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Journal of Clinical Microbiology, September 2004, p. 4323-4326, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4323-4326.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antimicrobial Susceptibilities of Invasive Pediatric Abiotrophia and Granulicatella Isolates

Xiaotian Zheng,1* Alexandra F. Freeman,1 Jay Villafranca,1 Dee Shortridge,2 Jill Beyer,2 William Kabat,1 Karen Dembkowski,1 and Stanford T. Shulman1

Children's Memorial Hospital/The Feinberg School of Medicine, Northwestern University, Chicago,1 Abbott Laboratories, Abbott Park, Illinois2

Received 12 March 2004/ Returned for modification 10 April 2004/ Accepted 27 May 2004


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ABSTRACT
 
Abiotrophia and Granulicatella species have been associated with various infections. Antimicrobial susceptibility data for these nutritionally variant streptococcus-like organisms, especially for pediatric isolates, are very limited. Little is known about the genetic bases of their resistance mechanisms. We report the results of identification to bacterial species level, antimicrobial susceptibility testing, macrolide resistance testing, and detection of genes encoding that resistance for a collection of 15 pediatric clinical isolates from normally sterile sites. Our results indicate that the prevalence of beta-lactam and macrolide resistance is high and that both erm and mef are found in these isolates.


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TEXT
 
Originally known as nutritionally variant streptococci, Abiotrophia and Granulicatella species are part of the normal human oral and intestinal flora. They were originally described in 1961 by Frenkel and Hirsch (8). In 1989, based on DNA-DNA hybridization studies, these organisms were classified into two groups: Streptococcus defectivus and Streptococcus adjacens (3). Based on 16S rRNA gene sequences and phenotypic characteristics, the two species subsequently were transferred to the new genus Abiotrophia in 1995 (10). Since then, three new species, A. elegans, A. balaenopterae, and "A. para-adiacens," have been added. Most recently, these species were proposed to be reclassified into two genera, Abiotrophia and Granulicatella (6, 7).

These bacteria have been associated with various infections including bloodstream infection and infective endocarditis (5, 16, 20). Other reported infections include otitis media, brain abscess, and septic arthritis (2, 9). Because of their unique growth requirements, relatively uncommon recovery from clinical specimens, and the lack of standardized testing methodology and interpretation, limited antimicrobial susceptibility data for these organisms, especially for pediatric isolates, are available. Little is known about the genetic basis of their resistance mechanisms. Here we report results of antimicrobial susceptibility testing and the detection of genes encoding resistance to macrolides for a collection of 15 pediatric clinical isolates from normally sterile sites collected at the Children's Memorial Hospital during the last 4 years.

Laboratory testing, results, and discussion. The 15 clinical isolates from normally sterile pediatric sites (12 from blood, two from cerebrospinal fluid [CSF], and one from peritoneal fluid) had been stored at –70°C before the study. All isolates grew on chocolate agar plates but not on 5% sheep blood agar plates (BBL). In addition, they all grew well as satellite colonies around Staphylococcus aureus colonies on 5% blood agar plates. Species-level identification was performed with conventional biochemical reaction tests and the API 20 Strep identification system (bioMerieux, Inc., Hazelwood, Mo.). The key biochemical reactions for species-level identification included hydrolysis of arginine and acid production from trehalose and sucrose (6).

Antimicrobial susceptibility testing was performed by standard broth microdilution methods (Microtech Medical Systems, Inc., Aurora, Colo.) with pyridoxal-supplemented medium (0.001%). To evaluate the MIC over a broader range of concentrations, erythromycin and clindamycin were tested with user-prepared microdilution plates that included 5% lysed horse blood and pyridoxal supplement (14, 19). Four isolates did not grow by 24 h in broth and were tested by Etest strips (AB Biodisk, Solna, Sweden) for these two drugs. Interpretation of antimicrobial susceptibility testing results for most drugs was based on the NCCLS interpretive criteria for the viridans group described in the table for Streptococcus spp. other than Streptococcus pneumoniae (14). Since there are no NCCLS breakpoints for ciprofloxacin and rifampin against these organisms, a MIC of ≥4 µg/ml was used as the level for ciprofloxacin resistance (4), and interpretation standards for S. pneumoniae were used for rifampin.

