Colistin Increases the Cidal Activity of Antibiotic Combinations Against Multidrug-Resistant Klebsiella pneumoniae : An In Vitro Model Comparing Multiple Combination Bactericidal Testing at One Peak Serum Concentration and Time

Abstract

The lack of treatment for multidrug-resistant (MDR) Enterobacteriaceae often leads to the use of double or triple antibiotic combinations to increase the option of clinical success. This study analyzes multiple combination bactericidal testing (MCBT) to screen double and triple antibiotic combinations, at standard peak serum concentration, for bactericidal activity against 21 MDR Klebsiella pneumoniae isolates. This method was compared with time–killing curves. The full bactericidal activity against all strains was obtained only by adding colistin. MCBT has a potential to become a rapid method for testing multiple antibiotic combinations for MDR microorganisms when colistin is used, providing clinicians with in vitro cidal data within 48 hr of strain isolation.

Introduction

Multidrug-resistant (MDR) Klebsiella pneumoniae is an increasingly important nosocomial pathogen that causes significant morbidity and mortality rates in critically ill patients.^18,21 The limited treatment options available for these patients because of the paucity of new agents available for clinical use have led to the use of double or triple antibiotic combinations to increase the option of clinical success.^5,10,17 Reports of antibiotic combination in in vitro and in vivo studies are increasing in the literature because of the global spread of MDR Gram-negative bacteria, and colistin (also known as polymyxin E), a previously abandoned polymyxin antibiotic, has re-emerged as a last-resort therapeutic option often used in combination therapy together with tigecycline and other drugs.^15,23 Thus, there has been widespread interest in using antimicrobial agents in combination to expand the antimicrobial spectrum, prevent the emergence of resistance, reduce toxicity, and provide synergistic activity.

There are multiple methodologies for synergy testing: among them checkerboard and time–killing (TK) curves are the most used even if time-consuming and labor-intensive²⁴ and multiple combination bactericidal testing (MCBT) have been used most often for cystic fibrosis isolates.² MCBT combines two or three drugs in microtiter wells and is relatively easy to perform in clinical microbiology laboratories.

In this study we report the use of an MCBT, which screens double and triple antibiotic combinations at peak serum level, comparing it with the TK method used as a reference.

Materials and Methods

Twenty-one well-characterized K. pneumoniae strains were used in this study. All strains were MDR or extensively drug-resistant clones, possessing different mechanisms of carbapenem resistance including, n.2 VIM-1, n.2 OXA-48, and n.17 K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains belonging to STs 258, 512, 101, and 307.^12,19 The strains were also selected on the basis of colistin resistance (12 out 21).

The following antibiotics were used in double and triple combinations for MCBT and the antibiotic concentrations were chosen on the basis of peak serum levels after standard intravenous administration: meropenem (120 mg/L) + ertapenem (70 mg/L) and/or with colistin sulfate (Sigma Chemical, St. Louis, MO) (20 mg/L); rifampin (6 mg/L) + tigecycline (0.9 mg/L) and/or with colistin (20 mg/L); and rifampin (6 mg/L) + colistin (20 mg/L).^4,7,22,27,28 For the same antibiotics, minimum inhibitory concentrations (MICs) were determined by microdilution in cation-adjusted Mueller-Hinton broth as recommended by CLSI 2012⁶ and results were interpreted according to EUCAST 2014 data.⁸

MCBT procedures were performed in triplicate according to previously published methods.¹ Plates were incubated at 35°C for 24 hr and wells were examined for turbidity. The contents of nonturbid wells at 24 hr were subcultured by streaking 10 μl of suspension onto a Mueller-Hinton cation-adjusted agar plate, which was then incubated for 24 hr at 35°C and examined for 99.9% kill the next day (bactericidal activity).

The single antibiotics and combinations were also evaluated by TK curves, at the same MCBT concentrations, following standard methods. Bactericidal activity of single antibiotic was defined as a ≥3 log₁₀ CFU/ml reduction of initial inoculum after 24 hr of incubation. Synergy was defined as a decrease of ≥2 log₁₀ CFU/ml between the combination and the most active single agent at 24 hr.⁴

Results

In this work we examined 21 MDR K. pneumoniae strains, most of them resistant to carbapenems, with the exception of the 1 OXA-48-producing K. pneumoniae strain, showing reduced susceptibility to meropenem, and both VIM-1 K. pneumoniae strains, susceptible to ertapenem, meropenem, and colistin. The MIC of rifampin ranged from 16 to 32 mg/L. Twelve out of 21 isolates were resistant to colistin (MIC range 4–32 mg/L) and most of all strains were susceptible to tigecycline (MIC range 0.03–1 mg/L). The results of the TK curves, expressed as difference between the initial inoculum and the number of residual viable colonies (CFU/ml) at 24 hr for each antibiotic, are shown in Table 1.

