To safely eliminate ARB from greywater, further studies should be carried out to understand how the transfer of ARGs occurs. Of particular importance is the determination of whether specific compounds abundant in greywater (e.g., detergents) lead to resistance evolution. This work dealt with the detection of Tetracycline-resistant bacteria and Tetracycline resistant genes on greywater, in the system and the soil also evaluating the potential multiple resistance. Preliminary genetic relations between the tet39 genes isolated showed a possible exchange between clinical and environmental strains. However, further study needs to be done to understand the clonal relations between the isolates better understand the clonal relations between the isolates and strengthen our results.

#### AUTHOR CONTRIBUTIONS

ET performed the experiments, analyzed the data, and wrote a draft manuscript. LB analyzed the data and supervised the writing of the draft manuscript. AG aided in interpreting the results and worked on the manuscript. ZR conceived the study and was in charge of the overall direction and planning, as well as writing the manuscript.

#### ACKNOWLEDGMENTS

This research was funded in part by the Zuck Maccabi Foundation. We would like to thank Dr. Tali Bruner and Chen HarGil for their technical assistance.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02666/full#supplementary-material

Agersø, Y., and Guardabassi, L. (2005). Identification of Tetracycline39, a novel class of tetracycline resistance determinant in Acinetobacter spp. of environmental and clinical origin. J. Antimicrob. Chemother. 55, 566–569. doi: 10.1093/jac/dki051

Alonso, A., Sanchez, P., and Martinez, L. (2001). Environmental selection of antibiotic resistance genes. Environ. Microbiol. 3, 1–9. doi: 10.1046/j.1462-2920.2001.00161.x

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Troiano, Beneduce, Gross and Ronen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# An Insight Into the Potentiation Effect of Potassium Iodide on aPDT Efficacy

Cátia Vieira1 , Ana T. P. C. Gomes1 \*, Mariana Q. Mesquita2 , Nuno M. M. Moura2 , M. Graça P. M. S. Neves2 , M. Amparo F. Faustino2 \* and Adelaide Almeida1 \*

1 Department of Biology and CESAM, University of Aveiro, Aveiro, Portugal, 2 Department of Chemistry and QOPNA, University of Aveiro, Aveiro, Portugal

#### Edited by:

Fabian Cieplik, Universitätsklinikum Regensburg, Germany

#### Reviewed by:

Tim Maisch, University of Regensburg, Germany Michael R. Hamblin, Massachusetts General Hospital and Harvard Medical School, United States Daniel Manoil, Université de Genève, Switzerland

#### \*Correspondence:

Ana T. P. C. Gomes<EMAIL_ADDRESS>M. Amparo F. Faustino<EMAIL_ADDRESS>Adelaide Almeida<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 23 July 2018 Accepted: 18 October 2018 Published: 19 November 2018

#### Citation:

Vieira C, Gomes ATPC, Mesquita MQ, Moura NMM, Neves MGPMS, Faustino MAF and Almeida A (2018) An Insight Into the Potentiation Effect of Potassium Iodide on aPDT Efficacy. Front. Microbiol. 9:2665. doi: 10.3389/fmicb.2018.02665 Antimicrobial photodynamic therapy (aPDT) is gaining a special importance as an effective approach against multidrug-resistant strains responsible of fatal infections. The addition of potassium iodide (KI), a non-toxic salt, is recognized to increase the aPDT efficiency of some photosensitizers (PSs) on a broad-spectrum of microorganisms. As the reported cases only refer positive aPDT potentiation results, in this work we selected a broad range of porphyrinic and non-porphyrinic PSs in order to gain a more comprehensive knowledge about this aPDT potentiation by KI. For this evaluation were selected a series of meso-tetraarylporphyrins positively charged at meso positions or at β-pyrrolic positions and the non-porphyrinic dyes Methylene blue, Rose Bengal, Toluidine Blue O, Malachite Green and Crystal Violet; the assays were performed using a bioluminescent E. coli strain as a model. The results indicate that KI has also the ability to potentiate the aPDT process mediated by some of the cationic PSs [Tri-Py(+)-Me, Tetra-Py(+)-Me, Form, RB, MB, Mono-Py(+)-Me, β-ImiPhTPP, β-ImiPyTPP, and β-BrImiPyTPP] allowing a drastic reduction of the treatment time as well as of the PS concentration. However, the efficacy of some porphyrinic and non-porphyrinic PSs [Di-Py(+)-Me opp, Di-Py(+)-Me adj, Tetra-Py, TBO, CV, and MG] was not improved by the presence of the coadjuvant. For the PSs tested in this study, the ones capable to decompose the peroxyiodide into iodine (easily detectable by spectroscopy or by the visual appearance of a blue color in the presence of amylose) were the most promising ones to be used in combination with KI. Although these studies confirmed that the generation of 1O2 is an important fact in this process, the PS structure (charge number and charge position), aggregation behavior and affinity for the cell membrane are also important features to be taken in account.

Keywords: antimicrobial photodynamic therapy, cationic porphyrins, phenothiazines, xanthenes, potassium iodide, bioluminescent E. coli

## INTRODUCTION

Antibiotics are among the most commonly prescribed drugs used in both human medicine and in farm animals, resulting in the selection of multiple drugs resistant (MDR) bacteria (Economou and Gousia, 2015; O'Neill, 2016). Infections with resistant bacteria are difficult to treat, causing severe illness and requiring costly and sometimes toxic alternatives, such as antibiotics of last resort.

Drugs of last resort, such as vancomycin against Gram-positive bacteria and colistin against Gram-negative bacteria, have been the most reliable therapeutic agents against MDR bacteria. However, bacterial strains resistant to these antibiotics have been isolated worldwide (Levine, 2006; Wang et al., 2018). This resistance can result from a chromosomal gene mutation, but comes mainly from horizontal transfer from external gene sources (Chambers and DeLeo, 2009; DeLeo et al., 2010; Gardete and Tomasz, 2014; Gao et al., 2016). The development of novel antibiotics is not likely to solve the problem and it is probably only a matter of time until they will be also ineffective. Bacteria will inevitably find ways of resisting to the conventional antibiotics, which is why alternative approaches are urgent.

Antimicrobial photodynamic therapy (aPDT) can be a very promising alternative to antibiotic treatment namely in localized infections (Dai et al., 2010). aPDT involves the use of a photosensitizer (PS) which in the presence of visible light and oxygen produces reactive oxygen species (ROS), such as singlet oxygen (1O2). These species are responsible for the oxidation of several cellular components conducting to rapid cell inactivation. This approach presents some advantages when compared with the use of antibiotics, such as being efficient independently of the microorganism antibiotic resistance profile (Jori et al., 2011), does not induce the development of resistance, even after several cycles of treatment (Giuliani et al., 2010; Tavares et al., 2010; Costa et al., 2011) and can be applied with efficacy against Gram-negative and Gram-positive bacteria. aPDT is considered more effective against Gram-positive bacteria due to their highly permeable cell walls allowing the easy diffusion of neutral, positive and negative charged PS into the cell. However, the impermeable external membrane of Gram-negative bacteria cell wall limits the anionic or neutral-charge PSs entrance (Minnock et al., 2000). This limitation is overcome by the use of cationic PS. These PSs are able to bind and penetrate into the cell wall by the "self-promoted uptake pathway" (Hancock et al., 1991; Merchat et al., 1996). Nevertheless, neutral PSs or PSs with low number of charges can be effective against this type of bacteria by coupling or combining them with positively charged entities such as poly-L-lysine, polyethylenimine and polymyxin B nonapeptide that act as membrane disruptors (Nitzan et al., 1992; Helander et al., 1997; Lounatmaa et al., 1998; Soukos et al., 1998). Ethylenediaminetetraacetic acid (EDTA) is also commonly used to destabilize the native organization of Gramnegative wall (Yoshimura and Nikaido, 1985; Jori et al., 2006). It has also been shown that different organic salts can improve the efficiency of aPDT against Gram-negative bacteria (Huang et al., 2012; Kasimova et al., 2014). Recently, some studies have demonstrated that aPDT can be potentiated by addition of several different inorganic salts, such as sodium bromide (Wu et al., 2016) sodium azide (Huang et al., 2012; Kasimova et al., 2014), sodium thiocyanate (St Denis et al., 2013) and potassium iodide (Vecchio et al., 2015; Zhang et al., 2015; Freire et al., 2016; Huang et al., 2016, 2017, 2018a,c; Hamblin, 2017; Reynoso et al., 2017; Wen et al., 2017). In fact, the addition of iodide has been shown to improve the efficiency of aPDT in several animal models of localized infection. This salt is non-toxic and is an approved drug for antifungal therapy (Hamblin, 2017). The studies involving the use of KI demonstrate that the combination of this salt with neutral porphyrins, fullerenes and other dyes gives rise to higher microbial inactivation rates when are compared to the use of the PSs alone. KI was firstly studied as potentiator of aPDT mediated by a C60 fullerene bisadduct (Zhang et al., 2015). The results showed that KI potentiated the ultraviolet A (UVA) or the white light-mediated killing of Gram-negative bacteria Acinetobacter baumannii, Grampositive methicillin-resistant Staphylococcus aureus and fungal yeast Candida albicans, increasing the effect in 1–2 logs. This extra killing effect was also observed in vitro and in vivo using a mouse model with an infected skin abrasion (Zhang et al., 2015). These promising results conducted to new studies concerning the mechanism of action involved. The KI effect using Methylene Blue (MB) as PS in the photoinactivation of Escherichia coli and S. aureus was also evaluated (Vecchio et al., 2015). The results showed that the addition of KI increased the bacterial killing in 4 and 2 logs for S. aureus and E. coli, respectively, in a dose-dependent manner. The authors also affirmed that the KI potentiator effect in these aPDT studies mediated by MB was probably due to the formation of reactive iodine species that were quickly produced with a short lifetime (Vecchio et al., 2015). Since then, some other examples of the potentiation of aPDT effect using combinations of PSs and KI were reported. For instance, MB and new methylene blue (NMB) were studied in the photoinactivation of oral C. albicans infection in a mouse model (Freire et al., 2016), Photofrin in the photoinactivation of several Gram-negative bacteria (Huang et al., 2017), BODIPY dyes in the photoinactivation of S. aureus, E. coli, and C. albicans (Reynoso et al., 2017). This approach was also efficient in aPDT of Gram-negative and Gram-positive bacteria mediated by Rose Bengal (Wen et al., 2017) and fullerenes (Huang et al., 2018b). Interestingly, an anionic porphyrin in the presence of KI was able to photoinactivate E. coli (Huang et al., 2018a). The combination of MB and KI was also efficient to treat an urinary tract infection in a female rat model (Huang et al., 2018c). All these reports helped to elucidate the mechanism of action of KI potentiation. It was proposed that the extra killing effect is caused by several parallel reactions initiated by the reaction of 1O2 with KI producing peroxyiodide (**Figure 1**), that can suffer further decomposition by two different pathways, which are dependent on the degree of binding of the PS to the microbial cells (Vecchio et al., 2015; Zhang et al., 2015; Freire et al., 2016; Gsponer et al., 2016; Hamblin, 2017; Huang et al., 2017, 2018a; Kashef et al., 2017; Reynoso et al., 2017; Wen et al., 2017). One of the pathways involves the formation of free iodine (I2/I− 3 ) and hydrogen peroxide (H2O2). Free iodine can kill microbial cells when generated in solution but needs to reach a sufficient threshold concentration to be microbicidal. The amount of free iodine produced depends on the amount of 1O2 produced, but also on the concentration of iodide anion present in solution (**Figure 1**). The other one involves a homolytic cleavage process producing reactive iodine radicals (I·− 2 ), which are much more toxic if generated very close to the target cells since these radicals have short diffusion distance (**Figure 1**).

The microbial killer role of the two species can be distinguished by observing the killing microbial curve profile. When the principal contribution for the killing is the free iodine, the curves assumes an abrupt threshold value. On the other hand, a gradual killing curve can be observed when the short-lived reactive iodine species are the mainly killing species (Huang et al., 2018a).

Until now, the literature survey only reported combinations of PSs and KI with a positive aPDT potentiation. Additionally, the possibility of extending the approach to cationic porphyrins was not evaluated. Consequently, in this work, in order to gain a more comprehensive knowledge about this type of potentiation, we decided to assess the effect of KI in the presence of a broad range of cationic porphyrinic and non-porphyrinic dyes as PSs (**Figure 2**). To achieve this objective and considering the high number of assays required to evaluate the different combinations of PSs with KI, the assays were performed using a bioluminescent E. coli strain as a bacterial model. It is well known that the bioluminescence approach can provide a sensitive and innocuous way to detect the viability state of microorganisms. Compared to the conventional plating count methodology, the use of bioluminescent strains in aPDT allows to monitor the process in real-time and it is a sensitive and cost-effective methodology to evaluate this effect. Moreover, the strong correlation between CFU and bioluminescent signal of the bioluminescent E. coli used in this work has already been proved and described (Alves et al., 2008, 2011a,b).

The structures of the selected PSs summarized in **Figure 2** comprise: (i) the five structurally related mesotetraarylporphyrins with one [**Mono-Py(**+**)-Me**], two [**Di-Py(**+**)-Me opp** and **Di-Py(**+**)-Me adj**], three [**Tri-Py(**+**)-Me**], and four [**Tetra-Py(**+**)-Me**] positives charges and a formulation (**Form**) based on these porphyrins; (ii) the three β-substituted porphyrins β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP** bearing positively charged imidazole units; and (iii) the non-porphyrinic dyes – methylene blue (**MB**), Rose Bengal (**RB**) and Toluidine Blue O (**TBO**), crystal violet (**CV**) and malachite green (**MG**).

In the selection of these three series of PSs was considered their different photoinactivation profile toward E. coli and their mechanism of action (Type I and Type II).