To assess the mechanism of macrolide resistance of these organisms, PCR was performed using primers specific for the known streptococcal resistance determinants mef(A), erm(A), erm(B), and tet(M) as previously described (1, 17, 18). Briefly, crude DNA lysate of each strain was prepared by boiling cell suspension for 10 min; following centrifugation to pellet cell debris, 1 µl of supernatant was used in the amplification reaction. PCRs were performed in a 25-µl volume with PCR Supermix as recommended by the manufacturer (Gibco BRL, Rockville, Md.) on a Gene Amp System 9700 (Applied Biosystems Inc., Foster City, Calif.). Primer pairs for resistance determinants examined along with the region of the gene amplified, annealing temperature, and primer reference are shown in Table 1. The thermal cycling profile was 30 cycles of amplification: denaturation at 94°C for 30 s, annealing at 45 to 52°C (temperatures for each primer pair are shown in Table 1) for 30 s, and extension at 72°C for 45 s. PCR products were detected and identified by electrophoresis through 2% agarose gels run at 100 V, followed by ethidium bromide staining and comparison to molecular weight standards (100-bp ladder; Gibco BRL). The primers were chosen with Oligo 5.0 (NBI Software, Plymouth, Minn.) from sequences deposited in GenBank (Bethesda, Md.). A positive control with chromosomal DNA previously shown to have the gene being tested and a negative amplification control were included in each PCR run.


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  		TABLE 1. PCR primer information

DNA sequencing for the determination of tet(M) was conducted as previously described (1). Briefly, the amplification product was sequenced using the Big Dye sequencing kit (Applied Biosystems Inc.). Sequencing reaction mixtures were purified with an Auto-Seq G-50 column (Amersham Pharmacia Biotech, Piscataway, N.J.), and reactions were run on an ABI 377 automated sequencer. The DNA sequence of amplified products from the strain in question was compared to the public sequence of the target gene (accession no. X04388) and found to be identical to the wild-type sequence. The region amplified did not include upstream regulatory regions; therefore, the reason for the lack of expression was not determined.

The API 20 Strep identification system gave the following identification codes: 0140000 (three isolates), 0540000 (three isolates), 0350451 (two isolates), 0140100 (two isolates), 0350011 (two isolates), 0350411, and 0140020. One isolate became nonviable, and the API 20 Strep test was not done. The system called the isolates tested either Abiotrophia adiacens or Abiotrophia defectiva, and most identifications were low discriminatory. The species-level identification in this study was based on conventional tests (Table 2). The 15 isolates included six of A. defectiva, six of G. adiacens, and three of G. elegans. Of these, two were susceptible (MIC, ≤0.12 µg/ml), 10 were in the intermediate category (MIC, 0.25 to 2 µg/ml), and three were resistant to penicillin, with MICs being ≥4 µg/ml (Table 3). For 13 of the 15 isolates the cefuroxime MIC was >2 µg/ml, 9 of 15 isolates were resistant to ceftriaxone (MIC of >2 µg/ml), 2 of 15 were resistant to tetracycline (MIC of >8 µg/ml), two were intermediate and 13 were susceptible to chloramphenicol, for one the trimethoprim-sulfamethoxazole MICs were 2 and 38 µg/ml, respectively, and for 14 isolates the MICs were <0.5 and 9.5 µg/ml, respectively. Given the clinical utility of rifampin, susceptibility testing was performed by Etest. For all the isolates tested, the MICs of rifampin were low (≤0.012 µg/ml). In addition, all isolates were susceptible to vancomycin (MIC, <1 µg/ml).


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  		TABLE 2. Clinical diagnosis and laboratory identification of the 15 casesa


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  		TABLE 3. Resistance of the clinical isolates to antimicrobial agents

Eight of 15 isolates (53%) were found to be macrolide resistant, with erythromycin MICs ranging from 2 to >64 µg/ml (Table 4). Three erythromycin-resistant isolates were also clindamycin resistant.


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  		TABLE 4. Macrolide and clindamycin susceptibilities and their genotypic determinants

As shown in Table 5, although there are a limited number of isolates in the present study, the resistance rates in the present study appear higher than those in the recently published data by Tuohy et al. (19) and Murray et al. (13). This is seen in various drug classes including beta-lactams, macrolides, clindamycin, and tetracycline. Possible reasons for the higher resistance rates are that the specimens in the present study were from pediatric patients and that children may possess flora that have been exposed to more antibiotics. A more recent article from Taiwan reported a very high prevalence of resistance to macrolides and clindamycin (12). For these organisms to date, there is only one fluoroquinolone-resistant isolate reported, from the blood of an adult febrile neutropenic patient who had received levofloxacin prophylaxis (13). All other isolates reported in three studies (12, 13, 19) were susceptible to levofloxacin. Among the 15 isolates in our study, the MICs of ciprofloxacin were low for 14, ranging from 0.25 to 1 µg/ml. One isolate for which the ciprofloxacin MIC was greater than 2 µg/ml was tested for levofloxacin by Etest, and the levofloxacin MIC was >32 µg/ml.


