Staphylococcus PDF

23 43 Mycobacterium Leprae Staphylococcus...

23 43 Mycobacterium Leprae Staphylococcus Staphylococcus aureus is the most important human pathogen. Introduction The other important human pathogens are coagulase- negative staphylococci (CONS), which include Staphylo- Family Micrococcaceae consists of Gram-positive cocci, which coccus epidermidis, Staphylococcus saprophyticus, Staphylococcus are aerobic and anaerobic, and are arranged in tetrads or clus- haemolyticus, Staphylococcus hominis, Staphylococcus warneri, ters. Micrococcaceae consists of four genera, Staphylococcus, Staphylococcus saccharolyticus, Staphylococcus schleiferi, and Micrococcus, Planococcus, and Stomatococcus. Differences between Staphylococcus lugdunensis. Staphylococci are capable of these genera are summarized in Table 23-1. acquiring resistance to many antibiotics and therefore Human infections caused by these genera are summarized can cause major clinical and epidemiological problems in in Table 23-2. Among these, Staphylococcus is the only genus of hospitals. medical importance. Staphylococcus aureus Staphylococcus S. aureus is an important human pathogen that causes a spec- The genus Staphylococcus consists of 32 species, most of which are trum of clinical diseases. These range from superficial skin animal pathogens or commensals. The bacteria belonging to this lesions like folliculitis to deep-seated abscess and various genus are aerobic and facultative anaerobic, catalase positive, pyogenic infections like endocarditis, osteomyelitis, etc. The oxidase negative, and are arranged in clusters, pairs, or tetrads. bacterium also causes toxin-mediated diseases, such as food poisoning, toxic shock syndrome (TSS), and staphylococcal Features distinguishing scalded skin syndrome (SSSS). TABLE 23-1 Staphylococcus, Micrococcus, and Planococcus Properties of the Bacteria Characters Staphylococcus Micrococcus Planococcus ◗ Morphology Arrangement of Clusters Clusters/ Tetrads Staphylococci show following features: bacteria Tetrads Presence of teichoic ⫹ ⫺ ⫺ They are Gram-positive cocci, measuring around 1 ␮m in acid diameter. Production of brown ⫺ ⫺ ⫹ They are nonmotile, nonsporing. pigment They are noncapsulated. They, however, contain a microcapsule, which can be visualized by electron micro- Glucose fermentation ⫹ ⫹ ⫺ scope only, but not by a light microscope. TABLE 23-2 Human infections caused by Staphylococcus, Micrococcus, and Stomatococcus Bacteria Diseases Staphylococcus aureus Skin infections—impetigo, folliculitis, furuncle, carbuncle, paronychia, wound infection Systemic infections—bacteremia, osteomyelitis, septic arthritis, endocarditis, pneumonia, meningitis, deep-seated abscess Toxin-mediated infections—food poisoning, toxic shock syndrome, staphylococcal scalded skin syndrome Staphylococcus epidermidis Opportunistic infections—intravenous catheter infections, CSF shunt infections, and catheter-associated peritonitis endocarditis in “immunocompromised” patients Staphylococcus saprophyticus Urinary tract infection particularly in sexually active young women Micrococcus spp. Opportunistic infections Stomatococcus spp. Opportunistic infections, bacteremia, endocarditis  174 BACTERIOLOGY The cocci are typically arranged in irregular grape-like clusters. Color Photo 12). Hemolysis is well marked on sheep or This appearance is due to incomplete separation of daughter rabbit blood agar, especially when incubated in an atmo- cells during successive divisions of bacteria, which takes place sphere of 20–25% CO2. Sheep blood agar is used for pri- in perpendicular planes. The grape-like clustering is seen when mary isolation. the bacteria are grown in solid media, but usually short chains Hemolysis is weak on horse blood agar. Human blood are seen when grown in liquid media. is not used, as it may contain antibiotics or other inhibi- In smears taken from pus, the cocci are present either singly tors. Other species of Staphylococcus do not produce or in pairs, in clusters, or in short chains of three or four cells. hemolysis. 3. MacConkey agar: S. aureus produces small pink colonies ◗ Culture due to fermentation of lactose. 4. Selective media: Mannitol salt agar, milk agar, and Staphylococci are aerobes and facultative anaerobes but can glycerol monoacetate agar are the commonly used selec- grow in the absence of oxygen also. They grow at a tempera- tive media for isolation of S. aureus from clinical specimens ture range of 10–42°C (optimum temperature 37°C) and a pH containing normal bacterial flora (e.g., stools). Mannitol range of 7.4–7.6 (optimum pH 7). salt agar contains 1% mannitol, 7.5% sodium chloride, and Culture on solid media: Staphylococci can grow on a wide 0.0025% phenol red indicator. Most strains of S. aureus fer- range of media including Mueller–Hinton agar, nutrient agar, ment mannitol with acid production, which gives rise to blood agar, and MacConkey agar. Primary isolation can be yellow zone formation around the colonies. made on nutrient agar and blood agar. Culture in liquid media: S. aureus produces turbidity in liquid 1. Nutrient agar: S. aureus produces round, convex, well- media and there is no production of pigment. defined colonies measuring 2–4 mm in diameter. The colo- Section III nies