Enterococcus

Background

Most enterococci are not beta-hemolytic and limited to a small percentage of isolates of E. faecalis and E. faecium. Resistance occurs when a susceptible strain of E. faecalis or E. faecium acquires a series of novel genes that enable it to construct a new cell wall that no longer contains the binding site for vancomycin. Resistance is usually the end result of several genes in an operon. In general, the phenotype of the organism usually correlates with the genotype.

OUTLINE

Disease Associations

Pathogenesis

Laboratory/Radiologic/Other Diagnostic Testing

Gross Appearance and Clinical Variants

Histopathological Features and Variants

Special Stains/Immunohistochemistry/Electron Microscopy

Differential Diagnosis

Prognosis

Treatment

Commonly Used Terms

Internet Links

DISEASE ASSOCIATIONS CHARACTERIZATION

PSEUDOMEMBRANOUS COLITIS

Clostridium difficile and vancomycin-resistant enterococcus: the new nosocomial alliance.

Poduval RD, Kamath RP, Corpuz M, Norkus EP, Pitchumoni CS.

Department of Medicine, Our Lady of Mercy Medical Center, Bronx, New York 10466, USA

Am J Gastroenterol 2000 Dec;95(12):3513-5 Abstract quote
OBJECTIVES: The aims of this study were to determine the frequency of the association between Clostridium difficile (C. difficile) and vancomycin-resistant Enterococcus (VRE) and delineate the role of C. difficile coinfection as a predictor of VRE infection versus colonization and adverse outcome.

METHODS: Patients with both C. difficile colitis and VRE (CD/VRE) were compared to patients with VRE alone with regard to demographics, comorbidity, prior antibiotic therapy, and coinfection with methicillin-resistant Staphylococcus aureus and funguria. C. difficile as a predictor of VRE infection (VRE-I) versus colonization (VRE-C) and adverse outcome was also studied.

RESULTS: Eighty-nine patients with VRE infection or colonization were studied. This included 31 cases of VRE-I and 58 VRE-C. C. difficile was isolated in 17 (19.1%) of patients; of these C. difficile was isolated before VRE in 9 patients and after VRE in 8. The two groups did not differ in age, residence, or comorbidity. C. difficile coinfection was not predictive of VRE-I versus VRE-C, nor was it associated with increased length of stay or mortality. However, the mortality rates in both groups was high, around 30%. A significant association was noted between the use of vancomycin and metronidazole (before the isolation of VRE) and C. difficile coinfection (p = 0.03 and p = 0.001, respectively). A high incidence of nosocomial coinfection with methicillin-resistant Staphylococcus aureus, funguria, and gram-negative sepsis was noted in both groups; the association with funguria was statistically significant (p = 0.029).

CONCLUSIONS: In conclusion, C. difficile coinfection is common in patients with VRE infection or colonization and is significantly associated with other nosocomial dilemmas like funguria. This may result in the emergence of highly virulent pathogens including vancomycin-resistant C. difficile, posing new challenges in the management of nosocomial diarrheas.

Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae from stools submitted for Clostridium difficile testing compared to results from a focused surveillance program.

Hacek DM, Bednarz P, Noskin GA, Zembower T, Peterson LR.

Department of Pathology, Clinical Microbiology Division, Northwestern Memorial Hospital and Northwestern University Medical School, Chicago, Illinois 60611, USA.
J Clin Microbiol 2001 Mar;39(3):1152-4 Abstract quote
It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for Clostridium difficile testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant Enterobacteriaceae (MDRE) with both methods.

Of the stools submitted for C. difficile testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for C. difficile testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for C. difficile testing.

Therefore, while screening stools submitted for C. difficile testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.

PATHOGENESIS CHARACTERIZATION

Resistance

vanA
High level of resistance (MIC>128 ug/ml)

vanB
Low level of resistance (16-64 ug/ml) is similar to vanA but operates at a lower efficiency

vanC
Low levels of resistance (4-16 ug/ml)
Cannot be transferred to other organisms (as of this writing)

Exceptions
vanA when present in E. raffinosus produces a MIC of only 16 ug/ml while some vanB clusters in isolates of E. faecium show resistance as high as 1024 ug/ml.

