Staphylococcus and Micrococcus PDF

The Gram-Positive Cocci of Medical Importance Maria Cyril Del Villar - Dalusong, RMT, MSMLS Staphylococci  First observed in human pyogenic lesions by von Recklinghausen in 1871. It was Sir Alexander Ogston, a...

The Gram-Positive Cocci of Medical Importance Maria Cyril Del Villar - Dalusong, RMT, MSMLS Staphylococci  First observed in human pyogenic lesions by von Recklinghausen in 1871. It was Sir Alexander Ogston, a Scottish surgeon, who established Introducti conclusively the causative role of the coccus in abscesses and on other suppurative lesions (1880). He also gave it the name Staphylococcus (Staphyle, in Greek, meaning ‘bunch of grapes’: kokkos, meaning a berry) due to the typical occurrence of the cocci “in grape- like clusters” in pus and in cultures.  Contains 33 defined species and 20 species found in man. Species Species of staphylococci are initially differentiated by the coagulase test and are classified into two groups: Coagulase-negative Coagulase-positive staphylococci staphylococci: (CONS): S. staphylococcus aureus epidermidis and S. (formerly also called saprophyticus are the Staph. pyogenes) is most clinically coagulase-positive. significant species in this group. Staphylococci General Characteristics Common inhabitant of the skin and mucous membranes Spherical cells arranged in irregular clusters Gram-positive, exhibit spherical cells (0.5 to 1.5 µm) © Eye of Science/Photo Researchers, Inc. © David M. Phillips/Visuals Unlimited Lack spores and flagella Views of S. aureus shape and May have capsules arrangement. Staphylococci General Characteristics Nonmotile, non–spore-forming, and aerobic or facultatively anaerobic, although a few strains can be obligate anaerobes Colonies produced after 18 to 24 hours of incubation are medium sized (4 to 8 mm) and appear cream-colored, white or rarely light gold, and “buttery-looking.” Rare strains of staphylococci are fastidious, requiring carbon dioxide, hemin, or menadione for growth. so-called small colony variants (SCVs) grow on media containing blood, forming colonies about one tenth the size of wild-type strains even after 48 hours or more of incubation. Staphylococci General Characteristics Some species are β-hemolytic. Are common isolates in the clinical laboratory and are responsible for several suppurative infections. Are normal inhabitants of the skin and mucous membranes of humans and other animals. Resemble some members of the family Micrococcaceae, such as the genus Micrococcus. Micrococci General Characteristics Catalase-producing, coagulase- negative Gram-positive cocci found in the environment and as members of the indigenous skin microbiota. Often recovered with staphylococci and can be differentiated easily from coagulase-negative staphylococci (CoNS) Some micrococci tend to produce a yellow pigment Other gram-positive cocci occasionally recovered from human clinical specimens include Rothia mucilaginosa, Aerococcus, Alloiococcus otitis (recovered from human middle ear fluid). Coagulase Positive Coagulase producing (coagulase-positive) staphylococci are: S. aureus, S. intermedius, S. pseudintermedius, S. hyicus, S. delphini, S. lutrae, S agnetis, and some strains of S. schleiferi. Isolates such as S. lugdunensis and S. schleiferi also can be occasionally mistaken for coagulase-positive staphylococci because of the presence of clumping factor. often animal-associated species and are less frequently isolated Majority of clinical staphylococcal isolates that are  Staphylococcus aureus most clinically significant species Morphology Spherical cocci, approximately 1 µm in diameter, arranged characteristically in grape-like clusters  cluster formation is due to cell division occurring in three planes, with daughter cells tending to remain in close proximity. May also be found singly, in pairs, and in short chains of three or four cells, especially when examined from liquid culture.  Long chains never occur. Nonspore forming, nonmotile and usually noncapsulated with the exception of rare strains. Stain readily with aniline dyes and are uniformly gram- positive.  Cultural Characteristics Cultural Characteristics Aerobes and facultative anaerobes. Optimum temperature for growth is 37°C ( range being 12–44°c). Optimum pH is 7.5. They can grow readily on ordinary media. Nutrient agar: Colonies are 1–3 mm in diameter and have a smooth glistening surface, an entire edge, a soft butyrous consistency and an opaque, pigmented appearance. Most strains produce golden-yellow (aureus) pigment. White-colony strains of S. aureus are fully virulent. Pigmentation is characteristic of this species when grown aerobically.  Pigmentation is enhanced on fatty media, such as tween agar, by prolonged incubation, and by leaving plates at room temperature.  