Comparative proteomics analysis of biofilms and planktonic cells of Enterococcus faecalis and Staphylococcus lugdunensis with contrasting biofilm-forming ability.

Biofilms make it difficult to eradicate bacterial infections through antibiotic treatments and lead to numerous complications. Previously, two periprosthetic infection-related pathogens, Enterococcus faecalis and Staphylococcus lugdunensis were reported to have relatively contrasting biofilm-forming abilities. In this study, we examined the proteomics of the two microorganisms' biofilms using LC-MS/MS. The results showed that each microbe exhibited an overall different profile for differential gene expressions between biofilm and planktonic cells as well as between each other. Of a total of 929 proteins identified in the biofilms of E. faecalis, 870 proteins were shared in biofilm and planktonic cells, and 59 proteins were found only in the biofilm. In S. lugdunensis, a total of 1125 proteins were identified, of which 1072 proteins were found in common in the biofilm and planktonic cells, and 53 proteins were present only in the biofilms. The functional analysis for the proteins identified only in the biofilms using UniProt keywords demonstrated that they were mostly assigned to membrane, transmembrane, and transmembrane helix in both microorganisms, while hydrolase and transferase were found only in E. faecalis. Protein-protein interaction analysis using STRING-db indicated that the resulting networks did not have significantly more interactions than expected. GO term analysis exhibited that the highest number of proteins were assigned to cellular process, catalytic activity, and cellular anatomical entity. KEGG pathway analysis revealed that microbial metabolism in diverse environments was notable for both microorganisms. Taken together, proteomics data discovered in this study present a unique set of biofilm-embedded proteins of each microorganism, providing useful information for diagnostic purposes and the establishment of appropriately tailored treatment strategies. Furthermore, this study has significance in discovering the target candidate molecules to control the biofilm-associated infections of E. faecalis and S. lugdunensis.

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types.

BACKGROUND AND PURPOSE: Our previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. METHODS: A comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. RESULTS: Forty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. CONCLUSION: Our findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

Impact of clonal lineages on susceptibility of Staphylococcus lugdunensis to chlorhexidine digluconate and chloride benzalkonium.

BACKGROUND: Little is known about susceptibility of Staphylococcus lugdunensis to antiseptics. The objective of this study was to evaluate, at the molecular and phenotypic level, the susceptibility of 49 clinical S. lugdunensis strains (belonging to the seven clonal complexes [CCs] defined by multilocus sequence typing) to two antiseptics frequently used in healthcare settings (chlorhexidine digluconate [CHX] and chloride benzalkonium [BAC]). RESULTS: The minimum inhibitory concentrations (MICs), by broth microdilution method, varied for BAC from 0.25 mg/L to 8 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L) and for CHX from 0.5 mg/L to 2 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L). The BAC and CHX minimum bactericidal concentrations (MBCs) varied from 2 mg/L to 8 mg/L (MBC50 = 4 mg/L, MBC90 = 8 mg/L) and from 2 mg/L to 4 mg/L (MBC50 and MBC90 = 4 mg/L), respectively. A reduced susceptibility to CHX (MIC = 2 mg/L) was observed for 12.2% of the strains and that to BAC (MIC ≥ 4 mg/L) for 4.1%. The norA resistance gene was detected in all the 49 isolates, whereas the qacA gene was rarely encountered (two strains; 4.1%). The qacC, qacG, qacH, and qacJ genes were not detected. The two strains harboring the qacA gene had reduced susceptibility to both antiseptics and belonged to CC3. CONCLUSION: The norA gene was detected in all the strains, suggesting that it could belong to the core genome of S. lugdunensis. S. lugdunensis is highly susceptible to both antiseptics tested. Reduced susceptibility to BAC and CHX was a rare phenomenon. Of note, a tendency to higher MICs of BAC was detected for CC3 isolates. These results should be confirmed on a larger collection of strains.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

fbl-typing and Antimicrobial Resistance Analysis of Staphylococcus lugdunensis.

