mdl 237 streptococci

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The Streptococci

With Emphasis on Streptococcus and Enterococcus

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Preliminary Grouping of Gram Positive Cocci

Gram Positive Coccus

Catalase

Rothia

StaphylococcusMicrococcus

PYR

+ _

+

“A Disk*

chains _

S.pyogenes“GAS”

*A disc contains bacitracin

Enterococcus

Streptococcus sp & otherGroup genera

+Other StrepGroup genera

S R

_See “Staph” PP

Note: SBA hemolysis as alt to PYR?


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Strep Group GeneraNOT on the test

• Streptococcus

• Enterococcus

• Aerococcus

• Leuconostoc

• Abiotrophia• Pediococcus

• Lactococcus

• Granulicatella

• Other “new” generawww.freelivedoctor.com

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Metabolism• Streptococci divide to form pairs and chains. Chain

length varies among species & depends upon growth conditions - broth generally favors chain formation

• The streptococci are catalase-negative Gram-positive cocci

• Streptococci conduct fermentation exclusively & therefore grow fairly slowly. As strict fermentors, their O2 requirement is described as aerotolerant anaerobes. Some grow better in the absence of oxygen, but virtually all streps will grow anywhere in regard to O2

• Streptococcal colonies are usually 1 mm or less after 24h incubation under optimum conditions, although this varies from species to species.


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Classification• Genetic analysis indicated a lack of relatedness between some

organisms previously in the Genus. The original genus Streptococcus has now been split into more than a dozen genera.

• Most notable of these were a few bile-resistant strep-like bacteria that inhabit the intestine of humans and many warm blooded animals, Streptococcus faecalis and Streptococcus faecium.

• The Genus Enterococcus was created for these “enteric”Streps, hence E. faecalis and E. faecium

• Fortunately for clinical microbiologists and physicians, the majority of the newly named genera contain few bacteria that are clinically significant

• The original genus Streptococcus and the genus Enterococcus comprise most of the human clinical isolates


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Classification• Below are some newly named genera that were

split from the genus Streptococcus:• Lactococcus• Aerococcus• Leuconostocnot important for test• Pediococcus• Gamella

• To reiterate, these are rarely of clinical significance but are occasionally misidentified as Streptococcus or Enterococcus


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Classification - hemolysis• In 1903 J.H. Brown grouped streps by their ability to lyse

RBCs

– Beta hemolysis( ): complete lysis of RBCs

– Alpha hemolysis ( ): K removal from RBCs resulting in an opaque greenish-brown zone around colonies

– Gamma hemolysis ( ): no effect on RBCs

– Alpha prime hemolysis ('): small zone of intact RBCs adjacent to the colony surrounded by a zone of complete hemolysis???

• Using the term “gamma hemolysis” is synonymous for having “no effect” - clinical microbiologists also call these organisms “non-hemolytic streptococci”


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Classification - Sherman• Sherman in the early 1900s placed the streps in “physiologic”

groups via physiological differences. His “pyogenic” group (to form pus) contained most of the pathogenic penicillin sensitive beta hemolytic species, usually isolated from pus.

• Another physiologic group was the bile tolerant penicillin resistant “gut” bacteria, removed from the genus Streptococcus and later becoming the genus Enterococcus

• It was he who reclassified Diplococcus pneumoniae (later officially changed to Streptococcus pneumoniae in the early 1970s) as the “pneumococcus” – only 1 species. This organism was hemolytic & originally penn sensitive

• It is (or at least was) by far the most common cause of community and hospital acquired bacterial pneumonia in humans. It is definitely the #1 cause of acute typical bacterial pneumonia.


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Classification - Sherman• Another of Sherman’s groups included the alpha hemolytic

bile and penicillin sensitive streptococci which are normal microbiota of the oral cavity – several species are included here including S. mutans and S. sobrinus

• He named them the “viridans” group because of their tendency to produce alpha hemolysis. Viridian is latin for a green tint.


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Classification - Lancefield• In 1927 Rebecca Lancefield discovered the ability to group

streptococci based upon differences in streptococcal cell wall polysaccharides, and the production of serum antibody in rabbits innoculated with these streptococcal “antigens.”

• Conducting “precipitin” tests in capillary tubes, Lancefield combined known serum antibodies with extracted streptococcal antigen, and identified which antigen(s) each species possessed.

