Gram Positive Organisms

Gram Positive Cocci

• coccus- single round/oval shaped bacteria

what shape of cocci is strep

chains

what shape of cocci is staph

clusters

Catalase positive cocci

• staphylococcus

coagulase positive staphylococcus

• S. Aureus

  • only relevant one for humans

Coagulase negative staph (CONS)

• s. epidermidis

• S. saprophyticus

catalase negative

• streptococcus

differentiating gram positive organisms

• staphlococcus

  • S. aureus

  • Coagulase negative staph

• Streptococcus species

  • differentiated by lancefield groups

  • differentiated by hemolysis

• Enterococcus species

  • primary human pathogens: E. faecalis and E. faecium

Staphylococci

• gram+ spheres in clusters

• common skin flora

  • can be colonizers or pathogens

• causes infection in wounds, weakened immune system, foreign devices

  • usually endogenous bacteria is infecting us

  • likes to stick to:

    • catheter

    • knee replacement

    • screws

    • chemo ports

    • pacemaker

    • mechanical valces

Staph vs other GPC

• staph = catalase positive

• streptococci and enterococci are catalase negative

• catalase

  • immune system makes a reactive oxygen species to kill bacteria

    • catalase can break those down

    • defense against oxidative stress= defense against our immune system

    • H₂O₂ → H₂O + O₂

    • result is the gas bubbles

S. Aureus

• the only coagulase positive staph we care about

• A major pathogen

  • very worrisome infection

  • can often be a contamination when shows in a lab result

  • if there are multiple coagulase positive staph in a result its contaminated

  • if S. aureus shows up in a lab test as real since it is a major pathogen

CONS

• S. epidermis, S. hominis, S. capitis, S. haemolyticus

  • often contaminants

  • sometimes pathogens

• S. saprophyticus

  • causes UTIs

• S. lugsunensis

  • pathogen

  • most virulent CONS

  • closely related to S. Aureus and should be treated like S. Aureus

Coagulase Agglutination test

• sample of the bacteria is mixed with plasma and either starts clumping immediately or does not

• Agglutination is the presence of bound coagulase

Staph on blood agar

• S. Aureus is B-hemolytic

  • complete/total lysis of RBC

• S. aurues destruction of RBCs

  • hemolysins break down hemoglobin

  • creates clear, colorless area around the bacteria

• Most CONS

  • are variably hemolytic

  • often alpha (green) or gamma (non) hemolytic

  • exceptions

    • S. ludgunensis is often beta-hemolytic

Lab tests to confirm S. Aureus

• gram stain- comes back gram positive

• Catalase- comes back catalase positive

• Coagulase- comes back positive (agglutination)

• Blood agar- comes back beta-hemolytic

Staph Aureus Virulence: Bug goals

• convert host tissue into nutrients

• inhibit immune response (neutrophils, complement, ROS, production)

Staph Aureus Virulence: inhibit immune response

• biofims form physical barrier to removal, suppress cytokine and anti-inflammatory response and secretes anti-immune toxins

  • protects from the immune system

  • lives in a nutrient sparing environment when in a biofilm

  • biofilms grow on

    • teeth

    • bones

    • native heart valves

    • foreign materials as well

  • Multiple bacteria growing in the same biofilm is really bad→ they start sharing resistance mechanisms

biofilms mechanism

• bacteria cells attach to a surface

  • through surface adhesions

  • conditioned surface interactions

  • initial attachment happens within hours

• Form a microcolony/ cell clusters

• Accumulation

  • exopolysaccharide

  • protein-protien interactions

• Mature biofilm

  • mature after a few days

  • once mature hard to clear

B-hemolysin

• S. Aurues virulence factor

• allows for staph to penetrate barriers in the body that bacteria shouldn’t get through