BACKGROUND: A broad variety of infections, ranging from skin infections to infective endocarditis can be caused by Staphylococcus lugdunensis. Bacterial virulence is often related to virulence genes, so we sought to investigate the relationship between virulence genes and the pathogenicity of S. lugdunensis and to explore an appropriate typing method to distinguish different pathogenic phenotypes of S. lugdunensis. METHODS: We describe the distribution of several virulence genes in different infection types in an attempt to find the relationship between virulence genes and pathogenicity. Subsequently, we make the Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) dendrogram and fbl-typing were performed using BioNumerics software, tried to compare the correlation between different methods and the different infectious diseases, and antimicrobial resistance of the strains, in order to obtain the epidemic characteristics and antimicrobial resistance information of S. lugdunensis based on a molecular approach. RESULTS: The results of virulence genes showed that the seven virulence genes we have described existed in most strains, and there was no significant correlation between virulence gene distribution and infection type. Compared with the MALDI-TOF MS dendrogram, we found that fbl-typing could better correspond to the pathogenic phenotype, with better recognition and reproducibility. In the phylogenetic tree constructed in the fbl R-region, we found a tendency for some infection types to be distributed in clusters, new type 3 was the most dominant fbl-type, followed by fbl47b. Bone and joint infection isolates and ear infection isolates were significantly clustered together, in addition, all the oxacillin-resistant isolates were concentrated in fbl-type fbl45j and fbl47b. CONCLUSIONS: In this study, we found no significant correlation between virulence genes from S. lugdunensis isolates and the site of infection. The fbl-typing has the characteristics of convenient operation, low cost, high repeatability, and is preferable to indicate the pathogenic phenotype. Based on fbl-typing, we described the epidemiological characteristics of S. lugdunensis in a hospital and supplemented the data for fbl-typing. We recommend that fbl-typing method be extended and supplemented.

Characterization of oxacillin-resistant Staphylococcus lugdunensis isolated from sterile body fluids in a medical center in Taiwan: A 12-year longitudinal epidemiological study.

BACKGROUND: In this study, our objective was to characterize Staphylococcus lugdunensis isolated from sterile body fluids (SBFs) in a medical center in Taiwan between 2009 and 2020. METHODS: We used MALDI-TOF MS, disk diffusion testing, agar dilution assay, SCCmec typing, and antibiotic resistance gene screening to identify and investigate the characteristics of oxacillin-resistant S. lugdunensis (ORSL). RESULTS: A total of 438 S. lugdunensis isolates were collected and 146 (33.3%) isolates were identified as ORSL. SCCmec type V was dominant (65.7%) in our ORSL isolates, followed by SCCmec type II (18.5%), and type IV (8.9%). After 2013, a slight increase in SCCmec types IV and V was revealed. Moreover, all ORSL isolates with type II and untypable SCCmec were highly resistant to oxacillin (MIC >32 µg/mL), compared to ORSL that had SCCmec types IV, V, and VT. All 146 ORSL isolates were resistant to penicillin and susceptible to teicoplanin and vancomycin. High resistance rates of ORSL to clindamycin (43.2%), erythromycin (43.2%), gentamicin (78.1%) and tetracycline (46.6%) was observed. Moreover, only two (1.4%) and six (4.1%) ORSL isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively. The erythromycin-resistant ORSL isolates mostly exhibited constitutive MLSB resistant phenotype (61/63, 96.8%) and contained either ermC alone (27/63, 42.9%) or a combination of ermC with ermA (28/63, 44.4%). CONCLUSION: Our present study showed a stable rate of ORSL from SBFs during 2009-2020. Moreover, teicoplanin, vancomycin, trimethoprim/sulfamethoxazole, and ciprofloxacin were shown to be highly efficient for the treatment of ORSL in vitro.

Narrowband ultraviolet B response in cutaneous T-cell lymphoma is characterized by increased bacterial diversity and reduced Staphylococcus aureus and Staphylococcus lugdunensis.

