antibiotics

Article Bioengineered Nisin Derivative M17Q Has Enhanced Activity against Staphylococcus epidermidis

Ellen Twomey 1, Colin Hill 2,3 , Des Field 2,3,* and Maire Begley 1,*

1 Department of Biological Sciences, Cork Institute of Technology, T12 P928 Cork, Ireland; [email protected] 2 School of Microbiology, University College Cork, T12 YN60 Cork, Ireland; [email protected] 3 APC Microbiome Ireland, University College Cork, T12 YN60 Cork, Ireland * Correspondence: des.fi[email protected] (D.F.); [email protected] (M.B.)

 Received: 24 April 2020; Accepted: 3 June 2020; Published: 6 June 2020 

Abstract: Staphylococcus epidermidis is frequently implicated in medical device-related infections. As a result of this, novel approaches for control of this opportunistic pathogen are required. We examined the ability of the natural nisin A, produced by , to inhibit S. epidermidis. In addition, a bank of 29 rationally selected bioengineered L. lactis strains were examined with the aim of identifying a nisin derivative with enhanced antimicrobial activity. Agar-based deferred antagonism assays revealed that wild type nisin A inhibited all 18 S. epidermidis strains tested. Larger zones of inhibition than those obtained from the nisin A producing L. lactis strain were observed for each derivative producer against at least one S. epidermidis strain tested. Six derivative producing strains, (VGA, VGT, SGK, M21A, M17Q, AAA), gave larger zones against all 18 strains compared to the wildtype producing strain. The enhanced bioactivity of M17Q was confirmed using well diffusion, minimum inhibitory concentration (MIC) and a broth-based survival assays. Biofilm assays were performed with plastic microtiter plates and medical device substrates (stainless-steel coupons and three catheter materials). The presence of nisin A significantly reduce the amount of biofilm formed on all surfaces. M17Q was significantly better at reducing biofilm production than nisin A on plastic and stainless-steel. Finally, M17Q was significantly better than nisin A at reducing bacterial numbers in a simulated wound fluid. The findings of this study suggest that nisin and bioengineered derivatives warrant further investigation as potential strategies for the control of S. epidermidis.

Keywords: ; antibacterial peptide; bioengineered peptide; nisin; Staphylococcus epidermidis; medical device related infections; biofilm

1. Introduction Staphylococcus epidermidis is a habitual member of the human commensal microbiota [1,2]. While most frequently found on the skin, this Gram-positive, coagulase negative organism has also been isolated from the mucous membranes of the nares [2]. Although benign when present on the skin of healthy individuals, S. epidermidis has been classed as an opportunistic pathogen [1,3]. Lee et al. have described it as a “formidable nosocomial pathogen,” due to its rapid spread, the development of resistance, its threat to immunocompromised individuals [1,3]. The ability of S. epidermidis to form biofilms on both biotic and abiotic surfaces and invade the body via postoperative skin wounds, complicates the recovery process and increases patient suffering. Easily transferred due to its ubiquitous presence on the skin, the production of an exopolysaccharide matrix permits planktonic S. epidermidis cells to adhere to the surfaces of indwelling medical devices, such as intramedullary pins, prosthetic devices, for example, artificial hips, catheters and cerebrospinal shunts [4,5]. Once the organism has invaded or been implanted into the body of the patient, development of a slime layer establishes a persistent source of infection within the body, where the most viable treatment option is the complete

Antibiotics 2020, 9, 305; doi:10.3390/antibiotics9060305 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 305 2 of 18 removal and replacement of the device [4,6]. By extracting and replacing contaminated devices such as prosthetic joints, pacemakers or shunts, afflicted individuals potentially face a secondary invasive surgery, as well as unnecessary pain and suffering. Such removals are coupled with a course of antibiotics to ensure the infection is cleared and to promote recovery [3]. However, a 2018 summary report released by the European Centre for Disease Prevention and Control remarks on the rapid emergence and spread of Multidrug Resistant S. epidermidis, (MRSE) [3]. This states that the efficacy of previously used successful drugs such as trimethoprim, fusidic acid, clindamycin and fluoroquinolones are in decline, with cases of resistance against the first line of defense, vancomycin, also being observed [3]. With the looming threat of resistant infections and a depleted antibiotic discovery pipeline, alternate treatment routes are being investigated. are ribosomally synthesized that are produced by some ; however, they usually operate selectively against closely related strains [7,8]. Nisin is a 34- bacteriocin produced by Lactococcus lactis with antimicrobial activity against a number of Gram-positive pathogens such as Methicillin Resistant (MRSA), streptococci and Vancomycin Resistant Enterococci [8]. Nisin exerts its antimicrobial effects by binding to a highly conserved component of the bacterial cell wall, lipid II [8,9]. By interfering with this integral element, it causes the formation of pores and prevents cell wall synthesis, triggering lysis and cellular death [8,9]. Nisin has been approved by both the United States Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) for use as a food in over 70 countries and is “generally regarded as safe” (GRAS) [10]. In recent years, studies have revealed nisin to be effective against an array of clinically relevant Gram-positive pathogens, including and drug resistant Staphylococcus aureus [8,11]. While not currently used in clinical settings, bacteriocins are demonstrating their capabilities at inhibiting the spread of infections in murine and other animal models. For example, nisin has been applied to wound dressings and catheters used on or in, cows undergoing routine surgical procedures and successfully reduced the likelihood of infections [12]. The bacteriocin lacticin 3147 has been found to inhibit the spread of Staphylococcus infections in a murine model [13]. Several naturally occurring variants of nisin have been found to be produced by L. lactis, with one Streptococcus species, (Streptococcus uberis) also possessing genes for another version of the peptide [14,15]. All variants discovered have identical modifications as the initially characterized nisin peptide nisin A but differ in their amino acid compositions. For example, nisin Z has histidine at position 27 in place of asparagine. Nisin Q contains a more varied sequence which differs by four amino acids, (valine at position 15, an asparagine at position 17, leucine at position 21 and another valine at position 30) [16,17]. All naturally occurring peptides are capable of inhibiting the growth of clinically relevant Gram-positive bacteria in agar based deferred antagonism and agar well diffusion assays [15–17]. In addition to the occurrence of natural variants of nisin, the gene encoded nature of the peptide has permitted extensive bioengineering with the hopes of creating novel nisin peptide derivatives with more desirable properties such as increased solubility, stability and antimicrobial activity. In our laboratory, we have employed site directed and site saturated mutagenesis approaches to generate an extensive and diverse bank of L. lactis strains that produce bioengineered nisin derivatives. These include strains genetically engineered to produced mutated versions of the wildtype nisin A peptide [8,14,18,19]. Several of the generated strains have displayed enhanced antimicrobial activity against selected pathogens when compared to the wildtype. For example, the bioengineered peptide I4V demonstrated a two to four-fold increase in activity (as determined by minimum inhibitory concentration assays) against three strains of Staphylococcus pseudintermedius a Gram-positive pathogen responsible for skin and soft issue infections in both humans and animal species [20]. Similarly, the bioengineered peptides M21A and M21V demonstrated a two-fold increased activity compared to the wildtype nisin A against the foodborne pathogen L. monocytogenes [21]. The ability of nisin to inhibit S. epidermidis has been reported. Davison et al. investigated nisin along with several other biocides against one S. epidermidis strain (RP62A) that is a strong biofilm producer [22]. The test was performed using capillary flow cells and confirmed that the bacteriocin Antibiotics 2020, 9, 305 3 of 18 possessed antimicrobial activity against the target, S. epidermidis RP62A [22]. Ghiselli et al. have studied the effect of nisin against S. epidermidis (two strains) graft infections in mice. Pre-treatment of the prosthetic graft material Dacron with nisin reduced bacterial counts by four logs [23]. Here we investigated the ability of nisin A to inhibit a larger number of S. epidermidis strains. The second objective was to subsequently interrogate a rationally selected bank of 29 L. lactis strains that produce bioengineered nisin derivatives (selected based on their ability to inhibit other Gram positive pathogens in separate studies in our laboratory) with the aim of identifying a derivative peptide with enhanced ability to inhibit S. epidermidis.

2. Results

2.1. Agar Based Deferred Antagonism and Well Diffusion Assays The ability of a wildtype nisin A-producing L. lactis strain and a bank of 29 L. lactis strains each producing a different variant of nisin A were examined for their ability to inhibit a collection of 18 S. epidermidis strains by performing agar based deferred antagonism assays (Figure1). Zones of inhibition were observed for the wildtype nisin A producer against all of the 18 S. epidermidis strains with representative zone sizes ranging from 9.8 to 16.3 mm (Table1). With the exception of the I4V producing strain against S. epidermidis 1, zones of inhibition were also observed for all of the 29 nisin derivative producers against all 18 S. epidermidis strains. Each nisin derivative producing strain produced at least one enhanced zone against a member of the bank of 18 strains when compared to the wildtype. Not all S. epidermidis strains were impacted equally, despite all being of the same species. For example, M21V had enhanced activity against 16 of the 18 strains used but against strains 26 and 1 it was found to possess activity equal to or less than nisin A. In all, it was noted that two derivative producing strains gave larger zones of inhibition than the wildtype against 17 S. epidermidis strains and six bioengineered derivative producing strains gave larger zones than the wildtype against all 18 S. epidermidis strains tested. Of these six (VGA, VGT, SGK, M21A, M17Q and AAA), the M17Q and AAA producers gave the largest zones of inhibition. Representative zone sizes ranging from 14 to 21 mm were observed for the AAA producing strain and representative zone sizes ranging from 13.1 to 21 mm were observed for the M17Q producing strain. The AAA derivative peptide was previously described as part of a study to investigate nisin derivatives containing small chiral amino acids within the hinge region [24]. This hinge region connects the N terminal of the nisin peptide, which contains one and two (β-methyl) lanthionine rings (A, B and C), to the C terminal which is composed of intertwined rings, D and E, (see Figure S1). Notably, although it exhibited enhanced bioactivity through deferred antagonism assays, the AAA derivative exhibited enhanced specific activity against just one of four strains it was tested against [24] revealing that in many cases it would appear that enhanced bioactivity may be attributable to enhanced diffusion through complex media, a phenomenon previously reported by Rouse et al., 2012 [25]. Given that M17Q represents a new enhanced derivative at a location that has not previously been identified by NMR as a ‘hotspot’ critical for the cellular adaptability of nisin [26], it was decided to focus on M17Q for the remainder of the study. In the M17Q peptide the methionine (M) at position 17, (i.e., M17), in the wildtype nisin is replaced with a glutamine (see Figure S1). For the M21V producing strain, larger zones of inhibition were obtained for 16 of the 18 S. epidermidis strains when compared to the wildtype producing strain. M21V has been the subject of a number of studies to evaluate its efficacy against a wide range of pathogenic bacteria, including L. monocytogenes MRSA, hVISA and C. difficile so it was decided to include it in subsequent experiments for comparison purposes [11,18,19,27]. Antibiotics 2020, 9, 305 4 of 18

Table 1. Comparison of the antimicrobial activity of wildtype nisin A (WT) producing L. lactis strain to that of 29 bioengineered nisin-derivative producing L. lactis strains through an agar based deferred antagonism assay. Each peptide producer was tested against 18 S. epidermidis strains in triplicate. The figures in the table are the results obtained for 1 repeat and are representative of the results obtained for all three biological repeats. Zone sizes are presented in millimeters. Values highlighted in bold indicate that the representative zone obtained from the bioengineered strain is larger than the zone obtained from the wildtype producer against that specific S. epidermidis strain.

S. epidermidis Strains 1 17 22 26 28 53 70 78 80 89 90 92 100 101 102 103 104 106 WT Nisin 11 14.9 12.3 16.3 11.2 11 9.8 10.8 11.7 12 11 11 11 12.3 12.3 10.7 13.3 13.6 Bioengineered L. lactis strains M21V 9.5 15.9 13.7 16 12.7 12.6 13 12 13 13 12.7 12 13.1 13.7 13.3 13.6 14 14 M17Q 14.3 21 19.4 21 19.9 15 13.1 17.7 19.4 17.8 17 16.4 15.7 18.3 16.8 15 20.3 19.7 M21S 11.3 16.6 12.8 16.7 13.6 12.8 11 16.7 14.3 14.2 11.2 11 12.6 15.4 13 12.7 14.7 13.7 M21V-H31K 9 15.5 12.8 15.7 13 12.4 9 12 14 11 12 11 11 12.1 13.2 12.5 13 12.7 M21K 10 13 9.8 15 12 10 11.3 12 10 14.5 11.7 9.7 11.8 15 12 12.5 12.7 12.8 VGA 15.7 18.2 16.8 18 14.5 17 10.2 15.7 15.2 18 13.8 18 15.7 15 18 15 19.3 20.7 K12A 10.7 13 13.4 15.9 14 13.3 10.9 10 15 15.2 13.3 13 11.5 14 15 11.9 15.3 13.7 K12S 10 16 12 16.7 15 12 8 17 14 13.4 12.3 11.4 13 14.5 14.6 12.5 17.7 14.7 K12T 10 17.7 10.5 15.1 10 10 8 10 9 10 12 9 11 10 12.5 12.3 13.8 11.3 K22T 10 16.5 11.2 18.2 13.6 11.5 13.6 10.8 13.3 14.6 13.3 10 11.4 15 17 12.7 16 13.6 VGV 10.1 14.9 13.6 16.1 13.3 13.3 11 9 12.4 8 12 15.7 11 13.7 11.2 13.5 18.5 18 VGT 12.2 18 16.4 18 14.2 16 11 11 14 15.1 14.3 11.7 11.7 13.3 15.8 13 19 14.6 PIT 11.7 15.7 11.3 17 13 13.5 11 11 10 16.2 11.6 10 12.7 11 15.2 12.1 16.3 16.1 PGA 13 19.7 14.3 19.4 19.6 14 12.7 10.6 16.4 9 16.3 11.5 14.7 15 15.5 14.9 18 15.7 T2L 11 10 9.5 12.2 10.5 10 8 11.8 10 12 11.8 16 14.1 10.7 14.3 14 14.9 15.1 G18Dhb 12.2 11 8 14.7 11 10.3 8 10 16.3 11.7 10 10 12 13 12 12 11.9 12.7 SGK 13.4 17 13.7 18.8 15.3 13.7 10 11 13.7 13.7 15 13.4 14 13 13.7 13.7 16 16.5 PAQ 10 12.5 11.7 18 12 11.8 10 14 9 13 14 12 11 12.7 14.3 12.3 17 15.3 HTK 11.6 16.5 10.4 17 14 10 10 14.3 14.5 13.7 12 11.7 12.3 13 16 12.3 15.9 15.9 I4V 0 14.6 11.7 18 15.9 11.5 9 11 10.7 12.7 13 12 13 11.7 13.7 11 15 14.7 N2OP 10 15.5 11.8 18 14.5 11.3 10 12.3 16.9 14.7 16 13.6 11.5 15 13.7 13.5 16.9 16.4 S29R 10 14 10.8 15.6 12.6 11 9 9 10.5 14 12.7 10 7 9 11.4 11 13.4 14 S29E 10 15.4 11.3 16.7 11.7 11 9 11 10 13.6 11.7 11 11 12 17 16.9 12 14.2 S29A 10 16 11.7 17.3 14 11.4 10 11 11.6 14.7 12.7 12 11.9 13.6 14 13 15.3 14 S29D 9 10.4 14 13.2 11.9 9.5 11.5 11 10.9 10 11.3 10.7 8 11 14.3 10 13 12.2 M21A 12 17.4 18.3 19.3 14.4 14.7 11 11.5 17.3 15.5 14 16.7 15 15.5 15.2 13.2 16.7 16.7 H31K 10.5 10.9 14 13 14 11.3 10.1 8.4 10 9.3 11.9 10.3 8 12 12.5 10.4 12.7 14 AAK 12.5 16 13.2 15.7 11.3 11.4 9 12 12.5 15 13.3 13.8 12.3 13.7 16.3 12 13.7 15 AAA 15 19.9 21 21 20 14.6 15.9 17.5 16 19 15.8 17 16 19 19.3 14 20 19 Antibiotics 2020, 9, 305 5 of 18 Antibiotics 2020, 9, x FOR PEER REVIEW 4 of 20

