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Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections
Diagnostic Microbiology and Infectious Disease 77 (2013) 216–219
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections Paul Ratcliffe a, Hong Fang a, Ellinor Thidholm a, Stina Boräng a, Katarina Westling b, Volkan Özenci a,⁎ a b
Division of Clinical Microbiology F 72, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 13 June 2013 Accepted 18 July 2013 Available online 10 September 2013 Keywords: Endocarditis Microbiology Granulicatella Abiotrophia NVS Antimicrobial susceptibility
a b s t r a c t Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Granulicatella and Abiotrophia spp., formerly known as nutritionally variant streptococci (NVS), are normal residents of the oral cavity but have been identified as agents of invasive infections (Ruoff, 2007). Because of both fastidious culturing and the unspecific colony morphology that they present on primary detection, such strains have caused major diagnostic difficulties. The NVS as a group have been estimated to cause 5–6% of all cases of streptococcal endocarditis (Brouqui and Raoult, 2001; Cargill et al., 2012; Roberts et al., 1979). However, it has been supposed that many culture-negative endocarditis could have been caused by these species, which could have lead to an underestimation as pathogens of infective endocarditis (Brouqui and Raoult, 2001; Cargill et al., 2012). Severe infections as infective endocarditis, neonatal infections, osteomyelitis, and endovascular infections, which were caused by streptococci, have been reported (Cargill et al., 2012). Infective endocarditis caused by NVS has in earlier studies been related with high bacteriologic failure (41%), a high rate of relapse after therapy (17%), and mortality at 17% (Stein and Nelson, 1987). In more recent surveys, mortality was 9.0% (Cargill et al., 2012; Giuliano et al., 2012). Combination therapy in the form ⁎ Corresponding author. Tel.: +46-8-5858-1147; fax: +46-8-5858-1125. E-mail address: [email protected] (V. Özenci). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.07.008
of penicillin G or amoxicillin (in cases of susceptible strain) with aminoglycosides is recommended in most treatment guidelines (Habib et al., 2009; Westling et al., 2007). The clinical significance of the uncommon but pathogenic bacteria in invasive infections underlines the need for reliable and rapid identification methods in microbiology laboratories. Identification of Granulicatella and Abiotrophia spp. is usually conducted by either biochemical testing and/or molecular confirmation (Cargill et al., 2012; Christensen and Facklam, 2001). However, the biochemical characteristics might be insufficient for accurate identification of NVS (Ruoff, 2007). Hitherto published studies showed that the biochemical testing might be misleading in identification of Granulicatella spp. Both Granulicatella adiacens and Granulicatella elegans were previously identified as Gemella spp., and it has been reported that G. elegans was misidentified as Streptococcus acidominimus, Gemella morbillorum, and Granulicatella adiacens (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007; Cargill et al., 2012). Although far from perfect in identification of NVS species, the Vitek 2 system is still an available approach for most clinical microbiology laboratories nowadays. However, the performance of Vitek2 for identification of these species has not previously been studied. A variety of molecular techniques have been developed to help accelerate the identification of routine-difficult bacteria, including Granulicatella and Abiotrophia spp. The increasing use of molecular
P. Ratcliffe et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 216–219
methods in the diagnosis of invasive infections, including 16S and 23S rRNA gene sequencing, may mean an improved recognition of formally rare causative organisms in infection (Breitkopf et al., 2005; Vollmer et al., 2010). However, by using the present sequencing method, 2 to 3 days is usually needed to get a result, and the personnel and reagent costs are higher than the biochemical testing. The development of matrix-assisted laser desorption ionization– time of flight mass spectrometry (MALDI-TOF MS) technology in the field of clinical microbiology shows promise for the rapid diagnosis of the previously difficult bacteria. There is, however, scarce data on identification of NVS by using this advanced technology. In publications where Granulicatella and Abiotrophia spp. were included, these species were regarded as rarely isolated species, and the number of isolates was limited (McElvania Tekippe et al., 2002; Neville et al., 2011; Schulthess et al., 2013). Here, we evaluated the performance of 2 MALDI-TOF MS systems, i.e., Bruker MS (Bruker Daltonik, Germany) and Vitek MS (bioMérieux, France), and the standardized phenotypic system Vitek 2 (bioMérieux, France) for the species determination of clinical NVS isolates. 16S rRNA gene sequencing was used as a reference method. The antimicrobial susceptibilities of these invasive isolates were determined by Etest. 2. Materials and methods 2.1. Bacterial strains A total of 14 clinical isolates were included in the study, consisting of 13 hematin isolates and 1 cerebral-spinal-fluid isolate, collected at the Department of Clinical Microbiology, Karolinska University Hospital, from 2006 to 2012. The 14 strains were identified by 16S rRNA gene sequencing as 10 G. adiacens, 1 G. elegans, and 3 Abiotrophia defectiva. 2.2. Culture conditions Hematin agar swirled with 5-mL 0.001% pyridoxal hydrochloride (PHC) was used for culturing Granulicatella and Abiotrophia. These bacteria do not grow well on agar media apart from hematin and grow best with the addition of PHC, which provides the bacteria with vitamin B6 necessary for their growth. The plates were incubated in CO2-enriched atmosphere for up to 5 days.
