INFECTION AND IMMUNITY, Feb. 2003, p. 882–890 Vol. 71, No. 20019-9567/03/$08.00�0 DOI: 10.1128/IAI.71.2.882–890.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Direct Continuous Method for Monitoring Biofilm Infection in aMouse Model

Jagath L. Kadurugamuwa,* Lin Sin, Eddie Albert, Jun Yu, Kevin Francis, Monica DeBoer,Michael Rubin, Carole Bellinger-Kawahara, T. R. Parr, Jr.,† and Pamela R. Contag

Xenogen Corp., Alameda, California 94501

Received 25 June 2002/Returned for modification 10 October 2002/Accepted 28 October 2002

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in amouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters.Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, weremade bioluminescent by insertion of a complete lux operon. These bacteria produced significant biolumines-cent signals for both in vitro studies and the development of an in vivo model, allowing effective real-timeassessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel andhighly correlated (S. aureus r � 0.98; P. aeruginosa r � 0.99) and could be maintained for 10 days or longer,provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation ofthe catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 3 to 10 5

CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding thecatheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from thecatheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the luxconstructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could bedetected directly on the support matrix, nondestructively, and noninvasively, this method is especially appeal-ing for the study of chronic biofilm infections and drug efficacy studies in vivo.

Microbial adhesion and biofilm formation on medical im-plants is a common occurrence and represents a serious med-ical problem. Since biofilm microorganisms are difficult toeradicate with antibiotic therapy, chronic, recurrent infectionsoften develop. With the increased use of prosthetic biomedicalimplants, chronic nosocomial infections have become preva-lent in recent years (9, 41). Bacterial colonization of indwellingdevices can be a prelude to both systemic infection and mal-function of the device.

A variety of techniques, such as direct microscopic enumer-ation, total viable count, metabolically active dyes, radiochem-istry, and fluorescence, have been used to investigate microbialbiofilms (1, 4, 8, 14, 17, 18, 23, 29, 33, 38). While some of thesemethods are useful for in vitro studies, they have not provedideal for the investigation of biofilms in experimental infectionmodels. The difficulty in analyzing biofilms in vivo lies in thelack of tools that allow noninvasive longitudinal study design.Assays developed to date, both direct and indirect, are time-consuming and laborious and involve the extraction of bacteriafrom support surfaces. To better understand and control bio-films on medical devices, rapid, direct, nondestructive, real-time quantitative monitoring methods that are adaptable tothe clinical situation are needed. These assays may be used todevelop new preventive and therapeutic methods to combatbiofilm related infections.

To this end, bioluminescent reporters offer a method oflabeling pathogens that is innocuous and allows the sensitive

detection of only live, metabolically active cells by optical bio-photonic imaging. Since bacterial luciferase requires reducedflavin mononucleotide for the generation of bioluminescencewithin the cell, it is a good indicator of the metabolic state ofcells (22). Many biological applications of bioluminescent andfluorescent reporter gene systems have been developed duringthe last few years (1, 3, 10, 14, 19–23, 26–28, 33, 35–38). Mon-itoring a disease process in a living animal by using biolumi-nescent-tagged pathogens was first demonstrated in 1995 byContag et al. (5). This noninvasive, rapid, real-time monitoringapproach has been applied to a myriad of animal infectionmodels and has proven to have significant advantages overconventional methods for studying disease and treatment inanimals (6, 7, 11, 12, 31).

Monitoring bioluminescence as a measure of metabolic ac-tivity provides a rapid, quantitative in situ measure of biofilmdevelopment and physiological activities of bacteria within bio-films. In this study, we describe the detection of bioluminescentstrains of Staphylococcus aureus and Pseudomonas aeruginosa,two of the most predominant biofilm-forming pathogens, usingreal-time monitoring to nondestructively, image catheter-based infections in an experimental animal model.

(Parts of the present study were presented at the 102ndGeneral Meeting of the American Society for Microbiology,abstr. J6, 2002.)

