Clinical Microbiology: Gram Stains, Antibiotic Stewardship and Key Pathogens

Gram positive stain color and why

PURPLE. Thick peptidoglycan holds the crystal violet. Mnemonic from lecture: Gram POSITIVE has more P's (Purple, Peptidoglycan thick)

Gram negative stain color and why

PINK. Thin peptidoglycan layer PLUS an outer membrane made of lipopolysaccharide (LPS); crystal violet washes off

Gram-positive cocci in CLUSTERS

Staphylococcus

Gram-positive cocci in PAIRS/CHAINS

Streptococcus and Enterococcus

Gram-positive RODS

Listeria monocytogenes, Corynebacterium

Gram-positive ANAEROBES

Peptostreptococcus, Cutibacterium, Clostridium spp. (associated with dental infections; can also cause intra-abdominal infections)

Gram-negative COCCI

Neisseria

Gram-negative COCCOBACILLI

Acinetobacter baumannii, Bordetella pertussis, Moraxella catarrhalis

Gram-negative ANAEROBES

Bacteroides fragilis (most clinically important), Prevotella, Fusobacterium

Gram-negative ENTERIC rods (colonize the gut)

Proteus mirabilis, E. coli, Klebsiella, Serratia, Enterobacter cloacae, Citrobacter

Gram-negative rods that do NOT colonize the gut

Pseudomonas aeruginosa, Haemophilus influenzae, Providencia

Where do anaerobes thrive?

Low-oxygen sites: oral cavity, GI tract, deep tissues

Commensal vs pathogen

Commensal = normal flora that isn't necessarily causing infection; pathogen = causes disease. Depends on SITE (E. coli in the gut = normal; E. coli in blood = infection)

Why collect cultures BEFORE starting antibiotics?

Antibiotics can negatively affect culture growth

Stepwise approach to ID (in order)

1) Identify infection type and likely source (community vs healthcare-acquired; anatomical site) 2) Collect cultures FIRST 3) Empiric therapy covering likely pathogens 4) De-escalate based on culture and sensitivity 5) Duration and follow-up (response, toxicity, prophylaxis)

Empiric vs targeted therapy

Empiric = chosen before culture results, based on infection type/likely organisms/patient factors. Targeted = based on culture and susceptibilities

Stewardship: the 4 D's of de-escalation (from lecture)

Right Diagnosis (is there an infection?), Drug, Dose (renally dose!), Duration (shortest effective); then de-escalate: narrow, stop, or switch IV to PO

Why are antibiotics "not benign"?

Adverse effects and C. difficile, increased hospital stay and cost, selective pressure for resistance

Selection pressure

Antibiotics kill susceptible bacteria and leave resistant organisms behind to multiply

Reading a coverage chart: what do +, 0, +/- mean?

+ = generally covers; 0 = does not reliably cover; +/- = variable/partial coverage (NOT reliable). ++ sometimes marks first-line. Coverage does not equal effectiveness at every site (PK/PD and penetration matter)

Antibiogram: what does it show and what's the minimum?

Percent of isolates susceptible to each drug; needs at least 30 isolates to be included

Susceptibility report: what do S, I, SDD, R mean?

S = Susceptible (likely responds at standard dose); I = Intermediate (borderline, may work at high concentrations such as urine); SDD = Susceptible-dose dependent (needs higher-than-standard dosing); R = Resistant (do not use)

MIC

Minimum inhibitory concentration: lowest antibiotic concentration that visibly inhibits bacterial growth in vitro. Compare to breakpoints (set by CLSI). You CANNOT compare MICs between different bug-drug pairs

Susceptibility lab workflow

Culture the sample, identify the organism, determine the MIC, compare the MIC to the breakpoint, report S/I/SDD/R

PK/PD target: Cmax/MIC (concentration-dependent)

Aminoglycosides (higher peak = more killing; large dose with extended interval; low troughs limit toxicity)

PK/PD target: Time > MIC (time-dependent)

Beta-lactams (keep concentration above MIC for most of the interval: more frequent dosing, extended infusion, or continuous infusion)

PK/PD target: AUC/MIC

Vancomycin (has elements of both concentration- and time-dependent killing)

Fluoroquinolone killing pattern

Concentration-dependent (slide 57)

Why give piperacillin/tazobactam as a 4-hour extended infusion?

