In Vitro Synergistic Activity of Colistin and Ceftazidime or Ciprofloxacin Against Multidrug-Resistant Clinical Strains of Pseudomonas aeruginosa

Abstract

Infections caused by multidrug resistant (MDR) Pseudomonas aeruginosa are difficult to treat. Antibiotic development is dwindling in recent years. In order to develop new alternate therapies antimicrobial activity of different antibiotic combinations are being studied in vitro and in vivo. Sub-inhibitory concentrations of colistin were tested in combination with ceftazidime or ciprofloxacin by the checkerboard method against 25 MDR strains of P. aeruginosa. Synergy was observed for ceftazidime or ciprofloxacin antibiotic combinations with colistin among 73.3% of MDR3 (R_{AMK, GEN, TOB} R_CAZ R_CIP) strains and 100% of MDR4 (R_{AMK, GEN, TOB} R_CAZ R_CIP R_TZP) strains. 6.6% strains of MDR3 and 14.3% strains of MDR5 (R_{AMK, GEN, TOB} R_CAZ R_CIP R_TZP R_IPM) phenotypes were inhibited by colistin and ceftazidime alone and 6.6% strains of MDR3 phenotypes were inhibited by colistin and ciprofloxacin alone. For the remaining strains, though synergy was not observed, significant reduction in minimum inhibitory concentration was evident. The results of this study are significant as sub-inhibitory concentrations of colistin have an advantage of reducing in vivo toxicity. These findings need further evaluation for clinical use.

Introduction

Over time, the treatment of infections caused by Pseudomonas aeruginosa has become very challenging to the clinicians. This is because of its extraordinary capacity in developing resistance to nearly all available antibiotics. The ability to develop resistance results from the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistant determinants, particularly those encoding metallo-beta-lactamases (MBLs) or extended-spectrum beta-lactamases. These resistant determinants are frequently transferred along with genes encoding aminoglycoside-modifying enzymes.⁶ In the last years, there have been no promising new antibiotics in the drug development pipeline to treat infections caused by multiresistant P. aeruginosa. Clinicians and microbiologists have been therefore forced to reappraise the clinical value of colistin.

Colistin, a relatively old polymyxin antibiotic was in clinical use in the 1950s and was withdrawn from treatment because of its toxicity. Recently, on-therapy resistance with colistin monotherapy has been reported.¹³ Colistin sulfate (polymyxin E) is one of the cyclic polypeptide antibiotics known to interact primarily with LPS and phospholipids present at the outer membrane of Gram-negative bacteria with also the ability to disturb permeability of the cytoplasmic membrane. This may allow the entry of hydrophobic and/or large molecules, which ultimately leads to leakage of cellular contents.¹⁵ Therefore, colistin can be used in combination with other antibiotics to obtain a synergistic effect. Synergy is defined as significantly greater activity provided by two agents combined, compared with that provided by the sum of each agent alone. Combined antimicrobial therapies can be used in clinical infections caused by bacterial strains that are susceptible to one or more antibiotics, or are resistant to all available antimicrobials.¹¹

Often, researchers may encounter high minimum inhibitory concentration (MIC) for antibiotics that are uninterpretable. The ability of the bacteria to survive at a high concentration of antibiotics clearly indicates that these strains have very strong mechanisms for overcoming the antibiotic effect and hence increasing the dose over time certainly will not be beneficial in treatment. Alternate treatment options with antibiotic combinations may be used for effective management of such infections. Hence, experiments were carried out to explore the potential for synergistic activity of colistin with ceftazidime or ciprofloxacin against multidrug-resistant (MDR) clinical strains of P. aeruginosa.

Materials and Methods

Bacterial strains and antibiotic susceptibility

Twenty-five MDR clinical strains of P. aeruginosa were selected for this study. The antibiotic susceptibility testing of these strains was done by Kirby-Bauer disc diffusion on Mueller-Hinton agar plates and the results were interpreted according to clinical and laboratory standards institute (CLSI, 2012) criteria.² Antimicrobials used in this test were amikacin (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), piperacillin-tazobactam (100/10 μg), tobramycin (10 μg), and colistin (10 μg) (HiMedia). P. aeruginosa ATCC 27853 was used as a quality control strain.

