# Presentation: 040723

## What’s New in the 2004 NCCLS Standards for Antimicrobial Susceptibility Testing?

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

****currently working with CDC’s Division of Laboratory Systems through an Interagency Personnel Agreement***

## At the conclusion of this talk, you will be able to......

**List the major changes in the ****2004 NCCLS tables (M100-S14)**
**Describe new test/report recommendations for *****Staphylococcus***** spp.**** including:**
**testing for ****inducible clindamycin resistance**
**use of ****cefoxitin disk test**** to detect oxacillin-resistant staphylococci**

## At the conclusion of this talk, you will be able to......(con’t)

**Discuss ****disk diffusion testing**** of *****Stenotrophomonas maltophilia***** and *****Burkholderia cepacia***
**Describe the new reference guide for ****QC testing frequency**** when various test components are modified**

## NCCLS Standards - 2004

**M100-S14 Tables ****(2004)**
**	****...****..to be used with text documents explaining how to perform the tests....**
**	****	****M2-A8   Disk Diffusion**** (2003)**
**	****	****M7-A6   MIC**** (2003)**
**	****	**

- New!

## Reference Terminology

**...****.when I refer to....**
**M100 ****-- this means the new tables (M100-S14)**
**M2**** -- this means the disk diffusion method (described in M2-A8)**
**M7**** -- this means the MIC method (described in M7-A6)****	****	**

## Major Changes 2004NCCLS M100-S14

- NCCLS

## Updated information in M100-S14  

**Updated information in M100-S14  **

## Major Changes

***Enterobacteriaceae***
**More on *****Salmonella***** spp. and using nalidixic acid to screen for fluoroquinolone resistance **
***Pseudomonas aeruginosa***** and other non-Enterobacteriaceae****	**
**Move levofloxacin from Test / Report Group “U” to Group “B”**
**Disk diffusion breakpoints for *****Stenotrophomonas maltophilia *****and***** Burkholderia cepacia***

## Major Changes (con’t)

***Staphylococcus***** spp.**
**Inducible clindamycin resistance testing/reporting**
**Cefoxitin disk test for *****mec*****A**
**Coagulase-negative***** *****staphylococci**
**More on *****mec*****A and oxacillin MIC results**
**More on reporting ********-lactams on oxacillin susceptible isolates**
***Enterococcus***** *****faecalis***
**Predicting imipenem susceptibility from ampicillin results**

## Major Changes (con’t)

**Quality Control**
**Reference Guide**** for QC testing frequency**
**QC ranges for *****E. coli***** ATCC 35218  and ********-lactam / ********-lactamase inhibitor combination agents when using Haemophilus Test Medium (HTM)**
**Oritavancin**** QC ranges**
***Staphylococcus aureus***** ATCC 29213**
***Enterococcus faecalis***** ATCC 29212**
***Streptococcus pneumoniae***** ATCC 49619**

## Major Changes (con’t)

**New breakpoints**
**Gemifloxacin***** ***
***Enterobacteriaceae *****(FDA-approved for***** Klebsiella pneumoniae)***
***Haemophilus *****spp*****.***
***Streptococcus pneumoniae***
**Telithromycin**
***Staphylococcus spp.***
***Haemophilus *****spp*****.***
***Streptococcus pneumoniae***

## Major Changes (con’t)

**Additions to Table 1A**** **
***Haemophilus***** spp.**
**Gemifloxacin (Group C)**
***Streptococcus pneumoniae***
**Gemifloxacin (Group (B)**

## Table 1A

**Suggested Agents **
**For Routine Testing and Reporting **
**(Fastidious Organisms)**

**Table 1A**

## Major Changes (con’t)

**Potential ****agents of bioterrorism**
**Separate tables for this group of organisms**
***Bacillus anthracis, Yersinia pestis, Burkholderia mallei, Burkholderia pseudomallei***

## Major Changes M100-S14GNR

- GNR

## Salmonella and Fluoroquinolones (FQ)

**“****FQ-susceptible strains of Salmonella that test resistant to ****nalidixic acid**** may be associated with clinical failure or delayed response in FQ-treated patients with extraintestinal salmonellosis. Extraintestinal isolates of Salmonella should also be tested for resistance to ****nalidixic acid.****  For isolates that test susceptible to FQs and resistant to ****nalidixic acid****, the physician should be informed that the isolate may not be eradicated by FQ treatment. A consultation with an infectious disease practitioner is recommended.”**

