Lecture 3Antimicrobials and Susceptibility

tests

Dr. Abdelraouf A. Elmanama

Islamic University-Gaza

Medical Technology Department

Lecture outlines

• Kirby-Bauer susceptibility test

• Antimicrobial profiles selection

• Reporting susceptibility test

What Does the Laboratory Need to Knowabout Antimicrobial Susceptibility Testing (AST)?

• Which organisms to test?

• What methods to use?

• What antibiotics to test?

• How to report results?

What Does a Laboratory Need to Know about AST? (con’t)

• How to determine the clinical significance of results?

• How to ensure accuracy of results?

–Quality control / quality assurance

• When to call the MD, infection control, public health?

What Does a Laboratory Need to Know about AST? (con’t)

• When to ask for help?

• Where to go for help?

Brief Review of Routine AST Methods

Routine Susceptibility Tests

• Disk diffusion (Kirby Bauer)

• Broth micro-dilution MIC

– NCCLS reference method

• Etest

Disk Diffusion

Test

Select colonies

Prepare inoculumsuspension

Mix well

Standardize inoculumsuspension

Swab plate

Remove sample

Add disks

Incubate overnight

Measure Zones

Transmitted Light Reflected Light

Zone Interpretive Criteria (mm)

Drug

Disk content

(ug)ResIntSusc

Cefazolin30 1415-17 18

Gentamicin10 1213-14 15

Flash presentation for summary

• Qualitative results

– Susceptible

– Intermediate – may respond if infection is at body site where drug concentrates (e.g. urine) or if higher than normal dose can be safely given

– Resistant

Disk Diffusion Test

Modify methods for fastidious Modify methods for fastidious bacteriabacteria

Clinical Conditions when MICs are Useful

• Endocarditis• Meningitis• Septicemia• Osteomyelitis• Immunosuppressed patients (HIV, cancer, etc.)• Prosthetic devices• Patients not responding despite “Sensitive results”

MIC

• Minimal inhibitory concentration• The lowest concentration of

antimicrobial agent that inhibits the growth of a bacterium

• Interpret:– Susceptible– Intermediate– Resistant

Inoculum Preparation MIC Testing

(NCCLS Reference Method)

• Standardize inoculum suspension

• Final inoculum concentration– 3 – 5 x 105 CFU/ml– (3 – 5 x 104 CFU/well)

Microdilution MIC tray

Prepare inoculumsuspension

Dilute & mix inoculumsuspension

Pour inoculuminto reservoir and inoculate MIC tray

Inoculate purity plate

Incubate overnight

Examining Examining purity platepurity plate

Reflected lightReflected light

Transmitted lightTransmitted light

Read MICs

- +

64

32

16

8

4

2

1

>64

0.5

MICs

>64

MIC on a MIC on a stripstrip

S. pneumoniaeS. pneumoniae Penicillin MIC = 3 Penicillin MIC = 3 g/mlg/ml

MIC Interpretive Criteria (g/ml)

DrugSuscIntRes

cefazolin 816 32

gentamicin 48 16

Empirical Treatment

Infants 1-3 mosAmpicillin + cefotaxime or ceftriaxone

Immunocompetent children > 3 mos and adults <55Cefotaxime or ceftriaxone +

vancomycin

Adults > 55 and adults of any age with alcoholism or other debilitating illnesses

Ampicillin + cefotaxime or ceftriaxone + vancomycin

Hospital-acquired meningitis, posttraumatic or postneurosurgery meningitis, neutropenic patients, or patients with impaired cell-mediated immunity

Ampicillin + ceftazidime + vancomycin

• Ceftazidime should be substituted for ceftriaxone or cefotaxime in neurosurgical patients and in neutropenic patients

Specific treatment

• N. meningitidis

– Penicillin sensitive Penicillin G or Ampicillin

– Penicillin-resistant Ceftriaxone or cefotaxime

Chemoprophylaxis for N. meningitidis

• Rifampin 600 mg every 12 h for 2 days in adults and 10 mg/kg every 12 h for 2 days in children >1 year

• Or

• One dose of ciprofloxacin (750 mg)

• One dose of azithromycin (500 mg)

• One intramuscular dose of ceftriaxone (250 mg)

• Rifampicin is not recommended in pregnant women.

• Pneumococci

– Penicillin-sensitive Penicillin G

– Penicillin-intermediate Ceftriaxone or cefotaxime

– Penicillin-resistant (Ceftriaxone or cefotaxime) + vancomycin

• Gram-negative bacilli (except Pseudomonas spp.) Ceftriaxone or cefotaxime

• Pseudomonas aeruginosa Ceftazidime

• Staphylococci spp.– Methicillin-sensitive Nafcillin– Methicillin-resistant Vancomycin

• Listeria monocytogenes Ampicillin + gentamicin• Haemophilus influenzae Ceftriaxone or

cefotaxime• Streptococcus agalactiae Penicillin G or

ampicillin• Bacteroides fragilis Metronidazole• Fusobacterium spp. Metronidazole

• Local Data and protocols should be observed and reviewed periodically

Thank you