HAEMOLYTIC STREPTOCOCCI This specimen was designated as a sample from a skin wound that was to be cultured, identified to species level and susceptibility tested [1-3]. The culture contained a Streptococcus agalactiae (serogroup B) often associated with cutaneous infections and serious invasive perinatal/neonatal infections [4]. This strain has several antimicrobial resistances (discussed below) and was distributed by API to survey participants as an Educational Sample (ES) challenge and susceptibility test sample. Participant grading was not performed. Responses of S. agalactiae and Streptococcus Group B (SGB) combined for 92.7% of acceptable responses. An additional ten answers were considered acceptable for a total of 98.9% of participant responses (799 of 808). This organism was a representative of a typical serogroup B β-haemolytic streptococcus encountered in current clinical practice. The original clinical source of this strain was a respiratory tract infection cultured during an international surveillance study in the Asia-Pacific (Japan). Organism Identification and Clinical Settings Colonies of streptococci usually appear light gray or almost white with moist or glistering surfaces. Colony size varies between the different β-haemolytic species and helps to distinguish groups. β- haemolytic streptococci of the pyogenic groups (S. agalactiae, S. pyogenes, and S. dysagalactiae subspecies equisimilis) form colonies of >0.5 mm after 24 hours of incubation. Among those, S. agalactiae produces the largest colonies with a relatively small zone of haemolysis. Non-haemolytic S. agalactiae strains can occur and look like enterococci [5]. Definitive identification of SGB is based on detection of the group B-specific cell wall antigen. For presumptive identification, the combination of a positive CAMP test with bacitracin susceptibility and a bile esculin negative reaction is adequate to differentiate group B from the other serogroups of β-haemolytic streptococci [5]. S. agalactiae was first described as a human pathogen in 1935 from three cases of fatal puerperal sepsis [6]. By the 1970 s, SGB had become the predominant pathogen causing septicemia and meningitis in neonates and infants younger than three months of age. Neonatal infections present as two different clinical entities: early-onset neonatal disease characterized by sepsis and pneumonia within the first seven days of life; and late-onset disease with meningitis and sepsis between seven days and three months of age. The most important risk factor for the development of invasive neonatal disease is the colonization of the maternal urogenital or gastrointestinal tract by S. agalactiae, which occurs in 10 to 30% of pregnant women [5]. The spectrum of SGB disease in adults includes pneumonia, bacteremia, endocarditis, urinary tract infections and osteomyelitis. In recent clinical trials for treatment of complicated
acute bacterial skin and skin structure infections, S. agalactiae was isolated in 3.5% of the microbiologically evaluable cases, and it was the fifth most common pathogen following S. aureus (68.1% of cases), S. pyogenes (10.5%), E. faecalis (4.5%) and E. coli (3.9%) [6]. Determination of Antimicrobial Susceptibility and Participant Performance The most commonly isolated β-haemolytic streptococcus species enjoy the reputation as being uniformly susceptible to penicillin, the recognized drug of choice for most clinical infections [5]. This may not be completely accurate as some investigators, as well as the CLSI working groups have examined evidence of some species having MIC results near established breakpoints (0.12 or 0.25 µg/ml ) [1]. The most likely species having higher MIC values to penicillins would be S. agalactiae and serogroup F isolates. When such organisms are encountered, they should be confirmed by repeated susceptibility testing and then forwarded to reference laboratories for further study [1]. This organism, originally cultured from a respiratory site in Japan, does have an elevated, non-wildtype MIC (0.12-0.25 µg/ml) to penicillin and some other β-lactams, see Table 1. Furthermore, resistances to macrolides (erythromycin, azithromycin, clarithromycin) and clindamycin were detected e.g. a constitutive ermb MLS B resistance. Other resistances were noted to the fluoroquinolones (FQ; ciprofloxacin and levofloxacin MIC, >4 µg/ml) and to tetracyclines (MIC, >8 µg/ml). Various parenterally delivered agents remain active including cefepime and ceftriaxone, but these cephalosporins had MIC values at their breakpoint concentration (0.5 µg/ml, by reference CLSI tests). Daptomycin, doripenem, linezolid, quinupristin/dalfopristin, glycopeptides (vancomycin, teicoplanin, telavancin) and telithromycin were also very active against this S. agalactiae strain. Regardless of penicillin susceptibility, other agents must be applied clinically due to patient penicillin allergy. Those antimicrobials most used have been the macrolides, clindamycin, FQs, and tetracyclines (doxycycline, minocycline, tetracycline HCL), all of which were not active against the challenge strain. Laboratories must be aware of these evolving resistances among β-haemolytic streptococci and be prepared to assist physicians to select appropriate treatment alternatives. The testing of this strain having marginal susceptibility to β-lactams was generally considered acceptable (Table 2) with the key resistances (R) detected by both tabulated methods [2, 3] at the following rates: FQs (98.0-100.0% R), tetracycline HCL (98.5-100.0% R), macrolides (azithromycin and erythromycin; 80.6-100.0% R) and clindamycin (70.7-100.0% R). A potential method-based problem was the low accuracy by some MIC systems in the defection of erythromycin resistance (19.4% false-susceptible or intermediate) and clindamycin resistance (29.3% false-susceptible or intermediate).
