Antimicrobial Susceptibility – Maximizing Clinical Microbiology Support for Serious Invasive Antimicrobial Resistant Streptococcal Infections

ANTIMICROBIAL SUSCEPTIBILITY – MAXIMIZING CLINICAL MICROBIOLOGY SUPPORT FOR SERIOUS INVASIVE ANTIMICROBIAL RESISTANT STREPTOCOCCAL INFECTIONS

Sample ES-03 was a simulated blood culture (probable endocarditis). Participants were instructed to identify the organism and perform antimicrobial susceptibility testing1-3 by the laboratory’s routinely utilized method/commercial system. The culture was a Streptococcus sanguinis4 strain in pure culture with an unusual antibiogram for the species, including a reduced potency against ribosomal-targeted agents (see Table 1). This specific strain was distributed as an educational challenge (ES series) to assess the problematic accuracy of contemporary identification methods for viridans group streptococci, and the current (CLSI M100-S23) breakpoints for all listed classes of antimicrobials that could be applied for streptococcal bacteremia and possible endocarditis treatment.

Table 1. Listing of expected susceptibility testing categorical results for a Streptococcus sanguinis (viridans group streptococcus from blood culture [endocarditis]) strain sent as specimen ES-03 (2013).

Antimicrobials listed by susceptibility category (Reference MIC in µg/mL)a: Susceptible Intermediate Resistant No breakpoint criteriaa Ampicillin (0.25) None Chloramphenicol (32) Amoxicillin/Clavulanate (≤1/2) Cefepime (≤0.5) Clindamycin (2) Ceftazidime (0.5) Ceftaroline (≤0.015) Doxycycline (8) Ciprofloxacin (1) Ceftriaxone (0.12) Linezolid (32) Gentamicin (4) Daptomycin (0.5) QD (4)b Imipenem (≤0.12) Doripenem (0.12) Tetracycline (32) Moxifloxacin (0.25) Erythromycin (≤0.12) Piperacillin/Tazobactam (≤0.5/4) Levofloxacin (1) Telavancin (0.06) Meropenem (0.12) Telithromycin (≤0.06) Penicillin (≤0.06) Tigecycline (0.03) Vancomycin (0.5) TMP/SMX (4)b a. Susceptibility categories determined by CLSI M100-S23 (2013) or by USA-FDA product package insert (tigecycline, telavancin). b. TMP/SMX=trimethoprim/sulfamethoxazole, and QD = quinupristin/dalfopristin.

The identification of Streptococcus viridans group (24.7% of responses); alpha streptococcus, not S. pneumoniae (24.5%); Streptococcus, alpha-hemolytic (9.5%); S. sanguinis (9.4%, the preferred result); S. mitis group (4.5%, correct group); Streptococcus spp (3.5%); Gram positive organism (2.8%); and eight other responses (1.0%) would all be considered acceptable performance. However, the overall accuracy was only 79.3%. The most frequent error was an identification of S. anginosus group (17.3%) followed by S. pneumoniae (1.0%) and S. mutans group (0.6%). This organism was identified as S. sanguinis by reference 16S sequencing and MALDI-TOF methods with very high confidence.4

Organism Identification

Streptococcus sanguinis (formerly S. sanguis) is a Gram positive coccus, usually of less than 2µm in size and most often described in Gram’s stain examination as being in chains of varying lengths. S. sanguinis is currently classified as a member of the viridans group streptococci in the S. mitis group.5,6 Viridans group streptococci are facultative anaerobic bacteria with complex nutritional requirements which are provided by the addition of blood or serum to the medium. The optimum growth temperature is

35-37°C with some strains requiring an atmosphere of 5% CO2 for growth, but all strains demonstrating enhanced growth in the presence of 5% CO2. Glucose and other carbohydrates are metabolized by fermentation, with lactic acid produced as the major metabolic end product.5 When cultured on routine isolation media, trypticase blood agar or chocolate agar, the colony morphology of viridans group streptococci can be variable but colonies are usually small- to medium-sized with a zone of alpha hemolysis. Alternatively, it is possible to see non-hemolytic colonies or smaller colonies (≤0.5mm) with beta hemolysis.