**M100-S14 (M2, M7); Table 2A**

## Salmonella spp. (blood)

**	****	****ampicillin****	****>32 R****	****	****ciprofloxacin****	**** ********0.25 S****	****ceftriaxone****	********0.5 S****	****	****trimeth-sulfa****	****>4/78 R**

***...******.Test nalidixic acid on extraintestinal isolates with ciprofloxacin MICs of 0.12–1.0 ************g/ml; a ciprofloxacin MIC of 2.0 ************g/ml is Intermediate and ************4.0 ************g/ml is Resistant***

_**MIC (**__****__**g/ml)**_

- Fresno 11/00

## Salmonella and Ciprofloxacin

**      ******** ****0.06 ****	****      S ****	****       ****	****    none ****	****	**** S**
**      ****0.12- 1  ****	****      S****	****	****     one****	****	**** R***
**        ******** ****4****	****	****      R****	****	****     two****	****	**** R**

**CIP**** ****MIC****	****    NCCLS****	****      Likely      Nalidixic **
**(********g/ml)    Interpretation      Mutation       acid**

**Threlfall et al. 2001. EID. 7:448.**
**Butt et al. 2003. EID 9:1621.**

***Some patients with extraintestinal infection with *****Salmonella *****spp.***** *****may fail FQ therapy; u****se nalidixic acid as a surrogate to detect single step mutants.**

## Salmonella spp. (blood)

**	****	****ampicillin****	****>32 R****	****	****ciprofloxacin****	****     ********0.25 S*****	****ceftriaxone****	********0.5 S****	****	****trimeth-sulfa****	****>4/78 R**

****....if nalidixic acid is resistant, add comment such as...... ***
**”****This isolate demonstrates reduced susceptibility to fluoroquinolones.  For some patients with extraintestinal Salmonella infections with such isolates, the isolates may not be eradicated by fluoroquinolone treatment.  ID consult suggested.”**

_**MIC (**__****__**g/ml)**_

- Fresno 11/00

## Salmonella and Ciprofloxacin

***“******Salmonella***** spp. isolated from sterile sites or from patients that have failed FQ therapy should be tested for the ****MIC of ciprofloxacin or susceptibility to nalidixic acid.**** Those isolates for which the ciprofloxacin MICs are ********0.125 ********g/ml or resistant to ****nalidixic acid**** should be considered to have reduced susceptibility to FQs and physicians should be warned that clinical failure or delayed response may be associated with FQ treatment of infections caused by these isolates”.****  **

**Poutanen and Low. 2003. CMN 25:97**

## Salmonella and Reduced Ciprofloxacin Susceptibility (MIC 0.12 g/ml )

**Isolates uncommon in ****USA**** **
**(**_**[www.cdc.gov/narms/annual/2001](http://www.cdc.gov/narms/annual/2001)**__**)**_
**UK ****study (1999) - 23% in *****S. typhi***
**Mostly travelers from India and Pakistan**
**	****Threlfall et al. 2001. EID. 7:448.**
**Nalidixic acid screen study - ****n=1010 Salmonella; 50 isolates w/ reduced ciprofloxacin susceptibility:**
**Sensitivity   100%**
**Specificity     87%**
**	****Hakanen et. al. 1999. JCM. 37:3572.**

## New Disk Diffusion Breakpoints

***Stenotrophomonas maltophilia***
**levofloxacin**
**minocycline**
**trimethoprim-sulfamethoxazole**
***Burkholderia cepacia***
**ceftazidime**
**meropenem**
**minocycline**

**35****°****C; ambient air; 20-24 h incubation**** **

## Excerpt from Table 2B (M2)....“Zone Diameter Interpretive Standards and Equivalent MIC Breakpoints for P. aeruginosa, Acinetobacter spp., S. maltophilia, and B. cepacia”

| Agent | R | I | S |   | Comments |
| --- | --- | --- | --- | --- | --- |
| Ceftazidime |  14
 17 | 15-17
18-20 |  18
 21 |  | 
For B. cepacia |
| Minocycline |  14 | 15-18 |  19 |  | May be reported for S. maltophilia and B. cepacia also |
| Trimeth-sulfa |  10 | 11-15 |  16 |  | May be reported for S. maltophilia also |