For the meso-tetraarylporphyrins with positive charges at the meso position the studies already performed demonstrated that their photodynamic efficiency was dependent on charge number, charge distribution, aggregation behavior and molecular amphiphilicity and the order of their efficacy was: **Mono-Py(**+**)-Me** < **Di-Py(**+**)-Me opp** < **Di-Py(**+**)-Me adj** < **Tetra-Py(**+**)-Me** < **Tri-Py(**+**)-Me**. Additionally, a formulation (**Form**) constituted by a non-separated mixture of Mono-Py(+)-Me (19%), Di-Py(+)-Me opp and Di-Py(+)-Me adj (20%) Tri-Py(+)-Me (44%) and Tetra-Py(+)-Me (17%) was also studied. This mixture has already proved to be efficient in the photoinactivation of S. aureus, E. coli and Pseudomonas syringae pv. actinidiae and is considered an excellent alternative to the highly efficient **Tri-Py(**+**)-Me** since the production costs and also the production time was reduced significantly (Marciel et al., 2018; Martins et al., 2018). The neutral 5,10,15,20-tetra-(4 pyridyl)porphyrin (**Tetra-Py**) precursor of the positively charged **Tetra-Py(**+**)-Me** was also included.

For the meso-tetraarylporphyrins with a positive charge at the beta-pyrrolic position (β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP**) a different efficacy profile in photoinactivation of E. coli at concentrations of 20 µM was observed in previous studies; however, at 5.0 µM none of the three PSs caused a significant decrease in bacterial activity (Moura et al., 2019).

Although porphyrins and porphyrins analogs comprise most of the PSs used in aPDT, several non-porphyrinic chromogens exhibit photodynamic activity (Ormond and Freeman, 2013). Thus, for this study were selected good 1O2 generators with positive charges that already proved their photodynamic efficiency in clinical trials such as the phenothiazinium salts **MB** and **TBO** (Abrahamse and Hamblin, 2016). In this study were also included two photoactive dyes that act mainly through type I mechanism (with lower 1O2 production rates), the **CV** and **MG**. In this evaluation the study was extended to the xanthene derivative **RB**. Combinations of KI with **RB** and with **MB** were

already studied and were introduced in this work to corroborate our results (Vecchio et al., 2015; Wen et al., 2017).

## MATERIALS AND METHODS

fmicb-09-02665 November 15, 2018 Time: 18:34 # 5

#### Photosensitizers: Stock Solutions and UV-Vis Spectra

Stock solutions of each porphyrin were prepared at 500 µM in dimethyl sulfoxide (DMSO) and stored in the dark. Stock solutions of non-porphyrinic dyes were prepared at 500 µM in phosphate buffer solution (PBS) and stored in the dark.

The porphyrins 5-(1-methylpyridinium-4-yl)-10,15,20 tris(pentafluorophenyl)-porphyrin mono-iodide [**Mono-Py(**+**)-Me**], 5,15-bis(1-methylpyridinium-4-yl)-10,20-bis(pent afluorophenyl)porphyrin di-iodide [**Di-Py(**+**)-Me opp**] 5,10 bis(1-methylpyridinium-4-yl)-15,20-bis(pentafluorophenyl)-po rphyrin di-iodide [**Di-Py(**+**)-Me adj**], 5,10,15-tris(1-methy lpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide [**Tri-Py(**+**)-Me**] and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide [**Tetra-Py(**+**)-Me**], the formulation (**Form**) of the non-separated porphyrins Mono-Py(+)- Me (19%), Di-Py(+)-Me opp and Di-Py(+)-Me adj (20%) Tri-Py(+)-Me (44%) and Tetra-Py(+)-Me (17%) and the neutral 5,10,15,20-tetra-(4-pyridil)porphyrin (**Tetra-Py**) were synthetized according with the literature (Simões et al., 2016; Marciel et al., 2018; Martins et al., 2018). The preparation of the mono-cationic porphyrins β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP** bearing an imidazole ring at the β-pyrrolic position were synthetized according with a procedure developed in our laboratory (Moura et al., 2019), Crystal Violet (**CV**) was purchased from Merck, Rose Bengal (**RB**) from Fluka AG, Malachite Green (**MG**) from Riedel-de-HaënTM, Methylene Blue (**MB**) and Toluidine Blue O (**TBO**) from Acros Organics. The UV-Vis spectra of the PSs are presented in **Supplementary Figure S2** (see Supporting Information).

## Light Sources

The potentiation of aPDT effect between the PS and KI was evaluated by exposing the bacterial suspension in the presence of each combination to a set of fluorescent PAR lamps which is constituted by 13 fluorescent lamps OSRAM 21 of 18 W each, PAR white radiation (380–700 nm) at an irradiance of 25 W m−2 . All the irradiances were measured with a Power Meter Coherent FieldMaxII-Top combined with a Coherent PowerSens PS19Q energy sensor.

## Bacterial Strains and Growth Conditions

The genetically transformed bioluminescent E. coli Top10 (Alves et al., 2011b) was grown on Tryptic Soy Agar (TSA, Merck) supplemented with 50 mg mL−1 of ampicillin (Amp) and with 34 mg mL−1 of chloramphenicol (Cm). Before each assay, one isolated colony was transferred to 10 mL of tryptic soy broth medium (TSB, Merck) previously supplemented with Amp and Cm and was grown overnight at 25◦C under stirring (120 rpm). An aliquot was transferred into 10 mL TSB under the same growth conditions till stationary growth phase was achieved. An optical density at 600 nm (OD600) of 1.6 ± 0.1 corresponded to ≈108 colony forming units (CFU) mL−1 .

The correlation between CFU mL−1 and the bioluminescent signal (in RLUs) of bioluminescent E. coli strain was evaluated. A fresh overnight bacterial culture was serially diluted (10−1 to 10−9 ) in PBS. Non-diluted and diluted aliquots were pourplated on TSA medium (0.5 mL) and, simultaneously, were read on a luminometer (0.5 mL) (TD-20/20 Luminometer, Turner Designs, Inc., Madison, WI, United States) to determine the bioluminescence signal. The results obtained are presented in **Supplementary Figure S1** (see Supporting Information).

### Antimicrobial Photodynamic Therapy (aPDT) Procedure

Bioluminescent E. coli culture was grown overnight and was tenfold diluted in PBS (pH 7.49), to a final concentration of ∼108 CFU mL−1 , which corresponds approximately to 108 RLU. The bacterial suspension was equally distributed in 50 mL sterilized and acid-washed beakers.

#### Bioluminescence Monitoring

All the experiments were carried out under PAR white light (380–700 nm) and the E. coli bioluminescence signal was measured in the luminometer at different times of light exposure. The assays were finished whenever the detection limit of the luminometer was achieved (c.a 2.3 log). Light control (LC), dark control (DC), and KI control, were also evaluated as described below.

#### Evaluation of the Inorganic Salt Effect on Tetra-Py(+)-Me Photodynamic Action

The first experiments were performed in order to assess the effect of different inorganic salts in the inactivation of E. coli through aPDT approach using the tetracationic porphyrin **Tetra-Py(**+**)- Me,** extensively studied in bacterial photoinactivation processes (Alves et al., 2008; Tavares et al., 2011; Simões et al., 2016). The selected inorganic salts were KI, NaI, KCl, NaCl, and NaBr and the assays were conducted with 50 mM of each salt and 5.0 µM of **Tetra-Py(**+**)-Me**. All the inorganic salts were purchased from Sigma-Aldrich (St. Louis, MO, United States) and stock solutions were prepared at 500 mM in PBS immediately before each experiment.

The assays were carried out by exposing the bioluminescent E. coli suspension to **Tetra-Py(**+**)-Me** at 5.0 µM with each salt added from the stock solution to achieve the final concentrations of 50 mM. Simultaneously, the following different controls were performed: one light control (LC) that contained a bacterial suspension exposed to the same light conditions as the samples, and dark controls (DC) that comprised a bacterial suspension incubated with the PS at 5.0 µM and with the distinct salts at 50 mM. DC were protected from light during all the procedure. The samples and controls were protected from light with aluminum foil and remained in the dark for 15 min to promote the porphyrin binding to E. coli cells before irradiation. Then, both samples and controls were exposed to the PAR

white light at 25 W m−2 under stirring (120 rpm) and placed on a tray; the beaker bottoms were covered with water to maintain the samples at constant temperature (25◦C). Finally, aliquots of 0.8 mL of samples and controls were collected at different times of light exposure and the bioluminescence signal was measured in the luminometer. Three independent experiments with two replicates were performed and the results were averaged.

#### Evaluation of the Antimicrobial Effect in the Presence of Different PSs and KI

The assays were carried out by exposing a final volume of 10 mL of a bioluminescent E. coli suspension to each PS at 5.0 µM and combinations of each PS at 5.0 µM and KI concentrations at 50 and 100 mM and for **RB**, **CV**, **MG** also at 25 mM. The samples were protected from light with aluminum foil and incubated in the dark for 15 min. Light and dark controls were also carried out simultaneously with the aPDT procedure: the light controls (LC) comprised a bacterial suspension and a bacteria suspension with KI at 100 mM exposed to the same light protocol; and the dark control (DC) comprised a bacterial suspension incubated with the PSs at 5.0 µM and KI at the higher concentration tested (100 mM) protected from light. The aPDT treatment was performed as described above. Three independent experiments with two replicates were performed and the results were averaged.

#### Detection of Iodine Formation

In a 96 wells microplate, appropriate volumes of each PS at 5.0 µM (1 µL) and combinations of each PS at 5 µM (1 µL) and KI at 100 mM (2 µL) were added to each well and irradiated with PAR white light at 25 W m−2 . The generation of iodine was monitored by reading the absorbance at 340 nm at irradiation times 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, and 120 min. As positive control it was used Lugol's solution diluted to 1:1000.

Another simple assay to detect iodine was also performed, for the different combinations of PS and KI, in the presence of amylose due to the well-known formation of a strong blue complex when these two species are present (Luallen, 2017). So, to the beakers containing a starch solution at a concentration of 2 mg L−1 , it was added each PS at 5 µM and KI at 100 mM. The samples were incubated in the dark for 15 min and afterward were exposed continuously and under stirring (120 rpm) to the same light source used in the aPDT assays. The color change was registered and photographed at different times of irradiation for each sample. At the same time, the following control assays were performed: PS + light; KI + light, PS + KI under dark.

#### Statistical Analysis

Three independent experiments with two replicates per assay for each condition were done. The statistical analysis was performed with GraphPad Prism. Normal distributions were checked by the Kolmogorov–Smirnov test and the homogeneity of variance was verified with the Brown Forsythe test. ANOVA and Dunnet's multiple comparison tests were applied to assess the significance of the differences between the tested conditions. A value of p < 0.05 was considered significant.

## RESULTS

The effect of KI for each series of PSs toward E. coli was evaluated using the same concentration of PS (5.0 µM) and KI concentrations of 50 and 100 mM (unless other concentrations were mentioned) under PAR white light at an irradiance of 25 W m−2 . These KI concentrations were selected considering the ones referred in similar studies and knowing that higher concentrations can limit the combined protocol application in clinic area due to osmotic stress. The PS, **TetraPy(**+**)-Me,** was selected to confirm the benefic effect of KI among other inorganic salts (NaI, NaCl, KCl, and NaBr). This well-known tetracationic porphyrin is extensively studied in bacterial photoinactivation processes and is considered an excellent reference when the efficacy of different cationic porphyrins are compared (Alves et al., 2008; Tavares et al., 2011; Simões et al., 2016). Low light doses ranging from 1.5 to 36 J/cm2 emitted by a fluorescent lamp set (380–700 nm) were selected based on their efficacy to inactivate a large range of microorganisms (Marciel et al., 2017; Moura et al., 2019). Additionally, this light source was able to accomplish the required overlap between PS absorption and light setup emission spectrum (see **Supplementary Figure S2**; Costa et al., 2010; Cieplik et al., 2015).

#### Evaluation of the Salt Effect on Tetra-Py(+)-Me Photodynamic Efficiency

The results presented in **Figure 3** show that the photoinactivation pattern of E. coli in the presence of **Tetra-Py(**+**)-Me** is strongly dependent on the anion used.

The results clearly indicate that when combinations of **Tetra-Py(**+**)-Me** with KI and NaI were used, a reduction of the bioluminescence signal of c.a. 4 log was observed after 30 min of irradiation. In the case of NaBr, KCl and NaCl no potentiation on

the aPDT effect was detected. Light and dark controls showed no significant variation in the bioluminescence produced by E. coli.

#### Evaluation of the KI Effect on the Photodynamic Action of Meso-Tetraarylporphyrins Bearing One to Four Positive Charges

The effects of KI at 50 and 100 mM in the photodynamic action of **Mono-Py(**+**)-Me**, **Di-Py(**+**)-Me opp**, **Di-Py(**+**)-Me adj**, **Tri-Py(**+**)-Me**, and **Tetra-Py(**+**)-Me** toward E. coli are summarized in **Figure 4**.

In the cases of the LCs (Bacteria and bacteria + KI irradiated) and DC (bacteria + PS + KI in the dark) no decrease in E. coli bioluminescent signal was detected. These results indicate that the viability of this recombinant bioluminescent bacterium was not affected by irradiation, by the presence of the salt or by any of the tested combinations of PS + KI in the dark.

The results shown in **Figure 4A** for the monocationic porphyrin [**Mono-Py(**+**)-Me**] demonstrated that its low efficacy is strongly improved by the presence of KI; the poor activity of this PS toward E. coli was previously related with its low water solubility leading to aggregation and, consequently, to low 1O2 generation. Under the conditions used in these assays this porphyrin maintained its low efficacy causing a decrease on E. coli bioluminescence signal of 0.9 log (p < 0.0001) after 240 min of irradiation. However, the addition of KI at 50 mM and 100 mM potentiated the effect of this mono-cationic porphyrin, causing bioluminescent signal reductions of c.a. 3.5 and 5.5 log (p < 0.0001) after 150 min of irradiation.

The dicationic porphyrins **Di-Py(**+**)-Me opp** and **Di-Py(**+**)- Me adj** without the presence of the coadjuvant promoted similar effects on the reduction (c.a. 6 log after, respectively, 150 and 120 min of irradiation) of E. coli bioluminescence signal (**Figures 4B,C**). However, when these two isomers were combined with KI the results obtained were significantly different. The combination of **Di-Py(**+**)-Me adj** with KI at 50 and 100 mM produced similar results in the photoinactivation of bioluminescent E. coli and no improvement in aPDT efficiency was detected (**Figure 4C**). In fact, in the last irradiation time, there were no significant differences in the E. coli bioluminescence signal promoted by **Di-Py(**+**)-Me adj** and the two combinations of **Di-Py(**+**)-Me adj** + KI. In the case of **Di-Py(**+**)-Me opp** (**Figure 4B**) the presence of KI (at 50 and 100 mM) led to a significant reduction on its efficacy. The maximum inactivation achieved for the combination of this PS with 100 mM of KI was 1.7 log (p < 0.0001).

