- Email: [email protected]
Misidentification of vancomycin-resistant Enterococcus faecium as E. avium
CFA and cell-wall peptidoglycan analysis (5). We also applied CFA analysis, but the decision to assign our isolate to the genus Aureobacterium was based on the results of simple and commonly available tests. Within the genus Aureobacterium, seven species have been described: A. barkeri, A. esteroaromaticum, A. flavescens, A. liquefaciens, A. saperdae, A. terregens, and A. testaceum (1,3,4), but no human isolates have been assigned to one of these species (3-5). The problem with differentiation of Aureobacterium strains to species level is the limited number of available case reports and that not all phenotypic data for every strain have been reported (3). It is assumed that correct species identification of an isolate can be established only by quantitative DNA-DNA hybridization or by 16s rRNA analysis of the strain (3,4). The patient described here suffered
only from a transient bacteremia, and she recovered without antimicrobial therapy. This is in contrast to previous reports (45) in which bloodstream invasion with Aureobacterium had a fulminant and lethal course, despite treatment with a broad variety of antibiotics with proven in vitro activity against the isolate. From the few published cases it appears that antimicrobial therapy has only little influence on the outcome of infection. It is not clear whether the outcome depends on bacterial virulence or on the immune competence of the patient. References 1. Holt, G.J. et al. (ed.), 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore. 2. Funke, G. et al. 1997. Clinical microbiology of coryneform bacteria. Clin. Microbial. Rev. 10:125-159. 3. Funke, G., A. von Graevenitz,and N. Weiss.
1994. Primary identification of Aureobacterium spp. isolated from clinical specimens as “Corynebacterium aquaticum.” J. Clin. Microbial. 32:1686-1691. Saweljew, P. et al. 1996. Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16s rRNA gene analysis. J. Clin. Microbial. 34:1540-1541. Nolte, ES. et al. 1996. Vancomycinresistant Aureobacterium species cellulitis and bacteremia with acute myelogenous leukemia. J. Clin. Microbial. 34:1992-1994. Clarridge, J.E. and CA. Spiegel. 1995. Corynebacterium and miscellaneous irregular gram-positive rods, Erysipelothrix, and Gardnerella, p. 357-378. In P.R. Murray et al. (ed.), Manual of Clinical Microbiology, 6th ed. ASM Press, Washington, DC. von Graevenitz, A. and G. Funke. 1996. An identification scheme for rapidly and aerobically growing gram-positive rods. Zbl. Bakt. 284:246-254.
Misidentification of Vancomycin-Resistant Enterococcus faecium as E. avium Eric D. Spitzer, M.D., Ph.D. Department of Pathology Annette Pirotta, M.S., SM(AAM), CIC Department of Healthcare Epidemiology Silvia G. Spitzer, Ph.D. Department of Pathology University Hospital Medical Centen SUNY at Stony Brook Stony Brook, New York 11794 Vancomycin-resistant enterococci (VRE) are an important cause of nosocomial infections and pose a serious therapeutic challenge. Limiting the spread of VRE is a major goal of infection control activities (1). Vancomycinresistant E. faecium (VR-E. faecium) and VR-E. faecalis are responsible for most VRE infections. Resistance is due to the acquisition of exogenous DNA sequences that harbor the vanA or vanB gene clusters (2). E. casselij7avus and E. gallinarum, both of which exhibit intrinsic, low-level resistance to vancomycin due to the vanC1 and vanC2 genes, have not been associated with nosocomial outbreaks. There have been only isolated reports of vancomycinresistant isolates of E. avium and E. durans (2). We recently encountered a VRE that was initially identified as E. Clinical
Microbiology
Newsletter
20: lo,1998
avium. Subsequent analysis indicated that it was an E. faecium. Misidentification of VREs can have important implications for infection control efforts.
Case Report As part of a VRE surveillance project, a VR-E. faecium was isolated from a rectal swab obtained from a 46-year-old man who had received an autologous bone marrow transplant. There was no evidence of active enterococcal infection. Seven days later a VRE was isolated from a repeat rectal culture. This isolate was initially identified as E. avium using a Vitek GPI card (bioMCrieux Vitek, Inc., Hazelwood, MO USA). The biotype number was 773476605 10 (92% E. avium, 6% E. faecium; Vitek software VTK-R05.03). It was resistant to ampicillin and vancomycin (GPS- TA card). The vancomycin MIC was greater than 256 pg/ml as determined by Etest (AB Biodisk NA Inc., Piscataway, NJ USA). Because of the rarity of VR-E. avium and the previous isolation of VR-E. faecium, additional tests were performed on the second isolate. Visual inspection of the GPI card suggested that the isolate was arginine dihydrolase positive although this reaction had been scored as negative 0 1998 Elsevier
Science
Inc.