Beta-lactams exhibit TIME-dependent killing; extended infusion keeps the concentration above the MIC longer

HNPEK

Haemophilus, Neisseria, Proteus, E. coli, Klebsiella (the more common GNs; respiratory + urinary)

SPACE

Serratia, Pseudomonas, Acinetobacter, Citrobacter, Enterobacter (nosocomial GNs; more resistant than HNPEK)

Respiratory gram negatives

H. influenzae, Moraxella catarrhalis, Neisseria meningitidis

Urinary gram negatives

E. coli, Klebsiella pneumoniae, Proteus mirabilis

Nosocomial gram negatives

SPACE (Serratia, Pseudomonas, Acinetobacter, Citrobacter, Enterobacter)

Which GN group is MORE resistant: HNPEK or SPACE?

SPACE

Staphylococcus: where does it live?

Skin and nares

Coagulase-positive Staph

S. aureus ("infects pretty much everything")

Coagulase-negative Staph (CoNS) examples

S. epidermidis (often a contaminant; forms biofilms on devices); S. saprophyticus (UTIs in young women)

MS vs MR

Methicillin-susceptible vs methicillin-resistant (applies to S. aureus and CoNS)

Staph epidermidis

CoNS; often a contaminant; forms biofilms on devices

Staph saprophyticus

CoNS; causes UTIs in young women

Strep pyogenes

Group A, beta-hemolytic. Pharyngitis, cellulitis, necrotizing fasciitis, rheumatic fever

Strep agalactiae

Group B, beta-hemolytic. Neonatal sepsis, obstetric infections

Strep anginosus

Abscesses

Strep pneumoniae

Meningitis, otitis media, pneumonia, sinusitis

Viridans group strep (mitis, mutans)

Endocarditis after dental procedures

Beta-hemolytic strep are named by...

Groups with letters: Group A (pyogenes) and Group B (agalactiae). Beta-hemolysis = complete lysis of red blood cells on blood agar

Enterococcus: where does it live and what does it cause?

Lives in the GI tract; causes UTI, endocarditis, biliary tree infections

E. faecalis vs E. faecium

faecalis = more common, LESS resistant. faecium = less common, MORE resistant (hospital; often VRE)

VRE

Vancomycin-resistant Enterococcus (usually E. faecium)

Gram-positive group that NO cephalosporin covers

Enterococcus (lecture: NO Enterococcus coverage!!!)

Four mechanisms of antibiotic resistance

Permeability, Enzymes, Binding site changes, Efflux pumps. [extra] Mnemonic: BEEP = Binding site, Enzymes, Efflux, Permeability

Main resistance defense of GRAM POSITIVES

Binding site changes (altered target so the drug no longer fits). GPs have no outer membrane and no periplasmic space

Why do GRAM NEGATIVES have more resistance options?

Two membranes: outer-membrane porins can be altered or deleted (permeability), efflux pumps pump the drug out, and enzymes (beta-lactamases) wait in the periplasmic space (the "moat")

Why can't gram positives harbor beta-lactamases easily?

They have no periplasmic space to hold the enzymes

Intrinsic vs acquired resistance

Intrinsic = built in (e.g., drug too big/bulky to pass GN porins). Acquired = e.g., shared bacterial DNA encoding resistance

Beta-lactamase mechanism

Hydrolyzes the beta-lactam ring, so the antibiotic loses activity. Most common resistance mechanism to beta-lactams

How do we fight beta-lactamases?

Pair the beta-lactam with a beta-lactamase inhibitor (BLI)

Beta-lactamase Class A

Serine: penicillinases, cephalosporinases, ESBLs

Beta-lactamase Class B

Metallo-beta-lactamases: NOT inhibited by any BLIs (cefiderocol is active against MBLs)

Beta-lactamase Class C

AmpC: can be inhibited by next-generation BLIs

Beta-lactamase Class D

OXA-type (includes carbapenemases): can be inhibited by durlobactam

Which class of beta-lactamase is not inhibited by any BLI?

Class B (metallo-beta-lactamases)

AmpC organisms

HECK YES: Hafnia alvei, Enterobacter cloacae, Citrobacter freundii, Klebsiella aerogenes

AmpC: key resistance feature

INDUCIBLE. May look susceptible to 3rd-gen cephalosporins at first, then upregulates AmpC after a few days of therapy and becomes resistant

AmpC drug of choice

Cefepime (stable against AmpC)

ESBL organisms

E. coli, Klebsiella pneumoniae, Proteus mirabilis (all HNPEK members)

ESBL: what does it destroy?

Penicillins, 3rd-generation cephalosporins, monobactams

ESBL drug of choice

Carbapenem

Why is ceftriaxone a bad choice for Enterobacter, Citrobacter, K. aerogenes (HECK YES)?