Biochemical tests which help confirm viridans group streptococcal identifications include leucine aminopeptidase (LAP) which is produced by all streptococci and enterococci, as well as a negative catalase reaction when exposed to 3% hydrogen peroxide. Pyrrolidonyl aminopeptidase (PYR) test is negative. No growth in 6.5% NaCl broth is observed, while growth on bile esculin agar is generally negative, except for the S. bovis group and some S. mitis group species.7 Bacitracin and optochin susceptibility are negative for most viridans group streptococci except S. pneumoniae, which like S. sanguinis is classified as part of the S. mitis group.

Currently the viridans group streptococci are classified into 5 major groups: the S. mutans group, S. salivarius group, S. anginosus group, S. mitis group, and the S. bovis group. Based on 16S rRNA gene sequence analysis the S. mitis group consists of S. mitis, S. oralis, S. sanguinis, S. parasanguinis, S. gordonii, S. pneumoniae, and S. pseudopneumoniae.5,6 A subgroup of the S. mitis group includes S. sanguinis, S. parasanguinis, and S. gordonii which are arginine and esculin positive, unlike the rest of the group. S. sanguinis is a commensal of the oral cavity commonly found in dental plaque leading to cavities. This pathogen is also the most common organism associated with native-valve bacterial endocarditis.

The S. anginosus group (the most common incorrect response) can produce beta-, alpha- or nonhemolytic colonies on blood agar plates.5,8 This group can demonstrate Lancefield antigens A, C, F, and G, and often are described to possess a characteristic “butterscotch” odor from isolation plates. Biochemically S. anginosus can be reliably separated from other viridans groups via positive reactions for three tests: acetoin production from glucose (Vogues-Proskauer), arginine, and sorbitol.7,8 A follow-up

questionnaire was forwarded to those 135 laboratories erroneously reporting the S. anginosus identification. All responses (100% of 55 total) stated they utilized the MicroScan System for identification of this organism.

Gene sequence identification by 16S rRNA remains the most reliable method for confirming viridans group streptococcus species. MALDI-TOF performs slightly better than conventional methods in identifying viridans group streptococci, but struggles with confidence on species, particularly among the S. mitis group.9

Antimicrobial Susceptibility Testing (Ungraded)

API survey participants were requested to perform antimicrobial susceptibility tests1-3 on the viridans group streptococcus S. sanguinis: see Tables 1-3. This well-characterized strain4 was selected to highlight the difficulty in accurate identification and susceptibility testing of these fastidious streptococci using commercial methods and contemporary clinical breakpoint criteria.3,10 This organism when isolated from a blood culture requires accurate identification and dependable determination of susceptibility to parenteral therapeutic agents, especially in the clinical context of endocarditis. However, current interpretations of MIC results3,10 can greatly differ between the two most applied sets of international breakpoint criteria (see below):

Clinical MIC breakpoint criteria (Susceptible / Resistant): Antimicrobial agent CLSI (µg/mL) EUCAST (µg/mL) Ampicillin ≤0.25 / ≥8 ≤0.5 / ≥4 Penicillin ≤0.12 / ≥4 ≤0.25 / ≥4 Ceftriaxone ≤1 / ≥4 ≤0.5 / ≥1 Meropenem ≤0.5 / NCa ≤2 / ≥4 Clindamycin ≤0.25 / ≥1 ≤0.5 / ≥1 Erythromycin ≤0.25 / ≥1 NC Chloramphenicol ≤4 / ≥16 NC Tetracycline ≤2 / ≥8 NC Vancomycin ≤1 / NC ≤2 / ≥4 Levofloxacin ≤2 / ≥8 NC Linezolid ≤2 / NC NC

a. NC = no criteria are published

An examination of the table above reveals only two identical breakpoints (9.1%) among a total of 22 compared interpretations. Furthermore, for this challenge organism (ES-03, 2013) suboptimal detection of high-level resistance was documented when testing chloramphenicol, clindamycin, and linezolid using either set of breakpoints with the disk diffusion and some commercial MIC methods (Table 2). The most

