Direct Continuous Method for Monitoring Biofilm Infection in a Mouse Model

Bacterial strains.The bacterial strains used in this study were P. aeruginosa ATCC 19660 and S. aureus ATCC 12600. These strains were engineered for bioluminescence and designated P. aeruginosa Xen 5 and S. aureus Xen 29. If not otherwise indicated, trypticase soy broth (TSB) supplemented with 0.25% glucose (TSBG) (Difco, Detroit, Mich.) was used as the growth medium to culture the bacterial pathogens.

Generation of bioluminescent S. aureus.S. aureus 12600 was transformed with a modified Photorhabdus luminescence lux operon (12) by using the gram-positive lux transposon plasmid pAUL-ATn 4001 luxABCDE Km ^r (11), which was introduced into the cells by electroporation as previously described (12, 34). Transformants were grown overnight in TSB containing erythromycin (5 μg/ml) and then plated onto TSB solid medium containing kanamycin (200 μg/ml) to select for clones in which the Tn 4001luxABCDE Km ^r cassette had transposed and inserted downstream of a promoter. Highly bioluminescent colonies were selected using an IVIS charge-coupled device camera (Xenogen Corp., Alameda, Calif.). One clone, designated S. aureus Xen 29, was selected and further characterized.

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

Generation of bioluminescent P. aeruginosa.P. aeruginosa ATCC 19660 was made bioluminescent by randomly introducing a Photorhabdus luminescence luxCDABE cassette into its chromosome by conjugating it with Escherichia coli S17-1 λ pir pUT mini-Tn 5 luxCDABE Tc ^r and allowing transposition of the lux operon to occur (40). To allow bioluminescent P. aeruginosa to be more readily distinguished from the bioluminescent E. coli donor strain, P. aeruginosa was initially made carbenicillin resistant by transforming it with plasmid p4027 (kindly provided by A. M. Kropinski, Queen's University, Kingston, Ontario, Canada). The E. coli-P. aeruginosa mixture was incubated overnight at 37°C, and then 100-μl volumes were spread onto Luria-Bertari plates containing 100 μg of carbenicillin per ml and 20 μg of tetracycline per ml. After overnight incubation at 37°C, the plates were screened for bioluminescent colonies and a highly bioluminescent clone was identified and designated P. aeruginosa Xen 5. Chromosomal DNA of bioluminescent P. aeruginosa Xen 5 was digested with ClaI or AatII and then independently self-ligated. The ligated fragments served as templates for inverse PCR amplification. Primers UTCF1 (5′GTG CAA TCC ATT AAT 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 by using Pfu DNA polymerase (Stratagene, La Jolla, Calif.). The resulting PCR fragments were purified using a PCR purification kit and sequenced with UTCF1 and UTCR as primers. The sequencing results were BLAST searched against the National Center for Biotechnology Information (NCBI) database.

Inverse PCR analysis of P. aeruginosa Xen 5 Tn 5 luxCDABE Tc ^r.Chromosomal DNA of bioluminescent P. aeruginosa Xen 5 was digested with ClaI or AatII and independently self-ligated. The ligated fragments were then used as templates for inverse PCR amplification of the lux fusion junction, which was amplified using primers UTCF1 and UTCR. The resulting PCR fragments were purified using a PCR purification kit and sequenced with UTCF1 and UTCR as primers. The sequencing results were BLAST searched against the NCBI database.

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

(ii) Catheter-associated biofilm.Biofilms of S. aureus and P. aeruginosa were developed on a 14-gauge Teflon intravenous catheter (Abbocath-T; Burns Vet Supply, Vancouver, Wash.). Briefly, the catheter was cut into 1-cm segments and each piece was sterilized with 70% ethanol and air dried. Bacterial biofilms were developed on the catheter by placing individual segments into tubes containing 1.0 ml of a cell suspension (10 ⁴ CFU/ml) in TSBG in the exponential phase of growth. After incubation for 2 to 3 h at 37°C, colonized catheters were recovered aseptically and rinsed once with TSBG to remove unbound bacteria. The catheters were then incubated further in fresh TSBG at 37°C for various time intervals. At specific time points, two or three catheters were removed for quantitative analysis of biofilm development. During the incubation time, the medium was decanted and replaced every 12 h with fresh TSBG. Control catheters were prepared as above but without the bacterial inoculum. Monitoring both bioluminescence and viable counts allowed assessment of the kinetics of biofilm formation on catheter material.