(A) (B)

Figure 1. Representative pictures of the agar based deferred antagonism assays that were performed with a wildtype producing L. lactis strain and 29 L. lactis strains that produce bioengineered nisin A derivatives. TheseThese strainsstrains werewere spotted spotted onto onto GM17 GM17 agar agar and and grown grown at 30at◦ 30C overnight°C overnight after after which which they werethey were overlaid overlaid with with tryptone tryptone soy agarsoy agar (TSA) (TSA) inoculated inoculated with with (A) Staphylococcus(A) Staphylococcus epidermidis epidermidis28and 28 and (B) Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 20 Staphylococcus(B) Staphylococcus epidermidis epidermidis53. Overlaid53. Overlaid plates plates were were incubated incubated for 18 for h at18 37h at◦C. 37 Zones °C. Zones of inhibition of inhibition from thefrom (1) the nisin (1) Anisin producing A producing strain strain (2) M21V (2) M21V producing producing strain andstrain (3) and M17Q (3) producingM17Q producing strain are strain circled. are circled. 2.2.2.2. Agar Agar Well Well Diffusion Diffusion Assays Assays,, Minimum Minimum Inhibitory Inhibitory Concentration Concentration Assays Assays and and Kill Kill Curve Curve NisinNisin A, A, M21V M21V and and M17Q M17Q peptides peptides were were purified purified from from the the relevant relevant L.L. lactis lactis producingproducing strains strains byby HPLC, HPLC, (see (see Figure Figuress S2 S2 and and S3). S3). The The ability ability of ofthe the three three peptides peptides to inhibit to inhibit two two representative representative S. epidermidisS. epidermidis strainsstrains (28 (28 and and 53; 53; chosen chosen as asthey they were were strong strong biofilm biofilm formers; formers; see see later) later) was was examined examined usingusing three three different different assays—agarassays—agar wellwell di diffusionffusion assay, assay, MIC MIC assay assay and and a broth-baseda broth-based kill kill curve. curve. As canAs canbe seenbe seen in Figure in Figure2, while 2, while all three all peptidesthree peptides produced produced distinct distinct zones of zones inhibition of inhibition in the well in ditheff usionwell diffusionassays, the assays, zones producedthe zones byproduced the derivative by the producing derivative strains producing appeared strains to beappeared enhanced to when be enhanced compare whento the compare wildtype. to the Using wildtype. a standard Using broth-based a standard MICbroth test,-based it was MIC observed test, it was that observed in the case that of in boththe caseS. epidermidis of both S.28 epidermidis and 53, M17Q 28 and and 53, M21V M17Q had and a M21V two-fold had reduction a two-fold in reduction MIC values in whenMIC values compared when to comparedthe wildtype to the nisin wildtype A peptide nisin (see A Table peptide2). The (see broth-based Table 2). The assay broth revealed-based thatassay the revealed concentration that the of concentrationnisin A employed of nisin resulted A employed in ~2 log resulted reduction in in~2 the log numbers reduction of in both theS. numbers epidermidis of bothstrains S. afterepidermidis the 1 h strainsincubation after time.the 1hr The incubation same concentration time. The same of M21V concentration caused ~2.5 of M21V log reduction caused ~2.5 in S. log epidermidis reduction28 in and S. epidermi~3 log reductiondis 28 and in ~3S. epidermidislog reduction53 numbers. in S. epidermidis A ~3 log 53 reduction numbers. of A both ~3 logS. epidermidis reduction strains of both was S. epidermidisobserved for strains M17Q, was the observed results offor which M17Q, are the presented results of in which Figure are3. presented in Figure 3.

Figure 2. Images are representative of the average zones of clearing obtained when 30 µL of a 30 µM Figure 2. Images are representative of the average zones of clearing obtained when 30 µL of a 30 µM stock of each peptide was added to well in Tryptone soya agar. Plates were incubated at 37 ◦C overnight. stock of each peptide was added to well in Tryptone soya agar. Plates were incubated at 37 °C overnight.

Table 2. Minimum inhibitory concentration (MIC) values for wild type peptide nisin A and its derivatives M21V and M17Q against S. epidermidis. All values in the Table are µM.

Nisin A M21V M17Q S. epidermidis 28 3.75 1.875 1.875 S. epidermidis 53 7.5 3.75 3.75

9 *** *** *** 8 *** *** *** 7 *** *** 6 *** *** 5 4

3 LogCFU/mL 2 1 0 Control Nisin A M21V M17Q

Antibiotics 2020, 9, x FOR PEER REVIEW 7 of 20

2.2. Agar Well Diffusion Assays, Minimum Inhibitory Concentration Assays and Kill Curve Nisin A, M21V and M17Q peptides were purified from the relevant L. lactis producing strains by HPLC, (see Figures S2 and S3). The ability of the three peptides to inhibit two representative S. epidermidis strains (28 and 53; chosen as they were strong biofilm formers; see later) was examined using three different assays—agar well diffusion assay, MIC assay and a broth-based kill curve. As can be seen in Figure 2, while all three peptides produced distinct zones of inhibition in the well diffusion assays, the zones produced by the derivative producing strains appeared to be enhanced when compare to the wildtype. Using a standard broth-based MIC test, it was observed that in the case of both S. epidermidis 28 and 53, M17Q and M21V had a two-fold reduction in MIC values when compared to the wildtype nisin A peptide (see Table 2). The broth-based assay revealed that the concentration of nisin A employed resulted in ~2 log reduction in the numbers of both S. epidermidis strains after the 1hr incubation time. The same concentration of M21V caused ~2.5 log reduction in S. epidermidis 28 and ~3 log reduction in S. epidermidis 53 numbers. A ~3 log reduction of both S. epidermidis strains was observed for M17Q, the results of which are presented in Figure 3.

Figure 2. Images are representative of the average zones of clearing obtained when 30 µL of a 30 µM Antibioticsstock2020 of, 9 each, 305 peptide was added to well in Tryptone soya agar. Plates were incubated at 37 °C6 of 18 overnight.

TableTable 2. 2. MinimumMinimum inhibitory inhibitory concentration concentration (MIC) (MIC) values values for for wild wild type type peptide peptide nisin nisin A A andand itsits derivativesderivatives M21VM21V andand M17Q M17Q against againstS. S. epidermidis epidermidis.. All All values values in in the the Table Table are areµ µM.M.

NisinNisin A A M21V M17Q M17Q S. epidermidisS. epidermidis28 28 3.753.75 1.875 1.875 1.875 S. epidermidisS. epidermidis53 53 7.57.5 3.75 3.75 3.75

9 *** *** *** 8 *** *** *** 7 *** *** 6 *** *** 5 4

3 LogCFU/mL 2 1

AntibioticsAntibiotics 2020, 9,0 x FOR2020 ,PEER 9, x FOR REVIEW PEER REVIEW 8 of 17 8 of 17 Control Nisin A M21V M17Q Figure 3.Figure 3. Figure 3. Counts of S. epidermidis 28 ( ) and S. epidermidis 53 ( ) after 1 h incubation in tryptone soya broth supplemented with 3.75µM nisin A, M21V or M17Q peptide. (*** = p < 0.001). 2.3. Inhibition2.3. Inhibition of Biofilm of Formation Biofilm Formation on Plastic on Plastic 2.3. Inhibition of Biofilm Formation on Plastic The abilityThe of ability the 18 of S. the epidermidis 18 S. epidermidis strains tostrains adhere to toadhere and formto and biofilm form biofilmon plastic on microtiterplastic microtiter Theplates ability wasp oflates theanalyzed was 18 S. analyzed epidermidisusing an using establishedstrains an established to adheremethod to method[28]. and All form [28]. 18 biofilmstrains All 18 were onstrains plastic found were microtiter to found be capable to be capableof of plates wasforming analyzedforming biofilms using biofilms to anvarying established to varyingdegrees method degreeson this [surface 28on]. this All (datasurface 18 strains not (data shown). were not foundshown). Strains to S.Strains be epidermidis capable S. epidermidis of 28 and S. 28 and S. formingepidermidis biofilmsepidermidis to 53 varying can be 53 considered degrees can be considered on strong this surface biofilm strong (data formersbiofilm not shown). formersalso based a Strainslso on based thisS. protocol. epidermidison this protocol. A 28comparison and A comparison of of S. epidermidisthe absorbances53the can absorbances be considered obtained obtained from strong the biofilmfrom negative the formers negative controls also controls and based the on andnisin this the A protocol. nisinwildtype A Awildtype comparisonpeptide, peptide, revealed revealed a a of the absorbancessignificantsignificant reduction obtained reductionfrom in biofilm the in negative formationbiofilm controls formation at all andthree at the all concentrations nisinthree Aconcentrations wildtype of nisin peptide, ofthat nisin were revealed that tested were a (1.875 tested, (1.875, significant3.75 reduction and3.75 7.5 µM)and in biofilm 7(Figure.5 µM) formation 4).(Figure When 4). atthe When all antibiofilm three the concentrations antibiofilm effects of effects M21V of nisin of and M21V that M17Q were and were M17Q tested compared (1.875,were compared to the to the 3.75 andwildtype 7.5 µM)wildtype (Figurea significantly4 a). significantly When enhanced the antibiofilm enhanced reduction ereductionff inects absorbance of M21V in absorbance andwas M17Q observed was were observed against compared bothagainst toS. the epidermidisboth S. epidermidis wildtype28 a and significantly 5328 at and both 53 enhanced 1.875 at both and 1.875 reduction 3.75 and µM. 3.75 inAgainst absorbance µM. Againststrain was53 strain at observed 7.5 53µMol, at 7.5 against the µMol, antibiofilm both the S.antibiofilm epidermidis forming forming capabilities28 capabilities and 53 atof both the derivatives 1.875of the and derivatives 3.75 wereµM. significantly Againstwere significantly strain enhanced 53 at enhanced 7.5 comparedµMol, compared the to antibiofilmnisin A,to nisin(p value forming A, (≤p 0.05). value capabilities However, ≤ 0.05). ofHowever, against against the derivativesS. epidermidis wereS. epidermidis significantly28 at 7.5 28 µM, at enhanced 7.5the µM,absorbances the compared absorbances of the to nisinwildtype of the A, wildtype ( pandvalue the andderivative0.05). the However,derivative peptides against peptides were similar. were similar. ≤ S. epidermidisOverall,28Overall, atM17Q 7.5 µ M,was M17Q the seen absorbances was to seennegatively to of negatively the affect wildtype biofilm affect and biofilmformation the derivative formation similar peptides tosimilar M21V were to that M21V similar. is, enhancedthat is, enhanced Overall,compared M17Qcompared was to seennisin toA.to negatively nisin A. affect biofilm formation similar to M21V that is, enhanced compared to nisin A. Figure 4.Figure 4.

2.4. Biofilm2.4. Inhibition Biofilm Inhibition on Stainless on Stainless Steel by M17QSteel by M17Q

Figure 5.Figure 5.

3. Discussion3. Discussion S. epidermidisS. epidermidis is an opportunistic is an opportunistic pathogen pathogen with an withability an to ability form tobiofilms form biofilms on medical on medicaldevices devices includingincluding cerebrospinal cerebrospinal shunts, catheters,shunts, catheters, artificial artificial hips and hips pacemakers and pacemakers [4,5]. Its [4,5].ubiquitous Its ubiquitous presence presence on skin meanson skin transfer means transferand contamination and contamination of indwelling of indwelling devices devicesis common is common [29]. The [29]. appearance The appearance of of MRSE furtherMRSE enhancesfurther enhances the threat the posed threat, as posed conventional, as conventional antibiotic antibiotic treatments treatments are losing are efficacy losing efficacy[3]. [3]. This increaseThis increase in antibiotic in antibiotic resistance resistance coupled coupled with the with expected the expected rise in medical rise in medicaldevice interventions device interventions due to andue increasing to an increasing elderly populationelderly population mean that mean alternative that alternative strategies strategies to control to S.control epidermidis S. epidermidis are are required.required. A major A focus major of focus research of research carried carriedout in our out laboratory in our laboratory is on the is identificationon the identification and and characterizationcharacterization of a group of a of group natural of natural microbially microbially produced produced antimicrobial peptides called called bacteriocins.bacteriocins. While traditionally While traditionally researched researched for their for ap plicationstheir applications as food preservativesas food it has become it has become increasinglyincreasingly evident evidentin recent in years recent that years bacteriocins that bacteriocins show promise show promise as therapeutic as therapeutic agents against agents against medicallymedically important important pathogens pathogens including including antibiotic antibiotic resistant resistant bacteria bacteria[30]. Of [30].particular Of particular interest interestis is the fact thatthe factbacteriocins that bacteriocins are gene are encoded gene encoded as this permits as this permitsthe generation the generation of bacteriocin of bacteriocin variants variants which which may possessmay possessmore desirable more desirable properties. properties. We have We extensively have extensively bioengineered bioengineered the bacteriocin the bacteriocin nisin A nisin A and haveand assembled have assembled a large banka large of banknisin ofA derivativenisin A derivative producing producing L. lactis strains.L. lactis Astrains. sub-bank A sub of- bank29 of 29 rationallyrationally selected selectedderivative derivative-producing-producing strains were strains the were focus the of focusthe current of the study.current study. Agar basedAgar deferred based deferred antagonism antagonism assays revealedassays revealed that the thatwildtype the wildtype nisin A nisinproducing A producing strain strain inhibitedinhibited all of the all 18 of S. the epidermidis 18 S. epidermidis strains that stra inswere that tested. were Six tested. of the Six 29 of nisin the 29derivative nisin derivative producers producers (VGA, VGT,(VGA, SGK, VGT, M21A, SGK, M17Q,M21A, AAA)M17Q, produced AAA) produced significantly significantly larger zones larger of zones inhibition of inhibition that the that the wildtypewildtype against allagainst 18 S. allepidermidis 18 S. epidermidis strains. Thestrains. remaining The remaining nisin derivative nisin derivative producers producers gave zones gave o fzones of various varioussizes; with sizes; enhanced with enhanced activity activityagainst aagainst large portiona large portionbut not butthe entirenot the set entire of S. set epidermidis of S. epidermidis strains used.strains The used. largest The zoneslargest were zones observed were observed for AAA for and AAA M17Q and producers. M17Q producers. We have We previously have previously reportedreported that the thatAAA the derivative AAA derivative exhibits exhibitsenhanced enhanced diffusion diffusion properties properties [24]. Indeed, [24]. Indeed,we have we also have also describeddescribed a similar a findingsimilar findingfor the hingefor the derivatives hinge derivatives NAK and NAK SVA and [25]. SVA Consequently, [25]. Consequently, AAA was AAA was not considerednot considered further andfurther subsequent and subsequent experiments experiments focused focusedon confirming on confirming the enhanced the enhanced bioactivity bioactivity of M17Q.of Three M17Q. se Threeparate se assaysparate were assays employed were employed and a well and characterized a well characterized nisin derivative nisin derivative M12V that M12V that has enhancedhas enhanced specific specificactivity activitywas included was included in these inexperiments these experiments for comparative for comparative purposes purposes [11,18]. [11,18]. A well A diffusion well diffusion assay revealed assay revealed that the that zones the obtained zones obtained with purified with purified M17Q pe M17Qptide pe wereptide were Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 20 Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 19 Figure 3. Counts of S. epidermidis 28 ( ) and S. epidermidis 53 ( ) after 1 h incubation in tryptone soya Figure 3. Counts of S. epidermidis 28 ( ) and S. epidermidis 53 ( ) after 1 h incubation in tryptone soya broth supplemented with 3.75µM nisin A, M21V or M17Q peptide. (*** = p < 0.001). broth supplemented with 3.75µM nisin A, M21V or M17Q peptide. (*** = p < 0.001). 2.3. Inhibition of Biofilm Formation on Plastic 2.3. Inhibition of Biofilm Formation on Plastic The ability of the 18 S. epidermidis strains to adhere to and form biofilm on plastic microtiter The ability of the 18 S. epidermidis strains to adhere to and form biofilm on plastic microtiter plates was analyzed using an established method [28]. All 18 strains were found to be capable of plates was analyzed using an established method [28]. All 18 strains were found to be capable of forming biofilms to varying degrees on this surface (data not shown). Strains S. epidermidis 28 and S. forming biofilms to varying degrees on this surface (data not shown). Strains S. epidermidis 28 and S. epidermidis 53 can be considered strong biofilm formers also based on this protocol. A comparison of epidermidis 53 can be considered strong biofilm formers also based on this protocol. A comparison of the absorbances obtained from the negative controls and the nisin A wildtype peptide, revealed a the absorbances obtained from the negative controls and the nisin A wildtype peptide, revealed a significant reduction in biofilm formation at all three concentrations of nisin that were tested (1.875, significant reduction in biofilm formation at all three concentrations of nisin that were tested (1.875, 3.75 and 7.5 µM) (Figure 4). When the antibiofilm effects of M21V and M17Q were compared to the 3.75 and 7.5 µM) (Figure 4). When the antibiofilm effects of M21V and M17Q were compared to the wildtype a significantly enhanced reduction in absorbance was observed against both S. epidermidis wildtype a significantly enhanced reduction in absorbance was observed against both S. epidermidis 28 and 53 at both 1.875 and 3.75 µM. Against strain 53 at 7.5 µMol, the antibiofilm forming capabilities 28 and 53 at both 1.875 and 3.75 µM. Against strain 53 at 7.5 µMol, the antibiofilm forming capabilities of the derivatives were significantly enhanced compared to nisin A, (p value ≤ 0.05). However, against of the derivatives were significantly enhanced compared to nisin A, (p value ≤ 0.05). However, against S. epidermidis 28 at 7.5 µM, the absorbances of the wildtype and the derivative peptides were similar. S. epidermidis 28 at 7.5 µM, the absorbances of the wildtype and the derivative peptides were similar. Overall, M17Q was seen to negatively affect biofilm formation similar to M21V that is, enhanced Antibiotics 2020, 9, 305 Overall, M17Q was seen to negatively affect biofilm 7formation of 18 similar to M21V that is, enhanced compared to nisin A. compared to nisin A.

