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.

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.

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.

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.

Characterization of a novel, type II staphylococcal cassette chromosome mec element from an endemic oxacillin-resistant Staphylococcus lugdunensis clone in a hospital setting.

BACKGROUND: Staphylococcus lugdunensis is a significant pathogen that causes community-acquired and nosocomial infections. The high prevalence of oxacillin-resistant S. lugdunensis (ORSL) is of major concern. Resistance to ß-lactams is caused by acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. The cassette is highly diverse, both structurally and genetically, among CoNS. Isolates carrying SCCmec II-ST6 are the major persistent clones in hospitals. OBJECTIVES: To investigate the structure and evolutionary origin of a novel type II SCCmec element in an endemic ST6 S. lugdunensis clone. METHODS: The structure of the SCCmec II element carried by ST6 strain CGMH-SL118 was determined by WGS and compared with those reported previously. RESULTS: A novel 39 kb SCCmec element, SCCmecCGMH-SL118, with a unique mosaic structure comprising 41 ORFs integrated into the 3' end of the rlmH gene, was observed. Some regions of SCCmecCGMH-SL118 were homologous to SCCmec IIa of the prototype MRSA strain N315. The structure of SCCmecCGMH-SL118 was similar to that of SCCmec IIb of the MRSA strain, JCSC3063, mainly lacking the aminoglycoside resistance determinant pUB110 in the J3 region but containing the insertion sequence IS256 in the J2 region. Notably, SCCmecCGMH-SL118 deletions in the J1 region compared with SCCmec types IIa and IIb, and a high homology to SCCmec elements of Staphylococcus aureus JCSC4610 and Staphylococcus haemolyticus strain 621 were found. CONCLUSIONS: The genetic diversity of the type II SCCmec element in ORSL suggests that CoNS is a potential reservoir for interspecies transfer of SCCmec to S. aureus in hospitals.

Staphylococcus lugdunensis: antimicrobial susceptibility and optimal treatment options.

Staphylococcus lugdunensis is a coagulase-negative staphylococcus (CoNS) with unusual pathogenicity resembling that of S. aureus. Unlike other CoNS, S. lugdunensis remains susceptible to most antibiotics. The resistance to penicillin varies widely (range, 15-87% worldwide), whereas methicillin resistance is still rare. We aimed to evaluate treatment options for infections caused by S. lugdunensis and more specifically to investigate whether penicillin G could be a better treatment choice than oxacillin. Susceptibility testing was performed using the disc diffusion method for penicillin G, cefoxitin, trimethoprim/sulfamethoxazole, erythromycin, clindamycin, gentamicin, norfloxacin, fusidic acid, rifampicin, and fosfomycin. Isolates susceptible to penicillin G were further tested with a gradient test for penicillin G and oxacillin. Of the 540 clinical isolates tested, 74.6% were susceptible to penicillin G. Among these penicillin-susceptible isolates, the MIC50 and MIC90 values for penicillin G were threefold lower than that for oxacillin. A majority of the isolates were susceptible to all other antibiotics tested. Breakpoints for fosfomycin have not yet been defined, and so no conclusions could be drawn. Two isolates were resistant to cefoxitin and carried the mecA gene; whole-genome sequencing revealed that both harbored the SCCmec element type IVa(2B). S. lugdunensis isolated in Sweden were susceptible to most tested antibiotics. Penicillin G may be a more optimal treatment choice than oxacillin. Although carriage of the mecA gene is rare among S. lugdunensis, it does occur.

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk.

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.

Comparative genomic analysis of Staphylococcus lugdunensis shows a closed pan-genome and multiple barriers to horizontal gene transfer.