NOTE: Both genes appear to be mobile to some degree on either plasmids or transposons

LABORATORY/RADIOLOGIC/
OTHER TESTS
CHARACTERIZATION

Molecular Typing
Pulsed field gel electrophoresis )PFGE) and ISPlasmids of the same molecular size has been found in multiple strains of enterococci and plasmids of different sizes found in a single enterococcal strain

Susceptibility testing

Best methods are broth microdilution incubated for a full 24 hours

Disk diffusion is acceptable but does have a greater minor error rate than do most broth methods

Brain Heart Infusion agar screen plate containing 6 ug/ml of vancomycin is effective for screening but does allow the growth of many E. gallinarum strains-thus enterococci that appear to be vanc susceptible by MIC but grow on agar screen plate should be tested for motility since E. gallinarum strains are motile but E. faecium and faecalis are not.

NOTE: The BHI is for pure cultures and should not be inoculated directly with stool or fecal material since it does not contain supplements to inihibit the growth of normal flora

NOTE: The zones of inhibition must be read carefully using transmitted instead of reflected lightAutomated methods such as VITEK cards are FDA approved

Bile Esculin Growth +
Can be alpha, beta, or gamma hemolysis Always identifies a gp. D Strep, by definition-now must decide whether it is enterococcus vs. non-enterococcus Growth in 6.5% NaCl + Gr. D enterococcus - Gr. D non-enterococci (eg S. bovis)

Media

Media containing 10 ug/ml of vancomycin usually preclude the growth of E. gallinarum and effectively eliminate the growth of E. casseliflavus and E. flavescens.

If higher concentrations are used, possibility of inhibition of some vanB strains-thus a major change could occur in the hospital's VRE strains and go undetected

NOTE: Use of amphotericin B in CAN may help reduce contamination with yeast but some vanB strains maybe susceptible to media containing both vancomycin and gentamycin since these drugs act synergistically against enterococci

Direct Plating vs. Enrichment broths

Easiest way to process rectal swabs is plate directly on selective media-may use a broth enrichment step such as bile esculin broth with a low level of vancomycin

Fecal swab can also have fecal material on swab suspended in 0.3-0.5 ml of broth or saline before plating-helpful if multiple media are inoculated

Temperature of plating
Many strains of E. faecium were inhibited by temperatures >42C, thus keep below 37C

Media QC

Should be performed on every new batch at least once a week

E. faecium and E. faecalis are recommended strains

VRE-Select uses biosensors to detect VRE collected on rectal swabs-some vanA and vanB strains were inhibited after direct inoculation

PCR

Can be used to detect vanA and vanB resistance genes from rectal swabsStrain typing is not possible and it is expensive.

However, 16 samples can be processed in 8 hour shift

Compared to standard broth enrichment method, this was 85% sensisitive, missing samples with <10 colonies

Surveillance

Little difference in recovery rates from swabs of rectum, perirectum, or perianal areaStool can also be used

Environmental samples are not warranted since they often produce misleading information about transmission of strains-VRE may remain viable for days to weeks

Appears to be no more resistant to disinfectants than vanc susceptible enterococciRoutine monitoring of Abx susceptibility patterns as part of lab surveillance

Testing of isolates obtained from normally sterile body sites and periodic screening of urine isolates of enterococci if no VRE have been recovered are important

ADDITIONAL TESTING

BROTH

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay.
Palladino S, Kay ID, Flexman JP, Boehm I, Costa AM, Lambert EJ, Christiansen KJ.

Department of Microbiology & Infectious Diseases, Royal Perth Hospital, Perth, Western Australia, Australia.
J Clin Microbiol. 2003 Jun;41(6):2483-6 Abstract quote
A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens.

One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively.

PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

MEDIA

Comparison of Two Commercially Available Selective Media to Screen for Vancomycin-Resistant Enterococci

Janet Shigei, MS, MT(ASCP)
Grace Tan, MT(ASCP)
Amy Shiao, MS, MT(ASCP)
Luis M. de la Maza, MD, PhD
and Ellena M. Peterson, PhD, MT(ASCP)

Am J Clin Pathol 2002;117:152-155 Abstract quote

Campylobacter medium (CAMPY, Becton Dickinson [BD] Microbiology Systems, Cockeysville, MD) and Vancomycin Screen Agar (VSA, BD) were compared for their ability to screen for vancomycin-resistant Enterococcus (VRE) from primary plates. A microdilution minimal inhibitory concentration (MIC) assay (Pasco, BD) served as the reference vancomycin MIC. In the random sample of 200 enterococcal clinical isolates tested, the distribution of isolates was as follows: Enterococcus faecium, 113 (97 VRE); Enterococcus faecalis, 71 (12 VRE); Enterococcus gallinarum, 10; and Enterococcus casseliflavus, 6.

Of the 75 vancomycin-susceptible strains, none grew on CAMPY and 4 grew on VSA, whereas all 109 VRE isolates grew on both screen plates. Of the 16 strains with a Van C phenotype, ie, E gallinarum and E casseliflavus, 2 grew on CAMPY and 14 on VSA.

Of the 899 clinical specimens plated onto both agars, 45 of 67 VRE were detected with both media, 20 were detected only with CAMPY, and 2 were not detected by either screen plate. CAMPY compared with VSA as a primary plating medium was more sensitive and, when used to screen for VRE isolates from primary plates, was more specific for strains displaying Van A and B phenotypes.

GROSS APPEARANCE/
CLINICAL VARIANTS CHARACTERIZATION

General

VARIANTS

Vancomycin resistant Enterococci (VRE) MMWR 1996;45:289-290

From 1989-1993, VREs reported to CDC have increased from 0.3% to 7.9% Susceptibility testing varied from Kirby-Bauer, Microscan, Vitek, Vancomycin screen agar, MIC panels, Sensititre system, Uniscept 47/61 sterile site isolates were from blood (77%)

Intrinsic low-level vancomycin resistance
E. casseliflavus and E. gallinarum Never found in pure culture and not currently considered pathogenetic
Contains the intrinsic VanC which cannot be transferred to other organisms and thus does not need to be treated or isolated patients who harbor this

NOTE: If one of these organsims is isolated from a sterile body site or were implicated as the etiologic agent of disease, current thinking is that this identification is incorrect and organism should be reidentified

E. gallinarum May have in vitro intermediate resistance to vancomycin Motile and nonpigmented

E. casseliflavus Motile and pigmented

E. faecium Nonmotile and nonpigmented

E. flavescens Pigmented

HISTOLOGICAL TYPES CHARACTERIZATION

General

Reporting Results Since VREF screening plates will pick up intrinsic low level variants E. casseliflavus and E. gallinarum, these must be identified to rule out important species of E. faecalis and faecium

Mixed cultures
If possible organism is likely to be casseliflavus or gallinarum, no reporting until ID is done

If it is one of these, report out as E. c or g, not considered to be important clinically. Susceptibility not performed

PROGNOSIS AND TREATMENT CHARACTERIZATION

COSTS

Vancomycin-sensitive and vancomycin-resistant enterococcal infections in the ICU: attributable costs and outcomes.

Pelz RK, Lipsett PA, Swoboda SM, Diener-West M, Powe NR, Brower RG, Perl TM, Hammond JM, Hendrix CW.

Department of Clinical Pharmacology, School of Medicine, Johns Hopkins University, Baltimore, MD 21287, USA.
Intensive Care Med 2002 Jun;28(6):692-7 Abstract quote
OBJECTIVES: To determine the economic and clinical outcomes associated with infection with vancomycin-resistant Enterococcus (VRE) and to compare these outcomes to those associated with infection with vancomycin-sensitive Enterococcus (VSE).

METHODS: During a 3-month, prospective, cohort study of 117 high-risk, critically ill patients we collected complete clinical and demographic and ICU cost data from all patients during their ICU stays.

