microbio - streptococcus, enteerococcus, and other Catalase-Negative gram positive cocci

today’s flow: reduce “jumping” by using one consistent diagnostic path

1. start broad

   1. GPC

   2. catalase negative

   3. pairs or chains

2. sort by plate

   1. beta hemolytic

   2. alpha hemolytic

   3. gamma hemolytic/ nonhemolytic

3. name likely groups

   1. GAS, GBS, C/G, S. pneumoniae, viridans, enterococcus/ group D

4. confirm

   1. bacitracin, PYR, CAMP, optochin, bile solubility, bile esculin, NaCl

5. interpret

   1. site + host + clinical significance

general characteristics: what makes these bacteria different from Staphylococci?

• microscopy

  • GPC in pairs or chains

  • may look more elongated than staph

• catalase

  • catalase NEGATIVE differentiates them from staph

  • avoid colonies from blood media - hemoglobin can cause FALSE POSITIVES

• growth

  • facultative anaerobes (some are CAPNOPHILIC)

  • growth is best on enriched media w blood/serum

• colony clues

  • small, transparent colonies are common

  • hemolysis pattern on BSA drives first major branch of identification

cell wall structure and Lancefield grouping

• core cell wall

  • thick peptidoglycan + teichoic acid + group carbohydrate in many clinically important species

• Lancefield groups

  • serologic groups by C carbohydrate; Group A, B, C, D, F, G, and others

  • Group A → S. pyogenes

  • Group B → S. agalactiae

  • Group D → Enterococcus/ S. gallolyticus (bovis)

  • NO Lancefield → S. pneumoniae/ viridans

• clinical shortcut

  • group + hemolysis + source often points toward the organism before full biochemical ID

hemolysis

• Beta-hemolysis: clear zone around colony

  • think: GAS, GBS, C/G groups

• alpha-hemolysis: green discoloration

  • think: S. pneumoniae vs Viridans

• nonhemolytic: no change in agar

  • think: Enterococcus/ Group D

• alpha prime: tiny intact RBC zone plus wider Beta zone - can be mistaken for beta/alpha mix of both

first pass identification algorithm

• GPC

• catalase negative

• read hemolysis

  • beta hemolytic: TAXO-A/ PYR/ CAMP/ Hippurate

  • alpha hemolytic: TAXO-P/ bile solubility

  • nonhemolytic: bile esculin/ 6.5% NaCl/ PYR

high yield anchors: organism → usual hemolysis → confirmatory clue

Beta hemolytic strep

• GROUP A - S. pyogenes

  • Throat, skin/soft tissue, invasive disease, post strep sequelae

  • TAXO-A/ PYR/ antigen testing

• GROUP B - S. agalactiae

  • pregnancy/newborn disease; screen vaginal + rectal swabs

  • CAMP/ hippurate/ NAAT

• GROUPS C/G

  • may cause pharyngitis, impetigo, pyogenic infections; Lancefield typing helps

  • normally tested w KITS instead of growth

Streptococcus pyogenes (Group A strep)

• identity

  • Lancefield group A

  • Beta hemolytic

  • PYR (+)

  • BACITRACIN SUSCEPTIBLE

• common disease

  • pharyngitis (strep throat)/ tonsilitis

  • Scarlet fever

  • impetigo/erysipelas/ cellulitis

• serious disease

  • necrotizing fasciitis

  • streptococcal TOXIC SHOCK SYNDROME

  • sequelae: RHEUMATIC FEVER, ACUTE GLOMERULONEPHRITIS

S. pyogenes virulence factors

• attach

  • M protein + lipoteichoic acid + fibronectin-binding protein help adherence to mucosal cells and keratinocytes

• avoid phagocytosis

  • M protein resists phagocytosis

  • hyaluronic acid capsule MASKS bacterial antigens

• damage cells

  • Streptolysin O is oxygen-labile (easily altered by Oxygen) and immunogenic

  • streptolysin S is oxygen stable

• spread

  • DNase, streptokinase, and hyaluronidase (spreading factor) help tissue invasion/spread

*antibodies to SLO can be measured in the ASO test to support evidence of recent GAS infection (+ does not mean current infection)

streptococcus pyogenes (GAS) clinical infections - throat

• pharyngitis and tonsillitis

  • primarily in children 5-15 yrs old

  • spread by droplets and close contact

  • symptoms: sore throat, fever, malaise, headache, ab pain, nausea, tender cervical lymph nodes

• scarlet fever

  • affects those w strep throat OR STREP SKIN INFECTIONS

  • streptococcal pyrogenic enterotoxins cause distinctive symptoms

    • scarlatiniform rash - red w small papules (sandpaper texture)

