# Presentation: 530529

## Detecting Resistance in Staphylococci and Enterococci!

**Janet Fick Hindler, MCLS MT(ASCP)***
**UCLA Medical Center**
**Los Angeles, CA**
**jhindler@ucla.edu**
*** ***

****also working as a consultant with the Association of Public Health Laboratories with support from CDC***

## At the conclusion of this talk, as related to staphylococci and enterococci, you will be able to......

**Discuss ****reliable methods**** for detecting resistance among staphylococci and enterococci.**
**Explain how to implement current ****CLSI ****antimicrobial susceptibility testing and reporting recommendations.**
**List results obtained from testing patient’s isolates that should be ****verified**** prior to reporting as part of ****quality assessment.****  **

## Staphylococcus aureus

- Staphylococcus

## Staphylococcus AST Issues

**Special tests**
**Induced ********-lactamase test**
**Oxacillin resistance detection:**
**Cefoxitin disk diffusion**
**Oxacillin-salt agar screen (*****S. aureus*****)**
***mecA*****, PBP2a**
**D zone test (inducible clindamycin resistance)**
**BHI-vancomycin screen for VISA and VRSA**
**Reporting**
***Warning*****: MRSA or MRCoNS are “R” to ALL ********-lactams**
**Predict any ********-lactam result using results from oxacillin and penicillin testing**

## Staphylococcus aureus

**oxacillin,**
** ****nafcillin**
**vancomycin**** **

**a cephalosporin, vancomycin, ß-lac/ß-lac inhibitor combo, carbapenem, macrolide, clindamycin, linezolid, quin-dalfo, fluoroquinolone**
**linezolid, quin-dalfo, fluoroquinolone, trim-sulfa**

**Oxacillin-S**

**Alternative Drugs**

**1st Choice Drugs**

**Organism**

**Sanford Guide, 2004**

**Oxacillin-R**

## Staphylococcus spp. Penicillin “MIC” Testing/Reporting*

***********-lactamase testing not necessary for disk diffusion!**

## Induced ß-lactamase Test

**Induced ß-lactamase Test**

**Oxacillin**
**(inducer)**

** ****Sub isolate to BAP**
** ****Drop oxacillin disk; incubate**
** ****Test cells from periphery of zone**
** ****Other methods**

## ß-lactamase Test

**Positive**

**Negative**

## Penicillinase-Stable Penicillins

**Oxacillin**** = group representative**
**Oxacillin ****performs best**** in in vitro test systems (see cefoxitin disk diffusion test)**
**Oxacillin results used to ****predict**** results of other penicillinase-stable penicillins:**
**Methicillin**
**Nafcillin**
**Dicloxacillin**
**MRSA**** = ORSA**

## Classic MRSA - Expression

***mecA***** ****= genetic determinant of MRSA**
***mec*****A codes for PBP2a**** which confers oxacillin resistance in MRSA and MR-CoNS (coagulase-negative staphylococci)**
**Heterogeneous**** ****expression**** - in MRSA (*****mecA***** +) population, some cells appear ****“S”**** and others appear ****“R”**** under standard test conditions**
**Homogeneous expression**** - in MRSA (*****mecA***** +) population, all cells appear ****“R”**

## Heterogeneous

**Heterogeneous**
**expression**

**Haze within zone due **
**to oxacillin-R cells**

**Homogeneous**
**expression**

**Confluent growth **
**up to disk**

## Measure Zones for Oxacillin and Vancomycin

_**USE**_** Transmitted**
**Light**

**NOT Reflected**
**Light**

## Primary Phenotypic Tests for mecA-mediated Resistance in Staphylococci

**Disk diffusion – ****test cefoxitin (30 ********g) disk (CX DD)**
***S. aureus***** - results comparable to oxacillin disk (OX DD)**
**Coagulase-neg staphylococci (CoNS) – results better than OX DD**
**Read with reflected light; easier to read than oxacillin**
**Report results for ****OXACILLIN****, not cefoxitin**
**MIC tests**** – test oxacillin**
**Oxacillin-salt agar**** screen for *****S. aureus***
**Incubation:***** *****33-35********C (do not exceed 35********C); full 24 h before reporting “S” **