show a butyrous consistency with a smooth glistening surface. ◗ Biochemical reactions S. aureus produces characteristic golden-yellow colonies S. aureus shows following reactions: due to production of a nondiffusible golden-yellow pig- ment. The pigment is believed to be a lipoprotein allied to It is coagulase positive. The production of coagulase is used carotene. The production of the pigment is enhanced by as a test to differentiate S. aureus from S. epidermidis and incubation at 22°C in the presence of oxygen. Milk agar and other CONS. Chapter 23 1% glycerol monoacetate agar are other media that facilitate It is phosphatase positive. Phosphatase production can also the production of pigment. On nutrient agar slopes, the be used to differentiate S. aureus from S. epidermidis, as the growth gives a characteristic “oil paint” appearance. latter either does not produce or has very weak phosphatase 2. Blood agar: S. aureus produces a clear zone of hemoly- activity. sis (beta-hemolysis) surrounding the colonies (Fig. 23-1, It is catalase positive. It produces enzyme catalase (unlike Streptococcus), which degrades H2O2 into nascent oxygen and water. It is oxidase negative. S. aureus ferments mannitol, sucrose, maltose, and trehalose under aerobic conditions, with the production of acid but no gas. Fermentation of mannitol is of diagnostic impor- tance, because most strains of S. aureus ferment manni- tol while those of S. epidermidis and S. saprophyticus do not ferment mannitol. It liquefies gelatin, hydrolyzes urea, reduces nitrate to nitrite, and is “Voges-Proskauer (VP)” and “methyl red (MR)” posi- tive but indole negative. ◗ Other properties Susceptibility to physical and chemical agents: The cocci withstand moist heat at 60°C for 30 minutes but are killed after 30 minutes. They are also killed rapidly by disinfectants, such as phenol, chlorhexidine, and hexachlorophene. The cocci are very sensitive to aniline dyes, such as crystal violet. The dye at a concentration of 1:500,000 inhibits the growth FIG. 23-1. Blood agar plate showing beta-hemolysis surrounding the of the cocci on blood agar medium but permits the growth of colonies of Staphylococcus aureus. streptococci.  STAPHYLOCOCCUS 175 Cell Wall Components and Antigenic ◗ Teichoic acid Structure Teichoic acid is the major antigenic determinant of the cell wall of S. aureus. It is a polymer of ribitol phosphate. Antibodies Cell wall associated proteins and polymers include the follow- to teichoic acids develop in endocarditis and in certain other ing (Fig. 23-2): staphylococcal infections. ◗ Cell wall peptidoglycan ◗ Protein A S. aureus cell wall is rich in peptidoglycans. Peptidoglycan is It is the major protein in the cell wall and has a molecular a polymer of the polysaccharide, which provides rigidity to weight of 13,000 Da. It is present in large quantities in the cell the cell wall of the bacteria. It has the characteristic penta wall of certain strains of S. aureus, such as the Cowan’s strain glycine bridges that link tetrapeptides to the muramic acid of S. aureus (SAPA). This is a group specific antigen. The anti- residues. gen is present in more than 90% strains of S. aureus. Protein A is absent in both the coagulase-negative staphylococci (CONS) and micrococci. Capsule or polysaccharide slime layer Pathogenesis and Immunity Clumping S. aureus causes disease by multiplying in tissues and causing TTeichoic factor acid inflammation, and also by liberating toxin. Section III Protein A Peptidoglycan layer ◗ Virulence factors Cytoplasmic membrane S. aureus produces several virulence factors (Table 23-3), which Cytoplasm include the following: Staphylococcal cell wall structure (a) Cell wall associated proteins and polymers (b) Extracellular enzymes FIG. 23-2. Cell wall structure of Staphylococcus aureus. (c) Toxins Chapter 23 TABLE 23-3 Virulence factors of Staphylococcus aureus Virulence factors Biological functions Cell wall associated polymers and proteins Peptidoglycan Inhibits chemotaxis of inflammatory cells Capsular polysaccharide Inhibits phagocytosis and chemotaxis Teichoic acid Mediates attachment of staphylococci to mucosal cell Protein A Chemotactic, anticomplementary, and antiphagocytic; causes platelet injury; and elicits hypersensitivity reactions Enzymes Coagulase The enzyme coats the bacterial cells with fibrin, rendering them resistant to opsonization and phagocytosis Catalase Produces nascent oxygen which causes oxidative damage to host tissue Hyaluronidase Hydrolyzes hyaluronic acids present in the matrix of the connective tissues, thereby facilitating the spread of bacteria in the tissues Penicillinase Inactivates penicillins Nuclease Hydrolyzes DNA Lipases Hydrolyzes lipids Toxins Toxic shock syndrome toxin Superantigen, stimulates the release of large amount of interleukins (IL-1 and IL-2) Enterotoxin Superantigen, acts by producing large amounts of interleukins (IL-1 and IL-2) Exfoliative toxin Splits intercellular bridges in the stratum granulosum of epidermis of the skin Leukocidin toxin Leukolysin is thermostable and causes lysis of leukocytes Hemolysin Causes lysis of erythrocytes  176 BACTERIOLOGY Cell wall associated proteins and polymers Differentiating features of coagulase TABLE 23-4 and clumping factors These