ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES

This specimen was presented as a blood culture (bacteremia) isolate and was to be identified and tested for antimicrobial susceptibility. The culture contained a epidermidis, a common isolate from blood cultures, some being considered skin contaminates. High rates of methicillin resistance are observed in coagulase-negative staphylococci (CoNS) such as S. epidermidis, thus requiring therapy with alternative agents such as vancomycin. This strain was selected to highlight the importance of accurate assessment of vancomycin activity, especially those strains at the extreme (MIC, 4 µg/ml) of the wildtype MIC distribution.

The strain was distributed by API to survey participants as an Educational Sample (ES) challenge and susceptibility testing specimen. Participant grading was not applied. Responses were S. epidermidis (83.6%); Staphylococcus, coagulase-negative (13.9%); aerobic growth, referred (1.0%); Gram-positive aerobe (0.4%); aerobic growth referred/Gram-positive aerobe (0.1%); and S. epidermidis, methicillin- resistant (0.1%). The overall accuracy rate was at 99.1%.

Organism Identification (ID)

S. epidermidis is a Gram positive, coagulase negative member of the family.1 Gram stain characteristics of S. epidermidis are typical of other members of Staphylococcaceae and will appear as Gram positive cocci that occur singly, in pairs, tetrads, short chains and grapelike clusters. The morphology of S. epidermidis when cultured on sheep blood agar plates often appears as a nonpigmented, smooth, glistening, and opaque colony of 2.5 to 6 mm diameter. Common identification characteristics that all S. epidermidis strains share are 1) inability to produce coagulase, 2) ability to grow anaerobically using standardized medium containing glucose, and 3) inability to ferment mannitol anaerobically. The key biochemical test for S. epidermidis is the coagulase test. The slide coagulase test using rabbit plasma can be performed to detect the presence of bound coagulase also known as "clumping factor". This result will be negative for S. epidermidis and present as a homogenous suspension after 10 seconds. A negative slide test should be confirmed with a tube coagulase test to detect the presence of bound and free coagulase. The tube coagulase test will result in a homogenous suspension (no clot formation) after 4 and 24 hours incubation for S. epidermidis. A positive test

(15% H2O2 concentration) will differentiate staphylococci isolates from enterococci. Proper technique is needed when performing the catalase test from colonies grown on blood agar to avoid false-positive results. Most commercially available automated identification systems can reliably identify S. epidermidis. When a CoNS isolate is recovered from a blood culture or other sterile body site, it is important to attempt to determine the identification to the species level. Differentiating a true CoNS bacteremic infection from a contaminate can be challenging. However, when a CoNS is isolated from two different blood draw sites or grows rapidly, with high intensity or from multiple samples, the probability of a true infection increases.

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Clinical Setting and Site of Infection

CoNS, particularly S. epidermidis, are among the most important pathogens involved in hospital- associated bloodstream infections. In the 2011 USA SENTRY Antimicrobial Surveillance Program, CoNS was the 5th most common organism isolated (5.7%) from bloodstream infections, and S. epidermidis represented approximately two-thirds of the speciated CoNS isolates. S. epidermidis is ubiquitous to healthy human skin and mucosal surfaces, readily colonizing newborns and remaining part of the normal microflora throughout life.2 S. epidermidis and other CoNS have long been regarded as culture contaminants but their important role as true pathogens and their increasing incidence have been recognized for many years.3,4 However, the clinical significance of a positive blood culture for CoNS may be difficult to assess. Various clinical and laboratory definitions have been proposed to differentiate true infection from contamination, and the most commonly used requires at least two positive blood cultures collected from two different venous sampling sites.5

CoNS rarely causes infections in healthy tissue,6 but has a pronounced ability to proliferate on surfaces of indwelling medical devices after surgical insertion,7 where it forms persistent multilayered agglomerations called biofilms. Because of these characteristics, CoNS are undoubtedly the most common cause of bacteremia related to indwelling devices and most of these infections are hospital-acquired. Other important infections caused by CoNS include central nervous system shunt infections, native or prosthetic valve endocarditis, urinary tract infections, bone and joint infections and endophthalmitis.8