• In this way, Lancefield originated the “Lancefield groups” of Streptococcus that we still use to this day. For example:

– S. pyogenes in Group A hemolytic

– S. agalactiae in Group B hemolytic

– S. zooepidemicus in Group C hemolytic

– S. faecalis in Group D variable


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Lancefield Capillary Precipitation

Antibody against astrep groupantigen

StrepAntigenExtract

No Precipitate(Negative Test)

Precipitate(Positive Test)

Ag-Abinterface

Ag-Abinterface

RabbitAnti-serum

RabbitAnti-serum

StrepAntigenExtract


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Classification - Lancefield• Lancefield realized that all species in each “group” generally (and

conveniently) shared clinically significant properties such as type of hemolysis, normal host, body system or tissue where indigenous, etc. For example:– Group A - S. pyogenes: human upper respiratory– Group B - S. agalactiate: human urogenital– Group C - S. zooepidemicus: from animal products– Group D - S. faecalis: bile-resistant, fecal origin

• Lancefield identified many other antigens, and proposed several Lancefield groups. Groups A, B, C, D, F, and G were the primary groups likely from human infections

• Lancefield later determined that viridans streps & pneumococci did NOT possess antigens that reacted with her antisera

• More recently, a new species, S. milleri was found to carry A,C, F & G antigens, and display all 3 types of hemolysis.


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Preliminary Grouping – NOT on test

StreptococcusEnterococcusLactococcusAerococcusLeuconostocPediococcusGamella

Brothch tet+ -+ -+ -- ++ -- ++ +

VaSS b

SSRRS

PYR -a

+ -b

+ - - +

LAP + + + - - + +/-

NaCl-++/-++/-+/- -

45oC +/- + - - - + -

Broth=growth in broth; ch=chains; tet=tetrads; Va=vancomycin; PYR=pyrrolidonyl arylamidase; LAP=leucine aminopeptidase; NaCl=growthin 6.5% sodium chloride; Growth at 45oCa Streptococcus pyogenes is PYR positiveb Occasional exception

Betahemolysis +/- - b - - - - -


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Cultural CharacteristicsStreptococcus species tend to be more fastidious than staphylococci and

Enterococcus SBA is ideal for growing Streptococcus as it contains various amino

acids, vitamins & minerals. Any Additional catabolizable carbs present in SBA can mask the normal appearance of hemolysis making it appear

Sheep’s blood has 3 advantages over other animal blood:1. Haemophilus species will not grow on SBA due to the presence of

NADase which neutralizes extracellular NAD (“V” factor). Hemolytic Haemophilus colonies on blood from other animals is easily confused with beta hemolytic Streptococcus species

2. Sheep’s blood gives a distinctly clear beta hemolysis that is obviously different from alpha hemolysis – not necessarily true of reactions on other animal bloods

3. Human blood can contain growth inhibiting antibodies, etc that prevent recovery of organisms from samples


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Cultural Characteristics• Growth of Streptococci can be enhanced by incubation in a CO2

rich environment (5-8%). Some strains, particularly some S. pneumoniae, will not grow at all without added CO2

• Some streptococci grow better anaerobically than under any other conditions, but remember that streps are aerotollerant

• As said earlier, streptococci are fermentors and therfore form small colonies (some species form “pin-point” colonies)

• Most species form colonies that are translucent, convex, shiny

• S. pneumoniae colonies vary. Some have slightly raised edges to form a low convex colony, others have a bump in the center (umbonate) and others have a smooth “dip” in the center (a concave or “umbilicate” colony)

• S. pneumoniae may have large capsules making their colonies “mucoid” – these rarely form umbilicate colonies www.freelivedoctor.com

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Cultural Characteristics• Enterococcus species and Streptococcus agalactiae

(group B streptococci or GBS) tend to grow larger colonies (and therefore more opaque) than other Streptococcus species

• Colonies of Enterococcus and GBS range form 1mm-1.5mm in diameter in 18-24h


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Identification• Streptococcal ID is important due to the potential severity of the

condition, but streps are fortunately fairly easy to ID to a point• Presumptive ID can be performed with a few conventional tests.

Isolation from any normally sterile body fluid or cavity necessitates definitive ID, usually by technical means such as serotyping, NA homology, etc.