1.4 *** 1.4 *** *** *** *** *** *** *** *** *** 1.2 *** *** *** *** *** *** *** *** 1.2 1 1 *** *** 0.8 *** *** 0.8 0.6 ** ** 0.6 ** ** - - 0.4 - -

Absorbance Absorbance 595nm @ 0.4

0.2 Absorbance 595nm @ 0.2 Antibiotics 2020, 09Antibiotics,Antibiotics x FORAntibioticsAntibiotics PEER 20 2020 20 20REVIEW,20 9,20 ,209 x,, ,xFOR9 9 ,FOR ,x x FOR FORPEER PEER PEER PEER REVIEW REVIEW REVIEW REVIEW 8 of 17 8 8of of 8178 17of of 17 17 Control Nisin A M21V0 M17Q Control Nisin A M21V M17Q Figure(A) 3. Figure Figure Figure Figure 3. 3. 3. 3. (A)

2.3. Inhibition 2.3.of2.3. Biofilm2.3. 2.3.Inhibition Inhibition InhibitionInhibition Formation of of Biofilm ofBiofilmof Biofilm Biofilmon PlasticFormation Formation FormationFormation on on Plastic ononPlastic PlasticPlastic

1.4 - *** -

The ability of the*** 18 S.*** epidermidis*** ***1.4 strains*** *** to*** adhere to and form biofilm on plastic microtiter

The ability ofThe Thethe The ability 18ability abilityS. epidermidisof of the ofthe the18 18 S. 18strainsS. epidermidis S.epidermidis epidermidis to adhere strains strains strains to toand to adhere to adhereform adhere biofilmto to*** and toand ***and form onform formplastic*** biofilm biofilm*** biofilm microtiter*** on on plastic onplastic*** plastic *** microtiter microtiter ***microtiter **

plates was analyzed1.2pplateslatespplateslates was wasusing waswas analyzed analyzed ananalyzedanalyzed established using using usingusing an an method establishedan anestablished establishedestablished [28]. method Allmethod method method** 18 strains [28]. [28]. [28].[28]. All Allwere AllAll18 18 strainsfound18 18strains strainsstrains towere were be werewere capablefound found foundfound to ofto be totobe capablebe becapable capablecapable of of ofof

** 1.2 ** forming biofilmsformingformingforming formingto varying biofilms biofilms biofilmsbiofilms degrees to to varying tovaryingto varyingonvarying this degrees degrees surface degreesdegrees on on (data this on onthis this this surfacenot surface surface surfaceshown). (data (data (data(data Strains not not notnotshown). shown). S. shown).shown). epidermidis Strains Strains StrainsStrains S.28 S. epidermidis andS.epidermidisS. epidermidisepidermidis S. 28 28 and 28 28and andand S. S. S.S. 1

epidermidis 53epidermidis canepidermidisepidermidisepidermidis be considered 53 53 can 5353can cancanbe bestrong considered be beconsidered consideredconsidered biofilm strong strongformers strongstrong biofilm 1biofilm a biofilmbiofilmlso basedformers formers formersformers on a thislsoalso aa lsobased lsoprotocol. based basedbased on on this onA onthis comparison this thisprotocol. protocol. protocol.protocol. A Aof comparison A comparisonA comparisoncomparison of of ofof

0.8 the absorbances obtained from the** negative controls and the nisin A wildtype peptide, revealed a the absorbancesthethe obtainedtheabsorbances absorbances absorbances from obtained obtained the obtained negative from from from thecontrols the thenegative negative negative and controlsthe controls controlsnisin*** and Aand wildtypeand the the thenisin nisin nisin peptide, A A wildtype wildtypeA wildtype revealed peptide, peptide, peptide, a revealed revealed revealed a a a 0.8 **

significant reductionsignificantsignificantsignificantsignificant in biofilmreduction reduction reductionreduction formation in in biofilm inbiofilmin biofilmbiofilm at allformation formation three formationformation concentrations at at all atallat three all allthree threethree concentrations concentrations of concentrationsconcentrations nisin that wereof of nisin ofnisinof tested nisinnisin that that that(1.875that were were werewere, tested tested testedtested*** (1.875 (1.875 (1.875(1.875, , ,,

0.6 ***

3.75 and 7.5 µM)3.753.753.75 3.75 (Figureand and andand 7 .57.5 4). 7µM)7 .5µM).5 When µM)µM) (Figure (Figure (Figure (Figurethe 4).antibiofilm 4). When 4). 4).When WhenWhen the the effects theantibiofilmthe0.6** antibiofilm antibiofilmantibiofilm of M21V effects effects and effectseffects ofM17Q of M21V ofM21Vof M21VM21V were and and andcomparedand M17Q M17Q M17QM17Q were were to werewere comparedthe compared comparedcompared to to the totheto thethe

