CMPT Clinical Bacteriology Program Innovation, Education, Quality Assessment, Continual Improvement www.cmpt.ca Challenge M142-4 August 2014 Blood culture: Granulicatella adiacens (Nutritionally variant streptococci) HISTORY MAIN EDUCATIONAL POINTS from M142-4 A simulated blood culture obtained from a 52 1. Granulicatella adiacens and other nutritionally variant streptococ- year old male in-patient with a heart murmur ci (e.g. Abiotrophia defectiva, other Granulicatella species) are was sent to category A laboratories. infrequent but important isolates from blood cultures. Participants were expected to isolate and iden- 2. Media or techniques (e.g. chocolate agar or a streak of Staphylo- tify Granulicatella adiacens. coccus aureus) that allow for these organisms to be isolated from blood cultures should be routinely used. CMPT QA/QC 3. The Gram stain may appear quite pleomorphic, but should not be CMPT control yielded a pure culture of Granu- mistaken for bacilli or for gram negative organisms. licatella adiacens, viable for 8 days. 4. Correct identification to a genus or species level may be achieved using identification systems in the laboratory. However, even in SURVEY RESULTS their absence, it is important for a laboratory to have the ability to cultivate and recognize a nutritionally variant streptococci. Reference Laboratories: 14/15 (93%) laborato- ries reported G. adiacens, Granulicatella spe- cies, or nutritionally variant streptococci (NVS). Taxonomically, Abiotrophia species is not cor- Grading One laboratory reported results using the incor- rect (discussed below), but was considered rect identifier. Consensus was reached, there- sufficiently close to merit partial grading. Maximum grade: 4 fore the challenge was considered suitable for ISOLATION and IDENTIFICATION Reporting Granulicatella, grading. Granulicatella/Abiotrophia, or Participants: 49/63 (78%) laboratories pro- Granulicatella species are catalase-negative nutritionally variant strepto- cocci was graded 4. cessing the sample correctly isolated and iden- gram positive cocci which grow as facultative tified the organism as G. adiacens, Granulica- anaerobes. They were first described as nutri- Reporting Abiotrophia species tella sp., or NVS; these laboratories received a tionally variant streptococci (NVS) because of or α-hemolytic streptococci grade of 4 (Table 1). their pyridoxal growth requirement.1,2 In 1995, refer, was downgraded to 3. all organisms classified as NVS were placed in One participant reported Abiotrophia/ Reporting gram positive cocci the genus Abiotrophia.3 Subsequently, 16S Granulicatella species and was graded as 4. was graded 1. rRNA gene sequencing has revealed further Three laboratories reported Abiotrophia sp. and phylogenetic divisions within the genus. They Reporting Streptococcus al- were graded as 3. have been split into two genera: Abiotrophia lactolyticus, gram variable bacilli, and Neisseria species, Participants also reported the identification as and Granulicatella, to which currently belong was graded 0. Streptococcus alactolyticus, β hemolytic strep- one (A. defectiva) and three (G. adiacens, G. tococci, gram positive cocci, gram variable ba- elegans, and G. balaenopterae) species, re- Failing to grow the bacteria cilli, and Neisseria species, among others. spectively.4 within the expected time Please refer to Table 1 for grading. frame was graded 0. Their Gram stain appearance varies depending Reporting results using the COMMENTS ON RESULTS on the culture media. In liquid media containing pyridoxal (e.g. thioglycolate broth), they appear wrong identifier was graded 0. The majority of participants were able to identi- as gram positive cocci in chains, indistinguisha- fy G. adiacens, or some taxonomic variant ble from other streptococci. More commonly, thereof, as a pathogen in this sample. when the Gram stain is performed on colonies from agar plates, especially those poor in pyri- Incorrect submissions may have been due to doxal, they may appear pleomorphic and even CMPT requires the full identifier errors in automated identification systems (e.g. in the results report form. Please gram variable.5 Extremely elongated forms may “S. alactolyticus”), in recognizing the distinctive be mistaken for bacilli, and swollen cells may note that the M is part of the Gram stain (e.g. “gram variable bacilli”), or in resemble spores, leading to confusion in inter- identifier and the sample should using appropriate media to achieve growth of be identified as M142-4 and not pretation of the stain.4,5 the organism (e.g. “anaerobic gram positive as 142-4 cocci”). Page 1 Copyright CMPT 2014 CMPT Clinical Bacteriology Program August 2014 M142-4 Table 1. Identification results Reported results Total Grade Granulicatella adiacens