A high-throughput system to rapidly assess the intracellular replication of Staphylococcus aureus has been developed utilizing S . aureus transformed with a dual gfp-luxABCDE reporter operonunder the control of a growth-dependent promoter . Replication of tagged bacteria internalized into bovine mammary epithelial cells [MAC-T] could be measured by monitoring fluorescence and bioluminescence from the reporter operon following removal of extracellular bacteria from the plates . Bacterial replicationinside cells was confirmed by a novel ex vivo time-lapse confocal microscopic method . This assay of bacterial replication wasused to evaluate the efficacy of antibiotics which are commonlyused to treat staphylococcal infections . Not all antibioticstested were able to prevent intracellular replication of S.aureus and some were ineffective at preventing replication ofintracellular bacteria at concentrations above the MIC determinedfor bacteria in broth culture . Comparison of the fluorescenceand bioluminescence signals from the bacteria enabled effectson protein synthesis and metabolism to be discriminated andgave information on the entry of compounds into the eukaryoticcell, even if bacterial replication was not prevented . Elevatedresistance of S . aureus to antibiotics inside host cells increasesthe likelihood of selecting S . aureus strains which are resistantto commonly used antimicrobial agents within the intracellularniche . The approach presented directly assesses intracellularefficacy of antibiotics and provides an evidence-based approachto antibiotic selection for prescribing physicians and medicalmicrobiologists.

 
Staphylococcus aureus is a versatile pathogen with a broad host range whose infections are causing considerable alarm within the medical community due to the increasing emergence of antibiotic-resistant strains . Infections associated with this organism range from minor wound infections to more serious diseases, including endocarditis, osteomyelitis, and septic shock . These infections are often life-threatening, so there is a pressing need to increase our understanding of staphylococcal disease and develop new antistaphylococcal agents . As S . aureus is also known for its ability to induce long-lasting persistent infections, it is important to ascertain the mechanisms the organism uses to evade host immune responses.

S . aureus has not traditionally been considered an intracellular pathogen; however, it is now well documented that S . aureus can internalize and survive within a wide variety of mammalian cells [2, 4, 19] . It has been suggested that the ability ofS . aureus to reside in an intracellular niche enables long-termcolonization of the host and the maintenance of a chronic infectivestate [4, 19, 49] . Bacterial uptake is triggered through specificinteractions between microbial surface components recognizingadhesive matrix molecules [12, 36], which include fibronectinbinding proteins in the bacterial cell wall, and the host cellthat ultimately leads to the active internalization of bacteriainto an endosome . S . aureus is able to escape from this endosome,where it is free to replicate in the host cell cytoplasm [4,40, 51].

The expression of many S . aureus microbial surface components recognizing adhesive matrix molecules is downregulated upon induction of the of the accessory gene regulator locus [agr] [30, 39, 42] . The agr locus is a quorum sensing-regulated system activated by autoinducing peptide pheromone [21, 26] . The two divergent transcriptional units of the agr locus, RNAII and RNAIII, are under the control of the P2 and P3 promoters, respectively [reviewed in reference 33] . RNAII is a polycistronic mRNA codingfor four peptides: AgrB and AgrD, required for the synthesisand export of autoinducing peptide, and AgrA and AgrC, whichconstitute a classical two-component signal transduction system responsible for sensing and responding to the autoinducing peptide.

RNAIII is the effector molecule in the agr regulon, acting primarily at the level of gene transcription . It is the transcriptionof RNAIII that is responsible for upregulation of secreted virulence factors as well as the downregulation of surface proteins [29, 34] . Different S . aureus strains produce autoinducing peptideswith distinct structures . Strains can be grouped on this basisbecause they will activate the agr response of strains withinthe same group and inhibit the agr response of strains fromdifferent groups by competitive inhibition [21, 27, 33] . Thisinhibitory action of autoinducing peptides on quorum sensinghas identified them as potential novel therapeutic and anti-infectiveagents for S . aureus; indeed a synthetic analogue of autoinducingpeptide, [Ala⁵]AIP-1, has been shown to be a potent inhibitorof agr in all S . aureus agr groups [27].