217
The strains were tested by depositing 1 bacterial colony on the target slide and followed by the addition of matrix solution (VITEK MS-CHCA) and air drying. The loaded slide was then inserted into the Vitek MS system. Microbial identification is achieved by obtaining spectra using MALDI-TOF technology and analyzing the spectra with the Vitek MS database. The peaks from these spectra are compared with the characteristic pattern for a species, genus, or family of microorganism, thus resulting in organism identification. The organisms were reported with a percentage-scaled confidence value as well as a confidence level. 2.5. Vitek 2 system Vitek 2 (bioMérieux, France) is an automatic phenotypic test system based on test cards that house a number of wells, which contain dehydrated biochemical substrates. Gram-positive cards were used for identifying the Granulicatella and Abiotrophia isolates. 2.6. 16S rRNA gene sequencing 16S rRNA PCR and sequencing were performed after heat extraction of the bacterial DNA. After initial PCR amplification, with primers 5′-AGA GTT TGA TCM TGG CTC AG-3′ and 5′-CCG TCA ATT CMT TTR AGT TT-3′ (Brosius et al., 1978; Harmsen et al., 2002), the PCR products were purified using Amicon Ultra-0.5 (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Sequencing of both strands was carried out, with the same primers as in the PCR, using an ABI PrismBigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequence cycling products were analyzed by capillary electrophoresis and fluorescence detection with an Applied Biosystems ABI 3130xl Genetic analyzer. The fluorescence data were analysed with the SeqScape program (version 2.6; Applied Biosystems) followed by BLASTN 2.2.2. for bacterial identification (Altschul et al., 1990). 2.7. Antimicrobial susceptibility testing The minimal inhibitory concentrations (MICs) of ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, gentamycin, levofloxacin, penicillin G, rifampicin, tetracycline, trimetoprim-sulphamethoxazole, and vancomycin were determined by Etest (bioMérieux, France). Antimicrobial susceptibilities were interpreted according to the guidelines established by the Swedish Reference Group for Antibiotics.
2.3. MALDI-TOF Bruker MS
3. Results
Measurements were performed with a microflex LT mass spectrometer (Bruker Daltonik, Germany) using FlexControl software (version 3.3). The spectra were imported into the integrated MALDI Biotyper software (version 3.0) and were analysed by standard pattern matching with default settings. The strains were tested without pretreatment. A colony from the hematin agar plate was directly spotted on the MALDI-plate, and then overlaid with 1 μL of matrix solution, and air-dried. The loaded plate was then applied to the instrument according to the manufacturer's instruction. The spectrum of each isolate was compared with those in the database and identification was provided with a score of reliability.
The 14 strains were identified as 10 G. adiacens, 1 G. elegans, and 3 A. defectiva by 16S rRNA sequencing, which was used as the reference method in the study (Table 1).
2.4. MALDI-TOF Vitek MS Vitek MS (bioMérieux, Marcy l'Etoile, France) is another automated microbial identification system based on MALDI-TOF technology. Myla™, a Web-based middleware application integrated in the Vitek systems, provides a platform for both slide composition consultation and results consultation for Vitek MS.
3.1. MALDI-TOF MS The Vitek MS gave correct identification for all 14 Granulicatella and Abiotrophia isolates to the species level with a confidence value of 99.9%. The Bruker MS could correctly identify 8 out of the 10 G. adiacens and all 3 A. defectiva isolates at the first analysis occasion. To Table 1 Clinical invasive Abiotrophia and Granulicatella isolates correctly identified by Bruker MS, Vitek MS, and Vitek 2. Bacteria
No. of isolates
Bruker MS at the first attempt (by repeated tests)
VITEK MS
VITEK 2
A. defectiva G. adiacens G. elegans Total
3 10 1 14
3 8 (2) 0 (1) 11 (3)
3 10 1 14
2 6 1 9
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Table 2 Phenotypic characteristics of clinical invasive Abiotrophia and Granulicatella isolates by Vitek 2. Biochemical test
Arginindihydrolase 1 (0.111 mg) Arginindihydrolase 2 (0.270 mg) D-Amygdalin D-Maltose D-Raffinose D-Trehalos Growth in 6.5% NaCl Lactose Leucin Arylamidase L-Pyrrolidonyl Arylamidase Phosphatase Pullulan Salicin Sucrose α-Galactosidase β-Galactosidase β-Glucuronidase (0.0018 mg) β-Glucuronidase (0.0378 mg)
Results for species (no. of isolates with positive test result/total no. of isolates) A. defectiva
G. adiacens
G. elegans
0/3 0/3 0/3 3/3 0/3 2/3 0/3 0/3 1/3 3/3 0/3 0/3 0/3 3/3 2/3 0/3 0/3 0/3
0/10 0/10 0/10 4/10 0/10 0/10 0/10 1/10 9/10 5/10 0/10 0/10 0/10 9/10 3/10 0/10 6/10 5/10
1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 0/1
reach a reliable score, 2 repeated tests were required for 2 of the G. adiacens isolates and 3 more tests for the G. elegans strain (Table 1). The scores obtained in the repeated tests were between 1.730 and 1.971. 3.2. Vitek 2 The Vitek 2 system could identify 7 of the 11 Granulicatella isolates and 2 of the 3 A. defectiva isolates at the species level but failed on identifying 4 G. adiacens and 1 A. defectiva isolate. One G. adiacens strain was defined as unidentified organism, 1 as G. morbillorum, and low discrimination was reported for the remaining 2 G. adiacens isolates and the A. defectiva isolate (Table 1). The relevant phenotypic characteristics obtained from Vitek2 were summarized in Table 2. 3.3. Antimicrobial susceptibility testing The antimicrobial susceptibilities of NVS isolates were presented in Table 3. In general, the NVS isolates in the present study were susceptible to clindamycin, levofloxacin, penicillin, and vancomycin but resistant to cefuroxime and gentamicin and, however, presented variable susceptibilities to ciprofloxacin, ceftriaxone, rifampicin, and tetracycline (Table 3).