Skin microbiota have been linked to disease activity in cutaneous T-cell lymphoma (CTCL). As the skin microbiome has been shown to change after exposure to narrowband ultraviolet B (nbUVB) phototherapy, a common treatment modality used for CTCL, we performed a longitudinal analysis of the skin microbiome in CTCL patients treated with nbUVB. 16S V4 rRNA gene amplicon sequencing for genus-level taxonomic resolution, tuf2 amplicon next generation sequencing for staphylococcal speciation, and bioinformatics were performed on DNA extracted from skin swabs taken from lesional and non-lesional skin of 25 CTCL patients receiving nbUVB and 15 CTCL patients not receiving nbUVB from the same geographical region. Disease responsiveness to nbUVB was determined using the modified Severity Weighted Assessment Tool: 14 (56%) patients responded to nbUVB while 11 (44%) patients had progressive disease. Microbial α-diversity increased in nbUVB-responders after phototherapy. The relative abundance of Staphylococcus, Corynebacterium, Acinetobacter, Streptococcus, and Anaerococcus differentiated nbUVB responders and non-responders after treatment (q<0.05). Microbial signatures of nbUVB-treated patients demonstrated significant post-exposure depletion of S. aureus (q=0.024) and S. lugdunensis (q=0.004) relative abundances. Before nbUVB, responder lesional skin harboured higher levels of S. capitis (q=0.028) and S. warneri (q=0.026) than non-responder lesional skin. S. capitis relative abundance increased in the lesional skin of responders (q=0.05) after phototherapy; a similar upward trend was observed in non-responders (q=0.09). Post-treatment skin of responders exhibited significantly reduced S. aureus (q=0.008) and significantly increased S. hominis (q=0.006), S. pettenkoferi (q=0.021), and S. warneri (q=0.029) relative abundances compared to that of no-nbUVB patients. Staphylococcus species abundance was more similar between non-responders and no-nbUVB patients than between responders and no-nbUVB patients. In sum, the skin microbiome of CTCL patients who respond to nbUVB is different from that of non-responders and untreated patients, and is characterized by shifts in S. aureus and S. lugdunensis. Non-responsiveness to phototherapy may reflect more aggressive disease at baseline.

Staphylococcus lugdunensis Uses the Agr Regulatory System to Resist Killing by Host Innate Immune Effectors.

Coagulase-negative staphylococci (CoNS) are frequently commensal bacteria that rarely cause disease in mammals. Staphylococcus lugdunensis is an exceptional CoNS that causes disease in humans similar to virulent Staphylococcus aureus, but the factors that enhance the virulence of this bacterium remain ill defined. Here, we used random transposon insertion mutagenesis to identify the agr quorum sensing system as a regulator of hemolysins in S. lugdunensis. Using RNA sequencing (RNA-seq), we revealed that agr regulates dozens of genes, including hemolytic S. lugdunensis synergistic hemolysins (SLUSH) peptides and the protease lugdulysin. A murine bacteremia model was used to show that mice infected systemically with wild-type S. lugdunensis do not show overt signs of disease despite there being high numbers of bacteria in the livers and kidneys of mice. Moreover, proliferation of the agr mutant in these organs was no different from that of the wild-type strain, leaving the role of the SLUSH peptides and the metalloprotease lugdulysin in pathogenesis still unclear. Nonetheless, the tropism of S. lugdunensis for humans led us to investigate the role of virulence factors in other ways. We show that agr-regulated effectors, but not SLUSH or lugdulysin alone, are important for S. lugdunensis survival in whole human blood. Moreover, we demonstrate that Agr contributes to survival of S. lugdunensis during encounters with murine and primary human macrophages. These findings demonstrate that, in S. lugdunensis, Agr regulates expression of virulence factors and is required for resistance to host innate antimicrobial defenses. This study therefore provides insight into strategies that this Staphylococcus species uses to cause disease.

