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Frontiers in Biology

ISSN 1674-7984

ISSN 1674-7992(Online)

CN 11-5892/Q

Front Biol    2012, Vol. 7 Issue (2) : 167-177    https://doi.org/10.1007/s11515-012-1183-5
RESEARCH ARTICLE
Enterococcus faecalis can be distinguished from Enterococcus faecium via differential susceptibility to antibiotics and growth and fermentation characteristics on mannitol salt agar
Maria L. G. Quiloan1, John Vu2, John Carvalho3()
1. Biology Program, Biology Department, California State University Dominguez Hills (CSUDH), Carson, CA 90747, USA; 2. M. S. Biology Program, Biology Department, California State University Dominguez Hills (CSUDH), Carson, CA 90747, USA; 3. Biology Department, California State University Dominguez Hills (CSUDH), Carson, CA 90747, USA
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Abstract

Enterococcus faecalis and Enterococcus faecium are both human intestinal colonizers frequently used in medical bacteriology teaching laboratories in order to train students in bacterial identification. In addition, hospitals within the United States and around the world commonly isolate these bacteria because they are a cause of bacteremia, urinary tract infections, endocarditis, wound infections, and nosocomial infections. Given that enterococci are becoming more of a world health hazard, it is important for laboratories to be able to distinguish these bacteria within hospitalized patients from other bacterial genera. In addition, laboratories must differentiate different species within the Enterococcus genus as well as different strains within each species. Though enterococci are differentiated from other bacterial genera via classical culture and biochemical methods, nucleic acid sequencing is required to differentiate species within the genus—a costly, time consuming, and technically challenging procedure for laboratory technicians that, in itself, does not necessarily lead to speedy identification of bactericidal antibiotics. In this study, we perform antibiogram analysis to show (1) that penicillin can be rapidly employed to distinguish strains and clinical isolates of E. faecalis from E. faecium, (2) E. faecalis is susceptible to ampicillin, and (3) that vancomycin resistance in enterococci shows no sign of abating. Additionally, we show that E. faecalis can grow on mannitol salt agar and ferment mannitol, while E. faecium lacks these phenotypes. These data reveal that we now have rapid, cost effective ways to identify enterococci to the species, and not just genus, level and have significance for patient treatment in hospitals.

Keywords enterococci      Staphylococcus      antibiotic resistance      bacteriology      microbiology     
Corresponding Author(s): Carvalho John,Email:jcarvalho@csudh.edu   
Issue Date: 01 April 2012
 Cite this article:   
John Vu,John Carvalho,Maria L. G. Quiloan. Enterococcus faecalis can be distinguished from Enterococcus faecium via differential susceptibility to antibiotics and growth and fermentation characteristics on mannitol salt agar[J]. Front Biol, 2012, 7(2): 167-177.
 URL:  
https://academic.hep.com.cn/fib/EN/10.1007/s11515-012-1183-5
https://academic.hep.com.cn/fib/EN/Y2012/V7/I2/167
BacteriaSpecial characteristicsStrain or clinical isolate
Enterococcus faecalis (18)Straina
Enterococcus faecalis (9)Strainb
Enterococcus faecalis (10)Strainb
Enterococcus faecalis (11)Strainb
Enterococcus faecalis (14)Strainb
Enterococcus faecalis (310)Weakened strainStrainb