Lastly, the higher MIC values of key therapeutic β-lactams (penicillins, ampicillin, cefepime, ceftriaxone, amoxicillin/clavulanate etc.) should be a concern to all clinical microbiology laboratories. Such strains should be referred to a reference laboratory or public health facility for confirmation, regardless of which β-haemolytic streptococcus was identified. Furthermore, a broad antibiogram must be generated for the treating doctor due to well documented resistances to other antimicrobial classes, example being this SGB strain. β-lactam Resistance mechanisms in β-haemolytic Streptococci SGB isolates are usually very susceptible to penicillin (MIC, 0.12 g/ml). However, SGB strains with reduced susceptibility to penicillin (MIC, 0.25 1 g/ml) have been reported sporadically [8, 9]. Similar to Streptococcus pneumoniae, decreased susceptibility to β-lactams in SGB is due to stepwise accumulation of amino acid alterations within the high-molecular-weight penicillin-binding proteins (PBPs), mainly in PBP1A, PBP2B and PBP2X [10, 11]. These alterations often occur within or adjacent to conserved motifs in the penicillin-binding (transpeptidase) domain, and have been associated with decreased binding affinity to β-lactam agents [11]. Recent publications studying these SGB isolates describe the resistance mechanisms in strains from the United States (CDC Active Bacterial Core Surveillance), Canada and Japan [8-11].
Table 1. Listing of expected susceptibility testing categorical results for a Streptococcus agalactiae (serogroup B; skin wound infection) strain sent as specimen ES-01 (2011). Antimicrobials listed by susceptibility category (Reference MIC in µg/ml) a Susceptible Non-susceptible Resistant Cefepime (0.5) b Ampicillin (0.5) b Ciprofloxacin (>4) Ceftriaxone (0.5) b Penicillin (0.25) b Clindamycin (>2) Daptomycin (0.25) Erythromycin (>4) Doripenem ( 0.06) b Levofloxacin (>4) Linezolid (1) Tetracycline (>8) Quinupristin/dalfopristin ( 0.5) Teicoplanin ( 1) Telithromycin ( 0.06) Vancomycin (0.5) a. Susceptibility categories determined by CLSI M100-S21 (2011) [3] or by USA-FDA product package insert (ceftaroline, tigecycline and telavancin, if utilized). b. Preferred therapeutic β-lactam drugs for β-haemolytic streptococcal infections. Note all β-lactams including the recently released ceftaroline would be considered active against β-haemolytic streptococci and non-susceptible results should be confirmed by a reference laboratory.
