| Abstract|| |
Introduction: The emergence of carbapenem-resistant Gram-negative pathogens poses a serious overwhelming threat to public health worldwide. The paucity in developing novel antimicrobials escalates the antimicrobial resistance problem, severely reducing the available therapeutic options. Tigecycline, a broad-spectrum glycylcycline, is considered the last-resort antimicrobial agent for the treatment of multidrug-resistant Gram-negative bacteria, except for Pseudomonas aeruginosa and Proteus spp. Objectives: This study was conducted to assess the activity of tigecycline against carbapenem-resistant Gram-negative clinical isolates. Materials and Methods: A total of 247 consecutive, nonrepeat carbapenem-resistant Gram-negative clinical isolates, detected by the VITEK 2 Compact system with advanced expert system, were obtained during the period from January 2012 to December 2015. Minimal inhibitory concentrations to tigecycline were determined using agar dilution method, and the results were interpreted using Food and Drug Administration (FDA) and EUCAST breakpoints. Results: The minimum inhibitory concentration (MIC) of tigecycline required to inhibit the growth of 90% of organisms (MIC90) varied from 1 to 8 μg/ml for the study isolates, except for P. aeruginosa and Proteus mirabilis where MIC90is ≥16 μg/ml. Using FDA breakpoint, sensitivity rates of tigecycline varied from 68% to 92%, except P. aeruginosa and P. mirabilis. However, the susceptibility rate for the same isolates remained within 58% to 90%, following EUCAST breakpoint. Conclusions: Tigecycline exhibited potent in vitro activity against Gram-negative carbapenemase producers, except P. aeruginosa and Proteus spp. Its broad-spectrum activity combining with stability against common resistance mechanisms and the lack of cross-resistance with other classes of antimicrobials make tigecycline a therapeutic agent for the multidrug-resistant microorganisms.
Keywords: Carbapenemases, Gram-negative isolates, tigecycline
|How to cite this article:|
Singh RM, Chongtham U, Huidrom S, Singh HL. Evaluation of in vitro activity of tigecycline against carbapenemase-producing Gram-negative clinical isolates. Ann Trop Med Public Health 2017;10:641-5
|How to cite this URL:|
Singh RM, Chongtham U, Huidrom S, Singh HL. Evaluation of in vitro activity of tigecycline against carbapenemase-producing Gram-negative clinical isolates. Ann Trop Med Public Health [serial online] 2017 [cited 2019 Nov 18];10:641-5. Available from: http://www.atmph.org/text.asp?2017/10/3/641/213126
| Introduction|| |
The emergence and dissemination of carbapenem-resistant (CR) Gram-negative microorganisms including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter spp. are a significant contributor to patient morbidity and mortality.,, Carbapenemase producers were generally resistant to the vast majority of antimicrobial agents available for clinical use, making the therapeutic options very limited. Tigecycline and colistin are among the few antimicrobials active and are commonly used for infections caused by CR Enterobacteriaceae and Acinetobacter baumannii.
Tigecycline, a semi-synthetic derivative of minocycline, is the first glycylcycline available for clinical use. In contrast with other tetracyclines, tigecycline has been shown excellent sustained in vitro activity against several Gram-positive and Gram-negative microorganisms, including multidrug-resistant strains, except P. aeruginosa and Proteus spp. Tigecycline has been approved by the United States Food and Drug Administration (FDA) as the agent of choice for treating complicated intra-abdominal infections, complicated skin and soft tissue infections, and community-acquired lower respiratory tract infections. Moreover, it evades ribosomal protection [tet (M)] and efflux [tet (A-E)] mechanisms that compromise classical tetracyclines.
