Galleria Mellonella As a Model to Study the Effect of Antimicrobial Surfaces on Contamination by Staphylococcus Aureus

Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076

Research Article

Galleria mellonella as a Model to Study the Effect of Antimicrobial Surfaces on Contamination by Staphylococcus aureus

Astrid Mayr1,2, Dorothea Orth-Höller1*, Holger Heinrich3, Guido Hinterberger1, Isabella Wille1,2, Verena Naschberger1, Cornelia Lass-Flörl1,2, Ulrike Binder1

1Department of Hygiene, Microbiology and Social Medicine, Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Innsbruck, Schoepfstr. 41, 6020 Innsbruck, Austria 2Christian Doppler Laboratory for Invasive Fungal Infections (Module 3 – self disinfecting surfaces), Austria 3EGGER Holzwerkstoffe Wismar GmbH & Co. KG, Am Haffeld 1, 23970 Wismar, Germany.

* Corresponding author Abstract Objective: The role of the inanimate environment in the transmission of Dorothea. Orth-Höller, Department healthcare-associated infections (HCAIs) is increasingly recognized. of Hygiene, Microbiology and Hence, several self-disinfecting surfaces have gained attention as new Social Medicine, Division of infection control strategies. The objective of this study was to assess the Hygiene and Medical antimicrobial efficacy of membrane-active polycation (maPK) surfaces Microbiology, Medical University against Staphylococcus aureus (S. aureus) applying the in vivo Galleria of Innsbruck, Innsbruck, mellonella (G. mellonella) contamination model. Schoepfstr. 41, 6020 Innsbruck,

Austria , Tel: +43 512 9003 70772; Methods: In the first set-up maPK surfaces were contaminated with S.

aureus and G. mellonella larvae were placed on the surface for 45 min. In E-mail: dorothea.orth@i- the second set- up larvae were contaminated with S. aureus and med.ac.at transferred to maPK surfaces. Finally, the cross-over rates to larvae and

Received: 23 August 2019; maPK-surface were determined. Additionally, log reduction values Accepted: 02 September 2019; (LRVs) of S. aureus on contaminated maPK and larvae were calculated. Published: 05 September 2019 Results: The first experimental set-up revealed a significantly higher

cross-over rate of S. aureus to the larvae from contaminated control

surface (~2 x 103 colony forming untis (cfu)/mL) when compared to maPK surface (~5.1 x 101 cfu/mL).

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 315

Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076

In the second set-up the cross-over rate of S. aureus from larvae to the control surface was significantly higher (~5.1 x 103 cfu/plate) than to the maPK surface (~2.1 x 101 cfu/plate).

In both experiments LRVs were superior on maPK with 98.1% (p=0.0117) and 99.5% (p=0.0001) reduction, respectively.

Conclusions: This in vivo study reveals the ability of maPK surfaces to reduce bacterial contamination and highlights the potential power to avoid cross-contamination from environmental surface.

Keywords: Healthcare-associated infections; Antimicrobial surfaces; Environmental contamination; Cross-transmission; Staphylococcus aureus; Galleria mellonella

Citation Astrid Mayr, Dorothea Orth-Höller, Holger Heinrich, Guido Hinterberger, Isabella Wille, Verena Naschberger, Cornelia Lass-Flörl, Ulrike Binder. Galleria mellonella as a Model to Study the Effect of Antimicrobial Surfaces on Contamination by Staphylococcus aureus. Archives of Clinical and Biomedical Research 3 (2019): 315- 325.