American Proficiency Institute – 2013 3rd Test Event ANTIMICROBIAL SUSCEPTIBILITY – MAXIMIZING CLINICAL MICROBIOLOGY SUPPORT FOR SERIOUS INVASIVE ANTIMICROBIAL RESISTANT STREPTOCOCCAL INFECTIONS (cont.) problematic drug-system accuracy issues were observed for chloramphenicol and clindamycin when tested by MicroScan, and for linezolid when tested by the Vitek Systems (Table 3). However, the small sample size for some Vitek and disk diffusion responses limits these analyses. Clearly greater harmonization among international interpretive criteria is a future priority, as well as re-evaluations of testing accuracy of some commercial and standardized methods (disk diffusion) regardless of criteria applied.1-3,10

Table 2. Participant performance for selected agents (≥50 responses by both test methods, except linezolid) listed by disk agar diffusion (DD) and quantitative MIC methods for ES-03 (2013), a S. sanguinis strain with an unusual resistance profile. DD MIC Antimicrobial agent Acceptable categorya No. % correct No. % correct Ampicillin Susceptible 6 83.3 186 98.4 Azithromycin Susceptible 0 - 78 100.0 Cefepime Susceptible 1 100.0 122 100.0 Cefotaxime Susceptible 8 100.0 175 100.0 Ceftriaxone Susceptible 36 100.0 210 100.0 Chloramphenicolb Resistant 9 55.6 76 75.0 Clindamycinb Resistant 33 48.5 187 42.8 Erythromycin Susceptible 36 100.0 192 97.9 Levofloxacin Susceptible 21 95.2 142 99.3 Linezolidb Resistant 2 50.0 8 75.0 Meropenem Susceptible 1 100.0 66 100.0 Penicillin Susceptible 32 90.6 238 97.5 Tetracycline Resistant 16 100.0 121 93.4 Vancomycin Susceptible 45 100.0 236 100.0 All drugs - 246 89.0 2,037 92.4 a. Correct categorical interpretation was determined by the reference MIC using the M07-A9 method and CLSI M100-S23 breakpoint criteria. b. Specimen selected to highlight detection of unusual resistances to these three agents.

Table 3. Categorical accuracy of selected methods or systems to detect resistances in a viridans group streptococcus for chloramphenicol, clindamycin and linezolid (ES-03, 2013).

No. response (% accuracy) by method: Antimicrobial agent Disk diffusion MicroScan BD Phoenix SensiTitre Viteka Chloramphenicol 9 (55.6) 63 (85.7) 1 (100.0) NDb 2 (100.0) Clindamycin 33 (48.5) 146 (28.8) 6 (100.0) 1 (100.0) 34 (91.2) Linezolid 2 (50.0) 1 (100.0) ND ND 7 (57.1) Tetracyclinec 16 (100.0) 96 (91.7) ND 1 (0.0) 24 (100.0) a. Includes Vitek 2 (89.6% of responses) and Vitek. b. ND = no data received; underline values = suboptimal detection using a 90% accuracy threshold. c. Co-resistance in the challenge strain due to an efflux-based mechanism conferred by tet(M).

Resistance Mechanisms

Resistance to macrolide, lincosamide and streptogramin (MLS) compounds is a common resistance phenotype displayed by streptococcal isolates, which involves target site modifications encoded by the ribose methylase genes (erm). The encoded protein methylates the adenine residue A2058 in the 23S rRNA domain V. This single alteration will confer resistance to macrolide, lincosamide, and streptogramin

B agents, the so-called MLSB phenotype often observed among Gram positive clinical isolates. Erm methylases add one or two methyl groups to adenine A2058 of the 23S rRNA, and the expression of the Erm-encoding gene can be inducible or constitutive. In the absence of an inducer molecule, the organism may remain susceptible in vitro to lincosamides and streptogramin B antibiotics.11