## Relocation of Levofloxacin in Table 1 for Pseudomonas aeruginosa and Other Non-Enterobacteriaceae

| ...new M100-S14 |  |
| --- | --- |
| Group B
Primary Test 
Report Selectively | ciprofloxacin 
levofloxacin |
|  |  |
| ...old M100-S13 |  |
| Group U 
Supplemental for Urine Only | levofloxacin or   lomefloxacin or 
norfloxacin or
ofloxacin |

**M100-S14 (M2, M7); Table 1 & 2B**

## Major Changes M100-S14Staphylococcus

- Staphylococcus

## Staphylococcus spp.

**“****Macrolide resistant isolates of *****S. aureus***** and coagulase-negative *****Staphylococcus***** spp. may have ****constitutive or inducible resistance to clindamycin**** [methylation of the 23S rRNA encoded by the *****erm***** gene also referred to as MLS****B**** ****(macrolide, lincosamide, and type B streptogramin) resistance] or may be resistant only to macrolides (efflux-mechanism encoded by the *****msrA***** gene).”**

**M100-S14 (M2, M7); Table 2C**

## Staphylococcus spp. Erythromycin / Clindamycin

| Mechanism | Determinant | Erythro | Clinda |
| --- | --- | --- | --- |
| Efflux | msrA | R | S |
| Ribosome alteration | erm | R | S* |
| Ribosome alteration | erm | R | R
constitutive |

***msrA*****	****= macrolide streptogramin resistance**
***erm*****	****= erythromycin ribosome methylase**
***requires induction to show resistance**

** **

***Staphylococcus***** spp. ****Erythromycin / Clindamycin**

## Staphylococcus aureus 

**  **
** ****	****	****    ****clindamycin****	****        S**
**	****	****	****erythromycin****	**** R**
**  ****	****	****    ****oxacillin****	****	****   ****	**** R**
** ****	**** ****	****    ****penicillin****	****        R**
** ****	****	****    ****vancomycin****	****   ****	**** S**

***If clindamycin-S and erythromycin-R, do not report clindamycin-S without performance of “D Test”***

## Staphylococcus aureus 

**  **
** ****	****  ****	****	****erythromycin****	**** R**
**  ****	****	****    ****oxacillin****	****	****   ****	**** R**
** ****	**** ****	****    ****penicillin****	****        R**
** ****	****	****    ****vancomycin****	****   ****	**** S**
**“****Contact laboratory if clindamycin results needed”**

***Optional reporting strategy***

## “D Test” – positive reaction

**clindamycin resistance**
**(*****erm-*****mediated)**
**Routine disk diffusion test:**
**Place 2 ********g clindamycin disk 15 mm to 26 mm from edge of 15 ********g erythromycin disk.**
**QC strain forthcoming!**

**“****D Test” – ****positive**** reaction**

## “D Test” – positive reaction

**Inducible **
**clindamycin resistance**
**(*****erm-*****mediated)**
**...****another example**

**“****D Test” – ****positive**** reaction**

**15 - 26 mm**

- Photos courtesy of J. Jorgensen and K. Fiebelkorn**.**

## “D Test” – negative reaction

**“****D Test” – negative reaction**

**NO induction**
**(*****msr*****A*****-*****mediated erythromycin resistance)**

## “D Test” – positive reaction

**E 15**

**CC 2**

**Routine purity plate:**
**  ****Streak 1/3 of plate       for confluent growth**
**  ****Place 2 ********g clindamycin disk 15 mm from edge of 15 ********g erythromycin disk**

**Inducible clindamycin resistance      (*****erm-*****mediated)**

## Staphylococcus aureus

**	****	****clindamycin****	****	**** R  ****	****	**
**	****	****erythromycin****	**** R**
** ****	**** ****	****oxacillin****	****	****	**** R**
** ****	****	****penicillin****	****	****	**** R**
** ****	****	****vancomycin     ****	**** S**

**“****This *****S. aureus*****  is presumed to be resistant based on detection of inducible clindamycin resistance.  Clindamycin may still be effective in some patients.”**