BACKGROUND: Coagulase negative staphylococci (CoNS) are commensal bacteria on human skin. Staphylococcus lugdunensis is a unique CoNS which produces various virulence factors and may, like S. aureus, cause severe infections, particularly in hospital settings. Unlike other staphylococci, it remains highly susceptible to antimicrobials, and genome-based phylogenetic studies have evidenced a highly conserved genome that distinguishes it from all other staphylococci. RESULTS: We demonstrate that S. lugdunensis possesses a closed pan-genome with a very limited number of new genes, in contrast to other staphylococci that have an open pan-genome. Whole-genome nucleotide and amino acid identity levels are also higher than in other staphylococci. We identified numerous genetic barriers to horizontal gene transfer that might explain this result. The S. lugdunensis genome has multiple operons encoding for restriction-modification, CRISPR/Cas and toxin/antitoxin systems. We also identified a new PIN-like domain-associated protein that might belong to a larger operon, comprising a metalloprotease, that could function as a new toxin/antitoxin or detoxification system. CONCLUSION: We show that S. lugdunensis has a unique genome profile within staphylococci, with a closed pan-genome and several systems to prevent horizontal gene transfer. Its virulence in clinical settings does not rely on its ability to acquire and exchange antibiotic resistance genes or other virulence factors as shown for other staphylococci.

Characterization of two novel variants of staphylococcal cassette chromosome mec elements in oxacillin-resistant Staphylococcus lugdunensis.

OBJECTIVES: Staphylococcus lugdunensis, a species of CoNS, has become an important hospital pathogen because of increasing resistance to ß-lactam antibiotics such as methicillin and oxacillin. Methicillin resistance is mainly due to the acquisition of the staphylococcal cassette chromosome (SCC) mec (SCCmec). Little is known about the structure of SCCmec in methicillin- or oxacillin-resistant CoNS. METHODS: WGS was performed to determine the structure of SCCmec elements of two clinical S. lugdunensis isolates: CMUH-22 and CMUH-25. RESULTS: These elements were found to be flanked by DRs and IRs with unique mosaic structures and a common integration site in the 3' end of the rlmH gene. The sequences of the regions located between rlmH and the ISSau4-like transposase genes of both elements were similar to those of SCCmec Vt of Staphylococcus aureus PM1. The SCCmec (type V, 5C2&4) of CMUH-25 harboured a novel ccrC complex and a C2-like mec complex in opposite orientations, similar to the type V SCCmec of S. aureus WIS. The sequences of the ccrA4B4 genes and J1 and J2 regions of CMUH-25 were similar to those of the SCC element of Staphylococcus haemolyticus NCTC 11042. In contrast, portions of the sequence of the J1 region of type Vt (5C2) SCCmec in strain CMUH-22 were highly similar to portions of those of Staphylococcus epidermidis RP62A and the composite SCCmec type V of S. aureus WAMRSA40. CONCLUSIONS: These observations suggest that the SCCmec elements of CMUH-25 and CMUH-22 evolved separately and assembled through different recombination events.

Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

Emergence of ileS2-Carrying, Multidrug-Resistant Plasmids in Staphylococcus lugdunensis.

Of 137 Staphylococcus lugdunensis isolates collected from two nephrology centers in Hong Kong, 10 (7.3%) and 3 (2.2%) isolates had high-level and low-level mupirocin resistance, respectively. Isolates with high-level resistance contained the plasmid-mediated ileS2 gene, while isolates with low-level resistance contained the mutation V588F within the chromosomal ileS gene. All but one of the ileS2-positive isolates belong to the predominating clone HKU1. Plasmids carrying the ileS2 gene were mosaic and also cocarry multiple other resistance determinants.

Clinical Features, Outcomes, and Molecular Characteristics of Community- and Health Care-Associated Staphylococcus lugdunensis Infections.

