- Email: [email protected]
In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives
J Infect Chemother xxx (2014) 1e4
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives Camila Rizek a, Juliana Rosa Ferraz a, Inneke Marie van der Heijden a, Mauro Giudice a, Anna Karina Mostachio a, Jorge Paez a, Claudia Carrilho b, Anna Sara Levin a, Silvia F. Costa a, * rias da Faculdade de Medicina da Universidade de Sa ~o Paulo, Av. Dr. En LIM-54, Departamento de Doenças Infecciosas e Parasita eas de Carvalho Aguiar 500, 1 andar, sala 112, CEP05403-000 Sao Paulo, SP, Brazil , Brazil University Hospital of University of Londrina, Parana
a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2014 Received in revised form 1 October 2014 Accepted 15 October 2014 Available online xxx
Background: The aim of this study was to evaluate the in vitro susceptibility of MDR gram-negatives bacteria to old drugs such as polymyxin B, minocycline and fosfomycin and new drugs such as tigecycline. Methods: One hundred and fifty-three isolates from 4 Brazilian hospitals were evaluated. Forty-seven Acinetobacter baumannii resistant to carbapenens harboring adeB, blaOxA23, blaOxA51, blaOxA143 and blaIMP genes, 48 Stenotrophomonas maltophilia including isolates resistant to levofloxacin and/or trimethoprim-sulfamethoxazole harboring sul-1, sul-2 and qnrMR and 8 Serratia marcescens and 50 Klebsiella pneumoniae resistant to carbapenens harboring blaKPC-2 were tested to determine their minimum inhibitory concentrations (MICs) by microdilution to the following drugs: minocycline, ampicillinsulbactam, tigecycline, and polymyxin B and by agar dilution to fosfomycin according with breakpoint criteria of CLSI and EUCAST (fosfomycin). In addition, EUCAST fosfomycin breakpoint for Pseudomonas spp. was applied for Acinetobacter spp and S. maltophilia, the FDA criteria for tigecycline was used for Acinetobacter spp and S. maltophilia and the Pseudomonas spp polymyxin B CLSI criterion was used for S. maltophilia. Results: Tigecycline showed the best in vitro activity against the MDR gram-negative evaluated, followed by polymyxin B and fosfomycin. Polymyxin B resistance among K. pneumoniae was detected in 6 isolates, using the breakpoint of MIC > 8 ug/mL. Two of these isolates were resistant to tigecycline. Minocycline was tested only against S. maltophilia and A. baumannii and showed excellent activity against both. Conclusions: Fosfomycin seems to not be an option to treat infections due to the A. baumannii and S. maltophilia isolates according with EUCAST breakpoint, on the other hand, showed excellent activity against S. marcescens and K. pneumoniae. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Polymyxin B Fosfomycin Tigecycline Minocycline MDR gram-negatives
1. Background Gram-negative bacteria have become the main problem of nosocomial infections in the last years, with an important increasing of multidrug resistant (MDR) infections in a scenario of no new drug with action against these microorganisms in the near future [1e4].
* Corresponding author. Tel.: þ55 1130617030; fax: þ55 1130617043. E-mail address: [email protected] (S.F. Costa).
Among the gram-negative bacteria, Acinetobacter baumannii and carbapenem-resistant Enterobacteriaceae (CRE) are important emerging MDR in the last years, and polymyxins B and E (colistin) and tigecycline are the only treatment option against these microorganisms [5]. Other pathogen problem is Stenotrophomonas maltophilia that exhibits intrinsic and acquired resistance to a wide variety of antimicrobial agents [6]. So far, trimethoprimsulfamethoxazole is the drug of choice to treat infections caused by this microorganism, however, during the past few years resistance to this antibiotic has been reporting [7]. Serratia marcescens is also a potential emerging MDR that shows intrinsic resistance to several drugs including polymyxins B and E [8].
http://dx.doi.org/10.1016/j.jiac.2014.10.009 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
2
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
The aim of this study was to evaluate the in vitro susceptibility of MDR gram-negative bacteria to old drugs such as polymyxin B, minocycline and fosfomycin and new drugs such as tigecycline. 2. Methods One hundred and fifty-three isolates (47 A. baumannii resistant to carbapenem, quinolones and aminoglycosides, 48 S. maltophilia with 67% of resistance to levofloxacin and/or trimethoprimsulfamethoxazole, 8 S. marcescens and 50 Klebsiella pneumoniae resistant to carbapenem; aminoglycosides and quinolones) from 4 Brazilian hospitals (03 of state of S~ ao Paulo and one of State of Paran a) were evaluated. The minimum inhibitory concentrations (MICs) by standard broth microdilution according to CLSI criteria to the following drugs: minocycline (SigmaeAldrich e St. Louis, USA), ampicillin-sulbactam (PfizereCarabobo, Venezuela), tigecycline (Pfizer Inc., Wyeth Pharmaceuticals, Philadelphia, USA), and polymyxin B (U.S. Pharmacopeia e Rockville, USA) and fosfomycin (SigmaeAldrich e St. Louis, MO 63103, USA) by agar dilution were performed. The bacteria were identified using miniaturized method API 20E rieux, Marcy L'Etoile, France). All and API 20NE (6.0 version, bio-Me A. baumannii isolates, 3 K. pneumoniae isolates, 9 S. maltophilia isolates, 4 S. marcescens isolates were isolated from bloodstream