The pigment is believed to be lipoprotein allied to carotene. Blood agar: Colonies have the same appearances as on nutrient agar, but may be surrounded by a zone of ß-hemolysis Hemolysis is more likely to be present if sheep, human or rabbit blood is used. Milk agar: Colonies are larger than those on nutrient agar and pigmentation is well- developed and easily recognized against the opaque white background. Skim milk agar cultured with S. aureus which appeared as glistening orange convex colonies (due to Staphyloxanthin production) with protease activity (shown as hydrolyzed clear zone surrounding the colonies) Phenolphthalein Phosphate Agar: An indicator medium and assists in the identification of S. aureus in mixed cultures. Colonies is similar to those on nutrient agar. Colonies become bright pink when culture plate is inverted over ammonia for a minute or so. Selective Salt Media: May be useful for the isolation and enumeration of staphylococci from materials, such as feces, food and dust, likely to contain a predominance of other kinds of bacteria. 7–10% of sodium chloride may be added to nutrient agar (Salt agar) or milk agar (salt milk agar); mannitol salt agar containing 1% mannitol, 7.5% NaCl, and phenol red in nutrient agar; and Ludlam’s medium containing lithium chloride and tellurite; and salt cooked meat broth (10% NaCl.). Biochemical Reactions Sugar fermentation: Ferments a range of sugars producing acid but no gas. Sugar fermentation is of no diagnostic value except for mannitol, which is usually fermented anaerobically by staph. S. aureus but not by other species. Catalase: Catalase positive (unlike streptococci). Coagulase: Lipolytic Characteristic: When grown on media containing egg- yolk, produce a dense opacity because most strains are lipolytic. Phosphatase Test : Useful screening procedure for differentiating S. aureus from S. epidermidis in mixed cultures, as the former gives prompt phosphatase reaction, while the latter is usually negative or only weakly positive. All strains of S. aureus produce phosphatase which liberates phenolphthalein from sodium phenolphthalein diphosphate S. Aureus become bright pink because phenolphthalein is pink in alkaline ph. Most other staphylococci form colonies that remain uncolored. Deoxyribonulease (DNase) Test : Produces a deoxyribonulease (DNase), and a heat-stable nuclease (thermonuclease). Other Biochemical Tests: Resistance S. aureus and the other micrococcaceae are among the hardiest of the non spore forming bacteria. They retain their viability for 3–6 months.  They have been isolated from dried pus after 2–3 months. It withstands moist heat at 60°C for 30 min but is killed after 60 min. Most strains grow in the presence of 10% NACl.  These features are of significance in food preservation. Resistance Readily killed by phenolic and hypochlorite disinfectants, and by antiseptic preparations as hexachlorophane, chlorhexidine and povidone iodine. Very sensitive to aniline dyes; thus they are inhibited on blood agar medium containing 1 in 500,000 crystal violet, which permits the growth of streptococci. Are uniformly resistant to lysozyme but some micrococci are sensitive to it.  Staphylococci are generally sensitive to lysostaphin—a mixture of enzymes produced by a particular strain of S. epidermidis. Antigenic Structure A. Cell-associated Polymers 1. Capsule: Capsular polysaccharide surrounding the cell wall inhibits opsonization. 2. Peptidoglycan: Polysaccharide peptidoglycan confers rigidity and structural integrity to the bacterial cell. It activates complement and induces release of inflammatory cytokines. 3. Teichoic acids: An antigenic component of the cell wall, facilitates adhesion of the cocci to the host cell surface and protects them from complement-mediated opsonization. B. Cell Surface Proteins 4.Protein A: a group-specific antigen unique to S. aureus strains.  Has many biologic properties including chemotactic, anticomplementary, and antiphagocytic and elicit its hypersensitivity reactions and platelet injury.  It is mitogenic and potentiates natural killer activity of human lymphocytes. B. Cell Surface Proteins Cytoplasmic membrane: serves as an osmotic barrier for the cell and provides an anchorage for the cellular biosynthetic and respiratory enzymes. Clumping factor (bound coagulase): component on the cell wall of S. aureus that results in the clumping of whole staphylococci in the presence of plasma (also called bound coagulase). Toxins and Enzymes A. Toxins - 1. Cytolytic Toxins : At least five cytolytic or membrane- damaging toxins are produced by S. aureus I. Alpha II. Beta III. Delta IV. Gamma V. Panton-Valentine [P-V] leukocidin. 2. Enterotoxins Responsible for the manifestations of staphylococcal food poisoning , nausea, vomiting and diarrhea 2–6 hours after consuming contaminated food containing preformed toxin. Commonly produced by about two-thirds of S. aureus strains, growing in carbohydrate and protein foods. Relatively heat stable, resisting 100°C for 10–40 minutes depending on the concentration of the toxin and nature of the medium. 