Enterococcus faecalis (313 VA-R)Vancomycin-resistantStrainb
Enterococcus faecalis (300A)Strainb
Enterococcus faecalis (411)Clinical isolatec
Enterococcus faecalis (412)Clinical isolatec
Enterococcus faecalis (413)Clinical isolatec
Enterococcus faecalis (414)Clinical isolatec
Enterococcus faecalis (415)Clinical isolatec
Enterococcus faecalis (416)Clinical isolatec
Enterococcus faecalis (417)Clinical isolatec
Enterococcus faecalis (418)Clinical isolatec
Enterococcus faecalis (419)Clinical isolatec
Enterococcus faecalis (420)Clinical isolatec
Tab.1  strains or clinical isolates used in this study
BacteriaStrain or Clinical Isolate
Enterococcus faecium (5)Straina
Enterococcus faecium (8)Straina
Enterococcus faecium (316)Clinical isolateb
Enterococcus faecium (152A)Clinical isolateb
Enterococcus faecium (401)Clinical isolatec
Enterococcus faecium (402)Clinical isolatec
Enterococcus faecium (403)Clinical isolatec
Enterococcus faecium (404)Clinical isolatec
Enterococcus faecium (405)Clinical isolatec
Enterococcus faecium (406)Clinical isolatec
Enterococcus faecium (407)Clinical isolatec
Enterococcus faecium (408)Clinical isolatec
Enterococcus faecium (409)Clinical isolatec
Enterococcus faecium (410)Clinical isolatec
Enterococcus faecium (421)Clinical isolatec
Enterococcus faecium (422)Clinical isolatec
Tab.2  strains or clinical isolates used in this study
Average disk diffusion sensitivities, zone diameter (mm)
VA 5*AM 10*TE 30AN 30P 10*E 15S 10B 10PB 300
E. faecalis (18)12.5*20.8*14.83.318.3*17.00.09.00.0
E. faecalis (9)10.0*24.0*20.012.018.0*16.00.010.010.0
E. faecalis (10)12.0*26.0*20.010.018.0*20.00.016.012.0
E. faecalis (11)10.024.0*8.08.018.0*16.00.010.010.0
E. faecalis (14)12.0*24.0*18.08.018.0*20.00.014.012.0
E. faecalis (300A)12.3*25.0*7.54.018.5*13.30.08.30.0
BoldItalic (310)11.7*22.3*22.025.317.0*22.715.322.07.3
E. faecalis (313-VA-R)0.0*21.3*16.70.010.7*0.00.010.30.0
E. faecalis (411)11.3*24.0*0.08.018.0*0.00.016.00.0
E. faecalis (412)11.3*24.0*6.010.018.0*20.00.014.00.0
E. faecalis (413)11.3*24.0*6.08.018.0*20.00.00.00.0
E. faecalis (414)11.3*24.0*6.010.018.0*0.00.012.00.0
E. faecalis (415)12.0*24.0*8.08.016.0*0.00.00.00.0
E. faecalis (416)12.0*30.0*10.020.020.0*22.010.016.00.0
E. faecalis (417)12.0*30.0*8.00.022.0*0.00.014.00.0
E. faecalis (418)10.0*24.0*20.012.020.0*24.00.010.00.0
E. faecalis (419)10.0*24.0*0.012.018.0*0.00.08.00.0
E. faecalis (420)10.0*24.0*0.012.018.0*16.00.010.00.0
E. faecium (5)14.0*20.0*20.010.08.0*10.00.010.00.0
E. faecium (8)0.0*0.0*20.08.00.0*0.00.014.00.0
E. faecium (316)13.7*23.0*18.34.08.0*15.00.017.00.0
E. faecium (152A)0.0*0.0*6.87.30.0*0.00.010.00.0
E. faecium (401)0.0*0.0*0.00.00.0*0.00.010.00.0
E. faecium (402)0.0*0.0*22.00.00.0*0.00.010.00.0
E. faecium (403)0.0*0.0*0.00.00.0*12.00.010.00.0
E. faecium (404)0.0*0.0*0.08.00.0*0.00.018.00.0
E. faecium (405)0.0*0.0*0.00.00.0*0.00.010.00.0
E. faecium (406)14.0*0.0*0.00.00.0*0.00.012.70.0
E. faecium (407)0.0*0.0*0.00.00.0*0.00.010.00.0
E. faecium (408)0.0*0.0*0.00.00.0*0.00.08.00.0
E. faecium (409)0.0*0.0*6.00.00.0*0.00.016.00.0
E. faecium (410)0.0*0.0*6.00.00.0*0.00.014.00.0
E. faecium (421)14.0*0.0*0.00.00.0*0.00.00.00.0
E. faecium (422)0.0*0.0*0.00.00.0*0.00.010.00.0
Tab.3  Antibiotic disk diffusion susceptibility testing for and
Fig.1  Zones of growth inhibition after 24 h for four representative enterococci. The arrows indicate the zone of inhibition following 24 h of incubation. The measurements of the arrows are found in Tables 3 and 4.