• can create fistulas

• can lead to a necrosis (flesh eating bacteria) situation

• will destroy WBC’s that try to engulf it

S. Aureus virulence factors: TSST-1

• toxic shock syndrome toxin

• associated with tampon use

• leads to skin breakdown or erythema

  • burn like appearance in kids

S. Aureus virulence factors: panton-valentine leucocidin

• destroys WBCs, and neutrophils

S. Aureus virulence factors: coagulase

• clots in plasma/blood

• used to determine if S. Aureus but is also a virulence factor

• protects from immune system

• may promote biofilm formation

• vegetation breaks off → dissemination of septic emboli

S. Aureus virulence factors: Catalase

• used to determine if Staph

• neutralizes H2O2 produced by phagocytes

Staphylococci Resistance

• majority of stap (98%+) in US produce beta-lactamase (an enzyme that lyses beta-lactam antibiotics) known as penicillinase

  • can’t use penicillin against staph spp.

    • ampicillin, amoxicillin, penicillin G

• Some contain Staphylococcal cassette chromosomes (SCC) mec elements

  • gene encoding production of protein PBP2a

  • importance

    • MEC gene makes methicillin resistnant

    • 44/46 beta lactams won’t work against MRSA

• S. Aureus has 4 PBPs

  • the MEC gene has modified a PBP that most antibiotics can no longer bind to

  • 5th gen cephalosporins are directly engineered to work against MRSA

PBP2a testing

• positive: means MRSA

• negative: means MSSA

• Can be performed in a short time span and can be done while waiting for other tests to confirm MRSA to guide treatment

How do we get MRSA?

• change in drug target site

• coded by SCC MecA/MecC gene

• Cassette chromosomal recombinase (ccr)

  • responsible for the movement of SCCmec between staph isolates

  • mobile resistance = horizontal gene transfer

• Many SCC subtypes carry additional resistance/virulence factors

• Type IV SCC = community-associated

community-acquired MRSA

• significant increase in the late 90’s and early 2000’s of MRSA acquired in the community

• linked to sports (football, wrestling, fencing), tattoo parlors, and more

Other common mechanisms of resistance

• if a drug works intracellularly there are 2 mechanisms of resistance

  • it gets effluxed back out of the cell

    • bacteria produces a pump to pump the antibiotic out of the cell

  • modifying the binding site

CONS

• common species

  • S. epidermis

  • S. Hominis

  • S. saphrophyticus (common cause of cystitis UTIs)

  • S. lugdunesis

• Frequently are culture contaminants

  • to treat or not to treat

    • treat as a real infection if in the CSF or in a sterile culture

  • 1 out 4 blood cultures is growing S. epidermidis

    • clue that it is a contaminant

    • if there is multiple CONS in one culture it is likely a contaminant

• commonly have MecA (methicillin resistance)

• generally, much less virulent than S. Aureus

S. Saprophyticus

• Novobiocin test- s. saprophyticus is resistant to

Staph testing summary: S. Aureus

• catalase: +

• coagulase: +

• Agglutination: + (clumps)

• Novobiocin: sensitive

• Virulence: very

• Methicillin resistance: mecA presence depends on patients/environmental risk factors

  • decreasing locally amongst S. aureus

Staph testing summary: CONS

• catalase: +

• coagulase: -

• Agglutination: -

• Novobiocin: S. saprophyticus will be resistant; other CONS will be senstive

• Virulence: less, often contaminants

• Methicillin resistance: very common

Clinical Syndromes seen in S. Aureus

• Skin and skin structure infections

  • boils, abscesses

  • necrotizing fasciitis

  • impetigo

  • furuncles, carbuncles

  • staph scalded skin

• Osteomyelitis

  • #1 causes of osteomyelitis

• pneumonia- hospital acquired/ventilator associated

  • not typically community acquired, however if so, necrotizing PNA)

  • can be seen in post influenza infection

  • causes pneumonia when in the hospital for 5+ days or if ventilated

• Bacteremia

  • secondary to skin/soft tissue infection

  • secondary to venous catheter

  • very concerning

  • tends to cause endocarditis

  • unlikely to cause UTIs so if showing up on urine culture test for bacteremia

• Endocarditis

  • on a native valve (strep is more likely to do it on a native valve

  • more likely to cause on a mechanical valve

  • risk factors: previous endocarditis, mechanical valve, needle/injection drug use