infections totalizing 63 isolates. The MIC50 and MIC90 were determined and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [9], in addition the European Committee on Antimicrobial Susceptibility Testing EUCAST fosfomycin breakpoint for Pseudomonas spp. [10] was applied for Acinetobacter spp, the Enterobacteria Food and Drug Administration (FDA) criteria for tigecycline was used for Acinetobacter spp and S. maltophilia [11,12]. and the Pseudomonas spp polymyxin B CLSI criterion was used for S. maltophilia (9). PCR for different resistance genes for each genus were performed: adeB, blaoxa23, blaoxa51, blaoxa143,d blaIMP, blaVIM, blaSIMand blaNDM for A. baumannii and blaKPC and blaNDM for S. marcescens and K. pneumoniae [13e18]; sul-1, sul-2 and qnrMR for S. maltophilia [19,20] The reactions were performed with specific primers for each one according to the authors. Sequencing of DNA of the PCR products was conducted using the MegaBACE 1000, a DNA analysis system of capillary 96 with GE Healthcare technology. Sequencing reactions were carried out in
accordance with the Protocol to the MegaBACE 1000, Journal ET Dye Terminator using Kit (with Thermo Sequenase DNA polymerase II™) code US81090. The sequences were analyzed by Sequence Analyser software using the Base Caller Cimarron 3.12. The genetic sequence was compared with database available on the internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/). 3. Results A total of 153 isolates were evaluated. Table 1 presents detailed results, 52% of A. baumannii were resistant to ampicillinsulbactam, however, all susceptible to minocycline, polymyxin B and tigecycline. Regarding fosfomycin, 97.9% (46/47) of A. baumannii were resistant using the EUCAST criteria for Pseudomonas spp. and none using CLSI breakpoint criteria. All isolates harbored blaOxA51 (genbank number KF356388eKF356391), 70% blaOxA143 (genbank number KF601337eKF601346), 18% blaOxA23 (genbank number KF601347eKF601348), 8% blaIMP (genbank number KF381487eKF381490), 66% adeB gene (genbank number JN646776eJN646777) and none harbored blaNDM. No difference in the susceptibility profile of all tested drugs was found when compared the isolates of A. baumannii harboring oxacilinases or metalo-beta-lactamase genes. All K. pneumoniae and S. marcescens isolates harbored the blaKPC gene (genbank number KF285575eKF285585 and KF436493eKF436495). The majority of K. pneumoniae were susceptible to all drugs tested; however, polymyxin B resistance was detected in 6 isolates, using the breakpoint of MIC > 8 ug/mL. Two of these isolates were resistant to tigecycline. All isolates of S. marcescens were susceptible to fosfomycin and intermediate to tigecycline (Table 1). The S. maltophilia isolates showed 8% resistance for tigecycline, 19% for fosfomycin and only one was intermediate for minocycline, but for polymyxin B, 52% were resistant (using the CLSI breakpoint for Pseudomonas spp.). One isolate presented resistance for both tigecycline and fosfomycin, but was susceptible to minocycline. Eighty percent of S. maltophilia isolates harbored the qnrMR gene (genbank number HQ711553.1), 30% sul-1 (genbank number KF307595eKF307596) and 4% sul-2 gene (genbank number KF307597). Of the trimethoprim-sulfamethoxazole resistant isolates, 87% (20/23) were also resistant to fosfomycin using EUCAST breakpoint criteria for Pseudomonas spp. and 21.7% (5/23) by CLSI
Table 1 In vitro susceptibility test of several drugs against multi-drug resistant gram-negative bacteria isolated from 4 Brazilian hospitals (MIC50 and MIC90 in mg/mL).
Sulbactam-ampicilin
Range MIC50 MIC90 Minocycline Range MIC50 MIC90 Tigecycline Range MIC50 MIC90 Fosfomycin* Range MIC50 MIC90 Polymyxin B Range MIC50 MIC90 Resistance genes detected by PCR
Acinetobacter baumannii (N ¼ 47)
Stenotrophomonas maltophilia (N ¼ 48)
Serratia marcescens (N ¼ 8)
Klebsiella pneumonia (N ¼ 50)
4 to 64 16 32 <¼0.06 to 0.5 <¼0.06 0.25 0.25 to 4 0.5 1 32 to 128 64 128 <¼0.25 to 2 <¼0.25 0.5 70% blaoxa143 18% blaoxa23 8% blaIMP 66% adeB
NT NT NT <0.25 to 8 0.5 2 0.25 to 16 1 4 32 to 256 128 256 0.25 to 64 8 16 80% qnrMR 30% sul-1 4% sul-2
NT NT NT NT NT NT 2 to 16 4 4 16 to 32 32 32 16 >16 >16 100% blaKPC
NT NT NT NT NT NT 0.06 to 8 0.5 2 0.5 to 64 16 32 1 to >16 2 16 100% blaKPC
NT: not tested; The MIC of ampicillin-sulbactam; minocycline, tigecyclin and polymyxin B was performed by microdilution and the fosfomycin by agar dilution.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
criteria. Only one strain was resistant to trimethoprimsulfamethoxazole and tigecycline and that particular isolates was also resistant to fosfomycin (EUCAST breakpoint) and presented two resistance genes searched: sul-1 and qnrMR (Table 1). 