Lugdunin production and activity in Staphylococcus lugdunensis isolates are associated with its genotypes

ABSTRACT Lugdunin produced by Staphylococcus lugdunensis has been shown to have broad inhibitory activity against Gram-positive bacteria; however, lugdunin activity among S. lugdunensis isolates and its association with different agr, SCCmec, and sequence types remain unclear. We used matrix-assisted laser desorption ionization-time-of-flight mass spectrometry to identify S. lugdunensis and collected 202 S. lugdunensis samples for further assays. Agar spot tests were performed to characterize S. lugdunensis lugdunin production and activity. Multilocus sequence typing, SCCmec, and agr genotyping were performed on S. lugdunensis. In all, 91 Staphylococcus aureus strains with varying vancomycin susceptibilities were used to examine lugdunin activity in S. lugdunensis. In total, 48 S. lugdunensis strains (23.8%) were found to be oxacillin-resistant S. lugdunensis (ORSL), whereas 154 (76.2%) were classified as oxacillin-sensitive S. lugdunensis (OSSL). Moreover, 16 (33.3%) ORSL and 35 (22.7%) OSSL strains showed antibacterial activity against S. aureus. Our data showed that most lugdunin-producing ORSL strains (14/48, 29.2%) were of ST3-SCCmec V-agr II genotypes, whereas most lugdunin-producing OSSL strains (15/154, 9.7%) were of ST3-agr II, followed by ST1-agr I (10/154, 6.5%). Our data also revealed that lugdunin exhibited weak inhibitory activity against the VISA ST239 isolate. In addition, we observed that ST239 VSSA was more resistant to lugdunin than ST5, ST59, and ST45 VSSA. Taken together, our data pioneered the epidemiology of lugdunin production in S. lugdunensis isolates and revealed its association with genotypes. However, further molecular and bioinformatics investigations are needed to elucidate the regulatory mechanisms of lugdunin production and activity. IMPORTANCE Lugdunin is active against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci by dissipating their membrane potential. However, the association of lugdunin activity with the genotypes of Staphylococcus lugdunensis has not been addressed. Here, we show the high prevalence of lugdunin-producing strains among ST1 (83.3%), ST2 (66.7%), and ST3 (53.3%) S. lugdunensis. Moreover, we identified the antibacterial activity of lugdunin-producing strains against VISA and hVISA. These results shed light on the potential application of lugdunin for the treatment of drug-resistant pathogens.

Therefore, urgent efforts and immediate development to discover new antibiotics from potentially underexplored niches are required.
Natural products play promising roles in the development of therapeutics against infectious diseases (3).Recently, the human microbiome has been recognized as a niche from which bioactive secondary metabolites can be isolated (3).Staphylococcus aureus is a disease-causing opportunistic pathogen that resides in the nasal cavities of 30% of the population and is prone to multidrug resistance (4,5).To combat S. aureus infections, a notable bacterium being studied for its potential probiotic activities is a coagulase-neg ative Staphylococcus, Staphylococcus lugdunensis (6,7).In recent years, clinical reports have shown that it can also be an emerging pathogen because it causes life-threatening invasive infections, such as endocarditis and periprosthetic joint infections (8).However, little attention has been paid to S. lugdunensis as a potential probiotic (6).In 2016, using functional and chemistry-based approaches, Zipperer et al. discovered that S. lugdu nensis produced a novel antibiotic called lugdunin, which exhibited broad bioactivity against Gram-positive bacteria, including methicillin-resistant S. aureus, vancomycin, and glycopeptide-intermediate-resistant S. aureus strains (3,9).S. lugdunensis IVK28 has a particularly strong capacity to prevent the growth of S. aureus (9).
Lugdunin is a thiazolidine-containing cyclic peptide antibiotic that is synthesized non-ribosomally and belongs to a new class of antibiotics (9).The analysis of a transpo son insertion site in the mutant M1 strain revealed the disruption of an uncharacterized gene encoding a putative non-ribosomal peptide synthetase (NRPS) (9).The operon responsible for lugdunin biosynthesis consists of four NRPS genes (lugA, B, C, and D) encoding adenylation domains for five amino acids and is exclusively found in S. lugdunensis (9).
Multilocus sequence typing (MLST) is the most widely used genotyping tool in epidemiological studies of S. lugdunensis to monitor its prevalence in hospitals (10)(11)(12).In addition, our previous study showed an association between ST and CRISPR-Cas types in S. lugdunensis (13).Among 130 CRISPR-Cas-positive S. lugdunensis isolates, type IIIA and type IIC CRISPR-Cas were found in 84 (64.6%) and 46 (35.4%) isolates, respectively (13).Isolates ST1, ST6, ST12, and ST15 harbored type IIIA CRISPR-Cas, whereas isolates ST4, ST27, and ST29 harbored type IIC CRISPR-Cas.None of the 60 ST3 isolates harbored type IIIA or type IIC CRISPR-Cas (13).Currently, clinical attention has been paid to S. lugdunensis because of its ability to resist β-lactam antibiotics such as oxacillin (oxacillinresistant S. lugdunensis, ORSL) (11).Thus, SCCmec typing is a "gold standard method" to determine resistance to β-lactam antibiotics (14,15).Most virulence factors in S. aureus are closely regulated by the agr locus, which encodes a two-component signaling pathway that can be divided into four major agr groups: types I-IV (16).The agr locus in S. lugdunensis was recently shown to play a role in biofilm formation and resist ance to host immune killing (17,18).Hence, despite well-reported evidence regarding the chemical structure and biosynthetic gene clusters of lugdunin, the epidemiology of lugdunin-producing strains and the association of lugdunin production with STs, SCCmec, and agr genotypes are still unknown.Thus, in the present study, we aimed to characterize lugdunin activity in S. lugdunensis clinical isolates collected from our hospital and investigate its association with different genotypes.