## Cefoxitin Disk for mecA-mediated Resistance in Staphylococci

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

**CLSI M100-S15 Table 2C (M2, M7)**

## Why does cefoxitin disk diffusion test work better than oxacillin disk in detecting isolates with mecA?

***mec*****A is expressed at higher levels in presence of ****cefoxitin**** as compared to oxacillin**

## Oxacillin-salt agar 

**Oxacillin-salt agar **
**screen for *****S. aureus***
**(MHA + 4% NaCl + 6 ********g/ml oxacillin)**

**- control**

**+ control**

**Positive**
**patient**

**+ control – *****S. aureus***** ATCC 43300**
**- control – *****S. aureus***** ATCC 29213**

**Negative**
**patient**

**Inoculum:****  **
**McFarland 0.5 standard**
**(1.5 x 10****8**** CFU/ml)**
** ****Use 1 mcl loop OR swab**

## Staphylococcus spp.

**“****Isolates of staphylococci that are shown to carry the *****mecA***** gene, or that produce ****PBP2a****, the gene product, should be reported as ****oxacillin resistant****.”**

**CLSI M100-S15 Table 2C (M2, M7)**

## MRSADetection of Gene or Gene Product

**PCR for *****mec*****A; testing time, 1-2 h**
**Colonies of *****S. aureus***
**Broth with GPC clusters – must look for *****S. aureus *****gene too**
**Nasal swab (FDA-cleared kit)**
**IDI-MRSA (Infectio Diagnostic)**
**PBP2a ****detection on colonies of *****S. aureus***

## PBP2a rapid latex agglutination assay kit

**- Perform on isolated colonies**
**- Extract PBP2a**
**- React with latex antibody to PBP2a**

## Utility of mecA or PBP2a Tests

**Rapid result**
**Communicate to physician rapidly to realize test’s value **
**Facilities with ****high incidence of MRSA:**
**Empiric therapy for *****S. aureus***** often vancomycin **
**Negative**** result: physician may**** ****discontinue vancomycin **
**Facilities with ****low incidence of MRSA:**
**Empiric therapy for *****S. aureus***** often ********-lactam**
**Positive**** result: physician may**** ****initiate vancomycin and contact isolation**

## Utility of mecA or PBP2a Tests (con’t)

**Usually not sufficient as a ****“stand alone”**** susceptibility test**
**Other drug results needed**
**Possible exception - MRSA surveillance cultures**
**Value in ****previous**** MRSA patients?**
**Good for ****arbitrating**** equivocal results from DD, MIC, or agar screen tests**

## Staphylococcus - Oxacillin

**	****	****	****	****	****	****  ****MIC (μg/ml)**** ****	****	****	****	****	****	**** **_**Susc**_**    ****	**** **_**Int**_**        **_**Res**_
***	******S. aureus*****	****  ****	****   ********2        ****	****  -   ****	****      ********4**
**	*****S. lugdunensis***
**	****CoNS****	****      ****	****	********0.25****	****  -          ********0.5**

**CLSI  M100-S15 Table 2C (M7)**

## Staphylococcus lugdunensis

**Coagulase**** ****	****	****	**
**	****Slide****	****	****	****	****    pos or neg**
**	****Tube coagulase ****	****          ****	****neg**
**Ornithine decarboxylase****	****pos**
**PYR****	****	****	****	****	****	****pos**
**Penicillin****	****	****	****	****     often susceptible**
**Sometimes misidentified as *****S. aureus***

**CMPH, 2004, ASM Press; MCM, 8****th**** ed, 2003 ASM Press**

## Staphylococcus lugdunensis 

**	**** ****	****	****	****	****	****MIC**** ****	****     ****M100   M100**
**	****	****	****	****	****     ****(********g/ml)    -S14    -S15**
**	****	****cefazolin****	****	********0.5****	****	****R*****	****    ****S****     ****	**
**	****	****clindamycin****	********0.5**** ****	**** ****	****S****	****    S**
** ****	**** ****	****erythromycin****	********0.5 ****	****	****S****	****    S**
** ****	**** ****	****oxacillin****	****	****  0.5****	****	****R****	****    S**
** ****	****	****penicillin****	****      ********0.06****	****	****R*****	****    S**
** ****	****	****vancomycin****	********0.5****	****	****S****	****    S**
** **

