Identification of Staphylococci to species level in veterinary microbiology is important to inform therapeutic intervention and management. We report on the efficacy of three routinely used commercial phenotypic methods for staphylococcal species identification, namely API Staph 32 (bioMérieux), RapID (Remel) and Staph-Zym (Rosco Diagnostica) compared to genotyping as a reference method to identify 52 staphylococcal clinical isolates (23 coagulase positive; 29 coagulase negative) from companion animals in Irish veterinary hospitals.
Genotyping of a 412 bp fragment of the staphylococcal tuf gene and coagulase testing were carried out on all 52 veterinary samples along with 7 reference strains. In addition, genotyping of the staphylococcal rpoB gene, as well as PCR-RFLP of the pta gene, were performed to definitively identify members of the Staphylococcus intermedius group (SIG). The API Staph 32 correctly identified all S. aureus isolates (11/11), 83% (10/12) of the SIG species, and 66% (19/29) of the coagulase negative species. RapID and Staph-Zym correctly identified 61% (14/23) and 0% (0/23) respectively of the coagulase-positives, and 10% (3/29) and 3% (1/29) respectively of the coagulase-negative species.
Commercially available phenotypic species identification tests are inadequate for the correct identification of both coagulase negative and coagulase positive staphylococcal species from companion animals. Genotyping using the tuf gene sequence is superior to phenotyping for identification of staphylococcal species of animal origin. However, use of PCR-RFLP of pta gene or rpoB sequencing is recommended as a confirmatory method for discriminating between SIG isolates.
Companion animals; Staphylococci species identification; Genotyping; tuf; rpoB
Coagulase negative staphylococci are the principal cause of prosthetic valve endocarditis but are a rare cause of native valve infections. However, the incidence of native valve endocarditis is increasing. Staphylococcus capitis is a coagulase negative staphylococcus with the capacity to cause endocarditis on native heart valves. Two cases of native valve endocarditis caused by S capitis are presented; both in patients with aortic valve disease. The patients were cured with prolonged intravenous vancomycin and rifampicin and did not need surgery during the acute phase of the illness. Five of the six previously described cases of endocarditis caused by this organism occurred on native valves and responded to medical treatment alone.
Keywords: Staphylococcus capitis; endocarditis; valvar disease; coagulase negative staphylococci
Coagulase negative staphylococci (CNS) were a rare cause of native valve endocarditis. However, they are emerging as an important cause of native valve endocarditis (NVE) in both community and healthcare settings. We describe a 64 yrs. old male who developed mitral valve endocarditis caused by coagulase negative staphylococci. There were no predisposing conditions or underlying cardiac disease that could have been the risk factor for the development of native valve infection. The patient had good recovery after six weeks of treatment with anti-staphylococcal antibiotics.
Coagulase negative staphylococcus; CoNS; Native valve endocarditis; NVE; Outpatient antimicrobial therapy; OPAT
A case of Staphylococcus lugdunensis endocarditis is presented with low back pain suggesting a secondary bone focus of infection. An umbilical skin lesion may have been an additional embolic phenomenon. The case highlights the aggressive nature of S lugdenensis endocarditis compared with other coagulase negative staphylococci and its association with native heart valves. In addition the importance of full identification of coagulase negative staphylococci isolated from patient samples in a case of suspected S lugdenensis infection is emphasised. Antibiotic treatment may be insufficient alone in the treatment of S lugdenensis endocarditis and early recourse to surgical intervention and valve replacement should therefore be considered.
