HACEK organisms exhibit low minimum inhibitory concentrations to ertapenem

HACEK organisms exhibit low minimum inhibitory concentrations to ertapenem

Journal of Global Antimicrobial Resistance 3 (2015) 149–150 Contents lists available at ScienceDirect Journal of Global Antimicrobial Resistance jou...

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Journal of Global Antimicrobial Resistance 3 (2015) 149–150

Contents lists available at ScienceDirect

Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar

Letter to the Editor HACEK organisms exhibit low minimum inhibitory concentrations to ertapenem Sir, The HACEK organisms are a group of fastidious, Gram-negative bacilli that are normal constituents of the oropharyngeal flora but also have a propensity to cause infective endocarditis. The HACEK grouping is comprised of Haemophilus spp., Aggregatibacter spp., Cardiobacterium spp., Eikenella corrodens and Kingella spp. It is estimated that HACEK organisms account for 5–10% of native valve, community-acquired endocarditis in non-intravenous drug users [1]. The American Heart Association guidelines endorse ceftriaxone, ampicillin/sulbactam or ciprofloxacin for treatment of HACEK endocarditis [2]. Carbapenem antibiotics are frequently considered as an alternative to ceftriaxone in this setting. Ertapenem is an attractive option because it is administered every 24 h, whilst the other US Food and Drug Administration (FDA)-approved carbapenem antibiotics must be administered every 6–8 h. In addition, ertapenem is stable in solution for 6 h at room temperature and for 24 h when refrigerated, which is important in the setting of outpatient or home-health therapy. The Clinical and Laboratory Standards Institute (CLSI) guidelines recommend microbroth dilution using cation-adjusted Mueller– Hinton broth with laked horse blood (2.5–5%, v/v) for susceptibility testing of HACEK isolates, with the exception of Haemophilus parainfluenzae [3] for which disk diffusion or microbroth dilution using Haemophilus Test Medium (HTM) is recommended [4]. Of note, breakpoints for HACEK organisms and ertapenem do not currently exist in the CLSI guidelines [3]. Compared with broth microdilution, gradient diffusion methods such as Etest are less labour intensive and have a longer shelf-life. Thus, gradient diffusion methods are an attractive alternative methodology for routine susceptibility testing

for clinical laboratories. The aims of this study were to determine ertapenem minimum inhibitory concentrations (MICs) for HACEK organisms and to determine whether agar-based testing is a viable methodology. To address the question of ertapenem susceptibility for HACEK organisms, the susceptibility of 73 stored isolates (Table 1) was studied using the Etest method. Following ca. 24 h of growth on blood or chocolate agar, isolates were re-suspended in 0.9% saline to achieve a 0.5 McFarland standard. All organisms were plated on Mueller–Hinton with blood (BMH) and HTM agars (Becton Dickinson, Franklin Lakes, NJ). An ertapenem Etest (bioMe´rieux, Durham, NC) was applied and plates were incubated at 35 8C with 5% CO2. MICs were read after 24 and 48 h. Quality control was performed in accordance with CLSI standards. Overall, the ertapenem MICs for all isolates were low, ranging from 0.002 to 0.250 mg/mL. Discrepancies between 24 and 48 h were attributed to poor growth at 24 h resulting in difficult interpretation. Growth was sufficient after 48 h allowing for straightforward interpretation. Forty-four isolates (60.3%) grew on BMH, whilst 58 isolates (79.5%) grew on HTM. All of the isolates were able to be analysed on at least one type of medium. Haemophilus spp. as well as Aggregatibacter segnis and all but one isolate of Aggregatibacter aphrophilus required HTM for growth, whilst Kingella kingae and 50% of E. corrodens isolates grew only on BMH. The remaining species grew on both media. The 48-h MICs are shown in Table 1. Organisms that grew on both BMH and HTM (n = 26) demonstrated comparable MICs between the two media. Goldstein et al. utilised agar dilution to evaluate the activity of ertapenem and 11 other antimicrobials against organisms recovered from bite wounds [5]. The MIC for all E. corrodens isolates (n = 16) was 0.03 mg/mL. Additional HACEK organisms were grouped with ‘fastidious Gram-negative bacilli’ and included five H. parainfluenzae and three Aggregatibacter spp. Ertapenem MICs for this group ranged from 0.015 mg/mL to 0.250 mg/mL but also

Table 1 Ertapenem minimum inhibitory concentrations (MICs; in mg/mL) at 48 h growth for HACEK organisms. Species


Mueller–Hinton agar with blood

Haemophilus Test Medium




MIC range




MIC range

Haemophilus parahaemolyticus Haemophilus parainfluenzae Aggregatibacter actinomycetemcomitans Aggregatibacter aphrophilus Aggregatibacter segnis Cardiobacterium hominis Eikenella corrodens Kingella denitrificans Kingella kingae