include capsular polysaccharide, protein A, peptido- glycan, and teichoic acid that contribute to pathogenesis of Coagulase Clumping factor staphylococcal diseases. Produced extracellularly Present on the surface Detected by tube test Detected by slide test Capsular polysaccharide: Few strains of S. aureus are capsulated. These strains are more virulent than the Heat labile Heat stable noncapsulated ones. Eight serotypes One serotype Needs CRF Does not need CRF The capsule protects the bacteria from phagocytosis. The capsule also facilitates adherence of the cocci to host Is a virulence factor Is not a virulence factor cells and to prosthetic implants. Protein A: Protein A is an important virulence factor Catalase: The enzyme catalase reduces H2O2 to nascent since it has non-specific interaction with Fc portion of the oxygen and water. This nascent oxygen causes oxidative damage immunoglobulin G (IgG) leaving the Fab portions free to of host tissue. This enzyme is produced after phagocytosis or combine with specific antigen. during metabolism of the bacteria. All strains of staphylococci It is chemotactic, anticomplementary, and antiphagocytic. produce catalase unlike streptococci. It causes platelet injury and elicits hypersensitivity reactions. Hyaluronidase: The enzyme hyaluronidase hydrolyzes the acidic mucopolysaccharides present in the matrix of the connective Peptidoglycan: It activates the complement, stimulates tissues, thereby facilitating the spread of bacteria in tissues. production of the antibodies, and inhibits chemotaxis by Section III inflammatory cells. Penicillinase: More than 90% of S. aureus produce enzyme penicillinase. The enzyme inactivates penicillin group of Teichoic acid: It mediates attachment of staphycocci to antibiotics, hence is responsible for widespread occurrence of mucosal cell. penicillin-resistant staphylococci. The gene for this enzyme is Extracellular enzymes acquired through plasmids. The enzymes include (a) coagulase, (b) catalase, (c) hyaluroni- Other enzymes: These include phosphatase, deoxyribo- nucleases, nucleases, proteases, phospholipase, and lipases. Chapter 23 dase, (d) penicillinase, and (e) other enzymes. Coagulase: S. aureus has a unique ability to clot a variety of Toxins mammalian plasma. Clotting of plasma is brought about by the action of the enzyme coagulase secreted by the pathogenic Toxins include (a) toxic shock syndrome toxin, (b) enterotoxin, strains of S. aureus. The enzyme coagulase is of two types: (c) exfoliative toxin, (d) leukocidins, and (e) hemolysins. (a) free coagulase and (b) bound coagulase. Toxic shock syndrome toxin: Toxic shock syndrome toxin (TSST) is a protein with a molecular weight of 22,000 Da A. Free coagulase: Free coagulase is a heat-labile and filter- and resembles enterotoxin F and exotoxin C. It is antigenic. able enzyme. It has eight antigenic types (A, B, C, D, E, F, G, Production of toxin is pH dependent and occurs at pH 7–8. The and H). Antigenic type A is produced by most human S. aureus toxin causes toxic shock syndrome (TSS). strains. The enzyme coagulase in association with coagulase- reacting factor (CRF) present in plasma converts fibrinogen S. aureus strains responsible for menstruation-associated to fibrin. In the absence of CRF, coagulase cannot bring about TSS and half of the strains responsible for non-menstruation clotting like in case of the guinea pig plasma. This fibrin coats associated TSS produce TSST-1. The strains producing the bacterial cells, rendering them resistant to opsonization TSST-1 belong to the bacteriophage group I. and phagocytosis and hence making bacteria more virulent. All TSST is a superantigen and hence a potent stimulant of T coagulase-producing staphylococci are, by definition, S. aureus. lymphocytes, resulting in release of large amount of inter- Coagulase production is demonstrated by tube coagulase test, leukins (IL-1 and IL-2) and tumor necrosis factor, ultimately which is an important test for the identification of S. aureus. manifesting in TSS. B. Bound coagulase: Bound coagulase is otherwise known as Enterotoxin: Enterotoxin is a heat-stable protein, capable of clumping factor. It is a heat-stable protein and is present in the resisting boiling for about 30 minutes. It is also gut-enzyme cell wall. This enzyme brings about clumping of the staphylococci resistant. The toxin is produced by nearly one-third of all the when mixed with plasma by directly acting on fibrinogen. Lysis strains of S. aureus, and these strains belong to bacterio- of the cell releases the enzyme. Unlike free coagulase, clumping phage group III (6/47). factor does not need CRF for its action; till date only one type has Nine antigenic types (A, B, C1,2,&3, D, E, G, H, I, and J) of been identified. Bound coagulase is not a virulence factor. enterotoxins have been described, out of which type A and B Differences between coagulase and clumping factors are sum- are most important. These proteins are of molecular weights marized in Table 23-4. ranging from 26,000 to 30,000 Da.  