Microbiology Chemotherapy 2014;60:151–156 DOI: 10.1159/000375440
Received: November 13, 2014 Accepted after revision: January 19, 2015 Published online: March 11, 2015
In vitro Susceptibility of Tigecycline against Multidrug-Resistant Gram-Negative Strains: Etest versus Agar Dilution Sezen Özkök Turhan Togan Ayşegül Yesilkaya Funda Timurkaynak Özlem Kurt Azap Hande Arslan Department of Infectious Diseases and Clinical Microbiology, Faculty of Medicine, Baskent University, Ankara, Turkey
Key Words Etest · Agar dilution · Tigecycline · Extended-spectrum β-lactamase · Bacteremia · Acinetobacter spp.
could be used for K. pneumoniae bacteremia treatment after determining its MIC value. Determining the MIC value by gold-standard methods is more appropriate due to the correlation between Etest and AD at high MIC values. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0009–3157/15/0603–0151$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Nosocomial infections are considered a major health problem worldwide due to both high mortality and morbidity rates [1]. Bacteremias are the second most common nosocomial infection in intensive care units [2]. Due to the high mortality (12–80%) in cases with bacteremia, early and appropriate antibiotic therapy is important [3]. High resistance in bacteremia agents observed in recent years brings about therapeutic challenges. At the same time, effective treatment options against these microorganisms have decreased considerably. Tigecycline, a member of the glycylcycline class, has been approved for the treatment of soft tissue, complicated intra-abdominal and community-acquired lower respiratory tract infections due to Gram-positive and Gram-negative bacteria by the Food and Drug Administration (FDA). Its broad spectrum of activity, including against resistant bacteria, makes tigecycline a good treatment choice for infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, penicillin-resistant Turhan Togan, MD Department of Infectious Diseases and Clinical Microbiology Konya Application and Research Center, Baskent University Hocacihan Mah, Saray Cad. 1, Selcuklu, TR–42080 Konya (Turkey) E-Mail drtogant @ gmail.com
Downloaded by: NYU Medical Center Library 128.122.253.212 - 4/29/2015 4:12:30 AM
Abstract Background and Aim: Tigecycline is a semi-synthetic tetracycline with activity against most multidrug-resistant (MDR) bacteria. Methods: We studied in vitro activity of tigecycline by agar dilution (AD) and Etest methods to evaluate their correlation. The study included 206 isolates of extendedspectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae and MDR Acinetobacter baumannii recovered from blood cultures of patients of Baskent University between 2008 and 2010. Results: ESBL-producing E. coli had MIC50/MIC90 values of 0.5/0.5 μg/ml by AD and 0.25/0.5 μg/ml by Etest. ESBL-producing K. pneumoniae had MIC50/MIC90 values of 1/2 μg/ml by AD and 0.75/2 μg/ml by Etest, whereas MDR A. baumannii had MIC50/MIC90 values of 4/4 μg/ml by AD and 2/4 μg/ml by Etest. The correlation between AD and Etest was weak for ESBL-producing E. coli and strong for ESBL-producing K. pneumoniae and MDR A. baumannii. Tigecycline MIC values for ESBL-producing E. coli were lower than the tigecycline concentration, while they were higher than the concentrations attainable by treatment doses for A. baumannii. Conclusion: Tigecycline is an appropriate agent in the treatment of E. coli bacteremia, but it is not for treating A. baumannii bacteremia. Tigecycline
Material and Methods The study was carried out on isolates collected from patients diagnosed with bacteremia in both the inpatient clinic and intensive care unit of Baskent University Hospital, which is a 288-bed tertiary care center. A total of 206 nonduplicate (one strain per patient) bacteremia strains of ESBL-producing E. coli, ESBL-producing K. pneumoniae and MDR A. baumannii were collected consecutively from January 2008 to October 2010. MDR A. baumannii was defined as resistance to at least three antibiotics from the following antibiotic groups: piperacillin/tazobactam, cephalosporin (3rd and 4th generation), carbapenems, aminoglycosides and quinolones. Gram-negative bacteria isolated from positive vials were identified by conventional methods. The combination disk method with ceftazidime/ceftazidime-clavulanic acid and cefotaxime/cefotaxime-clavulanic acid was used to detect ESBL production. Strains are considered ESBL positive if the inhibition zone for ceftazidime/ceftazidime-clavulanic acid and cefotaxime/cefotaxime-clavulanic acid disk is greater than the inhibition zone of ceftazidime and cefotaxime disk by at least 5 mm. A BBLTM CrystalTM Enteric/Nonfermenter Identification Kit (Becton Dickinson, Sparks, Md., USA) was used to identify Acinetobacter spp. strains at the species level. All isolates were stored at –20 ° C and subcultured before testing. E. coli ATCC 25922 (ESBL negative) and Enterococcus faecalis (ATCC 29212) were used as quality control strains.