• toxic shock syndrome

• hardware/prosthetic infections

Clinical Syndromes seen in CoNS

• hardware/prosthetic infections

• catheter-related bloodstream infections (although often a contaminant)

  • 1% mortality

• Skin and soft tissue infections

• endocarditis

  • S. lugdunensis concerning

    • should be treated the same as S. Aureus

  • often a prosthetic valve

• Cystitis (UTI)

Staph Cellulitis

• purulent (has puss)

• localized

• deep red/purple

• less clear margins

• central necrotic area

• follicle involvement

• IVDU

• penetrating trauam

Strep cellulitis

• often non-purulent

• rapid spread

• brighter red

• clear borders

• lymph node inflammation

• general breaks in skin

Necrotizing Fasciitis

• majority infections start superficial and burrow through layers of skin to the muscle

  • can create fistulas

• surgical emergency

• unusual but can start from the inside and work to the outer layers

• Can start as a little spot (furuncle) with lots of pain and turns in necrotizing fasciitis

Mechanical valve endocarditis

• method

  • MRSA obtains access to the blood stream

  • cell division and adherence of MRSA to (inflammed or damaged) valve surface

  • intracellular access to valve endothelium and MRSA persistence

  • proliferation of MRSA

  • Dissemination of MRSA

• As the mechanical valve opens it flicks bacteria off the valve and releases it into the blood stream

  • can be an emboli and block blood flow, to areas like the kidneys

Staph Diagnosis

• traditional bacterial culture w/ susceptibility

  • broth and incubation

  • agar plates with enhanced growth medium

• PCR

  • MRSA-specific PCR (nasal swab)

  • speeds up the process

• PBP2a agglutination assay

  • can take fluid from infection to determine if MSSA or MRSA

Blood culture

• blood draw from 2 sites

• 2 bottles each

  • 1 anaerobic

  • 1 aerobic

• 4 bottle total

MRSA nasal swab in pneumonia

• MRSA nasal swab PCR = great negative predictive value

  • if it is not in your nose it is not in your lungs

    • 98% sure it is not S. aureus pneumonia → don’t treat for S. aureus pneumonia

  • test sensitivity may decrease 24-48 hours after anti-MRSA treatment

    • still fairly reliable

  • Positive predictive value = low (30%)

    • just because it is in your nose does not mean it is causing pneumonia

    • if you know it is there we can decolonize patients before high risk procedures + decrease MRSA infection risk

      • will put mupirocin in the nose if it is positive for MRSA before surgery

  • PCR is not ordered unless you suspect or are concerned about MRSA

treatment: invasive MRSA infections

• daptomycin, vancomycin, and/or linezolid for certain infections

• typically considered drugs of choice

treatment: invasive MSSA infections

• Anti-staph penicillins (nafcillin or oxacillin)

• 1st gen cephalosporins (cefazolin)

treatment: oral agents vs MRSA

• doxycycline, minocycline, SMX/TMP, clindamycin, linezolid, tedizolid

treatment: oral agents vs MSSA

• 1st generation cephalosporins, anti-staph PCNs

• all the MRSA agents

treatment: decolonization

• mupirocin for nose (pre-op)

• chlorhexidine baths or clorox baths

• rarely antibiotic therapy

Streptococcus Species

• round

• catalase negative

• growth in chains

  • diplococci is S.pneumonia

• Hemolysis (clinically relevant strains)

• Aerobic

  • facultative anaerobe- grows in anaerobic or aerobic environments

• encapsulated (typically s. pneumonia)

Hemolysis

• beta-hemolysis

  • complete hemolysis; caused by hemolysins O and S

  • S= stable in oxygen

• Alpha-hemolysis

  • partial or green hemolysis caused by hydrogen peroxide

• Gamma-hemolysis

  • typically non-hemolytic (some browning of surrounding area)