Detection of metallo-beta-lactamase production

The strains were tested for MBL production by the double-disc synergy test (DDST) according to Qu et al.¹⁰ P. aeruginosa C-10 (VIM-2) and C-7 (IMP-7) were used as positive control strains.

Determination of MIC

The MIC of colistin, ciprofloxacin, and ceftazidime for each strain was determined by the broth macrodilution method⁹ using cation-adjusted Mueller-Hinton broth (HiMedia) and was interpreted following the CLSI standards. The concentrations of ceftazidime 0.5–2,048 mg/L, ciprofloxacin 0.25–64 mg/L, and colistin 0.25–4 mg/L were tested. P. aeruginosa ATCC 27853 was used as a control strain.

In vitro evaluation of antibiotic combination synergy

The effect of the combination of colistin with ciprofloxacin or ceftazidime was studied by the checker board broth microdilution assay in microtiter plates.⁹ Colistin was tested at its MIC and subinhibitory concentrations of ½, ¼, and ⅛ MIC with fractions of MICs of ceftazidime and ciprofloxacin. For strains showing an exceptionally high MIC for ceftazidime (>2,048 mg/L), a concentration equal to 2,048 mg/L was used as the cutoff value. Similarly, when the ciprofloxacin MIC exceeded 64 mg/L, a concentration equal to 64 mg/L was used as the cutoff value. To evaluate the effect of combinations, the fractional inhibitory concentration (FIC) was calculated for each antibiotic in each combination. The following formulae were used to calculate the FIC index: FIC of drug A=MIC of drug A in combination/MIC of drug A alone, FIC of drug B=MIC of drug B in combination/MIC of drug B alone, and FIC index=FIC of drug A+FIC of drug B. Synergy was defined as FIC index of ≤0.5, indifference was defined as FIC index of >0.5, but ≤4 and antagonism was defined as FIC index of >4.⁹

Results

Antibiotic resistance pattern of the strains

The prevalence of P. aeruginosa infection in our tertiary care hospital from August 2011 to July 2012 was found to be 20.7% (106/512).³ Among these, 25 strains were MDR having three different patterns, as shown in Table 1. All the isolates were susceptible to colistin and resistant to ciprofloxacin. Twenty-four isolates were resistant to ceftazidime and one isolate (PA16) was intermediately susceptible. The MICs of colistin, ceftazidime, and ciprofloxacin for all the strains are given in Table 1. Among the strains selected for the present study, only one (PA111) tested positive for MBL in the DDST.

Table 1.

Minimum Inhibitory Concentration of Colistin, Ceftazidime, and Ciprofloxacin and Fractional Inhibitory Concentration Results for the Antibiotic Combinations Tested Against Twenty-Five Multidrug-Resistant Strains and Control Strain (ATCC 27853) of Pseudomonas aeruginosa Tested In Vitro by the Checker-Board Method