4. Discussion Nowadays, with the increase of MDR gram-negative infections, older drugs such as polymyxins B and E (colistin) and fosfomycin have become options to treat infections caused by these agents. Few studies, however, had evaluated the in vitro activity of such drugs against well characterized MRD gram-negatives bacteria [1e4]. The present study evaluated the in vitro susceptibility of polymyxin B, fosfomycin, tigecycline, and minocycline against 153 MDR gram-negative bacteria. Minocycline was tested only against S. maltophilia and A. baumannii isolates, and showed excellent activity, all isolates were susceptible to this antibiotic. Regarding the others antibiotics, tigecycline in general showed good in vitro activity against the MDR gram-negative evaluated, except against S. marcescens (all isolates showed intermediate MIC) and some isolates of K. pneumoniae harboring KPC-2 and multidrug resistant S. maltophilia that were resistant. Generally, the polymyxins B and E show a good in vitro activity against the MDR gram-negative bacteria as the ones evaluated in the present study, except, against S. maltophilia e S. marcescens [21,22]. However, the susceptible profile depends on the breakpoint applied. In Brazil, the CLSI breakpoint criteria are the most frequent used [21,23]. Our data showed excellent activity of polymyxin B against A. baumannii harboring blaOxA143, blaOxA23 and blaIMP, all isolates were susceptible, and good activity against CRE harboring blaKPC-2, only 6 of 50 isolates were resistant to this antibiotic. Others studies have showed similar results [21,22]. Polymyxin B resistance, however, was identified in 6 of 50 K. pneumoniae isolates harboring KPC. Similar as previously described by Livermore et al. [24] that reported that only 3 of 52 Klebsiella spp. isolates were resistant to polymyxin E (colistin). Other drug that has been used recently as option to treat carbapenem resistant A. baumannii and CRE is tigecycline [5,25]. Our study showed a good in vitro activity of tigecycline against MDR gram-negative. Only S. maltophilia and S. marcescens showed MIC50 and MIC90 with intermediated sensibility. Few isolates of S. maltophilia, S. marcescens and K. pneumoniae were resistant according with the breakpoint of FDA. Authors have showed a good in vitro activity of tigecycline against carbapenem-resistant A. baumannii [25,26]. Resistance to tigecycline during treatment of Acinetobacter spp. infections due to efflux pump have been reported [27]. A study conducted by Behera et al. (2009) [28], showed that all K. pneumoniae and E. coli were susceptible to tigecycline; on the other hand 58% of A. baumannii were resistant (using the Enterobacteriaceae breakpoint). In our study, even the A. baumannii isolates that harbored adeB gene were susceptible to tigecycline. Fosfomycin has been recently used to treat MDR nosocomial infections [29]. However, there are few clinical studies with this antibiotic and no breakpoints consensus. Fosfomycin's suscepti de l’Antibility criteria range from less than 32 mg/mL (Comite te Française de Microbiologie) to less than biogramme de la Socie 128 mg/mL (British Society for Antimicrobial Chemotherapy). The most used criteria is the CLSI breakpoint to E. coli in urinary tract [30]. Thus, data in vitro of fosfomycin are need. The present study is the second study that evaluated the activity of fosfomycin against MDR gram-negative isolated in Brazil [31]. Most of the Enterobacteriaceae isolates were susceptible to this drug. All S. marcescens evaluated were susceptible using both CLSI and EUCAST criteria, however, the K. pneumoniae isolates showed a
3
broad range of MIC values, and S. maltophilia presented the higher MIC for fosfomycin, and 19% of S. maltophilia isolates were resistant to fosfomycin. The susceptible profile was different when using CLSI or EUCAST. Using CLSI criteria, the MIC50 achieved an intermediate breakpoint and with the EUCAST (breakpoint for Pseudomonas spp.) it would be already resistant. Regarding A. baumannii the breakpoint criteria used changes substantially the result. Using the CLSI breakpoint, none of the 47 isolates was resistant. However, with the EUCAST breakpoint, 97.9% (46/47) were resistant. In the present study, minocycline seems to be the drug with better in vitro activity against MDR S. maltophilia; however, the clinical experience with this antibiotic is still anecdotic. This study has several limitations. The in vitro results could not reflect the clinical behavior. Studies with a bigger sampling can show a different results and it can represent only a local susceptibility profile. In conclusion, in general, tigecycline showed good in vitro activity against the MDR gram-negative evaluated, except against S. maltophilia and S. marcescens. All isolates of A. baumannii were susceptible to polymyxin B, however, resistance was detected among K. pneumoniae. Fosfomycin showed high MIC to A. baumannii and S. maltophilia according with EUCAST breakpoint; on the other hand, it presented excellent activity against S. marcescens and K. pneumoniae harboring KPC. For MDR S. maltophilia, minocycline seems to be the drug with better in vitro activity. These results suggest that these drugs have a therapeutic potential against infections caused by MDR gram-negatives that can be useful in the future. Conflict of interest None. References [1] Livermore DM. Has the era of untreatable infections arrived? J Antimicrob Chemother 2009;64:i29e36. [2] Paksu MS, Paksu S, Karadag A, Sensoy G, Asilioglu N, Yildizdas D, et al. Old agent, new experience: colistin use in the paediatric intensive care unitea multicentre study. Int J Antimicrob Agents 2012;40:140e4. [3] Czihal P, Knappe D, Fritsche S, Zahn M, Berthold N, Piantavigna S, et al. Api88 is a novel antibacterial designer peptide to treat systemic infections with multidrug-resistant gram-negative pathogens. ACS Chem Biol 2012;20: 1281e91. [4] Cho YS, Yim H, Yang HT, Hur J, Chun W, Kim JH, et al. Use of parenteral colistin for the treatment of multiresistant gram-negative organisms in major burn patients in South Korea. Infection 2012;40:27e33. [5] Ku K, Pogue JM, Moshos J, Bheemreddy S, Wang Y, Bhargava A, et al. Retrospective evaluation of colistin versus tigecycline for the treatment of Acinetobacter baumannii and/or carbapenem-resistant Enterobacteriaceae infections. Am J Infect Control 2012;40:983e7. [6] Brooke JS. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 2012;25:2e41. [7] Hu LF, Chang X, Ye Y, Wang ZX, Shao YB, Shi W, et al. Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1 integron. Int J Antimicrob Agents 2011;37:230e4. E, Galisteo GJ, Jover L, Vinuesa T, Villa TG, Vin ~ as M. Comparison of [8] Fuste antibiotic susceptibility of old and current Serratia. Future Microbiol 2012;7: 781e6. [9] Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: 23rd informational supplement (M100eS23) CLSI. Wayne, PA: Clinical and Laboratory Standards Institute; 2013. [10] EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.0 (2013). EUCAST; 2013. Available at: http://www.eucast.org/ clinical_breakpoints/ [accessed 18.04.12]. [11] FDA. U.S. Food and Drug Administration. Tygacil (tigecycline) for injection. http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm228387.htm [accessed 18.04.12]. [12] FDA. U.S. Food and Drug Administration. Tygacil (tigecycline) iv injection label. http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/ 021821s021lbl.pdf [accessed 18.04.12]. [13] Lin L, Ling BD, Li XZ. Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
4
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
[14]
[15] [16]
[17]
[18]
[19] [20]
[21]
[22]
resistant clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex. Int J Antimicrob Agents 2009;33:27e32. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2006;27:351e3. Chen Y, Zhou Z, Jiang Y, Yu Y. Emergence of NDM-1-producing Acinetobacter baumannii in China. J Antimicrob Chemother 2011;66:1255e9. Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H. OXA-143, a novel carbapenem-hydrolyzing class D beta-lactamase in Acinetobacter baumannii. Antimicrobial Agents Chemother 2009;53:5035e8. Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, et al. Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 2007;45:544e7. Mostachio AK, Levin AS, Rizek C, Rossi F, Zerbini J, Costa SF. High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil. Int J Antimicrob Agents 2012;39: 396e401. Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006;70:296e316. S anchez MB, Hern andez A, Rodríguez-Martínez JM, Martínez-Martínez L, Martínez JL. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of QNR determinants. BMC Microbiol 2008;8:148. Gales AC, Jones RN, Sader HS. Contemporary activity of colistin and polymyxin B against a worldwide collection of gram-negative pathogens: results from the SENTRY antimicrobial surveillance program (2006e09). J Antimicrob Chemother 2011;66:2070e4. Lim TP, Tan TY, Lee W, Sasikala S, Tan TT, Hsu LY, et al. In-vitro activity of polymyxin B, rifampicin, tigecycline alone and in combination against carbapenem-resistant Acinetobacter baumannii in Singapore. PLoS One 2011;6:e18485.
[23] Nicodemo AC, Araujo MR, Ruiz AS, Gales AC. In vitro susceptibility of Stenotrophomonas maltophilia isolates: comparison of disc diffusion, Etest and agar dilution methods. J Antimicrob Chemother 2004;53:604e8. [24] Livermore DM, Warner M, Mushtaq S, Doumith M, Zhang J, Woodford N. What remains against carbapenem-resistant Enterobacteriaceae? Evaluation of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline. Int J Antimicrob Agents 2011;37:415e9. [25] 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;3:215. [26] Liao CH, Kung HC, Hsu GJ, Lu PL, Liu YC, Chen CM, et al. In-vitro activity of tigecycline against clinical isolates of Acinetobacter baumannii in Taiwan determined by the broth microdilution and disk diffusion methods. Int J Antimicrob Agents 2008;32:192e6. pez M, Ruiz de Alegría C, Ferna ndez-Cuenca F,Martínez[27] Rumbo C, Gato E, Lo Martínez L, Vila J, et al. Contribution of efflux pumps, porins, and b-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii. Antimicrobial Agents Chemother 2013;57:5247e57. [28] Behera B, Das A, Mathur P, Kapil A, Gadepalli R, Dhawan B. Tigecycline susceptibility report from an Indian tertiary care hospital. Indian J Med Res 2009;129:446e50. [29] Karageorgopoulos DE, Wang R, Yu XH, Falagas ME. Fosfomycin: evaluation of the published evidence on the emergence of antimicrobial resistance in gramnegative pathogens. J Antimicrob Chemother 2012;67:255e68. [30] Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum betalactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis 2010;10:43e50. [31] Perdig~ ao-Neto LV, Oliveira MS, Rizek CF, Carrilho CM, Costa SF, Levin AS. Susceptibility to fosfomycin of multiresistant gram-negative bacteria and performance of different susceptibility testing methods: preparing for clinical use. Antimicrobial Agents Chemother 2013;57.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original article
