Staphylococcus: General Characteristics & Laboratory Diagnosis

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Gram-positive cocci (staphylococcus) are frequently isolated in clinical microbiology laboratories. Although the majority of gram-positive cocci are members of the indigenous microbiota, some species are pathogens. In this article, we’re going to discuss the most common staphylococci, their characteristics, the infections they cause, and how to identify them in the lab. Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus lugdunensis, and Staphylococcus haemolyticus infections are highlighted.

General Characteristics of Staphylococcus

  • Staphylococcus is derived from the Greek term staphle, which means “bunches of grapes.”
  • Staphylococci are members of the Staphylococcaceae family.
  • Staphylococci are gram-positive, catalase-positive cocci.
  • They have spherical cells (0.5 to 1.5 m) that appear singly, in pairs, and in clusters on stained smears.
  • Staphylococci are nonmotile, nonspore-forming, aerobic or facultatively anaerobic, with the exception of a few strains that are obligate anaerobes.
  • Although the Gram stain can distinguish staphylococci from other gram-positive cocci, microscopy alone cannot distinguish staphylococci.
  • Colonies formed after 18 to 24 hours of incubation are medium in size (4 to 8 mm), cream-colored, white, or light gold in color, and “buttery-looking.”

Rare staphylococci strains are fastidious, requiring carbon dioxide, hemin, or menadione to grow. Even after 48 hours or more of incubation, these so-called small colony variants (SCVs) grow on blood-containing media, forming colonies one-tenth the size of wild-type strains.

  • Some species have β-hemolytic enzymes. Staphylococci are common clinical laboratory isolates and are responsible for a variety of suppurative infections. These organisms are common inhabitants of human and animal skin and mucous membranes.

The staphylococci are related to some members of the Micrococcaceae family, such as the genus Micrococcus. Micrococci are gram-positive cocci that produce catalase and are coagulase-negative. They are found in the environment and as members of the indigenous skin microbiota. They are frequently recovered with staphylococci and can be easily distinguished from coagulase-negative staphylococci (CoNS) by the characteristics listed in the table below. Some micrococci have a proclivity for producing a yellow pigment. Other gram-positive cocci found in human clinical specimens include Rothia mucilaginosa, Aerococcus, and Alloiococcus otitis (recovered from human middle ear fluid).

TestStaphylococciMicrococci
Modified oxidasea (a)+
Anaerobic acid production from glucose+-(b)
Growth on Furoxone–Tween 80–oil red O agar+
Anaerobic acid production from glycerol in the presence of erythromycin+
Resistance to bacitracin (0.04 units)R(c)S
Lysosome (50-mg disk)RS
Lysostaphin testS(c)R
R, Resistant; S, sensitive. (a) Commercially available, useful for presumptive identification. (b) Micrococcus kristinae and Micrococcus varians are positive. (c) Some stains show opposite reaction.
Micrococci growing on sheep blood agar showing
yellow pigment.
Micrococci growing on sheep blood agar showing
yellow pigment.
  • The coagulase test can be used to differentiate staphylococcal species; a positive test result is a clot formed in a tube containing plasma due to staphylocoagulase.
  • Staphylococci that produce staphylocoagulase (coagulase) include S. aureus, S. intermedius, S. pseudintermedius, S. hyicus, S. delphini, S. lutrae, S. agnetis, and some strains of S. schleiferi.
  • Because of the presence of clumping factor, isolates such as S. lugdunensis and S. schleiferi are occasionally misidentified as coagulase-positive staphylococci.

Clumping factor causes bacterial cells to agglutinate in plasma and was the inspiration for the slide coagulase test. This is a defunct test; only the tube coagulase test should be used for definitive testing.  The tube coagulase test will identify the majority of clinical staphylococcal isolates as S. aureus.

Other staphylococcal species that can be positive with the traditional coagulase test are frequently animal-associated and are less frequently isolated. A review of the patient’s history and antimicrobial susceptibility pattern can aid in distinguishing these less commonly isolated staphylococci species from S. aureus.

In many laboratories, the use of manual and automated commercial systems for identification has become standard practice. On consultation with the attending physician, unusual isolates may necessitate referral to reference laboratories.

The most clinically significant species is Staphylococcus aureus. It causes a variety of cutaneous infections as well as purulent abscesses. These infections of the skin and soft tissues can be superficial, such as impetigo or cellulitis. Cutaneous infections can develop into deeper abscesses, such as carbuncles, and involve other organ systems, resulting in bacteremia and septicemia. S. aureus is a common cause of infective endocarditis and toxin-induced diseases like food poisoning, and it has been linked to scalded skin syndrome (SSS) and toxic shock syndrome (TSS) (TSS).

Staphylococci that do not produce coagulase are known as coagulase-negative staphylococci (CoNS). The most clinically significant species in this group are S. epidermidis and S. saprophyticus. S. lugdunensis and S. haemolyticus are both present. S. epidermidis has been linked to a number of hospital-acquired or nosocomial infections, whereas S. saprophyticus has been linked to urinary tract infections (UTIs), primarily in adolescent girls and young women.

S. haemolyticus is occasionally recovered from wounds, septicemia, urinary tract infections (UTIs), and native valve infections. Because S. lugdunensis, like S. aureus, is slide coagulase positive, additional testing, including the tube coagulase test, is required for differentiation. S. lugdunensis has been linked to catheter-related bacteremia and endocarditis due to its aggressive ability to infect. Because of reporting criteria, S. lugdunensis should be identified to the species level when reporting antimicrobial susceptibilities in order to provide the appropriate treatment options. There are currently over 40 recognized CoNS species and subspecies. Those species isolated from humans are typically associated with skin and mucous membranes; however, some are found in very specific locations, such as the head (S. capitis) or ear (S. auris) (S. auricularis). Some are only found in animals, but others have been linked to human disease.