STAPHYLOCOCCUS 177 The toxins are superantigens and act by producing large Multiplying in tissues, amounts of interleukins, IL-1 and IL-2. Liberating toxins, and The enterotoxins are responsible for clinical conditions like Stimulating inflammation. staphylococcal food poisoning and pseudomembranous enterocolitis postantibiotic therapy. ◗ Host immunity Exfoliative toxin: Exfoliative toxin is of two types: (a) toxin S. aureus infection does not cause any life-long immunity. It A (molecular weight of 30,000 Da) and (b) toxin B (molecular causes repeated infections in a susceptible host. weight of 29,500 Da). Toxin A is heat stable, while toxin B is heat labile. The toxin is antigenic, and specific antibodies Clinical Syndromes against the toxin are protective. The strains producing this The diseases caused by S. aureus can be divided into two groups: toxin belong to bacteriophage group II. (a) inflammatory and (b) toxin-mediated staphylococcal diseases. The toxin breaks intercellular bridges in the stratum gran- ulosum of epidermis and causes its separation from the ◗ Inflammatory staphylococcal diseases underlying tissue, resulting in a blistering and exfoliating disease of the skin. These include the following conditions: Toxin in localized form causes bullous impetigo and in gen- Staphylococcal skin infections include impetigo, folliculitis, eralized form causes staphylococcal scalded skin syndrome furuncles, carbuncles, paronychia, surgical wound infection, (SSSS) in children below 4 years of age. blepharitis, and postpartum breast infection. S. aureus is the most common cause of boils. The infection is Leukocidins: Leukocidins include (a) alpha-lysin, (b) Panton- Section III acquired either by self-inoculation from a carrier site, such Valentine–leukocidin (PV–leukocidin), and (c) leukolysin. as the nose or through contact with another person harbor- The alpha-lysin is the most important leukocidin. It causes ing the bacteria. marked necrosis of the skin and hemolysis by damaging the Bacteremia and septicemia may occur from any localized cell membrane, leading to release of low-molecular-weight lesion, especially wound infection or as a result of intrave- substances from the damaged cells. nous drug abuse. PV–leukocidins are six in number, each consisting of two S. aureus is an important cause of acute bacterial endocardi- components. The molecular weight is around 32 kDa. These tis, of normal or prosthetic heart valves, which is associated Chapter 23 toxins cause death of human leukocytes and macrophages with high mortality. without causing any lysis. S. aureus is the most common cause of osteomyelitis in chil- Leukolysin is thermostable and causes lysis of leukocytes dren. The bacteria reach bone through blood stream or by and necrosis of tissues in vivo. direct implantation following trauma. S. aureus causes pneumonia in postoperative patients follow- Hemolysins: S. aureus produces four hemolysins: alpha (␣), ing viral respiratory infection, leading to empyema; it also beta (␤), gamma (␥), and delta (␦) hemolysins. leads to chronic sinusitis. S. aureus causes deep-seated abscesses in any organ after Alpha-hemolysin is a protein with a molecular weight of 33 bacteremia. kDa. It has lethal effects on a wide variety of cell types and lyses erythrocytes of several animal species. Beta-hemolysin is a sphingomyelinase that is active on ◗ Toxin-mediated staphylococcal diseases a variety of cells. It is a protein with a molecular weight of These include (a) staphylococcal food poisoning, (b) staphy- 35 kDa. It is a hot–cold hemolysin; i.e., its hemolytic proper- lococcal toxic shock syndrome, and (c) staphylococcal scalded ties are increased by exposure of the RBCs to cold temperature. skin syndrome. Delta-hemolysin is a protein with a molecular weight of Staphylococcal food poisoning: Staphylococcal food poison- 8 kDa. It acts primarily as a surfactant. ing is caused by enterotoxin. The enterotoxin is a preformed toxin, Gamma-hemolysin actually consists of three proteins. The already present in the contaminated food before consumption. three delta-hemolysin proteins interact with one of the two Milk and milk products and animal products like fish and meat PV–leukocidin proteins. kept at room temperature after cooking are mainly incriminated. When kept at room temperature, the contaminating staphylo- ◗ Pathogenesis of staphylococcal infections cocci multiply and produce toxin adequate enough (as little as S. aureus are pyogenic bacteria that cause localized lesions 25 ␮g of toxin B can lead to illness) to cause food poisoning. in contrast to streptococci that are spreading in nature. The toxin acts by stimulating the release of large amounts of Staphylococci adhere to the damaged skin, mucosa, or tis- interleukins IL-1 and IL-2. It is fairly heat resistant and so is not sue surfaces. At these sites, they evade defense mechanisms of inactivated by brief cooking. the host, colonize, and cause tissue damage. They produce Often a food handler, who either is a carrier of S. aureus disease by: (nose, skin) or is suffering from staphylococcal skin infection,  178 BACTERIOLOGY is the source of infection. The onset of symptoms is sudden, ◗ Reservoir, source, and transmission of infection appearing within 2–6 hours of ingestion of food. It is a self- Human cases and carriers are the important reservoir of limiting condition characterized by nausea, vomiting, abdomi- infection. nal cramps, and watery, nonbloody diarrhea. Staphylococcal