Microbiology: Diagnostic microbiology, antimicrobials and resistance

🦠 Diagnostic microbiology, antimicrobials and resistance. Doctors take samples and prescribe antibiotics every day. Doing both well - the right sample, collected correctly, interpreted in context, and the right antibiotic at the right dose for the right duration - improves outcomes and slows antimicrobial resistance, which already causes more than a million deaths worldwide each year. This chapter covers specimen collection and interpretation, the main antibiotic classes and their adverse effects, resistance mechanisms, antimicrobial stewardship and infection prevention and control.
Your four questions before prescribing: Is this a bacterial infection that needs antibiotics at all? Have the right samples been taken first (without delaying treatment in sepsis)? Which drug, dose, route and duration does the local guideline recommend for this site and this patient (allergy, renal function, pregnancy, interactions)? When will I review it (48–72 hours - start smart, then focus)?

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Taking the Right Samples

• Take samples before antibiotics whenever possible - but never delay antibiotics in sepsis or suspected meningococcal disease (give within 1 hour for high-risk sepsis).
• Blood cultures - two sets (aerobic and anaerobic bottles) from separate venepunctures, aseptic technique (2% chlorhexidine in 70% alcohol, allow to dry; do not palpate after cleaning), adequate volume (8–10 mL per bottle in adults - the biggest determinant of yield); fill aerobic bottle first with a butterfly needle; take from a new venepuncture and from each line lumen if line infection is suspected. Three sets over time for suspected endocarditis.
• Urine - mid-stream urine (MSU) or catheter specimen from the sampling port (not the bag); do not dipstick or culture urine in asymptomatic older people or catheterised patients - asymptomatic bacteriuria is common and should not be treated (except in pregnancy or before urological procedures).
• Sputum - deep cough specimen before antibiotics; AFB sputa (three samples, one early morning) for TB.
• Wound swabs - only when clinically infected; pus or tissue is better than a swab.
• Stool - for diarrhoea (C. difficile testing in hospital patients with type 5–7 stools); ova, cysts and parasites with travel history.
• CSF - before antibiotics if lumbar puncture can be done promptly and safely; otherwise give antibiotics first and do LP later; send for cell count, glucose (with paired blood glucose), protein, Gram stain, culture, PCR panel, ± AFB, cryptococcal antigen, oligoclonal bands.
• Nucleic acid amplification tests (PCR) - respiratory viruses, STIs (self-taken vulvovaginal swabs, first-void urine in men), meningitis panels, MRSA screening.
• Serology - paired acute and convalescent samples 10–14 days apart show a rise (fourfold); IgM for recent infection; IgG for immunity or past infection.

Interpreting Microbiology Results

• Contaminant or pathogen? - coagulase-negative staphylococci, diphtheroids, Bacillus species and Cutibacterium in one of several blood culture bottles are usually contaminants (unless a prosthetic device); S. aureus, S. pneumoniae, E. coli, Pseudomonas and Candida in blood are always significant.
• Colonisation versus infection - MRSA on a skin swab, Candida in sputum, Pseudomonas in a chronic leg ulcer, or bacteria in a catheter urine are often colonisation; treat the patient, not the culture.
• Sensitivity reports - S (susceptible), I ("susceptible, increased exposure" - use a higher dose), R (resistant); based on the minimum inhibitory concentration (MIC) compared with EUCAST breakpoints.
• Antibiotic levels and pharmacodynamics: time-dependent killing (β-lactams, vancomycin - keep levels above the MIC for as long as possible; frequent dosing or infusions) versus concentration-dependent killing (aminoglycosides, quinolones - high peaks; once-daily aminoglycoside dosing).
• Inflammatory markers - CRP is non-specific; procalcitonin is more specific for bacterial infection and can guide stopping antibiotics in some settings.
• Talk to the microbiologist for positive blood cultures, unusual organisms, multi-resistant organisms, and complex infections.

