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. 2024 Feb 15;170(2):001433. doi: 10.1099/mic.0.001433

Predatory bacteria prevent the proliferation of intraocular Serratia marcescens and fluoroquinolone-resistant Pseudomonas aeruginosa

Eric G Romanowski 1, Kimberly M Brothers 1, Rachel C Calvario 1, Nicholas A Stella 1, Tami Kim 2, Mennat Elsayed 2, Daniel E Kadouri 2,*, Robert M Q Shanks 1
PMCID: PMC10924457  PMID: 38358321

Abstract

Endogenous endophthalmitis caused by Gram-negative bacteria is an intra-ocular infection that can rapidly progress to irreversible loss of vision. While most endophthalmitis isolates are susceptible to antibiotic therapy, the emergence of resistant bacteria necessitates alternative approaches to combat intraocular bacterial proliferation. In this study the ability of predatory bacteria to limit intraocular growth of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus was evaluated in a New Zealand white rabbit endophthalmitis prevention model. Predatory bacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus were able to reduce proliferation of keratitis isolates of P. aeruginosa and to a lesser extent S. marcescens. However, it was not able to significantly reduce the number of intraocular S. aureus, which is not a productive prey for these predatory bacteria, suggesting that the inhibitory effect on P. aeruginosa and S. marcescens requires active predation rather than an antimicrobial immune response. Similarly, UV-inactivated B. bacteriovorus were unable to prevent proliferation of P. aeruginosa. Together, these data indicate in vivo inhibition of Gram-negative bacteria proliferation within the intra-ocular environment by predatory bacteria.

Keywords: predatory bacteria, Bdellovibrio bacteriovorus, Micavibrio aeruginosavorus, infection, antibiotic resistance, ocular infection, endophthalmitis, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus

Introduction

Alternative approaches to antibiotics have become a major focus of research due to increasing resistance among bacterial pathogens. One avenue of research for this purpose is the use of predatory bacteria as ‘living antibiotics’ [1–4]. Predatory bacteria such as Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus use a wide range of Gram-negative bacteria as a food source including numerous pathogens [5]. It has been demonstrated that Bdellovibrio bacteriovorus are indifferent to the antibiotic-resistance status of their prey [6, 7]. Moreover, it has been demonstrated that predatory bacteria are non-toxic to mammalian cells and in animal models and have the ability to attenuate Gram-negative bacteria in several in vivo models, including but not limited to airway and oral infections in rodents and central nervous system infection of zebrafish hindbrain ventricles [8–16].

Recent studies have evaluated the use of predatory bacteria for treatment of ocular surface infections [17–19]. These have demonstrated some efficacy against Escherichia coli [17] in a mouse model and Pseudomonas aeruginosa in rabbit models including the prevention of corneal perforations [19]. However freeze-dried B. bacteriovorus proved to be ineffective in treating Moraxella bovis in a large animal keratoconjunctivitis model [20]. The prior studies have focused on the anterior portion of the eye. In this study the goal was to evaluate the use of predatory bacteria in preventing bacterial proliferation in an endophthalmitis model using New Zealand white rabbits.

Endogenous endophthalmitis is frequently caused by Gram-negative bacteria that travel from the blood stream, through the blood-brain barrier and into the posterior portion of the eye [21, 22]. There bacteria can rapidly proliferate, induce a damaging immune response, and cause damage to the tissues crucial for vision such as the retina. This can lead to severe surgical interventions such as removal of the eye’s content (evisceration) or the entire globe (enucleation). Endophthalmitis caused by Gram-negative bacteria such as Klebsiella pneumonia is especially prominent in Asia [21–23]. At our hospital the most frequent Gram-negative bacteria that cause endophthalmitis are Pseudomonas aeruginosa and Serratia marcescens, whereas bacteria from the Staphylococcus and Streptococcus genera are the most prominent overall [24].