toxic shock syndrome: Staphylococcal toxic Human cases of cutaneous and respiratory infections shed shock syndrome (STSS) is caused by TSST. The toxin is a supe- large numbers of staphylococci into the environment for a rantigen, which causes STSS by stimulating the release of large prolonged period of time. Staphylococci colonize the skin amounts of interleukins IL-1 and IL-2 in the body. very early in life (in neonates on the umbilical stump). The STSS is an acute and potentially life-threatening con- Staphylococci shed by the patients and carriers contaminate dition similar to Gram-negative sepsis and septic shock. STSS handkerchiefs, bed linens, blankets, and other inanimate is a multisystem disease characterized by fever, hypotension, fomites and persist in them for weeks. myalgia, vomiting, diarrhea, mucosal hyperemia, and an ery- S. aureus found in the nose and sometimes on the skin, espe- thematous rash followed by desquamation of the skin, particu- cially in hospital staff and patients is the main source of larly on palms and soles. infection in hospitals. This condition was first documented in 1980 in the United Domestic animals, such as cows, can also be reservoirs of States among the menstruating women who used highly absor- staphylococcal infection. bent vaginal tampons; the vaginal swab from these women showed a heavy growth of S. aureus. This condition can also occur in other individuals, who have a local site of staphylococ- Key Points cal infection on skin or mucosa or on any other extragenital site. Staphylococcal infections may be acquired through: Section III Staphylococcal scalded skin syndrome: Staphylococcal Self-inoculation from nose or other sites in patients who scalded skin syndrome (SSSS) is caused by the exfoliative toxin, harbor staphylococci (endogenous infection) or exfoliatin. The condition is seen commonly in infants and chil- Direct contact with infected humans, carriers, and less fre- quently, animals (exogenous infection). Exogenous infection dren. It is associated with extensive exfoliation of the skin, in can also be acquired by close contact with infected fomi- which outer layer of the epidermis is separated from the under- tes or inhalation of air droplets in heavily contaminated lying tissue and is characterized by the appearance of extensive environment. bullae. These bullae when ruptured may leave behind scalded, red, tender skin. The lesion typically starts periorificially or in Chapter 23 skin folds. It usually resolves within 10 days’ time. Hospital-acquired S. aureus infections: This is the most com- Pemphigus neonatorum and bullous impetigo are the milder mon cause of hospital-acquired infections. Certain strains of S. forms, whereas Ritter’s disease in the newborn and toxic epidermal aureus causing hospital infections are known as hospital strains. necrolysis in the older persons are the severe forms of the SSSS. They exhibit certain properties, which are presented in Box 23-1. ◗ Bacteriophage typing Complications of Staphylococcal Diseases Strains of staphylococci can be typed by bacteriophage typ- Complications of staphylococcal diseases include bacterial ing, which is useful in epidemiological studies (Fig. 23-3). pneumonia, septicemia, arthritis, meningitis, etc. These com- Bacteriophage typing is based on the susceptibility of cocci to plications are frequently seen in persons with extreme of age, bacteriophages. This is carried out by pattern method, where debilitated persons, and immunosuppressed hosts. a set of 23 standard typing phages of S. aureus is used to type staphylococcal isolates and distinguish them from one another Epidemiology by their patterns of susceptibility to lysis. In this method, the ◗ Geographical distribution Staphylococcal infections are found throughout the world. Hospital strains of Nearly one-third of the adult population is asymptomatic car- Box 23-1 Staphylococcus aureus rier of staphylococci. Hospital infections caused by S. aureus are worldwide in distribution. Certain strains of staphylococci are the common causes of postopera- tive wound infections and other infections in the hospital environment. These strains are known as “hospital strains”. These hospital strains ◗ Habitat show following characteristics: Staphylococci are primary pathogens of humans and animals. 1. They are usually resistant to penicillin, methicillin, and other rou- They are present as commensals on skin, in the glands of the tinely used antibiotics. skin, and on mucous membranes. The cocci are commonly found 2. They belong to certain bacteriophage types. 3. Some of the strains (e.g., phage type 80/81) are known to cause in the intertriginous skin folds, perineum, axillae, and vagina. hospital infections throughout the world. Such strains are known Approximately, 35–50% of normal adults carry S. aureus in the as “epidemic strains”. anterior nares, 10% in the perineum, and 5–10% in the vagina.  