***“******D Test” positive and optional comment***

## Staphylococcus aureus 

**	****	****clindamycin****	****	**** S****  ****	****	**
**	****	****erythromycin****	**** R**
** ****	**** ****	****oxacillin****	****	****	**** R**
** ****	****	****penicillin****	****	****	**** R**
** ****	****	****vancomycin     ****	**** S**

**“****This *****S. aureus***** DOES NOT demonstrate inducible clindamycin resistance in vitro.”**

***“******D Test” negative and optional comment***

## Inducible Clindamycin Resistance - Incidence

**Varies**** considerably geographically **
**Community-associated MRSA**** **
**Frequently erythromycin-R  clindamycin-S**
**Often *****msrA*****-mediated mechanism (NOT inducible)**
**USA report 2002**
**617 *****S. aureus***** erythromycin-R clindamycin-S**
**50% NOT inducible resistance**
**	****Fiebelkorn et al. 2003. JCM. 41:4740.**

## Staphylococcus spp.

**“****The results of disk diffusion tests using a ****30 ********g cefoxitin disk**** and alternate breakpoints (see box at end of this table) can be used to predict *****mec*****A –mediated resistance in staphylococci.”**

**M100-S14 (M2, M7); Table 2C**

## Disk Diffusion Screen for mecA-mediated Resistance in Staphylococci

**Perform standard disk diffusion test with cefoxitin (30 ********g) disk**
**Incubate 24 h; however, results may be reported after 18 h, if resistant**
**Report results for ****OXACILLIN****, not cefoxitin**

## Staphylococcus spp.

***Staphylococcus***** spp.**

**Table 2C           (M2, M7)**

## Disk Diffusion Screen for mecA-mediated Resistance in Staphylococci (con’t)

** ****	****	****   ****	****                  **_**Cefoxitin zone (mm)**_** ****	****             **
***     ******S. aureus*****              ******** 19***** ****               ******** 20********
**     ****CoNS****	****                   ******** 24*********                ******** 25********
*******	****Report as oxacillin resistant**
********	****Report as oxacillin susceptible**
**CoNS, coagulase-negative staphylococci**

**M100-S14 (M2, M7); Table 2C**

## Staphylococcus - Oxacillin

**MIC (μg/ml):**** ****	****	****	****	****	****	****	****	****         ****	**** ****	**** **_**Susc**_**    ****	**** **_**Int**_**        **_**Res**_
***	******S. aureus*****	****  ****	****   ******** 2        ****	****  -   ****	****      ******** 4**
**	****CoNS****	****      ****	****	******** 0.25****	****  -          ******** 0.5**
**DD (mm):**
**	****        ****	**** ****	****	****  **_**Res**_** ****	**** **_**Int**_**        **_**Susc**_
***	******S. aureus*****	****  ****	**** ******** 10       11-12      ******** 13**
**	****CoNS****	****         ****	**** ******** 17           -          ******** 18**

**M100-S14 (M2, M7); Table 2C**

## Oxacillin Breakpoints Coagulase-Negative Staphylococci

**May ****overcall resistance**** for species other than *****S. epidermidis***** (e.g. *****S. lugdunensis, S. saprophyticus*****)**
**For ****serious infections**** with CoNS other than *****S. epidermidis*****, testing for *****mecA***** or PBP 2a may be appropriate for strains having oxacillin MICs of 0.5 – 2 ********g/ml or oxacillin zones ********17 mm**
**If *****mec*****A or PBP 2a negative, report as oxacillin susceptible **

**M100-S14 (M2, M7); Table 2C**

## Oxacillin Breakpoints Coagulase-Negative Staphylococci (con’t)

**For ****oxacillin-resistant**** strains (including PBP 2a or *****mec*****A positive strains), report all ********-lactams resistant**
**For ****oxacillin-susceptible**** strains, report any ********-lactams tested according to results generated**

**M100-S14 (M2, M7); Table 2C**

## Reporting Oxacillin MIC Results for Coagulase-Negative Staphylococci*

***For testing non-*****S. epidermidis***** from sterile sites**

## Reporting Oxacillin Disk Diffusion Results for Coagulase-Negative Staphylococci*

***For testing non-*****S. epidermidis***** from sterile sites**

## Major Changes M100-S14Enterococcus faecalis

- E. faecalis

## Enterococcus faecalis 

**“****Ampicillin susceptibility can be used to predict ****imipenem**** susceptibility provided the species is confirmed to be *****E. faecalis*****”.**