Experimental model of infection and monitoring of bioluminescent biofilms.The experimental foreign-body infection in murine model developed by Rupp et al. (32) was established with slight modifications. All experimental procedures were carried out according to the following protocol approved by the Institutional Animal Care and Use Committee. BALB/c female mice (Charles River, Wilmington, Mass.) weighing 18 to 22 g were anesthetized with Ketamine (Burns Vet Supply) at 100 mg/kg and Xylazine (Burns Vet Supply) at 5 mg/kg, their flanks 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 of at least 2 cm from the incision. The incision was then covered with intact skin and closed with surgical staples, and the skin was disinfected. One catheter segment was inserted on each side of each animal. Infection was induced by either implanting a precolonized catheter carrying a defined inoculum or by infecting the lumen of the sterile catheter with defined quantities of bacterial suspension in PBS postimplantation. The postimplant inoculum was introduced by injection into the catheter lumen via a 31-gauge needle, approximately 1 h after the implantation procedure. In each experiment, an extra group of animals was inoculated with vehicle (PBS), to serve as a negative control. Mice were imaged for a maximum of 5 min, at various time points following inoculation, using an IVIS camera system (Xenogen Corp.). Total photon emission from defined regions of interest within the images of each mouse was quantified using the LivingImage software package (Xenogen Corp.). The photon signals from the catheter were quantified from the ventral image of each mouse. After the final imaging time point, the mice were humanely killed and the infected catheters were surgically removed for enumeration of bacteria by both bioluminescence imaging and conventional plate count method. Bacteria recovered at the end of the experimental period were compared with the inoculated strain for bioluminescence.

Extraction and quantification of bacteria from the catheter biofilm assay. (i) In vitro.Two to three catheters were removed from the incubating chamber at the appropriate time according to each experimental objective. The catheters were 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 tubes were 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 biofilm bacteria from the support surface. To assess for complete removal of the biofilm bacteria, the catheters were imaged at different time intervals and the loss of bioluminescence signal was used to define the complete biofilm removal protocol. 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. Correlation 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 killed and the catheters were gently removed from the subcutaneous tissue by making a skin incision at approximately 2 cm from the implant wound. Harvested catheters were imaged and biofilm bacteria were detached from the catheters as described above, in addition to being enumerated by conventional colony count assay. Catheters from control mice were included in each experiment to assess the 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 inocula of 10 ³, 10 ⁵, 10 ⁶, 10 ⁷, and 10 ⁸ CFU. Each mouse in the group was subjected to implantation of two catheters. The mice were observed for 4 days, and the LD ₅₀ was determined using Reed and Muench proportional-distance calculation method (30). The potential lethal effect of implants or the surgical procedure was examined by implanting sterile catheters. None of the noninfected mice died throughout the observation period.

Characterization of S. aureus Xen 29.Southern blot analysis demonstrated only one copy of Tn 4001 luxABCDE Km ^r to be inserted in the chromosome of S. aureus Xen 29. The chromosomal integration site of Tn 4001 luxABCDE Km ^r was determined by BLAST analysis of the sequence obtained from the inverse PCR fragment. It was determined that the lux transposon had inserted after nucleotide 393 of a 455-nucleotide open reading frame, designated "SA2154' (analyzed against the S. aureus N315 genome located in the NCBI database).

Characterization of P. aeruginosa Xen 5.The inverse PCR results indicated that the transposon Tn 5 luxCDABE had inserted into the P. aeruginosa gene PA4974, which encodes a probable secretion protein similar to the outer membrane protein TolC of Vibrio cholerae and E. coli. Upstream of PA4974 was thiC and downstream was PA4975; both of these ORF were in the opposite direction to the PA4974.