2. Enterotoxins 8 serologically distinct staphylococcal enterotoxins (A-E, G-I) and 3 subtypes of enterotoxin C have been identified. Enterotoxin A is most commonly associated with disease. Enterotoxins C and D are found in contaminated milk products, and enterotoxin B causes staphylococcal pseudomembranous enterocolitis. Detection of the toxin sensitive serological tests, such as latex agglutination and ELISA are available. Toxin is potent, microgram amounts being capable of causing the illness. 3. Toxic Shock Syndrome Toxin-i (TSTT-1) Severe and often fatal disorder characterized by multiple organ dysfunction. (formerly called pyrogenic exotoxin c and enterotoxin F) is heat and proteolysis resistant, chromosomally mediated exotoxin. Is antigenic and most persons over 30 years of age have circulating antibodies. The enterotoxins and TSST-1 belong to a class of polypeptides known as superantigens which are potent activators of T lymphocytes leading to the release of cytokines such as interleukins and tumor necrosis factor. 4. Epidermolysis Toxins (Exfoliative Toxins) Also known as ET or ‘exfoliatin’ is responsible for the ‘staphylococcal scalded skin syndrome’ (SSSS). Two distinct forms of exfoliative toxin (ET-A and ET- B) have been identified, and either can produce disease.  ET-A is heat-stable and the gene is chromosomal, whereas ET-B is heat labile and plasmid-mediated. Seen mostly in young children and only rarely in older children and adults. B. Extracellular Enzymes Produces a number of enzymes such as: I. Coagulase catalase II. Hyaluronidase III. Fibrinolysin IV. Lipases V. Nucleases VI. Penicillinase. Epidemiology A normal component of man’s indigenous microflora and is carried asymptomatically in a number of body sites. About 10–30% healthy persons carry staphylococci in the nose and about 10% in the perineum and also on the hair. Vaginal carriage is about 5–10%, which rises greatly during menses, a factor relevant in the pathogenesis of TSS related to menstruation. Staphylococcal Diseases A. Cutaneous infections: Include wound and burn infection, pustules, furuncles or boils carbuncles, sty, impetigo and pemphigus neonatorum. B. Deep Infections: Include osteomyelitis, periostitis, tonsillitis, pharyngitis, sinusitis, bronchopneumonia, empyema, septicemia, meningitis, endocarditis, breast abscess, renal abscess and abscesses in other organs. C. Toxin-mediated Diseases 1) Food poisoning: food poisoning may follow 2–6 hours after the ingestion of food in which S. aureus has multiplied and formed enterotoxin. 2) Toxic shock syndrome (TSS): Toxin producing strains of S. aureus have been implicated in most cases of TSS, a multisystem disease that primarily afflicts young women. Most cases occur in menstruating women who use tampons. C. Toxin-mediated Diseases 3) Exfoliative diseases: lesions are produced by the strains of S. aureus which produce epidermolytic toxins. Responsible for the ‘staphylococcal scalded skin syndrome’ (SSSS), exfoliative skin diseases in which the outer layer of epidermis gets separated from the underlying tissues. Severe form of SSSS is known as Ritter’s disease in the newborn and toxic epidermal necrolysis in older patients. Milder forms are pemphigus neonatorum and bullous impetigo.  Bullous impetigo is a localized form of SSSS Laboratory Diagnosis 1. Specimens: Specimens to be collected depend on the type of lesion  Example: pus from suppurative lesions; sputum from respiratory infections; food remains and vomit from cases of food poisoning; nasal and perineal swabs from suspected carriers. Swabs of the perineum, pieces of hair and umbilical stump—may be necessary in special situations. 1. Specimen - Direct microscopy: Gram stained smears is useful in the case of pus, where cocci in clusters may be seen. This is of no value for specimens like sputum where mixed bacterial flora are normally present. 2. Culture: Cultured on a blood agar plate.  On blood agar plate, look for hemolysis around the colonies.  Plates are inspected for golden-yellow or white colonies. Selective media like Ludlam’s or Salt-milk agar or Robertson’s cooked meat medium containing 10% sodium. Smears are examined from the culture and coagulase test done when Stahylococci are isolated. 3. Identification: Simple biochemical tests can be used to differentiate S. aureus and the other staphylococci. I. Positive reactions for coagulase (clumping factor) II. heat-stable nuclease III. alkaline phosphatase IV. mannitol fermentation 4. Antibiotic Sensitivity Tests: As a guide to treatment antibiotic sensitivity tests should be performed appropriate to the clinical situation. This is important as staphylococci readily develop resistance to drugs. 5. Bacteriophage Typing: May be done if the information is desired for epidemiological purposes. Other typing methods include: I. Antibiogram pattern II. Plasmid profile III. DNA fingerprinting IV. Ribotyping V. PCR-based analysis for genetic pleomorphism. 6. Serological Tests: Sometimes be of help in the diagnosis of hidden deep infections. Anti-staphylolysin (anti- alphalysin) titers of more than two units per ml, especially when the titer is rising, may be of value in the diagnosis of deep seated infections, such as bone abscesses. Treatment Benzyl penicillin is the most effective antibiotic, if the strain is sensitive Cloxacillin, oxacillin, flucloxacillin are penicillinase resistant penicillin.  