5.6 Antimicrobial Resistance, Susceptibility Testing, Specimen Handling & Vaccines
Key Takeaways
Bacteria resist antibiotics by enzymatic inactivation, target alteration, reduced porin entry, efflux pumps or bypass pathways, and they spread resistance genes horizontally by conjugation, transformation and transduction.
The minimum inhibitory concentration (MIC) is the lowest drug concentration that prevents visible growth; larger disk-diffusion zones correspond to lower MICs, and breakpoints convert an MIC into susceptible, intermediate or resistant.
A D-shaped zone of inhibition on the D-test identifies inducible clindamycin resistance (erm methylation) in an erythromycin-resistant staphylococcus.
For diabetic foot infection, cultures should come from debrided deep tissue or bone, not superficial swabs, and tissue for culture must never be placed in formalin.
Live attenuated vaccines generally are avoided in pregnancy and significant immunosuppression, whereas inactivated, subunit, toxoid, conjugate and mRNA vaccines cannot cause the infection they prevent.
5.6 Antimicrobial Resistance, Susceptibility Testing, Specimen Handling & Vaccines
The microbiology outline groups emerging drug resistance (mechanisms and susceptibility testing), laboratory testing (principles, specimen collection and handling, culture and sensitivity) and infection prevention and treatment (sterilization, antimicrobials and vaccines). Sterilization is covered in 5.4 and individual antibiotic classes in 11.1; this section ties those topics to how resistance develops and how a result reaches the chart.
Mechanisms of Antimicrobial Resistance
| Mechanism | Example | Clinical consequence |
|---|---|---|
| Enzymatic inactivation | Penicillinase (blaZ) in staphylococci; extended-spectrum beta-lactamases (ESBLs, often CTX-M) in E. coli and Klebsiella; carbapenemases (KPC, NDM, OXA-48); aminoglycoside-modifying enzymes | ESBL producers need a carbapenem or another active agent; carbapenemase producers are often multidrug resistant |
| Target alteration | mecA-encoded PBP2a (MRSA); vanA D-Ala-D-Lac precursors (VRE); erm methylation of 23S rRNA; gyrA/parC point mutations; rpoB mutations | Loss of an entire drug class, for example all conventional beta-lactams in MRSA |
| Reduced entry | Loss of the OprD porin in Pseudomonas aeruginosa | Carbapenem (imipenem) resistance |
| Efflux pumps | MexAB-OprM in Pseudomonas; tet pumps | Multidrug or tetracycline resistance |
| Bypass pathways | Acquired sul or dfr genes encoding drug-insensitive folate enzymes | TMP-SMX resistance |
How resistance spreads. Spontaneous chromosomal mutations are passed vertically to daughter cells and are selected when antibiotic exposure kills susceptible neighbors. Horizontal gene transfer moves resistance between bacteria:
- Conjugation: transfer of plasmids through a sex pilus, the main route for ESBL and carbapenemase genes.
- Transformation: uptake of free DNA from lysed bacteria.
- Transduction: transfer by bacteriophages.
- Transposons and integrons: mobile elements that collect several resistance genes into one transferable unit.
Tolerance is not resistance. Bacteria inside biofilms on hardware or sequestra and dormant persister cells survive drug concentrations that kill free-floating (planktonic) cells of the same strain. They may still test susceptible in the laboratory. This is why chronic osteomyelitis and infected implants often need debridement or hardware removal.
Susceptibility Testing
- Minimum inhibitory concentration (MIC): the lowest antibiotic concentration that prevents visible growth, usually measured by broth microdilution in twofold dilutions (0.5, 1, 2, 4 mcg/mL and so on).
- Breakpoints: standard-setting bodies (CLSI in the United States and EUCAST in Europe) publish organism- and drug-specific cutoffs that classify an MIC as susceptible, intermediate (or susceptible-dose-dependent) or resistant. An MIC of 2 can be susceptible for one drug and resistant for another, so MIC numbers should not be compared across drugs.
- Disk diffusion (Kirby-Bauer): an antibiotic disk on a lawn of bacteria creates a zone of inhibition. A larger zone means a lower MIC.
- Gradient strips: a strip containing a continuous antibiotic gradient reads the MIC where the elliptical zone meets the strip.
- D-test: an erythromycin disk is placed near a clindamycin disk. Flattening of the clindamycin zone into a "D" shape shows inducible MLSB (macrolide-lincosamide-streptogramin B) resistance from an erm gene. Clindamycin may fail during therapy even though the isolate initially looks clindamycin-susceptible.
- MRSA detection: cefoxitin disk screening, PBP2a latex agglutination or mecA PCR.
- Rapid identification: MALDI-TOF mass spectrometry identifies organisms from colonies within minutes, and multiplex PCR panels detect resistance genes (mecA, vanA, KPC) directly from positive blood cultures.
