ENTEROCOCCAL SPECIES Sample ES-02 was a simulated blood culture isolate from a patient with symptoms of sepsis. Participants were asked to identify any potential pathogen and to perform susceptibility tests on that organism. 1-3 This culture contained Enterococcus gallinarum, a species that has been noted to be occasionally multidrugresistant (MDR) including resistance to glycopeptides (vancomycin and teicoplanin). This organism sample was sent to API participants as an Educational challenge and for susceptibility testing, grading was not performed. Responses of E. gallinarum, vancomycin-resistant (213); E. gallinarum (121), Enterococcus spp. (80); aerobic growth, referred (16); aerobic growth referred/gram-positive aerobe (3); and Gram-positive aerobe (3) were considered acceptable. However, only 436 participants (56.8%) had correctly identified the organism. The most common identification errors were calling the isolate an E. faecium (37.5%) or E. faecalis (3.4%). This strain should be quite easily identified and when using the Vitek card the probability of the organism being E. casseliflavus/gallinarum was 98%, further differentiated by pigment production. Using the Vitek 2 the result was 99% confidence for E. gallinarum. The actual clinical source of the organism used for this challenge was from a rectal swab screen for VRE by a western Ireland hospital (27 year old male patient on the Hematology/Oncology ICU service). Organism Identification E. gallinarum is a Gram-positive coccus that can be seen singly or in pairs and chains when looking at a Gram s stained slide of direct specimens. They are facultative anaerobes that grow well at 35 C in ambient air or CO 2 atmosphere on standard laboratory isolation media, producing catalase-negative results and non-pigmented colonies. 4 Many of the automated identification systems have had difficulty differentiating E. gallinarum, E. casseliflavus and E. faecalis due to their very similar biochemical reactions. 4-6 The ability of E. gallinarum to produce acid from methyl-α-d-glucopyranoside will assist differentiation from E. faecium and E. faecalis. A motility test can be used to differentiate the motile E. gallinarum from most other Enterococcus spp. including E. faecium for which E. gallinarum is commonly confused. The closely related E. casseliflavus species produces yellow pigmented colonies which distinguish it from E. gallinarum 4. Enterococci are ubiquitous and can be found in soil, water, plants, animals, and food. 4 They are normal inhabitants of the gastrointestinal tract where they occur in high numbers and they are less commonly found in the genitourinary tract or oral cavity. 7, 8 They are frequent causes of nosocomial urinary tract infection, wound infections and bacteremia. Enterococci have been implicated as the cause of up to 10% of all urinary tract infections (UTI) and 16% of nosocomial UTI. The SENTRY Antimicrobial Surveillance Program ranked Enterococcus as the 4th leading pathogen in North America (10.2%) and 5th in Europe
(7.2%) from bloodstream infections. 8 E. faecalis and E. faecium are the two most common enterococci found in clinical infections with E. gallinarum being an uncommon clinical isolate. 8 Although enterococci are an uncommon cause of central nervous system infection there have been rare reports of E. gallinarum involvement. 9 Early reports of vancomycin resistance in the enterococci involved E. gallinarum. 10 Further study indicated that E. gallinarum were more resistant to vancomycin than other Enterococci. This is due to the presence of vanc-1 which leads to an intrinsic low level resistance to glycopeptides. 11 Antimicrobial Susceptibility Testing (Ungraded) Participants were asked to perform antimicrobial susceptibility testing on this isolate of E. gallinarum. This strain was selected to challenge proper identification and to determine antimicrobial susceptibility across numerous classes of Gram-positive-active antimicrobial agents. The initial reference laboratory antimicrobial susceptibility testing was conducted by standardized reference broth microdilution methods 1 and susceptibility was determined based on CLSI document M100-S21 breakpoints. 2 The reference laboratory testing reported a total of 18 agents (Table 1) that demonstrated antimicrobial activity against this strain, however the very usually potent glycopeptides (vancomycin) were not active, having a MIC value of >16 µg/ml. Consensus accuracy criteria by susceptibility category are found in Table 2 for 16 antimicrobials having a significant sample of participant responses. Two agents (ciprofloxacin and quinupristin/dalfopristin) did not achieve consensus ( 80%) due to MIC results at or within one doubling dilution step of the susceptible-intermediate breakpoint, see Table 1. The disk diffusion (DD) results, 3 though much smaller numbers, had a range of 70.0 (erythromycin) to 100.0% (six drugs) correct responses with an overall average of 93.7% among only 126 results. Similarly, the MIC test results had an accuracy rate of 92.3 to 100% (average, 98.5% for 3,850 responses). Some participants continue to report antimicrobials for systemic infections(blood culture for this challenge) that are only active/indicated for urinary tract infections, such as nitrofurantoin (40), norfloxacin (5) and trimethoprim/sulfamethoxazole (9). This practice could result in compromised patient therapy. Also some laboratories reported susceptible results for the cephalosporins, six different agents. The E. gallinarum strain harbored an intrinsic van C1 and an acquired vana by PCR tests. These genes elevated the vancomycin MIC to >16 µg/ml as well as the teicoplanin MIC to > 8 µg/ml; the so-called VanA type resistance. This pattern, usually encountered in E. faecium strains, may have contributed to the high level of identification errors. However, using the entire antibiogram the ampicillin susceptibility