Interpretive criteria for in vitro susceptibility testing of tigecycline are currently based on the breakpoints approved by the FDA or the EUCAST., However, the Clinical and Laboratory Standards Institute (CLSI) has not yet published any tentative or approved guidelines. Differences in susceptibility results according to the minimum inhibitory concentration (MIC) interpretive criteria of the two guidelines and the testing methods have been emphasized in various reports.,
The present study was taken up with the objectives of evaluating the in vitro activity of tigecycline against carbapenem-resistant Gram-negative clinical isolates and also to assess the appreciable difference in susceptibility pattern using both the FDA and the EUCAST breakpoint criteria for tigecycline.
| Materials and Methods|| |
A total of 247 consecutive, nonduplicate carbapenem-resistant Gram-negative clinical isolates obtained over a period of 4 years (January 2012 to December 2015) from various clinical specimens such as urine (n = 101), blood (n = 50), pus (n = 65), wound swab (n = 35), sputum (n = 30), and intravenous catheters (n = 27) were included in this cross-sectioning study. It covered the patients of all age groups and both sexes.
Identification and antibiotic sensitivity pattern of the isolates were performed by the Vitek 2 system (bioMerieux, Marcy l'Etoile, France) using GN-ID and AST-280 cards following the manufacturer's instructions, on which 18 antimicrobials were assayed, respectively. This system also features a software called advanced expert system that interprets the antibiotic resistance patterns, validates the results and reports the resistance phenotype, even emerging or low-level resistance, and has proved useful in calculating MIC values.
Modified Hodge test (MHT) was employed for the detection of carbapenemases. Further, phenotypic differentiation was performed by combined disk test using ethylenediaminetetraacetic acid (EDTA) for metallo-beta-lactamases (MBLs), phenylboronic acid (PBA) for Klebsiella pneumoniae carbapenemases (KPCs), and disk enhancement test with both PBA and EDTA, for co-production of MBLs with KPCs.,
Tigecycline susceptibility testing
MICs to tigecycline were performed by agar dilution technique, following the CLSI guidelines. Mueller-Hinton agar (HiMedia, Mumbai, India) plates, containing serial twofold dilutions of tigecycline (Wyeth Pharmaceuticals, India) at concentrations ranging from 0.25 to 16 μg/ml, were inoculated as spots of each bacterial suspension with a wire loop calibrated to deliver 0.001 ml spread over a small area, and the final inoculum on the agar surface should be approximately 104 colony forming unit per spot. A control Mueller-Hinton agar plate without antibiotics was inoculated with the test strains. The test and the control plates were incubated at 35°C for 18–24 h. For Enterobacteriaceae, the US FDA breakpoint (susceptible ≤2 μg/ml, intermediate of 4 μg/ml, resistant ≥8 μg/ml) and EUCAST breakpoint (susceptible ≤1 μg/ml, resistant >2 μg/ml) were followed to interpret the result., However, the same MIC interpretive criteria for Enterobacteriaceae were applied to Acinetobacter spp. and P. aeruginosa.
Quality control was achieved using Escherichia More Details coli (ATCC 25922), P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 700603), carbapenemases positive K. Pneumoniae ATCC BAA-1705 and negative K. pneumoniae ATCC BAA-1706.
Data obtained from this study were analyzed using descriptive statistics such as percentage and proportion.
| Results|| |
Of the total 247 carbapenem-resistant clinical isolates, MHT identified 172 (69.64%) isolates as carbapenemase producers; metallo-beta-lactamases (MBLs) activity was detected in 132 (53.44%) isolates, KPC in 24 (9.72%), and coexistent of both KPC and MBL in 6 (2.43%) isolates. The coproductions of MBLs with extended-spectrum beta-lactamases and AmpC-β-lactamases were observed in 59 (23.89%) and 16 (6.48%) isolates, respectively [Table 1].
|Table 1: Distribution of carbapenemases among the Gram-negative isolates|
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The MIC values for tigecycline as obtained by agar dilution are shown in [Table 2]. Using FDA breakpoints for Enterobacteriaceae, sensitivity was highest for E. coli followed by Enterobacter aerogenes, Enterobacter cloacae, K. pneumoniae, and Citrobacter freundii. If the same breakpoints were applied to Acinetobacte r spp., the sensitivity rates were 76% and 68.42% for A. baumannii and Acinetobacter lwoffii, respectively. However, following EUCAST criteria, similar results were obtained, but the susceptibility rates were substantially decreased as shown in [Table 3].
|Table 2: Distribution of tigecycline minimum inhibitory concentrations for carbapenem-resistant isolates|
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|Table 3: Comparative susceptibility data to tigecycline among the carbapenem-resistant isolates|
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| Discussion|| |
At present, no interpretative criteria for tigecycline are made available from the CLSI. The FDA has recommended susceptibility breakpoint of MIC ≤2 μg/ml for Enterobacteriaceae, but EUCAST has mentioned a lower susceptibility (MIC ≤1 μg/ml) breakpoint., Moreover, there are no tigecycline breakpoints for Acinetobacter spp. recommended by the FDA and EUCAST. Therefore, the interpretation of categorical susceptibility for tigecycline is hampered by the lack of consensual breakpoint recommendations.