MATERIALS AND METHODS

Bacterial strains. The bacterial strains used in this study were P. aeruginosaATCC 19660 and S. aureus ATCC 12600. These strains were engineered forbioluminescence and designated P. aeruginosa Xen 5 and S. aureus Xen 29. If nototherwise indicated, trypticase soy broth (TSB) supplemented with 0.25% glu-cose (TSBG) (Difco, Detroit, Mich.) was used as the growth medium to culturethe bacterial pathogens.

* Corresponding author. Mailing address: 860 Atlantic Ave., Alam-eda, CA 94501. Phone: (510) 291-6246. Fax: (510) 291-6196. E-mail:Jagath.Kadurugamuwa@Xenogen.com.

† Present address: MicroGenomics Inc., Carlsbad, CA 92008.

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Generation of bioluminescent S. aureus. S. aureus 12600 was transformed witha modified Photorhabdus luminescence lux operon (12) by using the gram-positivelux transposon plasmid pAUL-ATn 4001 luxABCDE Km r (11), which was in-troduced into the cells by electroporation as previously described (12, 34). Trans-formants were grown overnight in TSB containing erythromycin (5 �g/ml) andthen plated onto TSB solid medium containing kanamycin (200 �g/ml) to selectfor clones in which the Tn 4001luxABCDE Km r cassette had transposed andinserted downstream of a promoter. Highly bioluminescent colonies were se-lected using an IVIS charge-coupled device camera (Xenogen Corp., Alameda,Calif.). One clone, designated S. aureus Xen 29, was selected and further char-acterized.

Southern blot and inverse PCR analysis of S. aureus ATCC 12600 Tn 4001luxABCDE Km r. To determine the number of integrations of Tn 4001 luxAB-CDE Km r into the chromosome of S. aureus Xen 29, Southern blot analysis wasconducted using the restriction endonucleases HindIII, SphI, and ClaI, with aPCR-amplified luxA gene fragment as a probe (33). To determine the site ofintegration, the sequence of the genomic DNA lying upstream of Tn 4001luxABCDE Km r was amplified by inverse PCR (25), using ClaI-digested genomicDNA and diverging oligonucleotide primers within the lux operon, IR2 (5� CGTTTC ATT ACC TCT GTT TGA 3�;

Generation of bioluminescent P. aeruginosa. P. aeruginosa ATCC 19660 wasmade bioluminescent by randomly introducing a Photorhabdus luminescenceluxCDABE cassette into its chromosome by conjugating it with Escherichia coliS17-1 � pir pUT mini-Tn 5 luxCDABE Tc r and allowing transposition of the luxoperon to occur (40). To allow bioluminescent P. aeruginosa to be more readilydistinguished from the bioluminescent E. coli donor strain, P. aeruginosa wasinitially made carbenicillin resistant by transforming it with plasmid p4027 (kind-ly provided by A. M. Kropinski, Queen’s University, Kingston, Ontario, Canada).The E. coli-P. aeruginosa mixture was incubated overnight at 37°C, and then100-�l volumes were spread onto Luria-Bertari plates containing 100 �g ofcarbenicillin per ml and 20 �g of tetracycline per ml. After overnight incubationat 37°C, the plates were screened for bioluminescent colonies and a highlybioluminescent clone was identified and designated P. aeruginosa Xen 5. Chro-mosomal DNA of bioluminescent P. aeruginosa Xen 5 was digested with ClaI orAatII and then independently self-ligated. The ligated fragments served as tem-plates for inverse PCR amplification. Primers UTCF1 (5�GTG CAA TCC ATTAAT TTT GGT G 3�) and UTCR (5� CAT ACG TAT CCT CCA AGC C 3�)were used to amplify the region upstream of the transposon insertion site byusing Pfu DNA polymerase (Stratagene, La Jolla, Calif.). The resulting PCRfragments were purified using a PCR purification kit and sequenced with UTCF1and UTCR as primers. The sequencing results were BLAST searched against theNational Center for Biotechnology Information (NCBI) database.

Inverse PCR analysis of P. aeruginosa Xen 5 Tn 5 luxCDABE Tc r. Chromo-somal DNA of bioluminescent P. aeruginosa Xen 5 was digested with ClaI orAatII and independently self-ligated. The ligated fragments were then used astemplates for inverse PCR amplification of the lux fusion junction, which wasamplified using primers UTCF1 and UTCR. The resulting PCR fragments werepurified using a PCR purification kit and sequenced with UTCF1 and UTCR asprimers. The sequencing results were BLAST searched against the NCBI data-base.