    • after rash, skin desquamation (peeling)

    • strawberry tongue

streptococcus pyogenes (GAS) clinical infections - skin

• impetigo

  • very young CHILDREN

  • small vesicles → weeping lesions that crust (no actual blister unlike herpes)

• erysipelas

  • in ELDERLY patients

  • spreading red rash with demarcated but IRREGULAR edge

• cellulitis

  • deep invasion of GAS leading to SEPSIS and GANGRENE (goes deep into tissues)

streptococcus pyogenes (GAS) clinical infections - serious

• necrotizing fasciitis (tissue death)

  • invasive infection characterized by rapidly progressing inflammation and necrosis of the skin, subq fat, and fascia

    • described as “flesh-eating disease”

  • life threatening disease

  • characterized as types 1, 2, 3

    • type 2 is Group A strep

• Streptococcal Toxic Shock Syndrome (TSS)

  • follows a severe initial strep infection

  • collapse of entire organ systems - gets access to bloodstream - sepsis can occur

streptococcus pyogenes (GAS) sequelae

• rheumatic fever

  • follows GAS PHARYNGITIS (can ONLY get from throat infection)

  • fever, inflammation of heart, joints, blood vessels, subq tissues

  • progressive damage to heart valves

    • antigenic cross reactivity btwn strep antigens and heart tissue

    • bacterial enterotoxins

    • actual bacterial invasion of the heart tissue

• acute glomerulonephritis

  • follows EITHER skin or strep throat infection

  • malaise, fever, nausea, nephrotic syndrome, hematuria, smoky brown urine

  • immune complexes deposit in glomeruli causing inflammation and damage

S. pyogenes clinical disease: organize by site and mechanism

• rheumatic fever and acute glomerulonephritis are COMPLICATIONS AFTER INFECTION, NOT direct bacterial invasion of those tissues

GAS lab diagnosis

• throat specimen → Beta colonies → confirmation

• specimen

  • swab POSTERIOR pharynx + each TONSIL

  • touch EXUDATE if present

  • AVOID tongue, uvula, cheeks

• culture

  • SBA - 35C for 18-24 hrs in CO2

    • if no growth → incubate + 24hrs

  • can use selective media: SBA containing SMZ, colistin, and polymyxin B

• confirmation

  • Lancefield typing preferred; bacitracin/PYR can support presumptive ID

• direct methods

  • rapid antigen detection tests and PCR are useful for throat swabs

  • culture remains important when confirmation needed

    • if kit is negative, still culture to confirm/dbl check

streptococcus agalactiae (GBS) - screening prevents newborn disease

• identity

  • group B antigen

  • Beta hemolytic

  • CAMP (+)

  • Hippurate (+)

• reservoir

  • NORMAL BIOTA of upper respiratory tract, GI tract, and vagina

    • pregnant women MUST be screened by 7 months (will be checked continuously from 7-9 months/until birth)

• risk

  • major cause of pneumonia, sepsis, and meningitis in NEWBORNS

• virulence

  • capsule containing sialic acid helps inhibit the alternative complement pathway

• other products

  • hemolysins, CAMP factor, neuraminidase, DNases, hyaluronidase, and proteases are useful to know for ID/context

GBS clinical infections

• organize by AGE and route

• EARLY onset newborn disease

  • < 7 days old

  • usually VERTICAL TRANSMISSION during birth (from mom)

  • associated w/ OBSTETRIC COMPLICATIONS

  • high mortality rate if not treated quickly

• LATE onset newborn disease

  • 7 days to 3 months

  • primarily MENINGITIS

  • less tied to obstetric complications

  • still serious

• ADULT disease

  • postpartum/post-abortion infections

  • endometriosis, wound infection, endocarditis

  • elderly or immunodeficient patients

*clinical logic: the same organism may be normal biota in one host but life threatening in another

GBS lab diagnosis

• pregnancy screen and confirmation tests

• specimen collection

  • vaginal + rectal swab at 36-27 weeks of pregnancy

• enrichment

  • inoculate in Lim broth, TransVag broth, or StrepB Carrot broth

  • incubate at 35C, 5% CO2 for 18-24 hrs

  • then subculture to SBA or chromogenic agar

• CAMP test

  • arrowhead of enhanced hemolysis near S. aureus = presumptive GBS

• Hippurate

  • purple after ninhydrin → hippurate positive = GBS

• NAATs may also be used, esp when RAPID detection is needed

Lab diagnosis: Beta hemolytic strep

• tests for detection/differentiation of GAS and GBS

  • bacitracin susceptibility (TAXO-A)