STAPHYLOCOCCUS 179 ◗ Specimens Specimens to be collected for demonstration of staphylococci depend on the nature of lesion (Table 23-6). ◗ Microscopy Demonstration of Gram-positive cocci arranged in clusters and pus cells in the Gram-stained smears of pus (Fig. 23-4, Color Photo 14), wound exudate, etc. are the characteristic features of pyogenic infection caused by S. aureus. It is noteworthy that microscopy: Alone is not adequate to differentiate various species of staphylococci or micrococci from one another. Is also of no value for sputum and other specimens where FIG. 23-3. Bacteriophage typing of staphylococci. mixed bacterial flora is present. Phage typing of human isolates of ◗ Culture TABLE 23-5 Staphylococcus aureus The identification of staphylococci is confirmed by culture and Group Phage other identification tests comprising a range of biochemical I 29, 52, 52A, 79, 80 and enzymatic tests followed by antibiotic sensitivity. The spec- Section III II 3A, 3C, 55, 71 imens are inoculated onto nutrient agar and blood agar and incubated at 37°C for 24 hours. On nutrient agar, large, circu- III 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 lar, smooth, convex, and glistening colonies showing golden- IV — yellow pigments can be observed. On blood agar, the colonies V 94, 96 show a zone of beta-hemolysis, which is not shown by any other Not allocated 81, 95 species of staphylococci. Specimens from heavily contaminated sources, such as vom- Chapter 23 itus and feces, are inoculated on selective media (e.g., manni- strain of S. aureus to be typed is inoculated on a nutrient agar tol salt agar or salt milk agar). These media inhibit growth of plate to produce a lawn culture. After drying the plate, various Gram-negative bacteria but allow the growth of staphylococci phages at their routine test dilution (RTD) are applied over and certain other Gram-positive cocci. marked squares on plate. Such plates are then incubated over- night at 30°C and observed for the presence or absence of lysis of the colonies by the phages. ◗ Identification of bacteria The phage type of a strain is known by designation of the The identifying features of S. aureus are summarized in phages that lyse it. Thus, if a strain is lysed by phages 83A, 84, Box 23-2. and 85, it is called phage type 83A/84/85. By this method, most of the strains of staphylococci can be classified and are divided Coagulase test into five lytic groups, while there are a few which cannot be Coagulase test is an important test carried out to detect S. classified and constitute the unclassified group (Table 23-5). aureus. The test is done in two ways: tube coagulase test and The national reference centre for staphylococcal phage typ- slide coagulase test. ing in India is located in the Department of Microbiology, Maulana Azad Medical College, New Delhi. ◗ Other typing methods Various specimens collected in TABLE 23-6 staphylococcal infections S. aureus has been classified into six biotypes (A, B, C, D, E, and F). Most human pathogenic strains belong to biotype A. Specimen Condition Other typing methods include (a) plasmid profile, (b) DNA Pus Suppurative lesions and fingerprinting, (c) ribotyping, and (d) PCR-based analysis of osteomyelitis genetic pleomorphism and (e) serotyping. Sputum Respiratory infections Blood Bacteremia Laboratory Diagnosis Feces and vomitus Food poisoning Laboratory diagnosis of staphylococcal infections is based on Urine Urinary tract infections the demonstration of staphylococci, in appropriate clinical Nasal and perineal swab Suspected carriers specimens, by microscopy and culture.  180 BACTERIOLOGY Human or rabbit plasma, which is rich in CRF, is used in the test. The plasma is collected in vials containing anticoagu- lants, such as oxalate, heparin, or EDTA. Citrated plasma is not used because if the specimen is con- taminated with Gram-negative bacilli, the latter may utilize the citrate and produce false positive reaction. Slide coagulase test: Slide coagulase test detects the bound coagulase or the clumping factor. The test is performed by mix- ing a dense suspension of the staphylococci with a loopful of undiluted rabbit plasma on a slide. In a positive test, clumping takes place within 10 seconds. Phosphatase test FIG. 23-4. Gram-stained pus smear showing staphylococci (⫻1000). The production of phosphatase can be demonstrated by culturing a mixed specimen on phenolphthalein phosphate agar and exposing the colonies to ammonium vapors. S. aureus Identifying features of colonies turn bright pink due to the release of phenolphthalein. Box 23-2 Staphylococcus aureus Novobiocin sensitivity 1. S. aureus are Gram-positive cocci arranged in irregular grape-like Novobiocin sensitivity is a simple disk diffusion test to differ- Section III clusters. 2. On nutrient agar, S. aureus colonies produce characteristic golden- entiate S. aureus from other staphylococci. This test is carried yellow colonies. out by using a 5-␮g novobiocin disk on an overnight culture of 3. On blood agar, S. aureus produces a clear zone of hemolysis staphylococci on Mueller–Hinton agar. Novobiocin sensitivity (beta-hemolysis). is shown by an inhibition zone of ⱖ16 mm. S. aureus is novobio- 4. S. aureus are coagulase positive. All coagulase-producing staphylo- cocci are, by definition, S. aureus. cin sensitive, while S. saprophyticus is novobiocin resistant. 