Keywords: Staphylococcus lugdunensis; endocarditis; coagulase negative staphylococci
Staphylococcus lugdunensis has gained recognition as an atypically virulent pathogen with a unique microbiological and clinical profile. S. lugdunensis is coagulase negative due to the lack of production of secreted coagulase, but a membrane-bound form of the enzyme present in some isolates can result in misidentification of the organism as Staphylococcus aureus in the clinical microbiology laboratory. S. lugdunensis is a skin commensal and an infrequent pathogen compared to S. aureus and S. epidermidis, but clinically, infections caused by this organism resemble those caused by S. aureus rather than those caused by other coagulase-negative staphylococci. S. lugdunensis can cause acute and highly destructive cases of native valve endocarditis that often require surgical treatment in addition to antimicrobial therapy. Other types of S. lugdunensis infections include abscess and wound infection, urinary tract infection, and infection of intravascular catheters and other implanted medical devices. S. lugdunensis is generally susceptible to antimicrobial agents and shares CLSI antimicrobial susceptibility breakpoints with S. aureus. Virulence factors contributing to this organism's heightened pathogenicity remain largely unknown. Those characterized to date suggest that the organism has the ability to bind to and interact with host cells and to form biofilms on host tissues or prosthetic surfaces.
Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology, and most CNS-related infections are hospital acquired. Distinguishing between these frequently multiple-antibiotic-resistant isolates is important for both treatment and transmission control. In this study we used isolates of methicillin-resistant coagulase-negative staphylococci (MR-CNS) that were selected from a large surveillance study of the direct spread of MR-CNS. This strain collection was used to evaluate (i) Raman spectroscopy as a typing tool for MR-CNS isolates and (ii) diversity between colonies with identical and different morphologies. Reproducibility was high, with 215 of 216 (99.5%) of the replicate samples for 72 isolates ending up in the same cluster. The concordance with pulsed-field gel electrophoresis (PFGE)-based clusters was 94.4%. We also confirm that the skin of patients can be colonized with multiple MR-CNS types at the same time. Morphological differences between colonies from a single patient sample correlated with differences in Raman and PFGE types. Some morphologically indistinguishable colonies revealed different Raman and PFGE types. This indicates that multiple MR-CNS colonies should be examined to obtain a complete insight into the prevalence of different types and to be able to perform an accurate transmission analysis. Here we show that Raman spectroscopy is a reproducible typing system for MR-CNS isolates. It is a tool for screening variability within a collection of isolates. Because of the high throughput, it enables the analysis of multiple colonies per patient, which will enhance the quality of clinical and epidemiological studies.
Native valve endocarditis caused by coagulase negative staphylococci has become more common. A study of 35 cases showed that the infections were usually acquired in the community and occurred in men (mean age 51 years). A pre-existing cardiac abnormality (mitral leaflet prolapse in a third of patients) was detected in 26 (74%). The source of the organisms in the community acquired infections was assumed to be the skin, though lesions were seldom demonstrated; most hospital acquired infections resulted from intravenous devices. Community acquired organisms were usually sensitive to penicillin, whereas those acquired in hospital were often multiresistant. Most infections were caused by Staphylococcus epidermidis. The frequency of acute presentation (26%) and of major neurological abnormality (23%), together with the need for valve replacement (often emergency) (51%) and the mortality (36%) suggest that coagulase negative staphylococci can be virulent aggressive pathogens, mimicking Staphylococcus aureus.
Staphylococcus lugdunensis is a member of the coagulase-negative staphylococci and commonly found as part of the human skin flora. It is a significant cause of catheter-related bacteremia and also causes serious infections like native valve endocarditis in previously healthy individuals. We report the complete genome sequence of this medically important bacterium.
To describe the patient populations and infections being treated with daptomycin, as well as the efficacy and safety outcomes.
Patients and methods
Data from the European Cubicin Outcomes Registry and Experience (EU-CORESM), retrospectively collected at 118 institutions between January 2006 and August 2008, were analysed.
Daptomycin treatment was documented in 1127 patients with diverse infections, including complicated skin and soft tissue infections (33%), bacteraemia (22%), endocarditis (12%) and osteomyelitis (6%). It was used empirically, before microbiological results became available, in 53% of patients. Staphylococcus aureus was the most common pathogen (34%), with 52% of isolates resistant to methicillin; coagulase-negative staphylococci and enterococci were also frequent, with 22% of Enterococcus faecium isolates resistant to vancomycin. Daptomycin was used as first-line therapy in 302 (27%) patients. When used second line, the most common reasons for discontinuation of previous antibiotic were treatment failure and toxicity or intolerance. The use of concomitant antibiotics was reported in 65% of patients. Most frequent doses were 6 mg/kg (47%) and 4 mg/kg (32%). The median duration of daptomycin therapy was 10 days (range 1–246 days) in the inpatient setting and 13 days (range 2–189 days) in the outpatient setting. The overall clinical success rate was 79%, with a clinical failure rate of <10% for all infection types. Low failure rates were observed in first- and second-line therapy (6% and 8%, respectively). Daptomycin demonstrated a favourable safety and tolerability profile regardless of treatment duration.