• The “ Streptococcus milleri” group, the new strep, carries A, C, F, and G antigens (47% of isolates type out as group F) or it may possess no Lancefield antigen at all. Also, isolates can be either beta, alpha, or nonhemolytic

• The name S. milleri has been challenged by researchers at the CDC who had call it Streptococcus anginosus

• Whatever name is finally assigned to it, this bacterium is definitely pathogenic and should be identified completely whenever isolated from normally sterile sites. SO, how to do it…


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Identification – S. milleri• Isolated from purulent deep tissue abscesses• Pinpoint colony which is often beta hemolytic

but could be non-hemolytic or alpha hemolytic• Distinct caramel / butterscotch odor - variable • Lancefield variable or non-groupable (47% F)• Bacitracin resistant (vs S. pyogenes)• Sulphonamide resistance (vs most other streps)• Optochin resistance (vs alpha hemolytic streps)• Tom: VP positive


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Identification - NVS• Another distinctive group of streptococci are Nutritionally

Variant Strep (NVS) – sometimes isolated from blood• This group cannot synthesize certain growth factors (e.g.

vitamin B6 and cysteine) which must be added to the culture medium. Common SBA does not contain these growth factors

• Detection of NVS can be accomplished by streaking SBA and then making a “band streak” of a Staph aureus culture across the plate (this is known as the “Staph streak”)

• NVS will grow as tiny colonies adjacent to the staph streak – S. aureus synthesizes and secretes the required growth factors

• Commercial supplemented blood agar media (such as Danish blood agar) contains the required growth factors for NVS

• Gram positive, catalase negative cocci that do not grow on sheep’s blood or chocolate agar should be suspected and tested using the “Staph streak” test


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Staph Streak For NVS

Specimen streakedFor isolated colonies Staph aureus culture

Band streak

NVS colonies“Satelliting” the Staph streak


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Identification - catalase• The catalase test generally differentiates the two major groups of

Gram-positive cocci (staph and strep), but it might not be straight forward as some streps produce “pseudocatalatase,” especially when grown on SBA. This is demonstrated as slight bubbling in the presence of hydrogen peroxide after a brief delay.

• Care must also be taken when growth is collected from SBA because blood itself contains catalase. All in all, it is better to grow cultures on non-blood media for catalase testing

• Cultures grown on SBA that show the characteristic pseuodocatalase reaction, should be sub-cultured on non-blood media, usually an agar slant. Drop H2O2 directly on the growth on the slant and monitor for bubbling. This is referred to as the “tube catalase test.”

• In a truly positive catalase test such as staph, there is an immediate appearance of very obvious bubblingwww.freelivedoctor.com

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Presumptive ID Tests - CAMP• The “CAMP” test (acronym for developers) is used to

differentiate S. agalactiae (GBS), which is positive, from other beta hemolytic strep, all of which are negative

• “CAMP factor” is a soluble hemolysin produced by GBS that combines in a synergistic way with a similar hemolysin of S. aureus to form an arrowhead zone of clearing. See image

• The staph is streaked perpendicularly to the unknown strep. After appropriate incubation time an “arrowhead-shaped” clear zone of hemolysis will appear


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Presumptive Identifying Tests• Several of the tests used to presumptively ID strep are

sensitivity tests for certain antibiotics (bacitracin, novobiocin, sulfomethoxizole, optochin, etc.).

• The bile-esculin test (several formats) which determines if organisms can hydrolyze esculin (to glucose) in the presence of bile, and the NaCl tolerance test, both indicate Enterococci which are +.

• The PYR (pyrrolidonyl peptidase) test is a rapid disk test performed on colonies growing on a agar media. Beta hemolytic GAS and Enterococcus are +


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Presumptive ID – NOT on test

Organism GASGBSGroups C,F,GEntero-coccusGroup D StrepViridansPneumo-cocci

Hemo-lysis a b

,,

,

,

Baci-tracin S R b

R b

NA

NA

NA NA

SXT R R S

NA

NA

NA NA

CAMP - + -

-

-

- -

PYR + - -

+

-

- -

BileEsculin - - -

+

+

- b

-

Opto-chin NA NA NA

R

R

R S

NaCl - - b

-

+

-

- -

NA=Not applicable; a Weak beta; b occasional exception


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Antigen Testing• Lancefield grouping can be used to ID strep as already discussed

• The original capillary precipitation test is tedious and time consuming, but recently developed methods utilize inert particles coated with group specific antibodies

• Antigens extracted from unknown streptococci are mixed with the antibody coated particles and observed for agglutination

• Unlike Lancefield’s difficult extraction technique of boiling 24h broth cultures in an acid solvent, recently developed extraction methods use enzymes or acid and take only minutes.

• GAS (from throat swabs) and GBS (colonies grown from vaginal or rectal swabs) antigens are sometimes detectable directly in clinical specimens or from enrichment broth cultures

• This GAS test is called the “rapid strep test.”