*** Edited to “R” because of oxacillin-R result**

**Interpretation**

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

***For testing non-*****S. epidermidis***** isolates from **
**sterile sites where CoNS is causing a serious infection**

## Recap of Tests for MRSA and MRCoNS   

***S. aureus***
**Tests for *****mec*****A, PBP2a, CX DD, OX DD or OX MIC work well **
**CX DD = OX DD = OX MIC**
**CoNS**
**Tests for *****mec*****A or PBP2a work well**
**OX MIC may overcall R in non-epidermidis CoNS**
**CX DD better than either OX MIC or OX DD**
**Addition of CX DD for strains with OX-R MICs eliminates some false OX-R (specificity increased from 61% to 91%)**

## Do We Need to Perform Multiple Tests to Confirm MRSA?

**Factors to consider****:**
**Technical****:**
**Reliability of your “routine” test system**
**Competency of staff**
**Incidence of errors**
**Patients / setting**
**Source of specimen**
**Patient history (including previous MRSA)**
**Incidence of MRSA**
**“****Consequences”**** of reporting or missing MRSA**

## Staphylococcus spp.Use Pen and Ox results to deduce results for other ß-lactams

**Pen****	****Ox****	****               Comments**
**  ****S****	**** S****	****S**** to penicillins, cephems, ****	****	****	****	****carbapenems**
**  ****R****	**** S****	****R**** to ß-lactamase-labile pens; ****  ****	****	****	****S**** to ß-lactamase-stable pens; ****	****	****	****S****  to ß-lac / ß-lac inhibitor ****	****	****	****	****combos, cephems, carbapenems**
**  ****R****	**** R****	****R**** to all ß-lactams **

**CLSI M100-S15 Table 1 (M2, M7)**

## Staphylococcus aureus 

**  **
** ****	****	**** ****	****   ****clindamycin       S****	****	**
** ****	****	**** ****	****   ****erythromycin****	****  S**
**  ****	****	**** ****	****   ****oxacillin****	****	****  S**
** ****	**** ****	**** ****	****   ****penicillin****	****	****  R**
** ****	****	**** ****	****   ****vancomycin       S**
**“****Cefazolin and other ß-lactams (except amoxicillin, ampicillin, and penicillins) are active against oxacillin-S and penicillin-R staphylococci.”**

***Consider adding comment to report to further explain ******ß-lactam***** *****results***

**Ucla example**

## Staphylococcus aureus 

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

**** For MRSA,***** *****If any ******ß-******lactam is tested and tests “S”,  do not report or change to “R”***

**“****Oxacillin-R staphylococci are resistant to all ß-lactams.  **
**MRSA isolated, please check Infection Control policies.”**

## Staphylococcus aureus 

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

***Historically, MRSA has been multiply resistant to ***
***other anti-staphylococcal agents.  However,***
***some MRSA, particularly community-associated strains ***
***are not multiply resistant.***

## Hospital-acquired (HA) vs. Community-associated (CA) MRSA

**Can’t distinguish by routine in vitro tests**
**Requires sophisticated laboratory tests + epidemiological information**

## HA-MRSA vs. CA-MRSA

| Parameter | HA-MRSA | CA-MRSA |
| --- | --- | --- |
| First reported | 1970s | 1990s |
| Infections | All types | Primarily skin & soft tissue |
| ß-lactam resistance | SCC mec I-III | SCC mec IV |
| Other antimicrobials | Multiply resistant | Often not   multiply resistant |
| Virulence factors | N/A | Sometimes PVL* |

***Panton-Valentine leukocidin**

## Staphylococcus aureus(OP wound – community-associated MRSA) 

**  ****	****	****clindamycin     ****	**** S****	****	**
** ****	**** ****	****erythromycin****	****	**** ****	****R**
** ****	**** ****	****oxacillin****	****	****	**** ****	****R**
** ****	****	****penicillin****	****	****	**** ****	****R**
**	****	****rifampin****	****	****	**** S**
**	****	****tetracycline****	****	**** S***
**	****	****trimeth-sulfa****	****        S**
** ****	****	****vancomycin     ****	**** S**