The intracellular location of S . aureus has important implications for its antibiotic susceptibility . In order to treat intracellular infections, the antibiotic needs to be capable of penetrating the eukaryotic cell to a sufficiently high concentration tobe effective against the intracellular bacteria [46] . So, penetrationand subcellular localization of antibiotics within phagocyticcells as well as the possibility of their intracellular inactivationmust be considered when assessing antibiotic efficacy [15]. Little is known about the intracellular pharmacodynamics of available antibiotics and how their accumulation within thecell relates to their activity . The intracellular milieu caninfluence both the metabolism of the intracellular bacteriumand the activity of the antibiotic; for example, intracellularenzymes or pH may inactivate certain antibiotics.

One way in which bacterial localization, growth, and gene expression can be studied is through the use of reporter and marker genes. A number of reporters are available for such investigations,for example, green fluorescent protein [GFP] and bacterial luciferase [Lux], which have their own particular advantages and disadvantages. The structural genes that encode the proteins necessary forbacterial bioluminescence are encoded by a polycistronic operonconsisting of five genes, luxAB [luciferase] and luxCDE [fattyacid reductase] [17, 28] . The bioluminescence process requiresenergy, in the form of FMNH₂ obtained from the bacterium's metabolism,a long-chain aldehyde synthesized and recycled by LuxCDE andmolecular oxygen . As the production of bioluminescence fromrecombinant bacteria containing the lux genes depends on cells'being biochemically active, it can be assumed that any compoundthat impairs the biochemistry and thus compromises cellularviability will lead to a rapid reduction in luminescence . Theeffect of antimicrobial compounds on lux-containing recombinantbacteria can therefore be assessed rapidly [43, 44, 48].

Previously bioluminescence emitted by recombinant S . aureus expressing a gfp-luxABCDE reporter operon under the controlof a growth-dependent promoter was used to track bacterial replication within mammalian cells in a 24-well microplate format . The useof the GFP-Lux dual reporter system provided GFP fluorescenceas an internal control for the luciferase signal and also facilitatedanalysis of fixed specimens by fluorescence microscopy duringthe infection process [40].

We have further developed this reporter-based approach to measure intracellular replication of S . aureus and utilized time-lapse confocal microscopy to confirm that the reporter signal is derived from intracellular staphylococci . The assay has been successfully applied to study the efficacy of commonly used therapeutic agents against intracellular S . aureus.

 
Bacterial strains and plasmids. The bacterial strains used throughout the study were S . aureus8325-4 [30], S . aureus RN6390 [32], and S . aureus RN6911 [22]. The plasmid pSB2030 [P_xylA::gfp-luxABCDE] confers chloramphenicolresistance and provides growth-dependent bioluminescence andfluorescence when used to transform S . aureus [40].

Preparation of the bacterial inoculum for invasion assays. S . aureus were grown overnight at 30°C statically in HEPES-buffered Dulbecco's modified Eagle's medium [DMEM] [Gibco] supplemented with 10% RPMI [Sigma], 5 µg ml^-1 chloramphenicol, and1.5 nM [Ala⁵]AIP-1 . Overnight cultures were washed twice inan equal volume of fresh medium to remove [Ala⁵]AIP-1 and then concentrated 10-fold . This cell suspension was used to inoculate fresh HEPES-buffered DMEM [Gibco] supplemented with chloramphenicol [5 µg ml^-1] to an OD₆₀₀ of approximately 0.08.

MIC determination. S . aureus was grown overnight at 30°C statically in HEPES-bufferedDMEM [Gibco] supplemented with 10% RPMI [Sigma] and 5 µgml^-1 chloramphenicol . Cultures were centrifuged at 3,000 x g for 5 min and resuspended in HEPES-buffered DMEM supplementedwith 10% RPMI and 5 µg ml^-1 chloramphenicol to an OD₆₀₀ of 0.08; 100 µl of this culture was used to inoculatethe wells of a 96-well plate containing 100 µl of HEPES-bufferedDMEM supplemented with 10% RPMI in which the antibiotics undertest had been serially twofold diluted . The plate was then incubatedat 37°C, and absorbance, luminescence, and fluorescencewere measured in a Tecan Genios Pro multilabel reader at 30-minintervals for 20 h.

MAC-T cell culture and cell invasion assays. The bovine mammary epithelial cell line MAC-T was routinelycultured in assay medium [DMEM with 10% fetal bovine serum,2 mM L-glutamine, 5 µg ml^-1 insulin, 1 µg ml^-1 hydrocortisone] supplemented with 100 U ml^-1 penicillin and 100 µg ml^-1 streptomycin [Sigma] as previously described [20] . Antibioticswere omitted from assay medium during internalization studies.MAC-T cells were seeded into 24-well [Costar] or 96-well clear-bottomed[Porvair] microtiter plates in assay medium and grown to confluenceas described previously [40], then infected with 1 ml [24-wellplate] or 200 µl [96-well plate] of the bacterial inoculum.Microtiter plates were centrifuged for 20 min at 1,000 x g tofacilitate interaction of the bacteria with the eukaryotic cellsurface . Plates were subsequently incubated for 1 h at 37°C.

