Staphylococcus and Related Organisms Ch 11, pg 539

Staphylococcus Micrococcus Rothia

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Preliminary Grouping of Gram Positive Cocci

Gram Positive Coccus
Catalase + Salt Tolerant Yes Facultative Yes Staphylococcus No _ Streptococcus Group

Rothia No
Micrococcus
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General characteristics
The Staphylococci belong to the family Micrococcocaceae (a.k.a. Staph group) Related bacteria encountered in human clinical specimens include Staphylococcus, Micrococcus, and Rothia mucilaginosus (formerly Stomatococcus mucilaginosus) At this time, there are ~40 recognized species of the genus Staphylococcus There are only a few recognized species of Micrococcus and of Rothia
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General characteristics
The Staph group is comprised of Grampositive cocci Based on planes of division they form various groupings which include clusters, tetrads, pairs, and even short chains When seen in pairs the longer axes are parallel rather than perpendicular (mutual)
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General characteristics
Species of the genus Staphylococcus divide in random planes and tend to form irregular clusters (Staph=grape-like clusters, coccus=round) Micrococcus species first divide in parallel planes and them perpendicular to that resulting in predominance of tetrads Rothia species form short chains and small clusters. Here, the longer axes are perpendicular rather than rather than parallel.
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Staph clusters Micrococcus tetrads

Rothia

General characteristics
Staphylococci have a thick multilayered peptidoglycan as the major component of their cell walls, the same as all Gram-positive bacteria Staphylococcus species contain teichoic acids in their cell walls Teichoic acids stabilize the cell wall, hold association with cell membrane, function in transport, etc. Cell walls of Micrococcus & Rothia species do not contain teichoic acids

General characteristics
Members of the staph group are mostly nonencapsulated and all clinically relevant ones are nonmotile All Staph group organisms that grow in air are catalase positive

Rothia is catalase negative (or weakly positive).

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General Clinical Significance

Staphylococcus aureus is the most virulent pathogenic species in the group It is implicated in a variety of infections including skin, respiratory tract, post-op infections and other systemic infections such as TSS. S. aureus also causes food poisoning. All Staph group species, including S. aureus, can lead a commensal existence (as an opportunistic pathogen) in the skin and mucous membranes of humans and many other animals, including domestic pets and farm animals.
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General Clinical Significance

Staphylococcal infections are generally acute, often involve inflammation and suppuration (fluid product of inflammation)

A small percentage of staphylococcal infections spread hematogenously to all regions of the body = systemic = bacteremia, and likely toxemia.
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 In addition to the coagulase positive S. aureus the most clinically significant species are: S. epidermidis S. saprophyticus Coagulase negative S. haemolyticus S. lugdunensis S. schleiferi S. intermedius Coagulase positive S. hyicus
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General Clinical Significance not on test

General Clinical Significance

S. intermedius is most often an inhabitant of dogs and S. hyicus is an inhabitant of swine S. intermedius infections in humans are usually associated with dog bites (and are probably identified incorrectly as S. aureus since the human clinical laboratories rely primarily on the coagulase test) Micrococcus species and R. mucilaginous are rarely pathogenic but must be differentiated from phenotypically similar Staphylococcus species
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General Growth Characteristics

Staphylococcus species are generally nonfastidious They grow well on media without blood or other special supplements Micrococcus species and R. mucilaginosa are mildly fastidious and grow more slowly than Staphylococcus sp.

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General Growth Characteristics

Staphylococcus and Micrococcus tolerate a high salt concentrations; they grow on media containing 5%-7.5% NaCl (e.g. Mannitol Salt Agar) Staphylococcus species are facultatively anaerobic, as is Rothia. Micrococcus is an obligate aerobe. Staphylococcus species produce a variety of hemolysins and other toxins
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General Cultural Characteristics

Some Staphylococcus species, most notably S. aureus, produce a hemolysin that completely lyses red blood cells of humans and some other mammals (sheep blood). This is referred to as beta hemolysis. None are alpha hemolytic (removal of potassium from RBCs) as is Streptococcus pneumoniae Morphology (color, shape, surface, etc) of staph colonies may change with age
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General Cultural Characteristics

Staphylococcus colonies have a convex profile and color ranges from white (ex: S. epidermidis, S. saprophyticus) to yellowish brown (S. aureus) Micrococcus colonies are usually not as shiny as Staphylococci and have a high convex profile The most commonly isolated Micrococcus species, M. luteus, is yellow
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General Cultural Characteristics