In the present study, tigecycline showed potent activity against carbapenemase-producing Enterobacteriaceae (75%–92%), a result similar to other studies.,, Among Enterobacteriaceae, the MIC90 was highest for Proteus mirabilis (16 μg/ml) and least for E. coli (1 μg/ml). Souli et al. and Castanheira et al. obtained MIC90 values of 0.5 μg/ml and 1 μg/ml for E. coli, respectively., We obtained higher MIC90 values for K. pneumoniae, Enterobacter spp., and Citrobacter spp. in comparison to other studies.,, Tan and Ng reported similar MIC90 of 2 and 4 for K. pneumoniae and Enterobacter spp., respectively. The vast majority of K. pneumoniae isolates are fully susceptible to tigecycline at typical MICs of 0.25–1 μg/ml; however, a few clinical strains with decreased tigecycline susceptibility exceeding MICs of 1 μg/ml, due to the expression of the AcrAB multidrug efflux pump, have been isolated.
Our study reflected a tigecycline MIC90 of 4 and 8 μg/ml for carbapenemase-resistant A. baumannii and A. lwoffii, respectively. Tan and Ng, Chen et al., and Ahmed et al. obtained a similar MIC90 (4 μg/ml) for Acinetobacter spp.,, However, Souli M et al., Gupta et al., and Farrell et al. showed excellent activity of tigecycline against Acinetobacter spp. inhibiting 99% of the isolates at a concentration of ≤2 μg/ml.,,
All the P. aeruginosa (MIC50 and MIC90 of 16 μg/ml) and P. mirabilis (MIC50 of 8 μg/ml and MIC90 of 16 μg/ml) exhibited resistance to tigecycline. Similar findings were demonstrated by Kelesidis et al., Gupta et al., and Dean et al.,, Tigecycline remains vulnerable to the chromosomally encoded multidrug efflux pumps of P. aeruginosa and Proteeae.,
The broth microdilution method was considered the reference method for in vitro susceptibility testing of the clinical isolates to tigecycline. In this study, MICs of tigecycline were determined by the agar dilution method. It should be noted that very few studies employed agar dilution technique to evaluate MICs to tigecycline., However, there is paucity of data to show that the MIC obtained by agar dilution is lower than that of broth dilution techniques. Like classical tetracyclines, tigecycline is prone to oxidation, and MIC values may vary with the testing conditions, and that either the storage of tigecycline-containing media or the use of aged broth may lead to unwarrantedly high MICs particularly for the most susceptible isolates, probably due to the inactivation by the dissolved oxygen. To avoid these problems, tigecycline should be incorporated into the media on the day of use, and that freshly-prepared or degassed broth should be used if MIC dilutions are performed in liquid media.
Our study demonstrated the differences in susceptibility rates obtained using FDA and EUCAST breakpoints. When the EUCAST breakpoint criteria were used, the percentage of tigecycline susceptibility decreased. We observed that the tigecycline susceptibility rates measured using FDA criteria for E. coli, K. pneumoniae, E. aerogenes, and Enterobacter cloacae were about 1%, 6%, 10%, and 22%, respectively, higher than those measured using EUCAST breakpoints. Similar differences in susceptibility rates for E. coli and K. pneumoniae were reported by Sader et al. (0.6% and 7.3%) and Chen et al. (1% and 10%)., Papaparaskevas et al. also obtained similar result for K. pneumoniae (8%). A major difference in susceptibility rates for Enterobacter spp. was reported in that study. However, the susceptibility rates determined by the two criteria for A. baumannii differed by about 12% which was lowered than that obtained by Chen et al. (30%) and Liu et al. (27%).,
| Conclusion|| |
Tigecycline showed potent activity against wide range of carbapenem-resistant Gram-negative clinical isolates in the present study, probably due to its broad-spectrum activity, stability against common resistance mechanisms, and lack of cross-resistance with other classes of antibiotics. The difference in tigecycline susceptibility rates as obtained by FDA and EUCAST interpretative breakpoint is also revealed.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Marchaim D, Chopra T, Perez F, Hayakawa K, Lephart PR, Bheemreddy S, et al.
