lactam that targets PBPs, became available in the 1940s, it was
particularly helpful for the treatment of S. aureus infections. However, S. aureus was one
of the first pathogens to develop penicillin resistance. The first reported S. aureus penicillin resistance mechanism was based on the extracellular penicillin-inactivating penicillinase. Penicillinase is usually encoded on plasmids, which are exchanged among pathogen clones, for instance, by transducing phages, thereby contributing to the fast dissemination of resistance. When penicillinase-insensitive β-lactams became available, the emergence of MRSA
led to the discovery of the alternative PBP2A, which confers broad β-lactam resistance.
Vancomycin: The most frequently used antibiotic for the treatment of MRSA infections is the
glycopeptide vancomycin, which inhibits peptidoglycan synthesis by binding to the terminal D-Ala-D-Ala amino acids in the stem peptide of the peptidoglycan precursor, thereby preventing the access of PBPs to their substrate. Vancomycin-intermediate-
esistant S. aureus (VISA) represents a problem of increasing concern because the VISA
phenotype is difficult to diagnose, and there are only very limited alternative therapy options available.
Daptomycin is a cyclic peptide antibiotic. It has been approved for use against S. aureus and is considered a mainstay of anti-MRSA therapy. aptomycin’s mode of action requires calcium. Daptomycin and calcium form a complex that behaves as a cationic peptide and oligomerises to form micelles. These micelles then penetrate the cell wall and insert into the lipid membrane by binding to phosphatidylglycerol. The micelles thereby disrupt the membrane causing depolarisation, permeabilisation and ion leakage. An MIC breakpoint that determines resistance has not been determined yet for daptomycin in S. aureus but a level for susceptibility has been set. An MIC of <1 mg/L is considered susceptible for bacteria and an MIC ≥1 mg/L is considered non-susceptible. See Mudgill
MRSA Biofilm Formation: Suggested approaches for management of S. aureus biofilm infections include treatment by antimicrobials alone/in combination along with surgical debridement or device removal, inhibiting biofilm formation by limiting bacterial attachment to medical devices with surface modification, application of newer anti-biofilm agents, or novel technologies like laser Shock waves. Antibiotic represent the cornerstone of therapy. Debriding and draining any soft tissue abscesses associated with the infection is recommended. Anti-MRSA antimicrobials include vancomysin, daptomycin, linezolid, trimethoprim-sulfamethoxazole, clindamycin, rifampin, long acting agents like dalbavancin, oritavancin, and telavancin and tetracylines such as doxycycline, minocyline, and tigecyline and some other novel antimicrobials like omadacycline (a tetracycline), lefamulin and delafloxacin. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024).
Chelators and Sulfydryl Compounds:
Cations (e.g., Mg2%, Fe2+, ca2+) play a crucial role in bacterial growth by promoting inter-bacterial inferactions and aggregation and are thought to be important for microbial adherence and biofilm formation. By sequestering these ions, high affinity emtal ion chelators such as ethylenediamine tetra acetic acid (EDTA), ethylene glycol tera acetic acid (EGTA) and tri-soium citrate (TSC) inhibit biofilm formation as well as bacterial adhesion to surfaces, thus showing useful antibacterial properties in vitro. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Nanoparticles:
The application of nanotechnology to the field of biofilm inhibition is a novel area of research. Nanoparticles (e.g., gold, silver, iron, copper and selenium) and nanomaterials have shwon enhanced fiofilm matrix penetration compared to free drug moelcules, interfering with biofilm formaiton and bacterial adhesion. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Antimicrobial Peptides (AMPs):
AMPs are peptides that are positively charged, amphipathic in nature and composed of fewer than 50 amino acids. More than 5000 AMPs have been described. AMPs can bind to and sirupt bacterail membranes and some possess anti-biofilm activity against S. aureus. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathogens (2024)
Phytochemicals:
Plant derived compounds, including different phytochemicals (e.g., tannic acid, ellagic acid, xanthohumol), several polypehnolic compounds, and favonoids are thought to act by affecting bacterial quorum sensing activity, thus interfering in the cell-cell interactions and in biofilm formation. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Stapahylococcal Phages:
A pahage can infect MRSA strains in both biofilm and planktonic phases. It was shwon that a triple combination of bacteriophae (Sb-1 phage), ceftaroline, and daptomycin effectively reduced S. aureus populations. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Antibodies/Vaccine Candidates:
Vaccine/antibody development for S. aureus has been challening, given the complix nature of staphylococcal infections and the production of a multitude of virulence factors. Although attempts are being made to use capsular polysaccharide (type 5 and 8), clumping factors A and B, fibronectin binding prtoein, adenosine triphosphate binding cassette transporter, and amidase as potential vacine candidates, and clumping factor A, adenosine triphosphate binding cassette transporter, and teichoic acids as therapeutic antibodies to preent and treat S. aureus infections, these are largely experimental. (Gupta, “Biofilm producing methicillin-resistant Staphylococcus auerus (MRSA) infections in humans: clinical implicaitons and management” Pathgoens (2024)
Prevention/Sterilization:
Introduction: S. aureus biofilm development is associated with four broad phases, namely attachment or adherence, proliferation (microcolony formation), maturation and dispersal. However, the mechanism of the first phase may depend on whether S. aureus attaches to an abiotic or biotic surface. Whereas attachment to abiotic surfaces such as glass, metals (Co-Cr, 316SS, titanium, etc.) and plastics (polyester, silicone, polyethylene, etc.) can be nonspecific, S. aureus adherence to biotic surfaces depends on bacterial MSCRAMM (the largest class of surface proteins anchored to cell wall peptidoglycan) recognition of host proteins. Thus, abiotic attachment is facilitated by Van der Waal’s forces, electrostatic and steric interactions.
Surface-anchored proteins play an obvious role in adherence to and invasion of host cells and tissues, as well as in biofilm formation, and therefore, these proteins represent critical factors that facilitate S. aureus colonization and survival during infection. Surface proteins that play a specific role in biofilm formation include Bap, clumping factors (ClfB), FnBPs, surface proteins SasC, SasG and protein A. ClfB, FnBPs and protein A are widely distributed
bacteriostatic and bactericidal coatings: Approaches currently being evaluated involve the development of bacteriostatic and bactericidal coatings in addition to engineering surfaces to prevent attachment of bacteria. For example, silver nanoparticle-coated catheters are being evaluated for use in preventing S. aureus attachment. Although in vitro studies have shown potential, there are concerns about the cytotoxicity of silver to host tissue due to accelerated thrombin formation and platelet activation, putting patients at higher risk for thrombosis. Nevertheless, these surfaces are now being tested for better host compatibility, making this approach promising for clinical use in the coming decade. Similarly, titanium, stainless steel and other commonly used implant materials are being coated with antibiotics such as vancomycin to prevent growth of S. aureus on these surfaces.
Another approach to reduce bacterial adhesion to abiotic surfaces is the development of materials that retard adhesion used in combination with the administration of antibiotics or antimicrobials. This dual strategy aims to prevent planktonic bacteria from easily attaching to the implant surface while allowing killing of this antibiotic susceptible population. The first strategy can be accomplished either by changing the surface physical properties (such as hydrophobicity/hydrophilicity, texture, charge and roughness) such that bacteria are no longer able to easily attach. A second strategy that facilitates the attachment of host cells to the implant has best been described by Gristina et al.as a “race for the surface” whereby the risk of developing infections on a surface can potentially be lowered by allowing host cells to competitively occupy it before bacteria are able to do so.