Clinically Significant Staphylococcus Species

Staphylococcus aureus

S. aureus is responsible for numerous infections, ranging from relatively mild to life-threatening. Infections can be categorized as suppurative or toxin-mediated disease. S. aureus can be recovered from almost any clinical specimen and is an important cause of nosocomial infections. S. aureus continues to increase in importance as a community-acquired pathogen, and increasing drug resistance is a concern with this common isolate.

Virulence Factors  of Staphylococcus aureus                        

S. aureus pathogenicity can be attributed to a variety of virulence factors, including enterotoxins, cytolytic toxins, and cellular components such as protein A. Several cytolytic and exfoliative toxins have been discovered. Despite these virulence factors, S. aureus has a high level of innate resistance and is classified as an opportunistic pathogen.

  • Enterotoxins

Enterotoxins produced by Staphylococcus aureus are heat-stable exotoxins that cause symptoms such as diarrhea and vomiting. There have been numerous serologically distinct enterotoxins identified, with the majority falling into groups A through E and G through J. Thirty to fifty percent of S. aureus isolates produce these toxins. Reheating contaminated food does not prevent disease because the enterotoxins are stable at 100° C for 30 minutes. Enterotoxins A, B, and D are the most common causes of staphylococcal food poisoning. TSS is linked to enterotoxins B and C, as well as G and I on occasion. Staphylococcal pseudomembranous enterocolitis has been linked to enterotoxin B. These toxins, along with toxic shock syndrome toxin-1 (TSST-1), are superantigens that can interact with a large number of T cells, triggering an aggressive, overreactive immune response.

  • Toxic Shock Syndrome Toxin-1

TSST-1, formerly known as enterotoxin F, is a chromosomal-mediated toxin that causes the majority of menstruating-associated TSS cases and approximately half of nonmenstruating cases. TSST-1 is a superantigen that stimulates T-cell proliferation and, as a result, the production of a large number of cytokines that cause the symptoms. TSST-1 causes leakage by endothelial cells at low concentrations and is cytotoxic to these cells at higher concentrations. TSST-1 is absorbed through the vaginal mucosa, resulting in the systemic effects seen in tampon-associated TSS.

  • Exfoliative Toxin

Epidermolytic toxin is another name for exfoliative toxin. Toxins are classified into two types: exfoliative toxin A and exfoliative toxin B. They cause the epidermal layer of the skin to slough off and have been linked to staphylococcal SSS, also known as Ritter disease. Ritter disease affects macrophages and causes extensive tissue damage. β-Hemolysin (sphingomyelinase C) is a lysin that acts on sphingomyelin in the plasma membrane of erythrocytes. It is also known as the “hot-cold” lysin. This toxin’s “hot-cold” property is characterized by increased hemolytic activity after incubation at 37° C and subsequent exposure to cold (4° C). This hemolysin is detected in the Christie, Atkins, and Munch-Petersen (CAMP) test, which is used to identify group B streptococci in the laboratory. δ-Hemolysin is less toxic to cells than α-hemolysin or β-hemolysin, despite being found in a higher percentage of S. aureus strains and some CoNS. γ-Hemolysin is only found in conjunction with Panton-Valentine leukocidin (PVL).

PVL is a toxic exotoxin that kills polymorphonuclear leukocytes. It has been linked to severe cutaneous infections and necrotizing pneumonia, as well as contributing to the organism’s invasiveness by suppressing phagocytosis. Although produced by a small number of S. aureus strains, it is frequently associated with community-acquired staphylococcal infections and may be a marker for such infections.

  • Protein A.

Protein A is one of several cellular components found in the cell wall of S. aureus. Protein A’s ability to bind the Fc portion of immunoglobulin G is likely its most important role in S. aureus infections (IgG). Binding IgG in this manner can inhibit phagocytosis and negate IgG’s protective effects.

α  HemolysinsDamages erythrocytes, platelets, and macrophages
β  Hemolysins                                            Also known as sphingomyelinase C, disrupts the erythrocyte plasma membranes. Responsible for CAMP assay effectiveness
δ HemolysinsFound in some CoNS strains as well as S. aureus. Less toxic than other hemolysins
γ HemolysinsAssociated with Panton-Valentine leukocidin
Panton-Valentine leukocidinPolymorphonuclear leukocyte toxicity
β-LactamaseEnzyme that cleaves the ring structure of penicillins and derivative antibiotics making them ineffective
Penicillin-binding protein 2Altered membrane binding protein
HyaluronidasePermits bacteria to spread through connective tissues
LipasesCommon to S. aureus and CoNS. Degrades lipids on skin surface making it more susceptible to bacterial entry into epidermal layers
StaphylocoagulaseResponsible for a positive tube coagulase test result. Also present in S. intermedius, S. pseudintermedius, S.hycius, S. delphini, S. lutrae, S agnetis , and some S. schleiferi
Toxic shock syndrome toxin-1A superantigen causing an overreactive immune response. Formerly known as enterotoxin F
Protein ABinds IgG and prevents phagocytosis
Enterotoxins A–E, G, JEnterotoxins A, B, and D are the cause of the majority of staphylococcal food poisoning cases; heat stable. Enterotoxins B and C, and rarely G and I, can cause enterocolitis
Exfoliative toxinsAlso known as epidermolytic toxin. Types A and B. Solely responsible for SSS and present in a minority of S. aureus species. May also cause bullous impetigo
Virulence Factors of Staphylococcus aureus
Key: CAMP, Christie, Atkins, and Munch-Petersen; CoNS, coagulase-negative staphylococcal; IgG, immunoglobulin G; SSS, scalded skin syndrome.