Following this, cell monolayers in 96-well plates were washed three times with fresh DMEM in an automated plate washer [Cellwash, Thermo Labsystems]; 24-well plates were washed manually . Monolayers were then incubated with lysostaphin [10 µg ml^-1; Sigma] in HEPES-buffered DMEM [Gibco] for 20 min at 37°C . Plateswere then washed again, and 1.5 ml [24-well plates] or 200 µl[96-well plates] of fresh HEPES-buffered DMEM [Gibco] was putinto all wells . The 24-well plates were incubated at 37°Cin a Victor 1420 multilabel counter [Perkin-Elmer Instruments],and the 96-well plates were incubated at 37°C in a Lucy1 luminometer [Anthos], where bioluminescence alone was monitored,or a Tecan Genios Pro multifunctional detector, to concomitantlymeasure luminescence and fluorescence.

Time-lapse confocal microscopy of intracellular S . aureus growth. Eukaryotic cells were seeded into 35-mm glass bottom microwell dishes [MatTek] in assay medium and grown to 80% confluence overnight in 5% CO₂ at 37°C [40] . The monolayers were thenwashed with DMEM prior to staining of cell membranes with 24mM FM4-64 [Molecular Probes], a membrane-selective fluorescent dye, for 30 s at room temperature . Cell monolayers were then washed three times in DMEM to remove excess dye . Stained cellswere then infected with 1 ml of the bacterial inoculum [OD₆₀₀ of 0.02]; plates were centrifuged at 1,000 x g for 20 min andthen incubated for 30 min to allow infection of the monolayers.Extracellular bacteria were then removed from the plates bylysostaphin treatment [40] . Specimens were placed on the heatedstage of a CO₂ incubator on a Zeiss LSM 510 confocal microscope.Image stacks with 1-µm slices were taken sequentiallyat 10-min intervals over an 8-h time course by a multitrackprotocol with a 488-nm laser with a band pass emission filterof 505 to 550 nm for GFP and a 543-nm laser with a long-pass emission filter of 560 nm for FM4-64 visualization.

 
High-throughput microplate assay of S . aureus intracellular replication. The assay developed previously [40] was sensitive to fluctuationsdue to differential expression of the agr regulon and variationin the rates of cell adhesion . To overcome this, the infectiousprocess has now been standardized by pretreatment of the bacterialinoculum with [Ala⁵]AIP-1 to repress agr expression and centrifugationof the inoculum onto the cell monolayer . After a 1-h incubationperiod, monolayers were washed and treated with lysostaphin,as this has been shown to effectively remove all extracellularand adherent bacteria [40] . The bioluminescence of the remainingintracellular replicating bacteria in 24-well plates was monitoredin a Victor 1420 multilabel counter [Perkin-Elmer Instruments].For assays in 96-well plates, bioluminescence was measured withan Anthos Lucy 1 luminometer . As the reporter plasmid in thestaphylococci [pSB2030] confers growth-dependent bioluminescence,an increase in bioluminescence can be used as a monitor of bacterialreplication . Nonreplicating bacteria do not produce light . Figure1 shows comparative data obtained from the 24-well and 96-wellformats from MAC-T cells infected with S . aureus 8325-4[pSB2030].

 
These data show that results obtained from both assay formatsand by both instruments are comparable . In each case replicationappears to begin approximately 3 h after inoculation [i.e., 1 h after the samples are incubated in the instruments] . Itcan be seen [Fig . 1] that at the beginning of the measurement period the bioluminescence from each well of the 96-well platestarts at a high level and then decreases rapidly . This initialhigh level of bioluminescence can be attributed to preformedLux proteins in the intracellular bacteria, as the residualvolume of wash buffer left in the wells following the postinfectionwashes is essentially free of bacteria . However, bioluminescencedecreases rapidly within the first hour due to the short half-lifeof luciferase, indicating that the bioluminescence observedafter this time point originates from de novo synthesis of thereporter proteins by replicating intracellular organisms.