RESULTS: After adjusting for variables in a stepwise multiple regression model VRE infections were associated with a median attributable increased ICU cost per patient of $33,251 (38,088 euros) and an increased length of hospital stay (LOS) of 22 days, while VSE infections were associated with an increased cost of $21,914 (25,102 euros) and an increased LOS of 27 days. The effect of VRE and VSE infections were not significantly different. Over the entire cohort the attributable cost per ICU patient day associated with VRE infection was $304 (348 euros).

CONCLUSIONS: The attributable cost of ICU care associated with VRE infection is $33,251 (38,088 euros) and per ICU patient day was $304 (348 euros). VRE and VSE infections do not differ in associated cost of ICU care, LOS, or mortality. Any VRE control strategy is be cost-effective if the overall cost per ICU patient-day is less than $304 (348 euros).

TREATMENT
Ann Intern Med 1995;123:250-259

Possible decrease if vancomycin usage is decreased but there was no change in VRE colonization rates in point prevalence studies

Clin Infect Dis 1996;23:1020-1025

If cefotaxime, ceftriaxone, and clindamycin use was also reduced, there was a decrease in prevalence-however this was a very short study period

ANTIBIOTICS

Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): results from the 2002 North American Vancomycin Resistant Enterococci Susceptibility Study (NAVRESS).

Zhanel GG, Laing NM, Nichol KA, Palatnick LP, Noreddin A, Hisanaga T, Johnson JL, The NAVRESS Group, Hoban DJ.

Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg; Departments of Medicine and Clinical Microbiology, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1R9.
J Antimicrob Chemother. 2003 Jul 29 Abstract quote
Background: The purpose of this study was to assess the prevalence of vancomycin-resistant enterococci (VRE) in urinary isolates in North America, and the activity of various antibiotics against VRE.

Materials and methods: Twenty-eight medical centres in the United States and 10 centres in Canada assessed the prevalence of VRE in urinary isolates in 2002. Each study site was asked to collect up to a maximum of 50 consecutive VRE (Enterococcus faecium, Enterococcus faecalis only) urinary isolates. Susceptibility was determined by NCCLS broth microdilution. The prevalence of vanA and vanB resistance genotypes was determined by multiplex PCR.

RESULTS: From the 28 US medical centres, a total of 697 VRE (616 [88.4%] E. faecium and 81 [11.6%] E. faecalis) were received. Approximately 75% of all VRE (E. faecium and E. faecalis) isolates demonstrated a VanA phenotype (resistance to both vancomycin and teicoplanin). PCR detection of vanA and vanB resistance determinants showed that the vanA genotype was present in 584 of 697 (83.8%) VRE isolates, whereas 113 (16.2%) isolates possessed the vanB gene. The most active agents were linezolid, nitrofurantoin and chloramphenicol, with 0.3%, 0.6% and 2.4% resistance, respectively. The majority (77.8%) of vancomycin-resistant E. faecium isolates displayed the VanA phenotype, and 538 of these 616 (87.3%) isolates were PCR-positive for vanA; the vanB genotype was detected in 78 (12.7%) isolates. Resistance was lowest with linezolid, chloramphenicol and nitrofurantoin at 0.3%, 0.3% and 0.5%, respectively. Only three genetically indistinguishable vanA-positive E. faecium were isolated from the 10 Canadian medical centres.

CONCLUSION: VRE urinary isolates are common in the United States, are primarily of the vanA genotype and are very susceptible to linezolid, nitrofurantoin and chloramphenicol. In Canada, VRE urinary isolates remain uncommon.

Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Rosai J. Ackerman's Surgical Pathology. Eight Edition. Mosby 1996.
Sternberg S. Diagnostic Surgical Pathology. Third Edition. Lipincott Williams and Wilkins 1999.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.
DeMay RM. The Art and Science of Cytopathology. Volume 1 and 2. ASCP Press. 1996.
Weedon D. Weedon's Skin Pathology Second Edition. Churchill Livingstone. 2002
Fitzpatrick's Dermatology in General Medicine. 5th Edition. McGraw-Hill. 1999.
Weiss SW and Goldblum JR. Enzinger and Weiss's Soft Tissue Tumors. Fourth Edition. Mosby 2001.