CSF Interpretation

Feature	Bacterial	Viral	TB	Fungal (cryptococcal)
Appearance	Turbid	Clear	Clear or fibrin web	Clear
White cells	Raised (100s–1000s) - neutrophils	Raised (10s–100s) - lymphocytes	Raised - lymphocytes	Raised or normal - lymphocytes
Protein	High (>1 g/L)	Normal or mildly raised	Very high	Raised
Glucose (CSF:serum ratio)	Low (<0.4)	Normal (>0.6)	Low	Low
Other	Gram stain, culture, PCR	Enterovirus, HSV, VZV PCR	AFB stain, culture, PCR - often negative	India ink, cryptococcal antigen; high opening pressure

• Normal CSF - white cells <5/µL (lymphocytes), protein 0.15–0.45 g/L, glucose about 60% of plasma, opening pressure 10–20 cmH₂O.
• Partially treated bacterial meningitis and early bacterial or Listeria meningitis can show lymphocytes - treat as bacterial if in doubt. HSV encephalitis may show red cells and lymphocytes.
• Traumatic tap - red cells fall across sequential tubes; xanthochromia (≥12 hours after onset) indicates subarachnoid haemorrhage.

Antibiotic Classes and Mechanisms

Target	Class	Examples	Spectrum notes
Cell wall	Penicillins	Benzylpenicillin, amoxicillin, flucloxacillin (staphylococci), co-amoxiclav (β-lactamase inhibitor), piperacillin–tazobactam (antipseudomonal)	Bactericidal; time-dependent
Cell wall	Cephalosporins	Cefalexin (1st), cefuroxime (2nd), ceftriaxone, cefotaxime (3rd - meningitis), ceftazidime (antipseudomonal), ceftazidime–avibactam, cefiderocol (resistant Gram-negatives)	No activity against enterococci, Listeria, MRSA (except ceftaroline); C. difficile risk
Cell wall	Carbapenems	Meropenem, ertapenem (no Pseudomonas cover)	Very broad - reserve for ESBL and severe infection
Cell wall	Glycopeptides	Vancomycin, teicoplanin	Gram-positives only (MRSA, C. difficile orally)
Cell membrane	Lipopeptides, polymyxins	Daptomycin (Gram-positive), colistin (resistant Gram-negative)	Daptomycin inactivated by surfactant - not for pneumonia; colistin nephrotoxic
Protein synthesis 30S	Aminoglycosides	Gentamicin, amikacin, tobramycin	Gram-negative, synergy in endocarditis; nephro- and ototoxic; concentration-dependent
Protein synthesis 30S	Tetracyclines	Doxycycline, minocycline, tigecycline	Atypicals, Lyme, chlamydia, rickettsiae, MRSA (some)
Protein synthesis 50S	Macrolides	Clarithromycin, erythromycin, azithromycin	Atypicals, penicillin allergy, Campylobacter
Protein synthesis 50S	Lincosamides, oxazolidinones	Clindamycin (anaerobes, toxin suppression), linezolid (MRSA, VRE)	Clindamycin - high C. difficile risk; linezolid - marrow suppression
DNA gyrase	Fluoroquinolones	Ciprofloxacin, levofloxacin, moxifloxacin	Ciprofloxacin - Gram-negatives including Pseudomonas; levofloxacin and moxifloxacin - respiratory
Folate synthesis	Sulfonamides, trimethoprim	Trimethoprim, co-trimoxazole	UTI, PCP, Stenotrophomonas, Nocardia, MRSA (some)
DNA damage	Nitroimidazoles	Metronidazole	Anaerobes, C. difficile (no longer first line), protozoa (Giardia, Trichomonas, amoeba)
Other	Nitrofurantoin, fosfomycin, pivmecillinam	-	Lower UTI only (urinary concentration)