Epidemiology of Staphylococcus aureus

The human nares is the primary reservoir for staphylococci, with colonization also occurring in the vagina, pharynx, axillae, and other skin surfaces. Nasal carriage is common in hospitalized patients. Because contact between patients and hospital personnel is common, organism transfer is common. As a result, increased colonization in patients and hospital workers is common. Hospital outbreaks can occur in patients in nurseries and burn units, as well as in those who have had surgery or other invasive procedures. S. aureus transmission can occur through direct contact with unwashed, contaminated hands as well as through contact with inanimate objects (fomites). Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in both health care and the community are a major health care concern. Decolonization protocols have been implemented to reduce colonization in specific populations, such as intensive care unit patients.

Infections Caused by Staphylococcus aureus

The development of staphylococcal infection, like most infections, is determined by the virulence of the strain, the size of the infectious do, and the state of the host’s immune system. Infections begin when staphylococci gain access to adjacent tissues or the bloodstream through a breach in the skin or mucosal barrier. Any event that weakens the host’s ability to fight infection promotes colonization and infection. Individuals with normal defense mechanisms are better able to combat the infection than those with weakened immune systems. Once the organism has passed through the initial barriers, it triggers the host’s acute inflammatory response, which causes polymorphonuclear cells to proliferate and become activated. However, organisms can resist the action of inflammatory cells by producing toxins and enzymes, resulting in the formation of a focal lesion.

  1. Skin and Wound Infections.

S. aureus infections are suppurative in nature. The abscess is typically filled with pus and surrounded by necrotic tissues and damaged leukocytes. Folliculitis, furuncles, and bullous impetigo are some of the more common, less serious skin infections caused by S. aureus. These opportunistic infections are typically caused by previous skin injuries such as cuts, burns, and surgical incisions. Folliculitis is a mild inflammation of a hair follicle or oil gland that causes the infected area to be raised and red. Furuncles (boils) are large, raised, superficial abscesses that can develop as a result of folliculitis.

Carbuncles form when multiple furuncles combine to form larger, more invasive lesions that can spread into deeper tissues. In contrast to patients with furuncles, patients with carbuncles frequently present with fever and chills, indicating that the bacteria has spread throughout the body. Staphylococcal pustules are larger and surrounded by a small zone of erythema, distinguishing it from streptococcal nonbullous impetigo, also known as impetigo contagiosa. Nonbullous impetigo is a highly contagious infection that can be transmitted through direct contact, fomites, or autoinoculation.

Staphylococcal infections can also occur as a result of skin diseases caused by a variety of factors. Infection thrives on dry, irritated skin, which is exacerbated by poor personal hygiene. Some of these infections manifest as a result of increased organism colonization in blocked hair follicles, sebaceous glands, and sweat glands. These superficial infections are sometimes misdiagnosed as insect or spider bites. Immunocompromised people, particularly those undergoing chemotherapy, suffering from chronic illnesses, or having invasive devices implanted, are predisposed to staphylococcal infections. Infections of the skin and soft tissues can progress to the point of being fatal.

  1. Scalded Skin Syndrome.

SSS is a bullous exfoliative dermatitis that primarily affects newborns and otherwise healthy young children. This syndrome is caused by a staphylococcal exfoliative or epidermolytic toxin produced by Staphylococcus aureus, which is most likely present at a lesion far from the site of exfoliation. Adults have also been diagnosed with the disease. Adults with chronic renal failure and patients with compromised immune systems are the most likely to develop SSS. Although the mortality rate in cases seen in children is low (0 to 7%), the rate in adults can be as high as 50%.

The disease’s severity ranges from a localized skin lesion in the form of a few blisters, known as pemphigus neonatorum, to a more widespread generalized condition affecting 90 percent of the body, known as Ritter disease. Purulent material is present in localized lesions. This lesion can progress to the generalized form, which is characterized by cutaneous erythema followed by profuse peeling of the skin’s epidermal layer. The erythema usually starts in the face, neck, axillae, and groin and then spreads to the trunk and extremities. The disease lasts only 2 to 4 days, with complete healing occurring after about 10 days. Children have a high rate of spontaneous recovery, and there is scarring. The kidneys metabolize and excrete the toxin. It is thought that an immature or compromised renal or immune system contributes to the higher incidence of SSS in children under the age of five and in adults.

Staphylococcal SSS must be distinguished from toxic epidermal necrolysis (TEN), a serious and potentially fatal disease. TEN can be caused by a variety of factors, but it is most commonly associated with drug reactions and has been linked to antimicrobials and anticonvulsants. The cause is unknown, but the symptoms appear to be the result of a hypersensitive reaction. Treatments differ, despite the fact that it has a very similar initial presentation to SSS. TEN can be resolved by administering steroids early in the course of the illness, whereas steroids aggravate SSS. The mortality rate associated with TEN is high, and suspected offending drugs should be discontinued as soon as possible.

  1. Toxic Shock Syndrome.

TSS is a rare but potentially fatal multisystem disease characterized by a sudden onset of fever, chills, vomiting, diarrhea, muscle aches, and rash, followed by hypotension and shock. It was first described in 1978 and was associated with women who used highly absorbent tampons, though men, children, and nonmenstruating women were also affected. TSS is classified into two types: menstruating-associated and nonmenstruating-associated. Despite the fact that nonmenstruating TSS has been linked to almost any staphylococcal infection, many cases have been linked to postsurgical infections.

Staphylococcal TSS is typically caused by a localized S. aureus infection; only the toxin TSST-1 is systemic. TSS manifests itself clinically as a high temperature, rash, and signs of dehydration, especially if the patient has had watery diarrhea and vomiting for several days. Patients in extreme cases may be severely hypotensive and in shock. The rash is mostly found on the trunk, but it can spread throughout the body. S. aureus cultures from focal lesions may be positive, but blood cultures are frequently negative. To confirm the diagnosis of TSS, S. aureus does not need to be isolated. Supportive therapy, as well as appropriate antimicrobial therapy, is administered to compensate for vascular volume loss. The majority of TSS patients recover, though 2 to 5% of cases may be fatal. The use of minimum absorbency tampons and FDA warning label requirements for tampon products have greatly reduced the risk of TSS.