by the instrument. The percent change in transmittance for the arginine well was 7 at 5 h, when the identification was made, but increased to 24 at 16 h (the corresponding values for the base control well were 2 and 1, respectively). Arginine positivity was demonstrated after overnight growth in Moeller decarboxylase broth with arginine. The GPI card was repeated and identified the isolate as E. faecium, although the arginine was again interpreted as negative (biotype # 75327260510,97% E. faecium, 1% E. avium). The multiresistant phenotype was also typical of VR-E. faecium isolates. To confirm that the isolate was E. faecium, the d-Ala-d-Ala ligase gene (ddl) was analyzed by PCR (3). DNA was released from 5 pl of broth culture by heating at 95°C for 5 min. PCR was performed with the F, and F, primers for the E. faecium ddl gene (4). This reaction produced the expected 550 bp product. The amplified product was gel purified and partially sequenced using a cycle sequencing protocol and automated sequencer (ABI Prism, Perkin-Elmer, Norwalk, CT). Comparison of this sequence to the GenBank NCBI database (http://www.ncbi.nlm.nih.gov) using the 0196-4399/96/$0.00
+ 19.00
89
Blast algorithm revealed a 253 bp segment that was 98% identical to the E. faecium ddl gene but only 79% identical to the corresponding E. avium ddl sequence. Multiplex PCR with vanA and vanB primers (5) produced a 783 bp product indicating the presence of vanA.
Discussion
decarboxylasebasewells suggestedthat a slow arginine reaction was responsible for the instrument’snegative interpretation of this reaction. Members of the genusEnterococcus can be readily distinguishedby differencesin the 16srRNA andaldlgenes (10). PCR-basedidentification of unusual VREs can be performed
in a clinically
Almost all nosocomial VRE infections are caused by E. faecium and E. faecalis containing either vanA or vanB, both of which are located on mobile genetic elements. The vanA cluster has been identified in several enterococcal species including E. avium (2,6), however, transferable vancomycin resistance remains rare in clinical isolates of enterococcal species other than E. faecium and E. faecalis. Inaccurate identification of isolates can further confuse the picture. A recent report described a pseudoepidemic of VR-E. durans resulting from misidentification of VR-E. faecium (7). The enterococci have been divided into three groups (I-III) basedon phenotypic reactions (8). The group II organisms,E. faecium, E. faecalis, E. casselijlavus, E. gallinarum, and E. mundtii, are all arginine positive which distinguishesthem from the other eight enterococcal species.The ability of the Vitek system to identify an isolate as E, faecium despite a negative arginine reaction servesasa reminder that identification schemesbasedon conventional biochemical testscannot always be used to interpret biotypes generatedby the Vitek system(9). Analysis of the transmissionreadingsof the arginine and
useful time frame using proceduresthat are available in an increasingnumber of clinical laboratories. Misidentification of VR-E. faecium asVR-E. avium can have significant consequences.Current infection control guidelinesfocus on E. faecium and E. faecalis but do not addresswhether patients with other VRE speciesrequire similar isolation precautions.In the case describedabove, misidentification of the isolatewould have raised the possibility that the patient had acquired a secondVRE, possibly from an external source,or that there had been in vivo transfer of the vanA cluster. Becauseof the confusion that can be causedby misidentification of VR-E. faecium asE. avium, putative isolatesof VR-E. avium should be confirmed with a conventional arginine broth test and/ or DNA-based confirmation procedure.
Editors: Mary JaneFerraro Paul A. Granato JosephineA. Morello R.J. Zabransky
General
0 1998 Elsevier
tion to the species level of clinically relevant enterococci by PCR. J. Clin. Microbial. 33:24-27. 4. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbial. 33:1434. 5. Free, L. and D.F. Sahm. 1996. Detection of enterococcal vancomycin resistance by multiplex PCR, pp. 150-155. In D.H. Persing (ed.), PCR Protocols for Emerging Infectious Diseases. ASM Press, Washington, DC. 6.
