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Microbiological diagnosis of Eggerthella lenta blood culture isolates in a Swedish tertiary hospital: Rapid identification and antimicrobial susceptibility profile
Anaerobe 38 (2016) 21e24
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Microbiological diagnosis of Eggerthella lenta blood culture isolates in a Swedish tertiary hospital: Rapid identification and antimicrobial susceptibility profile * € Karin Liderot, Paul Ratcliffe, Petra Lüthje, Ellinor Thidholm, Volkan Ozenci Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, Huddinge, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2015 Received in revised form 10 November 2015 Accepted 16 November 2015 Available online 22 November 2015
Eggerthella lenta is a Gram-positive anaerobic bacillus. Improved diagnostics and increased awareness of rare pathogens have revealed its potential to cause serious invasive infections. In this study, 18 clinical E. lenta isolates derived from positive blood cultures were included. Underlying problems of the patients were in the majority of cases related to the gastrointestinal tract. The performance of two MALDI-TOF MS systems, i.e. Bruker and Vitek MS, in identification of E. lenta was analyzed. In addition, the minimal inhibitory concentrations for clinically relevant antimicrobial agents were determined by routine procedures using E-test. 17 of the 18 E. lenta isolates investigated in this study were correctly identified to species level by the Bruker MS system, while the Vitek MS system identified all 18 isolates. Antimicrobial sensitivity towards the tested agents was in general good. However, high resistance rates were observed for penicillin G and piperacillin-tazobactam based on EUCAST breakpoints. © 2015 Elsevier Ltd. All rights reserved.
Keywords: MALDI-TOF MS Vitek Blood culture Anaerobic bacteria Antimicrobial susceptibility testing Etest
1. Introduction With an increasing number of immunocompromized and elderly patients and enhanced intensive care facilities, also the awareness for uncommon pathogens is increasing. Intensified efforts to detect and identify rarely occurring, fastidious organisms which might previously have been undetected, neglected or regarded innocuous are thus required. Eggerthella lenta is a nonsporulating, Gram-positive, obligate anaerobic rod-shaped bacterium within the family Coriobacteriaceae [1,2]. The species belonged previously to the heterogeneous genus Eubacterium; genetic analyses led to establishment of the distinct genus Eggerthella. Initially described as an intestinal commensal, improved diagnostics have revealed the potential of E. lenta to cause serious invasive infections associated with high morbidity and mortality in vulnerable patient groups [3e6]. Phenotypic, culture-based identification of slow-growing bacteria does not longer meet clinical requirements with respect to time and possibly accuracy. However, the infrequent detection of E. lenta even in large clinical laboratories hampers the
* Corresponding author. € E-mail address: [email protected] (V. Ozenci). http://dx.doi.org/10.1016/j.anaerobe.2015.11.005 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
implementation of rapid, reliable identification methods into clinical routine. In addition, recommendations regarding empirical antimicrobial therapy remain uncertain due to limited knowledge in terms of susceptibility profiles and resistance development. Continues collection and presentation of data from different laboratories is therefore valuable for validation of novel diagnostic approaches and to guide therapy based on compiled antimicrobial sensitivity data. The most recent larger reports originate from the USA [7,8], Australia [9] and Taiwan [6], while data concerning E. lenta isolated in Europe are scarce [10]. The geographic diversity and temporal trends of antimicrobial resistance among bacteria underline the importance of analyzing rare species causing invasive infections. We have compared two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, i.e. Bruker MS and Vitek MS, in species determination of this pathogen. In addition, we analyzed the antimicrobial susceptibility for 19 E. lenta isolates collected at Swedish tertiary care hospitals.
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K. Liderot et al. / Anaerobe 38 (2016) 21e24
2. Material and methods
2.4. Antimicrobial susceptibility testing
2.1. Bacterial isolates and patient characteristics
Antimicrobial susceptibility testing was performed using Etests rieux) on blood agar plates [Columbia blood agar base (bioMe (Alpha Biosciences), 5% horse blood, 0.1 mg/mL DL-tryptophan, pH 7.3 ± 0.2]. The minimal inhibitory concentrations (MICs) were recorded after 48 h incubation at 35 C in anaerobic conditions. Interpretive criteria provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, http://www.eucast. org/) and the Clinical and Laboratory Standards Institute (CLSI, http://www.clsi.org/) were applied.