  1. Food Poisoning

Enterotoxins from S. aureus, most commonly A (78%), D (38%), and B (10%), have been linked to gastrointestinal problems. Typically, the source of contamination is an infected food handler. Staphylococcal food poisoning is a type of intoxication caused by the consumption of a toxin produced outside the body. Food becomes contaminated with enterotoxin-producing strains of S. aureus due to improper handling and storage, allowing the bacteria to grow and produce toxin. An individual becomes ill after consuming enterotoxin-contaminated food.

Salads, particularly those containing mayonnaise and eggs, are frequently implicated in staphylococcal food poisoning, as are meat or meat products, poultry, egg products, bakery products with cream fillings, sandwich fillings, and dairy products. When contaminated with toxin-producing staphylococci, foods kept at room temperature are especially vulnerable to higher levels of toxin production. The enterotoxins have no discernible odor or alter the appearance or taste of the food. Symptoms appear quickly (between 2 and 8 hours after ingestion of the food) and disappear within 24 to 48 hours. Although there is no fever associated with this condition, it is common to experience nausea, vomiting, abdominal pain, and severe cramping. Diarrhea and headaches are also possible side effects. Death from staphylococcal food poisoning is uncommon, but it has occurred in elderly patients, infants, and severely disabled people.

  1. Other Infections.

Bacteremia caused by Staphylococcus aureus has been observed in intravenous drug users. The organisms enter the bloodstream through contaminated needles or a focal lesion on the skin, respiratory tract, or genitourinary tract. Any local S. aureus infection can develop into bacteremia, which can lead to secondary pneumonia, endocarditis, or bone infection. S. aureus can cause chronic infections in addition to acute, rapidly progressing disease. These chronic infections are frequently associated with SCVs. These variants are more difficult to detect in laboratory media because they are adapted for intracellular growth. Staphylococcal pneumonia is known to occur as a result of influenza virus infection. Although staphylococcal pneumonia is uncommon, it has a high mortality rate. Pneumonia is characterized by multiple abscesses and focal lesions in the pulmonary parenchyma and develops as a contiguous lower respiratory tract infection or as a complication of bacteremia. Infants and immunocompromised people, such as the elderly and patients receiving chemotherapy or immunosuppressants, are particularly vulnerable. S. aureus has also been linked to bone infections such as osteomyelitis, septic arthritis, and prosthetic joint infections. Staphylococcal osteomyelitis develops as a result of bacteremia. Bacteria can get stuck in the diaphysis of the long bones and cause an infection. Fever, chills, swelling, and pain in the affected area are all symptoms. S. aureus is a common cause of septic arthritis in children, especially after trauma to the extremities. Patients with a history of rheumatoid arthritis, diabetes mellitus, recent joint surgery, skin infections, or intravenous drug abuse are also at risk for septic arthritis. The organisms recovered from aspirated joint fluid may or may not be viable.

Staphylococcus epidermidis

S. epidermidis has been identified as an etiologic agent of disease. S. epidermidis is classified as normal skin biota, but it is a common source of hospital-acquired infections and is frequently a contaminant in improperly collected blood culture specimens. Instrumentation procedures such as catheterization, medical implantation, and immunosuppressive therapy are all risk factors for hospital-acquired infection. S. epidermidis is a common cause of UTIs acquired in the hospital. S. epidermidis is the most common cause of prosthetic valve endocarditis, but other CoNS, such as S. lugdunensis, have also been recovered in these cases. Infections with S. epidermidis have been linked to intravascular catheters, cerebrospinal fluid shunts, and other prosthetic devices. In immunocompromised patients, septicemia has been reported.

Infections caused by implanted devices, such as indwelling catheters and prosthetic devices, are frequently caused by isolates that have been shown to produce a biofilm. Biofilm formation is a critical step in bacterial pathogenesis and involves a complex interaction between the host, the indwelling device, and the bacteria. S. aureus adherence is aided by one bacterial factor. Epidermidis may be poly (γ-DLglutamic acid), which gives it an advantage against host defenses.

Staphylococcus saprophyticus

S. saprophyticus is linked to UTIs in young women; it is the second most common cause of uncomplicated cystitis in this population, after E. coli. This species adheres to the epithelial cells lining the urogenital tract more effectively than other CoNS. It is uncommon on other mucous membranes or skin surfaces. S. saprophyticus can be found in urine cultures in low numbers (10,000 colony-forming units per milliliter) and still be considered significant.

Staphylococcus lugdunensis

S. lugdunensis is capable of causing both community-associated and hospital-acquired infections. This organism is more virulent than others because it carries the gene mecA, which codes for oxacillin resistance. It is a significant pathogen in infective endocarditis, septicemia, meningitis, skin and soft tissue infections, urinary tract infections (UTIs), and septic shock. S. lugdunensis endocarditis is particularly aggressive, frequently necessitating valve replacement, and infections have a high mortality rate.

Other Coagulase-Negative Staphylococci

S. warneri, S. capitis, S. simulans, S. hominis, and S. schleiferi are less common but well-known opportunistic pathogens. These organisms have been linked to a variety of infections, including endocarditis, septicemia, and wound infections. Other CoNS species are found in the normal biota of humans and animals. Although they are not commonly recognized as pathogens, their role in certain infections is well established, and they cannot be dismissed as contaminants.