Etest Isolates stored at –20 ° C were thawed and subcultured to eosinmethylene blue agar (Becton Dickinson) and incubated at 37 ° C for 18 h. A suspension of 0.5 McFarland standard (108 cfu/ml) was
152
Chemotherapy 2014;60:151–156 DOI: 10.1159/000375440
prepared and inoculated onto freshly prepared BBL Mueller-Hinton agar (MHA; Becton Dickinson) plates. Commercial tigecycline Etest (bioMérieux SA, Marcy l’ Etoile, France) strips were applied to the agar surface. After incubation of the plates at 35 ° C for 18 h, the MIC value was determined as the value where the elliptical inhibition zone intersected the strip.
Agar Dilution Tigecycline laboratory powder was provided by Pfizer (Groton/New London, Conn., USA). The antibiotic stock solution for AD was studied according to CLSI guidelines [11]. Serial dilutions from this stock solution was added to freshly prepared molten BBL MHA (Becton Dickinson) in order to attain a final concentration of the plates of 0.0625–32 μg/ml. Bacterial suspension with an inoculum of 0.5 McFarland standard (108 cfu/ml) were inoculated onto the tigecycline-containing MHA plates. Plates were incubated at 35 ° C for 18–24 h. The lowest concentration of tigecycline that showed no bacterial growth was accepted as the MIC value. Although the MIC threshold value for tigecycline resistance in Enterobacteriaceae (including ESBL-producing E. coli and K. pneumoniae) determined by EUCAST was >2 μg/ml, no value has yet been determined for Acinetobacter spp. strains [4, 12]. The sensitivity threshold was accepted as ≤2 μg/ml for Acinetobacter spp. as in accordance with the previous studies [13–15].
Statistical Analysis Statistical analysis was performed using SPSS for Windows, version 15.0. Descriptive statistics, the Kruskal-Wallis test, MannWhitney test with Bonferroni correction and Spearman’s rank correlation analysis were used as appropriate. A p value
Received: November 13, 2014 Accepted after revision: January 19, 2015 Published online: March 11, 2015
In vitro Susceptibility of Tigecycline against Multidrug-Resistant Gram-Negative Strains: Etest versus Agar Dilution Sezen Özkök Turhan Togan Ayşegül Yesilkaya Funda Timurkaynak Özlem Kurt Azap Hande Arslan Department of Infectious Diseases and Clinical Microbiology, Faculty of Medicine, Baskent University, Ankara, Turkey
Key Words Etest · Agar dilution · Tigecycline · Extended-spectrum β-lactamase · Bacteremia · Acinetobacter spp.
could be used for K. pneumoniae bacteremia treatment after determining its MIC value. Determining the MIC value by gold-standard methods is more appropriate due to the correlation between Etest and AD at high MIC values. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0009–3157/15/0603–0151$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Nosocomial infections are considered a major health problem worldwide due to both high mortality and morbidity rates [1]. Bacteremias are the second most common nosocomial infection in intensive care units [2]. Due to the high mortality (12–80%) in cases with bacteremia, early and appropriate antibiotic therapy is important [3]. High resistance in bacteremia agents observed in recent years brings about therapeutic challenges. At the same time, effective treatment options against these microorganisms have decreased considerably. Tigecycline, a member of the glycylcycline class, has been approved for the treatment of soft tissue, complicated intra-abdominal and community-acquired lower respiratory tract infections due to Gram-positive and Gram-negative bacteria by the Food and Drug Administration (FDA). Its broad spectrum of activity, including against resistant bacteria, makes tigecycline a good treatment choice for infections caused by methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, penicillin-resistant Turhan Togan, MD Department of Infectious Diseases and Clinical Microbiology Konya Application and Research Center, Baskent University Hocacihan Mah, Saray Cad. 1, Selcuklu, TR–42080 Konya (Turkey) E-Mail drtogant @ gmail.com
Downloaded by: NYU Medical Center Library 128.122.253.212 - 4/29/2015 4:12:30 AM