Daptomycin has a relevant role in the treatment of Gram-positive infections.
cyclic lipopeptide; Gram-positive infections; registry
Staphylococcus capitis is a subtype of coagulase-negative staphylococci (CoNS) which could emerge as a significant pathogen causing infective endocarditis, prosthetic valve endocarditis, and late-onset sepsis. We isolated S. capitis strain QN1 from the skin swab sample of a female. Here we prepared a genome sequence for this strain consisting of 30 contigs totaling 2,430,101 bases and a GC content of 32.76%.
Endocarditis caused by Staphylococcus warneri and necessitating valve replacement occurred in a previously healthy 32-year-old patient following vasectomy. No sign of an underlying valvular defect was noted during the operation. S. warneri is a recently identified species of coagulase-negative staphylococci. Endocarditis caused by coagulase-negative staphylococci is uncommon in young, healthy patients with normal heart valves and has not previously been described as a complication of vasectomy. Similarly, infections caused by S. warneri have not previously been described in humans.
Optimal strategies for the prophylaxis and therapy of endocarditis caused by oxacillin-resistant, coagulase-negative staphylococci in patients with native or prosthetic valvular heart disease are not well defined. We compared the in vivo efficacies of ampicillin-sulbactam-based regimens with those of vancomycin-based oxacillin-resistant, beta-lactamase-producing coagulase-negative staphylococcal isolate (Staphylococcus haemolyticus SE220). Ampicillin-sulbactam (100 and 20 mg/kg of body weight, respectively, given intramuscularly in a two-dose regimen) was equivalent to vancomycin (30 mg/kg given intravenously in a two-dose regimen) in its prophylactic efficacy against the coagulase-negative staphylococcal strain (93 and 80%, respectively). The combination of ampicillin-sulbactam plus either rifampin or vancomycin did not enhance the prophylactic efficacy compared with that of ampicillin-sulbactam or vancomycin alone. In the therapy of established aortic valve endocarditis in rabbits caused by this same coagulase-negative staphylococcal strain, animals received 7-day ampicillin-sulbactam-based or vancomycin-based regimens with or without rifampin. All treatment regimens were effective at lowering intravegetation coagulase-negative staphylococcal densities and rendering vegetations culture negative compared with the coagulase-negative staphylococcal densities and vegetations of untreated controls, with ampicillin-sulbactam in combination with rifampin or vancomycin being the most active regimen. However, only the regimen of ampicillin-sulbactam in combination with vancomycin effectively prevented relapse of endocarditis posttherapy after a 5-day antibiotic-free period. For animals receiving rifampin-containing regimens, relapses of endocarditis were associated with the in vivo development of rifampin resistance among coagulase-negative staphylococcal isolates in the vegetation. Ampicillin-sulbactam was highly effective in the prevention of experimental endocarditis caused by a beta-lactamase-producing, oxacillin-resistant coagulase-negative staphylococcal strain. Ampicillin-sulbactam was also efficacious for the therapy of coagulase-negative staphylococcal endocarditis, especially when it was combined with vancomycin to prevent posttherapeutic relapses.
Coagulase negative staphylococci are skin commensals and are generally disregarded as contaminants in clinical specimens. Repeated isolation of coagulase negative staphylococci in blood cultures should warrant a species identification to recognize unusually virulent organisms that demand aggressive treatment, such as Staphylococcus lugdunensis. Staphylococcus lugdunensis is known to cause a wide variety of infections, including a predominant left-sided endocarditis. We report a rare case of native tricuspid valve Staphylococcus lugdunensis endocarditis in a non-intravenous drug user and include a brief literature review.