The study was performed at the Karolinska University Laboratory in Huddinge, Sweden, which serves the southern part of the greater Stockholm area and surrounding cities and suburbs. The laboratory receives blood culture specimens from three tertiary care hospitals: Karolinska University Hospital in Huddinge, Stock€derta €lje Hospital, holm, South General Hospital, Stockholm, and So € derta €lje, with a total of 1569 patient beds. Approximately 90,000 So blood cultures are processed each year. Usually, 4 blood culture bottles are received per one septic incident, but numbers might vary between 2 and 6 bottles. All blood cultures were incubated in the BacT/Alert 3D blood culture system for generally up to five days. Between 2008 and 2010, a total of 18 blood culture isolates initially identified as E. lenta by conventional methods were collected and stored at 70 C. Clinical information was collected from the Laboratory Information System. The isolates were retrieved from frozen stocks and cultured on blood agar plates [Columbia blood agar base (Alpha Biosciences, Baltimore, MD, USA), 5% horse blood, 0.1 mg/mL DL-tryptophan, pH 7.3 ± 0.2] under anaerobic conditions (0% O2, 10% CO2, 10% H2 and 80% N2; Anoxomat, Mart Microbiology, Drachten, The Netherlands) at 35 C for 2 days.
2.2. Vitek 2 system All isolates were re-identified by the automated Vitek 2 test rieux, France) using Vitek ANC ID cards for identisystem (bio Me fication of anaerobic and coryneformic bacteria following the recommendation of the manufacturer. Results were reported with a confidence value. In addition, identification was supported by Gram stain and basic conventional tests. One E. lenta isolate was sequenced for control purposes using the ABI Prism BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) with a GeneAmp 9700 thermocylcer (Applied Biosystems). A sequence of 393 base pairs showed 100% identity to E. lenta AUH-Julong365 (Accession number JN874873.1). The following species-specified sequences matched 100% (381/381) and 99% (381/382) with E. lenta.
2.3. Identification by MALDI-TOF MS For identification by MALDI-TOF MS, a colony from the agar plate was directly applied to the sample slide. Samples were overlaid with 1 mL of the supplier-specific matrix solution, air-dried and applied to the instrument according to the manufacturer's instruction.
2.3.1. MALDI-TOF Bruker MS Measurements were performed with a microflex MS from Bruker Daltonik, Germany. Spectra were analyzed by the integrated MALDI Biotyper software (version 3.0) with default settings; thresholds for species and genus identification were 2.0 and > 1.7, respectively.
2.3.2. MALDI-TOF Vitek MS The analysis was performed using the Vitek MS from Biorieux, France. Species identification with the Vitek MS system Me was based on the Vitek MS KnowledgeBase version 1.0; confidence values of 99% were considered reliable identification on species level.
3. Results and discussion 3.1. Isolates and patient characteristics Among clinical blood culture isolates between 2005 and 2011, 18 E. lenta isolates were identified and included in this study (2008, n ¼ 2; 2009, n ¼ 7; 2010, n ¼ 9). The median age of the 18 patients was 69 years (range 30e92 years), and half of them were female. The majority had underlying abdominal problems (n ¼ 11, 61%) and six of the 18 blood culture bottles (33%) showed polymicrobial growth. The present patients' data are similar to recently reported findings from Australia [9] and support that E. lenta should be regarded as significant pathogen, often associated with conditions located in the gastrointestinal tract. In bottles in which E. lenta was the sole microorganism detected, time to detection varied between 55 h and 109 h. Longer times to positivity was linked to patients under treatment with antimicrobials with good efficiency against E. lenta, i.e. metronidazole, meropenem and vancomycin. Short time to detection was noticed for blood cultures from patients treated with cefotaxime or piperacillin-tazobactam, which proved less efficient against E. lenta (see 3.3). 3.2. Identification of E. lenta 3.2.1. Phenotypic identification Re-analysis of the bacterial isolates recovered from storage confirmed their identity as E. lenta. Using the Vitek 2 system, probability scores of 99% were achieved for all isolates, confirming the previously established reliability of this identification method [11,12]. Colony and Gram stain morphology as well as conventional biochemical tests agreed with the description of the genus Eggerthella. In particular, all isolates were indole-negative and the majority produced catalase [1,13,14]. Concurring with the previously described species-specific variable catalase production by E. lenta [13,14], also few catalase-negative isolates (n ¼ 2) were detected. 3.2.2. Identification by MALDI-TOF MS The Bruker MS system identified 17 of the 18 isolates (94%) correctly to species level. One isolate could not be identified. Using the Vitek MS system, species identification with high confidence values (99.9%) was achieved for all isolates. Previous studies also reported excellent performance of the Bruker MS system for identification of E. lenta to genus level, with more variable results for species identification [9,15e17]. Studies using the Vitek MS system are less frequent and results are mixed, varying between 77% and 100% identification [9,18]. 3.3. Antimicrobial susceptibility All 18 isolates were tested for clinically relevant antimicrobial agents by Etest and MICs are depicted in Table 1. Knowledge regarding antimicrobial susceptibility among E. lenta is limited
K. Liderot et al. / Anaerobe 38 (2016) 21e24
23
Table 1 MIC distribution and resistance rates among 18 E. lenta isolates.