			MIC (mg/L)			MIC (mg/L) in combination		FIC			MIC (mg/L) in combination		FIC
MDR phenotype	No. of strains	Strain ID	COL	CAZ	CIP	COL	CAZ	COL	CAZ	COL+CAZ	COL	CIP	COL	CIP	COL+CIP
MDR 3	15	PA11	2	2048	16	0.5	256	0.25	0.125	0.375	0.5	2	0.25	0.125	0.375
R_AMK,GEN,TOB R_CAZ R_CIP		PA12	2	256	32	0.5	32	0.25	0.125	0.375	0.5	8	0.25	0.25	0.5
		PA16	2	16	64	0.25	4	0.125	0.25	0.375	0.25	16	0.125	0.25	0.375
		PA18	2	64	32	0.5	16	0.25	0.25	0.5	0.5	4	0.25	0.25	0.5
		PA22	2	128	64	0.5	32	0.25	0.25	0.5	0.5	8	0.25	0.25	0.5
		PA32	2	2048	4	0.5	256	0.25	0.125	0.375	0.5	0.5	0.25	0.125	0.375
		PA58	2	2048	16	0.5	256	0.25	0.125	0.375	0.5	4	0.25	0.25	0.5
		PA62	2	>2048	64	0.5	512	0.25	0.25	0.5	0.5	8	0.25	0.125	0.375
		PA86	2	64	>64	0.5	16	0.25	0.25	0.5	0.5	16	0.25	0.25	0.5
		PA89	2	>2048	>64	0.5	256	0.25	0.125	0.375	0.5	16	0.25	0.25	0.5
		PA92	2	512	>64	0.5	128	0.25	0.25	0.5	0.5	16	0.25	0.25	0.5
		PA98	2	512	32	0.5	64	0.25	0.125	0.375	1	16	0.5	0.5	1
		PA101	2	>2048	8	1	256	0.5	0.125	0.625	0.5	2	0.25	0.25	0.5
		PA133	2	2048	>64	0.5	1024	0.25	0.5	0.75	1	1	0.5	0.015	0.5015
		PA156	2	>2048	16	2	256	1	0.125	1.125	1	4	0.5	0.25	0.75
MDR 4	3	PA40
R_AMK,GEN,TOB R_CAZ R_CIP R_TZP		PA916	2	>2048	32	0.5	256	0.25	0.125	0.375	0.5	8	0.25	0.25	0.5
		PA977	2	>2048	16	0.5	256	0.25	0.125	0.375	0.5	4	0.25	0.25	0.5
MDR 5	7	PA57
R_AMK,GEN,TOBR_CAZ R_CIP R_TZP R_IPM		PA93	1	>2048	>64	1	256	1	0.125	1.125	0.5	1	0.5	0.015	0.5015
		PA959
		PA21
		PA24	1	>2048	>64	1	256	1	0.125	1.125	0.5	1	0.5	0.015	0.5015
		PA103
		PA111^MBL	1	>2048	>64	0.25	512	0.25	0.25	0.5	0.5	1	0.5	0.015	0.5015
MS	1	ATCC27853	2	2	0.5	2	2	1	1	2	2	0.5	1	1	2

Resistance interpreted according to current CLSI breakpoints (CLSI, 2012). MDR, multidrug-resistant—the number that appears after the MDR designation indicates the number of different antimicrobial classes to which isolates are resistant (e.g., MDR 3 indicates P. aeruginosa isolates that are resistant to at least 1 antimicrobial agent from 3 different classes.

AMK, amikacin; GEN, gentamicin; TOB, tobramycin; CAZ, ceftazidime; CIP, ciprofloxacin; TZP, piperacillin-tazobactam; IPM, imipenem; MS, multisensitive); MIC, minimum inhibitory concentration.

FIC for antibiotic combinations

Synergy was observed for ceftazidime or ciprofloxacin antibiotic combinations with colistin among 73.3% of MDR3 (R_{AMK, GEN, TOB} R_CAZ R_CIP) strains and 100% of MDR4 (R_{AMK, GEN, TOB} R_CAZ R_CIP R_TZP) strains. 6.6% strains of MDR3 and 14.3% strains of MDR5 (R_{AMK, GEN, TOB} R_CAZ R_CIP R_TZP R_IPM) phenotypes were inhibited by colistin and ceftazidime alone and 6.6% strains of MDR3 phenotypes were inhibited by colistin and ciprofloxacin alone. For the remaining strains, although synergy was not observed, significant reduction in MIC was evident.

Discussion

Antibiotic resistance by P. aeruginosa is increasing at an alarming rate globally and particularly in India. Data from the Surveillance of antimicrobial resistance in India (SARI) study group, revealed 42.6% resistance to ceftazidime, imipenem, or meropenem.⁷ Published data from our hospital reveal 6.6% resistance to imipenem.³ Carbapenem-resistant P. aeruginosa are a major concern among hospitals in India and resistance to carbapenems is often mediated by the production of MBL.

It is unfortunate to note that a majority of the MDR strains selected in this study showed very high MIC exceeding clinically achievable levels. In such cases, priority was given to select an effective antibiotic combination so as to reduce the MIC of ceftazidime or ciprofloxacin to clinically achievable levels, giving due consideration for the toxicity of both the antibiotics in combination. The results from checkerboard assay revealed interesting synergistic activity of antibiotic combinations against P. aeruginosa strains.