CoNS infections are characterized by their indolence and usually require the removal of the catheter or device to achieve cure. CoNS exhibit a high rate of methicillin resistance (76.4% in the global SENTRY Program, 2002-2010)9 and resistance to multiple other antimicrobial agents further complicates treatment of systemic infections. Although CoNS are usually susceptible to glycopeptides, increased MIC values for teicoplanin (29.9% at ≥4 μg/ml)9 and/or vancomycin (43.1% at ≥2 μg/ml)9 are being more frequently reported and may be related to poor clinical outcome.10,11 A recent retrospective study on MRSA bacteremia showed that patients with poor response to glycopeptide therapy (defined as persistent or a recurrent bloodstream infection episode) had 2.8- and 4.8-fold higher rates of isolates displaying elevated teicoplanin or vancomycin MICs (≥4 µg/ml), respectively, when compared to those patients with a favorable clinical response.12 Specific therapies for invasive CoNS infections may include vancomycin or linezolid or daptomycin with gentamicin or rifampicin added to prevent the emergence of resistance or to enhance bactericidal activity. More recently, the oxyimino cephalosporin ceftaroline was approved by the US Food and Drug Administration for treatment infections caused by S. aureus, including MRSA, and may represent a valuable option for treatment of CoNS infection.13

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Antimicrobial Susceptibility Testing (Ungraded)

Participants were asked to perform antimicrobial susceptibility testing on this S. epidermidis. This strain was selected to challenge proper identification and to determine antimicrobial susceptibility across numerous classes of agents, especially the Gram positive active antimicrobials. The initial reference laboratory antimicrobial susceptibility testing was conducted by standardized reference broth microdilution methods14 and susceptibility was determined based on CLSI document M100-S22, USA-FDA product package inserts or EUCAST breakpoints.15-17 The reference laboratory testing reported a total of 30 agents (Table 1) that exhibited activity varying from high potency (only eight drugs) to frank resistance (21 agents).

Consensus categorical susceptibility accuracy data are found in Table 2 for 26 antimicrobials having a significant sample of participant responses. All listed drugs achieved a category consensus (≥80% of results or a reference test); but one agent (vancomycin), due to MIC results at or within one doubling dilution step of intermediate breakpoints, required two categories for analysis. Furthermore, three active agents (Table 1) were not reported by a significant number of participants (ceftaroline, doxycycline, telavancin). Quinupristin/dalfopristin was not tested by the reference method, but was susceptible at ≤1 µg/ml by consensus participant results.

The acceptable performance by participants using the disk diffusion (DD) results18 ranged from 50.0% (cefazolin) to 100.0% (18 drugs). Lowest acceptable response rates were for some inactive β-lactams (50.0-66.7%) and rifampin (85.7%). Similarly, the commercial MIC system testing results had levels of acceptable performance at ≥98.3% (24 drugs), except for cefoxitin (80.0%; small sample at only 5 responses) and levofloxacin (76.0%). Nearly all (97.0%) of the incorrect levofloxacin intermediate results were generated by the Vitek 2 system.

The vancomycin results are shown in Table 3 listed by method of origin. The reference MIC value of 4 µg/ml should be categorized as susceptible and were correctly reported by 50.0% (Sensititre) to 99.5% (MicroScan) of participants. However, if this organism was a S. aureus the correct category would be consistent with a VISA strain e.g. vancomycin-intermediate.

Resistance Mechanisms

Although a commensal, CoNS acquired in the community are usually more susceptible, but those recovered from hospitalized patients often display a greater resistance phenotype to methicillin (oxacillin) and other anti-staphylococcal agents, except for vancomycin, teicoplanin, tigecycline, linezolid, and daptomycin.9,19 However, isolates exhibiting decreased susceptibility to glycopeptides have been reported in the USA and Europe since the early 1980s.11 In contrast to S. aureus, resistance to glycopeptides among CoNS applies almost exclusively to teicoplanin,20 a phenotype that can be observed

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.) in this specimen (a teicoplanin MIC value of 16 g/mL and a borderline MIC result for vancomycin [4 g/mL]). In addition, susceptibility to glycopeptides within different species of CoNS have been reported.9 Moreover, isolates resistant to teicoplanin often exhibit higher MIC results for vancomycin and daptomycin (Table 4).