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  		TABLE 5. Comparison of resistance to some antimicrobial agents in different studies

Macrolide resistance in streptococci can be caused by several different mechanisms. Ribosomal methylation (encoded by erm genes) and macrolide efflux [encoded by mef(A)] are among the most common mechanisms (11). Genotypic analysis by PCR for macrolide resistance genes was performed on all the isolates in this study (Table 2). Erythromycin-susceptible isolates were negative for these determinants tested. Among the eight resistant isolates, five were positive for mef(A) only and as expected were clindamycin susceptible. These included three G. adiacens and two A. defectiva isolates. Three other erythromycin-resistant isolates (two identified as G. adiacens and one as G. elegans) were positive for erm(B) and were also resistant to clindamycin, consistent with a constitutively expressed erm(B) gene. Interestingly, the erm(B)-positive G. elegans isolate was also positive for mef(A). The erythromycin MICs for these three erm(B)-positive isolates were 8 and >64 µg/ml.

The macrolide resistance observed in this small number of nutritionally variant isolates reflects the mechanisms and prevalence observed in S. pneumoniae and Streptococcus pyogenes in the United States. Efflux is the most common resistance mechanism among S. pneumoniae and S. pyogenes in the United States (60 to 70%), while erm(B) is less common (25%) (18, 18a). All three erm(B)-positive isolates were also tet(M) positive; two of the three were tetracycline resistant. For the third isolate (G. elegans) the tetracycline MIC was <1 µg/ml. The presence of tet(M) on this isolate was confirmed by DNA sequencing. In S. pneumoniae erm(B) and tet(M) are frequently found on the same transposon. Poyart et al. recently described similar findings in a clinical isolate of A. defectiva from a child with endocarditis (15). A Tn916-related element similar to the pneumococcal element Tn3872 was found to be responsible for its erythromycin and tetracycline resistance. In the present study, we speculate that a similar mechanism here is highly possible although the presence and type of a transposon (if any) were not characterized. In addition, unlike the A. defectiva isolate in the previous report, the isolates in this study were identified as two of G. adiacens (both were negative for trehalose fermentation) and one of G. elegans, expanding our understanding of the resistance mechanisms in this group of bacteria.

Even though this represents a limited number of pediatric clinical isolates, our data suggest that the prevalence of beta-lactam and macrolide resistance is high among recent invasive isolates of nutritionally variant streptococci. In addition, we demonstrated for the first time that the macrolide efflux resistance mechanism encoded by mef(A) that is common in other streptococcal species is also found in these species and that the prevalence of erm(B) and/or mef(A) in nutritionally variant streptococci parallels the reported distribution of these determinants in S. pneumoniae and S. pyogenes.


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FOOTNOTES
 
* Corresponding author. Mailing address: Children's Memorial Hospital, 2300 Children's Plaza, Box 53, Chicago, IL 60614. Phone: (773) 880-6910. Fax: (773) 880-4687. E-mail: x-zheng{at}northwestern.edu. Back