[6e10,14,19e24], and marked geographical variations in resistance pattern might exist. The rare detection and identification of isolates, and less frequent performance of extended antimicrobial susceptibility testing among anaerobes [25] contribute to the scarcity of information. In addition, data comparison might be impaired by different test methods applied, i.e. agar dilution method and Etest [26]. Most of the available data are from isolates collected outside Europe. Significant differences in resistance rates between published results and the data presented here were however essentially due to deviating breakpoints provided by EUCAST and CLSI, while pronounced variations in MIC distributions could not be established. Irrespectively, the clinical efficacy of penicillin G and piperacillin-tazobactam is questionable, considering the high MIC-values among the tested E. lenta isolates. Moreover, MIC distributions among isolates reported in studies published 2003e2006 [19e22,24] and 2009 or later [6,7,9] indicated a development towards reduced sensitivity against piperacillin-tazobactam. A similar trend over time could be noticed for moxifloxacin, although fewer studies were available for data comparison [6,9,10,22]. Consistent with the time span of sampling, sensitivity data among the isolates tested here are overall in accordance to data obtained in the later period. In line with our results, no resistance to imipenem has been reported so far, and only single isolates resistant to metronidazole [10,23,24] and vancomycin [8] have been detected. These data suggest that penicillin G and piperacillin-tazobactam are not recommended for empirical treatment of invasive infections with E. lenta. Resistance mechanisms in E. lenta have not been systematically studied. However, in a previous study, we determined high MIC-values for piperacillintazobactam in the absence of beta-lactamase production [12], indicating that alternative resistance mechanisms are active. Reduced sensitivity towards amoxicillin is moreover anticipated in case of beta-lactamase production. Since beta-lactam antibiotics might be a common choice for the treatment of infections caused by Gram-positive anaerobes, the general resistance profile of E. lenta highlights the importance of rapid identification of this pathogen.
4. Conclusions The present study shows that two MALDI-TOF MS systems and Vitek 2 are reliable methods in identification of E. lenta in the clinical routine. The observation on high resistance towards penicillin G and piperacillin-tazobactam is clinically relevant on the restrictive usage of these antimicrobials for empirical treatment of invasive infections with E. lenta. References [1] A. Kageyama, Y. Benno, T. Nakase, Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov, Int. J. Syst. Bacteriol. 49 Pt 4 (1999) 1725e1732. [2] W.G. Wade, J. Downes, D. Dymock, S.J. Hiom, A.J. Weightman, F.E. Dewhirst, et al., The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov, Int. J. Syst. Bacteriol. 49 Pt 2 (1999) 595e600. [3] I. Brook, E.H. Frazier, Significant recovery of nonsporulating anaerobic rods from clinical specimens, Clin. Infect. Dis. An Off. Publ. Infect. Dis. Soc. Am. 16 (1993) 476e480. [4] A.A. Venugopal, S. Szpunar, L.B. Johnson, Risk and prognostic factors among patients with bacteremia due to Eggerthella lenta, Anaerobe 18 (2012) 475e478. [5] S.K. Lau, P.C. Woo, A.M. Fung, K.M. Chan, G.K. Woo, K.Y. Yuen, Anaerobic, nonsporulating, gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing, J. Med. Microbiol. 53 (2004) 1247e1253. [6] M.R. Lee, Y.T. Huang, C.H. Liao, T.Y. Chuang, W.J. Wang, S.W. Lee, et al., Clinical and microbiological characteristics of bacteremia caused by Eggerthella, Paraeggerthella, and Eubacterium species at a university hospital in Taiwan from 2001 to 2010, J. Clin. Microbiol. 50 (2012) 2053e2055. [7] L.M. Ednie, P.C. Appelbaum, Antianaerobic activity of sulopenem compared to six other agents, Antimicrob. Agents Chemother. 