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DISEASE ASSOCIATIONS	CHARACTERIZATION
PSEUDOMEMBRANOUS COLITIS
Clostridium difficile and vancomycin-resistant enterococcus: the new nosocomial alliance. Poduval RD, Kamath RP, Corpuz M, Norkus EP, Pitchumoni CS. Department of Medicine, Our Lady of Mercy Medical Center, Bronx, New York 10466, USA	Am J Gastroenterol 2000 Dec;95(12):3513-5 Abstract quote OBJECTIVES: The aims of this study were to determine the frequency of the association between Clostridium difficile (C. difficile) and vancomycin-resistant Enterococcus (VRE) and delineate the role of C. difficile coinfection as a predictor of VRE infection versus colonization and adverse outcome. METHODS: Patients with both C. difficile colitis and VRE (CD/VRE) were compared to patients with VRE alone with regard to demographics, comorbidity, prior antibiotic therapy, and coinfection with methicillin-resistant Staphylococcus aureus and funguria. C. difficile as a predictor of VRE infection (VRE-I) versus colonization (VRE-C) and adverse outcome was also studied. RESULTS: Eighty-nine patients with VRE infection or colonization were studied. This included 31 cases of VRE-I and 58 VRE-C. C. difficile was isolated in 17 (19.1%) of patients; of these C. difficile was isolated before VRE in 9 patients and after VRE in 8. The two groups did not differ in age, residence, or comorbidity. C. difficile coinfection was not predictive of VRE-I versus VRE-C, nor was it associated with increased length of stay or mortality. However, the mortality rates in both groups was high, around 30%. A significant association was noted between the use of vancomycin and metronidazole (before the isolation of VRE) and C. difficile coinfection (p = 0.03 and p = 0.001, respectively). A high incidence of nosocomial coinfection with methicillin-resistant Staphylococcus aureus, funguria, and gram-negative sepsis was noted in both groups; the association with funguria was statistically significant (p = 0.029). CONCLUSIONS: In conclusion, C. difficile coinfection is common in patients with VRE infection or colonization and is significantly associated with other nosocomial dilemmas like funguria. This may result in the emergence of highly virulent pathogens including vancomycin-resistant C. difficile, posing new challenges in the management of nosocomial diarrheas.
Yield of vancomycin-resistant enterococci and multidrug-resistant Enterobacteriaceae from stools submitted for Clostridium difficile testing compared to results from a focused surveillance program. Hacek DM, Bednarz P, Noskin GA, Zembower T, Peterson LR. Department of Pathology, Clinical Microbiology Division, Northwestern Memorial Hospital and Northwestern University Medical School, Chicago, Illinois 60611, USA.	J Clin Microbiol 2001 Mar;39(3):1152-4 Abstract quote It has been suggested that a method of performing surveillance for vancomycin-resistant enterococci (VRE) is to screen specimens submitted for Clostridium difficile testing. We compared this approach to our focused surveillance program of high-risk units during October 1997 to compare the yield of VRE and multidrug-resistant Enterobacteriaceae (MDRE) with both methods. Of the stools submitted for C. difficile testing, 14% were positive for VRE or MDRE, whereas rectal swabs from routine surveillance yielded 11% VRE- or MDRE-positive results. Although stools submitted for C. difficile testing resulted in a higher percentage of positive cultures, 14 VRE- and 2 MDRE-positive patients from our high-risk population were missed because many patients had no stool submitted for C. difficile testing. Therefore, while screening stools submitted for C. difficile testing cannot replace our focused surveillance program, it appears advantageous to assess these stools at various intervals to detect new patient reservoirs of drug-resistant organisms that may benefit from routine surveillance cultures.