S. haemolyticus is a common CoNS isolate. It has been reported in wounds, bacteremia, endocarditis, and urinary tract infections (UTIs). Some S. haemolyticus isolates are resistant to vancomycin. S. pseudintermedius, a common cause of pyoderma in dogs as well as skin, ear, and postoperative infections in dogs and cats, has recently been linked to human infections. The first human case involved endocarditis following the implantation of a cardioverter-defibrillator device. Other human infections that have been reported sporadically since then have included surgical site infections, rhinosinusitis, and catheter-associated bacteremia. S. pseudintermedius zoonotic transmission threatens veterinary staff and pet owners. Many isolates have two SCCmec elements (II and III), which causes oxacillin resistance. In addition, resistance to several other antibiotic classes has been observed.

Laboratory Diagnosis of Staphylococcus species

Specimen Collection and Handling

The proper collection, transport, and processing of specimens are critical components in the correct diagnosis and interpretation of any bacterial culture result. To avoid drying, maintain the proper environment, and limit the growth of contaminating organisms, clinical materials collected from infected sites should be transported to the laboratory as soon as possible. Although no special procedures are required for staphylococci recovery, specimens should be taken from the site of infection after appropriate cleansing of the surrounding area to avoid contamination by skin microbiota. The physician can reduce normal skin biota contamination even further by submitting secretion aspirates, tissue samples, or blood culture specimens instead of swabs.

Microscopic Examination

Microscopic examination of stained smears prepared directly from clinical samples provides information useful in the early diagnosis and treatment of infection and should always be performed on appropriate specimens. When staphylococci are present, purulent exudates, joint fluids, aspirated secretions, and other body fluids contain a large number of gram-positive cocci as well as polymorphonuclear cells. Because microscopic morphology cannot identify the genus or species on its own, a culture should be performed regardless of the microscopic examination results. A single swab would be less satisfactory than an aspirate for both culture and smear results.

From an aspirated abscess in staphylococcus disease, numerous gram-positive cocci in clusters with many polymorphonuclear cells (Gram stain, original magnification 1000).
From an aspirated abscess in staphylococcus disease, numerous gram-positive cocci in clusters with many polymorphonuclear cells (Gram stain, original magnification 1000).

Isolation and Identification

Staphylococci grow quickly on standard laboratory culture media, particularly sheep blood agar (SBA). For heavily contaminated specimens, a selective medium such as mannitol salt agar (MSA), Columbia colistin–nalidixic acid agar (CNA), or phenylethyl alcohol (PEA) agar can be used. MSA’s high NaCl concentration (7.5%) makes it selective for Staphylococcus, whereas the presence of mannitol and phenol red distinguishes S. aureus from most CoNS. CHROMagar Staph aureus (Becton Dickinson, Franklin Lakes, NJ) is a proprietary selective and differential medium for Staph aureus isolation and identification. CHROMagar MRSA has a higher sensitivity than traditional oxacillin screening methods for further classifying S. aureus into MRSA or methicillin susceptible Staphylococcus aureus (MSSA) strains. For further classifying S. aureus into MRSA or methicillin susceptible Staphylococcus aureus (MSSA) strains, CHROMagar MRSA has a higher sensitivity than traditional oxacillin screening methods.

Cultural Characteristics of Staphylococccus

Staphylococci produce round, smooth, white, creamy colonies on SBA after 18 to 24 hours of incubation at 35° to 37° C. With prolonged incubation, S. aureus can produce hemolytic zones around colonies and may occasionally produce pigment (yellow). SCVs form nonpigmented, nonhemolytic pinpoint-size colonies that coexist with normal phenotype colonies.

Staphylococcus aureus colonies on sheep blood agar are β-hemolytic, creamy, and buttery in appearance.

Colonies of S. epidermidis are typically small to medium-sized, gray-to-white, nonhemolytic colonies. Some of them may be mildly hemolytic. S. saprophyticus grows in slightly larger colonies, with roughly half of the strains producing yellow pigment. S. haemolyticus grows in medium-sized colonies, causing moderate to weak hemolysis and variable pigment production. Colonies of S. lugdunensis are typically hemolytic and medium in size, though small colony variants can occur. The identification of Staphylococci should not be based solely on colony morphology.

Identification Methods

Staphylococci and micrococci have long been distinguished by oxidation-fermentation (O/F) reactions in O/F glucose medium. Staphylococci ferment glucose under anaerobic conditions, whereas Micrococci do not. The O/F tests, however, do not differentiate between weak acid producers such as Micrococcus kristinae and staphylococci that cannot grow or produce acid anaerobically such as S. saprophyticus, S. auricularis, S. hominis, S. xylosus, and S. cohnii . A modified oxidase test, such as the Microdase Disk (Remel, Lenexa, KS), can be used to quickly distinguish staphylococci from micrococci. Staphylococci are mostly negative, whereas micrococci are mostly positive. The table below outlines key characteristics for differentiating staphylococci from other gram-positive cocci.

+, ≥90% of strains positive; ±, ≥90% of strains weakly positive; −, ≥90% of strains negative; d, 11% to 89% of strains positive; (d), delayed reaction; R, resistant; S, Susceptible.
(a)A low but significant number (6% to 15%) of clinical isolates are alkaline phosphatase negative.
Modified from Bannerman TL: Staphylococcus, micrococcus, and other catalase: positive cocci that grow aerobically. In Murray PR, et al, editors: Manual of
clinical microbiology, ed 9, Washington, DC, 2007, ASM Press.
+, ≥90% of strains positive; ±, ≥90% of strains weakly positive; −, ≥90% of strains negative; d, 11% to 89% of strains positive; (d), delayed reaction; R, resistant; S, Susceptible.
(a)A low but significant number (6% to 15%) of clinical isolates are alkaline phosphatase negative.
Modified from Bannerman TL: Staphylococcus, micrococcus, and other catalase: positive cocci that grow aerobically. In Murray PR, et al, editors: Manual of
clinical microbiology, ed 9, Washington, DC, 2007, ASM Press.