Outcomes and genetic relatedness of carbapenem-resistant Enterobacteriaceae
at Detroit medical center. Infect Control Hosp Epidemiol 2011;32:861-71.
Lautenbach E, Synnestvedt M, Weiner MG, Bilker WB, Vo L, Schein J, et al.
Epidemiology and impact of imipenem resistance in Acinetobacter baumannii
. Infect Control Hosp Epidemiol 2009;30:1186-92.
Lautenbach E, Synnestvedt M, Weiner MG, Bilker WB, Vo L, Schein J, et al.
Imipenem resistance in Pseudomonas aeruginosa
: Emergence, epidemiology, and impact on clinical and economic outcomes. Infect Control Hosp Epidemiol 2010;31:47-53.
Carmeli Y, Akova M, Cornaglia G, Daikos GL, Garau J, Harbarth S, et al.
Controlling the spread of carbapenemase-producing gram-negatives: Therapeutic approach and infection control. Clin Microbiol Infect 2010;16:102-11.
Cai Y, Wang R, Liang B, Bai N, Liu Y. Systematic review and meta-analysis of the effectiveness and safety of tigecycline for treatment of infectious disease. Antimicrob Agents Chemother 2011;55:1162-72.
Fluit AC, Florijn A, Verhoef J, Milatovic D. Presence of tetracycline resistance determinants and susceptibility to tigecycline and minocycline. Antimicrob Agents Chemother 2005;49:1636-8.
Pillar CM, Draghi DC, Dowzicky MJ, Sahm DF.In vitro
activity of tigecycline against gram-positive and gram-negative pathogens as evaluated by broth microdilution and Etest. J Clin Microbiol 2008;46:2862-7.
European Committee on Antimicrobial Susceptibility Testing (EUCAST). Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 5.0, Valid from 01 January, 2015. 2015. p. 7. Available from: http://www.eucast.org/clinical_breakpoints/
. [Last accessed on 2015 Dec 21].
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Fifth Informational Supplement M100-S24. Wayne: Clinical and Laboratory Standards Institute; 2014. p. 56.
Papaparaskevas J, Tzouvelekis LS, Tsakris A, Pittaras TE, Legakis NJ; Hellenic Tigecycline Study Group.In vitro
activity of tigecycline against 2423 clinical isolates and comparison of the available interpretation breakpoints. Diagn Microbiol Infect Dis 2010;66:187-94.
Pérez-Vázquez M, Oliver A, Sánchez del Saz B, Loza E, Baquero F, Cantón R. Performance of the VITEK2 system for identification and susceptibility testing of routine Enterobacteriaceae
clinical isolates. Int J Antimicrob Agents 2001;17:371-6.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Twenty-Fifth Informational Supplement M100-S24. Wayne: Clinical and Laboratory Standards Institute; 2014. p. 114-22.
Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas
spp. and Acinetobacter
spp. J Clin Microbiol 2002;40:3798-801.
Tsakris A, Poulou A, Pournaras S, Voulgari E, Vrioni G, Themeli-Digalaki K, et al.
A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae
clinical isolates. J Antimicrob Chemother 2010;65:1664-71.
Sader HS, Farrell DJ, Jones RN. Tigecycline activity tested against multidrug-resistant Enterobacteriaceae
spp. isolated in US medical centers (2005-2009). Diagn Microbiol Infect Dis 2011;69:223-7.