An lnu(A)-Carrying Multi-Resistance Plasmid Derived from Sequence Type 3 Methicillin-Resistant Staphylococcus lugdunensis May Contribute to Antimicrobial Resistance in Staphylococci.

Methicillin-resistant Staphylococcus lugdunensis (MRSL) strains showing resistance to several common antibiotics have been reported recently. Sequence type (ST) 3 MRSL carrying SCCmec types IV, V, or Vt is the major lineage associated with health care-associated infections. We aimed to investigate the distribution and dissemination of antimicrobial resistance determinants in this lineage. Two representative ST3-MRSL strains, CGMH-SL131 (SCCmec V) and CGMH-SL138 (SCCmec IV), were subjected to whole-genome sequencing. Detection of antibiotic resistance genes and screening of susceptibility patterns were performed for 30 ST3-MRSL and 16 ST6-MRSL strains via PCR and standard methods. Except for mecA and blaZ, antimicrobial resistance genes were located within two plasmids: a 28.6 kb lnu(A)-carrying plasmid (pCGMH_SL138) in CGMH-SL138 and a 26 kb plasmid carrying non-lnu(A) resistance genes (pCGMH_SL131) in CGMH-SL131. Both plasmids shared common genetic features with multiple copies of IS257 flanked by genes conferring resistance to aminoglycoside (aacA-aphD and aadD), TET (tetk), and cadmium (cadDX) and tolerance to chlorhexidine (qacA/R); however, only pCGMH_SL138 harbored lnu(A) that conferred resistance to lincomycin and rep13 that encodes a replication initiation protein. Unlike ST6-MRSL, none of the ST3-MRSL isolates contained the ermA gene. Instead, most isolates harbored lnu(A) (20/30, 66.7%), and several other resistance genes found on pCGMH_SL138. These isolates and transformants containing pCGMH_SL138 exhibited susceptibility to ERY and higher MICs for lincomycin and aforementioned antibiotics. A novel lnu(A)-carrying plasmid, pCGMH_SL138, that harbored a multiresistance gene cluster, was identified in ST3-MRSL strains and may contribute to the dissemination of antibiotic resistance in staphylococci.

May Staphylococcus lugdunensis Be an Etiological Factor of Chronic Maxillary Sinuses Infection?

Staphylococcus lugdunensis is an opportunistic pathogen found in the healthy human skin microbiome bacterial community that is able to cause infections of diverse localization, manifestation, and course, including laryngological infections, such as necrotizing sinusitis. Chronic maxillary sinusitis is a disease present in up to one third of European and American populations, and its etiology is not fully described. Within this study, we aimed to characterize 18 S. lugdunensis strains recovered from maxillary sinuses and evaluate them as etiological agents of chronic disease. We performed MLST analysis, the complex analysis of both phenotypic and genetic virulence factors, antibiotic susceptibility profiles, and biofilm formation assay for the detection of biofilm-associated genes. Altogether, S. lugdunensis strains were clustered into eight different STs, and we demonstrated several virulence factors associated with the chronic disease. All tested strains were able to produce biofilm in vitro with numerous strains with a very strong ability, and overall, they were mostly susceptible to antibiotics, although we found resistance to fosfomycin, erythromycin, and clindamycin in several strains. We believe that further in-depth analysis of S. lugdunensis strains from different niches, including the nasal one, should be performed in the future in order to reduce infection rate and broaden the knowledge about this opportunistic pathogen that is gaining attention.

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms.

Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection indicating this bacterium overcomes host nutritional immunity. Despite this, there exists a significant knowledge gap regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis can usurp hydroxamate siderophores and staphyloferrin A and B from Staphylococcus aureus. These transport activities all required a functional FhuC ATPase. Moreover, we show that the acquisition of catechol siderophores and catecholamine stress hormones by S. lugdunensis required the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitated the ferrous iron transport system encoded by feoAB. Heme iron was acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice, we demonstrated that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci (isd, fhuC, sst-1, and feo), we demonstrate that only a strain deficient for all of these systems was attenuated in its ability to proliferate to high numbers in the murine kidney. We propose the concerted action of heme and non-heme iron acquisition systems also enable S. lugdunensis to cause human infection.

Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis. We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: ΔargA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the ΔargA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of ΔagrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H2O2 and its survival after 2 h in the presence of 1 mM H2O2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the ΔagrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the ΔagrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

Accurate detection of oxacillin-resistant Staphylococcus lugdunensis by use of agar dilution.

BACKGROUND/PURPOSE: Staphylococcus lugdunensis is a Gram-positive coagulase-negative bacterium and is recognized as a critical pathogenic species recently. Here, we aimed to evaluate the cefoxitin disk diffusion (CDD), oxacillin agar dilution (OAD), and mecA PCR for detecting oxacillin-resistant S. lugdunensis (ORSL) isolates. METHODS: Multilocus sequence typing (MLST) analysis was performed to determine the clonality of 117 S. lugdunensis isolates isolated between May 2009 and Jul 2014. CDD, OAD, and mecA PCR were used to identify oxacillin-resistant S. lugdunensis (ORSL). RESULTS: MLST results showed that the most common sequence type (ST) of our S. lugdunensis isolates was ST6 (35.9%) followed by ST3 (28.2%), ST27 (17.9%), and ST4 (6.8%). CDD and OAD showed that 39 and 43 isolates were ORSL, respectively. 4 ST3 CDD-susceptible S. lugdunensis (OSSL) isolates had MIC values ≥ 4 for oxacillin. mecA PCR results showed that 43 OAD-resistant S. lugdunensis and 3 OAD-susceptible ST27 S. lugdunensis had the mecA gene. Therefore, OAD was used as the gold standard to evaluate the performance of CDD and mecA PCR for identifying ORSL. The overall sensitivity, specificity, and accuracy of CCD for ORSL detection was 90.7%, 100%, and 96.8%, respectively. The sensitivity, specificity, and accuracy of mecA PCR for identifying ORSL was 100%, 95.9%, and 97.44%, respectively. CONCLUSION: Our results indicate that OAD shows higher accuracy for ORSL detection compared with CDD and mecA PCR.

Phylogenetic Distribution of CRISPR-Cas Systems in Staphylococcus lugdunensis.

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) genes (CRISPR-Cas) are present in many bacterial genomes with functions beyond adaptive immunity. We aimed to characterize the CRISPR-Cas system in the pathogenic Gram-positive bacterium Staphylococcus lugdunensis and determine its association with sequence types (STs) determined by multilocus sequence typing (MLST) and oxacillin susceptibility. Primers were designed to detect and sequence types IIIA and IIC CRISPR-Cas in 199 S. lugdunensis isolates. MLST and oxacillin susceptibility tests were also performed on the isolates. We found that 84 S. lugdunensis isolates had type IIIA CRISPR-Cas, while 46 had type IIC. The results showed a strong association between STs and CRISPR-Cas types. The ST1, ST6, ST12, and ST15 isolates had type IIIA CRISPR-Cas systems, and the ST4, ST27, and ST29 isolates had type IIC CRISPR-Cas. Interestingly, of 83 isolates containing type IIIA CRISPR-Cas, 17 (20.5%) were oxacillin-resistant S. lugdunensis (ORSL), and all of these ORSL isolates belonged to ST6 cluster 1. Moreover, spacers 23 and 21 were found in 16 and 17 ORSL isolates, respectively. In contrast, all 46 isolates with type IIC CRISPR-Cas were susceptible to oxacillin. Our results showed that 41.3% of CRISPR-Cas IIIA spacers were homologous to plasmids and 20.2% were homologous to phages. However, in type IIC CRISPR-Cas, 11.8% and 39.9% of spacers showed sequence homology with plasmids and phages, respectively. In conclusion, we found that the distribution and composition of the CRISPR-Cas system in S. lugdunensis was associated with STs and oxacillin susceptibility. IMPORTANCE CRISPR-Cas systems have been characterized as playing several biological roles in many bacterial genomes. Moreover, CRISPR-Cas systems are useful for epidemiological, diagnostic, and evolutionary studies of pathogenic bacteria. However, the characteristics of CRISPR-Cas systems in Staphylococcus lugdunensis have been rarely reported. In this study, we revealed that type IIIA CRISPR-Cas was dominant in S. lugdunensis isolates, followed by type IIC CRISPR-Cas. Moreover, the composition of CRISPR-Cas spacers was strongly associated with multilocus sequence typing and oxacillin susceptibility of S. lugdunensis. These results advance our understanding of the evolution of CRISPR-Cas systems; however, the biological functions of CRISPR-Cas systems in S. lugdunensis remain to be further characterized.