The **Tetra-Py(**+**)-Me** and **Tri-Py(**+**)-Me** were the most efficient porphyrins in the photoinactivation of bioluminescent E. coli, which is also in accordance with the literature (Simões et al., 2016). These porphyrins, when acting by themselves, showed to be potent PSs for the inactivation of bioluminescent E. coli, demanding short irradiation times (c.a. 70 min) to achieve total photoinactivation of this Gram-negative bacterium (**Figures 4D,E**). The combination of these PSs with KI at 50 and 100 mM increased dramatically the effect of these PSs in the photoinactivation of bioluminescent E. coli (**Figures 4D,E**). In the case of **Tri-Py(**+**)-Me**, it was observed an abrupt decrease in E. coli viability after 30 and 10 min of irradiation when the combinations of this PS with 50 mM and 100 mM of KI were used, respectively (**Figure 4D**). This sharp decrease was also observed for the combination of **Tetra-Py(**+**)-Me** and KI; after 30 and 10 min of irradiation no bioluminescent signal was detected for combinations **Tetra-Py(**+**)-Me** +KI 50 mM and **Tetra-Py(**+**)-Me** +KI 100 mM, respectively.

These results prompted us to study the effect of KI in the aPDT efficiency of the porphyrinic formulation (**Form**) described as an excellent alternative to the highly efficient **Tri-Py(**+**)-Me,** as it was mentioned above. The results summarized in **Figure 4F** show that this formulation at 5 µM in the absence of the coadjuvant and after 60 min of irradiation, promoted a decrease in the bioluminescence signal of E. coli of 4 log (p < 0.0001) (**Figure 4F**). When the assays were repeated in the presence of KI at 50 mM a more pronounced decrease in E. coli viability was detected after 40 min of irradiation, reaching the detection limit of the luminometer after 60 min. This rapid decrease in the viability of this bacterium occurred even sooner, after only 20 min of irradiation, when KI was used at 100 mM.

In order to check if the presence of positive charges is a required feature for the combination of KI with this series of porphyrins, the efficacy of the neutral 5,10,15,20-tetra (4-pyridyl)porphyrin (**Tetra-Py**) was evaluated in the presence of this salt at 50 and 100 mM. In **Figure 4G** are summarized the results obtained and it was verified that the low efficacy of this neutral porphyrin was not improved by the presence of the salt, suggesting that when an increment effect was observed in the presence of KI in this series of porphyrins, the presence of at least one positive charge is mandatory.

#### Evaluation of the KI Effect on the Photodynamic Action of Porphyrin Derivatives Bearing Cationic Imidazole Units at the β-Pyrrolic Position

The results obtained in the photoinactivation of bioluminescent E. coli with the monocationic porphyrins β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP** bearing an imidazole moiety at the β-pyrrolic position, both in the absence and in the presence of KI are presented in **Figure 5**. The low activity of these porphyrins at 5.0 µM in the photoinactivation of bioluminescent E. coli was improved in the presence of KI, although the inactivation increment was different. The combination of β**-BrImiPyTPP** and β**-BrImiPhTPP** with KI at 100 mM promoted a significant positive effect in the photoinactivation of E. coli with an increment on the bioluminescent reduction of 1.3 and 1.1 log for β**-ImiPhTPP** and β**-BrImiPyTPP** (p < 0.0001), respectively, after 240 min of irradiation when compared with the effect of these PSs in the absence of KI (**Figures 5A,C**).

A different profile was observed for porphyrin derivative β**-ImiPyTPP**. The best results were obtained with the combination of this PS with 100 mM of KI, promoting a significant decrease in E. coli viability (**Figure 5B**). The bioluminescence signal reduction reached the method detection limit after 240 min; when compared with the effect of these PS in

the absence of KI an increment on the bioluminescent reduction of 5.3 log in cell viability was observed (p < 0.0001).

#### Evaluation of the KI Effect in the Photodynamic Action of Non-porphyrinic Dyes

In **Figure 6** are summarized the effects of KI at 50 and 100 mM in the photodynamic inactivation of E. coli when using **RB** (A), **TBO** (B), **MB** (C), **CV** (D), and **MG** (F). Combinations of **RB** (**Figure 6A**) and **MB** (**Figure 6C**) at 5.0 µM and KI showed to have a potential effect in the photodynamic inactivation of E. coli, causing marked reductions in the E. coli viability when compared with the results obtained with these dyes alone. The PS **RB**, when acting alone, promotes a decrease of 1.3 log (p < 0.0001) in E. coli viability after 150 min of irradiation. When combined with KI, an efficient decrease in bioluminescent signal of E. coli was observed, even when KI at 25 mM was used. At this concentration, the combination of **RB** 5.0 µM + KI 25 mM, caused a sharp decrease in the E. coli viability after 90 min of irradiation, reaching the detection limit of the luminometer after 120 min. This marked effect was also observed when **RB** was combined with 50 mM of KI, but it was with the combination of **RB** 5.0 µM+ KI 100 mM that this effect became more noteworthy; after 20 min

of irradiation it was observed a decrease of 6 log (p < 0.0001) in E. coli viability and after 30 min no bioluminescent signal was observed.

A similar profile was observed with combinations of **MB** at 5.0 µM and KI. In the absence of KI, **MB** caused a decrease in the bioluminescence signal of E. coli of 5.5 log (p < 0.0001) after 180 min of irradiation, but when combinations of this PS with KI were used, an efficient decrease in the viability of this bacterium was also observed, after 30 and 60 min of irradiation, with KI at 100 and 50 mM, respectively.

In the cases of **TBO**, **CV**, and **MG**, a potentiation of their photodynamic action mediated by the presence of KI was not observed. In fact, **TBO** when acting alone at 5.0 µM revealed to be an excellent PS for the inactivation on bioluminescent E. coli, promoting a remarkable decrease in the bioluminescent signal of 6 log (p < 0.0001) after 60 min of irradiation. In the presence of KI, this reduction was only observed after 90 min of irradiation.

**CV** when acting alone caused a decrease in the bioluminescent signal of 3.2 log (p < 0.0001), however, in the presence of KI at 25, 50, and 100 mM the decrease did not go beyond 1.4, 2.2, and 2.7 log (p < 0.0001), respectively.

In the case of **MG** no significant effect was observed in the E. coli viability either when this dye was used alone or combined with KI.

#### Detection of Iodine Formation Mediated by the PS

In order to clarify if the photodynamic improvement was related with the iodine generation from KI by the PS, the different PSs (5.0 µM) were irradiated both in the absence and in the presence of that coadjuvant at 100 mM. To verify the generation of iodine, the absorbance at 340 nm was read after 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, and 120 min of irradiation. The results obtained are summarized in **Figure 7**.

The results had shown that the combination of KI with **Tri-Py(**+**)-Me**, **Tetra-Py(**+**)-Me**, and **Form** causes a higher production of I2, leading to a sharp increase in absorbance at 340 nm in the first 20 min of irradiation. On the other hand, the combination of KI with **Mono-Py(**+**)-Me**, **Di-Py(**+**)-Me adj**, **Di-Py(**+**)-Me opp** only was able to induce a gradual increase of the absorbance at 340 nm, thus indicating the lower ability to produce I2. The combination of **Tetra-Py** + KI did not produce I2.

The gradual increase in the absorbance at 340 nm was also observed in the case of mono-cationic porphyrins β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP**. However, in the case of β**-ImiPyTPP**, the absolute value of absorbance at 340 nm after 40 min of irradiation was higher than the values observed for the other PSs, indicating the formation of higher amounts of I2 in this case.

In the case of the non-porphyrinic dyes, the combination of KI with **MB** and **RB** demonstrated a higher ability to produce I2, with a sharp increase in the absorbance at 340 nm, after 30 min of irradiation. However, combinations of **TBO**+ KI and **CV**+ KI only produced a gradual increase in the absorbance, indicating the lower capability to produce I2. Combination of **MG**+ KI did not promote the formation of I2.

The visual appearance of the starch solutions after different irradiation periods are presented in **Figure 8**

(**Supplementary Tables S1**–**S3**) and the results corroborated that the time required for the formation of the complex between amylose and iodine was dependent on the PS used. In the presence of **Tri-Py(**+**)-Me**, **Tetra-Py(**+**)-Me**, and **Form**, the formation of the dark color (**Supplementary Table S1**) appeared just after 2–4 min of irradiation, while for **Di-Py(**+**)-Me adj** the iodine-amylose complex was observed after 45 min of irradiation. The formation of the colored complex was not observed for the neutral **Tetra-Py** after 240 min of irradiation and for **Mono-Py(**+**)-Me** and **Di-Py(**+**)-Me opp** after 75 min of irradiation a slight darkening of the solution was observed.

For the mono-cationic porphyrins β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP** the formation of the deep colored complex was only observed in the presence of β**-ImiPyTPP** after 60 min of irradiation (**Supplementary Table S2**).

In the assays performed with the non-porphyrinic dyes the combinations **MB**+KI and **RB**+KI promoted the formation of the dark complex after 2–5 min of irradiation and the combination **TBO**+KI after 30 min of irradiation. The combinations of **CV** and **MG** with KI were not able to produce the iodine-amylose complex even after 240 min of irradiation (**Supplementary Table S3**).

## DISCUSSION

Several studies have shown that aPDT combined with some inorganic salts, namely potassium iodide (Vecchio et al., 2015; Zhang et al., 2015; Huang et al., 2016, 2018a,b,c; Wen et al., 2017) can be potentiated. However, there is not any evidence until now that this potentiation can be observed for all types of PSs, namely cationic porphyrins. In order to gain a more comprehensive knowledge about the potentiation of aPDT by KI, a broad range of PSs were tested in this study.

We started our study by selecting the most effective salt and using as PS the widely studied tetracationic porphyrin 5,10,15,20 tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (**Tetra-Py**+**-Me**), which is frequently used as standard in aPDT studies. This can be considered a reference for all porphyrinic PSs,

5.0 µM and KI at 100 mM.

since this PS is extensively studied in bacterial photoinactivation processes (Alves et al., 2008; Tavares et al., 2011; Simões et al., 2016). The efficacy of bacterial inactivation by the combination of this PS and the salts KI and NaI was clearly higher than when the PS was used alone, showing that these combinations promoted an increase of the antimicrobial photodynamic efficiency of the PS. On the other hand, no effect was observed with the combinations of **Tetra-Py(**+**)-Me** with NaBr, KCl, and NaCl during the irradiation time. The loss of efficiency of this porphyrin in these cases could be explained by the fact that bromide and chloride ions retarded the 1O2 generation, and consequently its action as PS (Keum et al., 2003; Krumova and Cosa, 2016). Therefore, it was obvious that for this PS and under the tested conditions, only salts containing I− as counterion were capable of potentiate the antimicrobial photodynamic inactivation. Similar results were earlier observed when other PSs were tested (Hamblin, 2016). As the combinations PS + KI and PS + NaI were both effective to inactivate the E. coli, the potentiation of the others PSs was performed in the presence of the most studied salt KI.

Besides the difficulty of explaining which of the two proposed pathways of decomposition of peroxyiodide produced by the reaction of 1O2 and I− (see **Figure 1**) are responsible for the extra microbial killing when KI is present, it was assumed, as proposed previously in other studies, that some information can be taken by the profile of inactivation. If the inactivation curve shows a sharp decrease, free iodine is the main killing species, but if there is a more gradual increase in killing, then there is a contribution from short-lived reactive iodine species (Huang et al., 2018a). Considering the above, we tried to explain the results obtained with the two series of cationic porphyrins, including the neutral **Tetra-Py**, and with the non-porphyrinic PSs. In **Table 1** are summarized the results obtained concerning the inactivation profile observed for each combination of KI and PS at 5.0 µM in the photoinactivation of bioluminescent E. coli.

These results allow to classify the PSs studied as: (1) PSs in which its efficiency was potentiated by KI and it was observed a gradual decrease in the E. coli survival rate profile [**Mono-Py(**+**)- Me**, β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP**]; (2) PSs in

which its efficiency was potentiated by KI and it was observed an abrupt decrease in the E. coli survival rate profile [**Tri-Py(**+**)- Me, Tetra-Py(**+**)-Me**, **Form**, **RB**, and **MB**]; and (3) PSs in which its efficiency was not potentiated by KI [**Di-Py(**+**)-Me opp**, **Di-Py(**+**)-Me adj**, **Tetra-Py**, **TBO**, **CV**, and **MG**].

Based on the explanations given in previous works, we can assume that the mechanism of action of the combinations of KI and the PSs **Mono-Py(**+**)-Me**, β**-ImiPhTPP**, β**-ImiPyTPP**, and β**-BrImiPyTPP** is probably related to the preferential decomposition of the peroxyiodide to the iodine radicals (I·− 2 ) that, due to their short diffusion distance, cause a gradual decrease in the photoinactivation profile. In the case of **Tri-Py(**+**)-Me**, **Tetra-Py(**+**)-Me**, **Form**, **MB** and **RB** the preferential decomposition of the peroxyiodide leads to the formation of free iodine (I2/I− 3 ), which contributes significantly for the abrupt increase observed in the photoinactivation profile of the E. coli. This fact was confirmed by the formation of iodine, visible by spectroscopy (**Figure 7**) and by the color alteration during the irradiation in the presence of starch (**Figure 8**): PSs that cause a sharp decrease in the E. coli survival rate profile revealed higher ability to produce I2. On the other hand, the belatedly detection of I2 was observed for PSs that cause a gradual decrease in the E. coli survival rate profile.

In the cases of PSs in which the efficiency was not potentiated by KI, or was even reduced, we need also to look at other factors that can likewise contribute to this behavior.