Table 1.

Time–Killing Curves, Expressed as Difference Between the Initial Inoculum and the Number of Residual Viable Colonies (log₁₀ CFU/ml) After 24 hr of Incubation for Each Antibiotic at Peak Serum Concentration, and MIC Values Against 21 Strains of Klebsiella pneumoniae

	Single antibiotics mg/L
	Ertapenem		Meropenem		Colistin		Tigecycline		Rifampin
Strains (n)	70	MIC range	120	MIC range	20	MIC range	0.9	MIC range	6	MIC range
K. pneumoniae KPC-3 ST512 Col R (5)	4.2^4,a	256–512	4.4	512	4.3	16–32	0.5	0.5–1	3.2	32–64
K. pneumoniae KPC-3 ST307 Col R (2)	−3.4^b	16	−3.4	4–8	3.3	4	2.5	1–4	2.5	16–64
K. pneumoniae KPC-3 ST258 Col R (2)	4.2	32–128	4.3	16–64	4.5	8–32	3.2	1–2	3.7	64
K. pneumoniae KPC-3 ST258 Col S (2)	3.2	64–128	3.2	16–32	−5.2	0.06	3.3	1	3.2	32
K. pneumoniae KPC-2 ST101 Col S (3)	4.4	32–512	−5.5⁵	64–128	−3.2	0.03–0.12	4.2	1	4.2	32–64
K. pneumoniae KPC-2 ST101 Col R (3)	−3.2	256–1024	−4.2	32–256	4.2	4–16	3.5	0.25–2	2.2	32–64
K. pneumoniae VIM-1 Col S (2)	−5.2	0.012–1	−5.2	0.05–2	−5.3	0.015–0.06	0.3	0.03–0.25	4.2	32
K. pneumoniae OXA-48 Col S (2)	3.2	2–32	3.2	1–32	−3.2	0.015–0.03	0.2	2	2.2	32

Positive values indicate increase of growth.

Negative values indicate reduction of growth.

MIC, minimum inhibitory concentration; KPC, K. pneumoniae carbapenemase.

In time-killing analysis, neither tigecycline nor rifampin were cidal against all the studied MDR K. pneumoniae strains at peak serum concentration even for those strains that have peak serum values higher than MIC values. It is noteworthy that the activity of other antibiotics (ertapenem, meropenem, and colistin) was not bactericidal against some strains, although their peak serum concentration was higher than MIC values. The synergistic activity of various combinations tested and the agreement results between both methods are shown in Fig. 1. In the various combinations used, the double carbapenem combination was synergic for 9/21 strains on TK; full bactericidal activity against all strains was obtained only by adding colistin. The same was true for tigecycline+rifampin, cidal only against both VIM-1 K. pneumoniae strains: the addition of colistin rendered the combination strongly bactericidal. The rifampin+colistin combination was cidal against all strains with both methods. In all cases, adding colistin enhanced the effect of the double antibiotic combinations.

FIG. 1.

Agreement between time–killing and MCBT methods; synergistic activity of double and triple combinations against 21 Klebsiella pneumoniae. CST, colistin; ETP, ertapenem; MCBT, multiple combination bactericidal testing; MEM, meropenem; RIF, rifampin; TGC, tigecycline; TKC, TK curves.

All strains were tested by TK curves and MCBT. In all isolates the reference TK method revealed a high agreement of 100% with MCBT for rifampin+colistin, tigecycline+rifampin+colistin, and ertapenem+meropenem+colistin.

Of the 21 MDR K. pneumoniae strains, there was only a 43% concordance for ertapenem+meropenem whereas the MCBT and TK results agreed in 16 of the 21 isolates for tigecycline+rifampin. The best agreements were with combinations that included colistin.

Results of our study suggest that (1) MCBT is an easy and realistic method to study in vitro activity of combination therapies by using standard serum concentration of antibiotics, providing clinicians with in vitro cidal data within 48–72 hr of strain isolation; (2) MCBT can be used in place of TK curves because these methods showed the same discriminatory power and reproducibility (Fig. 1); (3) adding colistin as a third antibiotic to a nonbactericidal double-drug combination increased the likelihood of bactericidal effects; (4) these combinations combined with colistin are bactericidal in strains with a complex profile of resistance, including the same antibiotics combined.