Molecular Epidemiological Survey of Staphylococcus lugdunensis Isolates With Variable Number of Repeats in the von Willebrand Factor-Binding Protein Gene.

The von Willebrand factor binding protein in Staphylococcus lugdunensis (vWbl) comprises four major regions: the signal peptide (S), the non-repetitive (A) region, the repeat (R) region, and the wall-associated (W) region. Previous studies have demonstrated that the R region contains 10 copies of repeating sequences; however, we reveal that the copy number of repeats in the vWbl gene varies among different S. lugdunensis isolates. In this study, an epidemiological surveillance was conducted to determine whether the copy number of repeats in vWbl in different isolates of S. lugdunensis correlates with their infectivity. The number of repeats was estimated in a total of 212 isolates, consisting of 162 isolates of oxacillin-sensitive S. lugdunensis (OSSL) and 50 isolates of oxacillin-resistant S. lugdunensis (ORSL). Our data showed that 72.5% (116/162) of OSSL isolates contained 9 (25, 15.4%), 12 (43, 26.5%), or 13 (48, 29.6%) repeats, and 90% (45/50) of ORSL isolates had 9 (32, 64%) or 13 (13, 26%) repeats. In addition, 89.6% (26 of 29) of the sequence type (ST)27 strain had 12 repeats, and 86.8% (13 of 15) of the ST4 strain had 14 repeats. Twenty-seven of the 28 isolates with nine repeats were of the staphylococcal cassette chromosome mec (SCCmec) V or Vt type and belonged to ST3, and all isolates with 13 repeats were of SCCmec II type and belonged to ST6. All isolates with nine repeats had a stop codon at the 18th codon of the third repeat, suggesting that these isolates coded for nonfunctional vWbl. Further, western blot analysis confirmed that all strains translated vWbl, and only vWbl proteins coded by genes with nine repeats were exported outside the cell. These results suggest that number of vWbl repeats in S. lugdunensis have clonal specificities and may correlate with potential pathogenicity.

Identification of the iron-limitation stimulon in Staphylococcus lugdunensis.

During the infectious process, pathogens such as Staphylococcus lugdunensis have to cope with the condition of host-induced iron-limitation. Using the RNAseq approach, we performed the first global transcriptomic analysis of S. lugdunensis cells incubated in the absence and presence of iron chelator. One hundred and seventy-five genes were identified as members of the iron-limitation stimulon (127 up- and 48 downregulated). Six gene clusters known or likely required for the acquisition of iron have been identified. Among them, a novel Energy-Coupling Factor type transporter (ECF), homologous to the lhaSTA operon, has been found into a 13-gene putative operon and strongly overexpressed under iron-limitation condition. Moreover, the transcription of genes involved in resistance to oxidative stress (including catalase), virulence, transcriptional regulation, and hemin detoxification were also modified. These data provide some answers on the cellular response to the iron-limitation stress that is important for the opportunistic behavior of this pathogen.

Phenotypic and proteomic approaches of the response to iron-limited condition in Staphylococcus lugdunensis.

BACKGROUND: Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2'-dipyridyl (DIP). RESULTS: No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. CONCLUSIONS: This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.