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REFERENCES
 
    1
  1. Almer, L. S., V. Shortridge, A. Nilius, J. Beyer, N. Soni, M. Bui, G. Stone, and R. Flamm. 2002. Antimicrobial susceptibility and molecular characterization of community-acquired methicillin-resistant Staphylococcus aureus. Diagn. Microbiol. Infect. Dis. 43:225-232.[CrossRef][Medline]
    2. 2
  2. Biermann, C., G. Fries, P. Jehnichen, S. Bhakdi, and M. Husmann. 1999. Isolation of Abiotrophia adiacens from a brain abscess which developed in a patient after neurosurgery. J. Clin. Microbiol. 37:769-771.[Abstract/Free Full Text]
    3. 3
  3. Bouvet, A., F. Grimont, and P. A. D. Grimont. 1989. Streptococcus defectivus sp. nov. and Streptococcus adjacens sp. nov., nutritionally variant streptococci from human clinical specimens. Int. J. Syst. Bacteriol. 39:290-294.[Abstract/Free Full Text]
    4. 4
  4. Brueggemann, A. B., S. L. Coffman, P. Rhomberg, H. Huynh, L. Almer, A. Nilius, R. Flamm, and G. V. Doern. 2002. Fluoroquinolone resistance in Streptococcus pneumoniae in United States since 1994-1995. Antimicrob. Agents Chemother. 46:680-688.[Abstract/Free Full Text]
    5. 5
  5. Chang, H.-H., C.-Y. Lu, P.-R. Hsueh, M.-H. Wu, J.-K. Wang, and L.-M. Huang. 2002. Endocarditis caused by Abiotrophia defectiva in children. Pediatr. Infect. Dis. J. 21:697-700.[CrossRef][Medline]
    6. 6
  6. Christensen, J. J., and R. Facklam. 2001. Granulicatella and Abiotrophia species from human clinical specimens. J. Clin. Microbiol. 39:3520-3523.[Abstract/Free Full Text]
    7. 7
  7. Collins, M. D., and P. A. Lawson. 2000. The genus Abiotrophia (Kawamura et al.) is not monophyletic: proposal of Granulicatella gen. nov., Granulicatella adiacens comb. nov., Granulicatella elegans comb. nov. and Granulicatella balaenopterae comb. nov. Int. J. Syst. Evol. Microbiol. 50:365-369.[Abstract]
    8. 8
  8. Frenkel, A., and W. Hirsch. 1961. Spontaneous development of L form of streptococci requiring secretions of other bacteria or sulphydryl compounds for normal growth. Nature 191:728-730.
    9. 9
  9. Ince, A., B. Tiemer, J. Gille, C. Boos, and M. Russlies. 2002. Total knee arthroplasty infection due to Abiotrophia defectiva. J. Med. Microbiol. 51:899-902.[Abstract/Free Full Text]
    10. 10
  10. Kawamura, Y., X. Hou, F. Sultana, S. Liu, H. Yamamoto, and T. Ezaki. 1995. Transfer of Streptococcus adjacens and Streptococcus defectivus to Abiotrophia gen. nov. as Abiotrophia adiacens comb. nov. and Abiotrophia defectiva comb. nov., respectively. Int. J. Syst. Bacteriol. 45:798-803.[Abstract/Free Full Text]
    11. 11
  11. Leclercq, R. 2002. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin. Infect. Dis. 34:482-492.[CrossRef][Medline]
    12. 12
  12. Liao, C. H., L. J. Teng, P. R. Hsueh, Y. C. Chen, L. M. Huang, S. C. Chang, and S. W. Ho. 2004. Nutritionally variant streptococcal infections at a university hospital in Taiwan: disease emergence and high prevalence of ß-lactam and macrolide resistance. Clin. Infect. Dis. 38:452-455.[CrossRef][Medline]
    13. 13
  13. Murray, C. K., E. A. Walter, S. Crawford, M. L. McElmeel, and J. H. Jorgensen. 2001. Abiotrophia bacteremia in a patient with neutropenic fever and antimicrobial susceptibility testing of Abiotrophia isolates. Clin. Infect. Dis. 32:140-142.[CrossRef][Medline]
    14. 14
  14. NCCLS. 2004. Performance standards for antimicrobial susceptibility testing: 14th informational supplement. NCCLS document M100-S14. NCCLS, Wayne, Pa.
    15. 15
  15. Poyart, C., G. Quesne, P. Acar, P. Berche, and P. Trieu-Cuot. 2000. Characterization of the Tn916-like transposon Tn3872 in a stain of Abiotrophia defectiva (Streptococcus defectivus) causing sequential episodes of endocarditis in a child. Antimicrob. Agents Chemother. 44:790-793.[Abstract/Free Full Text]
    16. 16
  16. Ruoff, K. L. 1991. Nutritionally variant streptococci. Clin. Microbiol. Rev. 4:184-190.[Abstract/Free Full Text]
    17. 17
  17. Shortridge, V. D., G. V. Doern, A. B. Brueggemann, J. M. Beyer, and R. K. Flamm. 1999. Prevalence of macrolide resistance mechanisms in Streptococcus pneumoniae isolates from a multicenter antibiotic resistance surveillance study conducted in the United States in 1994-1995. Clin. Infect. Dis. 29:1186-1188.[CrossRef][Medline]
    18. 18
  18. Shortridge, V. D., R. K. Flamm, N. Ramer, J. Beyer, and S. K. Tanaka. 1996. Novel mechanism of macrolide resistance in Streptococcus pneumoniae. Diagn. Microbiol. Infect. Dis. 26:73-78.[CrossRef][Medline]
    19. 18
  19. Tanz, R. R., S. T. Shulman, V. D. Shortridge, W. Kabat, K. Kabat, E. Cederlund, J. Rippe, J. Beyer, S. Doktor, B. W. Beall, and the North American Streptococcal Pharyngitis Surveillance Group. Clin. Infect. Dis., in press.
    20. 19
  20. Tuohy, M. J., G. W. Procop, and J. A. Washington. 2000. Antimicrobial susceptibility of Abiotrophia adiacens and Abiotrophia defectiva. Diagn. Microbiol. Infect. Dis. 38:189-191.[CrossRef][Medline]
    21. 20
  21. Woo, P. C., A. M. Fung, S. K. Lau, B. Y. Chan, S. Chiu, J. L. Teng, T. Que, R. W. Yung, and K. Yuen. 2003. Granulicatella adiacens and Abiotrophia defectiva bacteraemia characterized by 16S rRNA gene sequencing. J. Med. Microbiol. 52:137-140.[Abstract/Free Full Text]

Journal of Clinical Microbiology, September 2004, p. 4323-4326, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4323-4326.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.




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