Important Adverse Effects and Interactions

• Penicillins - allergy, rash with amoxicillin in EBV, cholestatic jaundice with co-amoxiclav and flucloxacillin (may occur weeks after stopping), C. difficile.
• Cephalosporins and carbapenems - C. difficile, seizures (high-dose carbapenems; carbapenems reduce valproate levels dramatically).
• Macrolides - QT prolongation, GI upset, CYP3A4 inhibition (clarithromycin and erythromycin - statins → rhabdomyolysis, warfarin, DOACs, ciclosporin, digoxin via P-gp); azithromycin has fewer interactions.
• Fluoroquinolones (MHRA 2019 and 2024) - tendon rupture (older people, steroids), aortic aneurysm and dissection, QT prolongation, seizures, peripheral neuropathy, long-lasting disabling effects, C. difficile; use only when other antibiotics are inappropriate.
• Aminoglycosides - nephrotoxicity and ototoxicity (vestibular and cochlear, irreversible; m.1555A>G mitochondrial variant increases risk); avoid in myasthenia gravis; monitor levels.
• Vancomycin - nephrotoxicity (especially with piperacillin–tazobactam), infusion-related flushing ("vancomycin flushing syndrome").
• Tetracyclines - photosensitivity, oesophagitis (take upright with water), avoid in pregnancy and children under 12 (tooth staining, bone), idiopathic intracranial hypertension; reduced absorption with antacids, iron, calcium, milk.
• Trimethoprim - hyperkalaemia (blocks ENaC), raises creatinine without changing GFR, folate antagonism (avoid in the first trimester), marrow suppression with methotrexate (dangerous combination).
• Nitrofurantoin - avoid if eGFR <45 (can be used short-term down to 30 with caution); pulmonary fibrosis and hepatitis with long-term use; avoid at term (neonatal haemolysis).
• Metronidazole - metallic taste, disulfiram-like reaction with alcohol (avoid for 48 hours after), peripheral neuropathy with prolonged use, raises warfarin effect.
• Linezolid - thrombocytopenia (after 2 weeks), optic and peripheral neuropathy, serotonin syndrome with SSRIs (weak MAOI), lactic acidosis.
• Rifampicin - potent enzyme inducer (contraceptive failure, reduced warfarin, DOAC, steroid and antiretroviral levels), orange secretions, hepatitis.
• Clindamycin and broad-spectrum agents - C. difficile.

Prescribing in Special Situations

• Renal impairment - reduce doses of renally cleared drugs (aminoglycosides, vancomycin, many β-lactams, nitrofurantoin, co-trimoxazole, aciclovir); use creatinine clearance where the BNF advises.
• Liver disease - caution with co-amoxiclav, flucloxacillin, tetracyclines, rifampicin, isoniazid, azoles.
• Pregnancy - generally safe: penicillins, cephalosporins, erythromycin (preferred macrolide), nitrofurantoin (not near term), clindamycin. Avoid: tetracyclines, trimethoprim (first trimester), fluoroquinolones, aminoglycosides (unless life-threatening), co-trimoxazole (first trimester and near term), metronidazole high doses (caution).
• Breastfeeding - most penicillins and cephalosporins are compatible; check the BNF and UK Drugs in Lactation Advisory Service.
• Children - avoid tetracyclines under 12 and fluoroquinolones (cartilage) unless essential; weight-based dosing.
• Older people - higher risk of C. difficile, nephrotoxicity, QT and tendon effects, delirium; interactions with polypharmacy.
• Drug interactions - always check: warfarin (most antibiotics raise INR), statins with clarithromycin, methotrexate with trimethoprim, oral contraceptives with rifampicin and rifabutin (enzyme inducers; standard antibiotics do not reduce efficacy), theophylline with ciprofloxacin and clarithromycin, lithium.

Penicillin Allergy

• About 10% of patients carry a penicillin allergy label, but over 90% of them are not truly allergic (often childhood rash during a viral illness, or a known side effect such as nausea or diarrhoea).
• Allergy labels cause harm - more broad-spectrum antibiotics, more C. difficile and MRSA, longer stays, worse outcomes.
• Take a careful history: what happened, when, how soon after the dose, treatment needed, and whether penicillins have been tolerated since.
• Types: immediate (IgE-mediated - urticaria, angioedema, bronchospasm, anaphylaxis within 1 hour) versus delayed (rash days later - usually benign maculopapular, but severe cutaneous reactions - SJS/TEN, DRESS - are absolute contraindications to rechallenge).
• Cross-reactivity with cephalosporins is low (about 1–2%; mainly with similar side chains, e.g. cefalexin with amoxicillin); carbapenems <1%. Most patients with non-severe penicillin allergy can receive third-generation cephalosporins or carbapenems.
• De-labelling - direct oral amoxicillin challenge for low-risk histories, or referral for allergy testing (NICE CG183; BSACI). Record the reaction type accurately.

Mechanisms of Resistance

Mechanism	Example
Enzymatic inactivation	β-lactamases (penicillinase in S. aureus), ESBLs (cephalosporin-resistant E. coli, Klebsiella), carbapenemases (KPC, NDM, OXA-48, VIM - CPE), aminoglycoside-modifying enzymes
Altered target	PBP2a (mecA) in MRSA, altered PBPs in penicillin-resistant pneumococci, D-Ala-D-Lac in VRE, ribosomal methylation (macrolide resistance), DNA gyrase mutations (quinolone resistance), rpoB mutations (rifampicin)
Reduced permeability	Porin loss in Gram-negatives (carbapenem resistance in Pseudomonas)
Efflux pumps	Tetracycline and quinolone resistance; multidrug efflux in Pseudomonas
Bypass pathways	Altered dihydrofolate reductase (trimethoprim resistance)