***also “S” to doxycycline and minocycline**

**Linezolid?**

## http://www.lapublichealth.org/acd/MRSA.htm

**http://www.lapublichealth.org/acd/MRSA.htm**

**http://www.cdc.gov/ncidod/hip/aresist/mrsa_spotlight.htm**

## 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).”**

**CLSI M100-S15 Table 2C (M2, M7)**

## Staphylococcus spp. Erythromycin / Clindamycin

| Mechanism | Determinant | Erythro | Clinda |
| --- | --- | --- | --- |
| Efflux | msrA | R | S |
| Ribosome modification | erm | R | S* |
| Ribosome modification | 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  erythromycin-R and clindamycin-S, do not report clindamycin-S without performance of “D zone Test”***

## Staphylococcus aureus 

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

***Optional reporting strategy***

## “D Zone Test” – positive reaction

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

**15 - 26 mm**

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

**Inducible **
**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.**

## “D zone Test” – negative reaction

**“****D zone Test” – ****negative**** reaction**

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

## “D Zone Test” – positive reaction

**E 15**

**CC 2**

**Routine purity plate (e.g., BAP):**
**  ****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 Zone Test” positive and optional comment***

## Staphylococcus aureus

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

**“****This *****S. aureus*****  has inducible clindamycin resistance**
**(UCLA modification of CLSI comment)**

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

**Ucla example**

## Staphylococcus aureus 

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

**“****This *****S. aureus***** does not have inducible clindamycin resistance ”**

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

## D Zone Test QA/QC

**Daily / Weekly ****QC**
***S. aureus *****ATCC 25923 to QC erythromycin and clindamycin disks**
**QA ****(training, competency, validation)**** **
***S. aureus***** ATCC BAA-976 (*****msr*****A; ****not inducible****)**
***S. aureus***** ATCC BAA 977 (*****erm*****A; ****inducible****)**

## Erythromycin-R, Clindamycin-S S. aureus and D Zone Test

**Notes**
**Test applies to *****S. aureus***** and CoNS (OX-S and OX-R)**
**Most hospital-acquired MRSA are clindamycin-R**
**Many CA-MRSA are truly clindamycin-S**
**Clindamycin can be give orally**
**Clindamycin rarely used for CoNS**

## CLSI Interpretive Criteria Vancomycin - Staphylococcus spp.

| Method | Susceptible | Intermediate | Resistant |
| --- | --- | --- | --- |
| MIC (μg/ml) | 4  | 8-16 | 32 |
| Disk (30 μg) (mm) | 15 | - | - |

**VISA**

**VRSA**

**Warning:****  Some test methods ****DO NOT**** detect VISA or VRSA!!**
**Add BHI-vancomycin screen plate if you use a system that is problematic for detection of VISA / VRSA**

## Detection of VISA / VRSA

| Method | VISA | VRSA |
| --- | --- | --- |
| Disk diffusion | Not detected | May show subtle growth in zone |
| Automated systems | Inconsistent | Inconsistent  |
| CLSI broth microdilution reference MIC | Detected | Detected |
| CLSI agar dilution reference MIC | Detected | Detected |
| Etest (use 0.5 McFarland, 24h incubation) | Detected | Detected |

## VRSA(3 4 isolates encountered to date)

**Isolate ****	****Vanco MIC (****μ****g/ml) ****1**
**	**** ****1****	****	****            1024**
**	**** ****2****	****                        32 ****2**
**	**** ****3****	****	****	****     64 ****2**
**	**** ****4****	****	****	****   256 ****2 ****	**

**1  ****Reference broth microdilution MIC**
**2****  ****Missed or inconsistent results (some ****≤****2 μg/ml ) with                     ****	****automated methods**

**4/04 CDC recommendation: add vancomycin agar screen**
** ****if using automated method**

- x

## Marlowe, et al. 2001. JCM. 39:2637. 

**VISA colonies may be smaller and slower growing than typical *****S. aureus *****as shown on this 48h BAP; coagulase reaction may be weak.**

**MRSA (not VISA)**

**VISA**

**Marlowe, et al. 2001. JCM. 39:2637.**** **

**VISA:****13 cases to date in USA**

## BHI Vancomycin Agar Screen Test (BHI-V6)

**Brain heart infusion (BHI) agar with 6 **μ**g/ml vancomycin**
**Make suspension in broth or saline to ****0.5 McFarland**
**Use ****10-****μl**** micropipette or swab to inoculate BHI-V6 plate (up to 8 organisms / plate)**
**After absorption of inoculum, invert plates and incubate at ****35oC in ambient air**