Rosato,A. et al. 1995.Inducibleand constitutive expression of resistance to glycopeptides and vancomycin dependence in glycopeptide-resistant Enterococcus avium.Antimicrob. Agents Chemother. 39:830-833. Singer, D.A. et al. 1996. Pseudooutbreak of Enterococcus duransinfections and colonization associated with introduction of an automated identification system software update. .I. Clin. Microbial. 34:2685-2687. Facklam, R.R. and D.F. Sahm. 1995. Enterococcus, pp 308-314. In P.R. Murray, et al. (ed.), Manual of Clinical Microbiology, 6th ed. ASM Press,
Washington,DC. Ruoff, K.L. et al. 1990. Species identities of enterococci isolated from clinical specimens. J. Clin. Microbial. 28:435-437. 10. Evers, S. et al. 1996. Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes. J. Molec. Evol. 42:706-712.
Recommendations of the Hospital Infection Control Practices Advisory Committee (HICPAC). MMWR 44 (No. RR-12):1-19. 2. Leclercq, R. and P. Courvalin. 1997.
Information
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information
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This newsletter has been registered with the Copyright Clearance Center, Inc. Consent is given for copying articles for personal or internal use,or for the personal or internal use of specific clients. This consent is given on the condition that the copier pay through the Center the per-page fee stated in the code on the first page for copying beyond that permitted by the US Copyright Law. If no code appears on an article, the author has not given broad consent to copy and permission to copy must be obtained directly from the author. This
Science Inc
ISSN 0196-4399 CMNEEJ 20( 10)83-90,
References 1. Centersfor Disease Control. 1995. Recommendations for Preventingthe Spreadof VancomycinResistance
Resistance to glycopeptides in enterococci. Clin. Infect. Dis. 24545-556. 3. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identifica-
consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works. 1998
Address orders, changes of address, and claims for missing issues to Journal Fulfillment Department, Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Claims for missing issues can be honored only up to three months for domestic addresses and six months for foreign addresses. Duplicate copies will not be sent to replace ones undelivered due to failure to notify Elsevier of change of address.
Elsevier
Postmaster: Send address changes to Clinical of the Americas, New York, NY 10010. Editorials and letters printed in this newsletter necessarily reflect the opinions of the editors.
Microbiology
Newsletter;
are published
Elsevier
for the interest
llllll~~lllllllllll~lll~lllllllllllllllllll~lll Clinical Microbiology Newsletter is abstracted in Tropical Communicable Diseases and Current AIDS Literature.
0196-4399(19980515)20:10;1-D
90
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only from a transient bacteremia, and she recovered without antimicrobial therapy. This is in contrast to previous reports (45) in which bloodstream invasion with Aureobacterium had a fulminant and lethal course, despite treatment with a broad variety of antibiotics with proven in vitro activity against the isolate. From the few published cases it appears that antimicrobial therapy has only little influence on the outcome of infection. It is not clear whether the outcome depends on bacterial virulence or on the immune competence of the patient. References 1. Holt, G.J. et al. (ed.), 1994. Bergey’s Manual of Determinative Bacteriology, 9th ed. Williams and Wilkins, Baltimore. 2. Funke, G. et al. 1997. Clinical microbiology of coryneform bacteria. Clin. Microbial. Rev. 10:125-159. 3. Funke, G., A. von Graevenitz,and N. Weiss.
1994. Primary identification of Aureobacterium spp. isolated from clinical specimens as “Corynebacterium aquaticum.” J. Clin. Microbial. 32:1686-1691. Saweljew, P. et al. 1996. Case of fatal systemic infection with an Aureobacterium sp.: identification of isolate by 16s rRNA gene analysis. J. Clin. Microbial. 34:1540-1541. Nolte, ES. et al. 1996. Vancomycinresistant Aureobacterium species cellulitis and bacteremia with acute myelogenous leukemia. J. Clin. Microbial. 34:1992-1994. Clarridge, J.E. and CA. Spiegel. 1995. Corynebacterium and miscellaneous irregular gram-positive rods, Erysipelothrix, and Gardnerella, p. 357-378. In P.R. Murray et al. (ed.), Manual of Clinical Microbiology, 6th ed. ASM Press, Washington, DC. von Graevenitz, A. and G. Funke. 1996. An identification scheme for rapidly and aerobically growing gram-positive rods. Zbl. Bakt. 284:246-254.