• Spread of resistance - chromosomal mutation (vertical) or horizontal gene transfer (plasmids by conjugation, transposons, transformation, transduction) - resistance genes spread between species, especially in the gut and in hospitals.
• Drivers - overuse and misuse in humans and animals, poor infection control, broad-spectrum use, inadequate doses or durations, international travel and healthcare abroad.
• Key resistant organisms in the UK: MRSA, VRE, ESBL-producing Enterobacterales, CPE, multidrug-resistant Pseudomonas and Acinetobacter, Candida auris, drug-resistant gonorrhoea, MDR-TB. Screen patients transferred from hospitals abroad for CPE.
• UK National Action Plan (2024–2029) - "Confronting antimicrobial resistance": reduce human antimicrobial use, improve infection prevention, new diagnostics and antibiotics (subscription payment model).

Antimicrobial Stewardship

• "Start smart, then focus" (UKHSA):
  • Start smart - do not start antibiotics without clinical evidence of bacterial infection; take cultures first; follow local guidelines; document indication, drug, dose, route, duration or review date on the drug chart; give promptly in sepsis.
  • Then focus - review at 48–72 hours with results: stop, switch IV to oral (IVOS criteria - improving, tolerating oral intake, no deep-seated infection), change (narrow spectrum based on cultures), continue (with a documented review date), or OPAT (outpatient parenteral antibiotic therapy).
• Shorter courses are usually as effective - e.g. 5 days for community-acquired pneumonia, 3 days for uncomplicated cystitis in women, 7 days for male UTI, 7 days for most Gram-negative bacteraemias with source control.
• Delayed (back-up) prescribing and no antibiotic for self-limiting respiratory tract infections in primary care (NICE NG63 sore throat - FeverPAIN/Centor; NG91 otitis media; NG79 sinusitis; NG120 cough).
• TARGET toolkit (RCGP/UKHSA) for primary care; AWaRe classification (WHO - Access, Watch, Reserve).
• Patient education - antibiotics do not help colds; side effects; finish the agreed course (or stop when advised).

Infection Prevention and Control

• Standard precautions (for all patients): hand hygiene (WHO 5 moments - before patient contact, before aseptic tasks, after body fluid exposure, after patient contact, after contact with surroundings); bare below the elbows; PPE appropriate to the risk; safe sharps handling and disposal; safe management of linen, waste and blood spills; respiratory hygiene.
• Transmission-based precautions:

Route	Examples	Precautions
Contact	MRSA, C. difficile, CPE, norovirus, scabies	Single room, gloves and apron, dedicated equipment; soap and water for C. difficile and norovirus
Droplet	Influenza, meningococcus (first 24 h of treatment), pertussis, mumps, rubella, RSV	Single room, fluid-resistant surgical mask within 1–2 metres
Airborne	TB (pulmonary, smear-positive), measles, chickenpox, disseminated zoster, aerosol-generating procedures with respiratory viruses	Negative-pressure room, FFP3 respirator (fit-tested)

• Device-related infection bundles - peripheral and central lines (aseptic non-touch technique, daily review, remove when not needed - VIP score), urinary catheters (avoid if possible, remove early), ventilator-associated pneumonia bundles, surgical site infection prevention (NICE NG125 - no routine shaving, chlorhexidine skin prep, prophylactic antibiotics within 60 minutes before incision).
• Healthcare worker health - hepatitis B vaccination, seasonal influenza and COVID-19 vaccination, measles and varicella immunity, exclusion when infectious (48 hours after diarrhoea and vomiting), sharps injury management (wash, report, risk-assess, PEP for HIV, HBV vaccination/HBIG, HCV follow-up).

Notifiable Diseases

• Registered medical practitioners in England must notify the local UKHSA health protection team on clinical suspicion - do not wait for laboratory confirmation (Health Protection (Notification) Regulations 2010). Urgent cases by telephone.
• Examples: acute meningitis, meningococcal septicaemia, acute encephalitis, acute infectious hepatitis, food poisoning, infectious bloody diarrhoea, haemolytic uraemic syndrome, enteric fever, measles, mumps, rubella, whooping cough, scarlet fever, invasive group A streptococcal disease, diphtheria, tuberculosis, Legionnaires' disease, malaria, tetanus, botulism, cholera, plague, rabies, viral haemorrhagic fever, COVID-19, mpox, leprosy, yellow fever, anthrax, acute poliomyelitis, typhus, brucellosis.
• Not notifiable by clinicians (reported by laboratories or via other routes) - HIV, STIs (chlamydia, gonorrhoea, syphilis - GUM clinics report), influenza (unless novel), C. difficile (laboratory and mandatory hospital surveillance), MRSA bacteraemia (mandatory surveillance).