*** ** wildtype a significantly0.4wildtypewildtypewildtypewildtype a aenhancedsignificantly significantly aa significantlysignificantly reduction enhanced enhanced enhancedenhanced in absorbancereduction reduction reductionreduction in inwas absorbance inabsorbancein absorbanceobservedabsorbance was wasagainst waswas observed observed observedobserved both S.against against epidermidis againstagainst both both bothboth S. S. epidermidis S.epidermidisS. epidermidisepidermidis 28 and 53 at both2828 and28 281.875and and and 53 53 andat 53 53at both at bothat3.75 both both 1.875 µM.1.875 1.875 1.875 andAgainst and and and 3.75 3.75 3.75 3.75 strainµM. µM. µM. µM. Against Against53 0.4Against Againstat 7.5 strain strainµMol, strain strain 53 53 the at 53 53at 7.5antibiofilm at 7.5at 7.5µMol, 7.5 µMol, µMol, µMol, the the forming the antibiofilmthe antibiofilm antibiofilm antibiofilm capabilities forming forming forming forming capabilities capabilities capabilities capabilities 0.2 of the derivativesofof the oftheof were the derivativesthe derivatives derivatives derivativessignificantly were were were were enhancedsignificantly significantly significantly significantly compared enhanced enhanced 0.2enhanced enhanced to nisincompared compared compared compared A, (p valueto to nisin tonisinto ≤nisin nisin 0.05). A, A, ( A, pA,( pvalueHowever, ( value(pp value value ≤ ≤0.05). 0.05). ≤ ≤ against0.05). 0.05). However, However, However, However, against against against against S. epidermidis S.028S. epidermidis atS.epidermidisS. 7.5 epidermidisepidermidis µM, 28the 28 at 2828absorbancesat 7.5 at7.5at 7.5 µM,7.5 µM, µM,µM, the the theofabsorbancesthe absorbances the absorbancesabsorbances wildtype of of theand oftheof thewildtypethe thewildtype wildtypewildtype derivative and and andand the thepeptides thederivativethe derivative derivative derivative were peptides peptidessimilar. peptidespeptides were were werewere similar. similar. similar. similar. Overall, M17QOverall,Overall, wasOverall,Overall,Control seen M17Q M17Q M17QM17Qto negativelywas wasNisin waswas seen Aseen seenseen to affectto negatively totonegatively negatively negativelyM21Vbiofilm affect0 formationaffect affectaffect biofilm biofilmM17Q biofilmbiofilm similar formation formation formationformation to M21V similar similar similar similarthat to tois M21V, to toM21Venhanced M21VM21V that that thatthat is is, , enhancedis isenhanced,, enhancedenhanced (B) Control Nisin A M21V M17Q compared to comparednisincomparedcomparedcompared A. to to nisin tonisinto nisinnisin A. A. A. A. (B) Figure 4. Absorbances of biofilm formed by (A) S. epidermidis 28 and (B) S. epidermidis 53 on plastic Antibiotics 2020, 9, x FigureFOR PEER 4. AbsorbancesREVIEW of biofilm formed by (A) S. epidermidis 28 and (B) S. epidermidis 853 of on17 plastic Antibiotics 2020, 9, x FOR PEER REVIEWmicrotiter platesFigure in4. the presence Figure FigureFigureFigure 4 1.875. 4 . 4 4 . . µ M, ( Figure , ), 4 . 3.75 µ M, ( , ) and 8 of 7.5 17 µ M, ( , ) nisin microtiter plates in the presence 1.875 µM, ( , ), 3.75 µM, ( , ) and 7.5 µM, ( , ) nisin A and derivative peptides, M21V and M17Q. Control shows the amount of biofilm produced in the Figure 3. Figure 3. A and derivative peptides, M21V and M17Q. Control shows the amount of biofilm produced in the absence2.4. of Biofilm nisin, ( Inhibition2.4., 2.4.2.4. ).2.4.Biofilm Biofilm *** BiofilmonBiofilm denotes Stainless Inhibition Inhibition InhibitionInhibitionp <2.4. 0.001,Steel on onBiofilm Stainless onbyon **Stainless denotesStainlessM17QStainless Inhibition Steel Steelp < SteelSteel by0.005, byon M17Q byby M17QStainless * M17QM17Q denotes Steelp < 0.05 by M17Q and “-”denotes absence of nisin, ( , ). *** denotes p < 0.001, ** denotes p < 0.005, * denotes p < 0.05 and “-”denotes no statistical significance. 2.3. Inhibition2.3. of BiofilmInhibition Formation of Biofilmno statistical on Formation PlasticFigure significance. 5. on Plastic Figure FigureFigureFigure 5. 5. 5. 5. The remaining experiments focused on M17Q. Assessing the formation of biofilm on stainless 2.4. Biofilm Inhibition on Stainless Steel by M17Qsteel, (a material that can be used in artificial hips), was performed by calculating CFU/mL recovered The ability ofThe the ability 18 S. ofepidermidis the 18 S. strainsepidermidis to adhere strains to to and adhere form to biofilm and form on plasticbiofilm microtiter on plastic microtiter plates was analyzedplates was using analyzed Thean established remaining using an experimentsmethodestablished [28]. focusedmethod All 18on [28].strains M17Q. All were 18 Assessing strains found thewereto formationbe foundcapable to of ofbe biofilm capable on stainlessof steel, forming biofilmsforming to varying biofilms(a material degrees to varying3. that Discussion on can this degrees be surface used3. 3. Discussion on3.Discussion3. inDiscussion Discussion(datathis artificial surface not shown). hips), (data was notStrains performed shown). S. epidermidis Strains by calculating S. 28 epidermidis and S. CFU /28mL and recovered S. from epidermidis 53epidermidis can be considered scrapings53 can be strong takenconsidered obiofilmff the strong surface formers biofilm of a stainlesslso formers based steel on also this coupons based protocol. on following this A comparisonprotocol. 24 h incubation A comparisonof with S.of epidermidis. S. epidermidis S.isS. epidermidisan S.epidermidisS. epidermidisopportunisticepidermidis is is an isanis opportunistic an anpathogenopportunistic opportunisticopportunistic with pathogen pathogen an pathogenpathogen ability with with to withwith forman an ability an anability biofilms abilityability to to form totoformon formform medical biofilms biofilms biofilmsbiofilms devices on on medicalon onmedical medicalmedical devices devices devicesdevices the absorbancesthe absorbances obtainedThe from negative obtained the controlsnegative from the usedcontrols negative in this and experiment,controls the nisin and AS. thewildtype epidermidis nisin Apeptide, strainswildtype revealed in brothpeptide, withouta revealed nisin, a confirmed including cerebrospinalincludingincludingincludingincluding cerebrospinal shunts,cerebrospinal cerebrospinal cerebrospinal catheters, shunts, shunts, shunts, shunts, artificial catheters, catheters, catheters, catheters, hips artificialand artificial artificial artificial pacemakers hips hips hips hips and and and [4,5].and pacemakers pacemakers pacemakers pacemakers Its ubiquitous [4,5]. [4,5]. [4,5]. [4,5]. Its presenceIts ubiquitousIts Itsubiquitous ubiquitous ubiquitous presence presence presence presence significant reductionsignificant in thatreduction biofilmS. epidermidis formation in biofilm28 at andformation all 53three could concentrations at formall three substantial concentrations of nisin biofilms that ofwere and nisin adheretested that were(1.875 to the tested, surface (1.875 of the, stainless on skin meansonon transfer skinon onskin skinskin means means and meansmeans contaminationtransfer transfer transfertransfer and and andand contamination contamination of contaminationcontamination indwelling of devicesof indwelling ofindwellingof indwellingindwelling is common devices devices devicesdevices [29]. is is common isThecommonis commoncommon appearance [29]. [29]. [29].[29]. The The Theof Theappearance appearance appearanceappearance of of ofof 3.75 and 7.5 3.75µM) and (Figure 7.5steel µM) 4). couponsWhen (Figure the (Figure4). antibiofilm When5). the By effectsantibiofilm comparing of M21V effects these and controlsof M17Q M21V towere and the M17Qcompared strains were incubated to compared the with to nisin the A, it was MRSE furtherMRSE enhancesMRSEMRSEMRSE further further further furtherthe enhancesthreat enhances enhances enhances posed the the , the threattheas threat conventionalthreat threat posed posed posed posed, as, as , conventional, antibioticas conventionalas conventional conventional treatments antibiotic antibiotic antibiotic antibiotic are treatments treatmentslosing treatments treatments efficacy are are are losingare losing[3]. losing losing efficacy efficacy efficacy efficacy [3]. [3]. [3].[3]. wildtype a significantlywildtype aconfirmed significantly enhanced that reduction enhanced the wildtype in reduction absorbance peptide in couldwasabsorbance observed interfere was withagainst observed biofilm both against formationS. epidermidis both on S. this epidermidis surface type. Nisin This increase ThisinThis antibioticThisThis increase increase increaseincrease resistancein in antibiotic ininantibiotic antibioticantibiotic coupled resistance resistance resistanceresistance with coupled thecoupled coupledcoupled expected with with withwith therise the the expectedthe inexpected expectedmedicalexpected rise rise device rise risein in medical ininmedical interventions medicalmedical device device devicedevice interventions interventions interventionsinterventions 28 and 53 at both28 and 1.875 53 at andA both caused 3.75 1.875 µM. ~2-log and Against 3.75 reduction µM. strain Against in 53 strain at 7.5 strain 28 µMol, and 53 aatthe 1.5 7.5 antibiofilm log µMol, reduction the forming antibiofilm in strain capabilities 53forming counts, capabilities in both instances the due to an increasingduedueduedue to to an toelderlyanto increasing an anincreasing increasingincreasing population elderly elderly elderlyelderly mean population population populationpopulation that alternative mean mean meanmean that that strategies thatthat alternative alternative alternativealternative to controlstrategies strategies strategiesstrategies S. epto toidermidis control totocontrol controlcontrol S. areS. ep S. epS.idermidis idermidisepepidermidisidermidis are are areare of the derivativesof the were derivatives reductionsignificantly were was significantly enhanced found to becompared enhanced statistically to compared nisin significant A, (p to value (nisinp < 0.001).≤ A, 0.05). (p value M17QHowever, ≤ also0.05). against impacted However, S. epidermidisagainst biofilm required. A required. majorrequired.required.required. focus A A major of A A major majorresearch major focus focus focus focus carried of of researchof of research research research out in carried carried our carried carried laboratory out out out out in in our in in our is our our laboratory onlaboratory laboratory laboratory the identification is is on is ison on theon the the the identification identificationand identification identification and and and and S. epidermidisS. 28 epidermidis at 7.5 µM,formation 28 the at absorbances7.5in µM, a significant the absorbances of the way,wildtype causing of the and wildtype anthe approximate derivative and the peptides derivative 3-log lower were peptides recoverysimilar. were of bothsimilar.S. epidermidis characterizationcharacterizationcharacterization characterizationcharacterization of a group of of of aof naturala group a agroup group group microbially of of of naturalof natural natural natural producedmicrobially microbially microbially microbially antimicrobial produced produced produced produced antimicrobial antimicrobial peptides antimicrobial antimicrobial called peptides peptides peptides peptides called called called called Overall, M17QOverall, was seenM17Qstrains to was negatively when seen compared to affectnegatively tobiofilm the control.affect formation biofilm This logsimilar formation difference to M21V similar in numbers that to isM21V, comparedenhanced that is to, enhanced nisin A was deemed bacteriocins. Whilebacteriocins.bacteriocins.bacteriocins.bacteriocins. traditionally While While While While researchedtraditionally traditionally traditionally traditionally for researched researchedtheir researched researched applications for for for theirfor their their their asap ap foodplications ap plicationsapplicationsplications preservatives as as food as foodas food food preservatives itpreservatives has preservatives preservatives become it it has hasit it has hasbecome become become become compared tocompared nisin A. statisticallyto nisin A. significant (p < 0.01). increasingly evidentincreasinglyincreasinglyincreasinglyincreasingly in recent evident evident evidentevident years in in recentthat inin recent recent recent bacteriocins years years years years that that that that show bacteriocins bacteriocins bacteriocins bacteriocins promise show show as show show therapeutic promise promise promisepromise as as agents therapeutic as as therapeutic therapeutic therapeutic against agents agents agents agents against against againstagainst Figure 4. Figure 4 . medically important medically medically medically medically pathogens important important importantimportant including pathogens pathogens pathogenspathogens antibiotic including including includingincluding resistant antibiotic antibiotic antibiotic antibiotic bacteria resistant resistant resistantresistant [30]. bacteria Ofbacteria bacteriabacteriaparticular [30]. [30]. [30].[30]. Of interestOf Ofparticular Ofparticular particularparticular is interest interest interestinterest is is isis the fact that bacteriocinsthethe thefactthe fact fact factthat that that arethat bacteriocins bacteriocins gene bacteriocins bacteriocins encoded are are are geneare asgene gene thisgene encoded encoded permits encoded encoded as asthe this as thisas generation this thispermits permits permits permits the theof the generation thebacteriocin generation generation generation of variants of bacteriocin of bacteriocinof bacteriocin bacteriocin which variants variants variants variants which which which which 2.4. Biofilm Inhibition2.4. Biofilm on InhibitionStainless maySteelon Stainless bypossess M17Q Steel maymore maymay mayby possess desirablepossessM17Q possesspossess more more properties. moremore desirable desirable desirabledesirable We properties. properties. have properties.properties. extensively We We We Wehave have have havebioengineered extensively extensively extensivelyextensively bioengineered bioengineeredthe bioengineeredbioengineered bacteriocin the the nisin thethebacteriocin bacteriocin bacteriocin bacteriocinA nisin nisin nisinnisin A A AA and have assembledandandandand have have haveahave largeassembled assembled assembledassembled bank ofa alarge nisinlarge aa largelarge bank Abank derivative bankbank of of nisin ofofnisin nisinnisin Aproducing A derivative AderivativeA derivativederivative L. producinglactis producing producingproducing strains. L. L. lactis A L.lactisL. sub lactislactis strains. -strains.bank strains.strains. of A A 29sub AsubA sub-subbank-bank--bankbank of of 29 ofof29 29 29 Figure 5. Figure 5. rationally selectedrationallyrationallyrationallyrationally derivative selected selected selectedselected-producing derivative derivative derivativederivative strains-producing-producing--producingproducing were thestrains strains strainsfocusstrains were were of werewere the the the current thefocusthe focus focusfocus of study.of the oftheof thecurrentthe current currentcurrent study. study. study.study. Agar based deferred antagonism assays revealed that the wildtype nisin A producing strain Agar based deferred AgarAgarAgar based based antagonism based deferred deferred deferred assaysantagonism antagonism antagonism revealed assays assays thatassays revealed therevealed revealed wildtype that that that the nisinthe thewildtype wildtype A wildtype producing nisin nisin nisin A strainA producing producingA producing strain strain strain inhibited all ofinhibitedinhibited theinhibitedinhibited 18 S. all allepidermidis of all allof the ofofthe the18the 18 S. 18 stra18S. epidermidis S.epidermidisS.ins epidermidisepidermidis that were stra stra stra insstratested.ins insthatins that that that Sixwere were wereofwere tested.the tested. tested. tested.29 nisinSix Six SixofSix ofderivative the ofofthe the29the 29 nisin 29 29nisin producersnisinnisin derivative derivative derivativederivative producers producers producersproducers 3. Discussion3. Discussion (VGA, VGT, (VGA,SGK,(VGA,(VGA,(VGA, M21A, VGT, VGT, VGT,VGT, SGK, M17Q,SGK, SGK,SGK, M21A, M21A,AAA) M21A,M21A, M17Q, M17Q,produced M17Q,M17Q, AAA) AAA) AAA) AAA)significantly produced produced producedproduced significantlylarger significantly significantlysignificantly zones largerof larger inhibitionlargerlarger zones zones zoneszones of thatof inhibition ofofinhibition the inhibitioninhibition that that thatthat the the thethe wildtype againstwildtypewildtypewildtype wildtypeall 18 againstS. against epidermidis againstagainst all all 18 all all18 S.strains.18 18S. epidermidis S.epidermidisS. epidermidisepidermidis The remaining strains. strains. strains.strains. The The nisin The Theremaining remaining derivative remainingremaining nisin nisin producers nisinnisin derivative derivative derivative derivative gave producers producers zones producersproducers o fgave gave gavegave zones zones zones zones o of f ooff S. epidermidisS. is epidermidis an opportunistic is variousan opportunistic pathogen sizes;various variouswith withvariousvarious pathogen enhanced ansizes; sizes; ability sizes;sizes; withwith with activity withtowith enhancedan formenhanced enhancedabilityenhanced against biofilms activityto activity forma activityactivity largeon againstbiofilmsmedical against portion againstagainst a deviceson alarge butlarge a amedical largelarge not portion portion theportionportion devices entire but but but butnot notset notnotthe ofthe theS.theentire entire epidermidis entireentire set set setofset of S. ofofS. epidermidis S.epidermidisS. epidermidisepidermidis including cerebrospinalincluding cerebrospinal shunts, catheters,strains shunts, used. artificial catheters, strainsThestrainsstrainsstrains largesthips used. artificialused. and used.used. zones Thepacemakers The Thehips Thelargest werelargest largestandlargest observed zones pacemakers[4,5].zones zoneszones Itswere were for ubiquitous werewere observedAAA [4,5].observed observedobserved Itsand presence ubiquitous for M17Qfor forAAAfor AAA AAA AAAproducers. and presenceand andand M17Q M17Q M17QM17Q We producers. producers. have producers.producers. previously We We We Wehave have havehave previously previously previouslypreviously on skin meanson transferskin means and transfer contaminationreported and contamination that of indwellingreported thereportedreportedreported AAA thatof derivativethat devices indwelling thatthat the the theAAAthe AAA is AAA exhibitsAAAcommon devicesderivative derivative derivativederivative enhanced [29].is common exhibits exhibitsThe exhibitsexhibits diffusionappearance enhanced[29]. enhanced enhancedenhanced The properties appearance ofdiffusion diffusion diffusiondiffusion [24]. properties propertiesof Indeed, properties properties [24].we [24]. [24].have[24]. Indeed, Indeed, Indeed, Indeed,also we we wehavewe have havehave also also alsoalso MRSE furtherMRSE enhances further the enhances threatdescribed posed the threat, as a conventional similardescribed poseddescribeddescribeddescribed ,finding as conventionala aantibioticsimilar similar a a for similarsimilar the finding findingtreatments hinge finding antibioticfinding for derivativesfor forthefor arethe treatments thethehinge losinghinge hingehinge NAK derivatives efficacyderivatives are derivativesderivatives and losing SVA[3]. NAK NAKefficacy [25]. NAKNAK and andConsequently, and[3].and SVA SVA SVASVA [25]. [25]. [25].[25]. Consequently, Consequently, AAA Consequently,Consequently, was AAA AAA AAAAAA was was waswas This increaseThis in antibioticincrease inresistance antibioticnot consideredcoupled resistance notwithnot further not couplednotconsidered consideredthe consideredconsidered expected and with subsequent further further the rise furtherfurther expected inand and medical experiments andand subsequent subsequent rise subsequentsubsequent device in medical focusedexperiments experimentsinterventions experimentsexperiments device on confirming focused interventionsfocused focusedfocused on onthe confirming on onconfirming enhanced confirmingconfirming the bioactivitythe theenhancedthe enhanced enhancedenhanced bioactivity bioactivity bioactivitybioactivity due to an increasingdue to an elderly increasing populationof elderly M17Q. meanpopulation Threeof ofthat M17Q.se ofM17Q.of paratealternative M17Q.M17Q. mean Three Three assays ThreethatThree se strategiessealternativeparate were parateseseparateparate employed assays assays to assaysassaysstrategies control were were and werewere employedS. employed toa ep employedwell employedcontrolidermidis characterized and andS. are andand epa aidermidiswell well aa wellwell characterized nisincharacterized characterized characterizedare derivative nisin nisin M12V nisinnisin derivative derivative derivative derivativethat M12V M12V M12VM12V that that thatthat required. Arequired. major focus A major of research focushas enhanced of carried researchhas hasspecific outhas hasenhanced enhanced carried in enhancedenhanced ouractivity out laboratoryspecific specific was specificspecific in our includedactivity activity isactivity laboratoryactivity on was inwas the wasthesewas included identification included is includedincluded experiments on thein in these in identificationinthese andthesethese forexperiments experiments experimentscomparativeexperiments and for for forcomparativepurposesfor comparative comparativecomparative [11,18]. purposes purposes purposespurposes [11,18]. [11,18]. [11,18].[11,18]. characterizationcharacterization of a group of ofA a wellnatural group diffusion of microbiallyA A natural wellAA well assay well well diffusion diffusion microbially produceddiffusion diffusion revealed assay assay assay thatassay antimicrobial produced revealed revealed the revealed revealed zones antimicrobial that that peptides that obtainedthat the the the the zones zones called zones zones with peptides obtained obtained purified obtained obtained called with with M17Qwith with purified purified purified purifiedpeptide M17Q M17Q M17Qwere M17Q pe pe ptide peptide peptideptide were were were were bacteriocins.bacteriocins. While traditionally While traditionally researched for researched their applications for their ap asplications food preservatives as food preservatives it has become it has become increasinglyincreasingly evident in recentevident years in recent that bacteriocins years that bacteriocins show promise show as therapeuticpromise as therapeutic agents against agents against medically importantmedically pathogens important including pathogens antibiotic including resistant antibiotic bacteria resistant [30]. bacteria Of particular [30]. Of interest particular is interest is the fact that bacteriocinsthe fact that bacteriocinsare gene encoded are gene as this encoded permits as thisthe generationpermits the of generation bacteriocin of variants bacteriocin which variants which may possessmay more possess desirable more properties. desirable Weproperties. have extensively We have bioengineeredextensively bioengineered the bacteriocin the nisinbacteriocin A nisin A and have assembledand have a assembled large bank a of large nisin bank A derivative of nisin A producing derivative L. producing lactis strains. L. lactis A sub strains.-bank Aof sub29 -bank of 29 rationally selectedrationally derivative selected-producing derivative strains-producing were strains the focus were of the focuscurrent of study.the current study. Agar based Agardeferred based antagonism deferred antagonismassays revealed assays that revealed the wildtype that the nisin wildtype A producing nisin A strainproducing strain inhibited allinhibited of the 18 allS. epidermidisof the 18 S. stra epidermidisins that werestrains tested. that were Six of tested. the 29 Six nisin of thederivative 29 nisin producers derivative producers (VGA, VGT,(VGA, SGK, VGT,M21A, SGK, M17Q, M21A, AAA) M17Q, produced AAA) significantly produced significantly larger zones larger of inhibition zones of that inhibition the that the wildtype againstwildtype all 18 against S. epidermidis all 18 S. strains. epidermidis The strains.remaining The nisin remaining derivative nisin producers derivative gave producers zones ogavef zones of various sizes;various with enhancedsizes; with activity enhanced against activity a large against portion a large but portionnot the entirebut not set the of entire S. epidermidis set of S. epidermidis strains used.strains The largest used. zonesThe largest were zonesobserved were for observed AAA and for M17Q AAA producers.and M17Q Weproducers. have previously We have previously reported thatreported the AAA that derivative the AAA exhibits derivative enhanced exhibits diffusion enhanced properties diffusion [24]. properties Indeed, [24]. we haveIndeed, also we have also described a describedsimilar finding a similar for thefinding hinge for derivatives the hinge NAKderivatives and SVA NAK [25]. and Consequently, SVA [25]. Consequently, AAA was AAA was not considerednot furtherconsidered and furthersubsequent and experimentssubsequent experiments focused on confirmingfocused on theconfirming enhanced the bioactivity enhanced bioactivity of M17Q. Threeof M17Q. separate Three assays separate were assaysemployed were and employed a well characterized and a well characterized nisin derivative nisin M12V derivative that M12V that has enhancedhas specific enhanced activity specific was activity included was in includedthese experiments in these experiments for comparative for comparative purposes [11,18]. purposes [11,18]. A well diffusionA well assay diffusion revealed assay that revealed the zones that obtained the zones with obtained purified with M17Q purified peptide M17Q were pe ptide were Antibiotics 2020, 9, x FOR PEER REVIEW 9 of 20

2.4. Biofilm Inhibition on Stainless Steel by M17Q The remaining experiments focused on M17Q. Assessing the formation of biofilm on stainless steel, (a material that can be used in artificial hips), was performed by calculating CFU/mL recovered from scrapings taken off the surface of stainless steel coupons following 24 h incubation with S. epidermidis. The negative controls used in this experiment, S. epidermidis strains in broth without nisin, confirmed that S. epidermidis 28 and 53 could form substantial biofilms and adhere to the surface of the stainless steel coupons (Figure 5). By comparing these controls to the strains incubated with nisin A, it was confirmed that the wildtype peptide could interfere with biofilm formation on this surface type. Nisin A caused ~2-log reduction in strain 28 and a 1.5 log reduction in strain 53 counts, in both instances the reduction was found to be statistically significant (p < 0.001). M17Q also impacted S. epidermidis biofilm formation in a significant way, causing an approximate 3-log lower recovery of both S. epidermidis strains when compared to the control. This log difference in numbers compared to nisin A was deemed statistically significant (p < 0.01).

2.5. Biofilm Assay on Various Catheter Materials As with the stainless steel coupons, the degree of biofilm formation on catheters was assessed by determining bacterial counts recovered from the surface of each material type. The catheter tubes made of rubber, polyvinyl chloride and polyvinyl resin, were cut into even 2 cm pieces that were hollow on the inside. The negative controls, (catheter pieces incubated with S. epidermidis culture in the absence of nisin), confirmed that strains 28 and 53 could form biofilm and adhere to the three different surface types (Figure 5). When evaluating the impact of nisin A on S. epidermidis biofilm formation on catheters, it was observed that the wildtype peptide caused a single log reduction for both strain 28 and 53 on all material types when compared to the control. These decreases in CFU/mL were calculated to be statistically significant (p < 0.001). M17Q also caused a single log reduction in Antibioticsboth strains,2020, 9, 305 on all material types tested. This log reduction was also found to be statistically8 of 18 significant compared to the control (p < 0.001).