Colonies of R. mucilaginosus are distinctly convex and gray-white They usually are not as shiny as Staphylococcus Colonies of R. mucilaginosus are described as having a gumdrop consistency; they tend to adhere to an agar surface R. mucilaginosus is very difficult to emulsify when preparing smears for staining

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General Cultural Characteristics

Many strains of S. aureus have a golden color (aureus=golden) on certain media after prolonged incubation This pigmentation is usually more pronounced when the culture is kept at room temperature for several days This golden color is not unique to Staphylococcus aureus Additionally, many S. aureus isolates are white Pigmentation, except for the most common species of Micrococcus ( M. luteus), is therefore not a reliable criterion for identifying the Staphylococci
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Biochemical characteristic
Production of catalase is a defining characteristic of the Staphylococcus and Micrococcus species The catalase reaction of Rothia is weak or delayed and many are frankly catalase negative

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Biochemical characteristic
Staphylococcus species can be differentiated from Micrococcus species based upon oxygen requirements: Staph is facultative and Micrococcus species are obligate aerobes Rothia can be differentiated from Staphylococcus and Micrococcus by its lack of growth on a highsalt medium, its negative catalase reaction, and its tendency to adhere firmly to an agar surface

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Presumptive Genus Identification table not on test

6.5% Furazoli- Modified 0.04 U Salt done oxidase Bacitracin Micrococcus + R + S Staphylococcus + S R R. mucilaginosa NT NT NT
Not included: oxygen requirements

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Coagulase
Coagulase (staphylocoagulase) is a fibrinogen activating enzyme produced by some staph species - it has thrombin-like activity. In situ, coagulase combines with coagulase reacting factor (CRF) to catalyze the formation of fibrin clots around cells as a barrier to host immune components it is a virulence factor. Clinically significant staphylococci are usually divided into two groups: those that produce coagulase and those that do not Coagulase positive species include S. aureus, S. intermedius and S. hyicus S. intermedius and S. hyicus mostly inhabit animals and are only rarely found as a cause of human infections
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Coagulase
Normal coagulation of plasma Thrombin Fibrinogen (soluble )

Fibrin (insoluble)

Staphylococcus aureus coagulation of plasma* Free staphylocoagulase

Fibrinogen + CRF Fibrin

*EDTA Rabbit plasma is preferred for the free or tube coagulase test because it contains a large amount of CRF
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Coagulase testing
Since staphylocoagulase is synthesized and secreted into the medium in which it is growing, it is known as free coagulase (i.e. it is not bound to the cell that secreted it) The free coagulase test is performed by mixing a Staphylococcus colony or growth from a broth with a small amount of plasma in a test tube It is therefore usually referred to as the tube coagulase test (a free coagulase test and a tube coagulase test is the same thing)
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Coagulase testing
Rabbit plasma is preferred for the free (tube) coagulase test because it has more CRF than plasma from other species of animals including human plasma Cells of the test organism is mixed with rabbit plasma in a test tube and incubated at 35oC for up to 4 hours

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Coagulase testing
The tube is observed hourly during the four hour incubation period The formation of a fibrin clot or gel indicates a positive test

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Coagulase testing
S. aureus is the only coagulase positive species

found in human clinical specimens with any frequency (see previous discussion) In addition to staphylocoagulase some strains of S. aureus will produce staphylokinase (fibrinolysin) This enzyme will dissolve a fibrin clot (i.e. will have the opposite effect of coagulase)

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Coagulase testing
This could be a cause of false negative coagulase tests if tubes are not examined regularly over the four hour period If the plasma gels before 4 hours the test is read as positive and discarded Only catalase positive Gram positive cocci should be tested for tube coagulase as some other organisms such as Enterobacter & Klebsiella can give false positive results.
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Coagulase testing
Using rabbit plasma containing EDTA as anticoagulant will avoid the false positive tube coagulase tests caused by citrate consuming bacteria This will not be a problem if only catalase positive Gram-positive cocci are tested for coagulase

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Clumping Factor
95% of S.aureus isolates produce a separate enzyme that catalyzes the formation of fibrin from fibrinogen This enzyme is referred to as bound coagulase because it is an integral part of the cell wall of S. aureus Bound coagulase is not secreted into the surrounding medium Unlike free coagulase this enzyme does not require CRF in the plasma substrate
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Clumping Factor
It is also called slide coagulase or the clumping factor test. A clumping factor test, a bound coagulase test, and a slide coagulase test are synonymous. This is because the test is performed on a slide and the end point is the clumping of a heavy water or saline suspension of bacteria taken from an agar culture A loopful of plasma is thoroughly mixed with the heavy bacterial suspension on a slide and observed for clumping 31