Methicillin-resistant Staphylococcus aureus (MRSA) are also resistant to other penicillin and cephalosporins. Glycopeptides (vancomycin or teicoplanin) are the agents of choice in the treatment of systemic infection, but these agents are expensive and may be toxic. Treatment For mild superficial lesions, topical applications of drugs as bacitracin, chlorhexidine or mupirocin may be sufficient. The treatment of carriers is by local application of antibiotics such as bacitracin and antiseptics (chlorhexidine). In resistant cases, rifampicin along with another oral antibiotic may be effective.  Other Coagulase- positive Staphylococci  Other staphylo - coagulase producing (coagulase positive) staphylococci are; I. S. intermedius II. S. delphini III. S. lutrae IV. Some strains of S. hyicus. These are often animal-associated species and are infrequently isolated from human samples. Coagulase-negative Staphylococci (CONS) Staphylococcus epidermidis Invariably present on normal man skin. It is nonpathogenic ordinarily but can cause disease when the host defenses are breached. Has a distinct fondness for foreign bodies, such as artificial heart valves, indwelling intravascular catheters, central nervous system shunts, and hip prostheses. Their etiological role is proved by repeated isolation. Staphylococcus saprophyticus A common cause of urinary tract infections in sexually active young women. It may also cause urethritis in men and women, catheter associated urinary tract infections, prostatitis in elderly men, and rarely bacteremia, sepsis and endocarditis. Can be distinguished from S. epidermidis by its resistance to novobiocin and by its failure to ferment glucose anaerobically. It is nonhemolytic and does not contain protein A. Novobiocin sensitivity test S. epidermidis: white colonies, non- hemolytic S. saprophyticus: may be white or yellow, non-hemolytic. Other Coagulase- negative Staphylococci  S. haemolyticus has been reported in wounds, bacteremia, endocarditis, and UTIs. Other species include: I. S. lugdunensis II. S. warneri III. S. capitis IV. S. simulans, V. S. schleiferi. Sensitivity to Antibiotics 1. Production of Beta Lactamase (Penicillinase): Inactivates penicillin by splitting the beta lactam ring. Staphylococci produce four types of penicillinases, A to D. An inducible enzyme and its production is usually controlled by plasmids which are transmitted by transduction or conjugation.  Penicillinase plasmids are transmitted to the sensitive staphylococci by transduction and also possibly by conjugation. 2. Changes In Bacterial Surface Receptors: Reducing binding of beta-lactam antibiotics to cells. This change is normally chromosomal in nature and is expressed more at 30°C than at 37°C. This resistance also extends to cover beta lactamase resistant penicillin, such as methicillin and cloxacillins. May show resistance to other antibiotics and heavy metals also and cause outbreaks of hospital infection.  Strains have been called ‘epidemic methicillin- resistant Staphylococcus aureus’ or EMRM. 3. Development of Tolerance: Development of tolerance to penicillin, by which the bacterium is only inhibited but not killed. Methicillin-resistant Staphylococcus aureus (MRSA) Methicillin resistant strains of S. aureus (MRSA) became common, which were resistant not merely to penicillin, but also to all other beta lactam antibiotics. Isolates that are resistant have been traditionally termed methicillin-resistant staphylococci, with S. aureus being called MRSA and S. epidermidis referred to as MRSE. Methicillin-resistant Staphylococcus aureus (MRSA) Any staphylococcus isolated is identified as being resistant to methicillin, this implies that it is also resistant to nafcillin and oxacillin and to antibiotics, including the cephalosporins. Glycopeptides (vancomycin or teicoplanin) are the agents of choice in the treatment of systemic infection, but these agents are expensive and may be toxic. MRSA - Laboratory Diagnosis Oxacillin is generally used for detection of methicillin resistance.  The use of an oxacillin-salt agar plate, such as the oxacillin resistance screening agar can be used as a screening test for MRSA in clinical samples.  A high salt concentration (5.5% NACl) and polymyxin B make the medium selective for staphylococci. Gold standard for MRSA detection is the detection of the meca gene by using nucleic acid probes or polymerase chain reaction (PCR) amplification. Micrococci Introduction: Catalase-positive, gram-positive, coagulase- negative and usually oxidase-positive. They may occasionally colonize the skin or mucous membrane of humans, but they are rarely associated with infections. Only two species, Micrococcus luteus and Micrococcus lylae, remain in the genus. M. Luteus, have a tendency to produce a yellow pigmented colony.  Nine species of genus micrococcus have been described.  Micrococcus luteus, Micrococcus lylae,