Genome sequence of Staphylococcus lugdunensis N920143 allows identification of putative colonization and virulence factors

Staphylococcus lugdunensis is an opportunistic pathogen related to Staphylococcus aureus and Staphylococcus epidermidis. The genome sequence of S. lugdunensis strain N920143 has been compared with other staphylococci, and genes were identified that could promote survival of S. lugdunensis on human skin and pathogenesis of infections. Staphylococcus lugdunensis lacks virulence factors that characterize S. aureus and harbours a smaller number of genes encoding surface proteins. It is the only staphylococcal species other than S. aureus that possesses a locus encoding iron-regulated surface determinant (Isd) proteins involved in iron acquisition from haemoglobin.     

Biomaterial-associated staphylococcal peritoneal infections in a neutropaenic mouse model

Abstract Adhesion of staphylococcal cells to polyethylene with end point-attached heparin was quantified by bioluminescence. Staphylococcus epidermidis 3380 and the slime-producing S. epidermidis RP12 adhered to the highest extent, and S. lugdunensis 2342 to the least extent. Preincubation of the polymer with dialysis fluid reduced adhesion of S. epidermidis 3380 and RP12 but enhanced that of S. aureus , and preadsorption of the surface with fibronectin decreased subsequent adhesion of S. epidermidis and S. haemolyticus strains. When staphylococci were grown in the presence of a biomaterial their ability to activate peritoneal cells was decreased. The bactericidal activity was impaired, whereas ingestion of opsonized coagulase-negative staphylococci (CNS) strains was unaffected. With S. epidermidis RP12 the presence of biomaterial did not influence either phagocytosis or bactericidal effect of peritoneal cells. After intra-peritoneal challenge with staphylococcal strains, the organ uptake of S. aureus Cowan 1 was increased in normal mice whereas immunosuppressed mice died. CNS strains increased mainly in the peritoneal cavity of immunosuppressed mice. The uptake of bacteria in liver and kidneys was increased with S. epidermidis 3380, S. lugdunensis 2343 and S. schleiferi 667-88. Generally, CNS strains persisted in the peritoneal cavity of both normal and immunosuppressed mice. These data indicate that host defense mechanisms, mainly polymorphonuclear neutrophils, fail to eliminate CNS infections in the peritoneum, and that initial adhesion to an implanted biomaterial may be of lesser importance in the peritoneal cavity than in e.g. catheter-associated infections. There are strain-specific virulence factors of bacteria, and slime producing strains evade the host defense mechanisms more efficiently than non-slime producing strains.     

Phenotypic Variants of Staphylococci and Their Underlying Population Distributions Following Exposure to Stress

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4°C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL^-1), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4°C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4°C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.     

医院感染葡萄球菌菌种变迁与耐药性近况

目的：了解近9年来医院感染葡萄球菌菌种的变迁与近3年来葡萄球菌药状况。方法：1993年1月至2001年12月我院传染病科等13科室住院病人的各种标本采用血琼脂培养,所分离的葡萄球菌采用美国DADE公司生产的MICROSCAN WALKAY-40全自动微生物分析仪鉴定到种及其亚种。药敏试验药物有青霉素、苯唑西林、氨苄西林、哌拉西林、阿莫西林／克拉维酸（阿莫仙）、头孢唑啉、头孢噻肟、头孢哌酮／舒巴坦（舒普深）、庆大霉素、复方新诺明、环丙沙星、氧氟沙星、利福平、万古霉素、红霉素、林可霉素、四环素、伊米配能／西司他丁（泰能）共18种。采用液体稀释法测定每株葡萄球菌对受试药物的最小抑菌浓度（MIC）,操作按说明书进行。质控菌ATCC25923。依据新近NCCLS标准判读结果。结果：1993年至1998年分离的葡萄球菌中,金黄色葡萄球菌（金葡萄）占71.43%,凝固酶阴性葡萄球菌（CNS）占28.57%,包括表皮葡萄球菌（表葡菌）、腐生葡萄球菌、里昂葡萄球菌和头状葡萄球菌4种。1999年至2001年分离的424株葡萄球菌中,金葡菌仅占29.01%,CNS增至13种,占70.995,以表葡菌、溶血葡萄球菌、松鼠葡萄球菌为主。近3年来分离的各种葡萄球菌甲氧西林耐药率在73.03%-100%之间,除对舒普深、复方新诺明、利福平和万古霉素较敏感外,对其余抗菌药物的耐药率均超过60％,以金葡菌、溶血葡萄球菌、松鼠葡萄球菌的耐药率为最高。MRS耐药率普遍高于MSS,且均呈多重耐药。5.42%(23/424)菌株万古霉素MIC＞16mg/L,除1株为MSCNS外,其余22株均为MRS。结论：3年来医院感染葡萄球菌菌种构成比发生了显著变化,以CNS为主。对抗菌药物呈多重耐药,部分菌株对万古霉素敏感性降低,应予警惕。     