In vitro bacterial biofilm. (i) Microtiter plate assay. Early adherence to poly-styrene surfaces has been suggested to be an indication of the capacity of abacterial strain to form a biofilm. Thus, both parental strains and their biolumi-nescent derivatives were tested in a microtiter assay as described previously (4,16). Briefly, bacterial strains were cultivated overnight in TSBG. The cultureswere diluted in fresh TSBG to reach a standardized cell suspension (10 6 CFU/ml), and 100 �l of this suspension was used to inoculate sterile 96-well polysty-rene microtiter plates (Nalge Nunc International Corp., Naperville, Ill.). Aftercultivation for 24 h at 37°C, the wells were gently washed twice with 200 �l ofsterile phosphate-buffered saline (PBS). The plates were air dried, and theremaining surface-absorbed cells of the individual wells were stained with 100 �lof 0.1% safranin for 30 s. Absorbance was measured with a micro-ELISA auto-reader (Molecular Devices, Sunnyvale, Calif.) at 490 nm. Sterile TSBG lackingcells served as a control, and the value obtained with this well was subtractedfrom experimental readings. Each assay was performed in quadruplicate.

(ii) Catheter-associated biofilm. Biofilms of S. aureus and P. aeruginosa weredeveloped on a 14-gauge Teflon intravenous catheter (Abbocath-T; Burns VetSupply, Vancouver, Wash.). Briefly, the catheter was cut into 1-cm segments andeach piece was sterilized with 70% ethanol and air dried. Bacterial biofilms weredeveloped on the catheter by placing individual segments into tubes containing1.0 ml of a cell suspension (10 4 CFU/ml) in TSBG in the exponential phase ofgrowth. After incubation for 2 to 3 h at 37°C, colonized catheters were recoveredaseptically and rinsed once with TSBG to remove unbound bacteria. The cath-

eters were then incubated further in fresh TSBG at 37°C for various timeintervals. At specific time points, two or three catheters were removed forquantitative analysis of biofilm development. During the incubation time, themedium was decanted and replaced every 12 h with fresh TSBG. Control cath-eters were prepared as above but without the bacterial inoculum. Monitoringboth bioluminescence and viable counts allowed assessment of the kinetics ofbiofilm formation on catheter material.

Experimental model of infection and monitoring of bioluminescent biofilms.The experimental foreign-body infection in murine model developed by Rupp etal. (32) was established with slight modifications. All experimental procedureswere carried out according to the following protocol approved by the Institu-tional Animal Care and Use Committee. BALB/c female mice (Charles River,Wilmington, Mass.) weighing 18 to 22 g were anesthetized with Ketamine (BurnsVet Supply) at 100 mg/kg and Xylazine (Burns Vet Supply) at 5 mg/kg, theirflanks were shaved, and the skin was cleansed with Betadyne and alcohol. An 8-to 10-mm skin incision was made and dissected to create a subcutaneous tunnel,into which 1 cm segment of intravenous catheter was implanted at a distance ofat least 2 cm from the incision. The incision was then covered with intact skin andclosed with surgical staples, and the skin was disinfected. One catheter segmentwas inserted on each side of each animal. Infection was induced by eitherimplanting a precolonized catheter carrying a defined inoculum or by infectingthe lumen of the sterile catheter with defined quantities of bacterial suspensionin PBS postimplantation. The postimplant inoculum was introduced by injectioninto the catheter lumen via a 31-gauge needle, approximately 1 h after theimplantation procedure. In each experiment, an extra group of animals wasinoculated with vehicle (PBS), to serve as a negative control. Mice were imagedfor a maximum of 5 min, at various time points following inoculation, using anIVIS camera system (Xenogen Corp.). Total photon emission from definedregions of interest within the images of each mouse was quantified using theLivingImage software package (Xenogen Corp.). The photon signals from thecatheter were quantified from the ventral image of each mouse. After the finalimaging time point, the mice were humanely killed and the infected catheterswere surgically removed for enumeration of bacteria by both bioluminescenceimaging and conventional plate count method. Bacteria recovered at the end ofthe experimental period were compared with the inoculated strain for biolumi-nescence.