Misidentification of Vancomycin-Resistant Enterococcus faecium as E. avium Eric D. Spitzer, M.D., Ph.D. Department of Pathology Annette Pirotta, M.S., SM(AAM), CIC Department of Healthcare Epidemiology Silvia G. Spitzer, Ph.D. Department of Pathology University Hospital Medical Centen SUNY at Stony Brook Stony Brook, New York 11794 Vancomycin-resistant enterococci (VRE) are an important cause of nosocomial infections and pose a serious therapeutic challenge. Limiting the spread of VRE is a major goal of infection control activities (1). Vancomycinresistant E. faecium (VR-E. faecium) and VR-E. faecalis are responsible for most VRE infections. Resistance is due to the acquisition of exogenous DNA sequences that harbor the vanA or vanB gene clusters (2). E. casselij7avus and E. gallinarum, both of which exhibit intrinsic, low-level resistance to vancomycin due to the vanC1 and vanC2 genes, have not been associated with nosocomial outbreaks. There have been only isolated reports of vancomycinresistant isolates of E. avium and E. durans (2). We recently encountered a VRE that was initially identified as E. Clinical
Microbiology
Newsletter
20: lo,1998
avium. Subsequent analysis indicated that it was an E. faecium. Misidentification of VREs can have important implications for infection control efforts.
Case Report As part of a VRE surveillance project, a VR-E. faecium was isolated from a rectal swab obtained from a 46-year-old man who had received an autologous bone marrow transplant. There was no evidence of active enterococcal infection. Seven days later a VRE was isolated from a repeat rectal culture. This isolate was initially identified as E. avium using a Vitek GPI card (bioMCrieux Vitek, Inc., Hazelwood, MO USA). The biotype number was 773476605 10 (92% E. avium, 6% E. faecium; Vitek software VTK-R05.03). It was resistant to ampicillin and vancomycin (GPS- TA card). The vancomycin MIC was greater than 256 pg/ml as determined by Etest (AB Biodisk NA Inc., Piscataway, NJ USA). Because of the rarity of VR-E. avium and the previous isolation of VR-E. faecium, additional tests were performed on the second isolate. Visual inspection of the GPI card suggested that the isolate was arginine dihydrolase positive although this reaction had been scored as negative 0 1998 Elsevier
Science
Inc.
by the instrument. The percent change in transmittance for the arginine well was 7 at 5 h, when the identification was made, but increased to 24 at 16 h (the corresponding values for the base control well were 2 and 1, respectively). Arginine positivity was demonstrated after overnight growth in Moeller decarboxylase broth with arginine. The GPI card was repeated and identified the isolate as E. faecium, although the arginine was again interpreted as negative (biotype # 75327260510,97% E. faecium, 1% E. avium). The multiresistant phenotype was also typical of VR-E. faecium isolates. To confirm that the isolate was E. faecium, the d-Ala-d-Ala ligase gene (ddl) was analyzed by PCR (3). DNA was released from 5 pl of broth culture by heating at 95°C for 5 min. PCR was performed with the F, and F, primers for the E. faecium ddl gene (4). This reaction produced the expected 550 bp product. The amplified product was gel purified and partially sequenced using a cycle sequencing protocol and automated sequencer (ABI Prism, Perkin-Elmer, Norwalk, CT). Comparison of this sequence to the GenBank NCBI database (http://www.ncbi.nlm.nih.gov) using the 0196-4399/96/$0.00
+ 19.00
89
Blast algorithm revealed a 253 bp segment that was 98% identical to the E. faecium ddl gene but only 79% identical to the corresponding E. avium ddl sequence. Multiplex PCR with vanA and vanB primers (5) produced a 783 bp product indicating the presence of vanA.
Discussion
decarboxylasebasewells suggestedthat a slow arginine reaction was responsible for the instrument’snegative interpretation of this reaction. Members of the genusEnterococcus can be readily distinguishedby differencesin the 16srRNA andaldlgenes (10). PCR-basedidentification of unusual VREs can be performed
in a clinically
Almost all nosocomial VRE infections are caused by E. faecium and E. faecalis containing either vanA or vanB, both of which are located on mobile genetic elements. The vanA cluster has been identified in several enterococcal species including E. avium (2,6), however, transferable vancomycin resistance remains rare in clinical isolates of enterococcal species other than E. faecium and E. faecalis. Inaccurate identification of isolates can further confuse the picture. A recent report described a pseudoepidemic of VR-E. durans resulting from misidentification of VR-E. faecium (7). The enterococci have been divided into three groups (I-III) basedon phenotypic reactions (8). The group II organisms,E. faecium, E. faecalis, E. casselijlavus, E. gallinarum, and E. mundtii, are all arginine positive which distinguishesthem from the other eight enterococcal species.The ability of the Vitek system to identify an isolate as E, faecium despite a negative arginine reaction servesasa reminder that identification schemesbasedon conventional biochemical testscannot always be used to interpret biotypes generatedby the Vitek system(9). Analysis of the transmissionreadingsof the arginine and
useful time frame using proceduresthat are available in an increasingnumber of clinical laboratories. Misidentification of VR-E. faecium asVR-E. avium can have significant consequences.Current infection control guidelinesfocus on E. faecium and E. faecalis but do not addresswhether patients with other VRE speciesrequire similar isolation precautions.In the case describedabove, misidentification of the isolatewould have raised the possibility that the patient had acquired a secondVRE, possibly from an external source,or that there had been in vivo transfer of the vanA cluster. Becauseof the confusion that can be causedby misidentification of VR-E. faecium asE. avium, putative isolatesof VR-E. avium should be confirmed with a conventional arginine broth test and/ or DNA-based confirmation procedure.