## BHI Vancomycin Agar Screen Test (BHI-V6) (con’t)

**Examine after 16-18 hrs; if no growth, re-incubate and examine at ****24 hrs**
**Growth ****>1 colony**** is positive for VISA/VRSA**
**Confirm identification; perform non-automated MIC**
**QC **
**Vancomycin-susceptible:***** E. faecalis***** ATCC 29212**
**Vancomycin-resistant*****: E. faecalis***** ATCC 51299**

## CDC Criteria for Calling Isolate VISA

**Reference broth microdilution MIC:****	**
**8-16 ****μ****g/ml**
**Etest:**
**6-16 ****μ****g/ml **
**BHI-vancomycin (6 ****μ****g/ml) agar:**** **
**growth**
**Note:**** **
**VISA are usually MRSA but may be MSSA**

**VRSA **
**2****nd ****Case**

**From CDC Lab**

**VANCO**

##  Confirmation of VISA / VRSA

**If you suspect a VISA / VRSA:**
**See algorithm...... ****www.cdc.gov/ncidod/hip/Lab/images/VISA_VRSA**
**Repeat ID and susceptibility tests  **
**Contact your institution’s infection control department**
**Contact CDC at **_**[SEARCH@cdc.gov](mailto:SEARCH@cdc.gov)**_
**Contact your local health department**
**SAVE ISOLATE!!**

## Algorithm for Testing S. aureus with Vancomycin (VA)

**Algorithm for Testing *****S. aureus***** with Vancomycin (VA)**

**Acceptable**
**Primary Test Methods**
**Include:**

**Disk diffusion****2**** **_**plus**_** VA screen plate**
- (BHIA with 6 μg/ml of VA)

**VA MIC **_**<**_**2 ****μ****g/ml**
**And NO growth on**
**VA screen plate**

**VA MIC **_**<**_**2 ****μ****g/ml**
**AND GROWTH on**
**VA screen plate**
**(rare)**

**VA MIC **_**>**_**4 ****μ****g/ml**
**AND GROWTH on**
**VA screen plate**

**VA zone **_**<**_**14 mm**
** ****AND GROWTH on **
**VA screen plate**

**VA zone >14 mm **
**AND GROWTH on**
**VA screen plate**

**Report as VSSA****3**

**Report as VSSA****3**

**Possible VISA/VRSA**

**CHECK purity**

**SAVE ISOLATE**

**NOTIFY infection control, physician, local health department and CDC****5**** of “possible VISA/VRSA”**

**MIC method****1**** **_**plus**_** VA screen plate**
- (BHIA with 6 μg/ml of VA)

**Possible VISA/VRSA**

**VA zone >14 mm**
**and NO growth on**
**VA screen plate**

**CONFIRM isolate ID**

**RETEST using non-automated MIC method****4**

**SEND to reference laboratory for confirmation**

_**Important Footnotes**_
**1****Laboratories using automated susceptibility test methods should add a commercial vancomycin agar screen plate. **
**2****Disk diffusion alone is not sufficient to detect VISA.**
**3****If a laboratory is concerned about a result based on a patient’s history, MIC testing can be performed at CDC.****	****	**
**4 ****Non-automated methods: reference broth microdilution, agar dilution, agar gradient diffusion (Etest; use a 0.5 McFarland inoculum and Mueller-Hinton agar)**.
**5****Report to CDC by email: SEARCH@cdc.gov**

**April 2004**

- More VISA/VRSA info: www.cdc.gov/ncidod/hip/vanco/vanco.htm

## .....Inform Public Health Department (PH)

***S. aureus***
**Vancomycin MIC ****≥****4 ****μ****g/ml**
**Coagulase-negative staphylococci**
**Vancomycin MIC:**
**≥****32 ****μ****g/ml - not required but can send to PH and CDC will check to see if isolate has high MIC due to presence of *****vanA***** gene**
**4 - ****16 ****μ****g/ml – do not have to send to PH but may want to get isolate confirmed by reference lab if isolate from sterile body site, isolated multiple times, and patient has been on prolonged vancomycin.  PH lab or CDC can can do testing.**