7.5

*** *** *** *** *** *** 7 *** ***

6.5 - - * 6

5.5 *** Antibiotics 2020, 9, x FOR PEER REVIEW5Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 17 8 of 17

Log Log CFU/mL 4.5 Figure 3. Figure 3. 4

Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 17 Antibiotics 2020, 9, x FOR PEER2.3. InhibitionREVIEW of Biofilm Formation3.52.3. Inhibitionon Plastic of Biofilm Formation on Plastic 8 of 17 3 Figure 3. The ability of the 18 S. epidermidisTheC abilityNisA strains M17Q ofFigure Ctothe NisA adhere 18 M17Q3. S. epidermidis toC NisA and M17Q form C strains NisA biofilm M17Q to on adhere plastic to andmicrotiter form biofilm on plastic microtiter Antibiotics 2020, 9, x FOR PEER REVIEW Stainless Polyvinyl Polyvinyl 10 of 20 plates was analyzed using anp latesestablished was analyzed method using [28]. Allan established18 strains Rubberwere method found [28]. to Allbe 18capable strains of were found to be capable of (A) Steel Chloride Resin 2.3. Inhibition of Biofilmforming Formation biofilms on Plastic to varying degreesforming on biofilms this2.3. surface Inhibition to varying (data of Biofilmdegreesnot shown). Formation on this Strains surface on S.Plastic epidermidis (data not shown). 28 and S. Strains S. epidermidis 28 and S. epidermidis 53 can be considered7.5epidermidis strong biofilm ***53 can*** beformers considered also based strong on biofilm this protocol. formers A a comparisonlso based on of this protocol. A comparison of The ability of the 18 S. epidermidis strains to adhere to and formThe*** biofilmability*** ofon ***the plastic*** 18 S. microtiter epidermidis strains to adhere to and form biofilm on plastic microtiter 7 *** *** plates was analyzed theusing absorbances an established obtained method fromthe [28]. the absorbances Allnegative 18p strainslates controlsobtained was were analyzed and fromfound the the nisintousing negativebe A capablean wildtype established controls of peptide, and method the revealed nisin [28]. A Alla wildtype 18 strains peptide, were foundrevealed to abe capable of 6.5 forming biofilms to varyingsignificant degrees reduction on this in surface biofilmsignificant (data formation not reductionshown).forming at all three Strains biofilmsin biofilm concentrations- S. epidermidisto formation varying- of degrees28 atnisin and all* threethatS. on thiswere concentrations surface tested (data(1.875 of not, nisin shown). that were Strains tested S. epidermidis (1.875, 28 and S. 6 3.75 and 7.5 µM) (Figure 4). When3.75 and the 7 antibiofilm.5 µM)epidermidis (Figure effects 53 4). can Whenof beM21V considered the and antibiofilm M17Q strong were effects biofilm compared of formersM21V to and thealso M17Q based were on this compared protocol. to A the comparison of epidermidis 53 can be considered strong biofilm formers5.5 also based on this protocol. A comparison of the absorbances obtainedwildtype from a thesignificantly negative controlsenhancedwildtype and reduction the a significantly thenisin*** inabsorbances Aabsorbance wildtype enhanced obtained waspeptide, reductionobserved fromrevealed theagainstin absorbance negativea both controlsS. was epidermidis observed and the against nisin A both wildtype S. epidermidis peptide, revealed a 28 and 53 at both 1.875 and 3.75528 µM. and Against53 at both strain 1.875 53 and at 7.5 3.75 µMol, µM. Againstthe antibiofilm strain 53 forming at 7.5 µMol, capabilities the antibiofilm forming capabilities significant reduction in biofilm formation at all 4.5three concentrationssignificant of nisin reduction that were in biofilmtested (1.875 formation, at all three concentrations of nisin that were tested (1.875, of the derivatives were significantlyof the derivatives enhanced compared were significantly to nisin A, enhanced (p value compared≤ 0.05). However, to nisin againstA, (p value ≤ 0.05). However, against 3.75 and 7.5 µM) (Figure 4). When the antibiofilm Log CFU/mL 4 effects of M21V3.75 andand M17Q7.5 µM) were (Figure compared 4). When to the the antibiofilm effects of M21V and M17Q were compared to the S. epidermidis 28 at 7.5 µM, theS. absorbances epidermidis 28 of atthe 7.5 wildtype µM, the andabsorbances the derivative of the peptides wildtype were and thesimilar. derivative peptides were similar. wildtype a significantly enhanced reduction in 3.5absorbance waswildtype observed a significantlyagainst both enhancedS. epidermidis reduction in absorbance was observed against both S. epidermidis Overall, M17Q was seen to negativelyOverall, M17Q affect was biofilm seen formationto negatively similar affect to biofilmM21V thatformation is, enhanced similar to M21V that is, enhanced 28 and 53 at both 1.875 and 3.75 µM. Against strain3 53 at 7.5 µMol,28 theand antibiofilm 53 at both 1.875 forming and capabilities 3.75 µM. Against strain 53 at 7.5 µMol, the antibiofilm forming capabilities M17Q C NisA M17Q C NisA M17Q of the derivatives werecompared significantly to nisin enhanced A. comparedcompared toC nisinNisA to ofM17Qnisin A, the (p derivativesA.C value NisA ≤ 0.05). were However, significantly against enhanced compared to nisin A, (p value ≤ 0.05). However, against Stainless Polyvinyl Polyvinyl S. epidermidis 28 at 7.5 µM, the absorbances of the(B) wildtype andS. the epidermidis derivative 28 peptides at 7.5 µM, were theRubber similar.absorbances of the wildtype and the derivative peptides were similar. Figure 4. Antibiotics 20 20 FigureAntibiotics, 9 , x FORSteel 4 . PEER20 20 , 9 REVIEW,Chloride x FOR PEER ResinREVIEW 8 of 17 8 of 17 Overall, M17Q was seen to negatively affect biofilm formationOverall, similar M17Q to M21V was thatseen is to, enhancednegatively affect biofilm formation similar to M21V that is, enhanced Figure 5. Colony forming units recovered from (A) S. epidermidis 28 and (B) S. epidermidis 53 on compared to nisin A. Figure 3. Figure compared 3. to nisin A. 2.4. Biofilmstainless-steelFigure Inhibition 5. Colony and on Stainless catheter forming2.4. materials Steel unitsBiofilm byrecovered M17QInhibition in the absence from on Stainless (A of) nisinS. epidermidis Steel (C, by, M17Q28), and the presence(B) S. epidermidis of nisin A53 at on stainless-steel and catheter materials in the absence of nisin (C, , ), the presence of nisin A at Figure 4. Figure3.75 µ 5.M, ( , ) and derivative Figure peptide 5. M17Q Figure at 3.75 4 . µ M, ( , ). *** denotes p < 0.001, ** denotes 3.75 µM, ( , 2.3.) and Inhibition derivative2.3. of BiofilmInhibitionpeptide FormationM17Q of Biofilm at 3.75 on Formation µM, Plastic ( , on). Plastic *** denotes p < 0.001, ** denotes p < 0.005, * denotes p < 0.05 and—shows no statistical significance. p < 0.005, * denotes p < 0.05 and – shows no statistical significance. 2.4. Biofilm Inhibition on Stainless Steel by M17Q The ability ofThe 2.4.the ability Biofilm18 S. ofepidermidis Inhibitionthe 18 S. onstrainsepidermidis Stainless to adhere Steelstrains by to M17Qto and adhere form to biofilm and form on plasticbiofilm microtiter on plastic microtiter 2.5. Biofilm Assay on Variousplates was Catheter analyzedplates Materials was using analyzed an established using an establishedmethod [28]. method All 18 [28].strains All were 18 strains found wereto be found capable to ofbe capable of Figure 5. 2.6. Testing the Efficacy of Nisin A and M17Q Figurein a Simulated 5. Wound Fluid 3. DiscussionAs with the stainlessforming steel3. biofilmsDiscussion coupons,forming to varying biofilms the degree degrees to varying of biofilm on thisdegrees formation surface on this(data on surface catheters not shown). (data was notStrains assessed shown). S. epidermidis Strains S. 28 epidermidis and S. 28 and S. S. epidermidis 28epidermidis and 53 were53epidermidis can incubated be considered 53 can in be a simulated strongconsidered biofilm wound strong formers fluidbiofilm a withlso formers based or without on also this based nisin protocol. onto this A comparisonprotocol. A comparisonof of by determiningS. epidermidis bacterial is an opportunistic counts S. recovered epidermidis pathogen from is with thean opportunistic surfacean ability of to each form pathogen material biofilms with type. on an Themedical ability catheter devicesto form tubes biofilms on medical devices determine if antimicrobialthe absorbances activitythe absorbances wasobtained retained from obtained in thethis negative setting. from the Thecontrols negative negative and controls controls,the nisin and S.A epidermidisthewildtype nisin Apeptide, wildtype revealed peptide, a revealed a includingmade of cerebrospinal rubber, polyvinyl shunts,including chloride catheters, and cerebrospinal artificial polyvinyl hips shunts, resin, and pacemakers were catheters, cut into artificial [4,5]. even Its hips 2ubiquitous cm and pieces pacemakers presence that were [4,5]. Its ubiquitous presence strains cultured in significant the absence reductionsignificant of nisin, in reduction verified biofilm formation that in biofilm the viability at formation all three of strainsconcentrations at all three was notconcentrations of affected nisin that by of were nisin tested that were(1.875 tested, (1.875, 3. Discussion onhollow skin means on the transfer inside. Theand negativecontaminationon skin controls, means3. of Discussiontransfer (catheterindwelling and pieces contaminationdevices incubated is common with of indwellingS. [29]. epidermidis The appearancedevicesculture is incommon of the [29]. The appearance of absenceexposure of nisin),to the wound confirmed3.75 fluidand that 7 (Figure.5 3.75strainsµM) and 6).(Figure 28 The7 and.5 µM)addition 4). 53 When could(Figure of formthe nisin 4). antibiofilm biofilmWhen A at a the concen and effectsantibiofilm adheretration of toM21V of theeffects 3.75 three and µMof diM17QM21V resultedfferent were and M17Qcompared were to compared the to the MRSE further enhances the threatMRSE posed further, as enhances conventionalS. epidermidis the threat antibiotic is an posed opportunistic treatments, as conventional are pathogen losing antibiotic efficacy with an treatments [3]. ability to areform losing biofilms efficacy on medical[3]. devices S. epidermidis is ansurface opportunisticin ~1 log types reduction (Figure pathogen wildtype5in). counts When with a evaluatingofan significantlywildtype S. ability epidermidis to thea formsignificantly impactenhanced 28 biofilmsand of a nisinreduction reduction enhanced on A medical on inofS. reduction absorbance epidermidis~1.5 devices logs infor biofilmabsorbancewas S. epidermidis observed formation was against53 observed onin a both against S. epidermidis both S. epidermidis This increase in antibiotic resistanceThis increase coupledincluding in antibioticwith the cerebrospinal expected resistance rise shunts,coupled in medical catheters, with devicethe artificialexpected interventions hips rise and in medical pacemakers device [4,5]. interventions Its ubiquitous presence including cerebrospinalcatheters, shunts,simulated catheters, it waswound observed artificial fluid.28 and thatThe 53hips theatsame 28andboth wildtype and concentration pacemakers 1.875 53 at peptideand both 3.75 [4,5].1.875 of caused µM. M17Q Its and Against ubiquitous a 3.75 singlecaused µM. strain log ~2 Againstpresence reduction log 53 atreduction 7.5 strain µMol, for both53 in atthe counts strain7.5 antibiofilm µMol, 28of andboth the formingantibiofilm capabilities forming capabilities due to an increasing elderly populationdue to an increasing meanon skin that means elderly alternative transfer population strategies and contaminationmean to controlthat alternative S. of ep indwellingidermidis strategies are devices to control is common S. epidermidis [29]. The are appearance of on skin means transfer53 andstrains on contamination all materialin a simulated types ofof indwellingthe whenwound derivatives compared fluid.of devices the Whilewerederivatives to is the significantly commonboth control. nisinwere [29]. These significantlyA enhanced and The decreases M17Qappearance compared enhanced caused in CFU of toa / mLcompared statisticallynisin were A, ( calculatedp to value significantnisin ≤ A, 0.05). to (p value However, ≤ 0.05). against However, against required. A major focus of required. research carriedA MRSE major out further focus in our ofenhances research laboratory the carried threat is on posed out the in, identification as our conventional laboratory and antibiotic is on the treatments identification are losing and efficacy [3]. MRSE further enhancesbe thereduction statistically threat posedin bacterial significant, asS. conventional epidermidis numbers (p < 0.001).S. (28 pantibioticepidermidis

7.5 *** *** *** *** *** *** 7 *** *** 6.5 - - * 6 5.5 *** 5 4.5

LogCFU/mL 4 3.5 3 C NisA M17Q C NisA M17Q C NisA M17Q C NisA M17Q

Stainless Polyvinyl Polyviny (B) Rubber Steel Chloride l Resin Figure 5. Colony forming units recovered from (A) S. epidermidis 28 and (B) S. epidermidis 53 on stainless-steel and catheter materials in the absence of nisin (C, , ), the presence of nisin A at 3.75 µM, ( , ) and derivative peptide M17Q at 3.75 µM, ( , ). *** denotes p < 0.001, ** denotes p < 0.005, * denotes p < 0.05 and – shows no statistical significance.

2.6. Testing the Efficacy of Nisin A and M17Q in a Simulated Wound Fluid S. epidermidis 28 and 53 were incubated in a simulated wound fluid with or without nisin to determine if antimicrobial activity was retained in this setting. The negative controls, S. epidermidis

Antibioticsstrains cultured2020, 9, 305 in the absence of nisin, verified that the viability of strains was not affected9 of by18 exposure to the wound fluid (Figure 6). The addition of nisin A at a concentration of 3.75 µM resulted in ~1 log reduction in counts of S. epidermidis 28 and a reduction of ~1.5 logs for S. epidermidis 53 in a woundsimulated fluid. wound The samefluid. concentrationThe same concentration of M17Q caused of M17Q ~2 logcaused reduction ~2 log inreduction counts ofin bothcounts strains of both in astrains simulated in a woundsimulated fluid. wound While fluid. both While nisin both A and nisin M17Q A and caused M17Q a statistically caused a statistically significant significant reduction inreduction bacterial in numbers bacterial (p numbers< 0.001), ( thep < results0.001), obtainedthe results for obtained M17Q were for M17Q significantly were si lowergnificantly than thoselower obtainedthan those for obtained nisin A (forp < nisin0.001 A for (p strain< 0.001 28, forp