The mechanisms involved are not well understood, but CoNS with decreased susceptibility to glycopeptides show cell wall thickening and tend to form cellular aggregates.21 This mechanism observed in CoNS is similar to that documented for S. aureus strains with decreased susceptibility to vancomycin (VISA)22 and daptomycin.23 Several genetic pathways have been associated with these phenotypes.24 Like the development of decreased susceptibility to vancomycin in S. aureus (e.g., the VISA phenotype), the emergence of resistance to daptomycin in Enterococcus faecalis appears to be associated with modifications in the cell envelope as well, although the genes linked to these changes are distinct.25

In contrast to S. aureus, CoNS (mainly S. epidermidis) isolates exhibit high rates of recombination events and acquisition of foreign DNA (including resistance determinants), a feature that may explain the multidrug resistance (MDR) phenotype commonly observed among nosocomial strains.26 Recent data have suggested that some populations of S. epidermidis, particularly those hospital-acquired strains belonging to clonal complex (CC) 2-I, may be more prone to acquire, maintain and express resistance genes, and be able to cope with drug target site mutations associated with higher fitness cost.27 Lastly, in contrast to its community counterpart, nosocomial strains often produce biofilm, which combined with the characteristics described above, render S. epidermidis survival advantages during selective antimicrobial pressure.

References

1 Manual of Clinical Microbiology, 10th edition. Washington D.C.: ASM Press, 2011.

2 Kloos WE, Musselwhite MS. Distribution and persistence of Staphylococcus and Micrococcus species and other aerobic on human skin. Appl Microbiol Biotechnol. 1975; 30:381-385.

3 Von Eiff C, Peters G, Heilmann C. Pathogenesis of infections due to coagulase-negative staphylococci. Lancet Infect Dis. 2002; 2:677-685.

4 Widerstrom M, Wistrom J, Sjostedt A, Monsen T. Coagulase-negative staphylococci: update on the molecular epidemiology and clinical presentation, with a focus on Staphylococcus epidermidis and Staphylococcus saprophyticus. Eur J Clin Microbiol Infect Dis. 2012; 31:7-20.

5 Elzi L, Babouee B, Vogeli N, et al. How to discriminate contamination from bloodstream infection due to coagulase-negative staphylococci: A prospective study with 654 patients. Clin Microbiol Infect. 2012; 18:E355-E361.

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

6 Refsahl K, Andersen BM. Clinically significant coagulase-negative staphylococci: Identification and resistance patterns. J Hosp Infect. 1992; 22:19-31.

7 Uckay I, Pittet D, Vaudaux P, Sax H, Lew D, Waldvogel F. Foreign body infections due to Staphylococcus epidermidis. Ann Med. 2009; 41:109-119.

8 Casey AL, Lambert PA, Elliott TS. Staphylococci. Int J Antimicrob Agents. 2007; 29 Suppl 3:S23-32.

9 Sader HS, Jones RN. Antimicrobial activity of daptomycin in comparison to glycopeptides and other antimicrobials when tested against numerous species of coagulase-negative Staphylococcus. Diag Microbio Infect Dis. 2012; 73:212-214.

10 Tacconelli E, Tumbarello M, Donati KG, et al. Glycopeptide resistance among coagulase-negative staphylococci that cause bacteremia: epidemiological and clinical findings from a case-control study. Clin Infect Dis. 2001; 33:1628-1635.

11 Cremniter J, Slassi A, Quincampoix JC, et al. Decreased susceptibility to teicoplanin and vancomycin in coagulase-negative Staphylococci isolated from orthopedic-device-associated infections. J Clin Microbiol. 2010; 48:1428-1431.

12 Uckay I, Bernard L, Buzzi M, et al. High prevalence of isolates with reduced glycopeptide susceptibility in persistent or recurrent bloodstream infections due to methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2012; 56:1258-1264.

13 Ho TT, Cadena J, Childs LM, Gonzalez-Velez M, Lewis JS, 2nd. Methicillin-resistant Staphylococcus aureus bacteraemia and endocarditis treated with ceftaroline salvage therapy. J Antimicrob Chemother. 2012; 67:1267-1270.

14 CLSI. M07-A9. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: ninth edition. Wayne, PA: Clinical and Laboratory Standards Institute 2012.

15 CLSI. M100-S22. Performance standards for antimicrobial susceptibility testing: 22nd informational supplement. Wayne, PA: Clinical and Laboratory Standards Institute 2012.