Staphylococcus lugdunensis is a major cause of aggressive endocarditis, but it is also responsible for a broad spectrum of infections. The differences in clinical and molecular characteristics between community-associated (CA) and health care-associated (HA) S. lugdunensis infections have remained unclear. We performed a retrospective study of S. lugdunensis infections between 2003 and 2014 to compare the clinical and molecular characteristics of CA and HA isolates. We collected 129 S. lugdunensis isolates in total: 81 (62.8%) HA isolates and 48 (37.2%) CA isolates. HA infections were more frequent than CA infections in children (16.0% versus 4.2%, respectively; P = 0.041) and the elderly (38.3% versus 14.6%, respectively; P = 0.004). The CA isolates were more likely to cause skin and soft tissue infections (85.4% versus 19.8%, respectively; P < 0.001). HA isolates were more frequently responsible for bacteremia of unknown origin (34.6% versus 4.2%, respectively; P < 0.001) and for catheter-related bacteremia (12.3% versus 0%, respectively; P = 0.011) than CA isolates. Fourteen-day mortality was higher for HA infections than for CA infections (11.1% versus 0%, respectively). A higher proportion of the HA isolates than of the CA isolates were resistant to penicillin (76.5% versus 52.1%, respectively; P = 0.004) and oxacillin (32.1% versus 2.1%, respectively; P < 0.001). Two major clonal complexes (CC1 and CC3) were identified. Sequence type 41 (ST41) was the most common sequence type identified (29.5%). The proportion of ST38 isolates was higher for HA than for CA infections (33.3% versus 12.5%, respectively; P = 0.009). These isolates were of staphylococcal cassette chromosome mec element (SCCmec)type IV, V, or Vt. HA and CA S. lugdunensis infections differ in terms of their clinical features, outcome, antibiotic susceptibilities, and molecular characteristics.

Disk Diffusion Testing for Detection of Methicillin-Resistant Staphylococci: Does Moxalactam Improve upon Cefoxitin?

Disk diffusion testing is widely used to detect methicillin resistance in staphylococci, and cefoxitin is currently considered the best marker for mecA-mediated methicillin resistance. In low-inoculum diffusion testing (colony suspension at 106 CFU/ml), the addition of moxalactam in combination with cefoxitin has been reported to improve on cefoxitin alone for the detection of methicillin-heteroresistant staphylococci. However, moxalactam is absent from EUCAST and CLSI guidelines, which use high-inoculum diffusion testing (colony suspension at 108 CFU/ml), calling into question the potential interest of including moxalactam in their recommendations. The inhibition zone diameters of cefoxitin and moxalactam, alone and in combination, were evaluated for concordance with mecA and mecC positivity in a large collection of clinical Staphylococcus isolates (611 Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus saprophyticus isolates and 307 coagulase-negative staphylococci other than S. lugdunensis and S. saprophyticus isolates, of which 22% and 53% were mecA-positive, respectively) and in 25 mecC-positive S. aureus isolates using high-inoculum diffusion testing. Receiver operating characteristic, sensitivity, and specificity analyses indicated that the detection of mecA- and mecC-positive and negative isolates did not improve with moxalactam, either alone or in combination with cefoxitin, compared to cefoxitin alone. These findings were similar in both the S. aureus/S. lugdunensis/S. saprophyticus group and in the coagulase-negative staphylococci group. Our results do not support the use of moxalactam as an additional marker of methicillin resistance when testing with high-inoculum disk diffusion.

Important contribution of the novel locus comEB to extracellular DNA-dependent Staphylococcus lugdunensis biofilm formation.

The coagulase-negative species Staphylococcus lugdunensis is an emerging cause of serious and potentially life-threatening infections, such as infective endocarditis. The pathogenesis of these infections is characterized by the ability of S. lugdunensis to form biofilms on either biotic or abiotic surfaces. To elucidate the genetic basis of biofilm formation in S. lugdunensis, we performed transposon (Tn917) mutagenesis. One mutant had a significantly reduced biofilm-forming capacity and carried a Tn917 insertion within the competence gene comEB. Site-directed mutagenesis and subsequent complementation with a functional copy of comEB verified the importance of comEB in biofilm formation. In several bacterial species, natural competence stimulates DNA release via lysis-dependent or -independent mechanisms. Extracellular DNA (eDNA) has been demonstrated to be an important structural component of many bacterial biofilms. Therefore, we quantified the eDNA in the biofilms and found diminished eDNA amounts in the comEB mutant biofilm. High-resolution images and three-dimensional data obtained via confocal laser scanning microscopy (CSLM) visualized the impact of the comEB mutation on biofilm integrity. The comEB mutant did not show reduced expression of autolysin genes, decreased autolytic activities, or increased cell viability, suggesting a cell lysis-independent mechanism of DNA release. Furthermore, reduced amounts of eDNA in the comEB mutant biofilms did not result from elevated levels or activity of the S. lugdunensis thermonuclease NucI. In conclusion, we defined here, for the first time, a role for the competence gene comEB in staphylococcal biofilm formation. Our findings indicate that comEB stimulates biofilm formation via a lysis-independent mechanism of DNA release.