Clumping Factor
Clumping of the suspension within 10 seconds indicates a positive test Human plasma is preferable to rabbit plasma for the slide coagulase test because it yields more consistent results. Note Your textbook indicates that rabbit plasma is used for
both coagulase test; this is contrary to the Staphylococcus chapter in the bible (The Manual of Clinical Microbiology published by The American Society for Microbiology)
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Clumping Factor
The heavy cell suspension is first made in water or saline If clumping occurs prior to the addition of plasma this invalidates the test (false positive). Such strains are termed autoagglutinable and must be tested by the tube method. Since 5% of S.aureus produce free coagulase but not bound coagulase, an organism giving a negative clumping factor test must still be tested by the tube test
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Protein A
Protein A is unique to, and is an integral part of the cell wall surface of S. aureus. Protein A has an anti-phagocytic property (a virulence factor). Protein A also has the unusual ability to bind specifically to Fc fragments of IgG (a sort of antigen-antibody reaction) from several species of animals, including Homo sapiens. This makes it well adapted for another test

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Protein A clumping test

A diagnostic tool utilizes IgG adsorbed to some visible inert particle (such as latex beads) forming the basis of another clumping test for identifying S. aureus called the protein A clumping test. If you remember from Micro, this is an example of what we called an agglutination-type test. IgG coated particles are mixed with cells taken from an agar culture. The complex forms within 10 seconds in the presence of S. aureus appearing as large granular-firm (not stringy) clumps This test is claimed to be about 99% specific and sensitive for S. aureus
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Protein A Latex Test

S IgG L L

S
S S Fc L S L S S=S.aureus with Protein A L=Latex particle
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Protein A S

Protein A/Clumping factor

Some reports in the literature indicate that an occasional methicillin resistant S. aureus (MRSA) will give a negative protein A/clumping factor test Authors of these reports recommend that isolates that resemble colonies of S. aureus giving a negative protein A/clumping factor test and are also resistant to methicillin be tested by the tube coagulase method The Protein A test should only be used on isolates that presumptively ID (morphology, catalase, salt tollerance, mannitol fermentation, etc) as S. aureus since some organisms give a false positive reaction (Enterococcus, Micrococcus and rare strains of Staphylococcus saprophyticus). These false positive reactions are slower to develop, with clumps that are smaller and have a stringy consistency. A tube coagulase test should be ran on all presumptive S. aureus that give a negative Protein A test.
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Other S. aureus Characteristics

Mannitol fermentation is another useful characteristic it is unique to, and consistent among S. aureus strains. Virtually all strains of S. aureus ferment mannitol. Bright yellow colonies on a yellow background indicates mannitol fermentation on mannitol salt agar.

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Other S. aureus Characteristics

Another characteristic of most S. aureus cultures is the production of a heat stable enzyme that hydrolyzes RNA or DNA - a nuclease. Nucleases are heat stable, able to withstand 15 minutes of boiling water. Testing to see if the boiled growth medium contains an active enzyme that hydrolyzes DNA or RNA is called a thermonuclease test Nucleases that may be produced by most Staphylococcus species other that S. aureus are not stable after boiling (if they do produce a nuclease it is not resistant to heat)
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S. aureus diagnosis - summary

Clinical samples rarely contain pure cultures - should be assumed mixed. For this reason, culture on a selective medium for Gram positive bacteria such as Mannitol salt agar (MSA) or ColistinNalidixic Acid Agar (CNA). MSA: notice growth and agar turns yellow around colonies. Also conduct primary culture on sheep blood agar (SBA). See colonies that progress from small, covex and off-white to larger flatter opaque porcelain golden yellow colonies. Notice beta hemolysis. Innoculate plate by rolling swab (if culture is on swab) on surface of agar in the first quadrant, then streak the remaining quadrants for isolation with loop. Incubate 35oC for 24 hours. Preliminary ID: catalase positive, gram-positive coccus in tetrads and clusters. Additional: characteristic behavior on media and positive coagulase test. Can also use automated ID system and serological methods.
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Pathology - predisposing factors

This list is virtually true for all pathogens Immune system suppressed or otherwise compromised. Specifically Skin injuries (e.g. burns, surgical incisions, cuts, etc) Presence of foreign bodies (e.g intravenous lines, prosthetic devices, sutures, tampons-TSS) Pre-existing infections Chronic underlying conditions (e.g. auto-immune conditions, malignancies, alcoholism, heart disease, etc.) Compromised microbiota via antimicrobial therapy Infants susceptible: oral, skin: impetigo, scalded skin, respiratory, other
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S. aureus is a problem
S. aureus has been generally considered the #1 human pathogen since the 1980s. Why:
It is everywhere Nosocomial: big problem in hospitals Antibiotic resistance: 25% increase in MRSA isolates from 87-97 ONLY resistant to vancomycin Lots of toxins Good at immune evasion, rapid growth & spread = bacteremia Numerous types of infections
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Upper respiratory tract