At a Glance

Situation	Remember
Sepsis	Cultures then antibiotics within 1 hour - never delay
Asymptomatic bacteriuria	Do not treat (except pregnancy, before urological surgery)
CSF: neutrophils, low glucose	Bacterial meningitis
CSF: lymphocytes, very high protein, low glucose	TB meningitis
Ciprofloxacin	Tendon rupture, aortic aneurysm - last resort
Trimethoprim	Hyperkalaemia; never with methotrexate
Metronidazole	No alcohol
Clarithromycin	QT and CYP3A4 interactions (statins)
48–72 hours	Review: stop, switch, change, continue, OPAT
TB, measles, chickenpox	Airborne - negative pressure, FFP3

Spot the Problem

• An 85-year-old care home resident with a positive urine dipstick but no symptoms is prescribed trimethoprim. → Asymptomatic bacteriuria - should not be treated or tested.
• A man on simvastatin develops myalgia and dark urine after clarithromycin. → Rhabdomyolysis - CYP3A4 interaction.
• A woman on methotrexate becomes pancytopenic after trimethoprim for cystitis. → Combined folate antagonism - dangerous interaction.
• A 70-year-old on prednisolone develops heel pain after levofloxacin. → Achilles tendinopathy/rupture - stop the quinolone.
• Seizures in a patient with epilepsy on valproate after starting meropenem. → Carbapenems dramatically reduce valproate levels.

Exam-style Scenarios

• Scenario. A patient labelled "penicillin allergic" because of a childhood rash needs treatment for pneumonia. What do you do?
  Answer: Take a detailed history - a non-urticarial rash in childhood is low risk. Treat the current infection according to local guidance (a non-penicillin agent if needed), then arrange de-labelling (direct oral challenge or allergy referral) and document the reaction accurately. Avoid penicillins only if there was anaphylaxis, angioedema or a severe cutaneous reaction.
• Scenario. Day 3 of IV co-amoxiclav for pneumonia: afebrile, eating, CRP falling, cultures negative. What next?
  Answer: "Then focus" - switch to oral therapy (IV-to-oral switch criteria met), with a total course of 5 days for community-acquired pneumonia if clinically stable, and document the stop date.
• Scenario. Lumbar puncture: opening pressure 32 cmH₂O, lymphocytes 40/µL, protein 1.1 g/L, glucose ratio 0.3, Gram stain negative. HIV positive, CD4 40.
  Answer: Low glucose with lymphocytes - consider TB and cryptococcal meningitis. Request cryptococcal antigen and India ink (high opening pressure strongly suggests cryptococcus), AFB stain, TB PCR and culture. Treat empirically per specialist advice; therapeutic lumbar punctures for raised pressure.

Key Points

• Blood culture volume and asepsis determine yield; S. aureus is never a contaminant.
• Do not test or treat asymptomatic bacteriuria in older or catheterised patients.
• Learn CSF patterns: bacterial (neutrophils, low glucose), viral (lymphocytes, normal glucose), TB (lymphocytes, very high protein, low glucose).
• Fluoroquinolones carry MHRA warnings - use only when alternatives are inappropriate.
• Most penicillin allergy labels are wrong - de-label safely.
• Start smart, then focus - review at 48–72 hours with a documented plan.
• Notify on clinical suspicion; do not wait for confirmation.

Guidelines and Further Reading

• UKHSA: Start smart, then focus - antimicrobial stewardship toolkit for English hospitals; TARGET toolkit for primary care
• UK Standards for Microbiology Investigations (UKHSA)
• NICE NG15: Antimicrobial stewardship; NICE NG51: Sepsis
• NICE CG183: Drug allergy
• MHRA Drug Safety Update: Fluoroquinolone antibiotics - must now only be prescribed when other commonly recommended antibiotics are inappropriate (January 2024)
• UK National Action Plan: Confronting antimicrobial resistance 2024 to 2029
• National Infection Prevention and Control Manual for England (NHS England)