  • absence of hemolysis

Strep is grouped by hemolysis: Beta hemolysis

• GBS (S. agalactiae)

• GAS (S. pyogenes)

• Groups C and G

  • can occasionally cause strep throat but not common

Strep is grouped by hemolysis: Alpha hemolysis

• viridans group strep

  • S. mitis

  • S. mutans

• S. pneumoniae

  • can be alpha hemolytic or gamma hemolytic

Strep is grouped by hemolysis: gamma hemolysis

• S. bovis/ S. gallolyticus (group D)

• All enterococci too, although no longer considered group D strep

Lancefield Grouping

• based on carbohydrate antigens on Strep surface

• Group A- S. pyogenes

• Group B- S. agalactiae

Viridans Group Strep (VGS)

• typically alpha hemolytic but can be non-hemolytic

• Can colonize from mouth to anus but typically stays in the mouth and causes cavities

  • can cause endocarditis if in the blood stream

• 6 major groups

  • S. mutans

  • S. salivarius

  • S. anginosus

  • S. mitis

  • S. sanguinis

  • S. bovis (not grouped with the others)

  • Note: there is NO such thing as S, viridans

• Other group of non-beta hemolytic strep (technically not all officially in VGS but often lumped together)

  • S. constellatus, S. intermedius, S. milleri

Virulence of GAS

• Hyaluronidase

• M-protein

  • not only binding, limits phagocytosis and complement function

• Strepolysins

  • lyse natural immune response

  • Streptolysin O- lyses WBCs, platelets, RBCS

  • Streptolysin S- hemolytic zones on agar plates (non-immunogenic)

• Erythrogenic toxin

  • causes a rash (scarlet fever)

Virulence of S. pneumonia

• Encapsulated

  • protection from antibodies and phagocytic cells

  • spleen helps decapsulate and fight infection

    • if getting spleenectomy give penicillin to prevent strep and give pneumovax if planned procedure

  • Pneuomlysin (PLY)

    • lyses a variety of host cells

    • triggers a MASSIVE immune response

mechanisms of resistance

• beta-lactams: primary mode is mutations in PBPs

• second mechanism of non-PBP mutations has been identified essentially bypassing the need for PBPs in cell wall structures (rare but potent)

• macrolides: MLS resistance through 2 primary mechanisms- ERM (ribosomal binding site) and MEF (efflux pump)

Clinical syndromes and symptoms associated with strep

• Skin and Soft tissue infections (typically GAS, GBS)

  • Impetigo

  • cellulitis

• Necrotizing faciitis (GAS)

• Neonatal sepsis/meningitis (GBS)

  • moms get screened for vaginal group B strep

• Otitis media (S.pneumoniae)

• Sinusitis (multiple strep spp.)

• Strep throat (GAS)

• Community acquired pneumonia (S. pneumo)

• Bacteremia and endocarditis (virdians group, GAS)

  • endocarditis of native valves)

• Community acquired meningitis (S. pneumo #1 cause)

• Oral infections (virdians group)

Progression of Strep throat

• initial infection fever, sore throat with white patchy appearance

• can progress in 2-5 days to a strawberry tongue appearance (white patchy first, then red bumpy appearance) and total body rash

• Give antibiotics (penicillin or amoxicillin) to treat and stop further progession