16 European Committee on Antimicrobial Susceptibility Testing (2012). Breakpoint tables for interpretation of MICs and zone diameters. Version 2.0, January 2012. Available at http://www.eucast.org/clinical_breakpoints/. January 1, 2012.

17 Tygacil. Package Insert. 2011 Accessed at www.tygacil.com on June 2012.

18 CLSI. M02-A11. Performance standards for antimicrobial disk susceptibility tests; approved standard: eleventh edition. Wayne, PA: Clinical and Laboratory Standards Institute 2012.

19 Mendes RE, Sader HS, Farrell DJ, Jones RN. Update on the telavancin activity tested against European staphylococcal clinical isolates (2009-2010). Diagn Microbiol Infect Dis. 2011; 71:93-97.

20 Trueba F, Garrabe E, Hadef R, et al. High prevalence of teicoplanin resistance among Staphylococcus epidermidis strains in a 5-year retrospective study. J Clin Microbiol. 2006; 44:1922- 1923.

21 Nunes AP, Teixeira LM, Iorio NL, et al. Heterogeneous resistance to vancomycin in Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains:

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

characterisation of glycopeptide susceptibility profiles and cell wall thickening. Int J Antimicrob Agents. 2006; 27:307-315.

22 Cui L, Ma X, Sato K, et al. Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus. J Clin Microbiol. 2003; 41:5-14.

23 Cui L, Tominaga E, Neoh HM, Hiramatsu K. Correlation between reduced daptomycin susceptibility and vancomycin resistance in vancomycin-Intermediate Staphylococcus aureus. Antimicrob Agents Chemother. 2006; 50:1079-1082.

24 Shoji M, Cui L, Iizuka R, et al. walK and clpP mutations confer reduced vancomycin susceptibility in Staphylococcus aureus. Antimicrob Agents Chemother. 2011; 55:3870-3881.

25 Arias CA, Panesso D, McGrath DM, et al. Genetic basis for in vivo daptomycin resistance in enterococci. N Engl J Med. 2011; 365:892-900.

26 Miragaia M, Carrico JA, Thomas JC, Couto I, Enright MC, de Lencastre H. Comparison of molecular typing methods for characterization of Staphylococcus epidermidis: Proposal for clone definition. J Clin Microbiol. 2008; 46:118-129.

27 Mendes RE, Deshpande LM, Costello A, Farrell DJ. Analysis of the molecular epidemiology of Staphylococcus epidermidis clinical isolates from USA hospitals. Antimicrob Agents Chemother. 2012; 56:4656-4661.

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Table 1. Listing of expected susceptibility testing categorical results for a methicillin-resistant S. epidermidis (blood culture, bacteremia) strain sent as specimen ES-02 (2012).

Antimicrobials listed by susceptibility category (Reference MIC in µg/ml)a Susceptible Intermediate Resistant Ceftaroline (0.5) Teicoplanin (16) Cefoxitin (>8)b Daptomycin (0.5) Oxacillin (>2)b Doxycycline (0.5) Amoxicillin/clavulanate (>8/4) Linezolid (0.5) Ampicillin (>8) Telavancin (0.06) Cefepime (16) Tetracycline (2) Ceftazidime (>32) Tigecycline (0.12) Ceftriaxone (>8) Vancomycin (4)c Cefuroxime (>16) Ciprofloxacin (>4) Clindamycin (>2) Doripenem (8) Erythromycin (>16) Fusidic Acid (8) Gentamicin (>8) Imipenem (>8) Levofloxacin (>4) Meropenem (>8) Penicillin (>8) Piperacillin/tazobactam (16) Telithromycin (>8) TMP/SMX (>4) a. Susceptibility categories determined by CLSI M100-S22 (2012) or by USA-FDA product package insert (tigecycline, telavancin). b. Preferred testing drugs to determine MRSA isolates. Note all β-lactams with the exception of recently released ceftaroline would be considered inactive against this methicillin-resistant staphylococcus. c. MIC at breakpoint concentration; intermediate for S. aureus.

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Table 2. Participant performance for selected agents (≥50 responses by both test methods except cefoxitin with 14) listed by agar disk diffusion (DD) and quantitative MIC methods for ES-02 (2012), a methicillin-resistant S. epidermidis strain.