Incorporation of carbon from decomposing litter of two pioneer plant species into microbial communities of the detritusphere

For several Staphylococci, such as Staphylococcus aureus, Staphylococcus saprophyticus, and Staphylococcus epidermidis, invasion of eukaryotic cells has been described and this mechanism has been considered an important part of the infection process. The fibrinogen-binding protein (Fbl) of Staphylococcus lugdunensis, a homolog of the clumping factor A of S. aureus, has been described as fibrinogen-binding adhesin and might promote invasion of cells. We therefore characterized several clinical strains of S. lugdunensis in terms of whole cell fibrinogen and fibronectin binding and correlated these results with the invasion of epithelial and endothelial cells by S. lugdunensis. We described for the first time invasion of cells by S. lugdunensis. As invasion of cells by S. lugdunensis was only partly inhibited by cytochalasin D in contrast to a complete inhibition of invasion of cells by S. aureus, further invasion mechanisms are likely to be present in S. lugdunensis. In addition, the Fbl of S. lugdunensis is not involved in the invasion of cells as ruled out by an isogenic fbl mutant.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.     

Characteristics of atypical forms of staphylococci (SCVs) isolated from patients with osteomyelitis

Small-colony variants (SCVs), isolated from a population of the parental strains of Staphylococcus aureus, S. haemolyticus and S. epidermidis lost a number of features typical of the species and genus and were characterized by delayed growth, altered colony morphology, lack of pigmentation and changed carbohydrate consumption. Some SCVs of S. aureus had no plasmocoagulase and lecithinase activities. The analysis of 14 SCVs showed that they were auxotrophic for hemin and menadione and resistant to aminoglycoside antibiotics. Such aberrant phenotypic characteristics complicated or made it impossible their identification by the common clinical laboratory methods. The tRNA intergenic spacer length polymorphism analysis was used to identify the atypical forms of the staphylococci.     

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.     

High Genetic Variability of the agr Locus in Staphylococcus Species

The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.     

Cloning of an agr homologue of Staphylococcus saprophyticus

An agr homologue of Staphylococcus saprophyticus was identified, cloned and sequenced. The gene locus shows homologies to other staphylococcal agr systems, especially to those of S. epidermidis and S. lugdunensis. A putative RNAIII was identified and found to be differentially expressed during the growth phases. In contrast to the RNAIII molecules of S. epidermidis and S. aureus it does not contain an open reading frame that codes for a protein with homologies to the delta-toxin. Using PCR, the agr was found to be present in clinical isolates of S. saprophyticus.     

Neutrophil-stimulating activity of staphylococci based on reactive chemiluminescence data

The neutrophil-stimulating properties of 38 S. aureus strains and 32 S. epidermidis strains were studied in the reaction of luminol-mediated chemiluminescence. All S. aureus strains and 29 S. epidermidis strains were found to possess neutrophil-stimulating activity, the mean activity index for S. aureus being significantly higher. The stimulating activity of the strains varied within a wide range (the variation coefficient was 120.0 +/- 21.9%) and did not correlate with the content of protein A in bacterial cells and the degree of their hydrophoby. The opsonization of staphylococci with normal human serum enhanced the neutrophil reaction 1.5- to 100-fold and simultaneously leveled out the chemiluminescence indices in experiments with different strains (the variation coefficient was 8.0 +/- 1.5%). The nature of the neutrophil-stimulating effect of staphylococci and its relationship to the exploratory reactions of phagocytes are discussed.     