## Urine culture:>105 CFU/ml S. saprophyticus

**  **
**“*****Staphylococcus saprophyticus***** typically responds to urine concentrations of agents commonly used to treat acute, uncomplicated UTIs (e.g. nitrofurantoin, TMP-SMX, or a fluoroquinolone).”**

**CLSI M100-S15 Table 2C (M2, M7)**

## Staphylococcus ASTStrategy  

**Use reliable method for detection of ****MRSA**
**Do not report ANY ********-lactam as “S” for MRSA (or MRCoNS)**
**For ****erythromycin-R, clindamycin-S**** isolates, withhold clindamycin result; perform clindamycin induction test when requested**
**Use appropriate tests for detection of ****VISA/VRSA**
**For CoNS ****with OX MICs of 0.5-2 ****μ****g/ml from serious infections, perform CX DD and/or submit to reference lab for *****mecA***** or PBP2a**

## Enterococcus spp.

- Enterococcus

## Enterococcus spp. AST Issues

**Special tests**
**Aminoglycoside (gentamicin/streptomycin) synergy screens**
**BHI-vancomycin screen for VRE**
**Reporting**
***Warning*****: Enterococci are “R” to cephalosporins, aminoglycosides (low concentrations), clindamycin, trimeth-sulfa**

## Enterococcus spp. 

**endocarditis      pen or amp ****	****      vanc + gent   **
**	****              ****+  gent (or        ****	****         (or strep) **
**	****              ****strep) ****	**
**	****	****	****	**
**uncomplicated   amp or amox           nitrofurantoin; **
**  ****UTI       ****	****	****	****	****         fosfomycin**
**	****	****	****	**

**1st Choice Drugs**

**Alternative Drugs**

**Sanford Guide, 2004**

## Enterococcus spp. HLAR* (Synergy) Screening

**Disk diffusion**
**120 μg gentamicin; 300 μg streptomycin**
****** ****10 mm = NO HLAR**
**MIC screen**
**gentamicin:       500 μg/ml **
**streptomycin: 1000 μg/ml (broth)****	****	****	****	****                        2000 μg/ml (agar)**

*** high-level aminoglycoside resistance**

## Enterococcus spp. Aminoglycoside-Modifying Enzymes

**	****	****	****	****  **_**strep  gent  tob   amk/kan**_
**6-AAD****	****	****      +       -        - ****	****   -**
**3’APH****	****	****      -        -        -          +**
**2”APH/6’AAC****	****      -****	****       +****	****+****	****   +**
**6’AAC*********	****              -****	****       -****	****+****	****   +**
***      ******* *****found in all***** E. faecium******	***** **

**+ = enzyme inactivates drug; drug NOT synergistic**
**-**** = enzyme does not inactivate drug; drug synergistic**

## Enterococcus spp.

**Gent       Strep****	****        Choice of aminoglycoside**
**	****S****	****	****S****         ****	****	****      either **
**	****S****	****	****R****	****    ****	****           gentamicin**
**	****R****	****	****S****	****    ****	****	****streptomycin**
**	****R* ****	****	****R****	****    ****	****	****       none **
***HLAR to gentamicin confers HLAR to amikacin **
**and tobramycin**

**HLAR Test**

## Enterococcus spp. Predicting Synergy

**                             ****Susc****	****       Res**
**            ****Susc****	****    **_**“synergy“**_**	****   no synergy**
**             ****Res****	****    no synergy   no synergy**

**Aminoglycoside or HLAR screen**

**Cell wall **
**active agent***

***ampicillin, penicillin, or vancomycin**

## E. faecalis (blood)

**	****	****ampicillin ****	****     S**
**	****	****vancomycin        S**
**	****	****gent syn   ****	****     R**
**	****	****strep syn ****	****     S**
**“****Serious enterococcal infections need combination therapy with amp, pen, or vancomycin plus an aminoglycoside.  Synergy occurs only when both drugs in the combination are “S””**