The different behavior observed with the dicationic PSs **Di-Py(**+**)-Me opp** (the efficacy was lost in the presence of KI) and **Di-Py(**+**)-Me adj** (no potentiation with KI) (**Figures 4B,C**) is probably related with their structural features since both isomers have similar capability to generate 1O2 with high efficiency, as it was described by Simões et al. (2016). Consequently, it can be assumed that both compounds are able to promote the formation of peroxyiodide and its decomposition to iodine radical species (I·− 2 ). However, for **Di-Py(**+**)-Me opp** these radicals, with a short diffusion distance, probably were not generated close to the target cells and the depletion of 1O2 by the previous reaction was responsible by losing its previous efficacy. On the other hand, for **Di-Py(**+**)-Me adj** the formation of toxic radicals in close proximity to the target cells can justify the maintenance of its efficacy. However, the toxicity under these conditions was comparable to the previous one in the absence of iodide. The different charge distribution in the two di-cationic porphyrins can explain the different behavior in the presence of KI. A study of Alves et al. (2011a) showed the massive importance of the charge distribution in these two PS efficacies. In this work, the photodynamic inactivation of E. coli and Enterococcus faecalis using the two isomeric di-cationic porphyrins with different charge distribution showed that the porphyrin with adjacent cationic groups was significantly more active (for both bacteria) than the one with the cationic groups located in opposite meso positions. This fact was justified by the distortion of the macrocycle induced by the electrostatic repulsion between the neighboring charged groups in the porphyrin with adjacent cationic groups (Kessel et al., 2003). So, in the case of porphyrinic PSs with cationic groups located in opposite meso positions, accompanied by the preferential decomposition of the

, ,

peroxyiodide to the iodine radicals, as it was observed with **Di-Py(**+**)-Me opp**, the addition of KI can even impair the aPDT efficacy. With the porphyrin derivatives **Di-Py(**+**)-Me adj**, **Mono-Py(**+**)-Me**, and β**-ImiPyTPP** the asymmetric distribution of the charge allows the radicals to reach the bacterial cells more effectively. However, the potentiation of the aPDT processes mediated by **Mono-Py(**+**)-Me** and β**-ImiPyTPP** in the presence of KI but not by **Di-Py(**+**)-Me adj** can also be due to the higher production of free iodine by the two first porphyrins when compared with porphyrin **Di-Py(**+**)-Me adj**.

Neutral **Tetra-Py** revealed to be inefficient to photoinactivate E. coli, even when KI was used. This can be explained by the fact that this is a neutral PS, and consequently, is not capable to interact with the external membrane of the cell wall of this Gramnegative bacterium. Thus, even when 1O2 is produced in great amounts, the cytotoxic species will never be close enough to the bacterial cells to cause damage. It is also important to refer that this porphyrin tends to aggregate in aqueous media, making it difficult to act as a PS.

**CV** is known to have an efficient non-radiative deactivation route producing triplet species, such as 1O2, with low yield and acting mainly through an electron-transfer mechanism (Type I), which causes its bleaching (Docampo et al., 1983; Indig et al., 2000). The results clearly indicate its low efficiency in the photoinactivation of E. coli, either when acting alone or combined with KI. These results are justified by its poor 1O2 production rates allied to its photodegradation when irradiated. Such as in the case of **CV**, it was not surprising that **MG** did not produced any effect in the photoinactivation of bioluminescent E. coli, since this PS dye did not produce 1O2, acting only by the Type I mechanism (Zhuo, 2016). These two PSs dyes show the importance that 1O2 generation has in the potentiation of aPDT processes mediated by KI. The **TBO** acts mainly by Type II mechanism and, when acting alone inactivate efficiently the bacteria, as **MB** and **RB.** However, when combined with KI, no potentiation was observed. There is, however, a study in the literature reporting the potentiation of the effect of **TBO** by KI, but in this study the **TBO** was tested at 100 µM (Ghaffari et al., 2018). In our case, the concentration of **TBO** was 20 times lower (5.0 µM). These different experimental conditions can justify the differences observed in these two studies. Nevertheless, using NaN3 as potentiation agent, the aPDT effect of **TBO** was more effective when compared with the result without the NaN3 (Kasimova et al., 2014). **MB** used as the reference for all non-porphyrin dyes, once is the most commonly studied antimicrobial PS in the literature and has received regulatory approval to mediate photodynamic therapy (PDT) of several infectious diseases, acts mainly trough Type II mechanism (Marotti et al., 2010; de Oliveira et al., 2014). Moreover, its aPDT potentiation when combined with KI was already described (Vecchio et al., 2015). Besides that, and according with our results, **MB** can be designated as a PS reference for evaluate the potentiation of these dyes by KI.

It remains unanswered which factor determines whether the mechanism follows via formation of iodine radical species (I·− 2 ) or via formation of free iodine (I2/I− 3 ). To answer this question, we cannot neglect other factors that can also contribute for the efficiency of these PSs, such as 1O2 production, charge number and distribution, aggregation behavior, affinity for the cell membrane.

It is undeniable that the ability of KI to potentiate the aPDT process mediated by some cationic PSs, allows a drastic reduction of the aPDT treatment time as well as the reduction of the PS concentration. However, this potentiation is limited to some PSs and the addition of KI can even impair some PSs. This work helped to elucidate that for the series of compounds studied, the PSs capable to decompose the peroxyiodide into iodine (easily detectable by monitoring the formation of I2 through spectroscopy or by the visual appearance of a blue color in the presence of starch) are the promising ones in terms of complementing their efficacy with the action of iodine. Although these studies confirm that the generation of 1O2 is an important fact in this process, the PS structure, aggregation behavior and affinity for the cell membrane are also important features to take into account.

## AUTHOR CONTRIBUTIONS

CV performed the antimicrobial photodynamic evaluations assays of all PSs, analysis of biological results and contributed to the manuscript preparation. AG performed the analysis and interpretation of the biological results and contributed to the manuscript preparation. MM and NM performed the synthesis of the porphyrin derivatives. MN and MF were responsible for the supervision of the synthesis of the PSs and contributed in the analysis and interpretation of the biological results and in the manuscript preparation. AA was responsible for the supervision and the design of the antimicrobial photodynamic experiments and contributed in the analysis and interpretation of the biological results and in the manuscript preparation.

## FUNDING

Thanks are due to the University of Aveiro, to FCT/MEC for the financial support to Centre for Environmental and Marine Studies (CESAM) Unit (Project PestC/MAR/LA0017/2013) and QOPNA Research Unit (FCT UID/QUI/00062/2013), through national funds and when applicable co-financed by the FEDER, within the PT2020 Partnership Agreement and "Compete" 2020, and also to the Portuguese NMR Network. MM and NM thank to the Fundação para a Ciência e a Tecnologia FCT for their doctoral (SFRH/BD/112517/2015) and post-doctoral grants (SFRH/BPD/84216/2012), respectively.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 02665/full#supplementary-material

#### REFERENCES

fmicb-09-02665 November 15, 2018 Time: 18:34 # 15

photoinactivation of bacteria induced by a dicationic fulleropyrrolidinium derivative. Methods 109, 167–174. doi: 10.1016/j.ymeth.2016.05.019

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer TM and handling Editor declared their shared affiliation at the time of review.

Copyright © 2018 Vieira, Gomes, Mesquita, Moura, Neves, Faustino and Almeida. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Molecular Epidemiology and Risk Factors of Carbapenemase-Producing Enterobacteriaceae Isolates in Portuguese Hospitals: Results From European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE)

#### Edited by:

Patrícia Poeta, Universidade de Trás-os-Montes e Alto Douro, Portugal

#### Reviewed by:

Marie-Cecile Ploy, University of Limoges, France Alfonso Soler-Bistue, Instituto de Investigaciones Biotecnológicas (IIB-INTECH), Argentina

#### \*Correspondence:

Manuela Caniça<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 30 June 2018 Accepted: 05 November 2018 Published: 27 November 2018

#### Citation:

Manageiro V, Romão R, Moura IB, Sampaio DA, Vieira L, Ferreira E, the Network EuSCAPE-Portugal and Caniça M (2018) Molecular Epidemiology and Risk Factors of Carbapenemase-Producing Enterobacteriaceae Isolates in Portuguese Hospitals: Results From European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). Front. Microbiol. 9:2834. doi: 10.3389/fmicb.2018.02834 Vera Manageiro1,2, Raquel Romão1 , Inês Barata Moura1 , Daniel A. Sampaio3 , Luís Vieira3 , Eugénia Ferreira1,2, the Network EuSCAPE-Portugal and Manuela Caniça1,2 \*

1 National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal, 2 Centre for the Studies of Animal Science, Institute of Agrarian and Agri-Food Sciences and Technologies, University of Oporto, Oporto, Portugal, 3 Innovation and Technology Unit, Department of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal

In Portugal, the epidemiological stage for the spread of carbapenemase-producing Enterobacteriaceae (CPE) increased from sporadic isolates or single hospital clones (2010–2013), to hospital outbreaks, later. Here we report data from a 6 month study performed under the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). During the study period, 67 isolates (61 Klebsiella pneumoniae and 6 Escherichia coli) non-susceptible to carbapenems were identified in participant hospital laboratories. We detected 37 blaKPC−type (including one new variant: blaKPC−21), 1 blaGES−5, and 1 blaGES−6 plus blaKPC−3, alone or in combination with other bla genes. Bioinformatics analysis of the KPC-21-producing E. coli identified the new variant blaKPC−21 in a 12,748 bp length plasmid. The blaKPC−21 gene was harbored on a non-Tn4401 element, presenting upstream a partial ISKpn6 (1ISKpn6/1traN) with the related left IR (IRL) and downstream a truncated Tn3 transposon. PFGE and MLST analysis showed an important diversity, as isolates belonged to distinct PFGE and STs profiles. In this study, we highlighted the presence of the high-risk clone E. coli sequence-type (ST) 131 clade C/H30. This worldwide disseminated E. coli lineage was already detected in Portugal among other antibiotic resistance reservoirs. This study highlights the intra- and inter-hospital spread and possible intercontinental circulation of CPE isolates.

Keywords: carbapenemase-producing Enterobacteriaceae, KPC-21, EuSCAPE, Portugal, Klebsiella pneumoniae, Escherichia coli

## INTRODUCTION

fmicb-09-02834 November 24, 2018 Time: 16:19 # 2

Carbapenems, a class of β-lactam antibiotics with wide activity, are often the antimicrobials of last resort to treat infections associated to extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC (PMAβ)-producing Enterobacteriaceae isolates (Papp-Wallace et al., 2011; Rodríguez-Baño et al., 2018). Unfortunately, carbapenem non-susceptible Enterobacteriaceae (CNSE) have been reported worldwide mainly because of the acquisition of carbapenemaseencoding genes (Potter et al., 2016; Codjoe and Donkor, 2018). Since the first description of a carbapenemase-producing Enterobacteriaceae (CPE) in Europe in the 1990s, a large variety of carbapenemases has been identified in each of the four Ambler molecular classes, mainly the KPC-type (class A), VIM-, IMP-, and NDM-types (class B), and OXA-48-type (class D) (Grundmann et al., 2017; Logan and Weinstein, 2017). CPE isolates are usually resistant to many other β-lactam and non-β-lactam antibiotics, leading to multi-resistant isolates.

In Portugal, the epidemiological stage for the spread of CPE increased from sporadic isolates or single hospital clones, from April 2010 to February 2013, to sporadic hospital outbreaks later (Albiger et al., 2015; Manageiro et al., 2015b,c). Here we report data from a 6 month prevalence study performed under the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) with the collaboration of different Portuguese Laboratories.

## MATERIALS AND METHODS

#### Bacterial Isolation, Antibiotic Susceptibility, and Molecular Characterization

This study included a total of 104 clinical isolates (94 Klebsiella pneumoniae and 10 Escherichia coli) collected from November 2013 to April 2014 in 10 Portuguese hospitals. The first ten consecutive and non-replicated CNSE isolates obtained during this period, in each hospital, from blood, lower respiratory tract secretions, urine, puncture fluids, and wound secretions, of single patients, were sent to the National Reference Laboratory, in Lisbon, and were considered. Successive carbapenem-susceptible isolates of the same species were also preserved as controls whenever possible, accordingly to EuSCAPE protocol (Grundmann et al., 2017). Overall, 67 CNSE (61 K. pneumoniae and 6 E. coli) and 37 controls (33 K. pneumoniae and 4 E. coli) were analyzed.

In the context of the EuSCAPE study, all data were anonymized and collected in accordance with the European Parliament and Council decisions on the epidemiological surveillance and control of communicable disease in the European Community (Eur-Lex-31998D2119, 1998; Eur-Lex-32000D0096, 2000).

## Antibiotic Susceptibility and Molecular Characterization of Antimicrobial Resistance

Antimicrobial susceptibility was performed by disk diffusion method for 15 antibiotics (**Table 1**), and by broth microdilution method for tigecycline and colistin, using EUCAST guidelines1 . Clinical isolates with resistance or with decreased susceptibility to ertapenem were considered presumptively CPE. Isolates were considered multidrug resistant when presenting reduced susceptibility to three or more structurally unrelated antibiotics.

PCR and sequencing were applied to detect and identify the main CPE (blaKPC and blaGES from class A; blaIMP, blaVIM, and blaNDM from class B; and blaOXA−48 from class D)-, ESBL (blaTEM, blaSHV, blaOXA, blaCTX−M) – and PMAβ (blaCMY, blaMOX, blaFOX, blaLAT, blaACT, blaMIR, blaDHA, blaMOR, blaACC)-encoding genes, as previously described (Manageiro et al., 2015b). Plasmid-mediated colistin resistance-encoding genes (mcr-type) were also investigated (Manageiro et al., 2017).

#### Transfer Experiments

Transferability of blaKPC−21 from E. coli UR19829 was performed by broth mating out assays using sodium azide-resistant E. coli J53 as a recipient strain, and by transformation, as previously described (Manageiro et al., 2015b, 2017).

#### Molecular Typing

Clonal relatedness of 67 CNSE isolates was investigated by pulsed-field gel electrophoresis (PFGE) as previously described (Manageiro et al., 2017). Genetic diversity of the K. pneumoniae (n = 10, i.e., 1 representative of each PFGE cluster) and E. coli (n = 10) isolates was investigated by multilocus sequence typing (MLST) (Manageiro et al., 2015b). E. coli sequence type (ST) subclones were also analyzed on the basis of the E. coli fimH gene (Manageiro et al., 2015a).