PROGNOSIS AND TREATMENT	CHARACTERIZATION
COSTS
Vancomycin-sensitive and vancomycin-resistant enterococcal infections in the ICU: attributable costs and outcomes. Pelz RK, Lipsett PA, Swoboda SM, Diener-West M, Powe NR, Brower RG, Perl TM, Hammond JM, Hendrix CW. Department of Clinical Pharmacology, School of Medicine, Johns Hopkins University, Baltimore, MD 21287, USA.	Intensive Care Med 2002 Jun;28(6):692-7 Abstract quote OBJECTIVES: To determine the economic and clinical outcomes associated with infection with vancomycin-resistant Enterococcus (VRE) and to compare these outcomes to those associated with infection with vancomycin-sensitive Enterococcus (VSE). METHODS: During a 3-month, prospective, cohort study of 117 high-risk, critically ill patients we collected complete clinical and demographic and ICU cost data from all patients during their ICU stays. RESULTS: After adjusting for variables in a stepwise multiple regression model VRE infections were associated with a median attributable increased ICU cost per patient of $33,251 (38,088 euros) and an increased length of hospital stay (LOS) of 22 days, while VSE infections were associated with an increased cost of $21,914 (25,102 euros) and an increased LOS of 27 days. The effect of VRE and VSE infections were not significantly different. Over the entire cohort the attributable cost per ICU patient day associated with VRE infection was $304 (348 euros). CONCLUSIONS: The attributable cost of ICU care associated with VRE infection is $33,251 (38,088 euros) and per ICU patient day was $304 (348 euros). VRE and VSE infections do not differ in associated cost of ICU care, LOS, or mortality. Any VRE control strategy is be cost-effective if the overall cost per ICU patient-day is less than $304 (348 euros).
TREATMENT	Ann Intern Med 1995;123:250-259 Possible decrease if vancomycin usage is decreased but there was no change in VRE colonization rates in point prevalence studies
	Clin Infect Dis 1996;23:1020-1025 If cefotaxime, ceftriaxone, and clindamycin use was also reduced, there was a decrease in prevalence-however this was a very short study period
ANTIBIOTICS
Antibiotic activity against urinary tract infection (UTI) isolates of vancomycin-resistant enterococci (VRE): results from the 2002 North American Vancomycin Resistant Enterococci Susceptibility Study (NAVRESS). Zhanel GG, Laing NM, Nichol KA, Palatnick LP, Noreddin A, Hisanaga T, Johnson JL, The NAVRESS Group, Hoban DJ. Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg; Departments of Medicine and Clinical Microbiology, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, Manitoba, Canada R3A 1R9.	J Antimicrob Chemother. 2003 Jul 29 Abstract quote Background: The purpose of this study was to assess the prevalence of vancomycin-resistant enterococci (VRE) in urinary isolates in North America, and the activity of various antibiotics against VRE. Materials and methods: Twenty-eight medical centres in the United States and 10 centres in Canada assessed the prevalence of VRE in urinary isolates in 2002. Each study site was asked to collect up to a maximum of 50 consecutive VRE (Enterococcus faecium, Enterococcus faecalis only) urinary isolates. Susceptibility was determined by NCCLS broth microdilution. The prevalence of vanA and vanB resistance genotypes was determined by multiplex PCR. RESULTS: From the 28 US medical centres, a total of 697 VRE (616 [88.4%] E. faecium and 81 [11.6%] E. faecalis) were received. Approximately 75% of all VRE (E. faecium and E. faecalis) isolates demonstrated a VanA phenotype (resistance to both vancomycin and teicoplanin). PCR detection of vanA and vanB resistance determinants showed that the vanA genotype was present in 584 of 697 (83.8%) VRE isolates, whereas 113 (16.2%) isolates possessed the vanB gene. The most active agents were linezolid, nitrofurantoin and chloramphenicol, with 0.3%, 0.6% and 2.4% resistance, respectively. The majority (77.8%) of vancomycin-resistant E. faecium isolates displayed the VanA phenotype, and 538 of these 616 (87.3%) isolates were PCR-positive for vanA; the vanB genotype was detected in 78 (12.7%) isolates. Resistance was lowest with linezolid, chloramphenicol and nitrofurantoin at 0.3%, 0.3% and 0.5%, respectively. Only three genetically indistinguishable vanA-positive E. faecium were isolated from the 10 Canadian medical centres. CONCLUSION: VRE urinary isolates are common in the United States, are primarily of the vanA genotype and are very susceptible to linezolid, nitrofurantoin and chloramphenicol. In Canada, VRE urinary isolates remain uncommon.