and quinupristin/dalfopristin non-susceptibility findings were inconsistent with a E. faecium identification. Also the colonial morphology was discordant. All participants having submitted erroneous E. faecium or E. faecalis identifications, as well as producing susceptible results for vancomycin, should re-examine their methods for technical errors or procedural flaws. Resistance Mechanisms Observed in Enterococci The rate of vancomycin resistance among E. faecalis clinical isolates causing bloodstream infections (BSI) in USA hospitals has remained stable over the last decade (Table 3; unpublished data SENTRY Program). This VRE rate among E. faecium isolates has escalated from 57.1% in 2000 to 80.7% in 2010. Enterococcal isolates may acquire various types of glycopeptide resistance determinants via vanassociated genetic elements (vana/b/d/e/g/l/m/n). 12 Among these, vana and vanb are the most prevalent in clinically relevant species. 13 However, the prevalence of VanA type among E. faecalis and E. faecium causing BSI in the USA has increased over the years and currently accounts for approximately 77 and 97% of the VRE phenotypes observed, respectively (Table 3). In addition to acquired genes, lowlevel vancomycin resistance (2 32 g/ml) may be due to presence of intrinsic, chromosomally located vanc genes. vanc1-, vanc2- and vanc3-type are expressed constitutively in E. gallinarum, E. 13, 14 casseliflavus and E. flavescens, respectively. Overall, Van enzymes can be distinguished on the basis of the level and inducibility of glycopeptide resistance. The VanA type is characterized by acquired resistance to high levels of vancomycin and teicoplanin, and it is induced by both drugs. The VanB type is defined by acquired resistance to various concentrations of vancomycin but not to teicoplanin and is induced only by vancomycin. 13 However, isolates demonstrating a VanB phenotype with a vana genotype have been observed in the East Asia region (Japan, China, Korea and Taiwan) 15 and in the USA. These acquired van genes are carried by large 12, 13 transposon elements, which are usually associated with transmissible plasmids. These mobile elements contribute to genomic plasticity of bacterial organisms and explain the dissemination of van genes in E. faecalis, E. faecium and other enterococcal species, including E. avium, E. casseliflavus, E. durans, E. gallinarum and E. raffinosus. In addition, transfer of VanA resistance to non-enterococcal species such as Staphylococcus aureus, has been documented, thus producing VRSA. 12-14
References 1. CLSI. M07-A8. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: eighth edition. Wayne, PA: Clinical and Laboratory Standards Institute; 2009. 2. CLSI. M100-S21. Performance standards for antimicrobial susceptibility testing: 21st informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2011. 3. CLSI. M02-A10. Performance standards for antimicrobial disk susceptibility tests; approved standard: tenth edition, Wayne, PA: Clinical and Laboratory Standards Institute; 2009. 4. Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of Clinical Microbiology, 9th edition. Washington D.C.: ASM Press; 2007. 5. Jin WY, Jang SJ, Lee MJ, Park G, Kim MJ, Kook JK, Kim DM, Moon DS, Park YJ. Evaluation of VITEK 2, MicroScan, and Phoenix for identification of clinical isolates and reference strains. Diagn Microbiol Infect Dis 2011;70:442-7. 6. Ligozzi M, Bernini C, Bonora MG, De Fatima M, Zuliani J, Fontana R. Evaluation of the VITEK 2 system for identification and antimicrobial susceptibility testing of medically relevant Grampositive cocci. J Clin Microbiol 2002;40:1681-6. 7. Moellering RC, Jr. Emergence of Enterococcus as a significant pathogen. Clin Infect Dis 1992;14:1173-6. 8. Deshpande LM, Fritsche TR, Moet GJ, Biedenbach DJ, Jones RN. Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe: A report from the SENTRY Antimicrobial Surveillance Program. Diagn Microbiol Infect Dis 2007;58:163-70. 9. Khan FY, Elshafi SS. Enterococcus gallinarum meningitis: a case report and literature review. J Infect Dev Ctries 2011;5:231-4. 10. Kaplan AH, Gilligan PH, Facklam RR. Recovery of resistant enterococci during vancomycin prophylaxis. J Clin Microbiol 1988;26:1216-8. 11. Patel R, Piper KE, Rouse MS, Steckelberg JM, Uhl JR, Kohner P, Hopkins MK, Cockerill FR, 3rd, Kline BC. Determination of 16S rrna sequences of enterococci and application to species identification of nonmotile Enterococcus gallinarum isolates. J Clin Microbiol 1998;36:3399-407. 12. Hegstad K, Mikalsen T, Coque TM, Werner G, Sundsfjord A. Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium. Clin Microbiol Infect 2010;16:541-54.