Virulence factors among Staphylococcus lugdunensis are associated with infection sites and clonal spread.

Staphylococcus lugdunensis has emerged as a significant human pathogen, with distinct clinical and microbiological characteristics. Our goal was to identify the virulence factors in S. lugdunensis recovered from infected patients of two Greek hospitals during a six-year period (2008-2013). A collection of 38 S. lugdunensis was tested for biofilm formation, antimicrobial susceptibility, clonal distribution, virulence factors (ica operon, fbl, atlL, vwbl, slush) and antibiotic resistance genes (mecA, ermC) carriage. Strains were classified into pulsotypes by pulsed-field gel electrophoresis (PFGE) of SmaI DNA digests. The majority (22) was isolated from skin and soft tissue infections (SSTIs), nine from deep-sited infections (DSIs), including three bacteraemias and seven from prosthetic device-associated infections (PDAIs). All isolates were oxacillin-susceptible, mecA-negative and fbl-positive. The highest resistance rate was detected for ampicillin (50%), followed by erythromycin and clindamycin (18.4%). Fourteen isolates (36.8%) produced biofilm, whereas 26/38 (68.4%) carried the ica operon. Biofilm formation was more frequent in isolates from PDAIs. Thirty-six strains (94.7%) carried atlL and 31 (81.6%) carried vwbl, whereas slush was detected in 15 (39.5%). PFGE revealed a low level of genetic diversity: strains were classified into seven pulsotypes, with two major clones (C: 22 and D: nine strains). Type C strains recovered from all infection sites prevailed in biofilm formation and ermC carriage, whereas type D strains associated with SSTIs and DSIs carried more frequently vwbl, slush or both genes. Despite susceptibility to antimicrobials, the clonal expansion and carriage of virulence factors, combined with biofilm-producing ability, render this species an important pathogen that should not be ignored.

Multi-virulence-locus sequence typing of Staphylococcus lugdunensis generates results consistent with a clonal population structure and is reliable for epidemiological typing.

Staphylococcus lugdunensis is an emergent virulent coagulase-negative staphylococcus responsible for severe infections similar to those caused by Staphylococcus aureus. To understand its potentially pathogenic capacity and have further detailed knowledge of the molecular traits of this organism, 93 isolates from various geographic origins were analyzed by multi-virulence-locus sequence typing (MVLST), targeting seven known or putative virulence-associated loci (atlLR2, atlLR3, hlb, isdJ, SLUG_09050, SLUG_16930, and vwbl). The polymorphisms of the putative virulence-associated loci were moderate and comparable to those of the housekeeping genes analyzed by multilocus sequence typing (MLST). However, the MVLST scheme generated 43 virulence types (VTs) compared to 20 sequence types (STs) based on MLST, indicating that MVLST was significantly more discriminating (Simpson's index [D], 0.943). No hypervirulent lineage or cluster specific to carriage strains was defined. The results of multilocus sequence analysis of known and putative virulence-associated loci are consistent with a clonal population structure for S. lugdunensis, suggesting a coevolution of these genes with housekeeping genes. Indeed, the nonsynonymous to synonymous evolutionary substitutions (dN/dS) ratio, the Tajima's D test, and Single-likelihood ancestor counting (SLAC) analysis suggest that all virulence-associated loci were under negative selection, even atlLR2 (AtlL protein) and SLUG_16930 (FbpA homologue), for which the dN/dS ratios were higher. In addition, this analysis of virulence-associated loci allowed us to propose a trilocus sequence typing scheme based on the intragenic regions of atlLR3, isdJ, and SLUG_16930, which is more discriminant than MLST for studying short-term epidemiology and further characterizing the lineages of the rare but highly pathogenic S. lugdunensis.