Staphylococcus lugdunensis; coagulase negative staphylococci; skin commensals; left-sided endocarditis
Staphylococcus simulans is a common animal pathogen that occasionally can colonize human skin. Unlike other coagulase-negative staphylococci, S. simulans tends to cause more severe infections that resemble those caused by S. aureus. We present a case of vertebral osteomyelitis and endocarditis due to S. simulans. To the best of our knowledge, this is the first report of vertebral osteomyelitis associated with native valve endocarditis rather than orthopedic surgery.
A 46-year-old male butcher was admitted to the hospital with a 4-week history of high fever with profound sweating. He reported weakness in his legs and low back pain that compromised his walking ability. Blood cultures yielded Gram-positive cocci on Gram stain. These cocci were identified to the species level as S. simulans, a coagulase-negative staphylococcus. The patient was treated with antibiotics, which were discontinued after 6 months.
This case illustrates the importance of identifying coagulase-negative staphylococci to the species level. Accurate identification of S. simulans would further help investigations defining its pathogenic role in human infections.
Staphylococcus lugdunensis is a coagulase-negative staphylococcus (CNS). It is a major cause of prosthetic valve endocarditis; mitral valve prolapse (MVP) has emerged as a prominent predisposing structural cardiac abnormality. We describe a case of Staphylococcus lugdunensis endocarditis in an 18-year-old woman with preexisting mitral valve prolapse complaining of fever, a one-month history of continuous-remittent fever (Tmax 38.6°C). The transthoracic echocardiogram revealed large vegetation on the anterior mitral valve leaflet flopping from the atrial side to the ventricular side. Five sets of blood cultures were positive for coagulase-negative staphylococci. During hospitalization, after two weeks of antibiotic therapy, the patient complained of sudden pain in her right leg associated with numbness. Lower limb arterial Doppler ultrasound showed an arterial thrombosis of right common iliac artery. Transfemoral iliac embolectomy was promptly performed and on septic embolus S. lugdunensis with the same antibiotic sensitivity and the same MIC values was again isolated. Our patient underwent cardiac surgery: triangular resection of the A2 with removal of infected tissue including vegetation. Our case is an example of infective endocarditis by S. lugdunensis on native mitral valve in a young woman of 18 with anamnesis valve prolapse.
Aim: To develop and evaluate a rapid enzyme linked immunosorbent assay (ELISA) for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci.
Methods: Forty patients with a clinical and microbiological diagnosis of intravascular catheter related sepsis and positive blood cultures, caused by coagulase negative staphylococci, and 40 control patients requiring a central venous catheter as part of their clinical management were recruited into the study. Serum IgG responses to a previously undetected exocellular antigen produced by coagulase negative staphylococci, termed lipid S, were determined in the patient groups by a rapid ELISA.
Results: There was a significant difference (p = < 0.0001) in serum IgG to lipid S between patients with catheter related sepsis and controls. The mean antibody titre in patients with sepsis caused by coagulase negative staphylococci was 10 429 (range, no detectable serum IgG antibody to 99 939), whereas serum IgG was not detected in the control group of patients.
Conclusions: The rapid ELISA offers a simple, economical, and rapid diagnostic test for suspected intravascular catheter related sepsis caused by coagulase negative staphylococci, which can be difficult to diagnose clinically. This may facilitate treatment with appropriate antimicrobials and may help prevent the unnecessary removal of intravascular catheters.
enzyme linked immunosorbent assay; coagulase negative staphylococci; catheter infections
Human infections caused by coagulase-negative staphylococci have steadily increased in numbers and severity. Causes may be the use of artificial prostheses, immunocompromising chemotherapy and radiation therapy, and sophisticated surgical techniques, to name a few. Although the infectivity of coagulase-negative staphylococci as a group has been well documented for humans, attempts to study the pathogenesis of infections caused by individual species of coagulase-negative staphylococci have been hampered by the lack of an animal model that is not refractory to infection by these organisms. In the study reported here, a 2-day-old-mouse weight retardation test was used to assay the virulence of 60 clinical and reference strains of coagulase-negative staphylococci. These strains represented eight species of coagulase-negative staphylococci. The most virulent strains were demonstrated to be of the species Staphylococcus haemolyticus, S. saprophyticus, and S. epidermidis. The data further suggest that production of slime is a marker of virulence in S. epidermidis and that intraspecies differences in virulence occur.