Bacterial strains
A total of 202 S. lugdunensis isolates were collected between 2003 and 2014 at Chang Gung Memorial Hospital (Linkou), Taiwan.The sample sources and antimicrobial susceptibility results of 202 S. lugdunensis isolates are shown in Table S1.All S. lugdu nensis isolates were identified using a Bruker Biotyper (database 2.0) matrix-assisted laser desorption ionization-time-of-flight mass spectrometry system, according to the manufacturer's instructions, and S. lugdunensis isolates were stored in a tryptic soy broth with 20% glycerol at −80°C until further experiments.Vancomycin susceptibility was determined using the modified population analysis profile/area under the curve (19).In all, 91 S. aureus strains containing 28 ST5, 8 ST45, 27 ST59, and 28 ST239, with different vancomycin susceptibilities, were randomly selected and used as test pathogens to examine lugdunin activity in S. lugdunensis (20).

Screening of lugdunin activity by an agar spot test
Basic medium (BM) agar was prepared (1% soy peptone, 0.5% Kat yeast extract, 0.5% NaCl, 0.1% glucose, and 0.1% K 2 HPO4; pH 7.2) as previously described (21).A single colony of S. aureus HG001 was inoculated into a 5-mL BM medium tube (17 × 100 mm) and incubated at 37°C with shaking for 18-24 h.The BM agar was then melted and allowed to cool in a 50°C water bath for 0.5-1 h.The BM agar was then supplemented with 2,2′-dipyridyl (2-DP, 200 µM), an iron chelator, and the optical density (OD) 600 of S. aureus was adjusted to 0.125 and added together in the BM agar.The mixture was poured into a Petri dish and allowed to solidify.An S. lugdunensis overnight BM medium was prepared, and the S. lugdunensis culture was washed twice with 0.1× volume of phosphate-buffered saline and centrifuged at 4,000 rpm for 15 min at room temperature.The supernatant was discarded.The S. lugdunensis pellets were adjusted to OD 600 = 20 using the BM medium, and 10 µL was dropped at the center of the BM agar and allowed to dry.The BM agar was incubated for 48 h, and zone clearing or inhibition was observed.Clear zone inhibition was calculated as the inhibition zone diameter-colony diameter (cm), and the absence of a clear zone of inhibition was considered negative.Strains SL131 and SL118, with known genome sequences, were used as lugdunin-active positive and negative controls, respectively (22,23).Strain SL85, with strong lugdunin activity (zone size >0.2 cm), was used to examine anti-S.aureus activity.

SCCmec, STs, and agr genotyping
All protocols for SCCmec, STs, and agr genotyping were adopted from previous studies.For SCCmec typing, all ORSL isolates were performed using multiplex PCR (M-PCR 1 and M-PCR 2) and were interpreted as described previously (24).Briefly, the 10 µL reaction mixtures of M-PCR 1 contained 10 ng chromosomal DNA, M1 oligonucleotide primers (0.1 M), and Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, USA) at a final volume of 10 µL.A three-step thermal cycling protocol was used with an initial denaturation step (94°C, 2 min), followed by 30 cycles of denaturation (94°C, 1 min), an annealing step (50°C, 1 min), and an extension step (72°C, 1 min); and a final extension step at 72°C for 10 min.The 10 µL reaction mixtures of M-PCR 2 were the same as those for M-PCR 1 except that the annealing temperature was raised to 60°C for 1 min (24).For MLST typing of S. lugdunensis, seven housekeeping genes (aroE, dat, ddl, gmk, ldh, recA, and yqiL) were used, and the fragments were amplified using the primers as described previously (10).The selection of these housekeeping genes relied on their use in MLST schemes of S. aureus (25).The Pasteur MLST sequence definition database contains allele and profile data of S. lugdunensis (https://bigsdb.pasteur.fr/staphlugdunensis/).The ST of each S. lugdunensis isolate was determined using the search tool with a combination of S. lugdunensis loci (26).Two PCR primers (SL_agr-F and SL_agr-R) were designed to detect the agr genes of S. lugdunensis isolates (27).In addition, two forward primers and one reverse primer (SL_agr-1-F, SL_agr-2-F/SL_agr-R) were used to determine the agr type (27).

