In Vitro Activity of Minocycline Alone and in Combination with Cefoperazone-Sulbactam Against Carbapenem-Resistant Acinetobacter baumannii

Abstract

The aim of this study was to investigate the in vitro activity of minocycline combined with cefoperazone-sulbactam against carbapenem-resistant Acinetobacter baumannii (CRAB). A total of 53 nonduplicate CRAB were collected from inpatients of intensive care units in three hospitals in China from February 2009 to August 2011. Minimum inhibitory concentrations were determined by the broth microdilution method. The checkerboard method was used to determine whether combinations of the two agents act synergistically. Among the 53 A. baumannii, 64.2% of the isolates were susceptible to amikacin and 69.8% were susceptible to cefoperazone-sulbactam. More than 80% of isolates were resistant to the other agents and no isolates were found resistant to polymyxin B and minocycline. The combination of minocycline and cefoperazone-sulbactam demonstrated synergism in 39 isolates, partial synergism in 11 isolates, and indifference in 3 isolates. No antagonistic interactions were observed. Our study demonstrated that minocycline has good activity against CRAB and the combination of minocycline with cefoperazone-sulbactam had significant synergistic activity against these strains in vitro. The combination of minocycline and cefoperazone-sulbactam may be an alternative option for the treatment of infections caused by CRAB.

Introduction

A cinetobacter spp., especially Acinetobacter baumannii, has emerged worldwide as an important nosocomial and opportunistic pathogen, particularly in intensive care units (ICUs).³ The association of A. baumannii with pneumonia, urinary tract infections, bacteremia, wound infections, and meningitis has been well described.^6,12 Treatment of these pathogens is very difficult due to the high rate of multidrug resistance.⁸ Imipenem used to be considered as the “gold standard” therapy for severe infections. However, growing resistance to this agent has been reported in many countries.^3,10 In addition, the development of new antibiotic has not kept pace with the remarkable ability of A. baumannii to acquire different antibiotic resistance determinants. Therefore, the search of old antibiotics showing good synergistic activity against A. baumannii is probably of great implication.

Minocycline is an old antibiotic used for the treatment of common bacterial infections. Numerous of studies have indicated minocycline as a promising antibiotic against A. baumannii infections.^1,5 Sulbactam possesses the greatest intrinsic bactericidal activity against A. baumannii isolates of all β-lactamase inhibitors. Sulbactam alone is not available for clinical use, although sulbactam associated with cefoperazone (Sulperazon^®) is available and used clinically in China. Many antimicrobial combinations have been studied in vitro or in vivo against A. baumannii.^15,17 However, whether cefoperazone-sulbactam could enhance the bactericidal activity of minocycline against A. baumannii has not yet been determined. The present study aimed to evaluate the interaction between minocycline and cefoperazone-sulbactam against A. baumannii using the microdilution checkerboard method.

Materials and Methods

Bacterial isolates

A total of 53 nonduplicate carbapenem-resistant A. baumannii (CRAB) isolates were collected from inpatients of ICU in three hospitals of China from February 2009 to August 2011. The isolates were collected from tracheal aspirate cultures (n=29), sputum (n=14), bronchoalveolar lavage cultures (n=6), urine (n=2), and blood (n=1). The source of one isolate was not available. The isolates were identified with VITEK-2 system and API 20NE system (bioMérieux). Isolates exhibiting minimum inhibitory concentrations (MICs) for imipenem or meropenem equal to or exceeding 16 μg/ml were included in this study. Escherichia coli ATCC25922 was used as a control strain.

Antimicrobial agents

Laboratory-grade standard powders of amikacin, ceftazidime, cefepime, levofloxacin, rifampin, gentamicin, imipenem, meropenem, tigecycline, and minocycline were obtained from the National Institute for the Control of Pharmaceutical and Biological Products of China. Cefoperazone-sulbactam and piperacillin/tazobactam were obtained from Pfizer, Inc., and polymyxin B was obtained from Sigma, Inc. Stock solutions of all antimicrobial agents were prepared as recommended by the Clinical and Laboratory Standards Institute (CLSI).⁷

Antimicrobial susceptibility and synergy testing

MICs of antimicrobial agents were determined using the broth microdilution method according to the instructions of CLSI. The MIC of cefoperazone-sulbactam (≤16/8 mg/L) was defined as susceptible to Acinetobacter spp. based several reports, although no CLSI standard for sulbactam concentration currently exists.

Synergy tests of minocycline and cefoperazone-sulbactam were performed in 96-well microtiter plates, containing twofold dilutions of both antimicrobial agents dispensed in a checkerboard format. Cefoperazone-sulbactam was dispensed in the horizontal wells (concentration range, 1–512 μg/ml). Minocycline was dispended in the vertical wells (concentration range, 0.125–16 μg/ml). The last column was used for quality control. A bacterial suspension was dispended by 1:10 dilution after 6 hr of shaking culture (∼5×10⁵ CFU/ml of bacteria). Bacterial suspension (100 μl) was added into the wells and incubated for 20 hr at 35°C.

The fractional inhibitory concentration index (FICI) was calculated for each drug alone and in combination.

The FICI was calculated according to the formula⁹: FICI=FIC_A (MIC of drug A in combination/MIC of drug A alone) + FIC_B (MIC of drug B in combination/MIC of drug B alone). The FICI values were interpreted as synergy (FICI≤0.5), partial synergy (FICI>0.5 and <1), additive (FICI=1), indifference (FICI>1 and <4), and antagonism (FICI≥4).