*** *** 8 7.5 *** *** 7 *** 6.5 *** 6 5.5 5

4.5 Log CFU/mL 4 3.5

3 Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 17 Control Nisin A M17Q Antibiotics 2020, 9, x FOR PEER REVIEW 8 of 17 Figure 3. Figure 6. Kill curve performed in a simulated wound fluid where S. epidermidis 28 ( ) and S. epidermidis Figure 3. 53 ( ) were incubated for 1hr at 37 ◦C with nisin A and M17Q at a concentration of 3.75 µM. *** denotes Figure 6. Kill curve performed in a simulated2.3. Inhibition wound fluid of Biofilm where FormationS. epidermidis on Plastic28 ( ) and S. p < 0.001, ** denotes p < 0.005, * denotes p < 0.05 and “-” denotes no statistical significance. 2.3. Inhibition of Biofilm Formation on Plastic epidermidis 53 ( ) were incubated for 1hr at 37 °C with nisin A and M17Q at a concentration of 3.75 The ability of the 18 S. epidermidis strains to adhere to and form biofilm on plastic microtiter The ability of the 18 S. epidermidis3. strains DiscussionµM. to *** adhere denotes to p and< 0.001, form ** biofilm denotes onp < plastic 0.005,plates *microtiter was denotes analyzed p < 0.05 using and “an-” denotesestablished no statistical method [28]. All 18 strains were found to be capable of significance. plates was analyzed using an established methodS. epidermidis [28]. Allis an18 opportunisticstrains were found pathogen toforming be with capable anbiofilms ability of to to varying form biofilms degrees on on medical this surface devices (data not shown). Strains S. epidermidis 28 and S. forming biofilms to varying degrees on this surface (data not shown). Strains S. epidermidisepidermidis 28 and 53 S.can be considered strong biofilm formers also based on this protocol. A comparison of including3. Discussion cerebrospinal shunts, catheters, artificial hips and pacemakers [4,5]. Its ubiquitous presence epidermidis 53 can be considered strong onbiofilm skin meansformers transfer also based and on contamination this protocol. of Athe indwelling comparison absorbances devices of obtained is common from [ 29the]. negative The appearance controls of and the nisin A wildtype peptide, revealed a the absorbances obtained from the negativeMRSES. controls further epidermidis enhancesand isthe an nisin theopportunistic threat A wildtype posed, pathogen aspeptide, conventionalsignificant with revealed an reduction ability antibiotic a to inform treatments biofilm biofilms formation are on losing medical at e allfficacy devicesthree [3 concentrations]. of nisin that were tested (1.875, significant reduction in biofilm formationThisincluding at increase all three cerebrospinal in concentrations antibiotic shunts, resistance of catheters,nisin coupled that artificialwere with3.75 tested the and hips expected 7(1.875 .5and µM) pacemakers, rise (Figure in medical 4). [4,5]. When device Its theubiquitous interventions antibiofilm presence effects due of M21V and M17Q were compared to the 3.75 and 7.5 µM) (Figure 4). When the antibiofilmto an increasing effects elderly of M21V population and M17Q mean were that alternativecomparedwildtype ato strategies significantly the to control enhancedS. epidermidis reductionare in required. absorbance was observed against both S. epidermidis wildtype a significantly enhanced reductionA major in focusabsorbance of research was observed carried out against in our both laboratory28 S.and epidermidis 53 is at on both the 1.875 identification and 3.75 and µM. characterization Against strain 53 of at a 7.5 µMol, the antibiofilm forming capabilities 28 and 53 at both 1.875 and 3.75 µM. Againstgroup strain of natural 53 at 7.5 microbially µMol, the produced antibiofilm antimicrobial formingof the capabilities derivatives peptides called were bacteriocins.significantly enhanced While traditionally compared to nisin A, (p value ≤ 0.05). However, against of the derivatives were significantly enhancedresearched compared for their to applicationsnisin A, (p value as food ≤ 0.05). preservatives However,S. epidermidis it against has become 28 at 7.5 increasingly µM, the absorbances evident in recentof the yearswildtype and the derivative peptides were similar. S. epidermidis 28 at 7.5 µM, the absorbancesthat of bacteriocins the wildtype show and promise the derivative as therapeutic peptides agentsOverall, were against similar.M17Q medically was seen important to negatively pathogens affect including biofilm formation similar to M21V that is, enhanced Overall, M17Q was seen to negativelyantibiotic affect biofilm resistant formation bacteria similar [30]. Ofto particularM21V thatcompared interest is, enhanced is to the nisin fact A. that bacteriocins are gene encoded compared to nisin A. as this permits the generation of bacteriocin variants which may possess more desirable properties. We have extensively bioengineered the bacteriocin nisinFigure A 4 and. have assembled a large bank of nisin A Figure 4. derivative producing L. lactis strains. A sub-bank of 29 rationally selected derivative-producing strains 2.4. Biofilm Inhibition on Stainless Steel by M17Q were the focus of the current study. 2.4. Biofilm Inhibition on Stainless Steel by M17Q Agar based deferred antagonism assays revealedFigure that5. the wildtype nisin A producing strain Figure 5. inhibited all of the 18 S. epidermidis strains that were tested. Six of the 29 nisin derivative producers (VGA, VGT, SGK, M21A, M17Q, AAA) produced significantly larger zones of inhibition that the wildtype against all 18 S. epidermidis strains. The remaining nisin derivative producers gave zones of various sizes; with enhanced activity against a large3. Discussion portion but not the entire set of S. epidermidis strains 3. Discussion used. The largest zones were observed for AAA andS. epidermidis M17Q producers. is an opportunistic We have previously pathogen reported with an ability to form biofilms on medical devices S. epidermidis is an opportunistic pathogenthat the AAA with derivativean ability exhibitsto form enhancedbiofilms on di ffmedicalincludingusion properties devices cerebrospinal [24]. Indeed, shunts, we catheters, have also artificial described hips a and pacemakers [4,5]. Its ubiquitous presence including cerebrospinal shunts, catheters,similar artificial finding hips for and the pacemakers hinge derivatives [4,5]. Its NAK ubiquitouson and skin SVA presence means [25]. Consequently,transfer and contamination AAA was not of considered indwelling devices is common [29]. The appearance of on skin means transfer and contaminationfurther of indwelling and subsequent devices experiments is common focused [29]. The onMRSE appearance confirming further of theenhances enhanced the threat bioactivity posed of, as M17Q. conventional Three antibiotic treatments are losing efficacy [3]. MRSE further enhances the threat posedseparate, as conventional assays were antibiotic employed treatments and a wellare losing characterizedThis efficacy increase [3]. nisin in antibiotic derivative resistance M12V thatcoupled has enhancedwith the expected rise in medical device interventions This increase in antibiotic resistance coupledspecific with activity the expected was included rise in thesemedical experiments devicedue interventions to for an comparative increasing elderly purposes population [11,18]. A mean well dithatffusion alternative strategies to control S. epidermidis are due to an increasing elderly populationassay mean revealed that alternative that the strategies zones obtained to control with S.required. purifiedepidermidis M17QA are major peptide focus were of research significantly carried increased out in our laboratory is on the identification and required. A major focus of research carried out in our laboratory is on the identificationcharacterization and of a group of natural microbially produced antimicrobial peptides called characterization of a group of natural microbially produced antimicrobial peptidesbacteriocins. called While traditionally researched for their applications as food preservatives it has become bacteriocins. While traditionally researched for their applications as food preservativesincreasingly it has become evident in recent years that bacteriocins show promise as therapeutic agents against increasingly evident in recent years that bacteriocins show promise as therapeuticmedically agents against important pathogens including antibiotic resistant bacteria [30]. Of particular interest is medically important pathogens including antibiotic resistant bacteria [30]. Of particularthe fact interest that bacteriocins is are gene encoded as this permits the generation of bacteriocin variants which the fact that bacteriocins are gene encoded as this permits the generation of bacteriocinmay variants possess which more desirable properties. We have extensively bioengineered the bacteriocin nisin A may possess more desirable properties. We have extensively bioengineered the bacteriocinand have nisin assembled A a large bank of nisin A derivative producing L. lactis strains. A sub-bank of 29 and have assembled a large bank of nisin A derivative producing L. lactis strains. Arationally sub-bank selectedof 29 derivative-producing strains were the focus of the current study. rationally selected derivative-producing strains were the focus of the current study. Agar based deferred antagonism assays revealed that the wildtype nisin A producing strain Agar based deferred antagonism assays revealed that the wildtype nisin A producinginhibited allstrain of the 18 S. epidermidis strains that were tested. Six of the 29 nisin derivative producers inhibited all of the 18 S. epidermidis strains that were tested. Six of the 29 nisin derivative(VGA, producers VGT, SGK, M21A, M17Q, AAA) produced significantly larger zones of inhibition that the (VGA, VGT, SGK, M21A, M17Q, AAA) produced significantly larger zones of inhibitionwildtype that against the all 18 S. epidermidis strains. The remaining nisin derivative producers gave zones of wildtype against all 18 S. epidermidis strains. The remaining nisin derivative producersvarious gave zones sizes; owithf enhanced activity against a large portion but not the entire set of S. epidermidis various sizes; with enhanced activity against a large portion but not the entire set ofstrains S. epidermidis used. The largest zones were observed for AAA and M17Q producers. We have previously strains used. The largest zones were observed for AAA and M17Q producers. We havereported previously that the AAA derivative exhibits enhanced diffusion properties [24]. Indeed, we have also reported that the AAA derivative exhibits enhanced diffusion properties [24]. Indeed,described we have a alsosimilar finding for the hinge derivatives NAK and SVA [25]. Consequently, AAA was described a similar finding for the hinge derivatives NAK and SVA [25]. Consequently,not considered AAA was further and subsequent experiments focused on confirming the enhanced bioactivity not considered further and subsequent experiments focused on confirming the enhancedof M17Q. bioactivity Three separate assays were employed and a well characterized nisin derivative M12V that of M17Q. Three separate assays were employed and a well characterized nisin derivativehas enhanced M12V that specific activity was included in these experiments for comparative purposes [11,18]. has enhanced specific activity was included in these experiments for comparative purposesA well [11,18]. diffusion assay revealed that the zones obtained with purified M17Q peptide were A well diffusion assay revealed that the zones obtained with purified M17Q peptide were Antibiotics 2020, 9, 305 10 of 18 compared to those obtained for nisin A. MIC assays demonstrated that the MIC value of nisin A obtained for S. epidermidis was comparable to the MIC obtained for L. monocytogenes, the pathogen that is a primary target of nisin in the food industry [18]. MIC assays also revealed that there was a two-fold reduction in the MIC of M17Q. A broth-based survival assay showed that there was a significant reduction in bacterial counts following exposure of bacteria to M17Q in broth when compared to nisin A (i.e., there was up to 1.5 logs greater reduction in bacterial numbers in the presence of M17Q compared to wildtype nisin A). Altogether, the results confirm the enhanced activity of M17Q against S. epidermidis. Interestingly, it was a derivative at methionine 17 that provided the first evidence that nisin Z could be enhanced against particular targets when nisin Z M17Q/G18T demonstrated increased activity against a limited number of non-pathogenic strains (M. flavus, S. thermophilus)[14]. The basis of enhanced activity is currently unknown and we do not wish to speculate. However the amino acid substitution of glutamine for methionine at position 17 is located within C ring and it has been shown that this ring can adopt a number of conformations suggesting that this part of the peptide is involved in very specific interactions or may confer unique membrane-interacting properties upon the molecule. As the C ring is vital for the antimicrobial activity of nisin, any failure in its formation would result in an inactive peptide. Mass spectrometry analysis of the peptide confirmed the substitution of methionine, (131 amu), for glutamine, (128 amu) in a decrease of 3amu in the total mass of the peptide. The mass obtained for the derivative suggest that there were no changes to dehydration or cyclization compared to the wildtype peptide but this remains to be confirmed experimentally. Biofilms pose a growing threat as due to the nature of the exopolysaccharide and the slow growing state of cells, conventional antibiotics have difficulty crossing the slime layer and eradicating the organisms it protects [31,32]. The slow penetration of the biofilm by antibiotics can expose the cells within to sublethal concentrations and permitting them to develop resistance [32,33]. It has also been noted that once formed, antibiotics are incapable of removing a biofilm [34]. For this reason, prevention of biofilm formation is preferred rather than trying to treat the infection [34]. But with growing levels of resistance reducing the available options for treatment each year, the need for viable alternatives becomes more pertinent. The ability of nisin A to affect biofilm formation by several pathogens such as L. monocytogenes, S. aureus and S. pseudintermedius has been investigated and it has been shown that nisin A can impair biofilm formation as well as exhibit bactericidal activity as a result of its ability to access even the deepest part of a biofilm matrix [22,35]. However, it cannot remove previously established biofilms [21,36] unless it is used in combination with other antimicrobials [37]. To our knowledge, the only study in the literature that has examined the effect of nisin A exposure on biofilm formation by S. epidermidis is the study of Davison et al. where the authors only investigated its potential to remove established biofilms. While nisin A was shown to penetrate the biofilm, it did not cause any loss in biomass [22]. Taking these previous studies into account it was decided to focus our experiments on biofilm prevention rather than removal. Initial assays demonstrated that the S. epidermidis strains formed strong biofilms on plastic surfaces. When the experiments were repeated in the presence of nisin A, biofilm formation was significantly impaired. Biofilm production was further impaired in the presence of either M21V or M17Q. In previous biofilm studies in our laboratory with M21V against S. aureus the results obtained were similar to those obtained for the wildtype nisin A [36]. Therefore, in addition to reporting the enhanced anti-biofilm formation properties of M17Q, the enhanced anti-biofilm action of M21V is also a novel finding. Similar biofilm prevention experiments were then subsequently carried out using medical device-related materials. S. epidermidis has frequently been implicated in infections on medical devices made from these materials, for example, artificial hips (stainless steel) and catheters, (PVC, PVR and rubber). When stainless steel was employed as a substrate, nisin A reduced the number of S. epidermidis cells that were recovered compared to the control. While nisin A has previously been shown to reduce biofilm formation by L. monocytogenes on stainless steel [38], to our knowledge the ability of nisin A to affect biofilm formation by S. epidermidis on stainless steel has not previously been reported. Antibiotics 2020, 9, 305 11 of 18

The presence of M17Q caused a further impairment in biofilm production compared to the wildtype nisin A. Bower et al. assessed the in vitro and in vivo capacity of nisin A to hinder or prevent the growth of S. aureus, S. epidermidis and S. faecalis on Teflon intravenous catheters and PVC tracheotomy tubes [12]. The in vitro examination of Teflon and PVC revealed that when the tubes were incubated with overnight cultures of the selected pathogens for one hour and then placed in nutrient both, the bacteria showed good growth [12]. However, the same strains were noted to grow to a much lesser extent when the tubing was treated with the peptide [12]. The in vivo test, (which entailed inserting the tubing and catheters into animals prior to routine surgical procedures), showed that the skin around the insertion site of nisin treated intravenous Teflon catheters did not become infected, despite being left in for a week [12]. The same was observed for the nisin treated PVC tracheotomy tubes [12]. In our study, three catheter materials were exposed to S. epidermidis strains for 24 h in the absence and presence of nisin A or M17Q peptides. Similar to the experiments performed on stainless steel, a significant difference was observed between nisin A and the control. However, it was noted that while nisin A caused up to a 2.5 log reduction in bacterial numbers on stainless steel, it only caused a one log reduction on the catheter materials. Results obtained for M17Q were similar to those obtained for nisin A showing that M17Q did not possess enhanced anti-biofilm ability on the catheter materials under the conditions tested. Implanted medical devices are not the only cause of S. epidermidis related ailments, as this resident of the skin microbiome can also enter the body or establish a site of infection through breaks in the skin like surgical incision sites, burns and other general abrasions [3,39]. It was for this reason that kill curves in a simulated wound fluid were performed to determine whether nisin A and its derivative could maintain their antimicrobial characteristics in a medium representative of the environment of a skin wound. A study conducted in 2016 by Van Staden et al. observed through bioluminescent imaging that the number of S. aureus cells in an induced soft tissue wound declined significantly when exposed to the nisin peptide [40]. Although conducted in vitro, our study did find that nisin A and M17Q could inhibit the growth of the selected S. epidermidis strains in this alternative media, causing significant decreases in viable cells. Enhanced activity from the derivative peptide was also maintained despite the change from broth to a simulated wound fluid, as it produced an approximate one log greater reduction in bacterial numbers than the wildtype nisin A peptide against S. epidermidis 28 and 53. To date, nisin resistance has not been observed or reported in S. epidermidis strains. Alongside this, nisin A has also been found to promote wound healing, without any observable cytotoxic effects in mammalian cells when tested in animals [12,23,41]. This suggests that nisin A and other nisin derived peptides could be viable treatment options in the fight against and prevention of, medical device associated infections in the future. Nisin A, like many other antimicrobial agents, is not very effective at removing pre-established biofilm due to the nature of the slime layer structure [20]. It is for this reason that prevention rather than removal is preferable. By coating the nisin A or M17Q to the surfaces of medical devices prior to implantation, the peptides could be used as a pre-emptive defense against invading S. epidermidis cells. By removing the opportunity for a bacterium to bind to a nisin-coated surface, an infection could be prevented. Peptides such as M17Q could potentially improve patient well-being and reduce treatment costs. By opting to use these peptides in place of conventional antibiotics, the issue of damaging the resident microbiota, developing resistant infections and extended treatment time due to resistance related complications could be also be circumvented. Infections brought about by skin-dwelling S. epidermidis entering post-operative wounds or other abrasions could also be potentially be limited through the use of nisin variant-based ointments that could be applied topically. Antibiotics 2020, 9, 305 12 of 18

4. Materials and Methods

4.1. Bacterial Strains The biofilm forming strains of S. epidermidis were obtained from the Cork Institute of Technology Culture Collection, having originally being isolated from the blood of patients at Cork University Hospital. All Staphylococcal strains were grown in tryptone soy broth (TSB) or tryptone soy agar, (TSA), (Sigma Aldrich, Steinheim, Germany) and grown at 37 ◦C for 16–18 h. The TSB and TSA were supplemented with 1% (Sigma Aldrich, Steinheim, Germany), when performing biofilm assays. Lactococcus lactis NZ9800, a non-nisin producing strain with pDF05 (a pCI372 vector containing the nisin A insert) was cultured to harvest the wildtype peptide [19]. This strain will be referred to as the nisin A producing strain. The nisin A and nisin derivative producing strains were all obtained from the University College Cork Culture Collection and selected based on their ability to inhibit a range of Gram positive pathogens in separate studies in our laboratory [11,18,19,24,27,42,43]. L. lactis strains were grown as described by Field et al. [19].