This S. sanguinis clinical isolate displayed a multidrug resistance phenotype, which is a rarer phenotype among streptococcal clinical isolates. This resistance phenotype included high-level resistance to linezolid (32 g/mL), chloramphenicol (32 g/mL), clindamycin (>2 g/mL), and the streptogramin combination quinupristin/dalfopristin (4 g/mL). Elevated MIC results for the pleuromutilins, tiamulin (32 g/mL) and retapamulin (2 g/mL), were also reported.4 However, the S. sanguinis isolate remained susceptible to erythromycin (MIC ≤0.12 g/m). This resistance profile is consistent with that caused by the Cfr gene (i.e., decreased susceptibility to phenicol, lincosamide, oxazolidinone, pleuromutilin, and 12 streptogramin A [PhLOPSA] compounds).

The phenotype exhibited by the S. sanguinis isolate was not consistent with the presence of the erm gene, and further investigations demonstrated that the isolate also did not carry cfr. However, several mutations in the 23S ribosomal RNA were observed, including G2576T and C2610T, which are located within the peptidyl-transferase center (PTC) of the bacterial ribosome. These alterations are known to affect linezolid binding and to cause cross resistance to other ribosomal targeting agents, except for macrolides (erythromycin) which are most affected by pre and post translational modifications at residues

2058 and 2059 of 23S rRNA. This isolate also had elevated MIC results for tetracyclines due to the presence of an efflux-based mechanism conferred by tet(M).13 See Mendes et al. 20134 for additional information related to this clinical case.

Therapeutics for Invasive Viridans Group Streptococcal Disease

Viridans group streptococci (VGS) are a common cause of bacterial endocarditis and can represent 20-40% of cases.14 VGS can also be found in septicemia among immunocompromised hosts and are a leading cause of bacteremia in febrile and neutropenic patients in cancer centers.14,15 In a review of streptococcal bloodstream infections occurring during 2000-2011 at a comprehensive cancer care hospital, 55% of all streptococcal bloodstream infections were caused by VGS.15 The two most frequently isolated VGS species from blood cultures have been reported to be Streptococcus mitis and S. sanguinis.16 Less commonly, VGS may cause central nervous system infections such as meningitis and brain abscess, where they account for less than 1-5% of purulent meningitis cases.14,17,18 Invasive VGS infections other than endocarditis occur as part of a mixed aerobic-anaerobic infection rather than as a single-pathogen disease.14

Penicillin resistance (MIC ≥0.25 μg/mL, CLSI criteria) is a significant concern for patients infected with VGS isolates of nosocomial origin.14 It may range up to 50% of cases, with 10% highly penicillin-resistant (MIC ≥4 μg/mL). Community isolates, on the other hand, are frequently penicillin-susceptible. VGS exhibit variable activity to tetracycline, erythromycin, and clindamycin, with vancomycin and linezolid retaining excellent activity.14,19 Therapeutic regimens for septicemia caused by VGS include penicillin and/or vancomycin, and for meningitis, ceftriaxone and/or vancomycin.14,20 Therapy for endocarditis caused by VGS is based on penicillin-susceptibility level (MIC 0.12 μg/mL, MIC 0.25-0.5 μg/mL, or MIC >0.5 μg/mL). Treatments of varying lengths of time include an active β-lactam with or without an aminoglycoside (usually gentamicin), or alternatively vancomycin with or without an aminoglycoside, depending on the penicillin MIC and whether the infection is a native valve or prosthetic valve infection.14,20

Several other newer and investigational agents have potent activity against VGS isolates, such as oxazolidinones19 and various older or investigational lipoglycopeptides.21-23

References

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

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

3. Clinical and Laboratory Standards Institute. 2013. M100-S23. Performance standards for antimicrobial susceptibility testing: 23rd informational supplement. Clinical and Laboratory Standards Institute, Wayne, PA.

4. Mendes RE, Deshpande LM, Kim J, Myers DS, Ross JE, Jones RN. 2013. Streptococcus sanguinis displaying a cross resistance phenotype to several ribosomal RNA targeting agents. J. Clin. Microbiol. 51: 2728-2731.