#### Genomic Characterization of KPC-21-Producing E. coli

KPC-21-producing E. coli was genotypically characterized by whole-genome sequencing (WGS) as previously described (Manageiro et al., 2017). The assembled contigs were analyzed and studied for the presence of antibiotic resistance- and virulence-encoding genes, multi-locus sequence types, fim type, serotype, plasmid replicon types, and insertion sequences (ISs) using bioinformatics tools from the Center for Genomic Epidemiology2 and ISsaga (Varani et al., 2011).

The pUR19829-KPC21 plasmid structure was constructed based on the genetic organization of the closest plasmid

1http://www.eucast.org/clinical\_breakpoints/

2https://cge.cbs.dtu.dk/services/

sequences obtained by BLASTn, provided by NCBI3 , followed by contig neighbor's prediction from assembly information.

#### Statistical Analysis

fmicb-09-02834 November 24, 2018 Time: 16:19 # 3

OpenEpi software, version 3.01 was used for statistical analysis (Sullivan et al., 2009). Fisher exact test was used to assess differences in clinical and epidemiological risk factors for control and CNSE-carrying patients. One-tailed P values of ≤0.05 were considered to be statistically significant. Associations were determined by calculation of odds ratios with 95% confidence intervals.

#### Nucleotide Sequence Accession Number

The new blaKPC−21 nucleotide sequence was submitted to the NCBI GenBank Database with accession number NG\_049254 and the complete plasmid sequence of pUR19829-KPC21 with accession number MH133192.

### RESULTS AND DISCUSSION

During the study period, 67 isolates (61 K. pneumoniae and 6 E. coli) CNSE were identified in nine of the 10 Hospital Laboratories, with a non-susceptibility rate for meropenem and imipenem of 64 and 59%, respectively, for K. pneumoniae,

3http://blast.ncbi.nlm.nih.gov/Blast.cgi

and of 100% for E. coli. As expected, when compared with the control isolates, CNSE presented higher level of nonsusceptibility to all antibiotic classes tested (**Table 1**). Colistin and tigecycline MIC50 values for CNSE were similar than those obtained for control isolates. Eleven out of the 104 (16.3%) isolates were colistin resistant, without the presence of the plasmid-mediated mcr-1 or mcr-2 gene. However, MCR-1 determinant was already identified in different reservoirs in Portugal, such as vegetables, animals and humans (Jones-Dias et al., 2016; Beyrouthy et al., 2017; Kieffer et al., 2017).

Thirty-eight (56.7%) isolates (36 K. pneumoniae, 2 E. coli) were confirmed to be CPE; we identified 36 blaKPC−type (including one new variant: blaKPC−21), 1 blaGES−5, and 1 blaGES−6 plus blaKPC−3, alone or in combination with other bla genes (**Supplementary Figure S1**). The remaining 29 isolates were non-susceptible to carbapenems possibly due to porins deficiency with association of PMAβ (CMY-2 and DHA-1) and/or ESBL (mainly CTX-M-15) production (Martínez-Martínez, 2008).

The new blaKPC−21 gene differed from blaKPC−2 by one point mutation that leads to the amino acid substitution Trp105Arg; this position is involved in the binding and maintaining of the KPC catalytic activity (Papp-Wallace et al., 2010). In silico typing revealed an KPC-21-producing E. coli belonging to ST131 clade C/H30, associated with the fimbriae-encoding fimH allele 30, which become the most dominant lineage since the 2000s

TABLE 1 | Antimicrobial susceptibility of 67 (61 K. pneumoniae and 6 E. coli) CNSE and 37 (33 K. pneumoniae and 4 E. coli) control isolates.

∗Microdilution method.

(Nicolas-Chanoine et al., 2014; Pitout and DeVinney, 2017). Moreover, bioinformatics analysis of the KPC-21-producing E. coli identified this variant in a 12,748 bp length plasmid, with a mean coverage of 580-fold and GC content of 58.5% (**Figure 1**).

Dissemination of blaKPC has been mainly supported by the horizontal transfer of Tn4401-type transposon, which harbors tnpA encoding a transposase, tnpR encoding resolvase, and two insertion sequence elements (ISKpn7 and ISKpn6) bracketing the blaKPC gene (Cuzon et al., 2011). In this study, the blaKPC−21 gene was harbored on a non-Tn4401 element (Chen et al., 2014), presenting upstream a partial ISKpn6 (1ISKpn6/1traN) with the related left IR (IRL) and downstream truncated Tn3 transposon downstream (**Figure 1**). This region has 99.97% of identity with pKP1194a, a plasmid carried by a hospital-associated KPC-2-producing K. pneumoniae isolated in Brazil (Accession number KX756453) (**Figure 1**- gray region I); this suggest an intercontinental circulation of isolates and mobile genetic elements (MGE), and the consequent need of concerted actions against the spreading of antibiotic resistance, at a worldwide level. The pUR19829-KPC21 enclosed also an intact ISPsp7 element, an insertion sequence from IS30 family, firstly described in Pseudomonas spp. (Szuplewska et al., 2014). Furthermore, the pUR19829-KPC21 backbone contained a region coding for plasmid replication (IncQ2 repA, repC), and mobilization (mobA, mobC), showing >99.9% sequence identity to the corresponding regions of pKPSH169, a 7.7 Kbp qnrS2 harboring IncQ plasmid identified in municipal wastewater treatment facilities in Israel (Accession number KT896499) (**Figure 1**- gray region II); this similarity highlights the high level of promiscuity of isolates between clinical settings and environment, where both reservoirs play a role in the antibiotic resistance dissemination (Stokes and Gillings, 2011). However, the lack of conjugative elements or an oriT region, associated with the presence of a truncated oriV region (**Figure 1**) suggests that pUR19829-KPC21 plasmid is nonmobilizable (Smillie et al., 2010). This fact is corroborated by the absence of a successful plasmid conjugation or transformation.

The variables used in the evaluation of risk factors for infection or colonization of patients with CNSE or control

TABLE 2 | Evaluation of risk factors for patients with infections caused by carbapenem susceptible or CNSE bacteria.

OR, odds ratios; CI, 95% confidence intervals. (P) indicates a one-tail P-value for protective or negative association. One-tailed P values of ≤ 0.05 are underlined. LVT, Lisbon and Tagus Valley.

isolates are present in **Table 2**. When compared to the 37 control strains, only ESBL-production and the patient admission at a hospital in the center of Portugal were significantly associated with CNSE isolates in the period of the study. In the era of ESBL-producing Enterobacteriaceae, the antibiotic regimens suggested for severe health-associated infections are necessarily based on carbapenems (Rodríguez-Baño et al., 2018). Unfortunately carbapenem use has being described as a risk factor for CPE acquisition, only preceded by the use of medical devices (van Loon et al., 2018). In addition, the present study attests that Portugal, during the period of the study, has a different CNSE geographical distribution with the center of Portugal significantly associated with carbapenem non-susceptibility. This fact corroborates previous studies which indicated that in Portugal, in 2015, only sporadic isolates or single hospital cases were described (Albiger et al., 2015).

PFGE and MLST analysis showed an important diversity, with isolates belonging to distinct PFGE and STs (**Supplementary Figure S1**). With respect to K. pneumoniae (**Supplementary Figure S1A**), a total of 10 clusters and 25 unique PFGE profiles were generated using XbaI, indicating the that the circulating clones in that period were genetically diverse. However, carbapenemase-producing K. pneumoniae isolates were more clonal (six PFGE clusters including 69.4% of these isolates) than non-carbapenemase-producing K. pneumoniae (four PFGE clusters including 50.0% of these isolates). As shown in **Supplementary Figure S1**, both CNSE species showed intra- and inter-hospital spread (e.g., PFGE clusters KpI and KpIX), with some hospital-specific clones (e.g., PFGE clusters KpIV and KpVIII). However, as also showed in Spain in other EuSCAPE study (Esteban-Cantos et al., 2017), the carbapenem-non-susceptible K. pneumoniae population was more clonal than the carbapenem-susceptible population (data not shown). Ten different MLSTs were detected among carbapenemase-producing (ST14, ST15, ST45, ST231, and ST1513) and non-carbapenemase-producing (ST11, ST17, ST348, and ST395) K. pneumoniae isolates. At our knowledge,

this is the first description of ST17, ST395, and ST1513 K. pneumoniae in Portugal (Manageiro et al., 2015b; Rodrigues et al., 2016; Vubil et al., 2017). Noteworthy, the GES-5 enzyme was detected in a ST231 K. pneumoniae isolate as previously reported in Portugal, but in the same hospital, which shows its capacity to maintain in clinical settings due to the selection pressure of this environment (Manageiro et al., 2015b). Furthermore, ST45 was recently the cause of a hospitalbased outbreak caused by multidrug-resistant, KPC-3- and MCR-1-producing K. pneumoniae in Portugal (Mendes et al., 2018).

The high-risk clone carbapenemase-positive K. pneumoniae ST258 was not detected in this study or among clinical carbapenemase-producing K. pneumoniae isolates in Portugal (Manageiro et al., 2015b; Rodrigues et al., 2016; Vubil et al., 2017). However, concerning carbapenem-non-susceptible E. coli, besides the six different PFGE unique profiles, the isolates belongs all but two (ST405-fimH27 and ST23-fimH35) to the ST131 clade C/H30 high-risk clone disseminated worldwide (**Supplementary Figure S1B**) (Woodford et al., 2011; Pitout and DeVinney, 2017). Noteworthy, this clone was already detected in Portugal among other antibiotic resistance reservoirs, such as in an E. coli strain isolated from a dolphin housed at a Zoo Park (Manageiro et al., 2015a); in dogs and cats with urinary tract infection (Marques et al., 2018); and in E. coli strains from wastewater and gulls (Varela et al., 2015). Again, this shows that clinical settings and different environmental compartments may be considered communicating vessels through which bacteria and resistance genes are able to flow (Stokes and Gillings, 2011).

Portugal was one of the EuSCAPE participating countries that presented higher proportions of KPC-positive K. pneumoniae (Grundmann et al., 2017). The percentage of carbapenem nonsusceptible K. pneumoniae was low in invasive infections in the study period [2.4%, EARS-Net 2013]4 . However, although the consumption of carbapenems has declined by 13.3% between 2012 and 2016 (PPCIRA, 2017), Portugal is reporting since 2013 a significant increasing trend of carbapenem non-susceptible K. pneumoniae [6.4%, EARS-Net 2016]4 . The number of inter-institutional transmission is also increasing (Glasner et al., 2013; Albiger et al., 2015), being K. pneumoniae the principal cause of bacterial healthassociated infections in Portugal, as in other European countries (ECDC, 2013). Of concern is the fact that KPCproducing organisms cause infections with high morbidity and mortality (Porreca et al., 2018; Rodríguez-Baño et al., 2018). These results reinforces that reducing antibiotic use alone is likely insufficient for reversing resistance (Lopatkin et al., 2017). We strongly believe that the chain of transmission of isolates and genes in clinical settings will be reduced or broken, especially with containment measures rigorously implemented and followed at local level.

#### MEMBERS OF THE NETWORK EuSCAPE-PORTUGAL

North region: C.H. São João E.P.E. (J.T. Guimarães), H. Braga (C. Iglesias), C.H. Póvoa de Varzim e Vila do Conde E.P.E. (F. Fonseca). Centre region: C.H.U. de Coimbra/Covões (H. Oliveira), C.H.U. de Coimbra/HUC (L. Boaventura), C.H. Médio Tejo E.P.E. (Ana Soares). Lisbon and Tagus Valley region: C.H. Oeste Norte E.P.E. (A. Vicente). H. Garcia de Orta E.P.E. (J. Diogo), C.H. Lisboa Ocidental E.P.E. (E. Gonçalves), C.H. Lisboa Central E.P.E. (M. Pinto).

### AUTHOR CONTRIBUTIONS

VM designed the study, performed the molecular experiments, bioinformatics analysis, analyzed the data, and wrote the manuscript. RR, IBM, and EF performed the microbiological and molecular experiments, and analyzed the data. DAS and LV performed Illumina genome sequencing experiments. The Network EuSCAPE-Portugal participants acquired laboratory data. MC designed the study, wrote and reviewed the manuscript. All authors read and approved the final manuscript.

### FUNDING

VM was supported by Fundação para a Ciência e a Tecnologia (FCT) fellowship (Grant SFRH/BPD/ 77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank FCT for Project grant UID/MULTI/00211/2013.

## ACKNOWLEDGMENTS

The authors would like to thank Rafael Graça for his technical assistance.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2018. 02834/full#supplementary-material

FIGURE S1 | Pulsed-field gel electrophoresis (PFGE) dendrogram and genetic relatedness of 61 K. pneumoniae (A) and 6 E. coli (B) CNSE isolates. Isolate number, hospital code, year of isolation, carbapenems antibiotic susceptibility, detected carbapenemases, extended-spectrum β-lactamases (ESBL), inhibitor resistant SHV (IRS), and plasmid-mediated AmpC (PMAβ), Multilocus sequence typing (MLST) for selected isolates and PFGE profile types are shown. These profiles, from 0001 to 0035, were defined as forming clusters KpI to KpX, for K. pneumoniae, and from 0001 to 0006 for E. coli. For E. coli isolates, fim-type is also shown.