4. Discussion Accurate and rapid identification of Granulicatella and Abiotrophia spp. has been considered not easy in clinical microbiology laboratories (Cargill et al., 2012; Christensen and Facklam, 2001). In the present
study, we compared the performances of Bruker MS, Vitek MS, and Vitek 2 system in identification of invasive Granulicatella and Abiotrophia spp. The phenotypic characteristics may not always be sufficient for accurate identification of NVS (Ruoff, 2007). Biochemical testing has been reported to potentially lead to incorrect identification (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007; Cargill et al., 2012). In accordance with previous reports (Abdul-Redha et al., 2007; AlTawfiq et al., 2007; Cargill et al., 2012), 3/10 G. adiacens and 1/3 A. defectiva isolates, in the present study, were of low discrimination with G. morbillorum, G. elegans, and G. sanguinis by biochemical testing in Vitek 2 system. Our study demonstrates the limitations of the Vitek 2 system in the identification of NVS to the species level. 16S rRNA gene sequencing is proved to be effective in identification of Granulicatella and Abiotrophia in the study. However, the sequencing-based method is still not rapid enough to meet the clinical requirement. MALDI-TOF MS has been shown to be both accurate in the identification of bacteria and rapid, which is of proven benefit to patient care (Barenfanger et al., 1999; Doern et al., 1994; Eigner et al., 2009; Neville et al., 2011; Seng et al., 2009; van Veen et al., 2010). Nevertheless, data on the utility of MALDI-TOF for identification of NVS isolates are limited. In a recently published well-performed prospective study by Neville et al. (2011), where 927 isolates were investigated, only 1 NVS strain (G. adiacens) was included. The G. adiacens isolate in the Neville's study, by using MALDI-TOF Bruker MS, was identified to genus level in 1 of 3 tests and got unreliable identification at the remaining 2 tests. Similar phenomena were observed in 2 of the 10 G. adiacens isolates and 1 G. elegans isolate in our study, when the experiments were carried out with Bruker MS. In contrast, Vitek MS gave correct species identification for all 14 NVS isolates at the first analytic attempt. A possible explanation for the discrepancy is that the Vitek MS database houses at least 8 spectra for each species and that even uncommon spectra of the species are present. This approach ensures a high possibility for match for samples. Besides quality of the database, sample preparation methods could also play an important role in increasing the identification rate of gram-positive organisms (McElvania Tekippe et al., 2002; Schulthess et al., 2013). To pretreat the samples with formic acid will be investigated in our future study. The estimated time for analysis of 14 samples in duplicate is about 20–25 minutes for the Bruker system and 35–40 minutes for the Vitek MS. The Bruker system is smaller in size than Vitek MS, and reestablishment of vacuum is quicker for the Bruker MS. In contrast, it takes 3–8 hours for Vitek2 analysis, and 2 to 3 days is usually needed to get a result by the present sequencing method. The lack of standardized methods and interpretation criteria for antimicrobial susceptibility testing results, along with relatively small collections of clinical isolates for NVS, make it difficult to accurately assess antimicrobial susceptibility patterns. Our study demonstrates that Etest with pyridoxal hydrochloride-pretreated blood-supplemented Mueller-Hinton agar in a CO2-enriched atmosphere is a practically useful approach as susceptibility testing for
Table 3 Antimicrobial susceptibilities of Abiotrophia defectiva (n = 3) and Granulicatella (n = 11) isolates towards 11 antimicrobial agents. Bacteria/antibiotics
CIP
CLI
CTR
CXM
GEN
LEV
PEN
RIF
TET
TMP-SMX
VAN
MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) A. defectiva (resistant/total isolates) Granulicatella spp. (resistant/total isolates) Total (resistant/total isolates)
0.25–32 0.5 1.5 0/3 4/11 4/14
0.047–0.5 0.094 0.38 0/3 0/11 0/14
0.38–48 0.75 2 3/3 7/11 10/14
2–256 4 256 3/3 11/11 14/14
1.5–4 3 4 3/3 11/11 14/14
0.38–1.5 0.75 1 0/3 0/11 0/14
0.016–0.25 0.064 0.094 0/3 0/11 0/14
0.002–32 0.004 32 0/3 4/11 4/14
0.25–8 0.5 1 0/3 1/11 1/14
0.002–32 0.064 32 ND ND ND
0.25–2 0.5 1 0/3 0/11 0/14
CIP = ciprofloxacin; CLI = clindamycin; CTR = ceftriaxone; CXM = cefuroxime; GEN = gentamycin; LEV = levofloxacin; PEN = penicillin G; RIF = rifampicin; TET = tetracycline; TMP-SMX = trimetoprim-sulphamethoxazole; VAN = vancomycin; ND = not determined, the breakpoint is not available.