Extraction and quantification of bacteria from the catheter biofilm assay. (i)In vitro. Two to three catheters were removed from the incubating chamber atthe appropriate time according to each experimental objective. The catheterswere rinsed in fresh TSB and imaged to quantify the bioluminescence signal.They were then transferred to a separate tube containing 1 ml of TSB. The tubeswere placed in an ultrasonic bath at 38.5 to 40.5 kHz (VWR, San Francisco,Calif.), sonicated for 5 to 10 min, and votexed for 1 min to remove the biofilmbacteria from the support surface. To assess for complete removal of the biofilmbacteria, the catheters were imaged at different time intervals and the loss ofbioluminescence signal was used to define the complete biofilm removal proto-col. The suspension of bacteria that was removed from the catheter were diluted,plated on Trypticase soy agar, and incubated at 37°C for colony counting. Cor-relation between CFU counts and bioluminescent signal (relative light units,RLU) was determined by plotting RLU versus CFU.

(ii) In vivo. After the final imaging time point, the mice were humanely killedand the catheters were gently removed from the subcutaneous tissue by makinga skin incision at approximately 2 cm from the implant wound. Harvested cath-eters were imaged and biofilm bacteria were detached from the catheters asdescribed above, in addition to being enumerated by conventional colony countassay. Catheters from control mice were included in each experiment to assessthe adequacy of aseptic and surgical techniques.

Determination of LD 50. The dose of an individual strain that resulted in 50%lethality (LD 50) for mice was determined using the biofilm infection model.Briefly, groups of five mice were each implanted with catheters containing inoc-ula of 10 3, 10 5, 10 6, 10 7, and 10 8 CFU. Each mouse in the group was subjectedto implantation of two catheters. The mice were observed for 4 days, and the LD50 was determined using Reed and Muench proportional-distance calculationmethod (30). The potential lethal effect of implants or the surgical procedure wasexamined by implanting sterile catheters. None of the noninfected mice diedthroughout the observation period.

RESULTS

Characterization of S. aureus Xen 29. Southern blot analysisdemonstrated only one copy of Tn 4001 luxABCDE Km r to beinserted in the chromosome of S. aureus Xen 29. The chromo-

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somal integration site of Tn 4001 luxABCDE Km r was deter-mined by BLAST analysis of the sequence obtained from theinverse PCR fragment. It was determined that the lux trans-poson had inserted after nucleotide 393 of a 455-nucleotideopen reading frame, designated �SA2154’ (analyzed against theS. aureus N315 genome located in the NCBI database).

Characterization of P. aeruginosa Xen 5. The inverse PCRresults indicated that the transposon Tn 5 luxCDABE hadinserted into the P. aeruginosa gene PA4974, which encodes aprobable secretion protein similar to the outer membrane pro-tein TolC of Vibrio cholerae and E. coli. Upstream of PA4974was thiC and downstream was PA4975; both of these ORFwere in the opposite direction to the PA4974.

Comparisons of transformants and parental strains for bio-film formation. To assess the effect on biofilm formation ofinsertion of the lux transposon cassette into the bacterial chro-mosome, both wild-type and bioluminescent derivative strainswere tested for their ability to form biofilms in vitro by twodifferent quantitative methods. There was a slight increase inadherence of the lux constructs of both P. aeruginosa and S.aureus to polystyrene microtiter plates compared to the paren-tal strains. Consistent with these results, the lux constructs alsoshowed a slightly greater propensity to form biofilms on cath-eter material when assessed by the conventional colony countmethod (Table 1). The enhanced biofilm formation seen in thelux constructs is unlikely to be due to the growth rate, since thegeneration times between the strains were almost identical(Table 1). This slight increase in biofilm formation amonglux-containing strain merits further investigation, although wedo not think it significantly affected the reported results.