5 6 12 10 8 5 10 7 10

0 0 11 1 0 5 10 7 10

NG NG 0.125 0.016 NG 0.004 0.064 0.032 0.016

NG NG 0.125 0.016 NG 0.008 0.064 0.064 0.032

N/A N/A 0.064–0.125 0.016 N/A 0.004–0.008 0.032–0.064 0.032–0.064 0.008–0.032

5 6 12 10 8 5 5 7 0

0.016 0.032 0.125 0.064 0.064 0.004 0.064 0.016 NG

0.016 0.125 0.250 0.064 0.064 0.008 0.125 0.032 NG

0.004–0016 0.016–0.125 0.064–0.250 0.032–0.064 0.064 0.002–0.008 0.008–0.125 0.004–0.032 N/A











MIC50/90, MIC that inhibits 50 and 90% of the isolates, respectively; NG, no growth; N/A, not applicable. http://dx.doi.org/10.1016/j.jgar.2015.03.004 2213-7165/ß 2015 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.


Letter to the Editor / Journal of Global Antimicrobial Resistance 3 (2015) 149–150

included Capnocytophaga spp. and Weeksella virosa. Two additional studies evaluated the activity of imipenem and meropenem, but not ertapenem, against HACEK organisms [6,7]. MICs for imipenem and meropenem ranged from 0.002 to 16 mg/mL and from 0.002 to 0.25 mg/mL, respectively. Whilst imipenem and meropenem often predict susceptibility to ertapenem, this is not always true, as with non-fermenting Gram-negative bacilli. In a study by Coburn et al., 104 (59.8%) of 174 isolates failed to grow adequately in the control well using liquid medium containing 5% lysed horse blood [7]. Whilst a number of isolates in the current study only grew on BMH or HTM, none failed to grow on both media. These previous data together with the data in the current study suggest that HACEK organisms are sufficiently heterogeneous in their growth characteristics. It is plausible that agar-based testing may return a higher number of viable results, but additional investigation is required to validate this. In the future, it may be appropriate to have species-specific recommendations for antimicrobial susceptibility testing of these organisms. All of the isolates in this study had low MICs (0.25 mg/mL) for ertapenem, thus implicating ertapenem as a potential option for treatment of HACEK endocarditis. However, in vitro susceptibility testing requires correlation with clinical outcome studies before adopting ertapenem as standard treatment for these infections. Funding None. Competing interests CDB has received research support from bioMe´rieux and consulting fees from Thermo Fisher Scientific; MAP is the recipient of a bioMe´rieux Scholarship in Clinical Microbiology. Ethical approval

[2] Baddour LM, Wilson WR, Bayer AS, Fowler Jr VG, Bolger AF, Levison ME, et al. Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association: endorsed by the Infectious Diseases Society of America. Circulation 2005;111:e394–434. [3] Clinical Laboratory Standards Institute. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guideline – second edition. Document M45-A2. Wayne, PA: CLSI; 2010. [4] Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; twenty-third informational supplement. Document M100-S23. Wayne, PA: CLSI; 2013. [5] Goldstein EJ, Citron DM, Merriam CV, Warren YA, Tyrrell K, Fernandez H. Comparative in vitro activity of ertapenem and 11 other antimicrobial agents against aerobic and anaerobic pathogens isolated from skin and soft tissue animal and human bite wound infections. J Antimicrob Chemother 2001;48:641–51. [6] Kugler KC, Biedenbach DJ, Jones RN. Determination of the antimicrobial activity of 29 clinically important compounds tested against fastidious HACEK group organisms. Diagn Microbiol Infect Dis 1999;34:73–6. [7] Coburn B, Toye B, Rawte P, Jamieson FB, Farrell DJ, Patel SN. Antimicrobial susceptibilities of clinical isolates of HACEK organisms. Antimicrob Agents Chemother 2013;57:1989–91.

Morgan A. Pence1 Carey-Ann D. Burnham* Department of Pathology & Immunology, Division of Laboratory and Genomic Medicine, Washington University School of Medicine, St Louis, MO, USA *Corresponding author at: Department of Pathology & Immunology, Division of Laboratory and Genomic Medicine, Washington University in St Louis, 660 S. Euclid Avenue, Campus Box 8118, St Louis, MO 63110, USA. Tel.: +1 314 362 1547 E-mail addresses: [email protected] (M.A. Pence), [email protected] (C.D. Burnham). 1

None. References [1] Geraci JE, Wilson WR. Symposium on infective endocarditis III. Endocarditis due to Gram-negative bacteria Report of 56 cases. Mayo Clin Proc 1982;57:145–8.

Current address: Department of Laboratories, Cook Children’s Medical Center, 801 Seventh Avenue, Fort Worth, TX 76104, USA. 9 September 2014

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