In this study, B. bacteriovorus strain HD100 and M. aeruginosavorus strain ARL-13 were evaluated for the ability to prevent growth of bacterial pathogens within the eye and reduced proliferation of P. aeruginosa was reproducibly observed. Data suggests predatory bacteria can inhibit the proliferation of Gram-negative bacteria within the vitreous humour of the rabbit eye.

Methods

Strains and bacterial growth conditions

The predatory bacteria Bdellovibrio bacteriovorus HD100 (ATCC 15356) [25, 26] and Micavibrio aeruginosavorus ARL-13 [27] were used in the study. Predator lysates (co-cultures) were prepared as previously reported [28]. Predators B. bacteriovorus and M. aeruginosavorus were incubated at 30 °C with E. coli strain WM3064 (diaminopimelic acid auxotroph) [29] (1×109 CFU ml−1) for 24 and 72 h respectively. The resulting lysates were passed twice through a 0.45-µm pore-size filter (Millipore, Billerica, MA, USA). Predators were washed with phosphate buffered saline (PBS) and concentrated by three sequential 45 min centrifugations at 29 000 g . Finally, predator pellets were suspended in PBS to reach a final concentration of 1.7×1010 PFU ml−1 B. bacteriovorus and 3.5×109 PFU ml−1 M. aeruginosavorus. To confirm that predator samples were clear of microbial contaminants or residual E. coli prey, final predator preparations were tested on TSA blood agar, Nutrient agar and LB agar supplemented with 0.3 mM diaminopimelic acid. B. bacteriovorus UV inactivated cells were prepared by placing 1 ml of purified predator sample in a well of a 12 well plate and radiating the plate 20 times on the Auto Cross Link setting, while mixing the sample in-between each cross-link (UV Stratalinker 1800; Stratagene, San Diego, CA, USA). Lack of predator viability was confirmed by PFU plating, in which no plaque had developed. Structural integrity of the predator cells was confirmed by light microscopy (1000× magnification).

Pathogens used for this study were P. aeruginosa strain PaC, which is a fluoroquinolone resistant keratitis isolate [30]. S. marcescens strain K904 is a keratitis ocular isolate [31]. S. aureus strain E277 is an endophthalmitis isolate from The Charles T. Campbell Laboratory deidentified strain collection. These were streaked to single colonies on TSA blood agar plates from stocks stored at −80 °C. Single colonies were grown with aeration in lysogeny broth for 16–18 h and then adjusted in PBS to an inoculum of 5–10×103 CFU in 25 µl for injection into eyes.

Animal experiments

This study was approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee (Protocol #15025331 ‘The Use of Predatory Bacteria to Treat Ocular Infections’) and conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research.

Female New Zealand white rabbits weighing 1.1–1.4 kg were received from Charles River Laboratories’ Oakwood Rabbitry. Following systemic anaesthesia with 40 mg/kg of ketamine and 4 mg/kg of xylazine administered intramuscularly, and topical anaesthesia with 0.5 % proparacaine, the right eyes were inoculated in the vitreous via pars plana injection with 25 µl of the bacterial suspension or PBS depending on the group. Immediately following injections of the bacteria, the same eyes were injected with 0.1 ml (100 µl) of the predatory bacteria or PBS. Injection of the predatory bacteria or PBS was performed in a different location than the pars plana injection of bacteria. Rabbits were treated with 1.5 mg/kg ketoprofen, administered intramuscularly, to reduce pain after recovery from anaesthesia. At 24 h after inoculation, the rabbits were examined using a slit lamp and imaged. Rabbits were euthanized with an overdose of intravenous Euthasol solution following systemic anaesthesia with ketamine and xylazine administered intramuscularly. Vitreous humour taps were performed on the infected eyes by inserting a 23-gauge needle attached to a 1 cc syringe into the vitreous chamber about 4 mm from the limbus and removing about 0.2–0.3 ml of fluid. The vitreous humour was transferred to a sterile tube and placed on ice. Standard colony counts determinations were performed on the vitreous samples using TSA blood agar plates and incubated overnight at 37 °C. Cytokines were detected from vitreous humour using commercial kits IL-1β (Sigma-Aldrich), TNFα (Thermo Scientific). Clinical signs of endophthalmitis were determined by a masked reviewer and used a 10-point scale with a 0–2-point score given for discharge, redness of eye, chemosis, anterior eye involvement, and hypopyon.