Bioluminescence and viability during biofilm developmentin vitro catheter assays. Bioluminescent images of 2-day-oldbiofilms of P. aeruginosa Xen 5 and S. aureus Xen 29 on 1-cmsegment of 14-gauge Teflon intravenous catheters were re-corded using an IVIS camera and are shown in Fig. 1. Theintensity of the bioluminescent signal in P. aeruginosa Xen 5appeared to be higher than that in S. aureus Xen 29, whichcould be due to a number of factors particular to the organism.Also, P. aeruginosa Xen 5 exhibited a higher bioluminescenceactivity when grown in liquid media. No bioluminescence wasobserved in the wild-type strains or noncolonized (control)catheter. This finding confirms that bioluminescence is specificto the lux constructs; the signal could be detected even whenthe cells were embedded in a thick extracellular matrix (Fig. 2).

To assess the feasibility of using bioluminescence as a quan-

titative indicator of biofilm bacteria, studies were performed tocompare bioluminescence to the number of viable cells oncatheter material (Fig. 3). During log phase growth, the lumi-nescence output was proportional to the bacterial biomass, asdetermined by the number of culturable and bioluminescentcells. Bioluminescence was found to closely correlate with vi-able-cell count, yielding correlation coefficients of 0.99 and0.98 for P. aeruginosa Xen 5 and S. aureus Xen 29, respectively(Fig. 4). However, once the cultures reached stationary phase,the bioluminescence decreased as expected and was no longercorrelated with the number of cells, indicating a decrease ingrowth and metabolic activity of the population at stationaryphase (data not shown). This is probably due to oxygen and/orsubstrate limitations since the reduced expression of biolumi-nescence within the biofilm could be restored by replacementof the culture medium with fresh medium every 12 h. Thisprocedure not only replenished the growth medium but alsoremoved planktonic cells, leaving only the sessile bacterial pop-ulation embedded in the biofilm. Alternatively, this decrease inbioluminescence might be due to the accumulation of inhibi-tory molecules or to a combination of these factors. For P.aeruginosa and S. aureus, the bioluminescent signal and CFUreached a peak after 2 days of incubation, after which thenumbers remained relatively constant, presumably due to thebacterial population reaching the maximum size that was ableto colonize the catheter surface. The mean RLU of 6 � 106/catheter for P. aeruginosa Xen 5 and 8 � 10 5/catheter for S.aureus Xen 29 approximately corresponded to 1.8 � 10 7 CFU/catheter and 6 � 10 6 CFU/catheter for P. aeruginosa Xen 5and S. aureus Xen 29, respectively.

FIG. 1. Monitoring the level of bioluminescence activity of P.aeruginosa and S. aureus in a 2-day-old biofilm on catheter segments.Images were acquired with the IVIS camera and are displayed aspseudocolor images, with variations in color representing light inten-sity at a given location. Red represents the most intense light emission,while blue correspond to the weakest signal. Note the lack of lightsignal from the wild-type and control catheter, indicating the specificityof the detection system. The color bar indicates relative signal inten-sity.

TABLE 1. Comparative characteristics of parental andbioluminescent derivatives.

StrainIn vitro

doublingtime (min)

Biofilm formationon microtiter

platesaLD 50 (CFU)

S. aureusParent 23 0.8 � 0.2 1.6 � 108

lux mutant 24 1.3 � 0.3 9.9 � 107

P. aeruginosaParent 25 1.2 � 0.5 1.5 � 106

lux mutant 25 1.7 � 0.6 6.8 � 105

a Results are means � standard deviations.

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LD 50 studies in the biofilm in vivo model. The LD 50s forthe parental and lux derivatives in the biofilm model after 48 hare shown in Table 1. In terms of the LD 50 and survival time,there was no major difference between the wild-type and en-gineered strains, suggesting that the integration of the lux cas-sette into the bacterial chromosome had no effect on bacterialpathogenicity or survival in mice.