AmpC is inducible and causes resistance to 3rd-gen cephalosporins. Use cefepime

Why does piperacillin/tazobactam NOT reliably cover Enterobacter/Citrobacter?

AmpC breaks down penicillins and tazobactam is a 1st-gen BLI that is poor at inhibiting AmpC

The S. aureus exception

S. aureus is the major GRAM-POSITIVE that makes beta-lactamase (penicillinase). Most beta-lactamases come from gram negatives

PSSA vs MSSA vs MRSA

PSSA = penicillin-susceptible (no penicillinase). MSSA = makes penicillinase but is susceptible to penicillinase-resistant penicillins (nafcillin, oxacillin). MRSA = has mecA gene, resistant to essentially all beta-lactams

MRSA resistance mechanism

mecA gene codes for PBP2a (altered PBP2), a BINDING SITE change; beta-lactams barely bind it

MSSA resistance mechanism

Penicillinase (an enzyme)

Beta-lactam classes (4)

Penicillins, Cephalosporins, Carbapenems, Monobactam

Beta-lactam mechanism of action

Inhibit bacterial cell wall synthesis by preventing peptidoglycan synthesis

Beta-lactam adverse effects

Hypersensitivity, GI upset, seizures (if drug accumulates, e.g., elderly with poor renal function not renally dosed), cytopenias with prolonged use, interstitial nephritis

Natural penicillins (agents)

Penicillin G (IV), Penicillin G benzathine (IM), Penicillin VK (PO)

Natural penicillin spectrum

Beta strep, Strep anginosus. If susceptible: Strep pneumo, viridans strep, E. faecalis. Covers GP mouth anaerobes. NO gram-negative coverage

Drug of choice for syphilis

Penicillin G benzathine (IM)

[extra] Group A strep pharyngitis: drug class to think of

Natural penicillin (Penicillin VK PO) or amoxicillin, since natural penicillins reliably cover beta-hemolytic strep. (Extends lecture; confirm guideline in rotation)

Aminopenicillins (agents)

Ampicillin (IV), Amoxicillin (PO)

Aminopenicillin spectrum

Beta strep, Strep anginosus, E. faecalis, N. meningitidis. If susceptible: H. flu, E. coli, Proteus

DOC for E. faecalis

Ampicillin (aminopenicillin; 1st line). [extra] Mnemonic: E. faecAlis = Ampicillin

Aminopenicillin + BLI (agents)

Ampicillin/sulbactam (IV), Amoxicillin/clavulanate (PO)

What does adding a BLI to an aminopenicillin add?

Better HNPEK coverage, plus MSSA and anaerobes. Still does NOT cover SPACE

Anti-staphylococcal penicillins (agents)

Nafcillin, oxacillin (IV); dicloxacillin (PO)

Anti-staphylococcal penicillin spectrum

MSSA, MS-CoNS, beta strep, Strep anginosus. NO gram-negative coverage

DOC (1st line) for MSSA and MS-CoNS

Anti-staphylococcal penicillins (nafcillin, oxacillin, dicloxacillin) or 1st-gen cephalosporin (cefazolin)

Piperacillin/tazobactam spectrum

MSSA, MS-CoNS, strep, E. faecalis, HNPEK, Serratia, Pseudomonas, anaerobes. Starts to cover SPACE (Serratia, Pseudomonas) but NOT Enterobacter/Citrobacter (AmpC)

Piperacillin comes only with...

Tazobactam. IV product only

Cephalosporin coverage trend as generation goes up

Gram-positive coverage goes DOWN and gram-negative coverage goes UP (1st gen = best GP; 3rd gen = most GN of the first three). 4th gen is the broadest (adds Pseudomonas and AmpC organisms)

[extra] How the HNPEK GN coverage builds across generations

1st gen = PEK; 2nd gen = H + PEK; 3rd gen (X group) = full HNPEK. Just add letters as the generation goes up

1st-gen cephalosporins (agents)

Cefazolin (IV); cephalexin, cefadroxil (PO)

1st-gen spectrum

MSSA, MS-CoNS, beta strep, Strep anginosus, plus PEK (Proteus, E. coli, Klebsiella)

Cefazolin is 1st line for...

MSSA and MS-CoNS

Targeted therapy example from lecture: susceptible E. coli UTI

Cefazolin (1st gen; covers PEK)

2nd-gen cephalosporins (agents)

Cefuroxime (IV and PO); cephamycins cefotetan and cefoxitin (IV)

2nd-gen spectrum

MSSA, CoNS, strep (a little more strep than 1st gen), H. flu, PEK. Cephamycins ALSO cover anaerobes