Pharmacodynamic Targets
| Pattern | Drugs | Dosing logic |
|---|---|---|
| Time-dependent killing | Beta-lactams | Maximize time above MIC (frequent dosing or extended infusion) |
| Concentration-dependent killing | Aminoglycosides, fluoroquinolones, daptomycin | Maximize peak/MIC or AUC/MIC (once-daily aminoglycoside dosing) |
| Exposure (AUC)-dependent | Vancomycin | Current guidance targets an AUC24/MIC of about 400–600 rather than trough levels alone |
Specimen Collection, Handling & Culture
Laboratory results are only as good as the specimen:
- Obtain specimens before antibiotics whenever the patient is stable. Two sets of blood cultures from separate sites are drawn when bacteremia is possible.
- Diabetic foot infection: cleanse and debride the wound, then submit deep tissue obtained by curettage or biopsy. Superficial swabs of undebrided ulcers mostly grow colonizers and are discouraged.
- Osteomyelitis: bone biopsy (percutaneous or surgical, ideally through intact skin) gives the most reliable microbiology and allows histology. After resection, a proximal "clean margin" bone specimen helps decide whether residual infection remains.
- Separate the specimens: tissue for culture goes in a sterile container (with sterile saline if needed); tissue for histology goes in formalin. Formalin kills organisms and makes culture impossible.
- Anaerobes: use anaerobic transport media or tissue rather than dry swabs, and deliver promptly.
- Special requests: mycobacterial (AFB) and fungal cultures must be ordered specifically and can take weeks (up to 6–8 weeks for AFB).
- Interpretation: a Gram stain showing many neutrophils with one organism type supports true infection. One positive blood culture for coagulase-negative staphylococci out of two sets usually indicates skin contamination.
Infection Prevention, Surgical Prophylaxis & Vaccines
Prevention in Practice
- Hand hygiene: alcohol-based rub for most encounters; soap and water when hands are visibly soiled or for Clostridioides difficile, because alcohol does not kill spores.
- Standard precautions for all patients; contact precautions for MRSA, VRE, multidrug-resistant gram-negatives and C. difficile.
- Surgical site infection bundle: weight-based cefazolin (2 g for most adults, 3 g at or above 120 kg) given within 60 minutes before incision and redosed after about 4 hours or after major blood loss; hair clipping rather than shaving; alcohol-containing skin antisepsis such as chlorhexidine-alcohol; perioperative glucose control; normothermia.
Vaccine Types
| Type | Examples | Key point |
|---|---|---|
| Live attenuated | MMR, varicella, yellow fever, intranasal influenza | Strong cellular and humoral immunity; generally contraindicated in pregnancy and significant immunosuppression |
| Inactivated whole organism | Injectable influenza (most), inactivated polio, hepatitis A | Usually needs boosters |
| Subunit or recombinant | Hepatitis B, HPV, recombinant zoster vaccine | Cannot cause infection; recombinant zoster vaccine can be used in immunocompromised adults |
| Toxoid | Tetanus, diphtheria | Antibodies neutralize the exotoxin (wound management in 5.3) |
| Conjugate | Pneumococcal conjugate, Haemophilus influenzae type b, meningococcal | Protein carrier converts a T-independent polysaccharide into a T-dependent antigen with memory |
| mRNA | COVID-19 | Host cells translate the encoded antigen |
Healthcare personnel and hepatitis B. Podiatric clinicians perform sharp debridement and surgery, so they should complete a hepatitis B vaccine series and confirm protection with an anti-HBs titer of at least 10 mIU/mL. Vaccine eligibility and timing recommendations change, so check the current CDC schedules rather than memorizing a fixed calendar.
An erythromycin-resistant, clindamycin-susceptible Staphylococcus aureus isolate is tested by placing an erythromycin disk next to a clindamycin disk. The clindamycin zone is flattened on the side facing the erythromycin disk. What does this result indicate?
An erm-mediated ribosomal methylase that erythromycin induces, so clindamycin may fail during therapy
Efflux of macrolides only, so clindamycin therapy is fully reliable
Contamination of the plate with a second organism, so the test should be repeated before reporting
Production of PBP2a from the mecA gene, predicting resistance to all conventional beta-lactams
A diabetic patient has a moderate forefoot infection with probe-to-bone positive osteomyelitis of the second metatarsal head. Which specimen plan gives the most reliable microbiology?
Wound drainage collected on a superficial swab after starting empiric antibiotics
A dry cotton swab of the ulcer surface taken before any debridement, placed in formalin to preserve organisms
Debrided deep tissue and bone, with culture tissue in a sterile container and histology tissue in formalin
A single blood culture set drawn after the first antibiotic dose
A Pseudomonas aeruginosa isolate has a disk-diffusion zone of 32 mm to one antibiotic and 12 mm to another. What can be concluded about these zone sizes?
Zone size is unrelated to MIC and reflects only how fast the organism grows
The 32-mm zone suggests a lower MIC, but each zone must be read against that drug's own breakpoint
The 12-mm zone reflects a lower MIC because more drug remained bound to the paper disk after diffusion
Any zone larger than 20 mm proves susceptibility to every antibiotic class
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