Experimental mouse infection model. Figure 5 illustrates thereal-time biophotonic images of representative mice, infectedwith bioluminescent P. aeruginosa Xen 5 or S. aureus Xen 29,over a 20-day catheter-based infection. Both strains produceda significant bioluminescent signal in mice, allowing the pro-gression of infection to be monitored noninvasively in theexperimental model. The total photon emission from the in-fected sites was quantified using LivingImage software, andcumulative results are shown in Fig. 6. Following implantationof precolonized catheters, the bioluminescence measurementsincreased exponentially over 24 h. One day after implantation,

the bioluminescent signal reached approximately 10 5 RLU/catheter, and it remained moderately stable for all S. aureusand P. aeruginosa doses tested until the termination of theexperiment on day 20 (Fig. 6). The kinetics of biofilm devel-opment as a measure of bioluminescence in postimplantationinfection was also similar, except that the bioluminescent sig-nal reached a peak after 2 days as opposed to 1 day postim-plantation in the precolonized catheter model. The number ofCFU recovered from catheters following the 20-day imagingtime point had increased up to �10 6 to 10 7 CFU/catheterover all inoculating doses of 10 3 to 10 5 CFU/catheter (Table2). The strong bioluminescent signal detected from implantedcatheters suggests that the biofilm population remained met-abolically active throughout the experiment, and the increasein biomass, as measured by bioluminescence and CFUs, overthe starting inoculum confirmed that there was local prolifer-ation (in vivo) and colonization of biofilm bacteria on thecatheter matrix during the course of infection (Table 2). Suc-cessful infection is defined as the recovery of viable pathogensfrom the catheter in numbers greater than the initial infectivedose at the time of sacrifice. The inoculum studies showed that100% of the animals implanted with precolonized catheterswith all three doses (10 3, 10 4, or 10 5) of S. aureus or P.aeruginosa developed catheter-associated infection by the dayof sacrifice. An infection rate of 100% was reached with thehighest inoculum of either S. aureus or P. aeruginosa in thegroups where infection was started 1 h after implanting thecatheter (Table 2). In the groups of animals that were inocu-lated with 10 3 or 10 4 CFU of P. aeruginosa or S. aureus percatheter, the infection rate was 50 and 75% respectively, sug-gesting that the animals inoculated postimplantation withlower doses of bacteria were more capable of eliminating theinfection and thus preventing the establishment of a biofilm.The lower incidence of infection seen in this group is mostprobably explained by the actual dose inoculated in this groupbeing slightly lower than that in the group infected with pre-colonized catheters (Table 2). Local phagocytic activity mayalso have eradicated the infection before the pathogens wereable to colonize the catheter. This normal host defense may be

FIG. 2. Phase-contrast micrograph of a wet mount, showing 5-day-old biofilms of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B). A smalllump of catheter biofilm was detached from the catheter surface to show the bacterial aggregates.

FIG. 3. Growth and bioluminescence curves of P. aeruginosa Xen 5and S. aureus Xen 29 grown on catheter surfaces. Viable counts arereported as CFU per catheter, and bioluminescence is represented asRLU measured using the IVIS camera. Each data point is the meanand standard error for three or four catheters. Bioluminescence wasdetermined at each time point immediately prior to determination ofthe viable-cell count.

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especially effective in clearing the lower doses of infectingpathogens. Despite these differences, subcutaneous implanta-tion of the biofilm-containing catheter (pre- or postinfected)seemed to be well tolerated by the mice. An inoculum of �103 to 10 5 CFU of S. aureus and P. aeruginosa per catheterresulted in a reproducible, localized persistent infection sur-rounding the catheter until the termination of the experimenton day 20 (Fig. 5 and 6). Doses above 10 6 CFU/catheter for P.aeruginosa and 10 8 CFU/catheter for S. aureus resulted in100% mortality, whereas an inoculum of �10 5/catheter pro-duced a chronic infection. No bacteria could be cultured fromimplants that did not have a bioluminescent signal, suggesting

that monitoring of infection via bioluminescence is a reason-able measure of bacterial load. All the S. aureus and P. aerugi-nosa organisms recovered from catheters with 20-day infec-tions were shown to be bioluminescent, demonstrating thestability of lux constructs even after many days in vivo.

DISCUSSION

In this study, we have demonstrated the use of biophotonicimaging to improve the monitoring of catheter-associated bio-film infection in mice. Bioluminescent P. aeruginosa and S.aureus strains were generated using a complete bacterial luxoperon, and these strains were used to study the infectionprocess in real time in living animals. Unlike experiments withthe insect luciferase (luc), external addition of any substrate forbioluminescence is not a prerequisite in our system. Further-more, due to its linkage to cellular metabolism, luciferase ac-tivity in vivo indicates the physiological state of the intactorganism. Using an IVIS camera, we were able to visualize thebioluminescent, catheter-associated biofilm bacteria directlythrough the skin of live animals.