## VRE

**“****True VRE”**** - primarily *****E. faecium***
***van*****A***** *****- high level (MIC ******** 256 **μ**g/ml)**
***van*****B***** *****- intermediate level (MIC 8 - 1024 **μ**g/ml)**
**Low level vancomycin resistance**
***van*****C - (MIC 2 - 32 **μ**g/ml)**
**Intrinsic to***** E. gallinarum, E. casseliflavus***
**Strains ****do NOT**** cause nosocomial outbreaks, *****van*****C gene not readily transferred**
**Not considered “VRE”**

## Enterococcus spp.

**	****	****	****	****	****	****	****	****          ****Infx. Cntrl**
**Species****	****     Genotype   Motility  MGP*       Significance?**** ****	****	****	****	****	**
***E faecalis*****	****             vanA         -            -                 yes****	****	****	****           or vanB**
***E. faecium*****	****             vanA         -            -                 yes****	****	****	****	****  or vanB**
***E. casseliflavus*******  vanC         +           +                 no**
***E. gallinarum*****	****    vanC         +****	****       +                 no**

***acidification of methyl-a-D-glucopyranoside**
**** yellow pigment**

## Vancomycin Screen Agar for VRE 

**Vancomycin Screen Agar for VRE**** **
**(BHI + 6 ********g/ml vancomycin)**

**- control**

**+ control – *****E. faecalis *****ATCC 51299**
**- control – *****E. faecalis *****ATCC 29212**

**Positive**
**patient**

**+ control**

**Negative**
**patient**

## Enterococcus spp.(M100-S15; T-2D)

**> 1 colony = presumptive**
**Perform vancomycin MIC and tests for motility and pigment production to distinguish species with acquired resistance (VanA and VanB) from those with intrinsic, intermediate-level resistance to vancomycin (VanC) such as *****E. gallinarum or E. casseliflavus *****which often grow on the vancomycin screen plate.  In contrast to other enterococci, *****E. casseliflavus *****and *****E. gallinarum***** with vancomycin MICs of 8-16 ********g/ml (intermediate) differ from vancomycin-resistant enterococci for infection control purposes.**

## E. faecium (blood)

**ampicillin****	****>64 R**
**chloramphenicol****	****  8 S **
**ciprofloxacin****	****4 R**
**doxycycline ****	****2 S**
**linezolid****	****1 S**
**quin/dalfo ****	****0.5 S**
**rifampin****	****8 R**
**vancomycin****	****>32 R**
**gent synergy****	****R**
**strep synergy****	****R**

_**MIC (μg/ml)**_** **

**Example: supplemental **
**drugs that might be **
**reported on VRE**

## E. faecium (urine)

**ampicillin ****	****R**
**ciprofloxacin****	****R**
**nitrofurantoin****	****S**
**tetracycline****	****R**
**vancomycin****	****R**
**	****	**

## Enterococcus faecalis vs. Enterococcus faecium

***Enterococcus faecalis***
**Always ampicillin “S”**
**Occasionally can be VRE**
**Quinupristin-dalfopristin “R”**
***Enterococcus faecium*****	**
**>80% ampicillin “R”**
**Quinupristin-dalfopristin “S”**
**Most common species of VRE**

## E. casseliflavusE. gallinarum

**Rarely encountered in human specimens (other than rectal) **
**Can ****colonize**** human intestine**
**Can cause ****invasive infections**** in immunocompromised or chronically ill**
**When bacteremia, often ****polymicrobial**

## .....More Enterococcus spp.

**What about ****species of VRE**** other than *****E. faecium***** or *****E. faecalis***** with high vancomycin MICs?**
**If vancomycin MIC >16 ********g/ml for any *****Enterococcus***** spp. consider VRE (might have acquired *****van*****A gene); discuss case with infection control**
**Exception: Some *****E. gallinarum / E. casseliflavus***** with *****van*****C may have MICs of 32 ********g/ml; rare isolates with *****vanA***** (vanco MIC >64 ********g/ml) **
**What if an isolate only grows around a vancomycin disk or Etest?**
**This is probably a ****“vancomycin-dependent”**** enterococcus**

## Enterococcus spp.Strategy

**DD or MICs on ****sterile body site**** (+ other?) isolates**
**Include ****HLAR**** (synergy screen tests)**
**Do not report species that commonly have *****van*****C as ****“VRE”**

## Verification of Patient Results

**...****to follow in Session #4 (April 12)**

## Thank you!