Although both colistin combinations tested showed promising results against most of the MDR3 (73.3%) and all MDR4 P.aeruginosa strains, the colistin and ceftazidime combination was also effective against one additional strain of MDR3 (6.7%) and MDR5 (14.3%) phenotypes. The synergy observed may be due to the rapid permeabilization of the outer cell membrane, caused by colistin that allows enhanced penetration and activity of the other antibiotics in the combination.¹⁴ The interpretation of these results is limited with respect to MDR4 phenotype as only three strains were evaluated for the effect of antibiotic combinations. Gunderson et al. by a time-kill assay have shown an in vitro synergistic effect of colistin and ceftazidime on MDR P. aeruginosa and suggest that this combination may be effective on these isolates inspite of their elevated MIC.⁴ Timurkaynak et al. also observed an in vitro synergistic effect of the combination of colistin and rifampicin against MDR P. aeruginosa strains.¹⁴

In this study, 20% of MDR3 strains and 85.7% of MDR5 strains did not respond to colistin and ceftazidime combinations. The failure of certain strains to respond to colistin and ceftazidime combinations may be due to the notoriety of the pathogen to possess a wide array of other resistance determinants such as the chromosomally encoded AmpC cephalosporinase, plasmid-mediated beta-lactamases (PSE, VEB, GES, IBC, OXA), the loss of the OprD outer membrane porin, and the multidrug efflux pumps. In a study conducted by Lim et al., one of the strains known to possess VEB1and OXA10 in the absence of MBL totally failed to respond to combinations of two and three antibiotics tested.⁵

The combination of ciprofloxacin and colistin had a poor killing effect on all imipenem resistant and 20% strains of MDR3 phenotype. This may be partially due to the binding between the two antibiotics or due to the colistin's mechanism of action which is mediated by the binding of cations necessary to stabilize the bacterial membrane. This may have been altered in the presence of the fluoroquinolone interfering with cations in the solution.³ However, the combination of colistin and ciprofloxacin reduced the MIC for colistin and ciprofloxacin in all these 20% strains and when colistin was present at half of its MIC value in the combination, the ciprofloxacin-resistant strains turned to be susceptible, despite the lack of synergy. Mitsugui et al. have proposed that even with the lack of synergy, the reduction in the MICs are important in such instances as it may be possible to administer these agents in adjusted doses, allowing effective treatment as well as minimizing possible adverse effects.⁸ Sader and Jones state, the fact that all the strains have MIC values in the susceptible range for at least one of the antibiotics when they are tested in combination, demonstrates the clinical potential of these antibiotic combinations for the treatment of infections caused by MDR P. aeruginosa.¹² However, 13.3% strains of MDR3 phenotype showed only a reduction in the MICs by two- or fourfold for ciprofloxacin. Combined effect of mutations in quinolone resistance determining regions and over expression of efflux pumps also contribute to elevation of MIC of ciprofloxacin among clinical strains of P. aeruginosa.¹ Hence, further studies are warranted to establish an association between various mechanisms of fluoroquinolone resistance and the effectiveness of antibiotic combinations in reducing the MIC of ciprofloxacin.

Conclusion

Evaluation of the synergistic effect using the combination of colistin and ceftazidime or ciprofloxacin revealed that colistin at ¼ of its MIC value reduced the MIC of the latter. This observation is significant as it offers an advantage of reducing in vivo toxicity. The clinical application of in vitro results must be considered with caution in view of the variable susceptibilities of P. aeruginosa strains to antibiotic combinations and the pharmacokinetic characteristics of the antibiotics. The rate of synergy of combinations varies according to the strains and is not strictly associated with susceptibility or resistance to imipenem. The higher reliability of fluoroquinolones over cephalosporins in treating P. aeruginosa infections, suggest that the combination of colistin and ciprofloxacin could be a promising alternative for serious infections caused by MDR P. aeruginosa. Owing to the strain diversity, it is essential that every strain undergoes antibiotic combination testing before selecting an appropriate therapeutic option. Added significance is the in vitro finding that a small percentage of MDR3 phenotype strains exhibited resistance to antibiotic combinations. This may contribute to treatment failure in a few cases. The possibility of the evolution of multiple resistance mechanisms among clinical strains of P. aeruginosa may be the cause that needs to be analyzed.

Footnotes

Acknowledgment

This work was funded by Yenepoya University seed grant (YU/seed grant/2011–16). Ms. Beena Benita D'Souza acknowledges the JRF fellowship YU/JRF/004. Ethical approval: Yenepoya University ethics committee YUEC 217/29/8/11.

Disclosure Statement

All authors report no conflicts of interest relevant to this article.

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