The direct assessment of an in vivo biofilm in real timewithout exogenous sampling has several unique features. Thetechnique provides both spatial and sequential informationabout the progression of infection. Unlike using physical bio-chemical indicators, using luciferase as the reporter ensuresthat the signal observed is from viable metabolically activecells, as they exist in the biofilms. Because of its nondestructiveand noninvasive nature, the imaging procedure can be per-formed repeatedly, allowing each animal to be used as its owncontrol over time, overcoming the problem of animal-to-ani-mal variations. The biostatistics is improved through collectionof multiple data points from the same animal. Thus, the overallnumber of animals required is reduced. Furthermore, the abil-ity to monitor the pathogen burden quantitatively without ex-ogenous sampling considerably reduces the time and cost.

A number of animal models have been developed for study-ing aspects such as different biomaterials, pathogenesis, andtreatment of foreign-body infections (2, 13, 24, 32, 39, 41).These models have provided vital information about theprogress of infection and antibiotic pharmacodynamics. How-ever, they generally require the sacrifice of animals at eachsampling point and rely on traditional colony counting proce-dures, with lengthy incubation periods, for assessment of mi-crobial numbers. Such procedures also require the handling ofindividual samples, during which variability arises from thedifficulties in disrupting cell aggregates. These aggregates arenot released from the biofilm in the form of a homogeneoussuspension and thus cannot be easily recovered and quantified.Moreover, the ex vivo methods by which these events aretypically monitored require the removal of tissue and conse-quently the loss of contextual influences of the living animal.Analysis of biofilms in vivo is greatly enhanced by tools thatallow noninvasive quantitative study. The noninvasive ap-proach described here also allows for experimental protocolsthat are significantly more rapid and accurate than conven-tional techniques.

We observed that during in vitro growth, the biolumines-cence of P. aeruginosa and S. aureus was affected by the growthphase. There was a decline in bioluminescence as the biofilmbecame older, most probably due to a decreased cellular met-

FIG. 4. Scatter plots of viable cells and bioluminescence data todemonstrate the relationship between viable counts and biolumines-cence for P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B).

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FIG. 5. Real-time monitoring of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B) biofilms in a mouse model. Precolonized catheters wereimplanted at subcutaneous sites with doses ranging from 10 3 to 10 5 CFU, and growth of the biofilm was monitored by detecting photon emissionover a 20-day time course using an IVIS camera. Similar results were obtained when the catheter bed was inoculated with doses ranging from 103 to 10 5 CFU after subcutaneous implantation of sterile catheters.

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abolic activity. We were able to maintain a linear relationshipbetween bioluminescence levels and CFU by supplying freshmedia frequently to the biofilm bacteria, giving an indication ofdecreased substrate pools for the luciferase reaction. Interest-ingly, this was not an issue in our experimental infections,implying that an adequate supply of factors needed for thelight-emitting enzymatic reaction were available in vivo. Ex-traction of bacteria from infected mice confirmed a good cor-relation between bioluminescence data and number of viablebacteria in the catheter. Such a method of monitoring in vivo

bioluminescent organisms in living animals has been describedfor several other pathogens in acute infection and was dem-onstrated to correspond well to bacterial CFU data (5–7, 11,12, 31). However, it is important to also measure a nonlumi-nescent end point, since the emitted light can decrease withoutaffecting the viability of the cells (15). In the present study, wedemonstrated a good correlation between photon count imag-ing and viable counts in vivo (r 0.98). We also confirmed atleast a 2-log-unit increase in both light output and CFU for S.aureus and P. aeruginosa strains above the initial inoculating

FIG. 6. Growth and bioluminescence curves of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B) biofilms in mice infected with precolonizedcatheters carrying various inocula. Each data point is the mean and standard error for two or three mice. Each mouse was subjected to implantationof two catheters. The viable counts in each catheter were determined immediately after removal of the catheters from the implanted sites and areshown in the upper quadrants of the plot. Identical results were obtained when similar doses of bacteria (10 3, 10 4, and 10 5 CFU/catheter) wereinjected into the implant site after implantation, except that the RLU value peaked 2 days after infection.