P. Ratcliffe et al. / Diagnostic Microbiology and Infectious Disease 77 (2013) 216–219
NVS. The inhibition zones of the tested isolates were clear and easy to define after 20–24 hours of incubation. It has been known that there is a range of MICs of penicillin for Granulicatella and Abiotrophia isolates, with the majority of strains classified as either susceptible or relatively resistant. There is also variability in susceptibilities to aminoglycosides, but no cases of highlevel resistance have been reported (Ruoff, 2007). Similarly, the collection of NVS isolates in our study was susceptible to penicillin but presented low-level resistance to gentamycin with an MIC range at 1.5–4 mg/L. The isolates in our collection were also susceptible to clindamycin and levofloxacin, concordant to the findings by Tuohy et al. (2000). In line with the previous reports, high rates of cephalosporin resistance were observed among Granulicatella and Abiotrophia spp. in the present study (Tuohy et al., 2000; Zheng et al., 2004). Notably, we identified 4 rifampicin-resistant Granulicatella isolates with MICs at 32 mg/L, which has not been reported previously. Besides rifampicin, the isolates were also resistant to gentamycin, ceftriaxone, cefuroxime, erythromycin, or trimetoprimsulphamethoxazole. Although all 14 isolates were susceptible to vancomycin, elevated MICs (1–2 mg/L) were observed in 3 G. adiacens isolates, 2 of which were co-resistant to rifampicin. In conclusion, MALDI-TOF MS is a useful tool for rapid identification of NVS such as Granulicatella and Abiotrophia spp. The rapid and accurate diagnosis of such infective pathogens plays an important role in guiding early appropriate antibiotic therapy. The emergence of resistant NVS isolates from invasive infections indicates the necessity of monitoring the antimicrobial susceptibilities of such uncommon pathogens. References Abdul-Redha RJ, Prag J, Sonksen UW, Kemp M, Andresen K, Christensen JJ. Granulicatella elegans bacteraemia in patients with abdominal infections. Scand J Infect Dis 2007;39:830–3. Al-Tawfiq JA, Kiwan G, Murrar H. Granulicatella elegans native valve infective endocarditis: case report and review. Diagn Microbiol Infect Dis 2007;57:439–41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10. Barenfanger J, Drake C, Kacich G. Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J Clin Microbiol 1999;37: 1415–8. Breitkopf C, Hammel D, Scheld HH, Peters G, Becker K. Impact of a molecular approach to improve the microbiological diagnosis of infective heart valve endocarditis. Circulation 2005;111:1415–21. Brosius J, Palmer ML, Kennedy PJ, Noller HF. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A 1978;75:4801–5. Brouqui P, Raoult D. Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev 2001;14:177–207. Cargill JS, Scott KS, Gascoyne-Binzi D, Sandoe JA. Granulicatella infection: diagnosis and management. J Med Microbiol 2012;61:755–61.