DD MIC Acceptable Antimicrobial agent No. % correct No. % correct categorya Amoxicillin/clavulanate Resistant 12 100.0 231 99.6 Ampicillin Resistant 9 100.0 178 98.3 Ampicillin/sulbactam Resistant 3 66.7 206 98.5 Azithromycin Resistant 4 100.0 97 100.0 Cefazolin Resistant 6 50.0 195 100.0 Cefepime Resistant 0 - 54 100.0 Cefotaxime Resistant 1 100.0 72 100.0 Cefoxitin Resistant 9 100.0b 5 80.0b Ceftriaxone Resistant 9 100.0 197 100.0 Ciprofloxacin Resistant 21 100.0 528 99.8 Clindamycin Resistant 36 100.0 630 99.8 Daptomycin Susceptible 0 - 140 100.0 Erythromycin Resistant 28 100.0 633 99.7 Gentamicin Resistant 15 100.0 578 99.7 Imipenem Resistant 1 100.0 102 99.0 Levofloxacin Resistant 18 94.4 554 76.0 Linezolid Susceptible 4 100.0 500 99.8 Ofloxacin Resistant 3 100.0 51 100.0 Oxacillin Resistant 31 100.0b 675 99.9b Penicillin Resistant 21 100.0 447 99.3 Quinupristin/dalfopristin Susceptible 0 - 153 99.3 Rifampin Resistant 7 85.7 510 99.8 Tetracycline Susceptible 23 100.0 608 98.8 Tigecycline Susceptible 2 100.0 98 99.0 Trimethoprim/sulfamethoxazole Resistant 37 97.3 482 99.2 Vancomycin Susceptible- 30 96.6 698 98.8 Intermediatec a. Correct categorical interpretation was determined by the reference MIC using the M07-A8 (2009) method and CLSI M100-S22 breakpoint criteria (2012). b. Underlined values are the agent rates from which methicillin-resistant isolates are best detected. c. Two categories were needed to achieve ≥80% overall, all-method consensus for determining acceptable performance.

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Table 3. Distribution of vancomycin category participant responses for specimen ES-02 2012 (S. epidermidis), listed by method used.

No. of responses (%) by method: Category MicroScan BD Phoenix Vitek Vitek2 Sensititre Susceptible 379 (99.5) 7 (100.0) 4 (80.0) 217 (71.6) 1 (50.0) Intermediate 2 (0.5) - - 82 (27.1) - Resistant - - 1 (20.0) 4 (1.3) 1 (50.0)

American Proficiency Institute – 2012 2nd Test Event ANTIMICROBIAL SUSCEPTIBILITY - BACTEREMIC STAPHYLOCOCCAL INFECTIONS WITH STRAINS HAVING ELEVATED GLYCOPEPTIDE (VANCOMYCIN) MIC VALUES (cont.)

Table 4. MIC distribution of vancomycin, teicoplanin and daptomycin tested against coagulase-negative staphylococci (CoNS) submitted as part of the worldwide SENTRY Antimicrobial Surveillance Program (2002-2011).

Group/resistant subset MIC (g/mL) Number (cumulative %) inhibited at each oritavancin MIC (g/mL)a

Antimicrobial 50% 90% ≤0.06 0.12 0.25 0.5 1 2 4 8 16 >16

CoNS

Daptomycin (24,255) 0.25 0.5 378(1.6) 1840(9.1) 12000(58.6) 9059(96.0) 933(99.8) 39(>99.9) 6(100)

Vancomycin (2,201) 1 2 − 21(0.1) 132(0.6) 1900(8.5) 11612(56.3) 10454(99.5) 133(>99.9) 3(100)

Teicoplaninb (24,255) ≤2 4 − − − − − 17661(72.8) 4300(90.5) 1737(97.7) 457(99.6) 100(100)

CoNS with teicoplanin MICs at ≥16 g/mL

Daptomycin (557) 0.5 1 3(0.5) 10(2.3) 212(40.4) 275(89.8) 56(99.8) 1(100)

Vancomycin (557) 2 2 − 0(0.0) 0(0.0) 0(0.0) 90(16.2) 420(91.7) 46(>99.9) 1(100)

a Modal MIC values are in bold. Percentages of susceptible isolates according to the CLSI M100-S22 (2012) document are underlined. b Lowest concentration test, 2 g/mL.

American Proficiency Institute – 2012 2nd Test Event