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doses. This indicates significant bacterial growth on cathetersfollowing implantation. These observations can be especiallyappealing for the analysis of the efficacy of antimicrobial com-pounds in vivo, since the effectiveness could be rapidly moni-tored without the need for exogenous sampling and culturing.More importantly, once a good correlation between light signaland CFU is established, the bioluminescence could be used tomonitor the metabolic status and bacterial load at the site ofinfection.

In the present study, to maximize the clinical relevance, twoparameters were simulated: (i) the effective inocula required toproduce and maintain a stable foreign-body infection, and (ii)the assessment of timing of the infection (i.e., implantation ofestablished biofilm catheters versus infection after device im-plantation). For the first parameter, various doses of biofilmbacteria that would make it possible to maintain a stable in-fection in mice over a longer period was examined. For thesecond parameter, infection was produced either by implantinga precoated catheter with a defined inoculum or by infectingthe catheter lumen with a defined inoculum after implanting asterile catheter into the subcutaneous space of mice. Implant-ing infected catheters (with a standardized biofilm formed oncatheter) mimics intraoperative contamination with membersof the skin flora, e.g., infections that originate from bacterialseeding of the device. Delaying the infection until after deviceimplantation mirrors postoperative colonization of the im-planted device. Both approaches are clinically relevant andaddress the problems associated with device-related infections,as well as allowing the study of prevention and treatmentstrategies of infections related to medical devices.

A potential limitation of this model is that the implantedforeign body is not mechanically functioning to flush anyplanktonic bacteria present. However, the model presents sev-eral of the key characteristics of foreign-body infection thathave been observed clinically. First, the infection became lo-calized to the catheter so that distant spread was limited. Sec-ond, the infection became chronic, with only a slow increase inthe bacterial counts following establishment of the infection.Third, doses as low as 10 3 CFU were capable of producing astable chronic biofilm infection by both pathogens, thus dem-

onstrating success in reproducing infection without requiring alarge number of bacteria. The low inocula that were able toestablish a stable infection probably resemble the case in clin-ical situations. Another characteristic of our model is thatinfection can be induced in a reproducible manner with two ofthe bacterial species most commonly implicated in foreign-body infection.

This study presents a new model for studying chronic infec-tions by gram-positive and gram-negative bacterial biofilms.The method described here is rapid, uses fewer animals, andenables in vivo monitoring of the pathogen burden and themetabolic activity of a cell population under complex environ-mental conditions. Additionally, this model may be useful forthe study of the pathogenesis and antibiotic susceptibility ofboth gram-positive and gram-negative pathogens within bio-films or within the biofilm environment or in the context of abiofilm. Experiments intended to establish the applicability ofthe model in efficacy assessments of therapeutic agents areunder way.

ACKNOWLEDGMENTS

We thank E. Reynolds and C. Dalesio (Graphics and Communica-tions, Xenogen Corp.) for assistance with drawings.

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TABLE 2. P. aeruginosa Xen 5 and S. aureus Xen 29 catheter infection in mice

Infection conditions Inoculum(CFU/catheter) No. of mice No. of catheters

infected/total no.Mean RLU/catheter

at removalMean CFU/catheter

at removal

P. aeruginosa Xen 5Preinfected 2.5 � 103 2 3/3a 8.5 � 106 1.3 � 107

1.4 � 104 2 4/4 8.2 � 106 2.3 � 107

1.2 � 105 3 6/6 9.8 � 106 2.1 � 107

Postinfected 8.5 � 102 2 2/4 3.4 � 106 2.7 � 107

8.3 � 103 2 3/4 5.9 � 106 1.8 � 107

7.6 � 104 2 4/4 3.4 � 106 1.5 � 107

S. aureus Xen 29Preinfected 6.4 � 103 2 4/4 8.5 � 105 7.0 � 106

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a Four catheters were implanted, but only three were recovered.

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