Comparisons of transformants and parental strains for biofilm formation.To assess the effect on biofilm formation of insertion of the lux transposon cassette into the bacterial chromosome, both wild-type and bioluminescent derivative strains were tested for their ability to form biofilms in vitro by two different quantitative methods. There was a slight increase in adherence of the lux constructs of both P. aeruginosa and S. aureus to polystyrene microtiter plates compared to the parental strains. Consistent with these results, the lux constructs also showed a slightly greater propensity to form biofilms on catheter material when assessed by the conventional colony count method (Table 1). The enhanced biofilm formation seen in the lux constructs is unlikely to be due to the growth rate, since the generation times between the strains were almost identical (Table 1). This slight increase in biofilm formation among lux-containing strain merits further investigation, although we do not think it significantly affected the reported results.

Bioluminescence and viability during biofilm development in vitro catheter assays.Bioluminescent images of 2-day-old biofilms of P. aeruginosa Xen 5 and S. aureus Xen 29 on 1-cm segment of 14-gauge Teflon intravenous catheters were recorded using an IVIS camera and are shown in Fig. 1. The intensity of the bioluminescent signal in P. aeruginosa Xen 5 appeared to be higher than that in S. aureus Xen 29, which could be due to a number of factors particular to the organism. Also, P. aeruginosa Xen 5 exhibited a higher bioluminescence activity when grown in liquid media. No bioluminescence was observed in the wild-type strains or noncolonized (control) catheter. This finding confirms that bioluminescence is specific to the lux constructs; the signal could be detected even when the cells were embedded in a thick extracellular matrix (Fig. 2).

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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 as pseudocolor images, with variations in color representing light intensity at a given location. Red represents the most intense light emission, while blue correspond to the weakest signal. Note the lack of light signal from the wild-type and control catheter, indicating the specificity of the detection system. The color bar indicates relative signal intensity.

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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 small lump of catheter biofilm was detached from the catheter surface to show the bacterial aggregates.

To assess the feasibility of using bioluminescence as a quantitative indicator of biofilm bacteria, studies were performed to compare bioluminescence to the number of viable cells on catheter material (Fig. 3). During log phase growth, the luminescence output was proportional to the bacterial biomass, as determined by the number of culturable and bioluminescent cells. Bioluminescence was found to closely correlate with viable-cell count, yielding correlation coefficients of 0.99 and 0.98 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 longer correlated with the number of cells, indicating a decrease in growth and metabolic activity of the population at stationary phase (data not shown). This is probably due to oxygen and/or substrate limitations since the reduced expression of bioluminescence within the biofilm could be restored by replacement of the culture medium with fresh medium every 12 h. This procedure not only replenished the growth medium but also removed planktonic cells, leaving only the sessile bacterial population embedded in the biofilm. Alternatively, this decrease in bioluminescence might be due to the accumulation of inhibitory molecules or to a combination of these factors. For P. aeruginosa and S. aureus, the bioluminescent signal and CFU reached a peak after 2 days of incubation, after which the numbers remained relatively constant, presumably due to the bacterial population reaching the maximum size that was able to colonize the catheter surface. The mean RLU of 6 × 10 ⁶/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 5 and S. aureus Xen 29, respectively.

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FIG. 3.

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

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FIG. 4.

Scatter plots of viable cells and bioluminescence data to demonstrate the relationship between viable counts and bioluminescence for P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B).

LD ₅₀ studies in the biofilm in vivo model.The LD ₅₀s for the parental and lux derivatives in the biofilm model after 48 h are shown in Table 1. In terms of the LD ₅₀ and survival time, there was no major difference between the wild-type and engineered strains, suggesting that the integration of the lux cassette into the bacterial chromosome had no effect on bacterial pathogenicity or survival in mice.