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Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Comparison of MALDI-TOF MS and VITEK 2 system for laboratory diagnosis of Granulicatella and Abiotrophia species causing invasive infections Paul Ratcliffe a, Hong Fang a, Ellinor Thidholm a, Stina Boräng a, Katarina Westling b, Volkan Özenci a,⁎ a b
Division of Clinical Microbiology F 72, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden Department of Medicine, Division of Infectious Diseases, Karolinska Institutet, Karolinska University Hospital, Huddinge, SE 141 86 Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 1 February 2013 Received in revised form 13 June 2013 Accepted 18 July 2013 Available online 10 September 2013 Keywords: Endocarditis Microbiology Granulicatella Abiotrophia NVS Antimicrobial susceptibility
a b s t r a c t Granulicatella and Abiotrophia spp. were known as nutritionally variant streptococci (NVS). Such strains have caused major diagnostic difficulties due to fastidious culturing and unspecific colony morphology. The present study is aimed at comparing the performance of laboratory available diagnostic methods for NVS isolates and determining the antimicrobial susceptibility of these isolates. Fourteen clinical invasive isolates, consisting of 10 Granulicatella adiacens, 1 Granulicatella elegans, and 3 Abiotrophia defectiva were in parallel analyzed by 2 matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) systems, i.e., Bruker MS and Vitek MS, as well as Vitek 2 for the species determination. 16S rRNA gene sequencing was applied as a reference method. The Vitek MS gave correct identification for all 14 isolates. The Bruker MS could correctly identify 8/10 G. adiacens, 0/1 G. elegans, and 3/3 A. defectiva isolates at the first analysis occasion, and all 14 isolates became identifiable after repeated tests. The Vitek 2 system could identify 6/10 G. adiacens, 1/1 G. elegans, and 2/3 A. defectiva isolates at the species level. Antimicrobial susceptibilities of 11 antibiotics were determined by Etest. Resistance against ciprofloxacin, ceftriaxone, rifampicin, and tetracycline were observed in 4, 10, 4, and 1 isolates, respectively. In conclusion, MALDI-TOF MS is a useful tool for the rapid diagnosis of NVS. Phenotypic testing by Vitek 2 is only partially effective for the accurate identification of such strains. The emergence of resistant NVS isolates indicates the necessity of monitoring antimicrobial susceptibilities of such uncommon pathogens. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Granulicatella and Abiotrophia spp., formerly known as nutritionally variant streptococci (NVS), are normal residents of the oral cavity but have been identified as agents of invasive infections (Ruoff, 2007). Because of both fastidious culturing and the unspecific colony morphology that they present on primary detection, such strains have caused major diagnostic difficulties. The NVS as a group have been estimated to cause 5–6% of all cases of streptococcal endocarditis (Brouqui and Raoult, 2001; Cargill et al., 2012; Roberts et al., 1979). However, it has been supposed that many culture-negative endocarditis could have been caused by these species, which could have lead to an underestimation as pathogens of infective endocarditis (Brouqui and Raoult, 2001; Cargill et al., 2012). Severe infections as infective endocarditis, neonatal infections, osteomyelitis, and endovascular infections, which were caused by streptococci, have been reported (Cargill et al., 2012). Infective endocarditis caused by NVS has in earlier studies been related with high bacteriologic failure (41%), a high rate of relapse after therapy (17%), and mortality at 17% (Stein and Nelson, 1987). In more recent surveys, mortality was 9.0% (Cargill et al., 2012; Giuliano et al., 2012). Combination therapy in the form ⁎ Corresponding author. Tel.: +46-8-5858-1147; fax: +46-8-5858-1125. E-mail address: [email protected] (V. Özenci). 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.07.008
of penicillin G or amoxicillin (in cases of susceptible strain) with aminoglycosides is recommended in most treatment guidelines (Habib et al., 2009; Westling et al., 2007). The clinical significance of the uncommon but pathogenic bacteria in invasive infections underlines the need for reliable and rapid identification methods in microbiology laboratories. Identification of Granulicatella and Abiotrophia spp. is usually conducted by either biochemical testing and/or molecular confirmation (Cargill et al., 2012; Christensen and Facklam, 2001). However, the biochemical characteristics might be insufficient for accurate identification of NVS (Ruoff, 2007). Hitherto published studies showed that the biochemical testing might be misleading in identification of Granulicatella spp. Both Granulicatella adiacens and Granulicatella elegans were previously identified as Gemella spp., and it has been reported that G. elegans was misidentified as Streptococcus acidominimus, Gemella morbillorum, and Granulicatella adiacens (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007; Cargill et al., 2012). Although far from perfect in identification of NVS species, the Vitek 2 system is still an available approach for most clinical microbiology laboratories nowadays. However, the performance of Vitek2 for identification of these species has not previously been studied. A variety of molecular techniques have been developed to help accelerate the identification of routine-difficult bacteria, including Granulicatella and Abiotrophia spp. The increasing use of molecular
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methods in the diagnosis of invasive infections, including 16S and 23S rRNA gene sequencing, may mean an improved recognition of formally rare causative organisms in infection (Breitkopf et al., 2005; Vollmer et al., 2010). However, by using the present sequencing method, 2 to 3 days is usually needed to get a result, and the personnel and reagent costs are higher than the biochemical testing. The development of matrix-assisted laser desorption ionization– time of flight mass spectrometry (MALDI-TOF MS) technology in the field of clinical microbiology shows promise for the rapid diagnosis of the previously difficult bacteria. There is, however, scarce data on identification of NVS by using this advanced technology. In publications where Granulicatella and Abiotrophia spp. were included, these species were regarded as rarely isolated species, and the number of isolates was limited (McElvania Tekippe et al., 2002; Neville et al., 2011; Schulthess et al., 2013). Here, we evaluated the performance of 2 MALDI-TOF MS systems, i.e., Bruker MS (Bruker Daltonik, Germany) and Vitek MS (bioMérieux, France), and the standardized phenotypic system Vitek 2 (bioMérieux, France) for the species determination of clinical NVS isolates. 16S rRNA gene sequencing was used as a reference method. The antimicrobial susceptibilities of these invasive isolates were determined by Etest. 2. Materials and methods 2.1. Bacterial strains A total of 14 clinical isolates were included in the study, consisting of 13 hematin isolates and 1 cerebral-spinal-fluid isolate, collected at the Department of Clinical Microbiology, Karolinska University Hospital, from 2006 to 2012. The 14 strains were identified by 16S rRNA gene sequencing as 10 G. adiacens, 1 G. elegans, and 3 Abiotrophia defectiva. 2.2. Culture conditions Hematin agar swirled with 5-mL 0.001% pyridoxal hydrochloride (PHC) was used for culturing Granulicatella and Abiotrophia. These bacteria do not grow well on agar media apart from hematin and grow best with the addition of PHC, which provides the bacteria with vitamin B6 necessary for their growth. The plates were incubated in CO2-enriched atmosphere for up to 5 days.