In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives Camila Rizek a, Juliana Rosa Ferraz a, Inneke Marie van der Heijden a, Mauro Giudice a, Anna Karina Mostachio a, Jorge Paez a, Claudia Carrilho b, Anna Sara Levin a, Silvia F. Costa a, * rias da Faculdade de Medicina da Universidade de Sa ~o Paulo, Av. Dr. En LIM-54, Departamento de Doenças Infecciosas e Parasita eas de Carvalho Aguiar 500, 1 andar, sala 112, CEP05403-000 Sao Paulo, SP, Brazil , Brazil University Hospital of University of Londrina, Parana
a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 July 2014 Received in revised form 1 October 2014 Accepted 15 October 2014 Available online xxx
Background: The aim of this study was to evaluate the in vitro susceptibility of MDR gram-negatives bacteria to old drugs such as polymyxin B, minocycline and fosfomycin and new drugs such as tigecycline. Methods: One hundred and fifty-three isolates from 4 Brazilian hospitals were evaluated. Forty-seven Acinetobacter baumannii resistant to carbapenens harboring adeB, blaOxA23, blaOxA51, blaOxA143 and blaIMP genes, 48 Stenotrophomonas maltophilia including isolates resistant to levofloxacin and/or trimethoprim-sulfamethoxazole harboring sul-1, sul-2 and qnrMR and 8 Serratia marcescens and 50 Klebsiella pneumoniae resistant to carbapenens harboring blaKPC-2 were tested to determine their minimum inhibitory concentrations (MICs) by microdilution to the following drugs: minocycline, ampicillinsulbactam, tigecycline, and polymyxin B and by agar dilution to fosfomycin according with breakpoint criteria of CLSI and EUCAST (fosfomycin). In addition, EUCAST fosfomycin breakpoint for Pseudomonas spp. was applied for Acinetobacter spp and S. maltophilia, the FDA criteria for tigecycline was used for Acinetobacter spp and S. maltophilia and the Pseudomonas spp polymyxin B CLSI criterion was used for S. maltophilia. Results: Tigecycline showed the best in vitro activity against the MDR gram-negative evaluated, followed by polymyxin B and fosfomycin. Polymyxin B resistance among K. pneumoniae was detected in 6 isolates, using the breakpoint of MIC > 8 ug/mL. Two of these isolates were resistant to tigecycline. Minocycline was tested only against S. maltophilia and A. baumannii and showed excellent activity against both. Conclusions: Fosfomycin seems to not be an option to treat infections due to the A. baumannii and S. maltophilia isolates according with EUCAST breakpoint, on the other hand, showed excellent activity against S. marcescens and K. pneumoniae. © 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: Polymyxin B Fosfomycin Tigecycline Minocycline MDR gram-negatives
1. Background Gram-negative bacteria have become the main problem of nosocomial infections in the last years, with an important increasing of multidrug resistant (MDR) infections in a scenario of no new drug with action against these microorganisms in the near future [1e4].
* Corresponding author. Tel.: þ55 1130617030; fax: þ55 1130617043. E-mail address: [email protected] (S.F. Costa).
Among the gram-negative bacteria, Acinetobacter baumannii and carbapenem-resistant Enterobacteriaceae (CRE) are important emerging MDR in the last years, and polymyxins B and E (colistin) and tigecycline are the only treatment option against these microorganisms [5]. Other pathogen problem is Stenotrophomonas maltophilia that exhibits intrinsic and acquired resistance to a wide variety of antimicrobial agents [6]. So far, trimethoprimsulfamethoxazole is the drug of choice to treat infections caused by this microorganism, however, during the past few years resistance to this antibiotic has been reporting [7]. Serratia marcescens is also a potential emerging MDR that shows intrinsic resistance to several drugs including polymyxins B and E [8].
http://dx.doi.org/10.1016/j.jiac.2014.10.009 1341-321X/© 2014, Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
2
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
The aim of this study was to evaluate the in vitro susceptibility of MDR gram-negative bacteria to old drugs such as polymyxin B, minocycline and fosfomycin and new drugs such as tigecycline. 2. Methods One hundred and fifty-three isolates (47 A. baumannii resistant to carbapenem, quinolones and aminoglycosides, 48 S. maltophilia with 67% of resistance to levofloxacin and/or trimethoprimsulfamethoxazole, 8 S. marcescens and 50 Klebsiella pneumoniae resistant to carbapenem; aminoglycosides and quinolones) from 4 Brazilian hospitals (03 of state of S~ ao Paulo and one of State of Paran a) were evaluated. The minimum inhibitory concentrations (MICs) by standard broth microdilution according to CLSI criteria to the following drugs: minocycline (SigmaeAldrich e St. Louis, USA), ampicillin-sulbactam (PfizereCarabobo, Venezuela), tigecycline (Pfizer Inc., Wyeth Pharmaceuticals, Philadelphia, USA), and polymyxin B (U.S. Pharmacopeia e Rockville, USA) and fosfomycin (SigmaeAldrich e St. Louis, MO 63103, USA) by agar dilution were performed. The bacteria were identified using miniaturized method API 20E rieux, Marcy L'Etoile, France). All and API 20NE (6.0 version, bio-Me A. baumannii isolates, 3 K. pneumoniae isolates, 9 S. maltophilia isolates, 4 S. marcescens isolates were isolated from bloodstream infections totalizing 63 isolates. The MIC50 and MIC90 were determined and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) [9], in addition the European Committee on Antimicrobial Susceptibility Testing EUCAST fosfomycin breakpoint for Pseudomonas spp. [10] was applied for Acinetobacter spp, the Enterobacteria Food and Drug Administration (FDA) criteria for tigecycline was used for Acinetobacter spp and S. maltophilia [11,12]. and the Pseudomonas spp polymyxin B CLSI criterion was used for S. maltophilia (9). PCR for different resistance genes for each genus were performed: adeB, blaoxa23, blaoxa51, blaoxa143,d blaIMP, blaVIM, blaSIMand blaNDM for A. baumannii and blaKPC and blaNDM for S. marcescens and K. pneumoniae [13e18]; sul-1, sul-2 and qnrMR for S. maltophilia [19,20] The reactions were performed with specific primers for each one according to the authors. Sequencing of DNA of the PCR products was conducted using the MegaBACE 1000, a DNA analysis system of capillary 96 with GE Healthcare technology. Sequencing reactions were carried out in
