Guidelines for the Testing and Reporting of Antimicrobial Susceptibilities of Vancomycin Resistant Enterococci

Canadian External Quality Assessment
Advisory Group for Antibiotic Resistance

September 1998

ISBN 0-662-27498-9
© 1998

1.0 Introduction

These guidelines are produced under the auspices and authority of the Canadian External Quality Assessment - Advisory Group on Antibiotic Resistance (CEQA-AGAR). They represent a consensus of peer-reviewed information and expert opinion on the most appropriate ways to test for and report antimicrobial susceptibility and resistance. Clinical situations and individual laboratory operations may necessitate some variations from these guidelines. These guidelines are strongly recommended for use by all laboratories providing information for national surveillance programs on antimicrobial resistance.

2.0 Preamble

Although enterococci are relatively non-virulent, they have become important nosocomial pathogens, especially observed in clinical areas such as haematology-oncology and dialysis units. Over the past few years, enterococci have acquired increased resistance to antibiotics such as beta-lactams and aminoglycosides, the mainstay of therapy in serious infections. Infections with multi-drug resistant enterococci necessitate treatment with vancomycin, thus the appearance of vancomycin resistant enterococci (VRE) poses a serious threat to our ability to treat patients infected with these organisms^1-3. VRE have become widely disseminated in the United States within the last few years⁴, and have started to appear in most provinces in Canada^{5, 6,7,21}. The genes responsible for vancomycin resistance are mobile and may be present on plasmids or transposons. Thus, the threat of transfer of vancomycin resistance to other organisms, such as S. aureus is worrisome, and indeed, has already been accomplished in the laboratory⁸. VRE can spread quickly within an institution's environment, and is difficult to eradicate, as persons found to be colonized with VRE may remain colonized for a prolonged length of time (months to years). The exact nature of the epidemiology and natural history of VRE colonization and infection still remains to be determined ⁹.

3.0 Definitions

3.1 Vancomycin resistant enterococci are those enterococci that are resistant to vancomycin at a concentration greater than or equal to 32 mg/L (NCCLS).These enterococci will contain the genes vanA, vanB or vanD which confer resistance to vancomycin. Those resistant enterococci currently thought to be medically important are E. faecalis and E. faecium.

3.2 Other species of enterococcus (E. casseliflavus, E. gallinarum, E. mundtii, E. raffinosus) may exhibit low-level resistance to vancomycin (MIC <= 8 mg/L), but are not presently thought to be of consequence¹⁰.

3.3 Control organisms for susceptibility testing of Enterococci:
E. faecium ATCC 51299 - resistant to vancomycin (MIC 32 mg/L)*
E. faecalis ATCC 29212 - sensitive to vancomycin

^*Note that this strain may give an erroneous susceptible test result in gradient diffusion assays (E-Test^®).

4.0 What to Test

4.1 Enterococcal isolates, according to individual laboratory protocols, which are normally reported with susceptibility results, should be tested for vancomycin resistance. Some isolates of enterococci that are not normally reported, such as fecal isolates, may not be identified and tested for antibiotic resistance. Laboratories not participating in cross-Canada surveillance may only test those isolates that are thought to be clinically relevant or those from infection control/surveillance specimens. Those laboratories involved in national surveillance studies should test ALL enterococcal isolates for antimicrobial resistance. Therefore, these isolates may come from specimens taken for clinical reasons, including those specimens which would not normally be tested or specimens taken for surveillance/infection control purposes (and may include environmental specimens). The isolates that will be used to generate data for national surveillance should follow the laboratory protocol as detailed below (see Figure 1 - Flow Chart).

Figure 1: Flow Chart for Detection and Identification of VRE

4.2 Specimens with highest likelihood of containing VRE include:
· Perineal/rectal swab
· Feces
· Urine
· Drain sites
· Open Wounds
· Environmental sampling.

Patients, especially with diarrhea and incontinence readily shed VRE into their immediate environment. This contamination may be further spread upon the hands of caregivers and others, or upon equipment.

4.3 Some reference, central or regional laboratories may also receive isolates for testing from other laboratories.

5.0 Methods

5.1 Laboratory Identification and Interpretation (see Figure 1)

5.1.1 Inoculate specimens submitted for surveillance onto mEnterococcus agar or Bile Esculin Azide (BEA) agar (Enterococcosel agar) with 6 mg/L of vancomycin. Some environmental specimens may require enrichment, and should be incubated overnight in BHI or Enterococcosel broth¹¹ and then sub-cultured onto mEnterococcus agar with 6 mg/L of vancomycin.