4https://ecdc.europa.eu/

## REFERENCES

fmicb-09-02834 November 24, 2018 Time: 16:19 # 7

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Manageiro, Romão, Moura, Sampaio, Vieira, Ferreira, the Network EuSCAPE-Portugal and Caniça. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Combined Antibacterial Effects of Goat Cathelicidins With Different Mechanisms of Action

Pavel V. Panteleev1 , Ilia A. Bolosov1 , Alexander À. Kalashnikov1 , Vladimir N. Kokryakov2 , Olga V. Shamova2 , Anna A. Emelianova1 , Sergey V. Balandin1 and Tatiana V. Ovchinnikova1 \*

1 M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia, 2 Institute of Experimental Medicine, Saint Petersburg, Russia

#### Edited by:

José Luis Capelo, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal

#### Reviewed by:

César de la Fuente, Massachusetts Institute of Technology, United States Anzhela Galstyan, Westfälische Wilhelms-Universität Münster, Germany

#### \*Correspondence:

Tatiana V. Ovchinnikova<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 24 June 2018 Accepted: 19 November 2018 Published: 30 November 2018

#### Citation:

Panteleev PV, Bolosov IA, Kalashnikov AÀ, Kokryakov VN, Shamova OV, Emelianova AA, Balandin SV and Ovchinnikova TV (2018) Combined Antibacterial Effects of Goat Cathelicidins With Different Mechanisms of Action. Front. Microbiol. 9:2983. doi: 10.3389/fmicb.2018.02983 Being essential components of innate immune system, animal antimicrobial peptides (AMPs) also known as host-defense peptides came into sharp focus as possible alternatives to conventional antibiotics due to their high efficacy against a broad range of MDR pathogens and low rate of resistance development. Mammalian species can produce a set of co-localized AMPs with different structures and mechanisms of actions. Here we examined the combined antibacterial effects of cathelicidins, structurally diverse family of host-defense peptides found in vertebrate species. As a model we have used structurally distinct cathelicidins expressed in the leukocytes of goat Capra hircus. The recombinant analogs of natural peptides were obtained by heterologous expression in bacterial system and biological activities as well as the major mechanisms of antibacterial action of the peptides were investigated. As the result, the marked synergistic effect against wide panel of bacterial strains including extensively drug-resistant ones was observed for the pair of membranolytic α-helical amphipathic peptide ChMAP-28 and Pro-rich peptide mini-ChBac7.5Nα targeting a bacterial ribosome. ChMAP-28 was shown to damage the outer bacterial membrane at sub-inhibitory concentrations that could facilitate Pro-rich peptide translocation into the cell. Finally, resistance changes under a long-term continuous selective pressure of each individual peptide and the synergistic combination of both peptides were tested against Escherichia coli strains. The combination was shown to keep a high activity after the 26-days selection experiment in contrast to mini-ChBac7.5Nα used alone and the reference antibiotic polymyxin B. We identified the point mutation leading to amino acid substitution V102E in the membrane transport protein SbmA of the mini-ChBac7.5Nα-resistant strain obtained by selection. The experiments revealed that the presence of sub-inhibitory concentrations of ChMAP-28 restored the activity of mini-ChBac7.5Nα against this strain and clinical isolate with a weak sensitivity to mini-ChBac7.5Nα. The obtained results suggest a potential medical application of synergistic combinations of natural cathelicidins, which allows using a lower therapeutic dose and minimizes the risk of resistance development.

Keywords: antimicrobial peptide, cathelicidin, goat, proline-rich peptide, synergy, extensively drug-resistant, immune system

## INTRODUCTION

fmicb-09-02983 November 29, 2018 Time: 17:34 # 2

Over recent years, a growing number of bacterial species became resistant to clinically significant antibiotics. Host defense antimicrobial peptides (AMPs) came into sharp focus as possible alternatives to conventional antibiotics due to their high efficacy against a broad range of multiple drug-resistant pathogens, a rapid membranolytic mode of action and, as consequence, a low risk of resistance development. Cathelicidins, one of the major groups of animal AMPs, are known to be the key molecular factors of innate immunity of most vertebrate species, from hagfish to human (Ko´sciuczuk et al., 2012). Along with direct antimicrobial action, these peptides possess immunomodulatory activities, such as inhibition of apoptosis, cytokine stimulating, lipopolysaccharide (LPS) neutralizing, promotion of wound healing, and regulation of adaptive immune responses. All the above suggest that these compounds can be prototypes for novel therapeutics with complex mechanism of action (Steinstraesser et al., 2011). The precursors of cathelicidins are produced in immune and epithelia cells and contain the N-terminal part of 99–114 amino acid residues which is known as the cathelin domain. This structure is highly conserved among vertebrates, whereas the C-terminal domain, encoding the mature peptide, shows substantial heterogeneity. Interestingly, the cathelin domain does not exhibit a protease inhibitory function regardless of its high structural similarity to cystatins (Pazgier et al., 2013). Therefore, the question why the cathelin domain is highly conserved among vertebrate cathelicidins is still open. It is believed that the precursor proteins could play a role in the secretion, intracellular trafficking as well as prevent cytotoxicity of mature peptides and their proteolytic degradation. The potential toxicity of cathelicidins is also controlled by their compartmentalization in cytoplasmic granules of immune cells. In case of contact with pathogens AMPs are activated by fusion of procathelicidin-containing specific granules (or large granules of ruminant neutrophils) with the elastase/proteinase 3-containing azurophil granules and either the cytoplasmic membrane or phagosome (Graf et al., 2017). Secondary structures of mature cathelicidins include α-helices, β-hairpins, and extended linear regions enriched with Trp or Pro residues. Interestingly, neutrophils of some artiodactyls, including goats, do not contain defensin-like AMPs (Zhao et al., 1999), suggesting a key role of cathelicidins in the protection of these animals against pathogens. Study of artiodactyl cathelicidins can provide new molecular insight into their role in the host defense.

A number of studies on the synergy between AMPs and conventional antibiotics have been performed over the last years (Cassone and Otvos, 2010; Reffuveille et al., 2014; Simonetti et al., 2014; Ribeiro et al., 2015; de la Fuente-Núñez et al., 2016; Lázár et al., 2018). In contrast, the synergy between AMPs is not well investigated although this phenomenon might contribute to understanding of substantial peptide diversity at any host anatomic site. In most cases the repertoire of structurally diverse animal AMPs make possible both disturbing the membrane integrity of pathogenic microorganisms and inhibiting a number of metabolic processes via interaction with intracellular targets. Such a complex mechanism of action appears to prevent the development of resistance to AMPs. The present work is aimed to examine combined antibacterial effects of structurally distinct cathelicidins expressed in leukocytes of the domestic goat Capra hircus. Previously, we have isolated two novel AMPs mini-bactenecins, designated as mini-ChBac7.5Nα and mini-ChBac7.5Nβ, from leukocytes of the domestic goat (Shamova et al., 2016). These peptides are N-terminal fragments (22 and 21 aa, respectively) of the hypothetic ChBac7.5 peptide also classified as cathelicidin-3. Being Pro-rich AMPs, mini-bactenecins are thought to target intracellular structures such as the 70S ribosome and/or heat shock protein DnaK (Graf et al., 2017). In the study, we investigated a biological significance of the PRPRPR fragment localized at the C-terminus of mini-ChBac7.5Nα. For this purpose, a comparative testing of the wild-type peptide and its short derivative termed as mini-ChBac7.5Nα(1–16) was carried out. Earlier, bovine Bac7(1–16) was shown to be the minimal fragment of the native 60-residue peptide Bac7 displaying both antimicrobial activity in broth microdilution tests and ability to inhibit protein synthesis in vitro (Benincasa et al., 2004; Seefeldt et al., 2016). Along with mini-ChBac7.5Nα, the previously not investigated C. hircus myeloid AMP cathelicidin-6, designated as ChMAP-28, was chosen as the second component of the model system. The peptide primary structure was deduced from the deposited in GenBank mRNA sequence (AJ243126.1) coding the appropriate precursor protein. The novel cathelicidin has relatively high homology with the α-helical bovine cathelicidin BMAP-27 (**Figure 1**). ChMAP-28 contains eleven basic amino acid residues (Arg, Lys, His). As goat leukocytes were shown to simultaneously express mRNA for both cathelicidin-3 and -6 (Zhang et al., 2014), we supposed that the peptides were co-localized in the cells and could act synergistically during the immune response. The combined antibacterial effects of the goat cathelicidins were studied by a checkerboard titration method against a set of bacterial strains including the "ESKAPE" pathogens. The role of each cathelicidin in the synergistic cooperation and their predominant mechanisms of action were elucidated. Finally, antibacterial activity changes under a long-term continuous selective pressure of the individual peptides and their combination were investigated against Escherichia coli strains.

#### MATERIALS AND METHODS

All the bacterial strains used in this study are listed in **Table 1**. The clinical isolates were collected and provided by Sechenov First Moscow State Medical University hospital and Solixant LLC (Moscow, Russia). The resistant to conventional antibiotics strains were defined as extensively drug resistant (XDR) according to (Magiorakos et al., 2012).

## Expression and Purification of the Antimicrobial Peptides

The recombinant plasmids for expression of the goat cathelicidins were constructed with the use of pET-based vector as described previously (Panteleev and Ovchinnikova, 2017).

software (Sali and Blundell, 1993) by homology modeling on the basis of the NMR structure (D) of BMAP-27 (PDB 2KET) serving as a template. (E) Spatial structure of the mini-ChBac7.5Nα(1–16) fragment was modeled and overlaid on the basis of the crystal structure of the Bac7(1–16) bound to bacterial 70S ribosome (PDB 5F8K). Varying residues are marked with red for goat cathelicidin and blue for bovine cathelicidin. The structures were visualized by the Chimera software (Pettersen et al., 2004). (F) Amino acid frequency in mini-ChBac7.5Nα and its orthologs from mammalian species. The graph was plotted using the WebLogo server.

The target peptides were expressed in E. coli BL21 (DE3) as chimeric proteins that included 8 × His tag, the E. coli thioredoxin A with the M37L substitution (TrxL), methionine residue, and a mature cathelicidin. The ChMAP-28 amino acid sequence was translated from mRNA for the corresponding precursor protein (GenBank: AJ243126.1) as a 27-residue peptide without the C-terminal glycine, a common amidation signal in cathelicidins. The transformed E. coli BL21 (DE3) cells were grown up to OD600 1.0 at 37◦C in lysogeny broth (LB) containing 20 mM glucose, 1 mM MgSO4, and 0.1 mM CaCl2, 100 µg/ml of ampicillin and then were induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.3 mM. The cells were cultivated for 5 h at 30◦C with intense agitation. Then the cells were pelleted by centrifugation and sonicated in immobilized metal affinity chromatography (IMAC) loading buffer containing 6 M guanidine hydrochloride. The clarified lysate was applied on a column packed with Ni Sepharose (GE Healthcare). The recombinant protein was eluted with the buffer containing 0.5 M imidazole. Then the eluate containing the fusion protein was acidified (up to pH 1.0) and cleaved by 100-fold molar excess of cyanogen bromide over methionine for 20 h at 25◦C in the dark. The reaction products were lyophilized, dissolved in water, titrated to pH 5.0, and loaded on a semi-preparative Reprosil-pur C18-AQ column (10 mm × 250 mm, 5-µm particle size, Dr. Maisch GmbH). Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed with a linear gradient of acetonitrile in water containing 0.1% trifluoroacetic acid. The peaks were monitored at 214 and 280 nm. The collected fractions were analyzed by MALDI-TOF mass-spectrometry using Reflex III instrument (Bruker Daltonics). The fractions containing the target peptides were lyophilized and dissolved in water. The synthetic melittin (>98% pure) was kindly provided by Dr. Sergey V. Sychev (M.M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia). The recombinant tachyplesin-1 was obtained as described previously (Panteleev and Ovchinnikova, 2017). The peptides concentrations were estimated using UV absorbance.

#### TABLE 1 | Bacterial strains used in this study.

fmicb-09-02983 November 29, 2018 Time: 17:34 # 4

CI, clinical isolate; ∗ , no data available on strain source; XDR, extensively drug resistant strain; ESBL+, extended spectrum beta-lactamase producing strain; MBL+, metallo-beta-lactamase producing strain.

#### Circular Dichroism Spectroscopy and Structure Analysis

Secondary structures of the cathelicidins were analyzed in different environments by circular dichroism spectroscopy (CD) with the use of Jasco J-810 instrument (Jasco) at 25◦C. The experiment was performed in 10 mM sodium phosphate buffer (NAPB, pH 7.4), phosphate-buffered saline (PBS, pH 7.4), 50% TFE (Sigma), 30 mM DPC (Anatrace) micelles, and 30 mM SDS (Sigma) micelles. Final concentrations of the peptides were of 300 µM. Four consecutive scans were performed and averaged, followed by subtraction of the blank spectrum of the solvent. The CONTINLL program was used for data analysis (Provencher and Glöckner, 1981). Homology modeling was performed by MODELLER software (Sali and Blundell, 1993). The spatial structures were visualized by Chimera software (Pettersen et al., 2004).

#### Hemolysis and Cytotoxicity Assay

Hemolytic activity of the peptides was tested against the fresh suspension of human red blood cells (hRBC) using the hemoglobin release assay as described previously (Panteleev et al., 2016). Three experiments were performed with the hRBC from blood samples obtained from independent donors. The obtained data were represented as average means with standard deviations. The cytotoxicity of the peptides against HEK293T (transformed human embryonic kidney cells) and HEF (human embryonic fibroblasts) cell lines was studied using the colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay according to (Panteleev and Ovchinnikova, 2017). Three independent experiments were performed for each peptide. Half maximal inhibitory concentration (IC50) values were estimated as described previously (Kuzmin et al., 2018).

#### Antimicrobial Assay

Antimicrobial assay was performed as described previously (Panteleev et al., 2017b). Briefly, mid-log phase bacterial test cultures were diluted with the 2× Mueller-Hinton broth (MH, Sigma) supplemented with 1.8% NaCl or without it so that to reach a final cell concentration of 106 CFU/ml. 50 µl aliquots of the obtained bacterial suspension were added to 50 µl of the peptide solutions serially diluted with 0.1% water solution of bovine serum albumin (BSA) in 96-well flat-bottom polystyrene microplates (Eppendorf #0030730011). After incubation at 37◦C and 900 rpm for 24 h the minimum inhibitory concentrations (MIC) were determined as the lowest peptide concentrations that prevented growth of a test microorganism observed as visible turbidity. The results were expressed as the median values determined on the basis of at least three independent experiments performed in triplicate.

#### Checkerboard Assay

The peptides were twofold serially diluted with 0.1% BSA in 96-well microplates (Eppendorf #0030730011). Then, the peptide solutions were mixed in the new test plate crosswise in such a way that the resulting checkerboard contained each combination of the cathelicidins in eight doubly increasing concentrations, with wells containing the highest concentration of each peptide at opposite corners (Berditsch et al., 2015). Then, the antimicrobial assay was performed as described in the previous section. MICs were defined as the lowest concentrations of the peptides (when used individually or in the mix with another peptide at a sub-inhibitory concentration) that completely inhibited bacterial growth. The results were expressed as the median values determined on the basis of three independent experiments performed in duplicate. Estimation of synergistic effects of different cathelicidins was performed by calculating the fractional inhibitory concentration index (FICI) according to the equation: FICI = [À]/MICÀ + [B]/MICB, where MICÀ and MICB are the MICs of the individual substances, while [A] and [B] are the MICs of A and B when used together. A synergistic effect was defined at a FICI ≤ 0.5.