BacteriaGrowthFermentationBacteriaGrowthFermentation
E. faecalis (18)+ ++E. faecium (5)++
E. faecalis (9)+ ++E. faecium (8)+-
E. faecalis (10)+ ++E. faecium (316)+/ --
E. faecalis (11)+ ++E. faecium (152A)+/ --
E. faecalis (14)+ ++E. faecium (401)+-
E. faecalis (300A)+ ++E. faecium (402)+-
E. faecalis (310)+ ++E. faecium (403)+-
E. faecalis (313-VA-R)+ ++E. faecium (404)+-
E. faecalis (411)+ ++E. faecium (405)+-
E. faecalis (412)+ ++E. faecium (406)+-
E. faecalis (413)+ ++E. faecium (407)+-
E. faecalis (414)+ ++E. faecium (408)+-
E. faecalis (415)+ ++E. faecium (409)+-
E. faecalis (416)+ ++E. faecium (410)+-
E. faecalis (417)+ ++E. faecium (421)+-
E. faecalis (418)+ ++E. faecium (422)+-
E. faecalis (419)+ ++S. aureus+ + ++
E. faecalis (420)+ ++S. epidermidis+ + +-
Tab.4  Growth and fermentation characteristics of different enterococci on mannitol salt agar
Fig.2  Growth and fermentation patterns on mannitol salt agar. Plate shows the differing growth and fermentation patterns of selected enterococci on Mannitol Salt Agar, where fermentation is indicated by a change of the phenol red indicator from a red to yellow color. was used as a positive control for growth and fermentation while was used as a negative control. (1) , (2) , (3) (401), (4) (402), (5) (411), (6) (412).
1 Andersson D I (2003). Persistence of antibiotic resistant bacteria. Curr Opin Microbiol , 6(5): 452-456
doi: 10.1016/j.mib.2003.09.001 pmid:14572536
2 Archibald L K, Reller L B (2001). Clinical microbiology in developing countries. Emerg Infect Dis , 7(2): 302-305
doi: 10.3201/eid0702.010232 pmid:11294729
3 Blair E B, Emerson J S, Tull A H (1967). A new medium, salt mannitol plasma agar, for the isolation of Staphylococcus aureus. Am J Clin Pathol , 47(1): 30-39
4 Boe L, Gerdes K, Molin S (1987). Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance. J Bacteriol , 169(10): 4646-4650
pmid:3308847
5 Caldwell B A, Ye C, Griffiths R P, Moyer C L, Morita R Y (1989). Plasmid expression and maintanence during long-term starvation-survival of bacteria in well water. J Environ Microbiol , 55: 1860-1864
6 Carvalho J (2011). Importance of clinical microbiologists for U.S. healthcare infrastructure. Clin Lab Sci , 24(3): 136-141
pmid:21905578
7 Cetinkaya Y, Falk P, Mayhall C G (2000). Vancomycin-resistant enterococci. Clin Microbiol Rev , 13(4): 686-707
doi: 10.1128/CMR.13.4.686-707.2000 pmid:11023964
8 Chan E D, Iseman M D (2008). Multidrug-resistant and extensively drug-resistant tuberculosis: a review. Curr Opin Infect Dis , 21(6): 587-595
doi: 10.1097/QCO.0b013e328319bce6 pmid:18978526
9 Clark N C, Cooksey R C, Hill B C, Swenson J M, Tenover F C (1993). Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Antimicrob Agents Chemother , 37(11): 2311-2317
pmid:8285611
10 Courvalin P (2006). Vancomycin resistance in gram-positive cocci. Clin Infect Dis , 42(Suppl 1): S25-S34
doi: 10.1086/491711 pmid:16323116
11 Devriese L, Baele M, Butaye P (2006). The Genus Enterococcus. The Procaryotes . New York: Springer, 163-174
12 Domig K J, Mayer H K, Kneifel W (2003). Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria. Int J Food Microbiol , 88(2-3): 165-188
doi: 10.1016/S0168-1605(03)00178-8 pmid:14596988
13 Elzinga G, Raviglione M C, Maher D (2004). Scale up: meeting targets in global tuberculosis control. Lancet , 363(9411): 814-819