Editors: Mary JaneFerraro Paul A. Granato JosephineA. Morello R.J. Zabransky
General
0 1998 Elsevier
tion to the species level of clinically relevant enterococci by PCR. J. Clin. Microbial. 33:24-27. 4. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J. Clin. Microbial. 33:1434. 5. Free, L. and D.F. Sahm. 1996. Detection of enterococcal vancomycin resistance by multiplex PCR, pp. 150-155. In D.H. Persing (ed.), PCR Protocols for Emerging Infectious Diseases. ASM Press, Washington, DC. 6.
Rosato,A. et al. 1995.Inducibleand constitutive expression of resistance to glycopeptides and vancomycin dependence in glycopeptide-resistant Enterococcus avium.Antimicrob. Agents Chemother. 39:830-833. Singer, D.A. et al. 1996. Pseudooutbreak of Enterococcus duransinfections and colonization associated with introduction of an automated identification system software update. .I. Clin. Microbial. 34:2685-2687. Facklam, R.R. and D.F. Sahm. 1995. Enterococcus, pp 308-314. In P.R. Murray, et al. (ed.), Manual of Clinical Microbiology, 6th ed. ASM Press,
Washington,DC. Ruoff, K.L. et al. 1990. Species identities of enterococci isolated from clinical specimens. J. Clin. Microbial. 28:435-437. 10. Evers, S. et al. 1996. Evolution of structure and substrate specificity in D-alanine:D-alanine ligases and related enzymes. J. Molec. Evol. 42:706-712.
Recommendations of the Hospital Infection Control Practices Advisory Committee (HICPAC). MMWR 44 (No. RR-12):1-19. 2. Leclercq, R. and P. Courvalin. 1997.
Information
Subscription
information
can be found inside the front cover.
This newsletter has been registered with the Copyright Clearance Center, Inc. Consent is given for copying articles for personal or internal use,or for the personal or internal use of specific clients. This consent is given on the condition that the copier pay through the Center the per-page fee stated in the code on the first page for copying beyond that permitted by the US Copyright Law. If no code appears on an article, the author has not given broad consent to copy and permission to copy must be obtained directly from the author. This
Science Inc
ISSN 0196-4399 CMNEEJ 20( 10)83-90,
References 1. Centersfor Disease Control. 1995. Recommendations for Preventingthe Spreadof VancomycinResistance
Resistance to glycopeptides in enterococci. Clin. Infect. Dis. 24545-556. 3. Dutka-Malen, S., S. Evers, and P. Courvalin. 1995. Detection of glycopeptide resistance genotypes and identifica-
consent does not extend to other kinds of copying, such as for general distribution, resale, advertising and promotional purposes, or for creating new collective works. 1998
Address orders, changes of address, and claims for missing issues to Journal Fulfillment Department, Elsevier Science Inc., 655 Avenue of the Americas, New York, NY 10010. Claims for missing issues can be honored only up to three months for domestic addresses and six months for foreign addresses. Duplicate copies will not be sent to replace ones undelivered due to failure to notify Elsevier of change of address.
Elsevier
Postmaster: Send address changes to Clinical of the Americas, New York, NY 10010. Editorials and letters printed in this newsletter necessarily reflect the opinions of the editors.
Microbiology
Newsletter;
are published
Elsevier
for the interest
llllll~~lllllllllll~lll~lllllllllllllllllll~lll Clinical Microbiology Newsletter is abstracted in Tropical Communicable Diseases and Current AIDS Literature.
0196-4399(19980515)20:10;1-D
90
0196-4399/96/$0.00
+ 19.00
0 1998 Elsevier Science Inc.
Diseases
Bulletin,
Science Inc., 655 Avenue of the readers Abstracts
Clinical Microbiology
and do not
on Hygiene
and
Newsletter 20:10,1998