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 316 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076 Introduction Nosocomial infections represent a major problem The aim of this follow-up study was to further validate complicating patient care and result in increased the biocidal capacity of the maPK surface in an in vivo morbidity and mortality [1]. There is growing evidence model. For this purpose we used Galleria mellonella that exogenous sources such as surface contamination larvae and evaluated whether maPK surface prevents may serve as an important reservoir for pathogens and growth and cross-over of S. aureus ATCC 6538 from hence may contribute to their transmission [2-4]. High surface to larvae and vice versa. level of microbial contamination with relevant pathogens including multidrug-resistant microorganisms Methods distant and close to patients is reported [5-7]. Bacterial strains Environmental persistence has been shown for S. aureus (ATCC 6538) cultured on Columbia agar Staphylococcus aureus (S. aureus), methicillin-resistant containing 5% sheep blood (CBA; Becton Dickinson, S. aureus, vancomycin-resistant enterococcus, Heidelberg, Germany) at 35 ± 2°C was used as test- Acinetobacter baumannii, and Clostridium difficile [8, organism. The bacterial suspension was prepared by 9]. suspending a 24 h-old colony into 2 mL aqua destillata Cleaning and disinfection procedures display basic and adjusting it to a standard turbidity equivalent of 0.5 measures in infection control, however, multiple studies density units on the McFarland Scale (approximately show gaps in decontamination practices [10, 11]. 1.5 x 108 colony forming units (cfu)/mL) using Additionally, surfaces can rapidly become DensiCHECK™ Plus (bioMérieux, France). recontaminated after cleaning and disinfection [12]. Preparation of the larvae and evaluation of their Antimicrobial surfaces have been proposed as an cuticle microbiota additive tool to reduce environmental contamination and Sixth-instar larvae (SAGIP, Bagnacavallo, Italy) possibly prevent healthcare-associated infections weighing 0.3-0.4 g were kept at 18°C in the dark prior (HCAIs) [3, 13]. to use. Since the study protocol intended the use of S. aureus as test organism in the wax moth larvae model Polycationic coated surfaces which are microbicidal the natural occurrence of this microorganism on the upon contact were demonstrated to kill microbes in vitro exoskeletal cuticle of the insect was assessed in a pilot by causing physical damage to the microbe’s cell experiment. For this purpose, a modified protocol of the envelope [14]. We recently showed antimicrobial roll-plate culture method by Maki et al. [16] was activity of a surface coated with membrane-active performed. Briefly, five randomly chosen larvae were polycations (maPK) against S. aureus ATCC 6538 in each rolled back and forth across the surface of CBA vitro [15]. However, whether there is a real clinical (Becton Dickinson, Heidelberg, Germany) and Mannitol benefit is still a matter of debate. For various salt agar (MSA; Oxoid, Wesel, Germany) at least three antimicrobial surfaces implementation of antimicrobial times. After incubating the plates for 24h at 35 ± 2°C surfaces in the clinical setting has demonstrated several microorganisms were identified with standard microbial problems resulting in a reduced efficacy compared to in methods, including Matrix-assisted laser desorption vitro results.

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 317 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076 ionization time-of-flight mass spectrometry (MALDI- immersed separately into sterile phosphate buffered TOF®, Bruker, Bremen, Germany). saline (PBS) for 15 sec by slightly shaking. The suspension was serially diluted and plated onto MSA Determination of S. aureus burden on larvaes’ (Oxoid, Wesel, Germany) agar. The plates were cuticle after contamination incubated at 35 ± 2°C for 24h, bacterial load was As a further pilot experiment insects’ bioburden on the quantified in cfu/mL and larvae were disposed. cuticle after S. aureus contamination was ascertained. Larvae (n=5) were contaminated by immersing the latter Assessment of maPK using the Galleria mellonella into an inoculum containing approximately 1.5 x 108 model to simulate real life conditions cfu/mL of S. aureus for 5 sec. To determine residual To evaluate the antimicrobial potency of the surface two burden of S. aureus on the cuticle immediately after test proceedings were used (Figure 1). dipping, excess fluid was blotted off and larvae were

Figure 1: Flow chart regarding the principle of the two test procedures maPK: membrane-active polycation

In the first set-up maPK combined with an acrylate described previously [15]. Samples containing acrylate dispersion applied on melamine resin layer (Fritz Egger without maPK were used as control. Subsequently, five GmbH & Co. OG, Austria) was contaminated with S. larvae were simultaneously transferred onto the aureus (1.5 x 108 cfu/mL) on a 30 mm radius as contaminated maPK and control surfaces, respectively,