Statistical analysis

Data was analysed using GraphPad Prism software. All experiments were repeated at least twice. Kruskal-Wallis with Dunn’s post-test was used to compare medians and ANOVA with Tukey’s post-test was used to analyse means.

Results

Predatory bacteria prevent proliferation of P. aeruginosa

P. aeruginosa strain PaC is a fluoroquinolone resistant ocular clinical isolate that was chosen because it is susceptible to predation by predatory bacteria [32]. The ocular vitreous chamber of NZW rabbits was injected with P. aeruginosa (5.0×103 CFU) followed by B. bacteriovorus (4.3×108 PFU) or M. aeruginosavorus (8.8×107 PFU). Controls included injection of vehicle (PBS) or individual microbes. At 24 h eyes were examined by a slit lamp, imaged, and graded for clinical signs of inflammation. Eyes injected with predatory bacteria had increased inflammatory scores that were not significantly higher than the vehicle only control (PBS+PBS), (Fig. 1a). Eyes injected with P. aeruginosa only had a notable increase in inflammatory score (P<0.0001), by comparison, ocular inflammation was reduced in eyes injected with both P. aeruginosa and either predatory bacteria. Although, the clinical signs of inflammation were not significantly different between the PaC alone and the PaC with predatory bacteria, the eyes injected with both PaC and B. bacteriovorus HD100 were not significantly worse than the vehicle control eyes (Fig. 1a). Representative images of eyes from each group are shown in Fig. 1b.

Fig. 1.

Fig. 1.

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Effect of predatory bacteria on inflammation and P. aeruginosa (PaC) proliferation in a rabbit model of endophthalmitis. Each point represents a rabbit in all figures. HD100 indicates B. bacteriovorus; Mica signifies M. aeruginovorus. (a). Clinical inflammation scores for rabbits 24 h post-inoculation. Only the P. aeruginosa group (PAC) and P. aeruginosa with M. aeruginovorus groups were statistical different from the vehicle control, n=5–7. Medians and interquartile ranges (IQR) are shown. Asterisks indicate statistical differences from the PBS group. (b). Representative eyes from subject with median clinical scores are shown for each group. (c). Intraocular CFU ml-1 at 24 h post-injection (median and IQR, n=5–7). Asterisks indicate statistical differences from the PaC group, *, P<0.05. The dotted line indicates the inoculum of PaC introduced into the eye. (d). IL-1β cytokine measured by ELISA, means and standard deviations (SD) are shown, (n=5–7). (e). TNFα cytokine measured by ELISA, means and standard deviations (SD) are shown, (n=5–7). Asterisks indicate differences from the PBS group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

The trend toward reduced inflammation following addition of predatory bacteria to P. aeruginosa treated eyes correlated with a >95 % reduction in the P. aeruginosa bacterial burden in eyes injected with both predatory bacteria and P. aeruginosa compared to P. aeruginosa alone, P<0.05 (Fig. 1c). Unchecked, P. aeruginosa replicated from an inoculum of 5×103 to a burden 4.8×106 CFU in 24 h in the eye. This was 17-fold and 25-fold higher than what it achieved with B. bacteriovorus HD100 and M. aeruginosavorus respectively.

Damage associated pro-inflammatory cytokine IL-1β levels followed a similar trend to the clinical scores and were largely unaffected by predatory bacteria alone, elevated with P. aeruginosa alone, and significantly mitigated in eyes with both predatory bacteria and P. aeruginosa (Fig. 1d). A matching trend for pro-inflammatory cytokine TNFα was measured, although to a lesser extent where the predatory bacteria did not significantly reduce the cytokine levels compared to the P. aeruginosa alone (Fig. 1e).

Predatory bacteria reduce intraocular proliferation of S. marcescens

Fig. 2 shows experimental data in which predatory bacteria were evaluated for their ability to prevent intraocular growth of S. marcescens. Clinical presentation of S. marcescens infected eyes was less severe than that of P. aeruginosa and not notably changed by the addition of predatory bacteria (Fig. 2a). Representative images are shown in Figure S2B (available in the online Supplementary Material). S. marcescens was able to grow 85800-fold from 5.6×103 inoculum to 4.8×108 intraocularly in 24 h. This was reduced 2.2- and 2.7-fold by B. bacteriovorus and M. aeruginosavorus respectively, and was significantly reduced by B. bacteriovorus (Fig. 2c).

Fig. 2.

Fig. 2.

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Impact of predatory bacteria on S. marcescens (SM) proliferation and consequent cytokine production. (a). Clinical inflammation scores for rabbits 24 h post-inoculation, n=9. Medians and interquartile ranges (IQR) are shown. Asterisks indicate statistical differences from the PBS group. (b). Representative images with median clinical scores. (c). Intraocular CFU ml-1 at 24 h post-injection (median and IQR, n=9). Asterisks indicate statistical differences from the SM group (P<0.05). The dotted line indicates the inoculum of S. marcescens injected into the eye. (d). IL-1β cytokine measured by ELISA, means and standard deviations (SD) are shown, (n=5–8). (e). TNFα cytokine measured by ELISA, means and standard deviations (SD) are shown, (n=5–8). Asterisks indicate differences from the PBS group, or where indicated by brackets. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Similar to clinical presentation, IL-1β levels were induced less by S. marcescens than P. aeruginosa and were unaffected by co-incubation with predatory bacteria (Fig. 2d). By contrast, TNFα levels were higher in S. marcescens infected eyes than P. aeruginosa and were significantly reduced when S. marcescens was co-incubated with M. aeruginosavorus (Fig. 2e).

Predatory bacteria do not prevent S. aureus replication in the eye

Although B. bacteriovorus was shown to attach to the Gram-positive bacterium S. aureus, S. aureus does not support B. bacteriovorus or M. aeruginosavorus full predation or growth cycle [25, 33–35]. Inclusion of this bacteria should therefore give insight into whether the prevention of P. aeruginosa and S. marcescens intraocular growth by predatory bacteria is due to active predation or other mechanism, such as predatory bacteria-induced biosynthesis of antimicrobials or immune response triggered by the presence of the predators. The clinical evaluation showed modest inflammation with S. aureus alone that was not significantly altered with addition of predatory bacteria (Fig. 3a). Unchallenged by predatory bacteria, the S. aureus strain was able to grow 87-fold from 1.0×104 to 8.9×105 in 24 h. B. bacteriovorus was associated with a 2.26-fold reduction in S. aureus burden, whereas M. aeruginosavorus reduced less than two-fold (19 %) and neither of these changes were significantly different by ANOVA (Fig. 3b). IL-1β levels were not reduced by the presence of predatory bacteria (Fig. 3c).

Fig. 3.

Fig. 3.

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Differentiation between predation and induced antimicrobial host-response. (a, d). Clinical inflammation scores for rabbits 24 h post-inoculation, n=9–12. Medians and interquartile ranges (IQR) are shown. Asterisks indicate statistical differences from the PBS group. SA indicates S. aureus. (b, e). Intraocular CFU ml-1 at 24 h post-injection (median and IQR, n=9–12). Asterisks indicate statistical differences from the SA and PaC groups. The dotted line indicates the initial inoculum. (c, f). IL-1β cytokine measured by ELISA, means and standard deviations (SD) are shown, (n≥6). Asterisks indicate differences from the PBS group, or where indicated by brackets. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Viable predatory bacteria are required to reduce intraocular proliferation of P. aeruginosa

To further test whether the reduction in P. aeruginosa was mediated by predation, UV-inactivated B. bacteriovorus were tested. The UV-inactivated bacteria correlated with modestly increased clinical scores (Fig. 3d). Importantly, the P. aeruginosa inhibition by live B. bacteriovorus was absent for eyes using the UV-inactivated predator (Fig. 3e). Similarly, the reduction in P. aeruginosa induced IL-1β was reduced by live but not inactivated predatory bacteria (Fig. 3f).

Discussion

This proof-of-principle study determined whether predatory bacteria could inhibit growth of bacteria within the eye. This study demonstrated a clear ability of both tested predatory bacteria to reduce P. aeruginosa proliferation within the eye. While the reduction was less with S. marcescens, predation may be masked by the remarkably fast replication of S. marcescens strain K904 in the eye. While two different experiments differed in the extent to which P. aeruginosa caused clinical inflammatory signs (Fig. 1a versus Fig. 3a), the IL-1β levels were reproducibly reduced when predatory bacteria were added to P. aeruginosa infected eyes. IL-1 is associated with ocular tissue damage that triggers production of pro-inflammatory cytokines and reduces barrier function [36]. IL-1β is a marker of bacterial endophthalmitis associated with productive antimicrobial host-responses [37–39]. Unlike the tested pathogens, predatory bacteria failed to significantly induce intraocular cytokine levels above that of eyes injected with the PBS vehicle. This is in agreement with prior in vitro and in vivo studies [12, 13, 19, 20, 28, 32, 40–42] and, at least for B. bacteriovorus due to its unusual outer membrane composition and membrane-sheathed flagellum which reduce TLR4 and TLR5 activation [43, 44], which are major mediators of inflammation in bacterial endophthalmitis [45, 46].

Data from this study are consistent with the hypothesis that predatory bacteria actively prey upon P. aeruginosa and possibly S. marcescens in the vitreous chamber of the eye. This is based on three observations. First is that the predatory bacteria can prey upon the tested Gram-negative bacteria in vitro [32]. The second is that predatory bacteria failed to significantly reduce the proliferation of a bacteria that they are unable to prey upon, which suggests that an antimicrobial host response is not strongly induced by predatory bacteria. The third being that UV-inactivated predatory bacteria were unable to reduce intraocular P. aeruginosa. However, the presented data cannot rule out the alternative hypotheses that an elevated immune response might be triggered following predation as prey cell debris accumulates or the predatory bacteria could secrete inhibitory products capable of reducing bacterial growth. The reduced proliferation could also stem from a combination of factors. Consistently, a synergistic effect between the immune system and Bdellovibrio was previously suggested in a study monitoring zebrafish injected with Shigella [16].

In conclusion, this study strongly suggests that predatory bacteria can kill bacteria in the vitreous chamber of the eye. However, on its own would likely be insufficient to treat a clinical endophthalmitis. Further studies to determine whether predatory bacteria coupled with antibiotics would improve antimicrobial outcomes as has been previously tested in vitro [47].

Funding information

The author(s) received no specific grant from any funding agency.

Acknowledgements

This study was funded by National Institutes of Health grants R01EY027331 (to R.S.) and CORE Grant P30 EY08098 to the Department of Ophthalmology. The Eye and Ear Foundation of Pittsburgh and from an unrestricted grant from Research to Prevent Blindness, New York, NY provided additional departmental funding. This work was also funded by the U.S. Army Research Office and the Defence Advanced Research Projects Agency (DARPA) and was accomplished under Cooperative Agreement Number W911NF-15-2-0036 to DEK and RMQS. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Army Research Office, DARPA, or the U.S. Government. The U.S. Government is authorized to reproduce and distribute reprints for Government purposes notwithstanding any copyright notation hereon.

Conflicts of interest

The authors declare no conflicts of interest for this study.

Footnotes

Abbreviations: ARVO, Association for Research in Vision and Ophthalmology; CFU, colony forming unit; IL-1, interleukin 1; LB, lysogeny broth; PFU, plaque forming unit; TNF, tumor necrosis factor; TSA, trypticase soya agar.

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