Many commercial multitest systems now include these traditional biochemical tests. Molecular testing, plasmid typing, fatty acid analysis, and, more recently, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) have all been used to identify species and strains.

S. aureus is frequently identified using coagulation tests. In human, rabbit, or pig plasma, clumping factor, formerly known as cell-bound coagulase, causes agglutination. As shown on a glass slide with a heavy suspension of organism mixed with saline and a drop of plasma, clumping factor on the surface of bacterial cells directly converts fibrinogen to fibrin, which precipitates onto the cell surface, causing agglutination. Following a negative result, a tube coagulase test, as described later, was performed. Only the tube coagulase or agglutination tests should be used because this slide test is insensitive.

Tube coagulase test detects extracellular enzyme “free coagulase.” Top tube is coagulase positive.
Tube coagulase test detects extracellular enzyme “free coagulase.” Top tube is coagulase positive.

Staphylococcal coagulase, also known as free coagulase, is detected using the tube coagulase method. When bacterial cells are incubated with plasma, an extracellular molecule called staphylocoagulase forms a clot. The coagulase-CRF complex is formed when staphylocoagulase combines with coagulase-reacting factor (CRF), a thermostable thrombin-like molecule. The coagulase-CRF complex resembles thrombin and indirectly converts fibrinogen to fibrin. The clot that forms in the tube may undergo autolysis (due to fibrinolysin), giving the appearance of a negative outcome. Laboratories should look for clot formation after 4 hours of incubation at 37° C.

By testing for pyrrolidonyl arylamidase activity, S. aureus (negative) can be distinguished from S. lugdunensis, S. intermedius, and S. schleiferi (positive). Pyroglutamyl-naphthylamide (L-pyrrolidonyl—naphthylamide) is hydrolyzed to L-pyrrolidone and -naphthylamine, which react with p-dimethylaminocinnamaldehyde to form a red compound. Some laboratory scientists use the VogesProskauer (VP) test to distinguish S. aureus (positive) from S. intermedius (negative) (negative). Acetoin formation from glucose or pyruvate is a positive VP test result. S. intermedius is an animal pathogen, and animal bites cause the vast majority of human infections. VP is found in S. lugdunensis, S. haemolyticus, and S. schleiferi.

CoNS are staphylocoagulase-negative isolates. S. saprophyticus is found in urine isolates that do not coagulate. S. saprophyticus is thought to have been identified through novobiocin susceptibility testing with a 5-g novobiocin disk. S. saprophyticus is resistant to novobiocin, whereas the majority of other CoNS are susceptible. A schema for identifying clinically significant staphylococci is depicted in the image below. Although S. epidermidis, S. saprophyticus, and S. lugdunensis are the most clinically relevant CoNS, other species, such as S. haemolyticus, can be. The key tests for identifying clinically significant Staphylococcus spp., including some CoNS, are listed in Table below.

Key Tests for Identification of the Most Clinically Significant Staphylococcus Species
Key Tests for Identification of the Most Clinically Significant Staphylococcus Species
Schema for the identification of staphylococcal species. Note: Other Staphylococcus
spp. that are coagulase positive besides S. aureus include S. schleiferi and S. lugdunensis (which
can be slide test positive), S. intermedius, S. pseudintermedius, and S. hyicus (tube positive and
slide positive). R, Resistant; S, Susceptible.
Schema for the identification of staphylococcal species. Note: Other Staphylococcus
spp. that are coagulase positive besides S. aureus include S. schleiferi and S. lugdunensis (which
can be slide test positive), S. intermedius, S. pseudintermedius, and S. hyicus (tube positive and
slide positive). R, Resistant; S, Susceptible.

Rapid Methods of Identification

There are numerous commercial rapid agglutination test kits available for distinguishing S. aureus from CoNS. BBL Staphyloslide (BD-BBL, Sparks, MD), Staphaurex (Remel), and BACTiStaph are a few examples. These kits make use of plasma-coated carrier particles like latex. The plasma detects clumping factor (along with fibrinogen) and protein A in the cell wall of Staphylococcus aureus (with IgG). These kits frequently have higher specificity and sensitivity than traditional plasma slide tests and are faster than tube coagulase tests. As a result, they are frequently used in clinical laboratories. They are especially useful for identifying MRSA organisms, which frequently test weakly positive or negative in the slide coagulase test. Negative results should be confirmed using the tube coagulase test, nucleic acid amplification test, or MALDI-TOF analysis.

Third-generation agglutination kits contain antibodies that bind capsular antigens 5 and 8, as well as other surface molecules, in addition to detecting protein A and clumping factor. While these assays are more sensitive, they are generally less specific. Some CoNS, such as S. saprophyticus, S. hominis, and S. haemolyticus, have the potential to produce false-positive results. Some mistakes can be avoided by paying close attention to the source, colony morphology, and susceptibility pattern.

Although there are numerous automated and rapid multitest biochemical systems for the identification of staphylococci, their accuracy varies (70 percent to 90 percent ). Most systems are capable of correctly identifying S. aureus, most S. epidermidis, and S. saprophyticus strains, as well as some other staphylococcal species such as S. capitis, S. haemolyticus, S. simulans, S. lugdunensis, and S. intermedius.

In the clinical setting, molecular methods and other rapid identification systems have been introduced. Both MRSA and MSSA can be identified using real-time polymerase chain reaction (PCR). It has been used as a supplement to infection control practices to reduce MRSA infections, particularly in select targeted populations that would benefit from rapid testing results, such as patients in intensive care units and other select patient populations. As an aid in the diagnosis of sepsis and for targeted antimicrobial therapy treatment, molecular methods can directly identify staphylococci from a positive blood culture sample. Several commercial platforms are available, including the FilmArray BCID panel (BioFire, Durham, NC) based on multiplex PCR and the Verigene blood culture assay (Nanosphere, Northbrook, IL) that detects nucleic acids and proteins using a proprietary chemistry method. Within 1 to 2 hours of identifying a positive blood culture bottle, these systems detect Staphylococcus, S. aureus, and the mecA resistance gene for methicillin resistance detection.