Souli M, Kontopidou FV, Koratzanis E, Antoniadou A, Giannitsioti E, Evangelopoulou P, et al. In vitro
activity of tigecycline against multiple-drug-resistant, including pan-resistant, gram-negative and gram-positive clinical isolates from Greek hospitals. Antimicrob Agents Chemother 2006;50:3166-9.
Pliatsika V, Afkou Z, Protonotariou E, Sofianou D.In vitro
activity of tigecycline against metallo-beta-lactamase-producing Enterobacteriaceae
. J Antimicrob Chemother 2007;60:1406-7.
Castanheira M, Sader HS, Deshpande LM, Fritsche TR, Jones RN. Antimicrobial activities of tigecycline and other broad-spectrum antimicrobials tested against serine carbapenemase-and metallo-beta-lactamase-producing Enterobacteriaceae
: Report from the SENTRY Antimicrobial Surveillance Program. Antimicrob Agents Chemother 2008;52:570-3.
Kelesidis T, Karageorgopoulos DE, Kelesidis I, Falagas ME. Tigecycline for the treatment of multidrug-resistant Enterobacteriaceae
: A systematic review of the evidence from microbiological and clinical studies. J Antimicrob Chemother 2008;62:895-904.
Tan TY, Ng LS. Susceptibility of multi-resistant gram-negative bacilli in Singapore to tigecycline as tested by agar dilution. Ann Acad Med Singapore 2007;36:807-10.
Ruzin A, Visalli MA, Keeney D, Bradford PA. Influence of transcriptional activator RamA on expression of multidrug efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneumoniae
. Antimicrob Agents Chemother 2005;49:1017-22.
Chen YH, Lu PL, Huang CH, Liao CH, Lu CT, Chuang YC, et al.
Trends in the susceptibility of clinically important resistant bacteria to tigecycline: Results from the tigecycline in vitro
surveillance in Taiwan study, 2006 to 2010. Antimicrob Agents Chemother 2012;56:1452-7.
Ahmed NH, Baba K, Clay C, Lekalakala R, Hoosen AA.In vitro
activity of tigecycline against clinical isolates of carbapenem resistant Acinetobacter baumannii
complex in Pretoria, South Africa. BMC Res Notes 2012;5:215.
Gupta S, Aruna C, Nagaraj S, Dias M, Muralidharan S.In vitro
activity of tigecycline against multidrug-resistant gram-negative blood culture isolates from critically ill patients. J Antimicrob Chemother 2012;67:1293-5.
Farrell DJ, Turnidge JD, Bell J, Sader HS, Jones RN. The in vitro
evaluation of tigecycline tested against pathogens isolated in eight countries in the Asia-Western Pacific region (2008). J Infect 2010;60:440-51.
Dean CR, Visalli MA, Projan SJ, Sum PE, Bradford PA. Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa
PAO1. Antimicrob Agents Chemother 2003;47:972-8.
Ruzin A, Keeney D, Bradford PA. AcrAB efflux pump plays a role in decreased susceptibility to tigecycline in Morganella morganii
. Antimicrob Agents Chemother 2005;49:791-3.
Kumar S, Bandyopadhyay M, Mondal S, Pal N, Ghosh T, Bandyopadhyay M, et al.
Tigecycline activity against metallo-ß-lactamase-producing bacteria. Avicenna J Med 2013;3:92-6.
] [Full text]
Hope R, Warner M, Mushtaq S, Ward ME, Parsons T, Livermore DM. Effect of medium type, age and aeration on the MICs of tigecycline and classical tetracyclines. J Antimicrob Chemother 2005;56:1042-6.
Bradford PA, Petersen PJ, Young M, Jones CH, Tischler M, O'Connell J. Tigecycline MIC testing by broth dilution requires use of fresh medium or addition of the biocatalytic oxygen-reducing reagent Oxyrase to standardize the test method. Antimicrob Agents Chemother 2005;49:3903-9.
Liu JW, Wang LS, Cheng YJ, Hsu GJ, Lu PL, Liu YC, et al. In-vitro
activity of tigecycline against clinical isolates of Acinetobacter baumannii
in Taiwan. Int J Antimicrob Agents 2008;32 Suppl 3:S188-91.
Rajkumar Manojkumar Singh
Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal, Manipur
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]