or Granulicatella species, ± refer 40 4 nutritionally variant/deficient streptococci, ± refer 9 4 Abiotrophia/Granulicatella species, refer 1 4 Gram-positive cocci in chains, sent to reference lab to rule out Granulicatella and Abiotrophia spp. (NVS) 1 4 Abiotrophia species, refer ± probable 3 3 α-hemolytic streptococcus, refer 1 3 Streptococcus alactolyticus 1 0 anaerobic Gram-positive cocci sent to reference lab for identification 1 1 Gram-variable bacilli ± with spores, refer 2 0 Neisseria species, refer 1 0 Reported results with incorrect identifier 1 0 no growth in 5 days (received August 13 - CMPT QA/QC 1+-4+ growth) 1 0 no growth after 5 days incubation (sample lost received 2nd sample on 08/15 - CMPT QA/QC: no growth) 1 ungraded sample not normally processed 5 ungraded refer 2 ungraded Total 70 A defining characteristic of NVS is their inability ANTIMICROBIAL SUSCEPTIBILITY CMPT reminds participants to grow on pyridoxal-deficient media such as that they should send a report sheep blood agar, though this may vary de- Susceptibility testing methods and interpreta- for every sample received with pending on the specific composition.2,6 These tion guidelines for Abiotrophia and Granulica- the survey even if they do not bacteria grow on Brucella blood agar and choc- tella species have been published by the CLSI normally process it. olate agar (both contain pyridoxal), as well as in in the document M45-A2.10 The prescribed test If the laboratory does not nor- the broths used in current blood culture sys- is a broth microdilution assay, using pyridoxal mally process the sample, a tems.7 supplementation of cation-adjusted Mueller report should still be complet- Colonies of gram positive cocci that grow in Hinton broth with laked horse blood. ed indicating that is the case or that the sample would be blood culture bottles or on chocolate agar but Many laboratories may find it more practical to referred out. not on blood agar plates should trigger suspi- perform testing using E-test (or another antibi- cion for NVS. Demonstration of satellitism otic gradient strip) on a Mueller Hinton agar Failure to send a report will be around a streak of Staphylococcus aureus or a with blood, supplemented with pyridoxal.9 Re- considered “no report” and pyridoxal disk can be useful in identification. ports indicate, however, that E-tests may pro- graded 0 as CMPT does not duce MICs higher than those with broth microdi- keep records of what kind of Accurate determination of the different genera/ samples each laboratory pro- lution when testing penicillin G, potentially over- species in this group may pose a challenge for cess or not. 11 laboratories. Colonies test catalase negative. calling beta-lactam resistance. There are no These isolates can be identified on automated Kirby-Bauer disk breakpoints. identification systems, including the API Strep,8 There is limited antimicrobial susceptibility data Vitek 2, and MALDI-TOF platforms.9 However, available for NVS, as clinical isolates are rela- the accuracy of these tests is not perfect. Se- tively rare. It does appear that antimicrobial quencing of the 16S rRNA gene is the most resistance in Abiotrophia and Granulicatella is accurate way to distinguish between the spe- more common than in viridans group strepto- cies, though this may not be a practical ap- cocci.12 Penicillin resistance is relatively uncom- proach for many clinical laboratories.6,8 From a mon but still is seen in up to 20% of isolates, clinical standpoint, recognition that a bacteria but isolates appear to more likely be resistant belongs to the group of Abiotrophia/ to ceftriaxone (13 - 60%).13 Macrolide re- Granulicatella spp. is more important than sistance, mediated by mefA and ermB,13 is identification to the species level, as there may common: one study reported an azithromycin be ramifications in pathogenicity and antibiotic resistance rate of over 90%.14 Clindamycin re- susceptibilities. sistance has also been observed; however, no Page 2 Copyright CMPT 2014 CMPT Clinical Bacteriology Program August 2014 M142-4 vancomycin resistant isolates have been report- 7.Brouqui P, Raoult D. Endocarditis due to rare ed.12–14 and fastidious bacteria. Clin Microbiol Rev 2001;14(1):177–207. In summary, resistance testing of Abiotrophia 8.Woo PC-Y, Fung AM-Y, Lau SK-P, et al. and Granulicatella is logistically challenging, Granulicatella adiacens and Abiotrophia defec- but critical to guide therapy, as resistance to tiva bacteraemia characterized by 16S rRNA major groups of therapeutic agents is common. gene sequencing. J Med Microbiol 2003;52 (2):137–40. CLINICAL RELEVANCE 9.Ratcliffe P, Fang H, Thidholm E, Boräng S, Westling K, Özenci V. Comparison of MALDI- Abiotrophia and Granulicatella species are nor- TOF MS and