Newer technologies, including non–PCR-based molecular techniques, are currently being evaluated, such as the T2 Biosystems (Lexington, MA) and Qvella (Richmond Hill, ON) technologies for direct detection from patient blood samples, eliminating the need for the 24- to 48-hour growth incubation phase. The main advantage of these prototype technologies is the reduced time required to administer appropriate antimicrobial therapy, as well as the possibility of reduced mortality and morbidity. However, one significant disadvantage is that the clinical laboratory must be able to grow the organism if antibiotic susceptibilities are required, and thus cannot rely solely on molecular methods. Furthermore, in the case of a mixed infection, molecular methods may only identify one of the organisms present, necessitating the use of traditional methods in the majority of cases. When comparing molecular assays to conventional methods and the need to perform back-up conventional methods, the laboratory must consider the patient population’s requirements as well as the test’s sensitivity, specificity, positive predictive value, and negative predictive value.

Approaches to mass spectrometry, such as MALDI-TOF methods, are gaining traction in clinical microbiology laboratories. Technological advancements, such as the use of more molecular and mass spectrometry methods, such as MALDI-TOF, are expected to improve the ability to accurately and quickly identify staphylococci and other clinically relevant bacteria. The organism must be grown in a pure isolated colony for a specified period of time, at a specific incubation temperature, and on specific growth media. MALDI-TOF cannot distinguish between genetic differences such as MRSA and MSSA strains.

Antimicrobial Susceptibility

Routine testing of staphylococcal isolates in the laboratory is simple thanks to the Clinical and Laboratory Standards Institute (CLSI), formerly known as the National Committee for Clinical Laboratory Standards (NCCLS). When using commercial tests, laboratories must follow the manufacturer’s recommendations.

CoNS testing is dependent on the source and determining whether the isolate is a contaminant or a likely pathogen. When testing CoNS, the most recent interpretive standards for guidance and interpretation must be used. Susceptibility testing is required for serious infections caused by S. aureus and S. lugdunensis. Most S. aureus isolates are penicillin resistant due to the production of -lactamases (penicillinases), which break down the -lactam ring of many penicillins. The rise in resistance to alternative antimicrobial agents is a major source of concern.

Methicillin-Resistant Staphylococci

Penicillinase-resistant penicillins, such as nafcillin or oxacillin, must be used to treat penicillin-resistant strains. Although methicillin is no longer used in the United States, isolates resistant to nafcillin or oxacillin have traditionally been referred to as methicillin-resistant staphylococci, such as MRSA and methicillin-resistant Staphylococcus epidermidis (MRSE). While MRSA remains a serious health concern, some studies have found a reduction in deaths in health care settings due to invasive MRSA infections. This could be due to stricter infection control measures. However, incidences of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have increased since the 1990s (>50% of S. aureus isolates in some areas of the United States), and these infections can be found in patients who do not have traditional health care–associated risk factors, such as recent hospitalization, long-term care, hemodialysis, or indwelling device. CA-MRSA infections and outbreaks have been reported in athletes, correctional facility inmates, military recruits in close quarters, pediatric patients, and tattoo recipients. Although hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) rates have stabilized and even declined, continued vigilance is required to ensure that CA-MRSA rates are monitored, patient populations are educated, and appropriate control measures are implemented. According to the Centers for Disease Control and Prevention (CDC), 72,444 MRSA cases are reported each year.

MRSA infections, whether HA-MRSA or CA-MRSA, are costly and pose a serious threat to health care facilities. MRSA management necessitates strict adherence to infection control practices such as barrier protection, contact isolation, and handwashing compliance. The use of rapid MRSA tests may help in the control of this agent. Vancomycin remains the treatment of choice for MRSA infection, but concerns about glycopeptide resistance necessitate cautious use of these drugs and selective reporting by laboratories.

Previously, oxacillin was commonly used to detect methicillin resistance in staphylococcal species. The most recent CLSI M100 document recommends using cefoxitin to detect oxacillin (methicillin) resistance. Cefoxitin induces mecAmediated resistance more effectively. At the very least, the laboratory should report penicillin, cefoxitin, and/or oxacillin susceptibilities. S. lugdunensis has the same minimal inhibitory concentration breakpoint for cefoxitin as S. aureus, which distinguishes it from other CoNS. Testing for penicillin-binding protein 2a (PBP2a) or cefoxitin disk testing may be more appropriate for non–S. epidermidis strains that lack mecA. For correct interpretation, laboratory scientists must always refer to the most recent susceptibility guides. Except for fifth-generation cephalosporins with MRSA activity, MRSA isolates should be considered resistant to all -lactam antibiotics, including carbapenems.

MRSA populations are frequently heterogeneous in terms of resistance to beta-lactams, which means that one subpopulation is susceptible while another is resistant to methicillin. Despite the fact that nearly all cells have the genetic information to be resistant, only a small percentage (1 in 108 to 104 cells) express the resistance phenotype. The growth of the resistant subpopulation is accelerated by a neutral pH, a sodium chloride concentration of 2% to 4%, a cooler incubation temperature (30° to 32° C), and a longer incubation time (up to 48 hours).

To screen for MRSA in clinical samples, such as nasal specimens, media containing oxacillin, or preferably cefoxitin, can be used. These media can also distinguish MRSA isolates from isolates that are -lactamase hyperproducers or borderline oxacillin-resistant Staphylococcus aureus (BORSA) strains, which would not grow on these plates. MRSA Select (Bio-Rad Laboratories, Hercules, CA), Spectra MRSA (Remel), and CHROMagar MRSA are chromogenic selective differential media.