**M100-S14 (M2, M7); Table 2D**

## E. faecalis (blood)

**	****	****ampicillin ****	****       S**
**	****	****vancomycin          S**
**	****	****gent synergy   ****	****     R**
**	****	****strep synergy ****	****     S**
**“****Ampicillin-susceptible *****E. faecalis***** are imipenem susceptible”**

***If MD requests imipenem results on E. faecalis....***

## Major Changes M100-S14Quality Control

- QC

## Reference Guide to 

**Quality Control**
** ****Testing Frequency**

**Table 3B (M2, M7)**

## Excerpt from:  “Reference Guide to QC Testing Frequency” (for ATCC QC strains after 20-30 consecutive days of satisfactory daily testing)

| MIC test modification | 1 | 5 | 20 or 30 | Comments |
| --- | --- | --- | --- | --- |
| Use new shipment or lot number | X |  |  |  |
| Use new manufacturer of MIC test |  |  | X | In addition, perform in-house validation studies |
| Software update that affects AST results |  | X |  | Monitor all drugs not just those implicated in software modification |

**No. of days of consecutive QC testing required**

**M100-S14 (M2, M7); Table 3B**

## Excerpt from:  “Reference Guide to QC Testing Frequency” (con’t)

**Note 1:****  Addition of any NEW antimicrobial agent requires 20 or 30 consecutive days of satisfactory testing, prior to use of this guide.**
**Note 2:****  QC can be performed prior to or concurrent with testing patient isolates.  Patient results can be reported for that day if QC results are within the acceptable limits.**

**M100-S14 (M2, M7); Table 3B**

## Excerpt from:  “Reference Guide to QC Testing Frequency” (con’t)

**Note 3:****  Manufacturers of commercial or in-house prepared tests should follow their own internal procedures and applicable regulations.**
**Note 4:****  For troubleshooting out-of-range results, refer to M2-A8 or M7-A6, QC section.**
**Note 5:****  Broth, saline and/or water used to prepare an inoculum does not require routine QC.**

**M100-S14 (M2, M7); Table 3B**

## More Examples.....Application of Table 3C (consecutive days of daily QC required)

**Inoculum preparation, convert from:**
**Visual to Prompt – 20 or 30 days**
**Visual to photometer – 5 days**
**Prompt to photometer – 5 days**
**Instrument / software**
**Repair of instrument – 1 day (or more)**
**Replace hardware (e.g. reader-incubator) – 20 or 30 days**

## Haemophilus Test Medium (HTM)

**Addition of disk diffusion and MIC QC ranges for *****E. coli***** ATCC 35218**** (ß-lactamase producing strain) and ß-lactam / ß-lactamase inhibitor drugs**

## QC Ranges Using HTM

|  | H. influenzae ATCC 49247 | E. coli 
ATCC 35218 |
| --- | --- | --- |
| 
Zone (mm) |  |  |
| Amoxicillin-clavulanic acid | 15 - 23 mm | 17 - 22 mm |
| 
MIC (g/ml) |  |  |
| Amoxicillin | - |  256 |
| Amoxicillin-clavulanic acid | 2/1 – 16/8 | 4/2 - 16/8 |
| Ticarcillin-clavulanic acid | - | 16/2 – 64/2 |

**M100-S14 (M2, M7); Table 3A**

## Major Changes M100-S14Newer Antimicrobial Agents

- New Agents

## Newer Antimicrobial Agents...

| Agent | Drug class | Route of administration | FDA approved |
| --- | --- | --- | --- |
| Daptomycin
(Cubicin) | lipopeptide | IV | yes |
| Gemifloxacin
(Factive) | fluoroquinolone | PO | yes |
| Oritavancin | glycopeptide | IV | no |
| Telithromycin
(Ketek) | ketolide | PO | no |

**See Glossary in M100-S14**

## Daptomycin

**In vitro activity against gram-positive bacteria including MRSA and VRE**
**Mode of action**
**Bactericidal**
**Requires physiologic calcium**