4.2. Agar Based Deferred Antagonism Assays In order to determine whether the nisin producing L. lactis strains had antimicrobial activity against the bank S. epidermidis strains, deferred antagonism assays were performed following the protocol described by Field et al. [42]. The L. lactis strains were cultured for 16 h at 30 ◦C in GM17 broth. 5 µL of each strain was spotted onto GM17 agar and incubated overnight at 30 ◦C. Following this incubation period, the spots were UV treated in a CL-1000 Ultraviolet Crosslinker for 30 min. 10 mL volumes of sloppy TSA (at 0.75% w/v agar), was prepared and inoculated individually with 1% of an overnight culture of each S. epidermidis. The spotted plates were overlaid with the inoculated agar and incubated overnight at 37 ◦C. Zones of inhibition in the overlaid agar, indicative of antimicrobial activity, were measured using Vernier calipers, (resolution 0.05), recorded in millimeters and rounded to one decimal place. Each nisin producer was spotted in triplicate and overlaid with 3 separate cultures of each of the 18 S. epidermidis strains that is, biological repeats of all strains were tested. The results presented in Table1 are the results obtained for one repeat but are representative of the results that were obtained for all three repeats.

4.3. Purification of Nisin A and The Derivatives M21V and M17Q Nisin A, M17Q and M21V peptides were purified from overnight cultures of the relevant L. lactis strain as per the protocol set by Field et al. [42]. The strains were grown in 20 mL of GM17 broth, supplemented with chloramphenicol at 10 µg/mL, at 30 ◦C for 16 h and 1800 mL of sterile Tryptone Yeast broth was prepared to which 100 mL of a sterile 20% glucose solution (Sigma Aldrich Steinheim, Germany) and 100 mL of a sterile 20% β-glycerophosphate solution (Sigma Aldrich, Steinheim, Germany) were added to bring the final volume of broth to 2 L. 20 mL of GM17 overnight culture was used to seed the broth that is, a 1% inoculum. The strains were incubated at 30 ◦C for 18 h. After 18 h, the sample was then transferred to 500 mL centrifuge bottles and centrifuged at 7000 g for 15 min × using a Sorvall RC 6+ model centrifuge. The supernatant and the cell pellet were separated, to extract nisin from both elements. The supernatant was filtered through a 30 cm long column, with an internal diameter of 2.5 cm, packed with 60 g of Amberlite XAD-16 beads (Sigma Aldrich, Steinheim, Germany) and prewashed with 1 L of sterile water. After passing the supernatant through the column followed by washing with 500 mL 30% ethanol, bound nisin was eluted using 400 mL 70% 2-propanol 0.1% trifluoroacetic acid. The bacterial cell pellet material was resuspended in 300 mL 70% 2-propanol 0.1% trifluoroacetic acid (TFA) and stirred at room temperature for 3 h. After the 3-h period, the cell pellet was centrifuged again at 7000 g for 15 min and the supernatant was retained. This supernatant was × then combined with the eluted nisin from the column and placed into a rotary evaporator, (Buchi, Flawil, Switzerland). The 2-propanol was removed, concentrating the nisin in volume of approximately 300mL. The pH of the sample was adjusted to 4 and then applied to a 10 g (60 mL), SPE C-18 Bond Antibiotics 2020, 9, 305 13 of 18

Elute Column (Phenomenex, Cheshire, UK), pre-equilibrated with 60 mL methanol and 60 mL water and 120 mL of 30% ethanol was washed through the column prior to nisin being eluted into 60 mL of 70% 2-propanol 0.1% TFA. This 60 mL was separated into 15 mL samples, which were subjected to further rotary evaporation to reduce each fraction to a volume of 2 mL and 1.5 mL aliquots were run through a Phenomenex (Phenomenex, Cheshire, UK), C12 reverse phase (RP)-HPLC column (Jupiter 4u proteo 90 Å, 250 10.0 mm, 4 µm), equilibrated with 25% 2- propanol and 0.1% TFA. The nisin × sample moved through the column at a flow rate of 1.2mL per minute. The antimicrobial fractions were pooled and then placed in the rotary evaporator to remove any remaining acetonitrile. The peptides were freeze dried, (using a FreezeZone 6 bench top freeze dryer) and stored at 20 C. − ◦ 4.4. Agar Well Diffusion Assays

10 mL of TSB was inoculated with a single S. epidermidis colony and cultured for 18 h at 37 ◦C. 20 mL volumes of molten TSA were inoculated with 200 µL of the overnight bacterial culture and poured into sterile Petri dishes (depth of agar was ~4 mm). A sterile glass pipette was used to create wells (diameter of 6 mm), in the agar. 30 µL of each purified peptide (Nisin A, M17Q and M21V, each at a concentration of 30 µM), was pipetted into individual wells. The plates were incubated upright for 18 h at 37 ◦C. Following the incubation period, any observed zones of inhibition were measured using calipers and recorded in millimeters. The Nisin A, M17Q and M21V peptides were tested against three biological repeats of the selected S. epidermidis strains.

4.5. Minimum Inhibitory Concentration (MIC) Assays The MICs of peptides were determined as previously described [27]. MICs are broth-based assays that allow for clear confirmation of improved antimicrobial activity, as derivative peptides can appear artificially more effective than the wildtype due to better diffusion in agar based assays [24,25]. The assay was performed using flat bottom, non-tissue culture treated 96 well plates (Sarstedt, Nümbrecht, Germany). 200 µL volumes of PBS (Sigma Aldrich, Steinheim, Germany), containing 1% (wt/vol) Bovine Serum Albumin (Sigma Aldrich, Steinheim, Germany) was added to each well and incubated for 30 min at 37 ◦C. Wells were emptied then rinsed again with sterile PBS solution (Sigma Aldrich, Steinheim, Germany) and allowed dry. The wells were then filled with 100 µL of sterile TSB. The freeze-dried peptides were resuspended in TSB at a concentration of 30 µM and 100 µL of resuspended peptide was placed into the first well of the plate, before a twofold dilution was performed along the row of 12 wells. Strains S. epidermidis 28 and S. epidermidis 53, were grown overnight in 10mL of TSB. 400 µL of each overnight was sub-cultured in 10mL of fresh TSB and grown to an optical density of 0.5 at 600 nm (UV-1800 Shimadzu spectrophotometer). 20 µL of the culture was taken and inoculated into 980 µL of fresh TSB to obtain approximately 1 105 cells per mL dilution × was added into each well containing nisin peptides. The solution was pipetted up and down four times to ensure homogeneity. Once inoculated, the plates were incubated at 37 ◦C for 16 h. Following 16 h, the plates were visually inspected and the first clear well for each peptide was taken to be the MIC. The Nisin A, M17Q and M21V peptides were tested against the selected S. epidermidis strains in triplicate.

4.6. Kill Curve Assay Ten mL of sterile TSB was inoculated with a single colony of the selected S. epidermidis strain. The broth was then incubated for 18 h at 37 ◦C. Ten µL of overnight culture was added to a sterile Eppendorf tubes containing 990 µL sterile TSB supplemented with either nisin A, M21V or M17Q peptide (final concentration of 3.75 µM (12.52 µg/mL)). Tubes were vortexed, (VELP Scientifica, Usmate Velate, Italy) and placed into a 37 ◦C incubator. After 1 h the Eppendorf tubes were removed from the 1 incubator and 100 µL was taken and used to inoculate 900 µL of sterile 4 strength Ringers.’ From this, 1 5 a serial dilution was performed, vortexed and 100 µL of each dilution, (10− to 10− ), was plated onto TSA. The plates were incubated overnight at 37 ◦C after which they were counted and CFU/mL were Antibiotics 2020, 9, 305 14 of 18 calculated. The experiment was performed on three biological repeats of each strain (S. epidermidis 28 and S. epidermidis 53).

4.7. Biofilm Assays with Plastic Microtiter Plates Biofilm formation on plastic surfaces was assessed as per the protocol established by Mathur et al. [28]. Ten mL of TSB supplemented with 1% glucose was inoculated with a single colony of the selected S. epidermidis strains and grown for 18 h at 37 ◦C. 200 µL of sterile TSB + 1% glucose was added to a single well to act as a sample blank and negative control. 3 different concentrations of nisin A, M21V or M17Q peptides were individually resuspended in sterile TSB + 1% glucose (the final volume of broth was 990 µL and the final concentrations of peptides were 1.875 µM (6.28 µg/mL), 3.75 µM (12.52 µg/mL) and 7.5 µM (25.04 µg/mL)). To each concentration of peptide, 10 µL of overnight S. epidermidis culture was added (to reach a final concentration of 4.23 × 107 CFU/mL of S. epidermidis 28 and 3.11 107 CFU/mL of S. epidermidis 53). Tubes were vortexed and × 200 µL were transferred into individual wells of a 96 well plates (Sarstedt, Nümbrecht, Germany). The plates were incubated at 37 ◦C for 24 h. Each of the wells were washed gently with 200 µL of PBS three times (Sigma Aldrich, Steinheim, Germany). After washing, the cells were fixed to the plate by adding 200 µL of 2% sodium acetate (Sigma Aldrich, Steinheim, Germany). After removing the sodium acetate, 200 µL of 0.1% crystal violet (Prolab diagnostics, Bromborough, Birkenhead, UK), was added to each inoculated well and plates were left at room temperature for 10 min. The wells were rinsed with deionized water and allowed dry. When dry, the remaining crystal violet was resuspended using 200 µL of 95% ethanol (Sigma Aldrich, Steinheim, Germany). The absorbance reading of each well was taken using a calibrated Variskan Lux (Thermo Fischer, Darmstadt, Germany), A595nm. Nisin A, M17Q and M21V were tested against S. epidermidis 28 and 53 in triplicate, (i.e., three biological repeats).

4.8. Biofilm Assays on Stainless Steel All square stainless steel coupons used in this test had an area 2 cm2. A modified version of the method employed by Minei et al. was used to analyze the formation of S. epidermidis biofilm on the stainless steel coupons [38]. Stainless steel coupons were sterilized by autoclaving at 121 ◦C, at 15 p.p.i for 15 min after which they were placed in flat bottomed 6 well plates (Costar, Analab Limited, Lisburn, Co. Antrim). Nisin A, M21V or M17Q peptides were individually resuspended in fresh, sterile TSB + 1% glucose in sterile Eppendorf tubes (the final volume of broth in each tube was 1.80 mL and the final concentration of each peptide was 3.75 µM (12.57 µg/mL). 20 µL of an overnight culture (grown in 10 mL of TSB supplemented with 1% glucose for 18 h at 37 ◦C) of the relevant S. epidermidis strain was added to each tube bringing the final volume up to 2 mL. Tubes were vortexed and the entire 2 mL of contents was aseptically added to a well containing a sterile steel coupon. A sterility control was created by incubating an autoclaved coupon in 2 mL uninoculated TSB + 1% glucose. The 6 well plates were incubated for 24 h at 37 ◦C. After incubation, each stainless steel coupon was carefully removed from the coupon using sterile forceps and dipped into a sterile container of autoclaved, distilled water to displace any planktonic cells. Then coupons were individually placed into sterile 50 mL falcon 1 tubes with 5 mL of 4 strength Ringers’ solution and vortexed for 30 s at 35 Hertz using tabletop VELP Scientifica vortex, to dislodge any biofilm into the surrounding solution. The plates were removed using sterile forceps and inspected. Any remaining biofilm was removed via scrubbing with a sterile cotton swab, which was rubbed vigorously along each side and surfaces of the coupon 20 times. The tip 1 of the swab was cut and placed into the 10 mL solution of 4 strength Ringers and biofilm prior to the solution being vortexed. One hundred µL of the solution was then taken and placed into 900 µL of 1 1 6 sterile 4 strength Ringers solution. A serial dilution was performed and each dilution (10− to 10− ), was plated onto TSA and grown overnight at 37 ◦C. Plates were counted and CFU/mL were calculated. Nisin A, M17Q and M21V were tested against S. epidermidis 28 and 53 in triplicate, using biological repeats of each strain. Antibiotics 2020, 9, 305 15 of 18

4.9. Biofilm Assay on Various Catheter Materials Three different catheter materials, (rubber, polyvinyl chloride and polyvinyl resin), were cut into pieces, (2 cm in length with diameters of 4 mm). The catheter pieces were soaked in a 2% glutaraldehyde solution for 30 min to sterilize them after which they were rinsed thoroughly in sterile water. Each piece was then placed in an individual, sterile, 2 mL microtube (Sarstedt, Nümbrecht, Germany). Nisin A, M21V or M17Q peptides were individually resuspended in 1.80 mL fresh, sterile TSB + 1% glucose (the final concentration of each peptide was 3.75 µM (12.52 µg/mL). The peptide supplemented broth was added to the tubes containing catheter pieces and tubes were incubated at 37 ◦C, shaking at 100 rpm (Titramax 100, Heidolph Instruments, Walpersdorfer, Schwabach, Germany), for 24 h. Following incubation, the catheter pieces were aseptically removed from the tubes in a biological safety cabinet and dipped slowly in a container of sterile distilled water to remove any planktonic cells. Once rinsed, the pieces were transferred to a new, sterile, 2 mL microtube containing 1 1 mL of sterile 4 strength Ringers’ solution. Initially, samples were sonicated at 80% ultrasound power, using a desk top sonicating water bath at room temperature, (Transonic Digitals, Elma Ultrasonic, Singen, Germany) to detach the biofilm. The tubes were then vortexed at 40 hertz using a desk top VELP Scientifica desk top vortex, to loosen any remaining bound biofilm. When the biofilm was removed, 100 µL was taken from each tube, a serial dilution was performed and 100 µL of each dilution was plated onto fresh TSA plates. The plates were incubated for 18 h and CFU counts were carried out. This experiment was carried out in triplicate using three biological repeats of each S. epidermidis strain (S. epidermidis 28 and 53).

4.10. Testing the Efficacy of Nisin A and M17Q in a Simulated Wound Fluid One hundred mL of a simulated wound fluid was prepared in accordance to the method used by Bradford et al. [44], and 3.3 g of bovine serum albumin, 0.58 g of sodium chloride and 0.5 g of calcium chloride (all purchased from Sigma Aldrich, Steinheim, Germany), were dissolved in 100 mL of distilled water. The fluid was filtered through a 0.45 µm filter, (Sarstedt, Nümbrecht, Germany). The simulated wound fluid was then aliquoted into 10 mL volumes and stored at 4 ◦C prior to use. Freeze dried nisin and nisin derivative peptides were resuspended in sterile TSB supplemented with 1% glucose at a concentration of 30 µM. 87.5 µL of reconstituted peptide and 655 µL of simulated wound fluid were placed into sterile 1.5 mL Eppendorf tubes and then inoculated with 7.5 µL of selected S. epidermidis overnight culture, (an initial inoculum of 3.8 107 CFU/mL of S. epidermidis 28 × and 4.6 106 CFU/mL of S. epidermidis 53). This brought the final volume in each tube to 750 µL and × the concentration of each nisin peptide was 3.75 µM (12.52 µg/mL). Tubes were vortexed and incubated for 1 h at 37 ◦C. One hundred µL was taken from each tube, a serial dilution was performed and 100 µL of each dilution was plated on fresh TSA plates. The plates were incubated for 18 h at 37 ◦C after which colonies were counted and CFU/mL calculated. Each S. epidermidis strains (28 and 53) was tested in triplicate that is, three biological repeats.