53 (2009) 2163e2170. [8] E.J. Goldstein, D.M. Citron, K.L. Tyrrell, C.V. Merriam, Comparative in vitro activities of SMT19969, a new antimicrobial agent, against Clostridium difficile and 350 gram-positive and gram-negative aerobic and anaerobic intestinal flora isolates, Antimicrob. Agents Chemother. 57 (2013) 4872e4876. [9] B.J. Gardiner, A.Y. Tai, D. Kotsanas, M.J. Francis, S.A. Roberts, S.A. Ballard, et al., Clinical and microbiological characteristics of Eggerthella lenta bacteremia, J. Clin. Microbiol. 53 (2015) 626e635. [10] A. Liebetrau, A.C. Rodloff, J. Behra-Miellet, L. Dubreuil, In vitro activities of a new des-fluoro(6) quinolone, garenoxacin, against clinical anaerobic bacteria, Antimicrob. Agents Chemother. 47 (2003) 3667e3671.
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[11] E.H. Lee, J.E. Degener, G.W. Welling, A.C. Veloo, Evaluation of the Vitek 2 ANC card for identification of clinical isolates of anaerobic bacteria, J. Clin. Microbiol. 49 (2011) 1745e1749. € [12] K. Liderot, M. Larsson, S. Borang, V. Ozenci, Polymicrobial bloodstream infection with Eggerthella lenta and Desulfovibrio desulfuricans, J. Clin. Microbiol. 48 (2010) 3810e3812. [13] S.K. Lau, P.C. Woo, G.K. Woo, A.M. Fung, M.K. Wong, K.M. Chan, et al., Eggerthella hongkongensis sp. nov. and eggerthella sinensis sp. nov., two novel Eggerthella species, account for half of the cases of Eggerthella bacteremia, Diagn. Microbiol. Infect. Dis. 49 (2004) 255e263. [14] A. Mosca, P. Summanen, S.M. Finegold, G. De Michele, G. Miragliotta, Cellular fatty acid composition, soluble-protein profile, and antimicrobial resistance pattern of Eubacterium lentum, J. Clin. Microbiol. 36 (1998) 752e755. [15] D.P. Fedorko, S.K. Drake, F. Stock, P.R. Murray, Identification of clinical isolates of anaerobic bacteria using matrix-assisted laser desorption ionization-time of flight mass spectrometry, Eur. J. Clin. Microbiol. Infect. Dis. Off. Publ. Eur. Soc. Clin. Microbiol. 31 (2012) 2257e2262. [16] B.H. Schmitt, S.A. Cunningham, A.L. Dailey, D.R. Gustafson, R. Patel, Identification of anaerobic bacteria by Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry with on-plate formic acid preparation, J. Clin. Microbiol. 51 (2013) 782e786. [17] M. Barreau, I. Pagnier, B. La Scola, Improving the identification of anaerobes in the clinical microbiology laboratory through MALDI-TOF mass spectrometry, Anaerobe 22 (2013) 123e125. [18] W. Lee, M. Kim, D. Yong, S.H. Jeong, K. Lee, Y. Chong, Evaluation of VITEK mass spectrometry (MS), a matrix-assisted laser desorption ionization time-offlight MS system for identification of anaerobic bacteria, Ann. Lab. Med. 35 (2015) 69e75. [19] S.A. Roberts, K.P. Shore, S.D. Paviour, D. Holland, A.J. Morris, Antimicrobial susceptibility of anaerobic bacteria in New Zealand: 1999e2003,
J. Antimicrob. Chemother. 57 (2006) 992e998. [20] P.A. Bradford, D.T. Weaver-Sands, P.J. Petersen, In vitro activity of tigecycline against isolates from patients enrolled in phase 3 clinical trials of treatment for complicated skin and skin-structure infections and complicated intraabdominal infections, Clin. Infect. Dis. An Off. Publ. Infect. Dis. Soc. Am. 41 (Suppl. 5) (2005) S315eS332. [21] E.J. Goldstein, D.M. Citron, C.V. Merriam, Y.A. Warren, K.L. Tyrrell, H.T. Fernandez, In vitro activities of the new semisynthetic glycopeptide telavancin (TD-6424), vancomycin, daptomycin, linezolid, and four comparator agents against anaerobic gram-positive species and Corynebacterium spp, Antimicrob. Agents Chemother. 48 (2004) 2149e2152. [22] C.E. Edmiston, C.J. Krepel, G.R. Seabrook, L.R. Somberg, A. Nakeeb, R.A. Cambria, et al., In vitro activities of moxifloxacin against 900 aerobic and anaerobic surgical isolates from patients with intra-abdominal and diabetic foot infections, Antimicrob. Agents Chemother. 