accordance with the Protocol to the MegaBACE 1000, Journal ET Dye Terminator using Kit (with Thermo Sequenase DNA polymerase II™) code US81090. The sequences were analyzed by Sequence Analyser software using the Base Caller Cimarron 3.12. The genetic sequence was compared with database available on the internet (BLAST-http://www.ncbi.nlm.nhi.gov/blast/). 3. Results A total of 153 isolates were evaluated. Table 1 presents detailed results, 52% of A. baumannii were resistant to ampicillinsulbactam, however, all susceptible to minocycline, polymyxin B and tigecycline. Regarding fosfomycin, 97.9% (46/47) of A. baumannii were resistant using the EUCAST criteria for Pseudomonas spp. and none using CLSI breakpoint criteria. All isolates harbored blaOxA51 (genbank number KF356388eKF356391), 70% blaOxA143 (genbank number KF601337eKF601346), 18% blaOxA23 (genbank number KF601347eKF601348), 8% blaIMP (genbank number KF381487eKF381490), 66% adeB gene (genbank number JN646776eJN646777) and none harbored blaNDM. No difference in the susceptibility profile of all tested drugs was found when compared the isolates of A. baumannii harboring oxacilinases or metalo-beta-lactamase genes. All K. pneumoniae and S. marcescens isolates harbored the blaKPC gene (genbank number KF285575eKF285585 and KF436493eKF436495). The majority of K. pneumoniae were susceptible to all drugs tested; however, polymyxin B resistance was detected in 6 isolates, using the breakpoint of MIC > 8 ug/mL. Two of these isolates were resistant to tigecycline. All isolates of S. marcescens were susceptible to fosfomycin and intermediate to tigecycline (Table 1). The S. maltophilia isolates showed 8% resistance for tigecycline, 19% for fosfomycin and only one was intermediate for minocycline, but for polymyxin B, 52% were resistant (using the CLSI breakpoint for Pseudomonas spp.). One isolate presented resistance for both tigecycline and fosfomycin, but was susceptible to minocycline. Eighty percent of S. maltophilia isolates harbored the qnrMR gene (genbank number HQ711553.1), 30% sul-1 (genbank number KF307595eKF307596) and 4% sul-2 gene (genbank number KF307597). Of the trimethoprim-sulfamethoxazole resistant isolates, 87% (20/23) were also resistant to fosfomycin using EUCAST breakpoint criteria for Pseudomonas spp. and 21.7% (5/23) by CLSI
Table 1 In vitro susceptibility test of several drugs against multi-drug resistant gram-negative bacteria isolated from 4 Brazilian hospitals (MIC50 and MIC90 in mg/mL).
Sulbactam-ampicilin
Range MIC50 MIC90 Minocycline Range MIC50 MIC90 Tigecycline Range MIC50 MIC90 Fosfomycin* Range MIC50 MIC90 Polymyxin B Range MIC50 MIC90 Resistance genes detected by PCR
Acinetobacter baumannii (N ¼ 47)
Stenotrophomonas maltophilia (N ¼ 48)
Serratia marcescens (N ¼ 8)
Klebsiella pneumonia (N ¼ 50)
4 to 64 16 32 <¼0.06 to 0.5 <¼0.06 0.25 0.25 to 4 0.5 1 32 to 128 64 128 <¼0.25 to 2 <¼0.25 0.5 70% blaoxa143 18% blaoxa23 8% blaIMP 66% adeB
NT NT NT <0.25 to 8 0.5 2 0.25 to 16 1 4 32 to 256 128 256 0.25 to 64 8 16 80% qnrMR 30% sul-1 4% sul-2
NT NT NT NT NT NT 2 to 16 4 4 16 to 32 32 32 16 >16 >16 100% blaKPC
NT NT NT NT NT NT 0.06 to 8 0.5 2 0.5 to 64 16 32 1 to >16 2 16 100% blaKPC
NT: not tested; The MIC of ampicillin-sulbactam; minocycline, tigecyclin and polymyxin B was performed by microdilution and the fosfomycin by agar dilution.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
criteria. Only one strain was resistant to trimethoprimsulfamethoxazole and tigecycline and that particular isolates was also resistant to fosfomycin (EUCAST breakpoint) and presented two resistance genes searched: sul-1 and qnrMR (Table 1). 4. Discussion Nowadays, with the increase of MDR gram-negative infections, older drugs such as polymyxins B and E (colistin) and fosfomycin have become options to treat infections caused by these agents. Few studies, however, had evaluated the in vitro activity of such drugs against well characterized MRD gram-negatives bacteria [1e4]. The present study evaluated the in vitro susceptibility of polymyxin B, fosfomycin, tigecycline, and minocycline against 153 MDR gram-negative bacteria. Minocycline was tested only against S. maltophilia and A. baumannii isolates, and showed excellent activity, all isolates were susceptible to this antibiotic. Regarding the others antibiotics, tigecycline in general showed good in vitro activity against the MDR gram-negative evaluated, except against S. marcescens (all isolates showed intermediate MIC) and some isolates of K. pneumoniae harboring KPC-2 and multidrug resistant S. maltophilia that were resistant. Generally, the polymyxins B and