#### Biofilm Assay

Biofilm formation assay was performed as described previously (Panteleev et al., 2017a) with some modifications. Different E. coli strains and cultivating conditions were preliminary tested to achieve a strong biofilm formation (**Supplementary Figure S2**). The E. coli CI 214 cells were incubated in the trypticase soy broth (TSB) for 16 h at 37◦C and then were diluted 150-fold with the 2×M9 minimal medium supplemented with 50 mM

glucose, 10 µM thiamine, 2 mM MgSO4, 1 mM CaCl2, and the trace metals mixture. 50 µl of the obtained bacterial suspension were added to 50 µl aliquots of the peptide solutions serially diluted with sterilized water in 96-well microplates (Eppendorf #0030730011). The plates were incubated at 32◦C with gentle agitation (120 rpm) for 24 h to allow biofilm formation. Then, planktonic (unattached) cells were transferred into the new 96-well plate and OD620 of the cell culture was measured with the use of a microplate reader. The wells of the former plate were washed with PBS twice, and the formation of sessile biofilms was analyzed by crystal violet (CV) staining. Briefly, 160 µl of 0.1% crystal violet (CV, Sigma) solution was transferred to each well. The plates were incubated at 25◦C for 40 min and rinsed with distilled water to remove an excess of CV. Then the samples were dried for 10 min, and 160 µl of 96% ethanol (v/v) was added to the wells so that to dissolve the CV. 40 min later, the obtained extracts were transferred to a new 96-well plate. The absorption at 570 nm was measured with the use of a microplate reader. The experimental data were obtained from at least three independent experiments performed in triplicate. The results were reported relative to untreated bacteria served as a control. The results were analyzed using the GraphPad Prism 6.0 software.

#### Resistance Induction Experiments

Resistance induction experiments were performed using the previously described method (Chernysh et al., 2015) with some modifications. Briefly, on day 1, the overnight culture of wild-type bacteria was diluted with the 2× MH broth containing 1.8% NaCl so that to reach a final cell concentration of 106 CFU/ml. 50 µl aliquots of the obtained bacterial suspension were added to 50 µl of the peptide solutions serially diluted with 0.1% water solution of BSA in 96-well microplates (Eppendorf #0030730011). After incubation at 37◦C and 900 rpm for 22 ± 2 h, MICs were determined as described above. For each subsequent daily transfer, 5 µl of the inoculum taken from the first well with a sub-inhibitory drug concentration were diluted with 1 ml of the fresh 2× MH broth supplemented with 1.8% NaCl. Then, 50 µl of this suspension were sub-cultured into the next passage wells containing 50 µl aliquots of the peptide at concentrations from 0.25× to 16× of the current MIC of each agent. 26 repeated passages in the presence of antimicrobial agents were made for each bacterial strain during the experiment. Typically, the experiment was finished when the bacterial culture became resistant to antibiotic polymyxin B (Applichem) used as a control. Finally obtained cell cultures were passaged five times in the absence of antimicrobial agent to confirm that the acquired resistance is stable. Control serial passages in the absence of the agent were also performed. The obtained cultures showed unchanged MICs against antibacterial agents.

#### Bacterial Membranes Permeability Assay

To examine an ability of the peptides to affect the barrier function of outer and inner membranes of Gram-negative bacteria, we slightly modified the previously described procedure (Shamova et al., 2016) with the use of the E. coli ML-35p strain constitutively expressing cytoplasmic β-galactosidase and lacking lactose permease, and also containing β-lactamase in the periplasmic space. The state of the E. coli ML-35p outer and cytoplasmic membranes was assessed based on their permeability to chromogenic markers nitrocefin (Calbiochem-Novabiochem) and o-nitrophenyl-β-D-galactopyranoside (ONPG, AppliChem) which are the β-lactamase and β-galactosidase substrates, respectively. The cells were incubated in the TSB medium at 37◦C for 16 h, washed three times with 10 mM sodium phosphate buffer (pH 7.4) to remove residual growth media, diluted to the concentration of 2.5 × 108 CFU/ml. The experiments were performed in 10 mM sodium phosphate buffer with or without 0.9% NaCl. The final concentration of E. coli ML-35p cells was of 2.5 × 107 CFU/ml. The concentrations of ONPG and nitrocefin were of 2.5 mM and 20 µM, respectively. Peptide samples were placed in the wells of a 96-well plate with non-binding surface (NBS, Corning #3641), and the optical density (OD) of the solution rising due to the appearance of the hydrolyzed nitrocefin or ONPG was measured at 540 and 405 nm, respectively, using the Multiskan EX microplate reader (Thermo Fisher Scientific). The final volume in each well was 200 µl. The experiments were performed at 37◦C under stirring at 300 rpm. Control experiments were performed under the same conditions without addition of a peptide. Three independent experiments were performed, and the curve pattern was similar for all three series.

#### Flow Cytometry

The E. coli ML-35p cells were incubated in the TSB medium for 16 h at 37◦C and washed as described above. Bacterial cell suspensions were then incubated for 1 h at 37◦C with peptides at different concentrations prepared by twofold serial dilution. The assay was performed in the 96-well NBS microplates in 10 mM sodium phosphate buffer with or without 0.9% NaCl (pH 7.4). Then, SYTOX green (Life Technologies) was added to the treated cells at a final concentration of 2.5 µM and incubated for 10 min at room temperature in the dark. The SYTOX green does not penetrate live cells, but once inside the cell it binds to nucleic acids resulting in more than 500-fold enhancement of fluorescent emission. The fluorescence of the bacterial suspensions diluted 5-fold with PBS was measured (λExc = 488 nm, λEm = 530 nm) by NovoCyte flow cytometer (ACEA Biosciences). For each sample 105 events were recorded. Fluorescence signals were expressed as a percentage of two distinct cell groups: (1) healthy and partially damaged cells were deemed as totaling from 102 to 105 range of detection at 530 nm; (2) completely permeabilized (dead) cells were deemed as amounting ≥105 range of detection at 530 nm. Two independent experiments were performed, and the similar results were obtained.

## Cell-Free Protein Expression Assay

The cell lysate used for translation inhibition assay was prepared using the E. coli BL21 Star (DE3) cell culture grown at 30◦C in the 2x YTPG liquid medium (1% yeast extract, 1.6% tryptone, 0.5% NaCl, 22 mM NaH2PO4, 40 mM Na2HPO4, 0.1 M glucose). The chromosome of DE3 strains contains a gene encoding T7 RNA polymerase under control of the lacUV5 promoter. The bacterial culture was grown to OD600 0.8–1.0, then T7 RNA polymerase gene was induced by adding 0.2 mM IPTG. Bacteria were harvested at OD600 5.0–6.0 by centrifugation (3000 g,

30 min, 4◦C). The bacterial pellet was washed three times by suspending it in four volumes of wash buffer (10 mM Tris-acetate buffer, pH 8.2, 60 mM potassium glutamate, 14 mM magnesium acetate, 1 mM DTT), then resuspended in one volume of the same buffer (1 ml per 1 g of wet cell mass) and disrupted by sonication at 5–15◦C. The total cell lysate was centrifuged at 15000 g (30 min, 4◦C). The supernatant was split into aliquots and stored at −70◦C.

In order to investigate the effect of AMPs on the translation process, the peptides were added to a cell-free protein synthesis (CFPS) reaction mix with a plasmid encoding EGFP variant (F64L, S65T, Q80R, F99S, M153T, and V163A) under a control of the T7 promoter. The reaction mix consisted of the following components: 1.2 mM ATP, 0.8 mM UTP, 0.8 mM GTP, 0.8 mM CTP, 2 mM of each of 20 proteinogenic amino acids, 1.5 mM spermidine, 1 mM putrescine dihydrochloride, 0.06647 mM calcium folinate, 170 ng/ml tRNA from the E. coli MRE 600 strain, 0.33 mM NAD, 120 mM HEPES-KOH (pH 8.0), 10 mM ammonium glutamate, 175 mM potassium glutamate, 60 mM glucose, 15 mM magnesium glutamate, 2% PEG 8000, 25% E. coli BL21 Star (DE3) cell lysate, 10 ng/µl plasmid DNA. The reaction volume was 50 µl. The peptides were dissolved in PBS with the addition of 0.1% BSA. Streptomycin and erythromycin were used in the positive control reactions. Fluorescence of the sample without inhibitor was set as the 100% value. The reaction proceeded for 1.5 h in 96-well clear flat-bottom black polystyrene microplates (Corning #3340) sealed with Parafilm in a plate shaker (30◦C, 900 rpm). Fluorescence of the synthesized EGFP was measured with the microplate reader AF2200 (λExc = 488 nm, λEm = 510 nm). The experimental data were obtained from at least three independent experiments. IC50 values were determined by interpolation from non-linear regression curves using the GraphPad Prism 6.0 software.

#### Electrophoretic Mobility Shift Assay

The peptides binding to DNA was examined by electrophoretic mobility shift assay (EMSA) according to the previously described protocol (Panteleev et al., 2016). Briefly, the plasmid pUC19 was incubated with the tested peptides at increasing concentrations in the binding buffer containing 10 mM Tris-HCl (pH 8.0), 50 µg/ml BSA, 5% glycerol, 1 mM DTT, 150 mM NaCl, 20 mM KCl, and 1 mM EDTA, at 25◦C for 30 min. Then, the samples were analyzed by electrophoresis in 0.8% agarose gel. The DNA migration was detected by means of the ethidium bromide fluorescence tracking. The DNA-peptide (w/w) ratios were of 1:0 (negative control), 4:1, 2:1, 1:1, 1:2, respectively.

#### Genetic Analysis of Bacterial Strains

The sbmA and yaiW genes encoding the E. coli inner or outer membrane proteins, respectively, as well as a regulatory part of their common operon were amplified by polymerase chain reaction (PCR) using gene-specific primers (**Supplementary Figure S3**). Individual bacterial colonies of the tested strain were picked up from Petri dish and used as a template for PCR. The following components were mixed for the PCR: 2 µl of 10× Encyclo buffer (Evrogen), 0.4 µl of 50× Encyclo DNA polymerase (Evrogen), 10 µM forward primer, 10 µM reverse primer, 0.2 mM dNTPs, bacterial cells on inoculation loop, and water diluting to the total volume of 20 µl. Amplification was carried out on a thermocycler using: initial denaturation (95◦C, 10 min), 25 amplification cycles (94◦C, 30 s; 55◦C, 40 s; 72◦C, 90 s), and final elongation (72◦C, 10 min). The products were separated by electrophoresis on 1.5% agarose gel (4 V/cm) and visualized on a UV trans-illuminator. The PCR products were purified from agarose gel and inserted into pGEM-T vector (Promega). The ligation products were transformed into the chemically competent E. coli DH10B cells. Plasmid DNA was isolated from overnight cultures of single white colonies on LB agar plates supplemented with ampicillin (100 µg/ml), using Plasmid Miniprep kit (Evrogen). The plasmids were sequenced on both strands using the ABI PRISM 3100-Avant automatic sequencer (Applied Biosystems). At least two independent experiments were performed with each strain to prove the obtained results.

## RESULTS

#### Expression and Purification of the Recombinant Peptides

Natural goat cathelicidins do not undergo significant post-translational modifications, therefore heterologous expression in E. coli of the peptides fused with a carrier protein seems to be a reasonable approach for their production. The goat cathelicidins were produced using the same protocol. To facilitate the purification process and improve final yield, the recombinant peptides were obtained as fusion proteins with the N-terminal 8×His tag and thioredoxin A which was approved to be an effective carrier protein for different peptide scaffolds having antibacterial activity (Li, 2011). The peptides were purified by a downstream process including IMAC of the clarified total cell lysate, cleavage of the fusion protein with cyanogen bromide, and fine purification by RP-HPLC (**Supplementary Figure S1**). Final yields of ChMAP-28, mini-ChBac7.5Nα, and mini-ChBac7.5Nα(1–16) were 3.4, 9.2, and 7.5 mg per 1 l of the culture medium, respectively. The obtained recombinant cathelicidins were analyzed by MALDI-TOF mass-spectrometry. The measured m/z values of the cathelicidins matched the corresponding calculated molecular masses (**Supplementary Table S1**).

### Secondary Structure of Goat Cathelicidins

In this study, CD spectroscopy was used to analyze the secondary structure of the goat cathelicidin ChMAP-28. As shown in **Figure 1A**, the CD spectra of ChMAP-28 dissolved in phosphate buffer or phosphate-buffered saline showed a negative peak at the wavelength of 200 nm, which indicated that it mainly adopted random coil conformation. Therefore, the above conditions do not facilitate the peptide folding. In contrast, the CD spectra of ChMAP-28 interacted with SDS or DPC micelles showed a strong positive peak at 195 nm, and two negative peaks at 208 and 220 nm, which indicated that ChMAP-28 mainly adopted α-helix secondary structures in hydrophobic environments.

Indeed, the peptide has a relatively high homology to known α-helical cathelicidins from Bos taurus: 61% sequence identity with BMAP-27 and 42% – with BMAP-28 (**Figure 1B**). We performed homology modeling based on the BMAP-27 structure to visualize a probable spatial structure of ChMAP-28 in membrane-mimicking environment (**Figures 1C,D**). Significant homology between mini-ChBac7.5Nα and the N-terminal fragment of Bac7 suggests their similar structure, thus mini-ChBac7.5Nα was not analyzed by CD-spectroscopy. It is assumed that mini-ChBac7.5Nα, alike the peptide Bac7(1–16), adopts extended structures within the bacterial ribosomal exit tunnel (**Figure 1E**). Generally, mini-ChBac7.5Nα and its orthologs from mammalian species (artiodactyls and cetaceans) recorded in the Genbank showed a relatively high homology, especially between sequences at the N-terminal and central parts of the peptides (**Figure 1F**). Interestingly, recent studies revealed that the Bac7 homolog, isolated from the bottlenose dolphin Tursiops truncatus and designated as Tur1B, was enriched with Trp residues and displayed rather modest inhibitory effect on bacterial translation (Mardirossian et al., 2018).