In the 24-well plate format, bioluminescence levels are seento increase steadily throughout the experiment after the 1-htime period . This increase in bioluminescence is due to thereplication and overall increase in numbers of intracellularS . aureus . In the 96-well plates after 1 h, bioluminescenceincreases steadily throughout the experiment, again demonstratingthe ability of S . aureus to replicate in MAC-T cells . The generalpatterns in the infection processes and subsequent replicationof intracellular bacteria in both plate formats are similar.However, direct quantitative comparisons cannot be made, asdifferent instruments were used to perform the bioluminescentmeasurements for the different plate types.

Studying S . aureus intracellular replication by time-lapse confocal microscopy. In order to demonstrate that the increase in bioluminescenceobserved in the microtiter plate assay was due to intracellularreplication, S . aureus harboring the P_xylA-gfp-luxABCDE reporterwere used to infect cell monolayers previously treated with the dye FM4-64 to allow visualization of eukaryotic cell membranes [16] . Following infection of the monolayer, specimens were transferredto a Zeiss LSM 510 confocal microscope with a heated stage andCO₂ incubator at 37°C . Image stacks were taken as describedin Materials and Methods, and images were processed with a ZeissLSM Image browser.

As can be seen in Fig . 2, intracellular replication of S . aureusRN6390[pSB2030] is readily observable, with countable numbersand fluorescence output of GFP-tagged bacteria increasing overtime . Similar time-lapse imaging by the agr mutant S . aureusRN6911[pSB2030] shows that this strain is efficiently internalizeddue to high-level expression of cell surface adhesins . However,as expected, this strain does not replicate over the time courseof this experiment and the GFP signal is seen to degrade.

 
Efficacy of antibiotics on intracellularly replicating S . aureus. To investigate the effect of antibiotics on intracellularly replicating S . aureus, a selection of compounds were chosen that are used clinically for the treatment of staphylococcal infections . These different classes of antibiotics are all knownto act through different mechanisms on the bacterial cell.

Antibiotic concentrations effective at preventing replicationof planktonic S . aureus RN6390[pSB2030] were determined by microtiter broth dilution in tissue culture medium at 37°C . Replicationin these experiments was measured by absorbance, bioluminescence,and fluorescence as measured in a Genios Pro multifunctionaldetector . It has previously been shown for this reporter constructthat expression of the reporter operon occurs during bacterialreplication [40] . It can be seen in Fig . 3 that the signalsobtained from both Lux and GFP generally correlate well withthe growth of the reporter organism . Inhibition of GFP accumulationin antibiotic-treated samples was seen to correlate with inhibitionof replication in a dose-dependent manner [Fig . 3A and 3B].With most antibiotics tested, a reduction in bioluminescence[Lux] signal was seen to correlate with inhibition of replication[Fig . 3A and 3C]; however, with some antibiotics an increasein Lux signal was observed at subinhibitory concentrations [Fig.3C, panels 1 to 4] . Addition of fucidin to the bacterial culturessignificantly extended the lag phase of growth in a dose-dependentmanner.

 
To elucidate the effectiveness of antibiotics at arresting intracellular bacterial growth, 96-well invasion assays were performed as described above . Plates were incubated at 37°C for 3 h afterthe lysostaphin treatment and subsequent washes in DMEM . Afterthis period, when intracellular S . aureus were anticipated tobegin replicating, medium was aspirated from the wells and HEPES-buffered DMEM containing the test antibiotics was added to the wells. Following addition of the antibiotics, the plate was incubatedat 37°C in the Genios Pro, where bioluminescence and fluorescencewere monitored every 30 min . As illustrated in Fig . 4, the data indicate that the antibiotics enter the cells with different efficiencies, since the changes in patterns of bioluminescenceand fluorescence are different for each antibiotic, but in eachcase the ensuing effect on replication is very marked comparedto control wells incubated in medium alone.

 
It appears that fucidin is freely able to enter the cell, as bacterial replication is arrested upon addition of the antibiotic, indicated by a dose-dependent reduction of fluorescence and bioluminescence [Fig . 4A and B, panel 1] . This is unlike the control, where replication continues throughout the experiment. The effective concentration range of fucidin on intracellularS . aureus appeared similar to that on planktonic bacteria.