(BD-BBL) are capable of detecting MRSA directly from clinical samples. The media contains a high concentration of sodium chloride as well as antimicrobial compounds such as cefoxitin, which inhibit nonstaphylococci and non-MRSA isolates. MRSA isolates produce a colored colony after 24 or 48 hours of incubation, whereas MSSA and most other organisms are inhibited or produce a noncolored colony. MRSA can also be identified using automated antimicrobial susceptibility systems that use cefoxitin.

The majority of oxacillin resistance is caused by the gene mecA, which is carried on a mobile cassette called SCCmec. The gene encodes a modified penicillin-binding protein (PBP), PBP2a, also known as PBP2′. Because the altered PBP does not bind oxacillin, the drug is rendered ineffective. To detect these altered PBPs, latex agglutination tests are available, and they provide an alternative method for testing and confirming oxacillin resistance. This test is applicable to both CoNS and S. aureus.

The detection of the mecA gene using molecular nucleic acid probes or PCR amplification is the gold standard for MRSA detection. Many systems for direct detection from anterior nares swabs are available, including the BD GeneOhm MRSA assay and the Xpert MRSA assay (Cephid, Sunnyvale, CA) using the GeneXpert system, both of which use real-time PCR and produce results within several hours. Several molecular systems can distinguish between MRSA and MSSA in the same assay. Either type of test, with a shorter time to detection, has the potential to reduce HA-MRSA and CA-MRSA transmission within the hospital setting. These assays are currently moderate or high-complexity tests, but CLIA-waived versions may become available as point-of-care testing in the future and could be used in nursing homes and long-term health care facilities. Furthermore, some PCR assays can identify the most common CA-MRSA strains, which have a high mortality and morbidity rate and frequently carry the PVL genes. Recently, researchers in Europe discovered a new type of SCCmec derived from a different lineage of the previously described gene. Conventional and real-time PCR assays do not detect this new variant.

CA-MRSA can cause nosocomial infections, which could have significant implications for treatment and epidemiology. The Joint Commission, an accreditation organization for health care facilities, established safety goals for a laboratory-based alert system to detect MRSA, and many states have already enacted or are considering legislation requiring MRSA screening and reporting.

Vancomycin-Resistant Staphylococci

Vancomycin is the drug of choice, and in some cases the only drug available, for serious staphylococcal infections, and the emergence of vancomycin resistance has been a major source of concern in the medical community. The first vancomycin-intermediate Staphylococcus aureus (VISA) strains were found in Japan in 1996. Antimicrobial susceptibility testing methods that are automated may be unreliable in detecting these isolates. The disk diffusion procedure has limitations when it comes to detecting resistance. It has been suggested that clinical microbiology laboratories detect VISA using more than one method. Because VISA are difficult to identify, their prevalence may be underreported. True vancomycin-resistant Staphylococcus aureus (VRSA) isolates from patients receiving long-term vancomycin treatment were reported in the United States in 2002. So far, the majority of the isolates recovered in the United States have come from patients suffering from underlying conditions. These isolates should be detected using a reference method, and reporting should adhere to CDC guidelines. Adherence to infection control practices and CDC vancomycin resistance guidelines may help to limit the spread of this highly resistant organism.

Macrolide Resistance

Resistance to other classes of antimicrobials, such as macrolides, may not always be detectable through routine testing. Clindamycin, a macrolide, is commonly used to treat staphylococcal skin infections; additional testing using a modified double disk diffusion test (D-zone test) may be useful when macrolide test results are inconsistent (e.g., erythromycin resistant and clindamycin susceptible). The susceptibility to erythromycin and clindamycin is usually the same. Staphylococcal resistance to clindamycin, on the other hand, is occasionally inducible, which means it can be detected in vitro only when the bacteria are also exposed to erythromycin. It is critical for laboratories to stay current on antimicrobial resistance trends and to be aware of the limitations that can occur with susceptibility testing.

Disk diffusion can detect inducible clindamycin resistance by placing an erythromycin disk near a clindamycin disk and using the most recent performance standards for susceptibility testing. If an isolate has inducible clindamycin resistance, bacteria grow around the erythromycin disk and on the agar where the two drugs overlap. However, a zone of inhibition is observed on the side of the clindamycin disk that is further away from the erythromycin disk, flattening the clindamycin zone and resembling the letter “D.” Some automated methods are capable of detecting this resistance.

D-zone test–positive isolate showing flattening of
the clindamycin (CC) zone adjacent to the erythromycin (E) disk
and the characteristic D-like pattern.
D-zone test–positive isolate showing flattening of
the clindamycin (CC) zone adjacent to the erythromycin (E) disk
and the characteristic D-like pattern.

BIBLIOGRAPHY

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  • Becker, K., et al. (2014). Coagulase-negative staphylococci. Clinical Microbiology Reviews, 27, 870.
  • Becker, K., et al. (2015). Staphylococcus, Micrococcus, and other catalase-positive cocci. In J. Jorgenson, et al. (Eds.), Manual of clinical microbiology (ed. 11). Washington, DC: ASM Press.
  • Blake, W. B., et al. (2013). Multiplex identification of gram-positive bacteria and resistance determinants directly from positive blood culture broths: evaluation of an automated microarray-based nucleic acid test. PLoS Medicine, 10(7), e1001478.
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  • Centers for Disease Control and Prevention. Investigation and control of vancomycin-resistant Staphylococcus aureus (VRSA): 2015 update. Available at: https://www.cdc.gov/hai/pdfs/VRSA-Investigation-Guide -05_12_2015.pdf. (Accessed 27 March 2017).

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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