Experimental mouse infection model.Figure 5 illustrates the real-time biophotonic images of representative mice, infected with bioluminescent P. aeruginosa Xen 5 or S. aureus Xen 29, over a 20-day catheter-based infection. Both strains produced a significant bioluminescent signal in mice, allowing the progression of infection to be monitored noninvasively in the experimental model. The total photon emission from the infected sites was quantified using LivingImage software, and cumulative results are shown in Fig. 6. Following implantation of precolonized catheters, the bioluminescence measurements increased 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. aureus and P. aeruginosa doses tested until the termination of the experiment on day 20 (Fig. 6). The kinetics of biofilm development as a measure of bioluminescence in postimplantation infection was also similar, except that the bioluminescent signal reached a peak after 2 days as opposed to 1 day postimplantation in the precolonized catheter model. The number of CFU recovered from catheters following the 20-day imaging time point had increased up to ∼10 ⁶ to 10 ⁷ CFU/catheter over all inoculating doses of 10 ³ to 10 ⁵ CFU/catheter (Table 2). The strong bioluminescent signal detected from implanted catheters suggests that the biofilm population remained metabolically active throughout the experiment, and the increase in biomass, as measured by bioluminescence and CFUs, over the starting inoculum confirmed that there was local proliferation (in vivo) and colonization of biofilm bacteria on the catheter matrix during the course of infection (Table 2). Successful infection is defined as the recovery of viable pathogens from the catheter in numbers greater than the initial infective dose at the time of sacrifice. The inoculum studies showed that 100% of the animals implanted with precolonized catheters with all three doses (10 ³, 10 ⁴, or 10 ⁵) of S. aureus or P. aeruginosa developed catheter-associated infection by the day of sacrifice. An infection rate of 100% was reached with the highest inoculum of either S. aureus or P. aeruginosa in the groups where infection was started 1 h after implanting the catheter (Table 2). In the groups of animals that were inoculated with 10 ³ or 10 ⁴ CFU of P. aeruginosa or S. aureus per catheter, the infection rate was 50 and 75% respectively, suggesting that the animals inoculated postimplantation with lower doses of bacteria were more capable of eliminating the infection and thus preventing the establishment of a biofilm. The lower incidence of infection seen in this group is most probably explained by the actual dose inoculated in this group being slightly lower than that in the group infected with precolonized catheters (Table 2). Local phagocytic activity may also have eradicated the infection before the pathogens were able to colonize the catheter. This normal host defense may be especially effective in clearing the lower doses of infecting pathogens. Despite these differences, subcutaneous implantation of the biofilm-containing catheter (pre- or postinfected) seemed to be well tolerated by the mice. An inoculum of ∼10 ³ to 10 ⁵ CFU of S. aureus and P. aeruginosa per catheter resulted in a reproducible, localized persistent infection surrounding the catheter until the termination of the experiment on day 20 (Fig. 5 and 6). Doses above 10 ⁶ CFU/catheter for P. aeruginosa and 10 ⁸ CFU/catheter for S. aureus resulted in 100% mortality, whereas an inoculum of ≤10 ⁵/catheter produced a chronic infection. No bacteria could be cultured from implants that did not have a bioluminescent signal, suggesting that monitoring of infection via bioluminescence is a reasonable measure of bacterial load. All the S. aureus and P. aeruginosa organisms recovered from catheters with 20-day infections were shown to be bioluminescent, demonstrating the stability of lux constructs even after many days in vivo.

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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 were implanted at subcutaneous sites with doses ranging from 10 ³ to 10 ⁵ CFU, and growth of the biofilm was monitored by detecting photon emission over a 20-day time course using an IVIS camera. Similar results were obtained when the catheter bed was inoculated with doses ranging from 10 ³ to 10 ⁵ CFU after subcutaneous implantation of sterile catheters.

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FIG. 6.

Growth and bioluminescence curves of P. aeruginosa Xen 5 (A) and S. aureus Xen 29 (B) biofilms in mice infected with precolonized catheters carrying various inocula. Each data point is the mean and standard error for two or three mice. Each mouse was subjected to implantation of two catheters. The viable counts in each catheter were determined immediately after removal of the catheters from the implanted sites and are shown 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) were injected into the implant site after implantation, except that the RLU value peaked 2 days after infection.