Upon addition of the flucloxacillin [Fig . 4A and B, panel 2]intracellular bacteria are still observed to replicate at the concentrations tested, as indicated by an increase in fluorescence and bioluminescence . At 0.5 µg ml^-1 flucloxacillin appears to slow replication, as indicated by a significant drop in fluorescence. At concentrations below this, the fluorescence signal is slightly reduced; however, the Lux signal is significantly enhanced,as was observed with sublethal concentrations of flucloxacillinon planktonic S . aureus.

Vancomycin treatment of S . aureus RN6390[pSB2030] gave data similar to that seen with flucloxacillin [Fig . 4A and B, panel 3] . Bacterial replication continued in the presence of antibiotic, though at a lower rate than the control sample, as indicatedby the GFP signal . The level of bioluminescence was seen toexceed that expressed by the control sample at sublethal concentrationsof vancomycin . A vancomycin concentration of 4 µg ml^-1,which was sufficient to prevent replication of planktonic bacteria,was unable to prevent replication of the intracellular S . aureus.

The third cell wall synthesis inhibitor tested, teicoplanin,has effects on intracellular replicating S . aureus very similarto those observed with vancomycin [Fig . 4A and B, panel 4]. Reduction of growth rate was apparent from the GFP signal, while sublethal concentrations of the antibiotic provoked enhanced bioluminescence from the test samples . A teicoplanin concentrationof 2 µg ml^-1 was not able to fully arrest intracellularS . aureus replication but was sufficient to prevent growth of planktonic bacteria.

Gentamicin [Fig . 4A and B, panel 5] was clearly able to enterthe MAC-T cells, as shown by the reduction in intracellular growth rate measured by both Lux and GFP . The inhibitory levels measured via the reporters were similar to those observed on planktonic S . aureus, suggesting that this antibiotic is able to pass into the eukaryotic cells with ease.

Ciprofloxacin is also seen to influence intracellular bacterial replication in a dose-responsive manner [Fig . 4A and B, panel 6], with higher concentrations inhibiting replication to the greatest degree . Concentrations effective at arresting replicationof intracellular S . aureus were similar to those affecting replication of planktonic bacteria.

 
Previous studies by GFP-Lux reporters to elucidate agr expression and subsequent intracellular replication of S . aureus [40] useda 24-well plate assay, which monitored the bioluminescence of internalized staphylococci after addition of bacteria to a eukaryotic monolayer . The variability of the data achieved meant that many replicate samples were required in this assay format and thisdid not allow numerous specimens to be tested simultaneously.In this work the format has now been transferred to 96-wellmicroplates to allow greater numbers of concurrent samples tobe examined.

One of the main laboratory strains of S . aureus studies, S. aureus RN6390, has been shown to carry a deletion in rsbU which is proposed to affect virulence [5, 13, 14, 23] . The internalization capabilities of S . aureus rsbU⁺ and rsbU strains were comparedin the 96-well assay and revealed that intracellular replicationof both strains was similar, indicating that a deletion in rsbUdoes not appear to affect internalization and subsequent replicationof S . aureus by host cells [41] . Although bacterial replicationcould be measured, signals were often quite low and variablebetween replicate experiments in this assay format . This wasattributed to the fact that bacteria had internalized nonsynchronouslyduring the infection period.

Here we further developed the 96-well microtiter plate assaysby growing the initial inoculum with [Ala⁵]AIP-1 to prevent the induction of agr [27] . This ensures that expression of microbialsurface components recognizing adhesive matrix molecules, essentialfor cellular adhesion and uptake, is maximal [12, 36] and that bacteria are in a state optimal for host cell binding . Preliminary experiments showed the equivalence of infection by cycled cells previously used in these kind of experiments [40] and [Ala⁵]AIP-1-treated cultures . Infection will still not be synchronous if bacteria encounter host cells at different times, so to aid synchronicity, bacteria were centrifuged onto the epithelial cell monolayersseeded into 24- and 96-well plates . This ensured intimate contactbetween bacteria and host cells, encouraging internalizationto occur simultaneously and at higher frequency.

Following a 1-h incubation to allow internalization of S . aureus, external bacteria were removed by washing and lysostaphin treatment. The S . aureus used in these assays contain plasmid pSB2030, which confers growth-dependent bioluminescence [Lux] on the bacteria without the addition of exogenous substrate [40] . Thisenables bacterial growth to be monitored directly, without the need for sampling in luminometers with incubating abilities, providing an assay with great sensitivity and high reproducibility. Our data [Fig . 1] show that bacterial growth can be readily assessed in both 96- and 24-microwell formats.