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The strains were tested by depositing 1 bacterial colony on the target slide and followed by the addition of matrix solution (VITEK MS-CHCA) and air drying. The loaded slide was then inserted into the Vitek MS system. Microbial identification is achieved by obtaining spectra using MALDI-TOF technology and analyzing the spectra with the Vitek MS database. The peaks from these spectra are compared with the characteristic pattern for a species, genus, or family of microorganism, thus resulting in organism identification. The organisms were reported with a percentage-scaled confidence value as well as a confidence level. 2.5. Vitek 2 system Vitek 2 (bioMérieux, France) is an automatic phenotypic test system based on test cards that house a number of wells, which contain dehydrated biochemical substrates. Gram-positive cards were used for identifying the Granulicatella and Abiotrophia isolates. 2.6. 16S rRNA gene sequencing 16S rRNA PCR and sequencing were performed after heat extraction of the bacterial DNA. After initial PCR amplification, with primers 5′-AGA GTT TGA TCM TGG CTC AG-3′ and 5′-CCG TCA ATT CMT TTR AGT TT-3′ (Brosius et al., 1978; Harmsen et al., 2002), the PCR products were purified using Amicon Ultra-0.5 (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Sequencing of both strands was carried out, with the same primers as in the PCR, using an ABI PrismBigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The sequence cycling products were analyzed by capillary electrophoresis and fluorescence detection with an Applied Biosystems ABI 3130xl Genetic analyzer. The fluorescence data were analysed with the SeqScape program (version 2.6; Applied Biosystems) followed by BLASTN 2.2.2. for bacterial identification (Altschul et al., 1990). 2.7. Antimicrobial susceptibility testing The minimal inhibitory concentrations (MICs) of ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, gentamycin, levofloxacin, penicillin G, rifampicin, tetracycline, trimetoprim-sulphamethoxazole, and vancomycin were determined by Etest (bioMérieux, France). Antimicrobial susceptibilities were interpreted according to the guidelines established by the Swedish Reference Group for Antibiotics.
2.3. MALDI-TOF Bruker MS
3. Results
Measurements were performed with a microflex LT mass spectrometer (Bruker Daltonik, Germany) using FlexControl software (version 3.3). The spectra were imported into the integrated MALDI Biotyper software (version 3.0) and were analysed by standard pattern matching with default settings. The strains were tested without pretreatment. A colony from the hematin agar plate was directly spotted on the MALDI-plate, and then overlaid with 1 μL of matrix solution, and air-dried. The loaded plate was then applied to the instrument according to the manufacturer's instruction. The spectrum of each isolate was compared with those in the database and identification was provided with a score of reliability.
The 14 strains were identified as 10 G. adiacens, 1 G. elegans, and 3 A. defectiva by 16S rRNA sequencing, which was used as the reference method in the study (Table 1).
2.4. MALDI-TOF Vitek MS Vitek MS (bioMérieux, Marcy l'Etoile, France) is another automated microbial identification system based on MALDI-TOF technology. Myla™, a Web-based middleware application integrated in the Vitek systems, provides a platform for both slide composition consultation and results consultation for Vitek MS.