48 (2004) 1012e1016. [23] D.M. Citron, C.V. Merriam, K.L. Tyrrell, Y.A. Warren, H. Fernandez, E.J. Goldstein, In vitro activities of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria, Antimicrob. Agents Chemother. 47 (2003) 2334e2338. [24] D.B. Hoellman, L.M. Kelly, K. Credito, L. Anthony, L.M. Ednie, M.R. Jacobs, et al., In vitro antianaerobic activity of ertapenem (MK-0826) compared to seven other compounds, Antimicrob. Agents Chemother. 46 (2002) 220e224. [25] E.J. Goldstein, D.M. Citron, P.J. Goldman, R.J. Goldman, National hospital survey of anaerobic culture and susceptibility methods: III, Anaerobe 14 (2008) 68e72. [26] R.P. Rennie, L. Turnbull, C. Brosnikoff, J. Cloke, First comprehensive evaluation of the M.I.C. evaluator device compared to Etest and CLSI reference dilution methods for antimicrobial susceptibility testing of clinical strains of anaerobes and other fastidious bacterial species, J. Clin. Microbiol. 50 (2012) 1153e1157.
Contents lists available at ScienceDirect
Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Microbiological diagnosis of Eggerthella lenta blood culture isolates in a Swedish tertiary hospital: Rapid identification and antimicrobial susceptibility profile * € Karin Liderot, Paul Ratcliffe, Petra Lüthje, Ellinor Thidholm, Volkan Ozenci Division of Clinical Microbiology, Karolinska Institutet and Karolinska University Hospital, Huddinge, Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 August 2015 Received in revised form 10 November 2015 Accepted 16 November 2015 Available online 22 November 2015
Eggerthella lenta is a Gram-positive anaerobic bacillus. Improved diagnostics and increased awareness of rare pathogens have revealed its potential to cause serious invasive infections. In this study, 18 clinical E. lenta isolates derived from positive blood cultures were included. Underlying problems of the patients were in the majority of cases related to the gastrointestinal tract. The performance of two MALDI-TOF MS systems, i.e. Bruker and Vitek MS, in identification of E. lenta was analyzed. In addition, the minimal inhibitory concentrations for clinically relevant antimicrobial agents were determined by routine procedures using E-test. 17 of the 18 E. lenta isolates investigated in this study were correctly identified to species level by the Bruker MS system, while the Vitek MS system identified all 18 isolates. Antimicrobial sensitivity towards the tested agents was in general good. However, high resistance rates were observed for penicillin G and piperacillin-tazobactam based on EUCAST breakpoints. © 2015 Elsevier Ltd. All rights reserved.
Keywords: MALDI-TOF MS Vitek Blood culture Anaerobic bacteria Antimicrobial susceptibility testing Etest
1. Introduction With an increasing number of immunocompromized and elderly patients and enhanced intensive care facilities, also the awareness for uncommon pathogens is increasing. Intensified efforts to detect and identify rarely occurring, fastidious organisms which might previously have been undetected, neglected or regarded innocuous are thus required. Eggerthella lenta is a nonsporulating, Gram-positive, obligate anaerobic rod-shaped bacterium within the family Coriobacteriaceae [1,2]. The species belonged previously to the heterogeneous genus Eubacterium; genetic analyses led to establishment of the distinct genus Eggerthella. Initially described as an intestinal commensal, improved diagnostics have revealed the potential of E. lenta to cause serious invasive infections associated with high morbidity and mortality in vulnerable patient groups [3e6]. Phenotypic, culture-based identification of slow-growing bacteria does not longer meet clinical requirements with respect to time and possibly accuracy. However, the infrequent detection of E. lenta even in large clinical laboratories hampers the
* Corresponding author. € E-mail address: [email protected] (V. Ozenci). http://dx.doi.org/10.1016/j.anaerobe.2015.11.005 1075-9964/© 2015 Elsevier Ltd. All rights reserved.