5.1.2 Incubate for 72 hours in air at 35^o C.

5.1.3 From the mEnterococcus agar, E. faecalis isolates should appear magenta, and E. faecium isolates should appear a pale pink. This color change is not always distinct. From BEA agar, enterococci should appear brownish with a black halo.

5.1.4 Inoculate a positive and negative control organism on a vancomycin screen plate:

E. faecium ATCC 51299 - growth
E. faecalis ATCC 29212 - no growth

5.2 Identification of isolates (either from clinical specimens or from surveillance screen plates):

5.2.1 Gram stain organism to ensure it is a Gram positive coccus and test for catalase production (should be negative).

5.2.2 Subculture possible enterococcal isolates to blood agar and bile esculin agar plates and incubate at 35^o C in air for further testing. A bile esculin plate is not required if the isolate was recovered from BEA agar.

5.2.3 Inoculate a vancomycin screen plate (BHI with 6 mg/L vancomycin) with enterococcal isolates. Use control organisms defined above.

5.2.4 To confirm the isolates are enterococci, perform pyrrolidonyl arylamidase (PYR) and leucine aminopeptidase (LAP) tests, and interpret as per the following chart:

5.3 To differentiate the enterococcal species, do the following tests:

Pigment Production:
Check for pigment production by sweeping a white cotton swab across some colonies and observe for a bright yellow colour.

Acid production from sugars:
Inoculate xylose, arabinose, and pyruvate assays for detection of acid production, and incubate at 35^o C aerobically for 4 hours. Continue to incubate overnight if negative after 4 hours.

Motility:
Using a tube of motility medium (preferably with an indicator), inoculate with a heavy inoculum from the blood agar purity plate, and incubate aerobically at 30^o C for up to three days. Test is usually positive within 24 hours; note that a negative result at 72 hours may still not be a true negative result.

5.4 Expected results (see chart below)

5.5 Susceptibility Testing

For testing and reporting details refer to NCCLS documents M7-A4 (MIC), M2-A6 (Disc diffusion), and informational supplement M100-S9.

5.5.1 Vancomycin resistance is defined as an MIC >= 32 mg/L.

5.5.2 Vancomycin resistance profiles:

5.5.3 If isolate is resistant on vancomycin screen and is E. faecium or E. faecalis, an MIC for vancomycin should be performed (according to your laboratory protocol or sent to Reference Lab). Also test for beta-lactamase production, high-level aminoglycoside resistance and ampicillin resistance using the accepted laboratory protocol.

5.6 Molecular testing

Isolates may be tested for the presence of the genetic determinants of vancomycin resistance as well as typed for relatedness using pulsed-field gel electrophoresis.

5.6.1 Genotyping VRE

Molecular testing of VRE is intended to determine the resistance genotype for enterococci displaying Van A and Van B phenotypes. Testing is not required for isolates of E. gallinarum and E. casseliflavus demonstrating Van C phenotype.

Three gene clusters, containing vanA, vanB or vanD are commonly associated with vancomycin-resistance in E. faecium and E. faecalis. The most common gene sequence detected in the cluster encodes a ligase which synthesizes the depsipeptide D-alanyl-D-lactate (D-ala-D-lac). These ligases differ from the cellular ligase (ddl) which synthesizes the dipeptide D-alanyl-D-alanine (D-ala-D-ala). The nucleotide sequences for vanA¹², vanB¹³, and vanD¹⁴ have been determined and numerous PCR primer sets specific to each have been described in the literature^15,16. There are no primer sets recommended in this document, however, the use of published primer sets is recommended over the use of in-house primer sets.

A number of methods currently exist for strain typing enterococci. Tenover et al.¹⁷ have suggested that the reference method be DNA fingerprinting using pulsed-field gel electrophoresis. There is no standard PFGE protocol for typing enterococci in Canada. A number of PFGE methods for typing enterococci have been published^18,19. It is recommended that SmaI be used to restrict the DNA for PFGE analysis.

It is recommended that the general principles listed in the table below be followed when interpreting DNA fingerprints of potential outbreak strains. It should be noted that these principles are just a guide and genetic events may occur during an outbreak which may make interpretation difficult. For example, transposon insertion may alter the DNA fingerprint of a stain by up to 7 bands²⁰. In this event, a second strain typing method or additional epidemiological information may be required to determine relatedness to the outbreak strain. Furthermore, groups of epidemiologically related strains spanning no more than 3 months should be typed since the genetic variability of the organism may make relatedness determinations difficult.

When reporting the DNA fingerprints a letter/number system should be used. The letter indicates relatedness to the outbreak strain, that is, a different letter indicates an unrelated strain. The number following the letter indicates a subtype of the strain as determined by fingerprint analysis using the criteria in the table below:

6.0 Reporting

6.1 For internal/institutional reporting, report those isolates confirmed to be VRE according to your laboratory and institution's reporting and infection control policies.

