# Presentation: 454425

## Antimicrobial Susceptibility Testing for Bacteriology Laboratories

## Objectives:At the conclusion of this presentation participants will be able to...

**Explain how to implement current ****NCCLS**** antimicrobial susceptibility testing (AST) and reporting recommendations.  **
**Describe ****reliable methods**** for detecting resistance among:**
***Enterobacteriaceae***
***Pseudomonas aeruginosa***
***Staphylococcus aureus***
***Enterococcus spp.***
***Streptococcus pneumoniae *****and***** Streptococcus spp.***

## At the conclusion of this presentation Participants will be able to...(con’t)

**Describe ****effective reporting**** of AST results.**
**List steps that can be taken to ****verify AST results**** obtained on bacteria isolated from patients.**
**Discuss NCCLS-recommended ****QC procedures**** and how to trouble-shoot out-of-control QC results**

## What Does the Laboratory Need to Knowabout 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

- AST

## Routine Susceptibility Tests

**Disk diffusion (Kirby Bauer)**
**Broth micro-dilution MIC**** **
**NCCLS reference method**
**Etest**

## Disk Diffusion 
Test

**Test**

## Select colonies

**Select colonies**

**Prepare inoculum**
**suspension**

## Mix well

**Mix well**

**Standardize inoculum**
**suspension**

## Swab plate

**Swab plate**

**Remove sample**

## Add disks

**Add disks**

**Incubate overnight**

## Measure Zones

**Transmitted**
**Light**

**Reflected**
**Light**

## Measure Zones Transmitted Light*

***Staphylococci***** – oxacillin, vancomycin**
***Enterococci***** – vancomycin**

***Measure all other drug/bug **
**combinations with reflected light**

## Zone Interpretive Criteria (mm)

| 
Drug | Disk content (ug) | 
Res | 
Int | 
Susc |
| --- | --- | --- | --- | --- |
| cefazolin | 30 |  14 | 15-17 |  18 |
| gentamicin | 10 |  12 | 13-14 |  15 |

## Disk Diffusion Test

**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**

## Modify methods for fastidious bacteria

**Modify methods for fastidious bacteria**

## Clinical Conditions when MICs are Useful 

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

## 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 10****5**** CFU/ml**
**(3 – 5 x 10****4**** CFU/well)**

## Microdilution MIC tray

**Microdilution MIC tray**

**Prepare inoculum**
**suspension**

## Dilute & mix inoculum

**Dilute & mix inoculum**
**suspension**

## Pour inoculum

**Pour inoculum**
**into reservoir and **
**inoculate MIC tray**

## Inoculate 

**Inoculate **
**purity plate**

**Incubate overnight**

## Examining 

**Examining **
**purity plate**

**Reflected light**

**Transmitted light**

##  Optimal Use of Purity Plates

**Sub ****final test suspension**** to non-selective medium (after inoculating MIC test)**
**Streak for ****isolation**** (avoid several specimens per plate - may not reveal contaminants if no isolated colonies)**
**Examine ****before reading MIC**** (usually at 16-20 h)**
**Re-incubate**** if antibiogram questionable**

## Read MICs

**Read MICs**

## -

**-**

**+**

**64**

**32**

**16**

**  ****8**

**  ****4**

**  ****2**

**  ****1**

**>64**

** ****0.5**

**MICs**

**>64**

## Reporting Time: Disk Diffusion or MIC

***Staphylococci***** – oxacillin, vancomycin, cefoxitin**
***Enterococci***** – vancomycin**
**“****Resistance**** may be reported any time growth is observed after a minimum of 16 h incubation”**
**After minimum of 16 h, read test:**
**if ****“R”****, report**
**If ****“S”****, re-incubate and read again at 24 h**

**NCCLS M2-A8, M7-A6**

## MIC on a strip

**MIC on a strip**
**abbiodisk.com**

## Slide 32

***S. pneumoniae***** ****Penicillin MIC = 3 ********g/ml**

## MIC Interpretive Criteria (g/ml)

| 
Drug | 
Susc | 
Int | 
Res |
| --- | --- | --- | --- |
| cefazolin |  8 | 16 |  32 |
| gentamicin |  4 | 8 |  16 |

## MICs Recommended (NCCLS M100-S14)

**Viridans *****Streptococcus***** – penicillin**
***S. pneumoniae***** – penicillin, cefotaxime/ceftriaxone (sterile sites)**
***Enterococcus***** – vancomycin “Int” results**
***Staphylococcus***** – vancomycin zone ********14 mm**

## Credits

**Janet Fick Hindler, MCLS MT(ASCP)**
**UCLA Medical Center**
**Los Angeles, CA**