13. Woodford N. Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci. Microb Drug Resist 2001;7:229-36. 14. Reynolds PE, Courvalin P. Vancomycin resistance in enterococci due to synthesis of precursors terminating in D-alanyl-D-serine. Antimicrob Agents Chemother 2005;49:21-5. 15. Gu L, Cao B, Liu Y, Guo P, Song S, Li R, Dai H, Wang C. A new Tn1546 type of VanB phenotype-vana genotype vancomycin-resistant Enterococcus faecium isolates in mainland China. Diagn Microbiol Infect Dis 2009;63:70-5.
Table 1. Listing of expected susceptibility category results for the E. gallinarum sample found in ES-02 (2011). Antimicrobial agents by susceptibility category (reference MIC in µg/ml) a Susceptible Intermediate Resistant Amoxicillin/clavulanate ( 1) Quinupristin/dalfopristin (2) Vancomycin (>16) Ampicillin ( 1) Teicoplanin (>8) Penicillin (1) Cephalosporins Piperacillin/tazobactam (16/4) Chloramphenicol ( 8) Daptomycin (1) Erythromycin ( 0.25) Linezolid (1) Ciprofloxacin (1) Levofloxacin (1) Tetracycline (1) Tigecycline (0.12) Gentamicin ( 500) b Streptomycin ( 1000) b a. Categories assigned per CLSI M07-A8 and M100-S21 (2011) criteria or USA-FDA approved product package insert (tigecycline). b. Test used to predict synergy when combined with a cell-wall active agent (penicillin or vancomycin).
Table 2. Participant performance for selected agents ( 10 responses by one or both test methods) listed by agar disk diffusion (DD) and quantitative MIC methods for ES-02 (2011), an E. gallinarum strain with resistance to glycopeptides (VanA pattern). DD MIC Antimicrobial Agent Acceptable susceptibility category No. % correct a No. % correct a Amoxicillin/clavulanate Susceptible b 2 100.0 13 92.3 Ampicillin Susceptible 26 100.0 599 99.0 Chloramphenicol Susceptible 0-71 100.0 Ciprofloxacin Susceptible 9 22.2 244 80.3 Daptomycin Susceptible 0-119 100.0 Erythromycin Susceptible 10 70.0 316 96.8 Gentamicin Susceptible c 10 100.0 193 99.0 Levofloxacin Susceptible 9 88.9 275 99.3 Linezolid Susceptible 4 100.0 428 97.9 Penicillin Susceptible 22 90.9 519 98.7 Quinupristin/dalfopristin Intermediate 0-119 Rifampin Susceptible 1 100.0 194 100.0 Streptomycin Susceptible c 0-131 98.5 Tetracycline Susceptible 13 92.3 295 99.3 Tigecycline Susceptible 1 100.0 46 100.0 Vancomycin Resistant 28 96.4 651 98.2 d a. Proportion (%) conforming to 80% consensus of all participant s results and in agreement with reference laboratory MIC results interpreted by CLSI M100-S21 (2011) breakpoints. b. Category predicted by ampicillin test results. c. test used to predict synergy (susceptible) when combined with a cell-wall active agent (penicillin or vancomycin) for therapy. d. See text.
Table 3. Trends in VRE bacteremia rates in the SENTRY Antimicrobial Surveillance Program (USA; 2000-2010) hospitals, quantitating vancomycin resistance (VanA) patterns (7,424 strains). Vancomycin non-susceptible rate (% VanA a ): Surveillance year E. faecium E. faecalis (no. tested) (2,614) (4,810) 2000 57.1 (84.2) 4.0 (42.5) 2001 60.0 (88.2) 1.4 (35.7) 2002 70.7 (86.4) 2.9 (55.2) 2003 70.5 (88.5) 4.6 (34.8) 2004 68.6 (94.9) 2.6 (50.0) 2005 70.5 (95.6) 4.3 (51.2) 2006 69.7 (99.0) 4.8 (87.5) 2007 71.6 (97.6) 3.9 (89.7) 2008 75.3 (97.9) 6.5 (75.4) 2009 76.3 (97.6) 3.2 (65.6) 2010 80.7 (97.0) 5.3 (77.4) All years 71.7 (94.8) 4.0 (65.0) a The VanA phenotype was characterized by non-susceptibility to vancomycin (MIC, 8 g/ml) and teicoplanin (MIC, 16 g/ml).