implementation of rapid, reliable identification methods into clinical routine. In addition, recommendations regarding empirical antimicrobial therapy remain uncertain due to limited knowledge in terms of susceptibility profiles and resistance development. Continues collection and presentation of data from different laboratories is therefore valuable for validation of novel diagnostic approaches and to guide therapy based on compiled antimicrobial sensitivity data. The most recent larger reports originate from the USA [7,8], Australia [9] and Taiwan [6], while data concerning E. lenta isolated in Europe are scarce [10]. The geographic diversity and temporal trends of antimicrobial resistance among bacteria underline the importance of analyzing rare species causing invasive infections. We have compared two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems, i.e. Bruker MS and Vitek MS, in species determination of this pathogen. In addition, we analyzed the antimicrobial susceptibility for 19 E. lenta isolates collected at Swedish tertiary care hospitals.
22
K. Liderot et al. / Anaerobe 38 (2016) 21e24
2. Material and methods
2.4. Antimicrobial susceptibility testing
2.1. Bacterial isolates and patient characteristics
Antimicrobial susceptibility testing was performed using Etests rieux) on blood agar plates [Columbia blood agar base (bioMe (Alpha Biosciences), 5% horse blood, 0.1 mg/mL DL-tryptophan, pH 7.3 ± 0.2]. The minimal inhibitory concentrations (MICs) were recorded after 48 h incubation at 35 C in anaerobic conditions. Interpretive criteria provided by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, http://www.eucast. org/) and the Clinical and Laboratory Standards Institute (CLSI, http://www.clsi.org/) were applied.
The study was performed at the Karolinska University Laboratory in Huddinge, Sweden, which serves the southern part of the greater Stockholm area and surrounding cities and suburbs. The laboratory receives blood culture specimens from three tertiary care hospitals: Karolinska University Hospital in Huddinge, Stock€derta €lje Hospital, holm, South General Hospital, Stockholm, and So € derta €lje, with a total of 1569 patient beds. Approximately 90,000 So blood cultures are processed each year. Usually, 4 blood culture bottles are received per one septic incident, but numbers might vary between 2 and 6 bottles. All blood cultures were incubated in the BacT/Alert 3D blood culture system for generally up to five days. Between 2008 and 2010, a total of 18 blood culture isolates initially identified as E. lenta by conventional methods were collected and stored at 70 C. Clinical information was collected from the Laboratory Information System. The isolates were retrieved from frozen stocks and cultured on blood agar plates [Columbia blood agar base (Alpha Biosciences, Baltimore, MD, USA), 5% horse blood, 0.1 mg/mL DL-tryptophan, pH 7.3 ± 0.2] under anaerobic conditions (0% O2, 10% CO2, 10% H2 and 80% N2; Anoxomat, Mart Microbiology, Drachten, The Netherlands) at 35 C for 2 days.
2.2. Vitek 2 system All isolates were re-identified by the automated Vitek 2 test rieux, France) using Vitek ANC ID cards for identisystem (bio Me fication of anaerobic and coryneformic bacteria following the recommendation of the manufacturer. Results were reported with a confidence value. In addition, identification was supported by Gram stain and basic conventional tests. One E. lenta isolate was sequenced for control purposes using the ABI Prism BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) with a GeneAmp 9700 thermocylcer (Applied Biosystems). A sequence of 393 base pairs showed 100% identity to E. lenta AUH-Julong365 (Accession number JN874873.1). The following species-specified sequences matched 100% (381/381) and 99% (381/382) with E. lenta.