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 318 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076 and removed after 45 min. Residual microbial Microbial colonization on the larvaes’ cuticle (pilot contamination on the surface was determined by contact experiment) RodacTM plates containing MSA (Oxoid, Wesel, Investigation of the larvaes’ (n=5) exoskeletal cuticle Germany). To assess the crossover-rate of S. aureus revealed no S. aureus. This proved both, the suitability from surface to larvae, insects were finally immersed in of the model for our use and S. aureus as an appropriate sterile PBS each for 15 sec by slightly shaking. test organism. We could ensure that all S. aureus Suspensions were serially diluted with PBS and spread detected in following assays originated from intended on MSA (Oxoid, Wesel, Germany). contamination with S. aureus. Furthermore four bacterial genera including both, gram-positive In the second set of experiments the vice-versa situation (coagulase-negative staphylococci, average of 1 x 103 was examined by colonizing the larvae and measuring cfu/plate; enterococci, average of 4.5 x 102 cfu/plate, the spread of S. aureus from the larvae to the surface. Bacillus spp., average of 1.5 x 102 cfu/plate) and gram- Superficial colonization of G. mellonella was simulated negative (Escherichia coli, average of 4 x 102 cfu/plate) by immersing the larvae (n=5) into an inoculum bacteria were found on the larval cuticle. containing approximately 1.5 x 108 cfu/mL of S. aureus for 5 sec. Colonized larvae were placed to maPK and Residual S. aureus burden on the larvaes’ cuticle control surface for 45 min. Residual microbial after contamination (pilot experiment) contamination on the larvaes’ cuticle was determined by After contaminating the larvae (with 1.5 x 108 cfu/mL S. immersing the larvae in PBS each for 15 sec by slightly aureus) the residual burden of S. aureus was shaking, followed by spreading the solution on MSA determined. An average number of 5.1 x 104cfu/mL S. plates as described above. The cross-over rate from the aureus was identified in the suspension assuming that contaminated larvae to the surface was investigated by this amount of microorganisms was present on the contact RodacTM plates with MSA (Oxoid, Wesel, cuticle of the larvae after contaminating. Germany). All agar plates were incubated at 35 ± 2°C for 24h and Significant reduction of S. aureus burden on bacterial load was quantified in cfu/plate. contaminated maPK surfaces exposed to larvae- Test set-up 1 Definition and analysis Each experiment was repeated six times, and maPK

The bacterial cfu counts were log10 transformed to log surfaces were contaminated with S. aureus and densities (LD), and each LD was normalized to the subsequently larvae were exposed to these surfaces. initial bacterial LD, resulting in a log reduction value Bacterial load on larvae after exposure to the (LRV) that could then be used to compare reduction of contaminated surfaces revealed a colony count of ~5.1 x S. aureus on larvae, maPK and control surfaces (mean 101 cfu/mL (representing the contamination rate of the L R V ∆ ) . Percent reduction (PR) was evaluated by the larvaes’ cuticle) for maPK compared to a count of ~2 x formula PR=1-10-LRV∆ [17]. A p-value of < 0.05 was 103 cfu/mL after exposure to control surface (Table 1). considered statistically significant. Thus, a significantly higher cross-over rate of S. aureus Results from the control surface to larvae than from maPK surface to larvae was observed.

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 319 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076

Method Surface Type Recovery of S. aureus on:

larvae

S. aureus contamination of surface (set-up 1) maPK 5.1 x 101 *

control 2 x 103 * surface

S. aureus contamination of larvae (set-up 2) maPK 2.1 x 101 **

control 5.1 x 103 **

Table 1: Comparison of bacterial load on surfaces and larvae in both set-ups after exposure.

Note: maPK membrane-active polycation. *Colony forming units per mL; **Colony forming units per plate

Determination of residual microbial contamination of the surface by contact plates revealed a mean LRV of 2.56 for the antimicrobial surface compared to a mean LRV of 0.83 for the control surface (Table 2). These results represent a significant microbial growth reduction of 98.1% and a difference in LRVs of 1.73 (p=0.0117) for maPK compared to control.

Method Surface Type Mean LRV SD Mean LRV∆ * Mean PR p-value

Set-up 1 maPK 2.56 0.378 1.73 98.1% 0.0117 **

control 0.83 0.467 Set-up 2 maPK 3.46 0.148 2.35 99.5% 0.0001**

control 1.11 0.237

Table 2: Summary of the antimicrobial efficacy of maPK versus control surfaces in both set-ups against S. aureus

Note. maPK membrane-active polycation, LRV log reduction value, SD standard deviation, PR percent reduction * Presents the difference between LRVs of each experiment and was used to calculate the mean PR ** Indicating significance (p < 0.05).