Many bacterial species and viruses alike cause some manner of upper respiratory tract (URT) infection. S. aureus URT infection is fairly common with strep throat-like symptoms. Can coreside with S. pyogenes or respiratory viruses such as influenza or RSV. Often causes secondary infections following respiratory viral infection. Uncommon cause of accute sinusitis and otitis media
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Lower respiratory tract

S. aureus is an uncommon cause of community acquired pneumonia (both primary and secondary) which is a lower respiratory tract (LRT) condition, although nosocomial cases are not uncommon. In fact, a CDC study in 1990 said S. aureus was the #1 cause of nosocomial pneumonia! These cases have a high mortality rate due to the immune compromised status of the patient, and high degree of antibiotic resistance of strains in the hospital. Comments made above about S. aureus co-residing and secondary infections apply here as well. Over 50% of community acquired typical bacterial pneumonias are caused Streptococcus pneumoniae - #1 cause
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Lower respiratory tract

It is sometimes difficult to establish the cause of an LRT without the use of an invasive procedure because so many species of bacteria are picked up from the URT. Non-bacterial LRTs:
viruses are not an infrequent cause of LRTs fungal pathogens, especially Aspergillus sp,, and Pneumocystis carinii are found in immunocompromised patients
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Integument / wounds
S. aureus is a common resident of the skin and exposed mucus membranes: respiratory, genitourinary & gastrointestinal. S aureus is the most common cause of pathogenic integument infection in humans. S. aureus is the #1 cause of post-operative infection, whether it be introduced during the course of the operation or afterward. These initial infections often become systemic and have high mortality rates

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Integument / wounds
Less severe integumentary cases include styes, pimples, folliculitis, and other localized absecces. Folliculitis (infected hair follicles) can become more deep seated causing a furuncle (a.k.a. boil). Multiple furuncles coalesce into a carbuncle. In severe, case S. aureus can spread hematogenously from here to any body site. S. aureus & S. pyogenes cause impetigo, the most common skin infection in children highly contagious Also causes scalded-skin syndrome (Ritters syndrome) in infants via production of an exfoliating toxin fairly rare, at least in US
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Food poisoning
S. aureus is the #1 most common cause of food poisoning although it is comparatively mild in most cases. Symptoms include nausea, vomiting, diarrhea, abdominal cramping and mild fever. Symptom onset can be within minutes or hours of ingestion, with similar duration Foods: handled foods: wet, sugary or salty, handled after some preparation cooked, mixed, then served cold, at least initially
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S. aureus other pathology

*S. aureus is the classic cause of toxic shock syndrome, a highly acute and highly toxigenic condition super-infection & super-antigens result in organ destruction, shock, hypotension and death

Acute or chronic osteomylitis, mainly in children Endocarditis Septic arthritis Mastitis Meningitis Phlebitis / thrombophlebitis (clotting)
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Virulence Factors of S. aureus

S. aureus possesses many properties that contribute to its ability to cause disease Not all strains of S. aureus possess all of these virulence factors but most possess several: Capsule: Like many strains of S. epidermidis, S. aureus produces a slime layer that adheres firmly to plastic prosthetic devices like catheters, shunts, and plastics bags used for continuous ambulatory peritoneal dialysis (CAPD). Capsules are also anti-phagocytic
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 Protein A: because protein A binds to the Fc fragment of IgG, this can interfere with IgGs ability to function as an opsonin (initiate phagocytosis) ie protein A is also antiphagocytic Protein A also prevents IgG from activating complement thus preventing its various antimicrobial activities (e.g. complement mediates cytolysis, immune adherence, and initiation of inflammation) Teichoic acids in the cell wall of S. aureus also inhibits complement activation
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Virulence Factors of S. aureus

Virulence Factors of S. aureus

Enzymes/toxins: contribute to its virulence
Beta hemolysin Coagulase: hide Staphylokinse (fibrinolysin): escape Leukocidin: destroy PMNs Hyaluronidase: get between cells Nucleases, phospolipases, proteases Antibiotic-ases ex. Penicillinase and more..
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VF: more enzymes / toxins