doi: 10.1016/S0140-6736(04)15698-5 pmid:15016493
14 Emori T G, Gaynes R P (1993). An overview of nosocomial infections, including the role of the microbiology laboratory. Clin Microbiol Rev , 6(4): 428-442
pmid:8269394
15 Farmer P (2005). Pathologies of Power: Health, Human Rights, and the New War on the Poor. Berkley: University of California Press
16 Fisher K, Phillips C (2009). The ecology, epidemiology and virulence of Enterococcus. Microbiology , 155(6): 1749-1757
doi: 10.1099/mic.0.026385-0 pmid:19383684
17 Flint S J, Enquest L W, Krug R M, Racaniello V R, Skalka A M (2000). Principles of Virology: Molecular Biology, Pathogenesis, and Control, Washington D C: ASM Press
18 Forbes B A, Sahm D F, Weissfeld A S (2007). Laboratory methods and strategies for antimicrobial susceptibility testing (12th Ed), St. Louis: Mosby Elsevier, 188-189
19 Gerdes K, Rasmussen P B, Molin S (1986). Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells. Proc Natl Acad Sci USA , 83(10): 3116-3120
doi: 10.1073/pnas.83.10.3116 pmid:3517851
20 Godwin D, Slater J H (1979). The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K12. J Gen Microbiol , 111(1): 201-210
pmid:379278
21 Guerrier-Takada C, Salavati R, Altman S (1997). Phenotypic conversion of drug-resistant bacteria to drug sensitivity. Proc Natl Acad Sci USA , 94(16): 8468-8472
doi: 10.1073/pnas.94.16.8468 pmid:9238000
22 Harrison L (2007). StaphylococciTextbook of Diagnostic Microbiology (3rd Ed), St. Louis , MO: Saunders-Elsevier, 377-379
23 Huycke M M, Sahm D F, Gilmore M S (1998). Multiple-drug resistant enterococci: the nature of the problem and an agenda for the future. Emerg Infect Dis , 4(2): 239-249
doi: 10.3201/eid0402.980211 pmid:9621194
24 Jorgensen J H, Turnridge J D (2007). Susceptibility Test Methods: Dilution and Disk Diffusion Methods. Manual of Clinical Microbiology 9th Edition, Washington D C: ASM Press, 1152-1159
25 Kateete D P, Kimani C N, Katabazi F A, Okeng A, Okee M S, Nanteza A, Joloba M L, Najjuka F C (2010). Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob , 9(1): 23
doi: 10.1186/1476-0711-9-23 pmid:20707914
26 Kües U, Stahl U (1989). Replication of plasmids in gram-negative bacteria. Microbiol Rev , 53(4): 491-516
pmid:2687680
27 Leboffe M J, Pierce B E (2005). A Photographic Atlas for the Microbiology Laboratory (3rd Ed), Englewood, CO: Morton Publishing Company, 18-19
28 Lehman D C, Mahon C R, Swarna K (2007). Streptococcus, Enterococcus, and other catalase-negative gram-positive cocci. Textbook of Diagnostic Microbiology (3rd Ed), St Louis , MO: Saunders-Elsevier, 382-409
29 Lenski R E, Bouma J E (1987). Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B. J Bacteriol , 169(11): 5314-5316
pmid:3312174
30 Lightfoot N F, Scot R J D, Turnball P C B (1990). Antimicrobial susceptibility of Bacillus anthracis. Salisbury Med Bull , 68(Suppl): 95-98
31 Megran D W (1992). Enterococcal endocarditis. Clin Infect Dis , 15(1): 63-71
doi: 10.1093/clinids/15.1.63 pmid:1617074
32 Modi R I, Adams J (1991). Coevolution in bacterial-plasmid populations. Evolution , 45(3): 656-667
doi: 10.2307/2409918
33 Moellering R C Jr (1992). Emergence of Enterococcus as a significant pathogen. Clin Infect Dis , 14(6): 1173-1178
doi: 10.1093/clinids/14.6.1173 pmid:1623072