E show a good in vitro activity against the MDR gram-negative bacteria as the ones evaluated in the present study, except, against S. maltophilia e S. marcescens [21,22]. However, the susceptible profile depends on the breakpoint applied. In Brazil, the CLSI breakpoint criteria are the most frequent used [21,23]. Our data showed excellent activity of polymyxin B against A. baumannii harboring blaOxA143, blaOxA23 and blaIMP, all isolates were susceptible, and good activity against CRE harboring blaKPC-2, only 6 of 50 isolates were resistant to this antibiotic. Others studies have showed similar results [21,22]. Polymyxin B resistance, however, was identified in 6 of 50 K. pneumoniae isolates harboring KPC. Similar as previously described by Livermore et al. [24] that reported that only 3 of 52 Klebsiella spp. isolates were resistant to polymyxin E (colistin). Other drug that has been used recently as option to treat carbapenem resistant A. baumannii and CRE is tigecycline [5,25]. Our study showed a good in vitro activity of tigecycline against MDR gram-negative. Only S. maltophilia and S. marcescens showed MIC50 and MIC90 with intermediated sensibility. Few isolates of S. maltophilia, S. marcescens and K. pneumoniae were resistant according with the breakpoint of FDA. Authors have showed a good in vitro activity of tigecycline against carbapenem-resistant A. baumannii [25,26]. Resistance to tigecycline during treatment of Acinetobacter spp. infections due to efflux pump have been reported [27]. A study conducted by Behera et al. (2009) [28], showed that all K. pneumoniae and E. coli were susceptible to tigecycline; on the other hand 58% of A. baumannii were resistant (using the Enterobacteriaceae breakpoint). In our study, even the A. baumannii isolates that harbored adeB gene were susceptible to tigecycline. Fosfomycin has been recently used to treat MDR nosocomial infections [29]. However, there are few clinical studies with this antibiotic and no breakpoints consensus. Fosfomycin's suscepti de l’Antibility criteria range from less than 32 mg/mL (Comite te Française de Microbiologie) to less than biogramme de la Socie 128 mg/mL (British Society for Antimicrobial Chemotherapy). The most used criteria is the CLSI breakpoint to E. coli in urinary tract [30]. Thus, data in vitro of fosfomycin are need. The present study is the second study that evaluated the activity of fosfomycin against MDR gram-negative isolated in Brazil [31]. Most of the Enterobacteriaceae isolates were susceptible to this drug. All S. marcescens evaluated were susceptible using both CLSI and EUCAST criteria, however, the K. pneumoniae isolates showed a
3
broad range of MIC values, and S. maltophilia presented the higher MIC for fosfomycin, and 19% of S. maltophilia isolates were resistant to fosfomycin. The susceptible profile was different when using CLSI or EUCAST. Using CLSI criteria, the MIC50 achieved an intermediate breakpoint and with the EUCAST (breakpoint for Pseudomonas spp.) it would be already resistant. Regarding A. baumannii the breakpoint criteria used changes substantially the result. Using the CLSI breakpoint, none of the 47 isolates was resistant. However, with the EUCAST breakpoint, 97.9% (46/47) were resistant. In the present study, minocycline seems to be the drug with better in vitro activity against MDR S. maltophilia; however, the clinical experience with this antibiotic is still anecdotic. This study has several limitations. The in vitro results could not reflect the clinical behavior. Studies with a bigger sampling can show a different results and it can represent only a local susceptibility profile. In conclusion, in general, tigecycline showed good in vitro activity against the MDR gram-negative evaluated, except against S. maltophilia and S. marcescens. All isolates of A. baumannii were susceptible to polymyxin B, however, resistance was detected among K. pneumoniae. Fosfomycin showed high MIC to A. baumannii and S. maltophilia according with EUCAST breakpoint; on the other hand, it presented excellent activity against S. marcescens and K. pneumoniae harboring KPC. For MDR S. maltophilia, minocycline seems to be the drug with better in vitro activity. These results suggest that these drugs have a therapeutic potential against infections caused by MDR gram-negatives that can be useful in the future. Conflict of interest None. References [1] Livermore DM. Has the era of untreatable infections arrived? J Antimicrob Chemother 2009;64:i29e36. [2] Paksu MS, Paksu S, Karadag A, Sensoy G, Asilioglu N, Yildizdas D, et al. Old agent, new experience: colistin use in the paediatric intensive care unitea multicentre study. Int J Antimicrob Agents 2012;40:140e4. [3] Czihal P, Knappe D, Fritsche S, Zahn M, Berthold N, Piantavigna S, et al. Api88 is a novel antibacterial designer peptide to treat systemic infections with multidrug-resistant gram-negative pathogens. ACS Chem Biol 2012;20: 1281e91. [4] Cho YS, Yim H, Yang HT, Hur J, Chun W, Kim JH, et al. Use of parenteral colistin for the treatment of multiresistant gram-negative organisms in major burn patients in South Korea. Infection 2012;40:27e33. [5] Ku K, Pogue JM, Moshos J, Bheemreddy S, Wang Y, Bhargava A, et al. Retrospective evaluation of colistin versus tigecycline for the treatment of Acinetobacter baumannii and/or carbapenem-resistant Enterobacteriaceae infections. Am J Infect Control 2012;40:983e7. [6] Brooke JS. Stenotrophomonas maltophilia: an emerging global opportunistic pathogen. Clin Microbiol Rev 2012;25:2e41. [7] Hu LF, Chang X, Ye Y, Wang ZX, Shao YB, Shi W, et al. Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1 integron. Int J Antimicrob Agents 2011;37:230e4. E, Galisteo GJ, Jover L, Vinuesa T, Villa TG, Vin ~ as M. Comparison of [8] Fuste antibiotic susceptibility of old and current Serratia. Future Microbiol 2012;7: 781e6. [9] Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: 23rd informational supplement (M100eS23) CLSI. Wayne, PA: Clinical and Laboratory Standards Institute; 2013. [10] EUCAST. Breakpoint tables for interpretation of MICs and zone diameters. Version 3.0 (2013). EUCAST; 2013. Available at: http://www.eucast.org/ clinical_breakpoints/ [accessed 18.04.12]. [11] FDA. U.S. Food and Drug Administration. Tygacil (tigecycline) for injection. http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm228387.htm [accessed 18.04.12]. [12] FDA. U.S. Food and Drug Administration. Tygacil (tigecycline) iv injection label. http://www.accessdata.fda.gov/drugsatfda_docs/label/2010/ 021821s021lbl.pdf [accessed 18.04.12]. [13] Lin L, Ling BD, Li XZ. Distribution of the multidrug efflux pump genes, adeABC, adeDE and adeIJK, and class 1 integron genes in multiple-antimicrobial-
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009
4
C. Rizek et al. / J Infect Chemother xxx (2014) 1e4
[14]
[15] [16]
[17]
[18]
[19] [20]
[21]
[22]
resistant clinical isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus complex. Int J Antimicrob Agents 2009;33:27e32. Woodford N, Ellington MJ, Coelho JM, Turton JF, Ward ME, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int J Antimicrob Agents 2006;27:351e3. Chen Y, Zhou Z, Jiang Y, Yu Y. Emergence of NDM-1-producing Acinetobacter baumannii in China. J Antimicrob Chemother 2011;66:1255e9. Higgins PG, Poirel L, Lehmann M, Nordmann P, Seifert H. OXA-143, a novel carbapenem-hydrolyzing class D beta-lactamase in Acinetobacter baumannii. Antimicrobial Agents Chemother 2009;53:5035e8. Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, et al. Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 2007;45:544e7. Mostachio AK, Levin AS, Rizek C, Rossi F, Zerbini J, Costa SF. High prevalence of OXA-143 and alteration of outer membrane proteins in carbapenem-resistant Acinetobacter spp. isolates in Brazil. Int J Antimicrob Agents 2012;39: 396e401. Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006;70:296e316. S anchez MB, Hern andez A, Rodríguez-Martínez JM, Martínez-Martínez L, Martínez JL. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of QNR determinants. BMC Microbiol 2008;8:148. Gales AC, Jones RN, Sader HS. Contemporary activity of colistin and polymyxin B against a worldwide collection of gram-negative pathogens: results from the SENTRY antimicrobial surveillance program (2006e09). J Antimicrob Chemother 2011;66:2070e4. Lim TP, Tan TY, Lee W, Sasikala S, Tan TT, Hsu LY, et al. In-vitro activity of polymyxin B, rifampicin, tigecycline alone and in combination against carbapenem-resistant Acinetobacter baumannii in Singapore. PLoS One 2011;6:e18485.
[23] Nicodemo AC, Araujo MR, Ruiz AS, Gales AC. In vitro susceptibility of Stenotrophomonas maltophilia isolates: comparison of disc diffusion, Etest and agar dilution methods. J Antimicrob Chemother 2004;53:604e8. [24] Livermore DM, Warner M, Mushtaq S, Doumith M, Zhang J, Woodford N. What remains against carbapenem-resistant Enterobacteriaceae? Evaluation of chloramphenicol, ciprofloxacin, colistin, fosfomycin, minocycline, nitrofurantoin, temocillin and tigecycline. Int J Antimicrob Agents 2011;37:415e9. [25] 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;3:215. [26] Liao CH, Kung HC, Hsu GJ, Lu PL, Liu YC, Chen CM, et al. In-vitro activity of tigecycline against clinical isolates of Acinetobacter baumannii in Taiwan determined by the broth microdilution and disk diffusion methods. Int J Antimicrob Agents 2008;32:192e6. pez M, Ruiz de Alegría C, Ferna ndez-Cuenca F,Martínez[27] Rumbo C, Gato E, Lo Martínez L, Vila J, et al. Contribution of efflux pumps, porins, and b-lactamases to multidrug resistance in clinical isolates of Acinetobacter baumannii. Antimicrobial Agents Chemother 2013;57:5247e57. [28] Behera B, Das A, Mathur P, Kapil A, Gadepalli R, Dhawan B. Tigecycline susceptibility report from an Indian tertiary care hospital. Indian J Med Res 2009;129:446e50. [29] Karageorgopoulos DE, Wang R, Yu XH, Falagas ME. Fosfomycin: evaluation of the published evidence on the emergence of antimicrobial resistance in gramnegative pathogens. J Antimicrob Chemother 2012;67:255e68. [30] Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum betalactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis 2010;10:43e50. [31] Perdig~ ao-Neto LV, Oliveira MS, Rizek CF, Carrilho CM, Costa SF, Levin AS. Susceptibility to fosfomycin of multiresistant gram-negative bacteria and performance of different susceptibility testing methods: preparing for clinical use. Antimicrobial Agents Chemother 2013;57.
Please cite this article in press as: Rizek C, et al., In vitro activity of potential old and new drugs against multidrug-resistant gram-negatives, J Infect Chemother (2014), http://dx.doi.org/10.1016/j.jiac.2014.10.009