6.2 For external/national surveillance reporting purposes, for isolates confirmed to be VRE, report one isolate per patient. Isolates with MICs confirmed to harbor vanB should also be reported.

7.0 Summary

Enterococcal isolates may be obtained from clinical specimens or specimens taken for the purposes of surveillance. These isolates must be confirmed as either E. faecium or E. faecalis and their vancomycin MIC determined as well as the type of genetic determinant of resistance (vanA, vanB or vanD). Reporting of these isolates is determined internally at each institution, and for those institutions participating in external surveillance, one isolate per patient should be reported.

8.0 References

1. Spera RV Jr., Farber BF. Multiply resistant Enterococcus faecium. The nosocomial pathogen of the 1990s. JAMA 1992; 268(18): 2563-4.

2. Moellering RC JR. Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 1992; 14:1173-6.

3. Forward KR, Kennedy JK, Degagne PA, Bartlett KR, Harding GKM. The rapid emergency of high-level gentamicin resistance in enterococci. Can J Infect Dis 1990; 9:81-100.

4. Nosocomial enterococci resistant to vancomycin - United States 1989-1993. Morb Mortal Wkly Rep 1993 Aug 6; 42(30): 597-9.

5. Laboratory Proficiency Testing Program of Ontario Newsletter #164, October 17^th, 1995.

6. Berntson A, Fleming CA, Willey BM et al. Laboratory Proficiency Testing Program of Ontario Newsletter, #221, February 10^th, 1998

7. Clinical Microbiology Proficiency Testing (CMPT) Connections; , Volume 2, Summer 1998.

8. Noble WC et al. Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett 1992; 93:195-198.

9. Preventing the Spread of Vancomycin-resistant enterococci (VRE) in Canada. CCDR 1997; 23:suppl 8.

10. Tenover FC. Laboratory methods for sueillance of vancomycin-resistant enterococci. Clin Micro Newsletter 1998; 20(1):1-6.

11. Van Horn KG, Gedris CA, Rodney KM. Selective isolation of vancomycin-resistant enterococci. J Clin Micro 1996; 34:924-927.

12. Dutka-Malen S, Molinas C, Arthur M, Courvalin P. The VANA glycopeptide resistance gene is related to D-alanyl-D-alanine ligase cell wall biosynthesis enzymes. Mol Gen Genetc 1992;224:364-372.

13. Evers S, Sahm DF, Courvalin P. The vanB gene of vancomycin-resistant Enterococcus faecalis V583 is structurally related to genes encoding D-Ala-D-Ala ligases and glycopeptide-resistant proteins VanA and VanC. Gene 1993;124:143-144.

14. Perichon B, Reynolds P, Courvalin P. VanD-type glycopeptide-resistant Enterococcus faecium BM4339. Antimicrob Agents Chemother 1997; 2016-2018.

15. Miele A, Bander M, Goldstein BP. Use of primers selective for vancomycin

resistance genes to determine van genotype in enterococci and to study gene organization in vanA isolates. Antimicrob Agents Chemother 1995;39:1772-1778

16. Bell JM, Paton JC, Turnidge J. Emergence of vanocumycin-resistant enterococci in Austrailia: phenotypic and genotypic characteristics of isolates. J Clin Microbiol 1998; 2187-2190.

17. Tenover FC, Arbiet RD, Goering RV and the Molecular Typing Working Group. How to select and interpret molecular typing strain methods for epidemiologic studies of bacterial infections: A review for health care epidemiologists. Infect Control Hosp Epidemiol 1997;18:426-439.

18. Gordillo ME, Singh KV, Murray BE. Comparison of ribotyping and pulsed-field gel electrophoresis for subspecies differentiation of strains of Enterococcus faecalis. J Clin Micro 1992;30:577-580.

19. Thal LA, Silverman J, Donabedian S, Zervos MJ. The effect of Tn916 insertion on contour-clamped homogenous electrophoresis patterns of Enterococcus faecalis. J Clin Micro 1997;35:969-972.

20. Willey BM, Kreisworth BN, Simor AE, Williams G, Scriver SR, Phillips A, Low DE. Detection of vancomycin resistance in Enterococcus species. J Clin Micro 1992;30:1621-624.

21. Fortin, A., Milord, F., Guay, M., d'Halewyn, M.-A., Vigeant, P., and Leblanc. 1998. First known outbreak of colonizing vancomycin-resistant enterococci in Quebec. RMTC. 24-11:85-93.

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