Abstract
Aim: Development of a non-toxic, fluorescent-based, agar system for the screening of overexpressed bacterial efflux pump systems with common, inexpensive UV accessories. Materials and Methods: Wild type Gram-negative and positive bacteria expressing intrinsic efflux pumps and their progeny that overexpress a specific efflux pump were selected for evaluation of efflux pump activity in a Mueller-Hinton agar, containing increasing concentrations of the non-toxic fluorescent chromophore acridine orange (AO). The method is based on the same principle as the first-generation ethidium bromide method, according to which the concentration of the fluorescent dye that first produces fluorescence of the overlying bacterial colony represents the maximum concentration of the dye that the bacterium can extrude. The higher the concentration needed to produce fluorescence, the greater the ability of the bacterial efflux pump to extrude the dye. Results: Progeny of Escherichia coli, Salmonella enterica serovar Enteritidis and Staphylococcus aureus that over-expressed a given efflux pump fluoresced (i.e. accumulated AO) at concentrations of AO that were much greater than the ones required for the emission of fluorescence by their corresponding wild-type counterpart which expressed an intrinsic efflux pump. Conclusion: The AO agar method readily identifies strains of Gram-negative and Gram-positive bacteria that overexpress efflux pump systems compared to their wild-type progeny.
- Multidrug resistance
- Gram-negative and Gram positive bacteria
- Escherichia coli
- Salmonella enterica serovar Enteritidis
- Staphylococcus aureus
- acridine orange (AO)
- agar method
- evaluation of efflux pump activity
Multidrug resistance of Gram-negative clinical isolates is primarily due to the overexpression of efflux pumps such as the AcrAB-Tol C pump that extrude the antimicrobial agent before it reaches its intended target (1). Although overexpression of the efflux pump of the Gram-positive clinical isolate is less common (2), in the case of methicillin resistant Staphylococcus aureus (MRSA), most of the clinical isolates overexpress their main efflux pump NorA (3, 4). As previously predicted by Stuart Levy (5), Staphylococcus aureus which have survived continuous exposure to detergents and biocides, used routinely in a hospital setting, overexpress efflux pumps that extrude these agents and thereby render these strains resistant to these agents (5), and to divalent cations (6), and also to many commonly used antibiotics (7). The efflux pump responsible for the aforementioned resistance is due to the presence of a plasmid that carries the gene for the Qac efflux pump whose origins still remain a mystery (8).
The screening of clinical isolates for overexpression of efflux pumps has been successfully conducted with the aid of the ethidium bromide (EB) agar method (9-10). The automated evaluation of efflux pump activity on a real-time basis has also been successfully conducted with the use of EB and the Corbette 3000 thermocycler that provides temperature controls affording physiologically relevant conditions (11-13). Although both methods are user friendly, many countries rigorously control the use and disposal of EB since it is a toxic and mutagenic agent (14). Consequently, we have considered the development of similar fluorescence based methods with fluorescent chromophores that are non-toxic.
Acridine Orange (AO; N,N,N’,N’-tetramethylacridine-3,6-diamine) is a cationic fluorescent chromophore used for vital staining of eukaryotic cells. It is cell permeable, and interacts with DNA and RNA by intercalation and electrostatic attractions, respectively. When bound to DNA, it is spectrally very similar to fluorescein, with an excitation maximum at 502 nm and an emission maximum at 525 nm (green). When it associates with RNA, the excitation maximum shifts to 460 nm (blue) and the emission maximum shifts to 650 nm (red). AO has been shown to be a substrate of bacterial efflux pumps (15-17). Because AO does not present with toxicity or mutagenicity when employed at concentrations used for the demonstration of efflux pumps or vital staining of eukaryotic cells, we selected AO as the substrate for the screening of bacteria that overexpress their efflux pumps compared to their wild-type counterparts.
Concentrations of acridine orange which first promote fluorescence of Gram-negative bacterial strains. Note that the bacterial strains that over-express an efflux pump (highlighted) require higher concentrations than their parental counterparts in order to produce fluorescence.
Materials and Methods
Materials. Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) were purchased in powder form from Difco (Madrid, Spain). AO was purchased from Sigma (Madrid, Spain).