3.1. MALDI-TOF MS The Vitek MS gave correct identification for all 14 Granulicatella and Abiotrophia isolates to the species level with a confidence value of 99.9%. The Bruker MS could correctly identify 8 out of the 10 G. adiacens and all 3 A. defectiva isolates at the first analysis occasion. To Table 1 Clinical invasive Abiotrophia and Granulicatella isolates correctly identified by Bruker MS, Vitek MS, and Vitek 2. Bacteria
No. of isolates
Bruker MS at the first attempt (by repeated tests)
VITEK MS
VITEK 2
A. defectiva G. adiacens G. elegans Total
3 10 1 14
3 8 (2) 0 (1) 11 (3)
3 10 1 14
2 6 1 9
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Table 2 Phenotypic characteristics of clinical invasive Abiotrophia and Granulicatella isolates by Vitek 2. Biochemical test
Arginindihydrolase 1 (0.111 mg) Arginindihydrolase 2 (0.270 mg) D-Amygdalin D-Maltose D-Raffinose D-Trehalos Growth in 6.5% NaCl Lactose Leucin Arylamidase L-Pyrrolidonyl Arylamidase Phosphatase Pullulan Salicin Sucrose α-Galactosidase β-Galactosidase β-Glucuronidase (0.0018 mg) β-Glucuronidase (0.0378 mg)
Results for species (no. of isolates with positive test result/total no. of isolates) A. defectiva
G. adiacens
G. elegans
0/3 0/3 0/3 3/3 0/3 2/3 0/3 0/3 1/3 3/3 0/3 0/3 0/3 3/3 2/3 0/3 0/3 0/3
0/10 0/10 0/10 4/10 0/10 0/10 0/10 1/10 9/10 5/10 0/10 0/10 0/10 9/10 3/10 0/10 6/10 5/10
1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 0/1
reach a reliable score, 2 repeated tests were required for 2 of the G. adiacens isolates and 3 more tests for the G. elegans strain (Table 1). The scores obtained in the repeated tests were between 1.730 and 1.971. 3.2. Vitek 2 The Vitek 2 system could identify 7 of the 11 Granulicatella isolates and 2 of the 3 A. defectiva isolates at the species level but failed on identifying 4 G. adiacens and 1 A. defectiva isolate. One G. adiacens strain was defined as unidentified organism, 1 as G. morbillorum, and low discrimination was reported for the remaining 2 G. adiacens isolates and the A. defectiva isolate (Table 1). The relevant phenotypic characteristics obtained from Vitek2 were summarized in Table 2. 3.3. Antimicrobial susceptibility testing The antimicrobial susceptibilities of NVS isolates were presented in Table 3. In general, the NVS isolates in the present study were susceptible to clindamycin, levofloxacin, penicillin, and vancomycin but resistant to cefuroxime and gentamicin and, however, presented variable susceptibilities to ciprofloxacin, ceftriaxone, rifampicin, and tetracycline (Table 3).
4. Discussion Accurate and rapid identification of Granulicatella and Abiotrophia spp. has been considered not easy in clinical microbiology laboratories (Cargill et al., 2012; Christensen and Facklam, 2001). In the present
study, we compared the performances of Bruker MS, Vitek MS, and Vitek 2 system in identification of invasive Granulicatella and Abiotrophia spp. The phenotypic characteristics may not always be sufficient for accurate identification of NVS (Ruoff, 2007). Biochemical testing has been reported to potentially lead to incorrect identification (Abdul-Redha et al., 2007; Al-Tawfiq et al., 2007; Cargill et al., 2012). In accordance with previous reports (Abdul-Redha et al., 2007; AlTawfiq et al., 2007; Cargill et al., 2012), 3/10 G. adiacens and 1/3 A. defectiva isolates, in the present study, were of low discrimination with G. morbillorum, G. elegans, and G. sanguinis by biochemical testing in Vitek 2 system. Our study demonstrates the limitations of the Vitek 2 system in the identification of NVS to the species level. 16S rRNA gene sequencing is proved to be effective in identification of Granulicatella and Abiotrophia in the study. However, the sequencing-based method is still not rapid enough to meet the clinical requirement. MALDI-TOF MS has been shown to be both accurate in the identification of bacteria and rapid, which is of proven benefit to patient care (Barenfanger et al., 1999; Doern et al., 1994; Eigner et al., 2009; Neville et al., 2011; Seng et al., 2009; van Veen et al., 2010). Nevertheless, data on the utility of MALDI-TOF for identification of NVS isolates are limited. In a recently published well-performed prospective study by Neville et al. (2011), where 927 isolates were investigated, only 1 NVS strain (G. adiacens) was included. The G. adiacens isolate in the Neville's study, by using MALDI-TOF Bruker MS, was identified to genus level in 1 of 3 tests and got unreliable identification at the remaining 2 tests. Similar phenomena were observed in 2 of the 10 G. adiacens isolates and 1 G. elegans isolate in our study, when the experiments were carried out with Bruker MS. In contrast, Vitek MS gave correct species identification for all 14 NVS isolates at the first analytic attempt. A possible explanation for the discrepancy is that the Vitek MS database houses at least 8 spectra for each species and that even uncommon spectra of the species are present. This approach ensures a high possibility for match for samples. Besides quality of the database, sample