Results

Table 1 shows the MIC distribution of 13 antimicrobial agents against 53 isolates. Among the 53 A. baumannii, 64.2% of the isolates were susceptible to amikacin and 69.8% were susceptible to cefoperazone-sulbactam. More than 80% of isolates were resistant to the other agents and no isolates were found resistant to polymyxin B and minocycline. A total of 86.8% of the isolates were susceptible to minocycline and 13.2% to cefoperazone-sulbactam when used alone. The MIC₅₀ and MIC₉₀ for the tested strains were 4 and 8 μg/ml for minocycline and 128/64 μg/ml and 256/128 μg/ml for cefoperazone-sulbactam, respectively, when used alone. However, the MIC₅₀ and MIC₉₀ were 0.5 and 2 μg/ml for minocycline and 16/8 and 64/32 μg/ml for cefoperazone-sulbactam, respectively, when used in combination (Table 2).

Table 1.

Minimum Inhibitory Concentration (μg/ml) of 13 Antimicrobial Agents for 95 Strains of Carbapenem-Resistant Acinetobacter baumannii

Antimicrobial agent	MIC₅₀	MIC₉₀	%R
Polymyxin B	0.5	1	0
Amikacin	64	>256	64.2
Ceftazidime	128	>256	96.2
Cefepime	64	>256	98.1
Levofloxacin	64	>256	100
Rifampin	4	16	N/A
Gentamicin	256	>256	100
Piperacillin/tazobactam	256/4	>256/4	92.5
Tigecycline	1	2	N/A
Imipenem	64	128	100
Meropenem	32	256	100
Minocycline	4	8	0
Cefoperazone/sulbactam	128/64	256/128	69.8

MIC, minimum inhibitory concentration; %R, percent resistant; N/A, breakpoint not available.

Table 2.

Minimum Inhibitory Concentration (μg/ml) of Minocycline Combined with Cefoperazone-Sulbactam, Alone and in Combination, Among 53 Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

	Alone				Combination
Antimicrobial agent	MIC range	MIC₅₀	MIC₉₀	%S	MIC range	MIC₅₀	MIC₉₀	%S
Minocycline	1–8	4	8	86.8%	0.25–4	0.5	2	100.0%
Cefoperazone/sulbactam	8/4–256/128	128/64	256/128	7.6%	2/1–128/64	16/8	64/32	43.4%

%S, percent susceptible.

The interaction of minocycline and cefoperazone-sulbactam in combination (FICI≤0.5) was shown to be synergistic in 39 isolates (73.6%), partially synergistic (FICI>0.5 and <1) in 11 (20.8%) isolates, and indifferent (FICI>1 and <4) in 3 isolates (5.7%). No antagonistic interactions were observed. Figure 1 shows the cumulative inhibition ratio (CIR) of minocycline and/or cefoperazone-sulbactam against 53 CRAB isolates. The CIR curve was moved to the left when the two agents were used in combination, indicating the potential utility of this combination.

FIG. 1.

The cumulative inhibition ratio (CIR) of minocycline (MN) and/or cefoperazone-sulbactam (CS) against carbapenem-resistant Acinetobacter baumannii (n=53). The concentration of CS refers to the value of cefoperazone.

Discussion

The emergence of CRAB presents a serious challenge in the clinic. Infection due to multidrug resistance A. baumannii strains can lead to treatment failure, and is associated with an increased risk of death and overall cost of care.

Cefoperazone-sulbactam has good activity against A. baumannii and plays an important role in the control of Acinetobacter spp. strains. Yamaguchi et al. reported 99.5% in vitro susceptibility of Acinetobacter spp. strains in Japan to cefoperazone-sulbactam.¹⁸ Many studies have indicated that minocycline has activity against A. baumannii resistant to doxycycline or tetracycline and imipenem.^2,4 Moreover, minocycline exhibits favorable pharmacokinetic and pharmacodynamic features. It achieves very high serum and tissue concentrations that are above the MIC₉₀ values for A. baumannii.¹ Several studies have demonstrated that minocycline acts synergistically effect in combination with other antibiotics. Tan et al. identified 92.3% synergism of minocycline combined with colistin against imipenem-resistant A. baumannii using a time-kill method.^11,15

In the current study, most of the included isolates were expressed as multidrug resistant. Polymyxin and tigecycline B were the most active agents with MIC₉₀ of 1 and 2 μg/ml, respectively. Polymyxin has been described as the hopeful agent of last resort recent years.^13,15,16 Nevertheless, neurotoxicity and nephrotoxicity remain as main concerns for extensive use of polymyxins in an era of multidrug-resistant pathogens. Tigecycline is a new expanded-broad-spectrum glycylcycline and it has potent activity against Acinetobacter species. However, mean maximum serum concentration of tigecycline is only 0.63 μg/ml in normal dose due to the large volume of distribution, despite the outcome of tigecycline's in vitro activity is exciting.¹⁴

About 86.8% of CRAB isolates were susceptible to minocycline and only 13.2% were susceptible to cefoperazone-sulbactam when used alone. However, all of included strains were susceptible to minocycline and 43.4% to cefoperazone-sulbactam when used in combination. The MIC₉₀ of cefoperazone-sulbactam was reduced significantly from 256/128 μg/ml when used in combination. These data indicated synergy or partial synergy of the combination of the two agents against most A. baumannii isolates. No antagonistic interaction was observed.

In conclusion, the results of our study demonstrated that the CRAB isolates were resistant to most of the common antimicrobial agents. Minocycline, polymyxin B, and tigecycline have good activity against CRAB. The combination of minocycline with cefoperazone-sulbactam has significant synergistic activity against these strains in vitro. Thus, the combination of minocycline and cefoperazone-sulbactam may be an alternative option for the effective treatment of CRAB infections, although further studies are required to verify this synergistic activity in vivo and to elucidate the mechanisms underlying the effect.

Footnotes

Disclosure Statement

No competing financial interests exist.

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