As the reporter gene operon in pSB2030 also confers a fluorescent phenotype [GFP], we were able to use this, in combination withthe vital membrane stain FM4-64, to visualize eukaryotic membranesto develop a novel time-lapse confocal microscopy protocol toimage intracellular replication of S . aureus within living host cells . These data confirmed that S . aureus RN6390 was indeed replicating inside the eukaryotic cells [Fig . 2A] and provide temporal data comparable to that obtained from the in vitro assay in 96-well microplates . It was also noted that the GFPsignal from the replicating bacteria appears to increase overtime, commensurate with its expression from a growth-dependentpromoter.

The agr mutant S . aureus RN6911 is able to enter the eukaryotic cell; however, it is not able to escape the endosome and so cannot replicate . Wesson et al . [51] suggested that the increasedproduction of cell surface proteins by the agr mutant couldpromote the initial binding to the cell membrane and thus leadto higher numbers of internalized bacteria . However, they alsodemonstrated that while higher numbers of S . aureus RN6911 wereable to enter the cell, they were not capable of intracellulargrowth . Data from confocal time-lapse experiments illustratedin the present study [Fig . 2B] also demonstrate this lack ofintracellular replication by the agr mutant . S . aureus RN6911are seen to be present inside the cells in relatively high numbers,but no replication is seen over the time course of the experiment.It is also noticeable that the level of GFP in individual bacteriadiminishes over the period of the experiment . This is likelydue to photobleaching of the GFP protein in the bacteria . Asagr mutant S . aureus cells are unable to replicate intracellularly,they cannot replenish the damaged GFP, as the promoter drivingGFP expression is replication dependent [40] . What is also noticeablein these microscopy experiments is the fact that endosomal membranesappear to remain intact around S . aureus RN6911 cells . Thisfurther supports the hypothesis that agr expression is necessaryfor endosomal escape and subsequent replication within MAC-Tcells [40].

Many methods are available to evaluate the efficacy of antibacterial compounds in vitro . In these studies bacteria are generally extracellular and are exposed to a constant antibiotic concentration for the test period [approximately 18 h], after which the MICis determined . Two such standard test methods are the BritishSociety for Antimicrobial Chemotherapy agar dilution [50] and the microtiter broth dilution method [3] . While these experimentsprovide information on inhibition or the bactericidal activityof the compound, they do not provide any kinetic data on the killing rate during the incubation period [6, 8] . Our modificationof the latter method in which bacterial growth is measured byabsorbance, luminescence, and fluorescence provides additionalkinetic data on antibiotic efficacy . It is known that antibioticconcentrations in vivo fluctuate according to their pharmacokineticproperties [7]; therefore, the response of the antibiotic onbacteria located in an intracellular niche may differ from thoseresults obtained from in vitro experiments . Bacterial growthcannot be assessed intracellularly by absorbance readings; however,the use of Lux/GFP⁺ bacteria allows the kinetics of intracellulargrowth to be determined.

Most conventional assays that study the effect of antimicrobial agents on intracellular S . aureus use professional phagocytic cell lines . Generally macrophages are infected with S . aureus and following an infection period all extracellular bacteriaare eliminated by incubation in gentamicin [1, 46] or lysostaphin[45] . Following this, the intracellular growth is evaluatedin the presence and absence of the compound being tested . Aftervarious incubation times, the sum total of intracellular associatedbacteria is enumerated by plate count assays.

The use of the bioluminescence/fluorescence assay of S . aureus replication is readily adapted to screen mutants of S . aureus which cannot replicate intracellularly or to measure antibacterial efficacy without the need for painstaking bacterial count assays. To demonstrate the utility of this assay method, a number of antibiotics were tested having been chosen on the basis thatthey are widely used clinically as therapeutic agents againststaphylococcal infections . As it is believed that the intracellularlocation of S . aureus may be responsible for long-term chronicinfections [4, 19, 49], it is crucial to elucidate the intrinsicactivity of the drug on bacterial replication in an intracellularenvironment.

Prior to assessment of the action of antibiotics against intracellular S . aureus, the effects of the antibiotics on the growth and GFP/Lux reporter activity of planktonic S . aureus[pSB2030] were examined by a modification of the microtiter broth dilution method [3] . S . aureus[pSB2030] was incubated at 37°C in tissue culture medium with doubling dilutions of the test compounds in a 96-well plate . The absorbance, fluorescence, and luminescence intensity of these cultures were measured at 30-min intervals to assess bacterial growth and GFP and Lux expression . Dependingupon the mode of action of the antibiotics assayed, very differenteffects were seen upon Lux/GFP expression of the test organismwhen compared with the growth data.