Conclusions

Severe MDR infections are an increasing problem, and in areas where MDR microorganisms are endemic, the use of combination therapy with colistin has been an option in the treatment of serious infections.^11,14 However, evaluation of in vitro susceptibility testing methods for colistin has shown testing errors with various methods.^9,13,16 EUCAST recommends testing colistin with an MIC method with respect to the disk diffusion method.⁸ In agreement with results recently published, the broth microdilution method is considered more sensitive with respect to the gradient test.^9,16,18

Due to the limited options for the treatment of MDR K. pneumoniae, we report on the in vitro activity of the colistin combined with the most used drugs, showing improved antibacterial activity as demonstrated by different authors^20,25 in an attempt to assess synergy among two and three antibiotics using a simple and reproducible method and to compare the agreement between MCBT and TK curves using 21 clinical isolates of MDR K. pneumoniae.

Numerous methods used to detect in vitro synergy between antibiotics have been described: the gradient test and recently the two-dimensional gradient technique.^3,26 Both are relatively easy tests to routinely perform in the clinical microbiology laboratory.

Methodologically, MCBT is a rapid method requiring 48 hr to give results to the clinician, it is an easy method to use, requiring nonexpensive resources, and is easily performed in the laboratory. On the basis of our results, the MCBT method appears to be a possible alternative to TK curves because, using the peak serum concentration, we obtained the results in half the time with respect to TK curves.

In this context, our model appears robust at identifying a promising combination for the treatment of MDR KPC-producing isolates.

Because only a modest number of isolates were included in this study, it will be necessary to perform further MCBT studies for the other antibiotic combinations against MDR microorganisms. Therefore, the future direction of our work will aim to determine the possible veracity of this technique by developing other antibiotic associations.

In conclusion, the performance of MCBT is reliable when colistin is used in triple combinations against carbapenem-resistant strains both susceptible and resistant to colistin.

Footnotes

Acknowledgment

The authors express their gratitude to Scientific Bureau of the University of Catania for language support.

This work was supported by PON funding no. 01_02589.

Disclosure Statement

No competing financial interests exist.

References

Aaron

S.D.

, Ferris

, Henry

D.A.

, et al. 2000. Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia. Am. J. Respir. Crit. Care Med., 161:1206–1212.

Aaron

S.D.

, Vandemheen

K.L.

, Ferris

, et al. 2005. Combination antibiotic susceptibility testing to treat exacerbations of cystic fibrosis associated with multiresistant bacteria: a randomized, double-blind, controlled clinical trial. Lancet, 366:463–471.

Bonapace

C.R.

, White

R.L.

, Friedrich

L.V.

, et al. 2000. Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time-kill, and checkerboard methods. Diagn. Microbiol. Infect. Dis., 38:43–50.

Bulik

C.C.

, and Nicolau

D.P.

2011. Double-carbapenem therapy for carbapenemase-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother., 55:3002–3004.

Ceccarelli

, Falcone

, Giordano

, et al. 2013. Successful ertapenem-doripenem combination treatment of bacteremic ventilator-associated pneumonia due to colistin-resistant KPC-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother., 57:2900–2901.

Clinical and Laboratory Standards Institute. 2012. Methods for dilution of antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—ninth edition. CLSI document M07-A9. Clinical and Laboratory Standards Institute, Wayne, PA.

Dalfino

, Puntillo

, Mosca

, et al. 2012. High-dose, extended-interval colistin administration in critically ill patients: is this the right dosing strategy?. A preliminary study. Clin. Infect. Dis., 54:1720–1726.

European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, version 4.0. EUCAST 2014, Växjö, Sweden.

Gales

A.C.

, Reis

A.O.

, and Jones

R.N.

2001. Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. J. Clin. Microbiol., 39:183–190.

10.

Giamarellou

, Galani

, Baziaka

, et al. 2013. Effectiveness of a double-carbapenem regimen for infections in humans due to carbapenemase-producing pandrug-resistant Klebsiella pneumoniae. Antimicrob. Agents Chemother., 57:2388–2390.

11.

Gomez

, Sanchez

, Gul

, et al. 2011. Polymyxin combination therapy and the use of serum bactericidal titers in the management of KPC-producing Klebsiella pneumoniae infections: a report of 3 cases. Case Rep. Med., 2011:659769.

12.

Gona

, Barbera

, Pasquariello

A.C.

, et al. 2014. In vivo multiclonal transfer of bla_KPC-3 from Klebsiella pneumoniae to Escherichia coli in surgery patients. Clin. Microbiol. Infect., 20:633–635.

13.

Hindler

J.A.

, and Humphries

R.M.