4.11. Reproducibility and Statistical Analyses All experiments were performed in triplicate using three biological repeats. All CFU/mL data was converted to log10 prior to statistical analysis. All comparisons between data were based on the mean standard deviation. Statistical analysis was performed using MS Excel, version 16. In all cases, ± a p value < 0.05 was established as a threshold to consider a result as statistically significant. Asterisks indicating *, ** or *** designates statistically significant differences between samples (p < 0.05, p < 0.005 and p < 0.001, respectively).

5. Conclusions In conclusion, in addition to providing further evidence that nisin A can inhibit S. epidermidis and impact biofilm formation, we report the identification of a novel bioengineered nisin A derivative Antibiotics 2020, 9, 305 16 of 18

M17Q with enhanced antimicrobial and anti-biofilm activity. In our opinion, these findings suggest that nisin and its bioengineered derivatives warrant further investigation as a potential alternative strategy for the control of S. epidermidis.

Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/6/305/s1. Figure S1: (A) Structure of the nisin peptide indicating the M17Q substitution (B) Amino acid sequence of nisin peptide and M17Q derivative peptide. Figure S2: Reversed–phase high performance liquid (RP-HPLC) profile for (A) derivative peptide M21V, (B) derivative peptide M17Q and (C) wildtype peptide nisin A. Figure S3: Chromatogram displaying MALDI-TOF Mass Spectrometry of lyophilized peptides, wildtype nisin A and derivative peptide M17Q. Author Contributions: Conceptualization, M.B. and D.F.; formal analysis, E.T. and M.B.; investigation, E.T.; resources, C.H.; writing—original draft preparation, E.T., D.F. and M.B.; writing—review and editing, E.T., C.H., D.F. and M.B.; supervision, M.B and D.F.; funding acquisition, M.B., D.F. and E.T. All authors have read and agreed to the published version of the manuscript. Funding: Ellen Twomey is in receipt of a RÍSAM PhD Scholarship at Cork Institute of Technology. Acknowledgments: We would like to thank Hugh O’Donnell, Cork Institute of Technology for sourcing the medical device substrates. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Lee, J.Y.H.; Monk, I.R.; Gonçalves da Silva, A.; Seemann, T.; Chua, K.Y.L.; Kearns, A.; Hill, R.; Woodford, N.; Bartels, M.D.; Strommenger, B.; et al. Global Spread of Three Multidrug-Resistant Lineages of Staphylococcus epidermidis. Nat. Microbiol. 2018, 3, 1175–1185. [CrossRef][PubMed] 2. Chen, H.-W.; Liu, P.-F.; Liu, Y.-T.; Kuo, S.; Zhang, X.-Q.; Schooley, R.T.; Rohde, H.; Gallo, R.L.; Huang, C.-M. Nasal Commensal Staphylococcus epidermidis Counteracts Influenza Virus. Sci. Rep. 2016, 6, 27870. [CrossRef] [PubMed] 3. European Center for Disease Prevention and Control. Rapid Risk Assessment: Multidrug-Resistant Staphylococcus epidermidis. 2018. Available online: https://ecdc.europa.eu/sites/portal/files/documents/15- 10-2018-RRA-Staphylococcusepidermidis%2CAntimicrobialresistance-World_ZCS9CS.pdf (accessed on 11 March 2020). 4. Christensen, G.D.; Bisno, A.L.; Parisi, J.T.; McLaughlin, B.; Hester, M.G.; Luther, R.W. Nosocomial Septicemia Due to Multiply Antibiotic-Resistant Staphylococcus epidermidis. Ann. Intern. Med. 1982, 96, 1–10. [CrossRef] [PubMed] 5. Büttner, H.; Mack, D.; Rohde, H. Structural Basis of Staphylococcus epidermidis Biofilm Formation: Mechanisms and Molecular Interactions. Front. Cell. Infect. Microbiol. 2015, 5, 14. [CrossRef][PubMed] 6. Song, Z.; Borgwardt, L.; Høiby,N.; Wu, H.; Sørensen, T.S.; Borgwardt, A. Prosthesis Infections after Orthopedic Joint Replacement: The Possible Role of Bacterial Biofilms. Orthop. Rev. (Pavia) 2013, 5, 14. [CrossRef] 7. van der Meer, J.R.; Polman, J.; Beerthuyzen, M.M.; Siezen, R.J.; Kuipers, O.P.; De Vos, W.M. Characterization of the Lactococcus lactis Nisin a Operon Genes NisP, Encoding a Subtilisin-like Protease Involved in Precursor Processing, and NisR, Encoding a Regulatory Protein Involved in Nisin . J. Bacteriol. 1993, 175, 2578–2588. [CrossRef] 8. Field, D.; Cotter, P.D.; Hill, C.; Ross, R.P. Bioengineering for Therapeutic Success. Front. Microbiol. 2015, 6, 1363. [CrossRef] 9. Breukink, E.; Wiedemann, I.; van Kraaij, C.; Kuipers, O.P.; Sahl, H.G.; de Kruijff, B. Use of the Cell Wall Precursor Lipid II by a Pore-Forming Peptide Antibiotic. Science 1999, 286, 2361–2364. [CrossRef] 10. European Food Safety Authority. Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a Request from the Commission Related to the Use of Nisin (E 234) as a Food Additive. 2006. Available online: http://www.efsa.eu.int/science/afc/afc_opinions/catindex_en.html (accessed on 11 March 2020). 11. Campion, A.; Casey, P.G.; Field, D.; Cotter, P.D.; Hill, C.; Ross, R.P. In Vivo Activity of Nisin A and Nisin V against Listeria monocytogenes in Mice. BMC Microbiol. 2013, 13, 23. [CrossRef] Antibiotics 2020, 9, 305 17 of 18

12. Bower, C.; Parker, J.; Higgins, A.; Oest, M.; Wilson, J.; Valentine, B.; Bothwell, M.; McGuire, J. Protein Antimicrobial Barriers to Bacterial Adhesion: In Vitro and in Vivo Evaluation of Nisin-Treated Implantable Materials. Colloids Surf. B Biointerfaces 2002, 25, 81–90. [CrossRef] 13. Piper, C.; Casey, P.G.; Hill, C.; Cotter, P.D.; Ross, R.P. The Lantibiotic lacticin 3147 Prevents Systemic Spread of Staphylococcus aureus in a Murine Infection Model. Int. J. Microbiol. 2012, 2012, 1–6. [CrossRef][PubMed] 14. O’Sullivan, J.N.; O’Connor, P.M.; Rea, M.C.; O’Sullivan, O.; Walsh, C.J.; Healy, B.; Mathur, H.; Field, D.; Hill, C.; Ross, R.P. Nisin J, a Novel Natural Nisin Variant, Is Produced by Staphylococcus capitis Sourced from the Human Skin Microbiota. J. Bacteriol. 2020, 202, e00639-19. [CrossRef] 15. Wirawan, R.E.; Klesse, N.A.; Jack, R.W.; Tagg, J.R. Molecular and Genetic Characterization of a Novel Nisin Variant Produced by Streptococcus uberis. Appl. Environ. Microbiol. 2006, 72, 1148–1156. [CrossRef] 16. Mulders, J.W.M.; Boerrigter, I.J.; Rollema, H.S.; Siezen, R.J.; Vos, W.M. Identification and Characterization of the Lantibiotic Nisin Z, a Natural Nisin Variant. Eur. J. Biochem. 1991, 201, 581–584. [CrossRef][PubMed] 17. Zendo, T.; Fukao, M.; Ueda, K.; Higuchi, T.; Nakayama, J.; Sonomoto, K. Identification of the Lantibiotic Nisin Q, a New Natural Nisin Variant Produced by Lactococcus lactis 61-14 Isolated from a River in Japan. Biosci. Biotechnol. Biochem. 2003, 67, 1616–1619. [CrossRef][PubMed] 18. Field, D.; Quigley, L.; O’Connor, P.M.; Rea, M.C.; Daly, K.; Cotter, P.D.; Hill, C.; Ross, R.P. Studies with Bioengineered Nisin Peptides Highlight the Broad-Spectrum Potency of Nisin V. Microb. Biotechnol. 2010, 3, 473–486. [CrossRef] 19. Field, D.; Connor, P.M.O.; Cotter, P.D.; Hill, C.; Ross, R.P. The Generation of Nisin Variants with Enhanced Activity against Specific Gram-Positive Pathogens. Mol. Microbiol. 2008, 69, 218–230. [CrossRef] 20. Field, D.; Gaudin, N.; Lyons, F.; O’Connor, P.M.; Cotter, P.D.; Hill, C.; Ross, R.P. A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius. PLoS ONE 2015, 10, e0119684. [CrossRef] 21. Smith, M.K.; Draper, L.A.; Hazelhoff, P.-J.; Cotter, P.D.; Ross, R.P.; Hill, C. A Bioengineered Nisin Derivative, M21A, in Combination with Food Grade Additives Eradicates Biofilms of Listeria monocytogenes. Front. Microbiol. 2016, 7, 1939. [CrossRef] 22. Davison, W.M.; Pitts, B.; Stewart, P.S. Spatial and Temporal Patterns of Biocide Action against Staphylococcus epidermidis Biofilms. Antimicrob. Agents Chemother. 2010, 54, 2920–2927. [CrossRef] 23. Ghiselli, R.; Giacometti, A.; Cirioni, O.; Dell’Acqua, G.; Mocchegiani, F.; Orlando, F.; D’Amato, G.; Rocchi, M.; Scalise, G.; Saba, V. RNAIII-Inhibiting Peptide And/Or Nisin Inhibit Experimental Vascular Graft Infection with Methicillin-Susceptible and Methicillin-Resistant Staphylococcus epidermidis. Eur. J. Vasc. Endovasc. Surg. 2004, 27, 603–607. [CrossRef][PubMed] 24. Healy, B.; Field, D.; O’Connor, P.M.; Hill, C.; Cotter, P.D.; Ross, R.P. Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives. PLoS ONE 2013, 8, e79563. [CrossRef][PubMed] 25. Rouse, S.; Field, D.; Daly, K.M.; O’Connor, P.M.; Cotter, P.D.; Hill, C.; Ross, R.P. Bioengineered Nisin Derivatives with Enhanced Activity in Complex Matrices. Microb. Biotechnol. 2012, 5, 501–508. [CrossRef] [PubMed] 26. Medeiros-Silva, J.; Jekhmane, S.; Paioni, A.L.; Gawarecka, K.; Baldus, M.; Swiezewska, E.; Breukink, E.; Weingarth, M. High-Resolution NMR Studies of Antibiotics in Cellular Membranes. Nat. Commun. 2018, 9, 1–10. [CrossRef][PubMed] 27. Field, D.; Daly, K.; O’Connor, P.M.; Cotter, P.D.; Hill, C.; Ross, R.P. Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes. Appl. Environ. Microbiol. 2015, 81, 2762–2769. [CrossRef][PubMed] 28. Mathur, T.; Singhal, S.; Khan, S.; Upadhyay, D.J.; Fatma, T.; Rattan, A. Detection of biofilm formation among the clinical isolates of Staphylococci: An evaluation of three different screening methods. Indian J. Med. Microbiol. 2006, 24, 25–34. [CrossRef][PubMed] 29. Chen, Y.E.; Fischbach, M.A.; Belkaid, Y. Skin Microbiota-Host Interactions. Nature 2018, 553, 427–436. [CrossRef] 30. Field, D.; Cotter, P.D.; Ross, R.P.; Hill, C. Bioengineering of the Model Lantibiotic Nisin. Bioengineered 2015, 6, 187–192. [CrossRef] 31. Dunne, W.M.; Mason, E.O.; Kaplan, S.L., Jr.; Kaplan, S.L. Diffusion of Rifampin and Vancomycin through a Staphylococcus epidermidis Biofilm. Antimicrob. Agents Chemother. 1993, 37, 2522–2526. [CrossRef] Antibiotics 2020, 9, 305 18 of 18

32. Jefferson, K.K.; Goldmann, D.A.; Pier, G.B. Use of Confocal Microscopy to Analyze the Rate of Vancomycin Penetration through Staphylococcus aureus Biofilms. Antimicrob. Agents Chemother. 2005, 49, 2467–2473. [CrossRef] 33. Walters, M.C.; Roe, F.; Bugnicourt, A.; Franklin, M.J.; Stewart, P.S. Contributions of Antibiotic Penetration, Oxygen Limitation, and Low Metabolic Activity to Tolerance of Pseudomonas aeruginosa Biofilms to Ciprofloxacin and Tobramycin. Antimicrob. Agents Chemother. 2003, 47, 317–323. [CrossRef][PubMed] 34. Qin, Z.; Zhang, J.; Hu, Y.; Chi, Q.; Mortensen, N.P.; Qu, D.; Molin, S.; Ulstrup, J. Organic Compounds Inhibiting S. epidermidis Adhesion and Biofilm Formation. Ultramicroscopy 2009, 109, 881–888. [CrossRef] [PubMed] 35. Okuda, K.; Zendo, T.; Sugimoto, S.; Iwase, T.; Tajima, A.; Yamada, S.; Sonomoto, K. and Mizunoe, Y. Effects of Bacteriocins On Methicillin-Resistant Staphylococcus aureus Biofilm. Antimicrob. Agents Chemother. Am. Soc. Microbiol. J. 2013, 57, 5572–5579. [CrossRef] 36. Field, D.; Connor, R.O.; Cotter, P.D.; Ross, R.P.; Hill, C. In Vitro Activities of Nisin and Nisin Derivatives Alone and in Combination with Antibiotics against Staphylococcus Biofilms. Front. Microbiol. 2016, 7, 508. [CrossRef] 37. Mathur, H.; Field, D.; Rea, M.C.; Cotter, P.D.; Hill, C.; Ross, R.P. Fighting Biofilms with Lantibiotics and Other Groups of Bacteriocins. NPJ Biofilms Microbiomes 2018, 4, 1–13. [CrossRef] 38. Minei, C.C.; Gomes, B.C.; Ratti, R.P.; D’angelis, C.E.M.; De Martinis, E.C.P. Influence of Peroxyacetic Acid and Nisin And Coculture with Enterococcus faecium On Listeria monocytogenes Biofilm Formation. J. Food Protection. 2008, 71, 634–638. [CrossRef] 39. Natsis, N.E. and Cohen, P.R. Coagulase-Negative Staphylococcus Skin And Soft Tissue Infections. Am. J. Clin. Dermatol. 2018, 19, 671–677. [CrossRef] 40. van Staden, A.D.P.; Heunis, T.; Smith, C.; Deane, S.; Dicks, L.M.T. Efficacy of Lantibiotic Treatment of Staphylococcus aureus—Induced Skin Infections, Monitored by In Vivo Bioluminescent Imaging. Antimicrob. Agents Chemother. 2016, 60, 3948–3955. [CrossRef] 41. Mouritzen, M.V.; Andrea, A.; Qvist, K.; Poulsen, S.S.; Jenssen, H. Immunomodulatory Potential Of Nisin A With Application In Wound Healing. Wound Repair Regen. 2019, 27, 650–660. [CrossRef] 42. Field, D.; Begley, M.; O’Connor, P.M.; Daly, K.M.; Hugenholtz, F.; Cotter, P.D.; Hill, C.; Ross, R.P.Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens. PLoS ONE 2012, 7, e46884. [CrossRef] 43. Molloy, E.M.; Field, D.; O’Connor, P.M.; Cotter, P.D.; Hill, C.; Ross, R.P. Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity. PLoS ONE 2013, 8, e58530. [CrossRef][PubMed] 44. Bradford, C.; Freeman, R.; Percival, S.L. In Vitro Study of Sustained Antimicrobial Activity of a New Silver Alginate Dressing. J. Am. Col. Certif. Wound Spec. 2009, 1, 117–120. [CrossRef][PubMed]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).