Abstract Background and Aim: Tigecycline is a semi-synthetic tetracycline with activity against most multidrug-resistant (MDR) bacteria. Methods: We studied in vitro activity of tigecycline by agar dilution (AD) and Etest methods to evaluate their correlation. The study included 206 isolates of extendedspectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae and MDR Acinetobacter baumannii recovered from blood cultures of patients of Baskent University between 2008 and 2010. Results: ESBL-producing E. coli had MIC50/MIC90 values of 0.5/0.5 μg/ml by AD and 0.25/0.5 μg/ml by Etest. ESBL-producing K. pneumoniae had MIC50/MIC90 values of 1/2 μg/ml by AD and 0.75/2 μg/ml by Etest, whereas MDR A. baumannii had MIC50/MIC90 values of 4/4 μg/ml by AD and 2/4 μg/ml by Etest. The correlation between AD and Etest was weak for ESBL-producing E. coli and strong for ESBL-producing K. pneumoniae and MDR A. baumannii. Tigecycline MIC values for ESBL-producing E. coli were lower than the tigecycline concentration, while they were higher than the concentrations attainable by treatment doses for A. baumannii. Conclusion: Tigecycline is an appropriate agent in the treatment of E. coli bacteremia, but it is not for treating A. baumannii bacteremia. Tigecycline
Material and Methods The study was carried out on isolates collected from patients diagnosed with bacteremia in both the inpatient clinic and intensive care unit of Baskent University Hospital, which is a 288-bed tertiary care center. A total of 206 nonduplicate (one strain per patient) bacteremia strains of ESBL-producing E. coli, ESBL-producing K. pneumoniae and MDR A. baumannii were collected consecutively from January 2008 to October 2010. MDR A. baumannii was defined as resistance to at least three antibiotics from the following antibiotic groups: piperacillin/tazobactam, cephalosporin (3rd and 4th generation), carbapenems, aminoglycosides and quinolones. Gram-negative bacteria isolated from positive vials were identified by conventional methods. The combination disk method with ceftazidime/ceftazidime-clavulanic acid and cefotaxime/cefotaxime-clavulanic acid was used to detect ESBL production. Strains are considered ESBL positive if the inhibition zone for ceftazidime/ceftazidime-clavulanic acid and cefotaxime/cefotaxime-clavulanic acid disk is greater than the inhibition zone of ceftazidime and cefotaxime disk by at least 5 mm. A BBLTM CrystalTM Enteric/Nonfermenter Identification Kit (Becton Dickinson, Sparks, Md., USA) was used to identify Acinetobacter spp. strains at the species level. All isolates were stored at –20 ° C and subcultured before testing. E. coli ATCC 25922 (ESBL negative) and Enterococcus faecalis (ATCC 29212) were used as quality control strains.
Etest Isolates stored at –20 ° C were thawed and subcultured to eosinmethylene blue agar (Becton Dickinson) and incubated at 37 ° C for 18 h. A suspension of 0.5 McFarland standard (108 cfu/ml) was
152
Chemotherapy 2014;60:151–156 DOI: 10.1159/000375440
prepared and inoculated onto freshly prepared BBL Mueller-Hinton agar (MHA; Becton Dickinson) plates. Commercial tigecycline Etest (bioMérieux SA, Marcy l’ Etoile, France) strips were applied to the agar surface. After incubation of the plates at 35 ° C for 18 h, the MIC value was determined as the value where the elliptical inhibition zone intersected the strip.
Agar Dilution Tigecycline laboratory powder was provided by Pfizer (Groton/New London, Conn., USA). The antibiotic stock solution for AD was studied according to CLSI guidelines [11]. Serial dilutions from this stock solution was added to freshly prepared molten BBL MHA (Becton Dickinson) in order to attain a final concentration of the plates of 0.0625–32 μg/ml. Bacterial suspension with an inoculum of 0.5 McFarland standard (108 cfu/ml) were inoculated onto the tigecycline-containing MHA plates. Plates were incubated at 35 ° C for 18–24 h. The lowest concentration of tigecycline that showed no bacterial growth was accepted as the MIC value. Although the MIC threshold value for tigecycline resistance in Enterobacteriaceae (including ESBL-producing E. coli and K. pneumoniae) determined by EUCAST was >2 μg/ml, no value has yet been determined for Acinetobacter spp. strains [4, 12]. The sensitivity threshold was accepted as ≤2 μg/ml for Acinetobacter spp. as in accordance with the previous studies [13–15].
Statistical Analysis Statistical analysis was performed using SPSS for Windows, version 15.0. Descriptive statistics, the Kruskal-Wallis test, MannWhitney test with Bonferroni correction and Spearman’s rank correlation analysis were used as appropriate. A p value