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 320 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076 Significant reduction of pathogen cross-over from The second experimental set-up demonstrated efficacy contaminated larvae to maPK surface-Test set-up 2 of the antimicrobial surface also when larvae were Transmission of the microorganism from colonized contaminated with S. aureus and transferred to the larvae to surfaces was simulated and each experiment surface by a reduced transference of microorganisms was repeated six times. from the larvae to the antimicrobial active surface, and a significantly reduced residual colonization (99.5%; Investigation of the surfaces after contact with p=0.0001) of the larvae. contaminated larvae revealed a higher prevalence of S. aureus on the control surface compared to maPK (~5.1 To the best of our knowledge, this is the first study that x 103 versus ~2.1 x 101 cfu/plate), and substantiates a investigated the biocidal efficacy of surfaces in a more higher transmission of S. aureus from larvae to control practical assessment involving this model. (Table 1). Since the transmission of nosocomial pathogens from Determination of S. aureus on larvae after exposure to hospital surfaces is evident and even potentiated by the surfaces revealed a LRV of 3.46 for larvae unsatisfactory adherence to infection control guidelines transferred to maPK and a LRV of 1.11 for larvae [18-20], the implementation of antimicrobial surfaces as exposed to control (Table 2). Comparison of the two an approach to minimize contamination in the hospital LRVs showed a difference of 2.35 (p=0.0001) setting is eligible [8, 21]. representing a significant PR of 99.5% of the test- organism on larvae in case of exposure to maPK Several studies have shown effectiveness and potential surface. of self-disinfecting surfaces in in-vitro assays [22, 23]. However, when implementing these surfaces in the Discussion hospital setting, various problems became evident, We recently demonstrated in vitro antimicrobial activity which were not predictable after their in-vitro of maPK surfaces against S. aureus ATCC 6538 [15]. evaluation, demonstrating that the transfer of data This follow-up investigation confirmed the previously obtained by laboratory models into real-life setting is described activity in a Galleria mellonella larvae model challenging. For example, copper surfaces showed used to mimic a more “real life” condition by applying a antimicrobial potency with long term effectiveness in living organism on the surface. Here, we demonstrate vitro [24]. However, impact of organic soiling on the that larvae exposed to the contaminated surface showed antimicrobial effect of copper and acquired copper a lower rate of microbial transference in case of resistance in gram-positive and in gram-negative exposure to the antimicrobial active surface. This data bacteria has been reported and thus limiting its was supported by a significant reduction (98.1%; application in the hospital setting [25, 26]. Micro- p=0.0117) of the bacterial load on the antimicrobial patterned surfaces, designed to prevent microbial surface after contaminating the surface with S. aureus adhesion to the surface were evaluated in vitro and in and exposure of Galleria mellonella larvae on this the clinical setting and demonstrated inhibition of surface. surface contamination and transfer of clinical relevant organisms in both settings [21, 27]. In this case the issue

Archives of Clinical and Biomedical Research Vol. 3 No. 5 – October 2019. [ISSN 2572-9292] 321 Arch Clin Biomed Res 2019; 3 (5): 315-325 DOI:10.26502/acbr.50170076 of cost, applicability and stability was not considered in In addition, this study did not address the impact of the initial studies. Hence, translational pre-clinical in potential compromising factors on biocidal activity of vivo trials, like the Galleria model, might be useful to the maPK surface, like organic soiling or microbial further validate the biocidal capacity of surfaces prior to resistance to the biocide [26]. The main challenge ahead their implementation in the hospital setting [17]. is to evaluate whether our tested surface has any in vivo impact in terms of reducing HCAI. We demonstrate, that maPK surfaces possess Conclusion antimicrobial efficacy both, in vitro and in vivo, In conclusion, the maPK surface showed antimicrobial although surfaces displayed a stronger killing effect in efficacy in a larvae, in vivo model. Thus, this surface vitro (LRV of 8 in vitro versus 1.73 and 2.35 in vivo). may offer a complementing tool to reduce microbial Besides demonstrating a reduction of bacterial surface burden in the hospital setting and delay recontamination contamination in the larvae model we also show a in a more sustained manner compared to periodic reduction of bacterial transfer from the larvae to the cleaning and disinfection alone. surface and vice-versa in the present study. These observations lead to the hypothesis that maPK surfaces List of abbreviations might also reduce pathogen transfer from and onto CBA: Columbia agar 5% sheep blood; HCAI: surfaces via through healthcare workers’ (HCWs) Healthcare-associated infection; HCW: Healthcare hands, which is a major concern for microbial worker; LD: Log density; LRV: Log reduction value; transmission in the hospital setting [18, 19]. maPK: membrane- active polycation; MSA: Mannitol salt agar; PBS: Phosphate buffered saline; PR: Percent Galleria larvae are mainly used as alternative model reduction; SD: Standard deviation organisms to simulate systemic forms of disease and have been adapted for a broad range of bacterial and Declarations also fungal pathogens [28]. Ethics approval and consent to participate T hese organisms bear several advantages in comparison The use of G. mellonella does not require ethic to other invertebrate models, i.e. they are easy to keep, approval. cheap to purchase, can be maintained at temperatures up to 37°C and the larvae are not subject to the ethical Consent for publication limitations of mammalian models [28]. Not applicable.

Limitations of the present study include that contact- Availability of data and material plate sampling was performed with agar plates which All data generated or analyzed during this study are didn’t contain neutralizing compounds, but earlier data included in this published article. displayed no influence. Furthermore, the experiments were performed only with S. aureus ATCC 6538 using Competing interests one inoculum. The effectiveness against other relevant The authors declare that they have no competing microorganisms and a broader range of inocula has to interests. be investigated.

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