5. Versalovic J, Carroll KC, Funke G, Jorgensen J, Landry ML, Warnock DW. 2011. Manual of Clinical Microbiology. 10th ed. ASM Press, Washington, DC.

6. Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA. 2006. Gram positive pathogens. 2nd ed. ASM Press, Washington, DC.

7. Garcia LS, ed. 2010. Clinical Microbiology Procedures Handbook. 3rd ed. ASM Press, Washington, D.C.

8. Doern CD, Burnham CA. 2010. It's not easy being green: the viridans group streptococci, with a focus on pediatric clinical manifestations. J. Clin. Microbiol. 48: 3829-3835.

9. Davies AP, Reid M, Hadfield SJ, et. al. 2012. Identification of clinical isolates of alpha-hemolytic streptococci by 16S rRNA gene sequencing, matrix-assisted laser desorption ionization-time of flight mass spectrometry using MALDI Biotyper, and conventional phenotypic methods: a comparison. J. Clin. Microbiol. 50: 4087-4090.

10. The European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2013. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.1, February 2013. Available at http://www.eucast.org/clinical_breakpoints/. Accessed August 2013.

11. Schwendener S, Perreten V. 2012. New MLSB resistance gene erm(43) in Staphylococcus lentus. Antimicrob. Agents Chemother. 56: 4746-4752.

12. Long KS, Poehlsgaard J, Kehrenberg C, Schwarz S, Vester B. 2006. The cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics. Antimicrob. Agents Chemother. 50: 2500-2505.

13. Connell SR, Tracz DM, Nierhaus KH, Taylor DE. 2003. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob. Agents Chemother. 47: 3675-3681.

14. Schlossberg D, ed. 2008. Clinical Infectious Disease. Cambridge University Press, New York, NY.

15. Shelburne SA 3rd, Tarrand J, Rolston KV. 2013. Review of streptococcal bloodstream infections at a comprehensive cancer care center, 2000-2011. J. Infect. 66: 136-146.

16. Bochud PY, Calandra T, Francioli P. 1994. Bacteremia due to viridans streptococci in neutropenic patients: a review. Am. J. Med. 97: 256-264.

17. Liu YT, Lin CF, Lee YL. 2013. Streptococcus sanguinis meningitis following endoscopic ligation for oesophageal variceal haemorrhage. J. Med. Microbiol. 62: 794-796.

18. Carley NH. 1992. Streptococcus salivarius bacteremia and meningitis following upper gastrointestinal endoscopy and cauterization for gastric bleeding. Clin. Infect. Dis. 14: 947-948.

19. Flamm RK, Mendes RE, Ross JE, Sader HS, Jones RN. 2013. Linezolid surveillance results for the United States: LEADER Surveillance Program 2011. Antimicrob. Agents Chemother. 57: 1077-1081.

20. Gilbert DN, Moellering Jr. RC, Eliopoulos GM, Chambers HF, Saag MS, editors. 2013. The Sanford Guide to Antimicrobial Therapy. 43rd edition. Antimicrobial Therapy, Inc., Sperryville, VA.

21. Jones RN, Stilwell MG. 2013. Comprehensive update of dalbavancin activity when tested against uncommonly isolated streptococci, Corynebacterium spp., Listeria monocytogenes and Micrococcus spp. (1,357 strains). Diagn. Microbiol. Infect. Dis. 76: 239-240.

22. Sader HS, Flamm RK, Jones RN. 2013. Daptomycin activity when tested against uncommonly isolated streptococcal and three other Gram-positive species groups [abstract #P65]. 28th International Congress of Chemotherapy and Infection. June 6-9, 2013, Yokohama, Japan.

23. Mendes RE, Sader HS, Flamm RK, Jones RN. 2013. Activity of oritavancin tested against uncommonly isolated Gram-positive pathogens responsible for documented infections in hospitals worldwide [abstract #C2-094]. 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC). September 10-13, 2013, Denver, CO, USA.

American Proficiency Institute – 2013 3rd Test Event