  • CAMP test

  • sodium Hippurate hydrolysis

  • PYR (if necessary) - not always accurate

  • antigenic testing

B-hemolytic strep tests: TAXO-A

• TAXO-A

  • for the presumptive ID of S. pyogenes

  • disk containing 0.04 IU bacitracin

• test

  • streak 1 colony of B-strep over ½ SBA, add disk onto streaked surface, incubate overnight at 35C in CO2

• results

  • susceptible (ANY zone of inhibition) = GAS

  • resistant (NO zone of inhibition) = NOT GAS

B-hemolytic strep tests: CAMP

• CAMP test (Christine, Atkins, and Munch-Petersen)

  • for the presumptive ID of Group B Strep

  • GBS produce beta hemolysis that reacts SYNERGISTICALLY with S. aureus

• test

  • streak vertical straight line of S. aureus, streak B-hemolytic strep to be tested at a right angle (but not touching) , and incubate at 35C overnight in CO2

• results

  • arrowhead of hemolysis @ junction (+) = Group B strep

  • NO arrowhead of hemolysis @ junction (=) = NOT group B strep

B-hemolytic strep tests: Hippurate

• sodium hippurate hydrolysis

  • for differentiating S. galactiae from other beta strep

  • S. agalctiae possess hippuricase enzyme

    • hydrolyzes NaHipp to form sodium benzoate and glycine

• test

  • add bacteria to tube of NaHip, incubate overnight at 35C, add 3-2 drops of Nihydrin and incubate at 37C for 10 mins

• results

  • clear turns purple color (+) = GBS

  • NO color change (=) = other beta strep

B-hemolytic strep tests: PYR

• PYR

  • disk containing PYR substrate (Pyrrolidonyl-α-naphthylamide)

  • GAS is able to hydrolyze substrate

• test

  • add 1 drop of saline to PYR disk, add 1 colony to disk, incubate RT 5 min, and then add 1 drop of color developer (1 min)

• results

  • color change (red/pink) = positive - GAS

  • no color change = negative - NOT GAS

other beta hemolytic streptococcal groups: do not stop at beta=GAS/GBS

• groups C and G

  • large colony isolates often S. dysgalactiae subsp. equismillis

  • infection sites can resemble GAS

• S. anginosus group

  • small colony isolates may be Beta or Alpha or nonhemolytic

  • frequently linked to abscesses and pyogenic infections

• KEY TESTING POINT

  • Lancefield typing is the best way to separate groups C/G from GAS when morphology overlaps

alpha hemolytic strep: S. pneumoniae vs Viridans strep

• S. pneumoniae

  • pneumococcus

  • Lancet shaped diplococci

  • opportunistic pathogen of the RESPIRATORY tract

• confirm pneumococcus

  • optochin SUSCEPTIBLE

  • bile soluble

  • quelling reaction can identify capsule

• Viridans strep

  • NORMAL BIOTA of ORAL cavity, URT, GI and female genital tract

  • opportunistic infection

• confirm viridans

  • optochin RESISTANT

  • bile insoluble

  • Lancefield grouping is usually of little value

S. pneumoniae capsule

• Identity

  • alpha hemolytic pneumococcus

  • part of normal URT flora

  • lancet shaped diplococci

• capsule

  • abt 90 capsule types

  • capsule is antigenic and a MAJOR VIRULENCE FACTOR

• QUELLUNG RXN

  • anti-capsular serum causes CAPSULE SWELLING

  • useful conceptually for capsular typing

• other products

  • hemolysin, IgA protease, neuraminidase, and hyaluronidase also contribute

pneumococcus study anchor

• alpha hemolytic

• optochin susceptible

• bile soluble

• capsulated diplococcus

s. pneumoniae clinical infections

• pneumococcal lobar pneumonia

  • common in elderly + underlying disease & can follow another illness as a secondary infection

  • fluid facilitates spread to adjacent alveoli → reaches fibrous septa that separates major lung lobes

  • SYMPTOMS: chills, cough, dyspnea, edema, hypoxia

• meningitis

  • occurs in all groups (50% of bacterial meningitis cases in the US)

  • can follow ear infection or pneumonia

  • rapid course w HIGH mortality

  • SYMPTOMS: headache, lethargy, vomiting, fever, nuchal rigidity

  • COMPLICATIONS: cranial nerve sign, seizures, bacteremia, coma

• other infections

  • most frequently isolated in children under 3 with recurrent ear infections

  • bacteremia, endocarditis, peritonitis, empyema, and HUS-like complications

• prevention/treatments

  • vaccines area available- routine for children under 5, elderly, and college students

  • penicillin is drug of choice, though resistance is a concern