Fucidin, an inhibitor of peptide translocation, at lower concentrations [0.125 to 1 µg ml^-1] gives a marked dose-dependent increase in the lag phase of the culture [Fig . 3A, panel 1] . At the MIC[2 µg ml^-1] no growth is seen . These growth data derivedfrom absorbance are closely mirrored by the GFP and Lux signals[Fig . 3B and C, panel 1] . The Lux signal from bacteria grownwith lower concentrations of fucidin [0.125 to 0.5 µgml^-1], however, is seen to exceed that of the control samplewithout antibiotic . As the Lux signal is influenced by bothprotein synthesis and the metabolic state of the cell, the high bioluminescence can be attributed to uncoupling of respiratory metabolism from other cellular processes . This effect is commonly seen when challenging Lux⁺ bacteria with sublethal concentrations of xenobiotics [47] . In contrast the GFP signal, which is notinfluenced by metabolic activity, never exceeds control values.This observation stresses the advantage of using a GFP-Lux dualreporter operon, not only by providing an internal control for possible reporter-dependent data, but also by giving more insights into the physiological effects of compounds on bacteria.

The cell wall-synthesis inhibitors flucloxacillin, vancomycin,and teicoplanin all provide similar data, with little effectseen upon the growth of the bacteria until the MIC is reached[Fig. 3A, panels 2 to 4] . The growth data are closely comparable to that obtained from the GFP signal [Fig . 3B, panels 2 to 4].Once again, an enhancement of the Lux signal over the control, indicative of respiratory uncoupling, is observed at lower concentrations of these antibiotics.

The effect of gentamicin on GFP and Lux expression by S . aureus[pSB2030] shows good dose dependency [Fig . 3B and C, panel 5], with noindication of respiratory uncoupling at concentrations below the MIC [1 µg ml^-1] from the Lux data . The growth rate at intermediate concentrations of gentamicin [0.25 to 0.5 µgml^-1] appears to be variable . This may be attributed to error-proneprotein synthesis due to mRNA misreading [9] affecting the fitness of the cells . Misreading of the GFP-Lux mRNA would result in nonfunctional reporter proteins, so little or no reporter signalis seen at these concentrations.

A dose-dependent inhibition of replication by ciprofloxacin,a DNA gyrase inhibitor, can be closely correlated with boththe GFP and Lux signals [Fig . 3A, B, and C, panel 6] . Littleor no reporter signal is measured with ciprofloxacin concentrationsabove the MIC of 0.5 µg ml^-1 . Reporter signals at lower concentrations are lower than the antibiotic-free control indicative of sublethal injury of the bacteria.

The addition of fucidin to S . aureus[pSB2030] infected MAC-T cells shows a dose-dependent reduction in fluorescence and bioluminescence [Fig . 4A and B, panel 1] indicative of entry of the antibioticinto the cell where it can repress replication of the bacteria.A full reduction of both GFP and Lux reporter signals at 1 µgml^-1, a concentration below the MIC of 2 µg ml^-1, suggeststhat S . aureus may be under stress in the intracellular milieuwhich makes them more susceptible to this agent . Alternatively,the signal generated by the low number of intracellular bacteriarelative to that found in broth-grown cultures may be belowthe sensitivity of the assay.

Flucloxacillin, vancomycin, and teicoplanin all provide similar data when used to challenge intracellular staphylococci [Fig. 4A and B, panels 2 to 4] . A dose-dependent reduction is seen in the accumulation of GFP with all of the compounds . On internalized bacteria with concentrations of vancomycin of 1 to 4 µgml^-1 [MIC = 4 µg ml^-1], teicoplanin of 0.5 to 2 µgml^-1 [MIC = 2 µg ml^-1] and flucloxacillin of 0.125 to0.5 µg ml^-1 [MIC = 0.25 µg ml^-1], GFP and Lux signalsare readily measurable indicating the bacteria can still replicate.This may be due an inability of these antibiotics to penetratethe eukaryotic cells . However, an increase in Lux signal abovethe control [Fig. 4 B, panels 2 to 4], indicative of uncouplingfor each of these antibiotics, suggests the agents are capableof penetrating the cells to some extent . The activity of both teicoplanin and vancomycin against S . aureus residing in human neutophils has previously been investigated [37] . In this studyit was demonstrated that at a concentration of 10 µg ml^-1 each of these antibiotics had an inhibitory effect on bacterial replication; however, a total arrest of bacterial replicationwas not observed.