Various other S. aureus exotoxins: Enterotoxins: work mainly on small intestine result in active transport of ions = diarrhea TSST-1: also called superantigen, is pyrogenic and results in release of cytokines that are strongly vasoactive = vessel damage = leaking = dramatic BP drop & organ failure Exfoliating toxins others
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 Penicillin resistance (possession of penicillnase) is coded on a plasmid the enzyme is also known as beta-lactamase: inactivates the betalactam ring of penicillins and other beta-lactam antibiotics such as the cephalosporins Semi-synthetic drugs (modified penicillins) such as methicillin and oxacillin were developed for treating beta lactamase positive S. aureus infections Some strains are now resistant to these drugs MRSA, etc. A recent survey indicated that as many as 34% of S. aureus isolates were MSRA.
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Antimicrobial Susceptibility

Antimicrobial Susceptibility
MRSA is not only resistant to methicillin and most other penicillins and cephalosporins, but they are often resistant to almost all other antibiotics except vancomycin Although vancomycin is the drug of choice for treating MRSA infections, there are now vancomycin resistant (VRSA) S. aureus strains. The first VRSA strain was identified in Japan in 1997, and 8 cases were confirmed in the US in 2002.
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Antimicrobial Susceptibility
A high percentage of CoNS are also resistant to methicillin. Even though these isolates may be responsible for a variety of infections, methicillin resistant varieties are no more likely to be associated with nosocomial infections than are methicillin sensitive isolates. Hospital and nursing home epidemiological surveillance programs are routinely conducted for MRSA but NOT for methicillin resistant coagulase negative staphylococci
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Coagulase Negative Staphylococcus

Coagulase negative staphylococci (CoNS) are generally less virulent that S. aureus CoNS are inherently difficult to speciate even using modern clinical products and methods Unless a CoNS isolate is cultured repeatedly from a normally sterile body site (e.g.blood, CSF), identification to species level is usually not attempted Isolates that must be definitively identified to species level are sent to reference labs
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CoNS S. epidermidis
The most common CoNS species in clinical samples is S. epidermidis, comprising 50-80% of these isolates. S. epidermidis can be presumptively differentiated from other Staph species on the basis of the following observations It does not ferment mannitol or trehalose Coagulase negative It is sensitive to novobiocin It is resistant to polymyxin B Pathology of S. epidermidis is alsmost exclusively associated with skin penetration in the hospital setting
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CoNS S. epidermidis
S. epidermidis produces a capsule that adheres to plastic devices such as intravenous catheters, prosthetic heart valves, and shunts S. epidermidis and other CoNS are cause of native valve endocarditis

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CoNS S. saprophyticus
Staphylococcus saprophyticus is a CoNS associated with urinary tract infections, mostly in females, especially college age women S. saprophyticus is one of the few frequently isolated CoNS that is resistant to Novobiocin Novobiocin resistant staphylococci causing significant bacteruria can be presumptively identified as S. saprophyticus

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Coagulase Negative Staphylococcus

Following S. epidermidis and S. saprophyticus, S. hemolyticus, S. schleifferi and S. lugdunensis are the next most commonly isolated clinically significant CoNS ???? The PYRase test is a test that can aid in differentiating S. hemolyticus, S. schleifferi and S. lugdunensis (PYRase positive) from other staphylococci.

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Micrococcus species
Micrococcus colonies are highly convex, yellow, not as glistening as Staph, and usually not as large as those of Staph Microscopically individual Micrococcus cells are somewhat larger than staphylococci and the predominant spatial arrangement is tetrads Tetrads and pairs of cells can set adjacent to form right angle geometric patterns like dominos
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Micrococcus species
Refer to slide 21 of this Power Point for a few tests used in differentiating the three genera of the Micrococcaceae. Being obligate aerobes, Micrococcus species produce acid only in the open tube (O tube O reaction) of the Oxidation/Fermentation test medium.

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Rothia mucilaginosus
Rothia mucilaginosus is most abundant in the oral cavity Microscopically Rothia species form short chains and small clusters. Here, the longer axes are perpendicular rather than parallel The texture of R. mucilagenosus colonies is unique: When pressure is applied using a loop, needle, or wooden applicator stick, R. mucilagenosus adheres tenaciously to the agar surface When further pressure is applied the upper portion of the colony peels off leaving the bottom portion sticking to the agar surface The most reliable test for differentiating R. mucilaginosus from staph and micrococci is strict salt sensitivity on salt agar (e.g.5-7.5% NaCl) such as mannitol salt agar. Micrococcus doesnt grow well, but it grows a little bit.
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Presumptive Genus Identification table not on test

6.5% Furazoli- Modified 0.04 U Salt done oxidase Bacitracin Micrococcus + R + S Staphylococcus + S R R. mucilaginosa NT NT NT

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