Statistical analysis
We used chi-square tests to compare categorical variables.All statistical analyses were conducted using SPSS version 22.0 (IBM, Armonk, NY, USA).A P-value of <0.05 was considered to indicate a statistically significant difference.

DISCUSSION
In this study, we determined the prevalence of lugdunin-producing S. lugdunensis strains and found that the distribution of lugdunin-producing strains was associated with SCCmec, agr, and ST genotypes.Our lugdunin screening using the agar spot test showed that not all S. lugdunensis strains produced lugdunin under the given conditions, strongly suggesting that lugdunin production may be strain specific and associated with STs or other genotypes that could affect lugdunin production and regulation.We analyzed its association with different genotypes and found that the of the ORSL lugdunin-producing strains belonged to ST3 and harbored the agr II genotype.This ST type was similar to that of the reference strain IVK28 used by Zipperer et al., which also belongs to ST3.The SCCmec typing of ORSL revealed that most strains (27, 56.3%) harbored SCCmec type V, strongly suggesting that it was community acquired.However, 16 patients (33.3%) had SCCmec type II, indicating nosocomial infections.Owing to the low prevalence of ORSL in lugdunin production, the association between SCCmec types and lugdunin production remains unclear.The majority of the OSSL lugdunin-pro ducing strains also belonged to ST3-agr II.Regarding the association between lugdu nin production and STs, our data strongly suggest that the activity of the lugdunin operon differed among STs.However, the organization of the lugdunin operon and the relative gene expression in lugdunin-non-producing ST3-SCCmec V-agr II are unclear.Our previous study showed a strong association between the CRISPR/Cas system and STs in S. lugdunensis (13).These findings strongly suggest an association between the CRISPR-Cas system and lugdunin activity in S. lugdunensis, although the role of the CRISPR-Cas system in S. lugdunensis remains unknown.
Similar to what we observed in the lugdunin-producing group, a different prevalence rate of lugdunin non-production was observed among STs.This led us to speculate why some S. lugdunensis strains failed to produce lugdunin.There are three possibili ties: (i) the expression of the lugdunin operon genes in the lugdunin-non-producing group may be low and may not produce lugdunin to exhibit antibacterial activity.(ii) Single-nucleotide polymorphisms or other mutations may occur in the lugdunin operon, leading to truncated or inactive enzymes for lugdunin synthesis.(iii) Other novel gene clusters, ligand enhancers, or inhibitors may also directly or indirectly regulate lugdunin production and activity.Thus, further investigation is required.
The quorum-sensing agr system is a major element in pathogenicity regulation and biofilm formation in S. aureus (28).S. lugdunensis shares genomic features with S. aureus (29), and our results showed that the agr system might play a role in lugdunin production.To date, the agr locus in S. lugdunensis has been shown to play a role in biofilm formation and resistance to host immune killing; however, its role in lugdunin production has not been investigated (17,18).Therefore, our study provides a potential role of agr in the regulation and production of S. lugdunensis lugdunin.However, the regulatory mechanisms for lugdunin production by agr remain to be investigated.
It is also worth noting that lugdunin produced by SL85 was active against various sequence types of S. aureus with different vancomycin susceptibilities.Our results showed that lugdunin had relatively weak antibacterial activity against ST239 VISA compared with that against hVISA and VSSA.However, it is promising baseline data to prompt the use of lugdunin in clinical practice.The synergistic effect of lugdunin antibacterial activity combined with other antibiotics is worth investigating in the future.Hort et al. reported that the alteration of the cell wall architecture with a thick cell wall and low cross-linking provided decoy binding sites for vancomycin and led to a decrease in the susceptibility of S. aureus to vancomycin (30).The antibacterial activity of lugdunin targets bacteria by dissipating their membrane potential (31); therefore, changes in the cell wall architecture of VISA and hVISA may also contribute to their higher resistance to lugdunin.Taken together, we demonstrated the epidemiology of lugdunin production among S. lugdunensis isolates in Taiwan and revealed its association with genotypes.However, the regulatory mechanism by which lugdunin production is regulated remains unclear.

TABLE 1
Distribution of lugdunin activity in S. lugdunensis with different oxacillin susceptibility, SCCmec, agr, and MLST types

TABLE 2
Summary of inhibition zone size and molecular types

TABLE 3
Antibacterial activity of lugdunin against different sequence types and vancomycin susceptibility of S.