2.3. Identification by MALDI-TOF MS For identification by MALDI-TOF MS, a colony from the agar plate was directly applied to the sample slide. Samples were overlaid with 1 mL of the supplier-specific matrix solution, air-dried and applied to the instrument according to the manufacturer's instruction.
2.3.1. MALDI-TOF Bruker MS Measurements were performed with a microflex MS from Bruker Daltonik, Germany. Spectra were analyzed by the integrated MALDI Biotyper software (version 3.0) with default settings; thresholds for species and genus identification were 2.0 and > 1.7, respectively.
2.3.2. MALDI-TOF Vitek MS The analysis was performed using the Vitek MS from Biorieux, France. Species identification with the Vitek MS system Me was based on the Vitek MS KnowledgeBase version 1.0; confidence values of 99% were considered reliable identification on species level.
3. Results and discussion 3.1. Isolates and patient characteristics Among clinical blood culture isolates between 2005 and 2011, 18 E. lenta isolates were identified and included in this study (2008, n ¼ 2; 2009, n ¼ 7; 2010, n ¼ 9). The median age of the 18 patients was 69 years (range 30e92 years), and half of them were female. The majority had underlying abdominal problems (n ¼ 11, 61%) and six of the 18 blood culture bottles (33%) showed polymicrobial growth. The present patients' data are similar to recently reported findings from Australia [9] and support that E. lenta should be regarded as significant pathogen, often associated with conditions located in the gastrointestinal tract. In bottles in which E. lenta was the sole microorganism detected, time to detection varied between 55 h and 109 h. Longer times to positivity was linked to patients under treatment with antimicrobials with good efficiency against E. lenta, i.e. metronidazole, meropenem and vancomycin. Short time to detection was noticed for blood cultures from patients treated with cefotaxime or piperacillin-tazobactam, which proved less efficient against E. lenta (see 3.3). 3.2. Identification of E. lenta 3.2.1. Phenotypic identification Re-analysis of the bacterial isolates recovered from storage confirmed their identity as E. lenta. Using the Vitek 2 system, probability scores of 99% were achieved for all isolates, confirming the previously established reliability of this identification method [11,12]. Colony and Gram stain morphology as well as conventional biochemical tests agreed with the description of the genus Eggerthella. In particular, all isolates were indole-negative and the majority produced catalase [1,13,14]. Concurring with the previously described species-specific variable catalase production by E. lenta [13,14], also few catalase-negative isolates (n ¼ 2) were detected. 3.2.2. Identification by MALDI-TOF MS The Bruker MS system identified 17 of the 18 isolates (94%) correctly to species level. One isolate could not be identified. Using the Vitek MS system, species identification with high confidence values (99.9%) was achieved for all isolates. Previous studies also reported excellent performance of the Bruker MS system for identification of E. lenta to genus level, with more variable results for species identification [9,15e17]. Studies using the Vitek MS system are less frequent and results are mixed, varying between 77% and 100% identification [9,18]. 3.3. Antimicrobial susceptibility All 18 isolates were tested for clinically relevant antimicrobial agents by Etest and MICs are depicted in Table 1. Knowledge regarding antimicrobial susceptibility among E. lenta is limited
K. Liderot et al. / Anaerobe 38 (2016) 21e24
23
Table 1 MIC distribution and resistance rates among 18 E. lenta isolates.
[6e10,14,19e24], and marked geographical variations in resistance pattern might exist. The rare detection and identification of isolates, and less frequent performance of extended antimicrobial susceptibility testing among anaerobes [25] contribute to the scarcity of information. In addition, data comparison might be impaired by different test methods applied, i.e. agar dilution method and Etest [26]. Most of the available data are from isolates collected outside Europe. Significant differences in resistance rates between published results and the data presented here were however essentially due to deviating breakpoints provided by EUCAST and CLSI, while pronounced variations in MIC distributions could not be established. Irrespectively, the clinical efficacy of penicillin G and piperacillin-tazobactam is questionable, considering the high MIC-values among the tested E. lenta isolates. Moreover, MIC distributions among isolates reported in studies published 2003e2006 [19e22,24] and 2009 or later [6,7,9] indicated a development towards reduced sensitivity against piperacillin-tazobactam. A similar trend over time could be noticed for moxifloxacin, although fewer studies were available for data comparison [6,9,10,22]. Consistent with the time span of sampling, sensitivity data among the isolates tested here are overall in accordance to data obtained in the later period. In line with our results, no resistance to imipenem has been reported so far, and only single isolates resistant to metronidazole [10,23,24] and vancomycin [8] have been detected. These data suggest that penicillin G and piperacillin-tazobactam are not recommended for empirical treatment of invasive infections with E. lenta. Resistance mechanisms in E. lenta have not been systematically studied. However, in a previous study, we determined high MIC-values for piperacillintazobactam in the absence of beta-lactamase production [12], indicating that alternative resistance mechanisms are active. Reduced sensitivity towards amoxicillin is moreover anticipated in case of beta-lactamase production. Since beta-lactam antibiotics might be a common choice for the treatment of infections caused by Gram-positive anaerobes, the general resistance profile of E. lenta highlights the importance of rapid identification of this pathogen.