2013. Colistin MIC variability by method for contemporary clinical isolates of multidrug-resistant Gram-negative bacilli. J. Clin. Microbiol., 51:1678–1684.

14.

Hirsch

E.B.

, Guo

, Chang

K.T.

, et al. 2013. Assessment of antimicrobial combinations for Klebsiella pneumoniae carbapenemase-producing K. pneumoniae. J. Infect. Dis., 207:786–793.

15.

Humphries

R.M.

, Kelesidis

, Bard

J.D.

, et al. 2010. Successful treatment of panresistant Klebsiella pneumoniae pneumonia and bacteraemia with a combination of high-dose tigecycline and colistin. J. Med. Microbiol., 59:1383–1386.

16.

Lo-Ten-Foe

J.R.

, de Smet

, Diederen

B.M.W.

, et al. 2007. Comparative evaluation of the VITEK 2, disk diffusion, Etest, broth microdilution, and agar dilution susceptibility testing methods for colistin in clinical isolates, including heteroresistant Enterobacter cloacae and Acinetobacter baumannii strains. Antimicrob. Agents Chemother., 51:3726–3730.

17.

Meredith

G.J.

, Press

E.G.

, Nguyen

M.H.

, et al. 2012. The combination of doripenem and colistin is bactericidal and synergistic against colistin-resistant, carbapenemase-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother., 56:3395–3398.

18.

Mezzatesta

M.L.

, Caio

, Gona

, et al. 2014. Carbapenem and multidrug resistance in Gram-negative bacteria in a single centre in Italy: considerations on in vitro assay of active drugs. Int. J. Antimicrob. Agents, 44:112–116.

19.

Mezzatesta

M.L.

, Gona

, Caio

, et al. 2013. Emergence of an extensively drug-resistant ArmA- and KPC-2-producing ST101 Klebsiella pneumoniae clone in Italy. J. Antimicrob. Chemother., 68:1932–1934.

20.

Michail

, Labrou

, Pitiriga

, et al. 2013. Activity of tigecycline in combination with colistin, meropenem, rifampin, or gentamicin against KPC-producing Enterobacteriaceae in a murine thigh infection model. Antimicrob. Agents Chemother., 57:6028–6033.

21.

Munoz-Price

L.S.

, Poirel

, Bonomo

R.A.

, et al. 2013. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Dis., 13:785–796.

22.

Novelli

, Adembri

, Livi

, et al. 2005. Pharmacokinetic evaluation of meropenem and imipenem in critically ill patients with sepsis. Clin. Pharmacokinet., 44:539–549.

23.

Pournaras

, Vrioni

, Neou

, et al. 2011. Activity of tigecycline alone and in combination with colistin and meropenem against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae strains by time-kill assay. Int. J. Antimicrob. Agents, 37:244–247.

24.

Sopirala

M.M.

, Mangino

J.E.

, Gebreyes

W.A.

, et al. 2010. Synergy testing by Etest, microdilution checkerboard, and time-kill methods for pan-drug-resistant Acinetobacter baumannii. Antimicrob. Agents Chemother., 54:4678–4683.

25.

Tascini

, Tagliaferri

, Giani

, et al. 2013. Synergistic activity of colistin plus rifampin against colistin-resistant KPC-producing Klebsiella pneumoniae. Antimicrob. Agents Chemother., 57:3990–3993.

26.

van Belkum

, Halimi

, Bonetti

E.J.

, et al. 2015. Meropenem/colistin synergy testing for multidrug-resistant Acinetobacter baumannii strains by a two-dimensional gradient technique applicable in routine microbiology. J. Antimicrob. Chemother., 70:167–172.

27.

Yzerman

E.P.

, Boelens

H.A.

, Vogel

, et al. 1998. Efficacy and safety of teicoplanin plus rifampicin in the treatment of bacteraemic infections caused by Staphylococcus aureus. J. Antimicrob. Chemother., 42:233–239.

28.

Zhanel

G.G.

, Baudry

P.J.

, Tailor

, et al. 2009. Determination of the pharmacodynamic activity of clinically achievable tigecycline serum concentrations against clinical isolates of Escherichia coli with extended-spectrum beta-lactamases, AmpC beta-lactamases and reduced susceptibility to carbapenems using an in vitro model. J. Antimicrob. Chemother., 64:824–828.

Colistin Increases the Cidal Activity of Antibiotic Combinations Against Multidrug-Resistant Klebsiella pneumoniae : An In Vitro Model Comparing Multiple Combination Bactericidal Testing at One Peak Serum Concentration and Time–Kill Method