Vancomycin resistance has been reported in clinical isolates of both coagulase-negative staphylococci and Staphylococcus aureus. The emerging threat of widespread vancomycin resistance poses a serious public health concern given the fact that vancomycin has long been the preferred treatment of antibiotic-resistant gram-positive organisms. Though major efforts are now being focused on improving our understanding of vancomycin resistance, there is much that remains unknown at this time. This article reviews the major epidemiologic, microbiologic, and clinical characteristics of vancomycin resistance in both coagulase-negative staphylococci and S. aureus. The review begins with a discussion of issues common to both coagulase-negative staphylococci and S. aureus, such as definitions, laboratory detection of vancomycin resistance, and infection control issues related to vancomycin-resistant staphylococci. The rest of the article is then devoted to a discussion of issues unique to each organism, including epidemiology, risk factors for infection, mechanisms of resistance, and management options.
Coagulase-negative staphylococci (CoNS) have been increasing in importance as a cause of native valve endocarditis (NVE). Most cases of NVE caused by CoNS are attributable to Staphylococcus epidermidis. NVE caused by CoNS acquired in a nosocomial setting may differ from cases acquired in the community in several ways. It may be associated with hemodialysis, the presence of a long-term indwelling central catheter or pacemaker, or a recent invasive procedure; nosocomial cases may have a higher rate of methicillin resistance among CoNS isolates, and so be more likely to be treated with vancomycin. Unfortunately, NVE caused by methicillin-resistant CoNS has been associated with significantly higher rates of persistent bacteremia and in-hospital mortality than methicillin-susceptible isolates. The poor outcomes in these cases point to the need for alternative therapies with potent activity against methicillin-resistant CoNS. In our medical center, a 76-year-old man presented with native-valve endocarditis and positive blood cultures for methicillin-resistant Staphylococcus epidermidis (MRSE). During each of three 6-week courses of treatment with vancomycin, blood cultures were negative, but they once again became positive for MRSE when vancomycin was discontinued. The minimum inhibitory concentration of the MRSE isolates for vancomycin remained stable at 2 μg/mL. Eventually, treatment with daptomycin was initiated (500 mg [7 mg/kg]) 3 times/week for 6 weeks. Over the following year, no positive cultures for MRSE were detected.
Nosocomial bloodstream infections (nBSIs) are an important cause of morbidity and mortality. Data from a nationwide, concurrent surveillance study, Brazilian SCOPE (Surveillance and Control of Pathogens of Epidemiological Importance), were used to examine the epidemiology and microbiology of nBSIs at 16 Brazilian hospitals. In our study 2,563 patients with nBSIs were included from 12 June 2007 to 31 March 2010. Ninety-five percent of BSIs were monomicrobial. Gram-negative organisms caused 58.5% of these BSIs, Gram-positive organisms caused 35.4%, and fungi caused 6.1%. The most common pathogens (monomicrobial) were Staphylococcus aureus (14.0%), coagulase-negative staphylococci (CoNS) (12.6%), Klebsiella spp. (12.0%), and Acinetobacter spp. (11.4%). The crude mortality was 40.0%. Forty-nine percent of nBSIs occurred in the intensive-care unit (ICU). The most frequent underlying conditions were malignancy, in 622 patients (24.3%). Among the potential factors predisposing patients to BSI, central venous catheters were the most frequent (70.3%). Methicillin resistance was detected in 157 S. aureus isolates (43.7%). Of the Klebsiella sp. isolates, 54.9% were resistant to third-generation cephalosporins. Of the Acinetobacter spp. and Pseudomonas aeruginosa isolates, 55.9% and 36.8%, respectively, were resistant to imipenem. In our multicenter study, we found high crude mortality and a high proportion of nBSIs due to antibiotic-resistant organisms.
Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, are increasingly important causes of nosocomial infection. Microbiologists and clinicians no longer can afford to disregard clinical isolates of coagulase-negative staphylococci as contaminants. Accurate species identification and antimicrobial susceptibility testing, in a clinically relevant time frame, are important aids in the diagnosis and management of serious coagulase-negative staphylococcal infections. Emphasis in the clinical laboratory should be placed on the routine identification of S. epidermidis and Staphylococcus saprophyticus, with identification of other species of coagulase-negative staphylococci as clinically indicated. The application of newer techniques, such as plasmid analysis and tests for slime production and adherence, contribute to our understanding of the epidemiology and pathogenesis of coagulase-negative staphylococci and may also be helpful in establishing the diagnosis of infection.
Geographical differences in the genetic diversity of Helicobacter pylori isolates were examined by analyzing rpoB sequences. An extremely high level of allelic diversity among H. pylori strains was found. The rpoB sequences of Asian and non-Asian (North and South American, European, and South African) strains were found to differ. An amino acid polymorphism (alanine and threonine RpoB types) was found at the 497th residue by deduced amino acid analysis. RpoB with a threonine residue (RpoBThr) was uniquely present in East Asian countries, and two-thirds of the H. pylori isolate population in this region was RpoBThr; however, this type was rare or absent in Western countries, where RpoBAla predominated. RpoBThr strains induced a much larger amount of interleukin-8, a chemokine that plays an important role in chronic inflammation, than RpoBAla strains in cultured MKN45 cells.
We used amplification of the 16S rRNA gene followed by sequencing to evaluate the persistence of bacterial DNA in explanted heart valve tissue as part of the routine work of a clinical microbiology laboratory, and we analyzed the role of this persistence in the relapses observed in our center. We enrolled 286 patients treated for infective endocarditis (IE) who had valve replacement surgery and were diagnosed according to the modified Duke’s criteria described by Li et al. from a total of 579 IE cases treated in our center. The patients were grouped based on the infecting bacteria, and we considered the 4 most common bacterial genus associated with IE separately (144 were caused by Streptococcus spp., 52 by Enterococcus spp., 58 by Staphylococcus aureus and 32 by coagulase-negative Staphylococcus). Based on our cohort, the risk of relapse in patients with enterococcal prosthetic valve infections treated with antibiotics alone was 11%. Bacterial DNA is cleared over time, but this might be a very slow process, especially with Enterococcus spp. Based on a comprehensive review of the literature performed on Medline, most reports still advise combined treatment with penicillin and an aminoglycoside for as long as 4–6 weeks, but there has been no consensus for the treatment of enterococcal infection of prostheses in IE patients.
With the increase in nosocomial infections caused by coagulase negative staphylococci (CNS), laboratory diagnosis of CNS with reduced susceptibility to glycopeptides (vancomycin and teicoplanin) has become important. This study was designed to determine the glycopeptide susceptibility of clinical isolates of methicillin resistant coagulase negative staphylococci (MRCNS) at the department of microbiology, government medical college and hospital, Amritsar, India.
A total of 250 CNS isolated from various clinical specimens were speciated and their methicillin resistance was detected by studying the minimum inhibitory concentration (MIC) of oxacillin by macrobroth dilution method. Glycopeptide susceptibility of 130 methicillin resistant strains obtained was determined for vancomycin by vancomycin screen agar test, MIC of vancomycin by macrobroth dilution/and E test. Teicoplanin susceptibility was determined using teicoplanin disc diffusion test and MIC was determined by macrobroth dilution method.
All the MRCNS isolates were found to be susceptible to vancomycin and teicoplanin. MIC of vancomycin ranged between ≤0.5 µg/ml to 1 µg/ml and of teicoplanin from ≤0.5 µg/ml to 2µg/ml.
Continuous monitoring of MIC of vancomycin in MRCNS is required to prevent the emergence of vancomycin resistance in these multidrug resistant organisms.
Glycopeptides; Minimum inhibitory concentration
Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory.