Clearance of Gram-Negative Bacterial Pathogens from the Ocular Surface by Predatory Bacteria
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antibiotics Article Clearance of Gram-Negative Bacterial Pathogens from the Ocular Surface by Predatory Bacteria Eric G. Romanowski 1 , Shilpi Gupta 2, Androulla Pericleous 2, Daniel E. Kadouri 2 and Robert M. Q. Shanks 1,* 1 Charles T. Campbell Eye Microbiology Laboratory, Department of Ophthalmology, The Eye and Ear Institute, University of Pittsburgh School of Medicine, 203 Lothrop Street, Room 1020, Pittsburgh, PA 15213, USA; [email protected] 2 Department of Oral Biology, Rutgers School of Dental Medicine, 110 Bergen St, Newark, NJ 07103, USA; [email protected] (S.G.); [email protected] (A.P.); [email protected] (D.E.K.) * Correspondence: [email protected]; Tel.: +1-(412)-647-3537 Abstract: It was previously demonstrated that predatory bacteria are able to efficiently eliminate Gram-negative pathogens including antibiotic-resistant and biofilm-associated bacteria. In this proof-of-concept study we evaluated whether two species of predatory bacteria, Bdellovibrio bacteri- ovorus and Micavibrio aeruginosavorus, were able to alter the survival of Gram-negative pathogens on the ocular surface. Clinical keratitis isolates of Pseudomonas aeruginosa (strain PAC) and Serratia marcescens (strain K904) were applied to the ocular surface of NZW rabbits followed by application of predatory bacteria. At time intervals, surviving pathogenic bacteria were enumerated. In addi- tion, B. bacteriovorus and S. marcescens were applied to porcine organ culture corneas under contact lenses, and the ocular surface was examined by scanning electron microscopy. The ocular surface Citation: Romanowski, E.G.; Gupta, epithelial layer of porcine corneas exposed to S. marcescens, but not B. bacteriovorus was damaged. S.; Pericleous, A.; Kadouri, D.E.; Using this model, neither pathogen could survive on the rabbit ocular surface for longer than 24 h. Shanks, R.M.Q. Clearance of Gram-Negative Bacterial Pathogens M. aeruginosavorus correlated with a more rapid clearance of P. aeruginosa but not S. marcescens from from the Ocular Surface by Predatory rabbit eyes. This study supports previous evidence that predatory bacteria are well tolerated by Bacteria. Antibiotics 2021, 10, 810. the cornea, but suggest that predatory bacteria do not considerably change the ability of the ocular https://doi.org/10.3390/antibiotics surface to clear the tested Gram-negative bacterial pathogens from the ocular surface. 10070810 Keywords: ocular infection; predatory bacteria; Bdellovibrio; Micavibrio; Pseudomonas aeruginosa; Academic Editors: Mark Willcox, Serratia marcescens; conjunctivitis; keratitis Fiona Stapleton, Debarun Dutta and Ilias Karaiskos Received: 21 May 2021 1. Introduction Accepted: 1 July 2021 Published: 3 July 2021 Predatory bacteria including Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative bacteria that prey upon other Gram-negative bacteria [1,2]. These Publisher’s Note: MDPI stays neutral species are able to prey on a wide range of antibiotic-resistant bacteria including many with regard to jurisdictional claims in human pathogens [3,4] such as ocular isolates of Pseudomonas aeruginosa and Serratia published maps and institutional affil- marcescens [4]. B. bacteriovorus has a broad host-range by which it invades the bacterial cell iations. and replicates in the bacterial periplasm, whereas M. aeruginosavorus exhibits a narrower host-range and acts as an epibiotic predator as it attaches to the outside of prey bacteria [5]. These predators were shown to be highly effective against bacteria in biofilms, which are notoriously recalcitrant to traditional antibiotic therapy [6–9]. We previously postulated that predatory bacteria could be used as a topical treatment Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. for bacterial infection of the eye and demonstrated that predatory bacteria are not toxic to This article is an open access article human ocular surface cell lines and well tolerated on the ocular surface of rabbits [4,10]. distributed under the terms and Other groups have found similar tolerability of predatory bacteria on leporine and bovine conditions of the Creative Commons ocular surfaces [11,12]. Furthermore, intravenous and intranasal inoculation of Micavibrio Attribution (CC BY) license (https:// and Bdellovibrio species, even at high numbers, caused no morbidity or mortality in mice, creativecommons.org/licenses/by/ although they did mildly increase production of proinflammatory cytokines IL-6, TNF- 4.0/). alpha, and chemokine CXCL-1 [13], and numerous mammalian cell lines were unperturbed Antibiotics 2021, 10, 810. https://doi.org/10.3390/antibiotics10070810 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 810 2 of 9 Antibiotics 2021, 10, x FOR PEER REVIEW 2 of 9 by Bdellovibrio strains [4,14,15]. Together, these data suggest that Bdellovibrio and Micavibrio species can be safely used as an experimental therapeutic. Additionally, in vivo studies had reviled that predatory bacteria have potential as “living antimicrobials” for control of kinespathogens. IL-6, TNF-alpha,B. bacteriovorus and chemokinehave shown CXCL-1 efficacy [13], and in numerous limiting Klebsiella mammalian pneumoniae cell lines and Yersinia werepestis unperturbedproliferation by inBdellovibrio airway and strains systemic [4,14,15]. rodent Together, infection these models data suggest [16,17 that]. Similarly, they Bdellovibrio and Micavibrio species can be safely used as an experimental therapeutic. were able to prey upon Shigella flexneri in the hindbrain of zebrafish, promoting the survival Additionally, in vivo studies had reviled that predatory bacteria have potential as “living antimicrobials”of the zebrafish for larvaecontrol [of18 pathogens.]. B. bacteriovorus have shown efficacy in limiting KlebsiellaOcular pneumoniae infections and Yersinia caused pestis by Gram-negative proliferation in airway bacteria, and such systemic as keratitis, rodent in- are associated fectionwith contactmodels lens[16,17]. use Similarly, and can they lead were to aable loss to of prey ocular upon acuity Shigella [19 flexneri–21]. Leadingin the causes of hindbrainthese infections of zebrafish, include promotingPseudomonas the survival aeruginosa of the zebrafishand Serratia larvae [18]. marcescens [22–24]. Antibiotic resistanceOcular infections has been caused noted by among Gram-negative keratitis bacteria, isolates such and as kera is correlatedtitis, are associated with worse clinical withoutcomes contact [ 24lens–29 use]. Dueand can to the lead need to a forloss new of ocular approaches acuity [19–21]. to treat Leading resistant causes microbial of infections, thesewe evaluated infections include the ability Pseudomonas of B. bacteriovorus aeruginosa andand SerratiaM. aeruginosavorus marcescens [22–24].to promoteAntibiotic the clearance resistance has been noted among keratitis isolates and is correlated with worse clinical outcomesof keratitis [24–29]. isolates Due of toS. the marcescens need for newand appr fluoroquinolone-resistantoaches to treat resistant microbialP. aeruginosa infec- and from the tions,ocular we surface evaluated using the ability a rabbit of ocularB. bacteriovorus surface and occupancy M. aeruginosavorus model. to promote the clearance of keratitis isolates of S. marcescens and fluoroquinolone-resistant P. aeruginosa and2.Results from the ocular surface using a rabbit ocular surface occupancy model. 2.1. Scanning Electron Microscopy Visualization of B. bacteriovorus 109J with Porcine Corneas 2.Ex Results Vivo 2.1. ScanningAs a first Electron step Microsco in the studypy Visualization we visualized of B. bacteriov the interactionorus 109J with of Porcine predatory Corneas bacteria and the Excornea Vivo in order to determine whether predatory bacteria could adhere to the corneal surface andAs whether a first step there in the was study any we clear visualized impact the of interaction this interaction of predatory using bacteriaB. bacteriovorus and 109J as thearepresentative cornea in order to strain determine of predatory whether pred bacteria.atory bacteria Although could a adhere previous to the study corneal demonstrated surface and whether there was any clear impact of this interaction using B. bacteriovorus the absence of a clinical inflammatory response by rabbits, it did not evaluate the ocular 109J as a representative strain of predatory bacteria. Although a previous study demon- stratedsurface the at absence a microscopic of a clinical level. inflammatory Figure 1response depicts by ex rabbits, vivo porcineit did not corneasevaluate the from an organ ocularculture surface model at a where microscopicB. bacteriovorus level. Figurestrain 1 depicts 109J ex wasvivo inporcine contact corneas with from the an ocular surface organunder culture a contact model lens where for B. 3 h.bacteriovorus The predatory strain 109J bacteria was in could contact adhere with the to theocular ocular surface, surfacebut failed under to a produce contact lens any for clear 3 h.epithelial The predatory damage bacteria similar could to adhere the mock to the treated ocular (no bacteria) surface,samples. but failed By comparison, to produce any when clear usingepithelial a sample damage Gram-negativesimilar to the mock pathogen, treated (no S. marcescens bacteria)strain K904, samples. under By thecomparison, same experimental when using a conditions, sample Gram-negative adherent bacteria pathogen, were S. present and marcescens strain K904, under the same experimental conditions, adherent bacteria were