Table 2. Participant performance for selected agents ( 50 responses by both test methods listed by agar disk diffusion (DD) and quantitative MIC methods for ES-01 (2011), a S. agalactiae with an elevated penicillin MIC [1-3]. DD MIC Antimicrobial agent Acceptable category a No. % correct No. % correct Ampicillin b Susceptible-Intermediate 13 100.0 366 99.5 Azithromycin Resistant 2 100.0 61 95.1 Cefepime Susceptible 0-58 100.0 Cefotaxime Susceptible 1 100.0 79 100.0 Ceftriaxone Susceptible 15 100.0 40 99.1 Chloramphenicol Susceptible 2 100.0 55 100.0 Clindamycin Resistant 26 100.0 338 70.7 Daptomycin Susceptible 0-58 100.0 Erythromycin Resistant 29 96.6 206 80.6 Levofloxacin Resistant 16 100.0 449 98.0 Linezolid Susceptible 2 100.0 293 100.0 Oxacillin c - - - - - Penicillin b Susceptible-Intermediate 24 87.5 402 98.8 Quinupristin/dalfopristin Susceptible 0-74 100.0 Tetracycline Resistant 13 100.0 206 98.5 Tigecycline Susceptible 1 100.0 46 100.0 Vancomycin Susceptible 23 100.0 382 99.5 a. Correct categorical interpretation was determined by the reference MIC using the M07-A8 (2009) [1] method and CLSI M100-S21 breakpoint criteria (2011) [3]. b. Two categories (susceptible [S] and intermediate or non-s) were needed to achieve 80% overall, allmethod consensus for determining acceptable performance. c. Surrogate marker class representative (penicillins) often used for other streptococcal species (S. pneumoniae) to detect strains with elevated MIC values. However, no criteria have been established for β-haemolytic streptococci.
References: 1. Clinical and Laboratory Standards Institute. M07-A8. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard: eighth edition. Wayne, PA. CLSI, 2009 2. Clinical and Laboratory Standards Institute. M02-A10. Performance standards for antimicrobial disk susceptibility tests; approved standard: tenth edition. Wayne, PA. CLSI, 2009 3. Clinical and Laboratory Standards Institute. M100-S21. Performance standards for antimicrobial susceptibility testing: 21st informational supplement. Wayne, PA. CLSI, 2011 4. Moet GJ, Jones RN, Biedenbach DJ, Stilwell MG, Fritsche TR. Contemporary causes of skin and soft tissue infections in North America, Latin America, and Europe: Report from the SENTRY Antimicrobial Surveillance Program (1998-2004). Diagn Microbiol Infect Dis. 2007; 57: 7-13. 5. Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA. Manual of Clinical Microbiology, 9 th edition. Washington D.C.: ASM Press, 2007. 6. Lancefield RC, Hare R. The serological differentiation of pathogenic and non-pathogenic strains of hemolytic streptococci from parturient women. J Exp Med. 1935; 61: 335-349. 7. Corey GR, Wilcox M, Talbot GH, Friedland HD, Baculik T, Witherell GW, et al. Integrated analysis of CANVAS 1 and 2: Phase 3, multicenter, randomized, double-blind studies to evaluate the safety and efficacy of ceftaroline versus vancomycin plus aztreonam in complicated skin and skin-structure infection. Clin Infect Dis. 2010; 51: 641-650. 8. Dahesh S, Hensler ME, Van Sorge NM, Gertz RE, Jr., Schrag S, Nizet V, et al. Point mutation in the group B streptococcal pbp2x gene conferring decreased susceptibility to beta-lactam antibiotics. Antimicrob Agents Chemother. 2008; 52: 2915-2918. 9. Kimura K, Suzuki S, Wachino J, Kurokawa H, Yamane K, Shibata N, et al. First molecular characterization of group B streptococci with reduced penicillin susceptibility. Antimicrob Agents Chemother. 2008; 52: 2890-2897. 10. Longtin J, Vermeiren C, Shahinas D, Tamber GS, McGeer A, Low DE, et al. Novel mutations in a patient isolate of Streptococcus agalactiae with reduced penicillin susceptibility emerging after long term oral suppressive therapy. Antimicrob Agents Chemother. 2011: In press. 11. Nagano N, Nagano Y, Kimura K, Tamai K, Yanagisawa H, Arakawa Y. Genetic heterogeneity in pbp genes among clinically isolated group B Streptococci with reduced penicillin susceptibility. Antimicrob Agents Chemother. 2008; 52: 4258-4267.