Disease Associations
Pathogenesis
Laboratory/Radiologic/Other Diagnostic Testing
Gross Appearance and Clinical Variants
Histopathological Features and Variants
Special Stains/Immunohistochemistry/Electron Microscopy
Differential Diagnosis
Prognosis
Treatment
Commonly Used Terms
Internet Links

PATHOGENESIS	CHARACTERIZATION
Resistance
vanA	High level of resistance (MIC>128 ug/ml)
vanB	Low level of resistance (16-64 ug/ml) is similar to vanA but operates at a lower efficiency
vanC	Low levels of resistance (4-16 ug/ml) Cannot be transferred to other organisms (as of this writing)
Exceptions	vanA when present in E. raffinosus produces a MIC of only 16 ug/ml while some vanB clusters in isolates of E. faecium show resistance as high as 1024 ug/ml. NOTE: Both genes appear to be mobile to some degree on either plasmids or transposons

LABORATORY/RADIOLOGIC/ OTHER TESTS	CHARACTERIZATION
Molecular Typing	Pulsed field gel electrophoresis )PFGE) and ISPlasmids of the same molecular size has been found in multiple strains of enterococci and plasmids of different sizes found in a single enterococcal strain
Susceptibility testing	Best methods are broth microdilution incubated for a full 24 hours Disk diffusion is acceptable but does have a greater minor error rate than do most broth methods Brain Heart Infusion agar screen plate containing 6 ug/ml of vancomycin is effective for screening but does allow the growth of many E. gallinarum strains-thus enterococci that appear to be vanc susceptible by MIC but grow on agar screen plate should be tested for motility since E. gallinarum strains are motile but E. faecium and faecalis are not. NOTE: The BHI is for pure cultures and should not be inoculated directly with stool or fecal material since it does not contain supplements to inihibit the growth of normal flora NOTE: The zones of inhibition must be read carefully using transmitted instead of reflected lightAutomated methods such as VITEK cards are FDA approved
Bile Esculin Growth +	Can be alpha, beta, or gamma hemolysis Always identifies a gp. D Strep, by definition-now must decide whether it is enterococcus vs. non-enterococcus Growth in 6.5% NaCl + Gr. D enterococcus - Gr. D non-enterococci (eg S. bovis)
Media	Media containing 10 ug/ml of vancomycin usually preclude the growth of E. gallinarum and effectively eliminate the growth of E. casseliflavus and E. flavescens. If higher concentrations are used, possibility of inhibition of some vanB strains-thus a major change could occur in the hospital's VRE strains and go undetected NOTE: Use of amphotericin B in CAN may help reduce contamination with yeast but some vanB strains maybe susceptible to media containing both vancomycin and gentamycin since these drugs act synergistically against enterococci
Direct Plating vs. Enrichment broths	Easiest way to process rectal swabs is plate directly on selective media-may use a broth enrichment step such as bile esculin broth with a low level of vancomycin Fecal swab can also have fecal material on swab suspended in 0.3-0.5 ml of broth or saline before plating-helpful if multiple media are inoculated
Temperature of plating	Many strains of E. faecium were inhibited by temperatures >42C, thus keep below 37C
Media QC	Should be performed on every new batch at least once a week E. faecium and E. faecalis are recommended strains VRE-Select uses biosensors to detect VRE collected on rectal swabs-some vanA and vanB strains were inhibited after direct inoculation
PCR	Can be used to detect vanA and vanB resistance genes from rectal swabsStrain typing is not possible and it is expensive. However, 16 samples can be processed in 8 hour shift Compared to standard broth enrichment method, this was 85% sensisitive, missing samples with <10 colonies
Surveillance	Little difference in recovery rates from swabs of rectum, perirectum, or perianal areaStool can also be used Environmental samples are not warranted since they often produce misleading information about transmission of strains-VRE may remain viable for days to weeks Appears to be no more resistant to disinfectants than vanc susceptible enterococciRoutine monitoring of Abx susceptibility patterns as part of lab surveillance Testing of isolates obtained from normally sterile body sites and periodic screening of urine isolates of enterococci if no VRE have been recovered are important
ADDITIONAL TESTING
BROTH
Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay. Palladino S, Kay ID, Flexman JP, Boehm I, Costa AM, Lambert EJ, Christiansen KJ. Department of Microbiology & Infectious Diseases, Royal Perth Hospital, Perth, Western Australia, Australia.	J Clin Microbiol. 2003 Jun;41(6):2483-6 Abstract quote A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.
MEDIA
Comparison of Two Commercially Available Selective Media to Screen for Vancomycin-Resistant Enterococci Janet Shigei, MS, MT(ASCP) Grace Tan, MT(ASCP) Amy Shiao, MS, MT(ASCP) Luis M. de la Maza, MD, PhD and Ellena M. Peterson, PhD, MT(ASCP)	Am J Clin Pathol 2002;117:152-155 Abstract quote Campylobacter medium (CAMPY, Becton Dickinson [BD] Microbiology Systems, Cockeysville, MD) and Vancomycin Screen Agar (VSA, BD) were compared for their ability to screen for vancomycin-resistant Enterococcus (VRE) from primary plates. A microdilution minimal inhibitory concentration (MIC) assay (Pasco, BD) served as the reference vancomycin MIC. In the random sample of 200 enterococcal clinical isolates tested, the distribution of isolates was as follows: Enterococcus faecium, 113 (97 VRE); Enterococcus faecalis, 71 (12 VRE); Enterococcus gallinarum, 10; and Enterococcus casseliflavus, 6. Of the 75 vancomycin-susceptible strains, none grew on CAMPY and 4 grew on VSA, whereas all 109 VRE isolates grew on both screen plates. Of the 16 strains with a Van C phenotype, ie, E gallinarum and E casseliflavus, 2 grew on CAMPY and 14 on VSA. Of the 899 clinical specimens plated onto both agars, 45 of 67 VRE were detected with both media, 20 were detected only with CAMPY, and 2 were not detected by either screen plate. CAMPY compared with VSA as a primary plating medium was more sensitive and, when used to screen for VRE isolates from primary plates, was more specific for strains displaying Van A and B phenotypes.