As the cell wall's function is to preserve cell integrity by withstanding the internal osmotic pressure, it may be that osmotic protection afforded the bacteria by the eukaryotic cytosol mayhelp S . aureus resist cell wall synthesis inhibitors and continue replicating . The growing prevalence of methicillin-resistantS . aureus has led to an increased reliance on the glycopeptides vancomycin and teicoplanin, to which methicillin-resistant S. aureus strains have been uniformly susceptible . However, these data suggest that while these drugs are prescribed to overcome staphylococcal infections they may only show a limited efficiencyin inhibiting intracellular S . aureus replication.

If during staphylococcal infections bacteria are residing inan intracellular environment, the fact these antibiotics appearto have lower efficacy inside cells could cause increased problemslong term . As bacteria are exposed to sublethal concentrationsof antibiotics, it provides a selective environment for theemergence of antibiotic resistant staphylococcal strains againstthe prescribed drug.

The effect of ciprofloxacin on intracellular S . aureus [Fig. 4A and B, panel 6] shows a clear dose-dependent reduction inGFP and Lux signals . The effective range of concentration isequivalent to that seen in planktonic cells indicating thatciprofloxacin is readily able to enter the MAC-T cells and retainsfull activity in the cell's cytosol . This correlates well withdata obtained of the intracellular killing of S . aureus in humanneutophils, where a concentration of 1 µg ml^-1 was foundto kill 90% of phagocytosed bacteria in comparison to untreatedneutrophils [31, 38].

Challenge of intracellular S . aureus with gentamicin at concentration of 0.06 to 0.25 µg ml^-1 reveals a dose-dependent reduction of both the GFP and Lux signals [Fig . 4A and B, panel 5], indicativeof arrest of replication and showing that this antibiotic canreadily enter the MAC-T cells.

The data obtained studying the effects of gentamicin have proved to be very interesting . Gentamicin is an aminoglycoside that interferes with bacterial protein synthesis by binding to the ribosomal 30S subunit . Until recently, it has been assumed that aminoglycosides are poorly effective against treating intracellular pathogens due to their poor ability to penetrate the eukaryoticcell membrane [25] . Indeed in conventional assays to enumerate numbers of intracellular S . aureus, gentamicin is used to remove extracellular bacteria from samples . Our data have clearly demonstrated that gentamicin is able to penetrate the cell and inhibit replication of intracellular S . aureus . Indeed, when concentrations of gentamicinof 100 µg ml^-1 or 50 µg ml^-1, which are routinelyused in traditional intracellular replication assays, were addedto monolayers infected with Lux⁺ S . aureus, no bioluminescencewas observed, indicating that bacterial replication had beencompletely abolished [data not shown] . This observation concurswith recent reports that have demonstrated that gentamicin iscapable of entering macrophages and killing intracellular bacteria[10, 11, 15, 35] . Hamrick et al . [15] demonstrated that evenat concentrations of 5 µg ml^-1 gentamicin was able toaffect intracellular bacterial viability . We have demonstratedthat concentrations as low as 0.06 µg ml^-1 affected replicationof intracellular S . aureus tagged with a gfp-lux dual reporter.These results suggest that a reevaluation of the use of gentamicinin the experimental study of persistence and replication ofbacterial pathogens within host cells is well overdue.

The ability of intracellular bacteria to be protected againstthe killing actions of antibiotics that have a poor abilityto penetrate the eukaryotic cell as well as intracellular inactivationof the compound could be responsible for the therapy failureand persistent recurrent infections that occur within the host[15, 18, 24] . Our data suggest that for efficient antistaphylococcaltherapy, it would be pertinent to include antibiotics that canbe demonstrated to be effective against intracellular S . aureus.

.

 
We thank the United Kingdom Medical Research Council for funding [G9219778], Vyv Salisbury for providing antibiotics, and CathRees for helpful discussions and critical reading of the manuscript.

 
* Corresponding author . Mailing address: University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, United Kingdom . Phone: 44 115 951 6169 . Fax: 44 115 951 6162 . E-mail: phil.hill@nottingham.ac.uk .

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