34 Murray P R, Rosenthal K S, Pfaller M A (2009). Enterococcus and other gram-positive cocci. Medical Microbiology (6th Ed) , Philadelphia: Mosby Elsevie243-246
35 Noble W C, Virani Z, Cree R G (1992). Co-transfer of vancomycin and other resistance genes from Enterococcus faecalis NCTC 12201 to Staphylococcus aureus. FEMS Microbiol Lett , 93(2): 195-198
doi: 10.1111/j.1574-6968.1992.tb05089.x pmid:1505742
36 Pang T, Peeling R W (2007). Diagnostic tests for infectious diseases in the developing world: two sides of the coin. Trans R Soc Trop Med Hyg , 101(9): 856-857
doi: 10.1016/j.trstmh.2007.04.014 pmid:17544047
37 Rhee J I, Ricci J C D, Bode J, Schugerl K (1994). Metabolic enhancement due to plasmid maintenance. Biotechnol Lett , 16: 881-884
doi: 10.1007/BF00128618
38 Rhode C (1995). Technical information sheet No. 12: plasmid isolation from bacteria: some fast procedures. World J Microbiol Biotechnol , 11(3): 367-369
doi: 10.1007/BF00367126
39 Rice L B (2006). Antimicrobial resistance in gram-positive bacteria. Am J Med , 119(6 Suppl 1): S11-S19, discussion S62-S70
doi: 10.1016/j.amjmed.2006.03.012 pmid:16735146
40 Seo J H, Bailey J E (1985). Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli. Biotechnol Bioeng , 27(12): 1668-1674
doi: 10.1002/bit.260271207 pmid:18553628
41 Smith M A, Bidochka M J (1998). Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size. Can J Microbiol , 44(4): 351-355
doi: 10.1139/w98-020 pmid:9674107
42 Stetler H C, Granade T C, Nunez C A, Meza R, Terrell S, Amador L, George J R (1997). Field evaluation of rapid HIV serologic tests for screening and confirming HIV-1 infection in Honduras. AIDS , 11(3): 369-375
doi: 10.1097/00002030-199703110-00015 pmid:9147429
43 Sung J M L, Lindsay J A (2007). Staphylococcus aureus strains that are hypersusceptible to resistance gene transfer from enterococci. Antimicrob Agents Chemother , 51(6): 2189-2191
doi: 10.1128/AAC.01442-06 pmid:17371826
44 Teixeira L M, Carvalho M G S, Shewmaker P L, Facklam R R (2011). Manual of Clinical Microbiology. Washington , D C: ASM Press, 350-364
45 Usdin M, Guillerm M, Calmy A (2010). Patient needs and point-of-care requirements for HIV load testing in resource-limited settings. J Infect Dis , 201(s1 Suppl 1): S73-S77
doi: 10.1086/650384 pmid:20225951
46 Valenzuela M S, Ikpeazu E V, Siddiqui K A (1996). E. coli growth inhibition by a high copy number derivative of plasmid pBR322. Biochem Biophys Res Commun , 219(3): 876-883
doi: 10.1006/bbrc.1996.0339 pmid:8645273
47 Vu J, Carvalho J (2011). Enterococcus: review of its physiology, pathogenesis, diseases and the challenges it poses for clinical microbiology. Front Biol , 6(5): 357-366
doi: 10.1007/s11515-011-1167-x
48 Wilkinson D, Wilkinson N, Lombard C, Martin D, Smith A, Floyd K, Ballard R (1997). On-site HIV testing in resource-poor settings: is one rapid test enough? AIDS , 11(3): 377-381
doi: 10.1097/00002030-199703110-00016 pmid:9147430
49 Woodford N, Johnson A P, Morrison D, Speller D C (1995). Current perspectives on glycopeptide resistance. Clin Microbiol Rev , 8(4): 585-615
pmid:8665471
50 Yamahara K M, Layton B A, Santoro A E, Boehm A B (2007). Beach sands along the California coast are diffuse sources of fecal bacteria to coastal waters. Environ Sci Technol , 41(13): 4515-4521
doi: 10.1021/es062822n pmid:17695890
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