#### Cytotoxic Properties of Goat Cathelicidins

To estimate cytotoxic effect of the cathelicidins, human red blood cells (hRBC) as well as adhesive cell lines of human embryonic fibroblasts (HEF) and human embryonic kidney cells (HEK293T) were used. Melittin known as a potent cytolytic peptide was used as a positive control. It is known that most Pro-rich AMPs have no pronounced toxicity to mammalian cells. Earlier, we showed that cytotoxicity of mini-bactenecins at concentrations up to 30 µM against a set of mammalian cell lines after 24 h was quite modest (Shamova et al., 2016). However, the peptides are not completely non-toxic. The data analysis revealed that both mini-ChBac7.5Nα and its shortened analog showed cytotoxic activity against mammalian cell lines at concentrations >25 µM (**Figure 2**). Mini-ChBac7.5Nα almost lacked hemolytic activity and lysed only 2% of red blood cells at the concentration of 100 µM. In contrast, a half maximal hemolysis concentration (HC50) of ChMAP-28 was of ∼100 µM, and the peptide had the IC50 against HEK293T cells of ∼3.5 µM. Interestingly, its bovine ortholog BMAP-28 possessed the IC50 against murine 3T3 cells and HC50 of <3.75 and ∼20 µM, respectively (Ahmad et al., 2009). Melittin was proved to be significantly more toxic than α-helical cathelicidins and completely damaged all the cells tested at concentrations of <2.5 µM.

## Antimicrobial Activity of Goat Cathelicidins

Amphiphilic AMPs are known to be adsorbed on plastic surfaces (Wiegand et al., 2008). For these reason, serial dilutions of the peptides were performed in the presence of BSA in the growth medium in order to minimize this effect. MICs of goat cathelicidins and melittin against Gram-positive and Gram-negative bacteria are presented in **Table 2**. It was reported that Pro-rich AMPs have high antimicrobial activity against Gram-negative bacteria and are less active or

inactive against most Gram-positive bacteria. In whole, our results confirmed this. It is noteworthy that the medium formulation as well markedly affects the activity values of Pro-rich AMPs. Antibacterial activities of some insect Pro-rich AMPs was low when tested in the presence of a salt, which might inhibit absorption of the peptides to the bacterial surface (Gennaro et al., 2002). Therefore, the salt influence on the antibacterial activity was investigated in this study. Indeed, the presence of 0.9% NaCl resulted in several-fold decrease in the activity of mini-bactenecins against all the strains tested. The shortened analog mini-ChBac7.5Nα(1–16) was shown to be less active and more salt-sensitive as compared with the wild-type mini-ChBac7.5Nα. Interestingly, antibacterial activities of the peptides were similar when tested in a salt-free medium against Gram-negative bacteria E. coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Enterobacter cloacae. In contrast to the other tested strains, these bacteria have the ABC transport system based on the homodimeric cytoplasmic membrane protein SbmA. In E. coli the cytoplasmic membrane protein SbmA and outer membrane lipoprotein YaiW participate in transport of some Pro-rich AMPs and bacteriocins (Arnold et al., 2014). Mutation or deletion of either SbmA or YaiW significantly decreased the ability of the Bac7 to internalize, and significantly reduced susceptibility to the peptide (Arnold et al., 2014). Our results TABLE 2 | Antibacterial activity of goat cathelicidins and melittin.

fmicb-09-02983 November 29, 2018 Time: 17:34 # 8

∗Antibacterial testing was performed in the Mueller-Hinton broth at 37◦C. ∗∗The presence of genes encoding the membrane transporters SbmA and YaiW that affect sensitivity to proline-rich antimicrobial peptides (Arnold et al., 2014).

indicated that the presence of the C-terminal fragment PRPRPR did not influence the efficiency of the peptide translocation via SbmA transporter in a salt-free medium, but could play a key role when acting against SbmA-deficient bacteria (e.g., Gram-positive bacteria) or applying in the presence of a salt. Previous study of the Pro-rich pig cathelicidin PR-39 revealed that an activity of the full length peptide was hardly affected by 100 mM NaCl while the shortened peptide derivatives lacked most of their antimicrobial properties under the same conditions (Veldhuizen et al., 2014). It is likely that the observed effect occurs due to electrostatic interactions between positively charged peptides and negatively charged bacterial membranes. Antimicrobial activity of mini-bactenecins seems to be a function of a total charge of the peptide rather than of a charge density and overall hydrophobicity, since mini-ChBac7.5Nα(1–16) has both higher charge-to-length ratio and longer retention time in reversed-phase HPLC (**Supplementary Figure S1**) than the wild-type mini-ChBac7.5Nα. A total charge could be important at initial stages of Pro-rich AMPs interaction with bacteria, i.e., during the primary electrostatic attraction followed by displacement of divalent cations cross-bridging LPS on the cell surface, that destabilized the membrane and led to the peptide self-promoted uptake.

Cathelicidin ChMAP-28 exhibited significantly more potent antibacterial activity (≥16-fold higher) than melittin against most strains tested. ChMAP-28 was shown to be less sensitive to high ionic strength as compared with mini-bactenecins. ChMAP-28 and last line antibiotics polymyxin B and meropenem were tested against extensively drug resistant clinical isolates of Gram-negative bacteria which belong to "ESKAPE" pathogens: E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, E. cloacae, P. mirabilis (**Supplementary Table S2**). Generally, ChMAP-28 exhibited a potent antimicrobial activity comparable with that or even higher than that of the above mentioned control antibiotics. The peptide was shown to effectively kill all the bacteria including polymyxinand meropenem-resistant strains, thus arguing against cross-resistance to the peptide.

#### Synergy Between Goat Cathelicidins

Antimicrobial activity of most Pro-rich AMPs including mini-bactenecins is reduced at physiological salt concentrations. In view of this, interaction with other co-localized membrane-active molecules may enhance or restore the activity of Pro-rich AMPs. To check the assumption, antibacterial effects of the combination of the goat cathelicidins mini-ChBac7.5Nα and ChMAP-28 were evaluated in the medium containing a physiological concentration of NaCl (**Table 3**). A set of Gram-negative bacterial species and one Gram-positive strain S. aureus 209P sensitive to mini-bactenecins were used as the test microorganisms. To reduce adsorption of AMPs on plastic surfaces while testing antimicrobial activity in vitro, we used 0.1% BSA for serial dilutions (Wiegand et al., 2008; Bolosov et al., 2017). In combination with ChMAP-28 at sub-inhibitory concentrations, mini-ChBac7.5Nα exhibited antimicrobial activity with more than fourfold decreased MIC values that led to FICI values of ≤0.375 against different E. coli strains. The peptides showed a strong synergistic effect against K. pneumoniae, E. cloacae, A. baumannii with at least an eightfold decrease in MICs for both agents and FICI values of 0.25, 0.25, and 0.133, respectively. Interestingly, the presence of ChMAP-28 either completely restored or slightly increased the activity of mini-ChBac7.5Nα as compared with that evaluated in a salt-free medium against these bacterial strains (**Table 2**), including the clinical isolate of E. coli CI 214 with a weak sensitivity to mini-ChBac7.5Nα (the MIC values were of 4 µM in a salt-free medium and >64 µM in the presence of 0.9% NaCl). As described above, all the mentioned strains normally have the SbmA transport system. It suggests that ChMAP-28 acting at sub-inhibitory concentrations may promote translocation of

∗The estimation of synergistic effects between cathelicidins was performed by calculating the fractional inhibitory concentration index (FICI) according to the equation: FICI = FICA + FICB = [À]/MICÀ + [B]/MICB, where MICÀ and MICB are the MICs of individual peptides, while [A] and [B] are the MICs of A and B when used together. A synergistic effect was defined at a FICI ≤ 0.5.

mini-ChBac7.5Nα through the outer membrane, which is an obstacle to Pro-rich AMPs when electrostatic interactions are affected by increased ionic strength. Inside the periplasmic space mini-ChBac7.5Nα can effectively use cytoplasmic membrane transporters to get into the cell. At the same time, ChMAP-28 did not restore the activity of mini-ChBac7.5Nα against P. aeruginosa, and no synergy was observed. These findings are consistent with the previous study that revealed the lack of synergy between Pro-rich AMPs and the membranolytic peptide CRAMP while testing antimicrobial activity against P. aeruginosa (Knappe et al., 2016). Surprisingly, a pronounced synergistic effect was observed against S. aureus 209P with MICs of both peptides almost identical to those measured in a salt-free medium, thus resulting in FICI of 0.188. This observation allowed us to speculate that some Gram-positive bacterial strains might have transport systems for Pro-rich AMPs. On the other hand, the presence of Pro-rich AMPs could interact with the structures of cell wall teichoic acids, the anionic glycopolymers, and thereby helped ChMAP-28 molecules to reach lipid bilayer.

#### Analysis of Membrane-Permeabilizing Activity

Antimicrobial peptides can realize their biological functions by damaging membrane integrity and specifically inhibiting intracellular processes. One of the most important objectives in functional study of AMPs is to elucidate a mechanism of their antimicrobial action. The effect of the goat cathelicidins on E. coli ML-35p membrane integrity was characterized by monitoring both the SYTOX Green uptake by flow cytometry and permeability to chromogenic markers – ONPG and nitrocefin. The membranolytic peptide melittin was used as a positive control. The flow cytometry data show that mini-ChBac7.5Nα does not influence the E. coli cytoplasmic membrane integrity regardless of salt concentration (**Figure 3A**). This is in agreement with our previous data (Shamova et al., 2016). In contrast to the longer peptide Bac7(1–35) (Podda et al., 2006), mini-ChBac7.5Nα did not significantly damage membranes at higher concentrations than the MIC values. The cathelicidin ChMAP-28 was shown to damage bacterial membrane at nanomolar concentrations that led to the appearance of bacterial subpopulations with increased fluorescence intensity by one or two orders of magnitude vs. a control (**Figure 3B**). These shifted peaks on the graph may represent cells with qualitatively different grades of membrane damage.

Synergy between two different AMPs could result from either facilitation of translocation of one of them into the cell by another peptide or cooperative augmentation of the membrane damage, as was shown for cathelicidins and defensins (Nagaoka et al., 2000). To decide between these scenarios, a comparative analysis of the ability of the cathelicidins to disrupt the integrity of inner and outer bacterial membranes was conducted in a wide range of concentrations. Interestingly, mini-ChBac7.5Nα was shown to effectively damage outer membrane in a salt-free environment (**Figure 4A**). However, the addition of 0.9% NaCl reduced the activity to a modest effect at 8–32 µM (**Figure 4B**), that could explain a weak antibacterial activity of mini-bactenecins in the presence of salt (**Table 2**). Translocation of mini-ChBac7.5Nα into periplasmic space likely depends on ability to disrupt the outer membrane, which becomes an impassable barrier in the presence of NaCl. At the same time, ChMAP-28 was proved to damage the outer membrane in a salt-containing medium at concentration of 0.008 µM (**Figure 4C**) that was equal to the fractional MIC of the synergy combination with mini-ChBac7.5Nα (see **Table 3**). The data presented in **Figure 4D** allowed us to rule out the effect of potentiating the cytoplasmic membrane permeabilization: in most cases the presence of mini-ChBac7.5Nα did not significantly affect or even decreased the ability of ChMAP-28 to damage the membrane. The same was true when we tested the peptide mixtures on the E. coli ML-35p outer membrane (graphical data not shown). Taken together, these results suggest that ChMAP-28 at sub-inhibitory concentrations promotes translocation of mini-ChBac7.5Nα into the periplasmic space rather than enhances its membrane activity.

#### Inhibition of in vitro Protein Synthesis in E. coli

Taking into account the reported data on the mechanism of action of proline-rich AMPs and the inability of mini-ChBac7.5Nα to disrupt cytoplasmic membrane integrity, we tested an ability of this peptide and other antimicrobial compounds to inhibit protein biosynthesis in vitro. The experiment was carried out using the bacterial cell-free protein synthesis system expressing the green fluorescent protein (GFP). The results obtained for streptomycin with IC50 value of 0.2 µM and a full inhibition of >1 µM correspond with the published data (Krizsan et al., 2014) (**Figure 5A**). IC50 for mini-ChBac7.5Nα was of ∼1 µM which is comparable to that of conventional inhibitors of bacterial translation – streptomycin and erythromycin. Apart from that, the values of IC50 for mini-ChBac7.5Nα were similar to those of its homologs – the Bac7 fragments (Seefeldt et al., 2016), and also to the previously determined MICs against E. coli (see **Table 2**). It should be noted that the mini-ChBac7.5Nα(1–16) fragment inhibits biosynthesis twice less effectively than the wild-type mini-ChBac7.5Nα that might account for the reduction of antibacterial activity. Interestingly, the cathelicidin ChMAP-28 also affects protein biosynthesis, but at much higher concentrations than its MIC. It seems that ChMAP-28 ability to inhibit translation is due to a non-specific interaction with nucleic acids. This assumption is supported by the fact that tachyplesin-1, which is known to bind DNA (Yonezawa et al., 1992), demonstrates a comparable level of inhibition. For several cationic AMPs, e.g., for indolicidin (bovine tryptophan-rich cathelicidin), binding to DNA is considered to be one of the mechanisms of their antimicrobial action. AMP-DNA binding induces aggregation and interferes with the process of replication (Hsu et al., 2005). It was shown that both goat cathelicidins bound plasmid DNA at a mass ratio of 1:1 (**Figures 5B,C**). In addition, ribosome-binding is supposed to be the main factor responsible for bacterial growth inhibition by the Bos taurus cathelicidin Bac7. Comparing our results with known data on Bac7 and bearing in mind a high homology degree between mini-ChBac7.5Nα and the N-terminal fragment of Bac7, we assume that the main target for mini-ChBac7.5Nα is also the 70S ribosome. Data obtained allow us to conclude that two goat cathelicidins – ChMAP-28 and mini-ChBac7.5Nα possess essentially different mechanisms of antimicrobial action: ChMAP-28 preferentially acts by increasing cytoplasmic membrane permeability, while mini-ChBac7.5Nα specifically inhibits bacterial translation.

#### Anti-biofilm Activity of Goat Cathelicidins