preparation methods could also play an important role in increasing the identification rate of gram-positive organisms (McElvania Tekippe et al., 2002; Schulthess et al., 2013). To pretreat the samples with formic acid will be investigated in our future study. The estimated time for analysis of 14 samples in duplicate is about 20–25 minutes for the Bruker system and 35–40 minutes for the Vitek MS. The Bruker system is smaller in size than Vitek MS, and reestablishment of vacuum is quicker for the Bruker MS. In contrast, it takes 3–8 hours for Vitek2 analysis, and 2 to 3 days is usually needed to get a result by the present sequencing method. The lack of standardized methods and interpretation criteria for antimicrobial susceptibility testing results, along with relatively small collections of clinical isolates for NVS, make it difficult to accurately assess antimicrobial susceptibility patterns. Our study demonstrates that Etest with pyridoxal hydrochloride-pretreated blood-supplemented Mueller-Hinton agar in a CO2-enriched atmosphere is a practically useful approach as susceptibility testing for
Table 3 Antimicrobial susceptibilities of Abiotrophia defectiva (n = 3) and Granulicatella (n = 11) isolates towards 11 antimicrobial agents. Bacteria/antibiotics
CIP
CLI
CTR
CXM
GEN
LEV
PEN
RIF
TET
TMP-SMX
VAN
MIC range (mg/L) MIC50 (mg/L) MIC90 (mg/L) A. defectiva (resistant/total isolates) Granulicatella spp. (resistant/total isolates) Total (resistant/total isolates)
0.25–32 0.5 1.5 0/3 4/11 4/14
0.047–0.5 0.094 0.38 0/3 0/11 0/14
0.38–48 0.75 2 3/3 7/11 10/14
2–256 4 256 3/3 11/11 14/14
1.5–4 3 4 3/3 11/11 14/14
0.38–1.5 0.75 1 0/3 0/11 0/14
0.016–0.25 0.064 0.094 0/3 0/11 0/14
0.002–32 0.004 32 0/3 4/11 4/14
0.25–8 0.5 1 0/3 1/11 1/14
0.002–32 0.064 32 ND ND ND
0.25–2 0.5 1 0/3 0/11 0/14
CIP = ciprofloxacin; CLI = clindamycin; CTR = ceftriaxone; CXM = cefuroxime; GEN = gentamycin; LEV = levofloxacin; PEN = penicillin G; RIF = rifampicin; TET = tetracycline; TMP-SMX = trimetoprim-sulphamethoxazole; VAN = vancomycin; ND = not determined, the breakpoint is not available.
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NVS. The inhibition zones of the tested isolates were clear and easy to define after 20–24 hours of incubation. It has been known that there is a range of MICs of penicillin for Granulicatella and Abiotrophia isolates, with the majority of strains classified as either susceptible or relatively resistant. There is also variability in susceptibilities to aminoglycosides, but no cases of highlevel resistance have been reported (Ruoff, 2007). Similarly, the collection of NVS isolates in our study was susceptible to penicillin but presented low-level resistance to gentamycin with an MIC range at 1.5–4 mg/L. The isolates in our collection were also susceptible to clindamycin and levofloxacin, concordant to the findings by Tuohy et al. (2000). In line with the previous reports, high rates of cephalosporin resistance were observed among Granulicatella and Abiotrophia spp. in the present study (Tuohy et al., 2000; Zheng et al., 2004). Notably, we identified 4 rifampicin-resistant Granulicatella isolates with MICs at 32 mg/L, which has not been reported previously. Besides rifampicin, the isolates were also resistant to gentamycin, ceftriaxone, cefuroxime, erythromycin, or trimetoprimsulphamethoxazole. Although all 14 isolates were susceptible to vancomycin, elevated MICs (1–2 mg/L) were observed in 3 G. adiacens isolates, 2 of which were co-resistant to rifampicin. In conclusion, MALDI-TOF MS is a useful tool for rapid identification of NVS such as Granulicatella and Abiotrophia spp. The rapid and accurate diagnosis of such infective pathogens plays an important role in guiding early appropriate antibiotic therapy. The emergence of resistant NVS isolates from invasive infections indicates the necessity of monitoring the antimicrobial susceptibilities of such uncommon pathogens. References Abdul-Redha RJ, Prag J, Sonksen UW, Kemp M, Andresen K, Christensen JJ. Granulicatella elegans bacteraemia in patients with abdominal infections. Scand J Infect Dis 2007;39:830–3. Al-Tawfiq JA, Kiwan G, Murrar H. Granulicatella elegans native valve infective endocarditis: case report and review. Diagn Microbiol Infect Dis 2007;57:439–41. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403–10. Barenfanger J, Drake C, Kacich G. Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J Clin Microbiol 1999;37: 1415–8. Breitkopf C, Hammel D, Scheld HH, Peters G, Becker K. Impact of a molecular approach to improve the microbiological diagnosis of infective heart valve endocarditis. Circulation 2005;111:1415–21. Brosius J, Palmer ML, Kennedy PJ, Noller HF. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A 1978;75:4801–5. Brouqui P, Raoult D. Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev 2001;14:177–207. Cargill JS, Scott KS, Gascoyne-Binzi D, Sandoe JA. Granulicatella infection: diagnosis and management. J Med Microbiol 2012;61:755–61.
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