GROSS APPEARANCE/ CLINICAL VARIANTS	CHARACTERIZATION
General
VARIANTS
Vancomycin resistant Enterococci (VRE)	MMWR 1996;45:289-290 From 1989-1993, VREs reported to CDC have increased from 0.3% to 7.9% Susceptibility testing varied from Kirby-Bauer, Microscan, Vitek, Vancomycin screen agar, MIC panels, Sensititre system, Uniscept 47/61 sterile site isolates were from blood (77%)
Intrinsic low-level vancomycin resistance	E. casseliflavus and E. gallinarum Never found in pure culture and not currently considered pathogenetic Contains the intrinsic VanC which cannot be transferred to other organisms and thus does not need to be treated or isolated patients who harbor this NOTE: If one of these organsims is isolated from a sterile body site or were implicated as the etiologic agent of disease, current thinking is that this identification is incorrect and organism should be reidentified
E. gallinarum	May have in vitro intermediate resistance to vancomycin Motile and nonpigmented
E. casseliflavus	Motile and pigmented
E. faecium	Nonmotile and nonpigmented
E. flavescens	Pigmented

HISTOLOGICAL TYPES	CHARACTERIZATION
General
Reporting Results	Since VREF screening plates will pick up intrinsic low level variants E. casseliflavus and E. gallinarum, these must be identified to rule out important species of E. faecalis and faecium
Mixed cultures	If possible organism is likely to be casseliflavus or gallinarum, no reporting until ID is done If it is one of these, report out as E. c or g, not considered to be important clinically. Susceptibility not performed