  • usually better with in 24 hours

• 5% of patients will experience a post-strep glomerulonephritis

  • regardless of treatment

  • it is not toxin mediated but immune mediated

  • not permenant

  • goes unnoticed in most younger patients

  • happens with group A strep

  • patients are 20% more likely to need dialysis or have CKD in the future

Strep throat to rheumatic fever

• can occur just a few weeks after initial infection- causes chronic issues

• process

  • cell wall M-protein of GAS is highly antigenic

  • antibodies form against M-protein

  • antibodies react with cardiac myofiber protein and smooth muscles

  • causes release of pro-inflammatory cytokines

  • leading to cardiac tissue damage and disease

• Resulting Cardiac changes

  • erosion or the valve leaflets

  • thicking of tissues/valves

  • stenosis and regurgitation

  • often affects the left ventricle

Sequale of untreated strep throat

• chorea

  • involuntary movement of extremities

  • usually several months after initial infection and last for months

  • doesn’t last forever

• Subcutaneous nodules

  • deposits of antibodies

• Arthritis

  • antibodies gather in joints and cause stiffness

otitis media antibiotic use

• many cases of otitis media is caused by self-limiting viruses (strep is the most common bacterial cause)

• typically symptomatology 48-72 hours

  • can be managed with analgesics, anti-inflammatory agents

    • either systemic or topical

• Provide Rx and instruct to wait for 48-72 hours to fill and only if persistent symptoms

  • ~40% decline in antibiotic use, no difference in hard clinical endpoints

Diagnosis

• traditional bacterial culture and susceptibilities

  • susceptibilities are not often conducted on GAS, GBS, viridans group because GAS and GBS are 100% susceptible to penicillin and viridans is 90-100% susceptible

• Rapid diagnositics with PCR

  • antigen-antibody test in the back of the throat

• Pneumococcal antigen testing

  • cheap and reliable when positive

  • 40% sensitive so patients can still have strep pneumo if comes back negative

Strep Treatment

• antibiotic therapy- targeted to organism/susceptibility

  • GAS, GBS are universally susceptible to PCN (amox and amp)

    • many viridans group strep are as well but not all

  • S. pneumoniae

    • ceftriazone (often DOC) levofloxacin, some isolates are susceptible to penicillin as well

• Vaccination against S. pneumoniae

• antibiotic treatment of pregnant women at time of delivery

  • ampicillin

• 
  

when Strep Bovius and Strep Galaliticus is detected in the blood stream what do you do?

prompt for a GI/colon cancer screening

Enterococcus Species

• round

• cocci

  • diplococci

  • chains

• commensal

  • lives in the GI tract

• Growth in 6.5% NaCl to differentiate from S. Bovis

• gamma hemolysis

• formerly group D strep

enterococcus infections

• 90% of human infections come from

  • E. faecalis

  • E. faecium

• other less common causes of human infections

  • E. avium

  • E. gallinarum

  • E. casseliflavus

Clinical syndromes

• intra-abdominal infections

  • although not nearly as common as gram-negative enterbacterales or gram-negative anaerobes

  • has to be translocated from a different part of the gut

• blood stream infections

  • secondary to centeral venous catheter

  • secondary to cystitis

  • secondary to IAI

  • can also go to the heart and cause endocarditis

• Urinary tract infections (cystitis)

  • foley catheter often involved

• Dose not really cause respiratory tract infections

• very small chance of causing skin infections (will be polymicrobial)

• does not cause meningitis

resistance mechanisms

• VanA, VanB, VanC, VanD, VanE, and Van E genes

  • linked to vancomycin resistance

  • VanA most common, VanB second most common

  • passed through two transferrable operons

  • Subsititution of a amino acid (lactate for alanine) not allowing vancomycin to bind

    • changes from d-ala d-ala to d-ala d-lactate so vanc has no where to bind to

• Risk to S. Aureus

  • can share the vanc resistant genes with other bugs like S. aureus which is very dangerous bc it could create VRSA (has not happened yet)

• Change in PBP

  • intrinsic resistance to all cephalosporins (no binding sites for cephalosporins)

Diagnosis for enterococcus

• traditional bacterial cultures and susceptibilities

• rapid diagnostics (PCR, MALD-TOF)

  • consistent with other gram-positive organisms discussed

Enterococcus Treatment

• ampicillin (or PCN) is DOC

  • addition of an aminoglycoside or even ceftriaxone for synergy for invasive infections

• Vancomycin is alternative

• several options for vancomycin resistant enterococci (VRE)

  • daptomycin, linezolid