Bacteria. The following bacteria were employed in this study: Escherichia coli AG100 K12 which has its main efflux pump acrAB intact, was generously provided by Professor H Nikaido, E. coli AG100A which has its main efflux pump gene acrAB deleted (ΔacrAB) and their progeny induced to high level resistance to tetracycline (TET) by prolonged and continuous exposure to increasing concentrations of the antibiotic (18), that result in the over-expression of genes that regulate and code for the AcrAB efflux pump (E. coli AG100TET8) and for the overexpression of the AcrEF efflux pump (E. coli AG100ATET8) (18). Salmonella enterica serovar Enteritidis NCTC 13349, the clinical strain Salmonella enterica serovar Enteritidis 104 and Salmonella enterica serovar Enteritidis 5408 were also used. Salmonella enterica serovar Enteritidis 104 and Salmonella enterica serovar Enteritidis 5408 have been exposed to increasing concentrations of ciprofloxacin (Salmonella enterica serovar Enteritidis 104CIP and Salmonella enterica serovar Enteritidis 5408CIP, respectively) and were overexpressing their AcrB transporter of the AcrAB-TolC efflux pump by 6-fold over that of their respective parents (19). These strains were generously provided by Professor S Fanning. Staphylococcus aureus wild-type ATCC 25923, S. aureus NCTC 25923 that was adapted to 50 mg/l of ethidium bromide (20) and the clinical strain HPV-107 Staphylococcus aureus (21) which contains a plasmid that carries the operon that codes for the Qac efflux pump (8) were also used as examples of Gram-positive bacteria. The HPV-107 strain was generously provided by Professor H. de Lencastre. MRSA1 clinical strain was previously identified and characterized in our laboratory (10, 23) and was also used in this work. All bacteria were cultured on MHA, colonies isolated and cultured over night in MHB at 37°C.
AO assay. Bacteria were grown on MHB until mid-log phase (optical density of 0.6 at 600 nm). Single sterile swabs were dipped into these cultures, the swabs touched the inner sides of the culture tubes to remove excess fluid and then plates of MHA containing increasing concentrations of AO were swabbed from the centre to the periphery of the plate. The cartwheel design of swabbing affords as many as 12 individual streaks of culture (9). An example of this design, with four strains, is provided in Figure 1. The swabbed plates were incubated for 16 h and fluorescence of the overlying bacterial streak was determined with a source of UV illumination. A hand-held UV lamp suffices. The concentration of AO that first produced evidence of green fluorescence of the bacterial streak was recorded for each strain. The plates were then photographed with an electronic camera, maintained at a constant distance from the UV illuminated plate.
Results
It was observed that parental strains that do not overexpress efflux pumps began to fluoresce at lower concentrations than those that acquired resistance by overexpression of their efflux systems.
Among the Gram-positive strains (Figure 1), it was observed that S. aureus NCTC 25923 and its progeny adapted to 50 mg/l of EB began to fluoresce at 5 mg/l of AO while the multidrug resistant clinical strains S. aureus HPV 107 and MRSA1 did not fluoresce at concentrations as high as 20 mg/l of AO.
Among the Gram-negative strains (Table 1), one can observe that S. enteritidis NCTC 13349, S. enteritidis 104, S. enteritidis 5408 and E. coli AG100A began to fluoresce at 1 mg/L of AO. The strain E. coli AG100 began to fluoresce at 5 mg/l and at 10 mg/l fluorescence can be observed for the strains with overexpression of efflux pumps, namely, E. coli AG100TET, S. enteritidis 104CIP and S. enteritidis 5408CIP. E. coli AG100ATET did not fluoresce at concentrations as high as 20 mg/l of AO. This latter strain lacks an AcrAB efflux pump and overexpresses its AcrEF efflux pump by more than 80-fold over than the one of its parental strain AG100A, which also lacks an AcrAB efflux pump (15).
Accumulation of acridine orange by Gram-positive bacteria Staphylococcus aureus strains cultured on agar containing increasing concentrations of the fluorescent chromophore.
Discussion
Our results demonstrate that AO is a suitable replacement for EB in the agar cartwheel method (9) for the identification of multidrug resistant strains that overexpress their efflux pumps. Since the replacement of EB with AO eliminates the risks associated with the use of EB, the AO agar method bypasses the restrictions that have limited the use of EB in many countries. The basic concept behind the AO agar method is identical to the one using EB agar, and allows rapid and relatively easy screening of large numbers of strains whose multidrug resistance is due to overexpression of an efflux pump system. The benefits afforded by the EB agar method, such as its simplicity, consumption of inexpensive reagents, the use of inexpensive accessories normally found in a clinical laboratory, the lack of need for expensive instrumentation, etc., are retained in the AO agar method, and therefore facilitate its use in a clinical microbiology laboratory.
Acknowledgements
A. Martins was supported by TÁMOP-4.2.1/B-09/1/KONV-2010-0005 – Creating the Center of Excellence at the University of Szeged supported by the European Union and co-financed by the European Regional Fund. L. Amaral was supported by BCC grant SFRH/BCC/51099/2010 provided by the Fundačão para a Ciência e a Tecnologia (FCT) of Portugal. This work was supported by EU-FSE/FEDER-PTDC/BIA-MIC/105509/2008 and EU-FSE/FEDERPTDC/SAU-FCF/102807/2008 from the Fundačão para a Ciência e a Tecnologia (FCT) of Portugal.
- Received November 28, 2011.
- Revision received December 21, 2011.
- Accepted January 4, 2012.
- Copyright © 2012 The Author(s). Published by the International Institute of Anticancer Research.