4. Conclusions The present study shows that two MALDI-TOF MS systems and Vitek 2 are reliable methods in identification of E. lenta in the clinical routine. The observation on high resistance towards penicillin G and piperacillin-tazobactam is clinically relevant on the restrictive usage of these antimicrobials for empirical treatment of invasive infections with E. lenta. References [1] A. Kageyama, Y. Benno, T. Nakase, Phylogenetic evidence for the transfer of Eubacterium lentum to the genus Eggerthella as Eggerthella lenta gen. nov., comb. nov, Int. J. Syst. Bacteriol. 49 Pt 4 (1999) 1725e1732. [2] W.G. Wade, J. Downes, D. Dymock, S.J. Hiom, A.J. Weightman, F.E. Dewhirst, et al., The family Coriobacteriaceae: reclassification of Eubacterium exiguum (Poco et al. 1996) and Peptostreptococcus heliotrinreducens (Lanigan 1976) as Slackia exigua gen. nov., comb. nov. and Slackia heliotrinireducens gen. nov., comb. nov., and Eubacterium lentum (Prevot 1938) as Eggerthella lenta gen. nov., comb. nov, Int. J. Syst. Bacteriol. 49 Pt 2 (1999) 595e600. [3] I. Brook, E.H. Frazier, Significant recovery of nonsporulating anaerobic rods from clinical specimens, Clin. Infect. Dis. An Off. Publ. Infect. Dis. Soc. Am. 16 (1993) 476e480. [4] A.A. Venugopal, S. Szpunar, L.B. Johnson, Risk and prognostic factors among patients with bacteremia due to Eggerthella lenta, Anaerobe 18 (2012) 475e478. [5] S.K. Lau, P.C. Woo, A.M. Fung, K.M. Chan, G.K. Woo, K.Y. Yuen, Anaerobic, nonsporulating, gram-positive bacilli bacteraemia characterized by 16S rRNA gene sequencing, J. Med. Microbiol. 53 (2004) 1247e1253. [6] M.R. Lee, Y.T. Huang, C.H. Liao, T.Y. Chuang, W.J. Wang, S.W. Lee, et al., Clinical and microbiological characteristics of bacteremia caused by Eggerthella, Paraeggerthella, and Eubacterium species at a university hospital in Taiwan from 2001 to 2010, J. Clin. Microbiol. 50 (2012) 2053e2055. [7] L.M. Ednie, P.C. Appelbaum, Antianaerobic activity of sulopenem compared to six other agents, Antimicrob. Agents Chemother. 53 (2009) 2163e2170. [8] E.J. Goldstein, D.M. Citron, K.L. Tyrrell, C.V. Merriam, Comparative in vitro activities of SMT19969, a new antimicrobial agent, against Clostridium difficile and 350 gram-positive and gram-negative aerobic and anaerobic intestinal flora isolates, Antimicrob. Agents Chemother. 57 (2013) 4872e4876. [9] B.J. Gardiner, A.Y. Tai, D. Kotsanas, M.J. Francis, S.A. Roberts, S.A. Ballard, et al., Clinical and microbiological characteristics of Eggerthella lenta bacteremia, J. Clin. Microbiol. 53 (2015) 626e635. [10] A. Liebetrau, A.C. Rodloff, J. Behra-Miellet, L. Dubreuil, In vitro activities of a new des-fluoro(6) quinolone, garenoxacin, against clinical anaerobic bacteria, Antimicrob. Agents Chemother. 47 (2003) 3667e3671.
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