5. S. aureus are phosphatase positive, DNAase positive, and mannitol positive. Polymyxin B resistance Chapter 23 6. S. aureus are novobiocin and polymyxin B sensitive. Polymyxin B sensitivity is again a simple disk diffusion test to differentiate S. aureus from other staphylococci. This is carried out by using a 300-U polymyxin B disk on an overnight culture of staphylococci on Mueller–Hinton agar. Polymyxin resistance Coagulase test is shown by an inhibition zone of ⬍10 mm. S. aureus is usually polymyxin resistant. Treatment Skin and soft tissue infections are treated best with local wound care with or without topical antibiotics (e.g., neomycin). Spontaneous or surgical drainage of pus and debride- ment of necrotic tissue is an effective mode for treatment of staphylococcal abscess. Systemic antibiotics are necessary for Positive Negative deep-seated and systemic infections. Key Points Benzyl penicillin is the drug of choice for penicillin- FIG. 23-5. Tube coagulase test. sensitive strains of S. aureus. Erythromycin, vancomycin, or first-generation cephalospo- rins are recommended for patients with allergy to penicillin. Tube coagulase test: Tube coagulase test is carried out to detect free coagulase. In this test, 0.1 mL of an overnight broth culture is mixed with 0.5 mL of a 1:10 dilution of human or rabbit plasma. The plasma-broth culture mixture is incu- ◗ Penicillin resistance in staphylococci bated in a water bath at 37°C for 3–6 hours. In a positive test, Penicillin resistance in the bacteria is increasingly recognized the plasma is coagulated and does not flow (Fig. 23-5, Color since 1945. Nearly 80% or more strains of S. aureus are resistant Photo 13). to penicillin. It is of three types:  STAPHYLOCOCCUS 181 1. Plasmid-mediated resistance: This type of resistance may be control of S. aureus infection. Treating with nasal creams contain- due to the production of enzyme penicillinase (beta-lactamase), ing neomycin or bacitracin prevents recurrent infections in cases which is plasmid mediated. This enzyme inactivates penicillin of nasal carriers of S. aureus. Topical application of antimicrobial by splitting the beta-lactam rings. Staphylococci produce four agents prevents dissemination of infection from the abscesses. types of penicillinases (A, B, C, and D). Penicillinase plasmids are transmitted to the staphylococci by both transduction and con- jugation. The plasmids also carry markers of resistance to heavy Coagulase-Negative Staphylococci metals, such as arsenic, cadmium, mercury, lead, and bismuth as well as to other antibiotics, such as erythromycin and fusidic acid. Coagulase-negative staphylococci (CONS) are the normal flora 2. Chromosomal-mediated resistance: This type of resis- of the skin. CONS are opportunistic bacteria. They cause infec- tance has also been documented. Reduction in the affinity of tions in debilitated or immunocompromised patients and in the penicillin-binding proteins (PBPs; present on the cell wall patients fitted with urinary catheters, cardiac valves, pacemak- of the staphylococci) to the beta-lactam antibiotics also con- ers, and artificial joints. tributes to the resistance of the bacteria to penicillins and other They form white nonpigmented colonies, morphologically beta-lactam antibiotics. similar to those of S. aureus. 3. Tolerance to penicillin: Staphylococci developing tolerance They are differentiated from S. aureus by their failure to coag- to penicillin are only inhibited but not killed. Penicillin-resistant ulate the plasma due to the absence of the enzyme coagulase. strains can be treated with beta-lactamase-resistant penicillins, e.g., oxacillin, flucloxacillin, cloxacillin, methicillin, or vancomycin. CONS of medical importance include (a) S. epidermidis, (b) S. saprophyticus, (c) S. haemolyticus, (d ) S. saccharolyticus, (e) S. hominis, ( f ) S. schleiferi, ( g) S. lugdunensis, and (h) Staphylococcus Section III ◗ Methicillin-resistant staphylococci simulans. Methicillin-resistant S. aureus (MRSA) denotes resistance of S. aureus to penicillin, as well as to all other beta-lactam anti- biotics including the third-generation cephalosporins and car- Staphylococcus epidermidis bapenems. Resistance to methicillin is due to the production S. epidermidis forms white colonies on blood agar. It is catalase of a novel PBP, designated as PBP 2a. PBPs are the targets of positive, coagulase negative, and does not ferment mannitol. It beta-lactam antibiotics. tolerates salt, survives drying, and is highly antibiotic resistant. Chapter 23 Infections caused by MRSA are being increasingly reported It is a normal skin commensal. Carriage rate is as high as 100%. worldwide since 1980. The infection is also being increasingly This bacterium is transmitted by self-inoculation or by contact reported now, from different hospitals. MRSA usually colo- with infected patients and hospital personnel. nizes the broken skin and can cause a wide range of local and Ability to produce slime is an important virulence factor of systemic staphylococcal infections. the bacterium. S. epidermidis causes infection by adhering itself Hospital staffs harboring MRSA are the chief source of to the surface of the intravenous plastic catheters and pros- infection for the patients. These strains can cause a wide range thetic devices. The adherence is believed to be facilitated by of infections including bacteremia, endocarditis, and pneu- polysaccharide glycocalyx known as slime, produced in large monia. These strains are increasingly recognized as important quantities by the bacteria. Slime also inhibits the action of lym- agents of hospital-acquired infection in hospitalized patients phocytes and neutrophils. S. epidermidis is an important agent undergoing prosthetic heart valve surgery. of hospital-acquired infection. It causes: MRSA strains can be treated with glycopeptide antibiot- infection in compromised hosts, such as neutropenic ics, such as vancomycin and teicoplanin in serious systemic patients, particularly in association with intravenous cath- infections, such as pneumonia, bacteremia, and endocarditis. eters and other prosthetic devices, such as heart valves. MRSA are sensitive to one or more of the second-line drugs, endocarditis in patients with prosthetic valves, intravenous which include erythromycin, clindamycin, quinolones, fusidic catheter infections, CSF shunt infections, catheter-associated acid, trimethoprim, chloramphenicol, tetracycline, and rifampi- peritonitis and endocarditis. cin. However, ciprofloxacin, rifampicin, and fusidic acid are not sepsis in neonates, osteomyelitis, wound infections, vascular used simply because of the possibility of emergence of resistance. graft infections, and mediastinitis. Proper hand-washing and use of topical agents, such as mupirocin and chlorhexidine on skin and nose to eradicate Vancomycin is the drug of choice for treatment of infection the agents are effective to prevent and control nosocomial caused by S. epidermidis. infections caused by MRSA. Staphylococcus saprophyticus Prevention and Control S. saprophyticus forms white colonies on blood agar. It is catalase There is no effective immunization with toxoids or bacterial positive, coagulase negative, and does not ferment mannitol. It vaccines against staphylococcal infection. Cleanliness, frequent normally inhabits the skin and genital mucosa. The bacterium hand-washing, and aseptic management of lesions help in the causes:  182 BACTERIOLOGY Differences between Staphylococcus Other Coagulase-Negative Staphylococci TABLE 23-7 aureus, Staphylococcus epidermidis, There are many other coagulase-negative staphylococci that and Staphylococcus saprophyticus have been reported recently to cause human infections. These Test S. aureus S. epidermidis S. saprophyticus include the following: Coagulase ⫹ ⫺ ⫺ 1. S. haemolyticus causes bacteremia, endocarditis, urinary Clumping factor ⫹ ⫺ ⫺ tract infection, and wound infection. Heat-stable nuclease ⫹ ⫺ ⫺ 2. S. saccharolyticus causes endocarditis. Urease Variable ⫺ ⫹ 3. S. hominis causes bacteremia in cancer patients. 4. S. schleiferi causes wound infections, bacteremia, and ␤-galactosidase ⫺ ⫺ ⫹ indwelling catheter infections. Alkaline ⫹ ⫹ ⫺ 5. S. lugdunensis causes endocarditis, peritonitis, osteomyeli- phosphatase tis, and breast abscesses. Polymyxin B Resistant Resistant Sensitive 6. S. simulans causes septicemia, osteomyelitis, and septic arthritis. Novobiocin Sensitive Sensitive Resistant Acid from mannitol ⫹ ⫺ ⫺ Acid from trehalose ⫹ ⫺ Micrococcus Acid from mannose ⫹ ⫹ ⫺ Micrococci in comparison to staphylococci are larger and PYR test ⫺ ⫺ ⫹ measure up to 2 ␮m in diameter. In smears, they appear as Gram-positive cocci arranged in tetrads. On culture, they pro- Section III duce colonies with yellow, pink, or red pigments. It is doubtful that they are human pathogens. Urinary tract infection by endogenous spread in colonized women. It adheres to the epithelial cells lin- ing the urogenital tract. It causes dysuria, pyuria, and Planococcus hematuria. Urethritis, catheter-associated urinary tract infections, pros- They are Gram-positive cocci most commonly found in seawater, Chapter 23 tatitis in elderly men, and rarely, sepsis and endocarditis. prawns, and shrimp. They are distinguished from other Gram- positive cocci by their ability to grow in a higher salt concentra- Urinary tract infection caused by S. saprophyticus can be tion of 12% sodium chloride. They are nonpathogenic to humans. treated with quinolones (such as norfloxacin) or with trimethoprim–sulfamethoxazole. S. epidermidis and S. saprophyticus are distinguished from each Stomatococcus other by their reaction to antibiotic novobiocin—S. epidermidis is sensitive, while S. saprophyticus is resistant. The differences They are capsulated Gram-positive cocci arranged in pairs or between S. aureus, S. epidermidis, and S. saprophyticus are summa- clusters. On culture, they produce white and mucoid colonies. rized in Table 23-7. It is doubtful that they are human pathogens. CASE STUDY A group of 25 students of 11–12 years of age studying in a higher secondary school in Haripur were admitted to a hospital with complaints of severe vomiting and diarrhea within 3 hours of consuming the food prepared in their school. History revealed that a new cook appointed a few days back in the school prepared the food. All the students consumed the same food. Children were treated and were discharged after observing them overnight in the hospital. What is the possible cause of this food poisoning? What is the possible reservoir for this organism that was responsible for this outbreak? What are the tests you will perform to establish the etiological diagnosis of the condition? What steps you will take to prevent this infection?