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Clinical Significance• S. pyogenes (GAS) causes several diseases some of which have

been previously discussed

– *Streptococcal pharyngitis (…+ otitis media & sinusitis)

– *Scarlet fever

– *Rheumatic fever

– *Necrotizing fasciitis

– Streptococcal toxic shock syndrome

– Meningitis

– Glomerulonephritis

– Impetigo

– Cellulitis

…and more


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Clinical Significance• S. agalactiae (GBS)

– *Neonatal meningitis / septicemia

– Cystitis and pyelonephritis

– Postpartum endometritis

– Postpartum septicemia

– Suture site infections in women following caesarian section


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Clinical Significance• Streptococcus pneumoniae

– *Community acquired typical bacterial pneumonia

– Meningitis (any age group but predominates in adults over 60)

– Sinusitis and otitis media

– Bacteremia and septicemia (usually associated with meningitis and pneumonia)


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Clinical Significance• Enterococcus sp.

– *Upper and lower urinary tract infections (mainly nosocomial & nursing homes)

– Native valve and prosthetic valve endocarditis

– Intra-abdominal and pelvic infections

– Wound infections

– Septicemia and meningitis in neonates and rarely other age groups


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Clinical Significance• Beta hemolytic groups C, F and G• Recent reports suggest these organims may be

emerging pathogens connected to various pathologies– ?Acute pharyngitis

– ?Acute otitis media

– ? Endocarditis

– ? Meningitis

– ? Toxic shock syndrome

– ? Rheumatic fever

– ? Catheter-related septicemia


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Clinical Significance• Viridans streptococci

– *A leading cause (#1 strep) of bacterial native valve and prosthetic valve endocarditis – not pre-empted by streptococcal pharyngitis

– *Dental caries: capsule allows the bacterium (mainly S. mutans & S. sobrinus) to adhere firmly to the teeth. Adherent bacteria produce acid fermentation products that dissolve the enamel of the teeth


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Clinical Significance - other• *Streptococcus “milleri” (S.

anginosus) is associated with deep tissue and intra-abdominal abscesses

• *The nutritionally variant streptococci (NVS) are associated with bacterial endocarditis


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Antimicrobial Susceptibility• The antibiotic of choice for treating most streptococcal

infections has always been penicillin G.

• GAS and GBS are uniformly susceptible to it, although GAS penicillin resistance is present and growing

• Until recently virtually all S. pneumoniae were also susceptible to penicillin G. A small percentage has developed complete pen resistance and others are “intermediately” resistant (requiring a high dose of penicillin G to effect a cure)

• Enterococcus sp. are often moderately or completely resistant to penicillin G and other beta lactam antibiotics (Keflex, etc)

• Until recently the drug of choice was ampicillin in enterococcal UTIs & non-urinary infections - this resistance is increasing


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Antimicrobial Susceptibility• Vancomycin is now the drug of choice for beta-lactam resistant

Enterococcus species

• Very recently however, even vancomycin resistance Enterococcus (VRE) have been detected in hospital outbreaks including Centra Health facilities

• Surveillance for VRE is conducted in all accredited medical facilities.

• A higher percentage of E. faecium isolates are VR than are E. faecalis isolates (E. faecium is the #1 most VR bacterium), however E. faecalis is a much more common human clinical isolate, so there is no agreement as to which is worse.

• Presently there does not seem to be a good antimicrobial alternative available for treating serious infections caused by VRE


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Virulence Factors - GAS• Capsule: anti-phagocytosis & specific attachment to specific

tissues• M protein in cell wall: anti-phagocytosis & specific attachment to

specific tissues• Streptolysin O and S: leukocidins vs neutrophils & macrophages• Streptococcal pyrogenic exotoxins (SPE): “superantigens”

directly stimulate T-cells (~1000X more than a normal immune response) and cause them release high levels of the cytokines that are responsible for fever and shock

• SPE is also responsible for various tissue destruction, including skin lesions, and perhaps scarlet fever

• Hyaluronidase: dissolves the hyaluronic acid that holds cellular basement membranes together (Hyaluronidase is therefore a “spreading factor”)


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Virulence Factors• S. pneumoniae

– Capsule: anti-phagocytosis & specific attachment to specific tissues

– Pneumolysin: anti-phagocytosis; it also interferes with the action of tracheal cilia – results in a croup-like cough

• Other GBS– Capsule: anti-phagocytosis & specific attachment to

specific tissues• Enterococcus (some strains)

– Intrinsic resistance to beta lactam antibiotics due to the production of beta lactamases


mdl 237 streptococci

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