## Daptomycin (con’t)

**Susceptibility testing media requirements**
**Mueller-Hinton broth – 50 mg/L calcium chloride**
**Mueller-Hinton agar – 28 mg/L calcium chloride**
**Currently, no NCCLS breakpoints; FDA breakpoints available in product literature**
**NCCLS QC ranges available**
**No resistance reported to date among *****S. aureus***** or Group A or B Streptococcus**

## Gemifloxacin

**Active against ****respiratory pathogens**** including *****Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila***
**Inhibits DNA synthesis**** through inhibition of both DNA gyrase and topoisomerase IV (dual target)**

## Oritavancin

**Bactericidal in vitro**
**Active against most gram-positive pathogens including VRE**

## Telithromycin

**Active against ****respiratory pathogens**** (*****S. pneumoniae, H. influenzae, M. catarrhalis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila)***
** ****Active against other ****gram-positive bacteria**** that have inducible MLS****B**** mechanism of resistance and does not induce resistance**
**Active against *****S. pneumoniae***** resistant to erythromycin and clarithromycin regardless of resistance mechanism**

## Major Changes M100-S14Bioterrorism Agents

- Bioterrorism

## Bioterrorism Agents

**Table 2K (M7)**

**Bioterrorism Agents**

## Potential Agents of Bioterrorism

**Bacteria included in M100-S14**
***Bacillus anthracis***
***Burkholderia mallei***
***Burkholderia pseudomallei***
***Yersinia pestis***
**New Tables  **
**1B (Antimicrobial agents to test/report)**
**2K (MIC interpretive standards)**

## Very last pages of M100-S14!

**Summary of Comments and **
**Subcommittee Responses**

## Recap of ..“Summary of Comments and Subcommittee Responses”

**ESBLs testing**
**Report ****cefepime**** as resistant for ESBL producers**
**There is limited data on use of ****cephamycins ****(e.g. cefoxitin, cefotetan) for treating infections caused by ESBL producers**
**If ****ESBL confirmatory test negative****, report results as tested (do not override to resistant)**
**Currently, ****only *****E. coli***** and *****Klebsiella***** spp.**** are addressed in NCCLS ESBL testing rules**

## Recap of..“Summary of Comments and Subcommittee Responses” (con’t)

**Other GNR**
**There are no specific NCCLS recommendations for testing for inducible ********-lactamases.  To help detect resistance to 3****rd**** generation cephalosporins resulting from selection of derepressed mutants, repeat testing after 3-4 days is suggested for *****Enterobacter*****, *****Citrobacter*****, *****Serratia*****.**
***mec*****A**** and coagulase-negative staphylococci**
**Discussed above**

## Recap of..“Summary of Comments and Subcommittee Responses” (con’t)

**Incubation temperature**** **
**range 33-35********C **
**oxacillin – *****Staphylococcus***** spp., 33-35********C (not >35********C)**
***Haemophilus***** spp.**
******-lactamase testing only would not detect BLNAR strains**
**MIC testing –frequency of performing ****colony count**** to QC inoculum**
**Perform at least quarterly**
**QC of commercial ****McFarland standards**
**Follow manufacturers recommendations**

## Some Issues Under Discussion by NCCLS 

***Staphylococcus***** spp****. - re-evaluate moxifloxacin, gatifloxacin, levofloxacin, ciprofloxacin breakpoints**
**Acinetobacter**** – examine correlation of disk and MIC results for ********-lactams and tetracyclines**
**Development of ****new Guideline**** for testing bacteria not currently addressed in NCCLS AST standards (e.g. *****Corynebacterium*****, HACEK, etc.)**

## Enterobacteriaceae -Lactam Breakpoints and ESBL Issues

**Re-evaluation of ********-lactam breakpoints**** for Enterobacteriaceae**
**Example: cefotaxime**
**Current – Susceptible at ******** 8 ********g/ml**
**Proposed – Susceptible at ******** 1 or ******** 2 ********g/ml**
**Substantial data needed**
**Goal is to more accurately detect all ********-lactamase and other ********-lactam resistance mechanisms with revised breakpoints **
**Changing breakpoints – ****commercial systems**** project it will take 3 years ...much $$$$$!**

## Thank you!

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