Staphylococci in orthopaedic surgical wounds.

From 50 infected surgical wounds of orthopaedic patients, 43 (86%) staphylococcal strains were isolated. 34 of all these staphylococci belonged to Staphylococcus aureus species (i.e. 68 % of all isolates from surgical wounds; 79% of staphylococcal isolates); 9 were coagulase-negative staphylococci (i.e. 21% of all isolates from surgical wounds; 18% of staphylococcal isolates). Among microorganisms isolated from the wounds we also found 2 (4%) of the Enterobacteriaceae family; 2 (4%) of the Pseudomonas genus; 3 (6%) of the Streptococcus genus. Thus, orthopaedic surgical wounds were infected by staphylococci (mainly S. aureus) more frequently than by other micro-organisms. All the staphylococcal strains were screened for methicillin resistance by agar disk diffusion testing and for the presence of mecA gene responsible for methicillin resistance by PCR. 32% of the S. aureus and 33% of the S. epidermidis strains resulted methicillin resistant and mecA-positive. The data confirm the diffusion of methicillin resistant S. aureus in surgical site infections and shows that the so-called "new pathogens", i.e. S. epidermidis and other coagulase-negative staphylococci, also exhibit a frequent and hazardous methicillin-resisting ability.     

Phenotype Switching Is a Natural Consequence of Staphylococcus aureus Replication

The pathogen Staphylococcus aureus undergoes phenotype switching in vivo from its normal colony phenotype (NCP) to a slow-growing, antibiotic-resistant small-colony-variant (SCV) phenotype that is associated with persistence in host cells and tissues. However, it is not clear whether phenotype switching is the result of a constitutive process that is selected for under certain conditions or is triggered by particular environmental stimuli. Examination of cultures of diverse S. aureus strains in the absence of selective pressure consistently revealed a small gentamicin-resistant SCV subpopulation that emerged during exponential-phase NCP growth and increased in number until NCP stationary phase. Treatment of replicating bacteria with the antibiotic gentamicin, which inhibited NCP but not SCV replication, resulted in an initial decrease in SCV numbers, demonstrating that SCVs arise as a consequence of NCP replication. However, SCV population expansion in the presence of gentamicin was reestablished by selection of phenotype-stable SCVs and subsequent SCV replication. In the absence of selective pressure, however, phenotype switching was bidirectional and occurred at a high frequency during NCP replication, resulting in SCV turnover. In summary, these data demonstrate that S. aureus phenotype switching occurs via a constitutive mechanism that generates a dynamic, antibiotic-resistant subpopulation of bacteria that can revert to the parental phenotype. The emergence of SCVs can therefore be considered a normal part of the S. aureus life cycle and provides an insurance policy against exposure to antibiotics that would otherwise eliminate the entire population.     

Identification of Micrococcaceae in Clinical Bacteriology

The cellular morphology, identifying physiological characteristics, and a key to the human genera of Micrococcaceae are presented with flow charts for identification of aerobic and anaerobic isolates. These flow charts can be amended as desired, depending upon the degree of accuracy desired. Micrococcaceae isolates in a 350-bed private general hospital during a 15-week period are tabulated to show relative numbers of the different genera and species, with their probable relationship to infection or contamination. Only 11 of the 220 Micrococcaceae isolates were not Staphylococcus; no Sarcina or Peptococcus were isolated. Of the Staphylococcus isolates, 61% were S. epidermidis. Almost 18% of the S. aureus isolates were coagulase-negative. Of the S. aureus isolates, 80% of the coagulase-positive isolates were infecting agents, as were 67% of the coagulase-negative S. aureus isolates, compared to only 48% of S. epidermidis isolates. Two of four Gaffkya isolates but only one of seven Micrococcus isolates were infecting agents. If coagulase production is used as the sole criterion for speciation of staphylococci, and Micrococcus is not differentiated from Staphylococcus, the term "coagulase-negative staphylococci" does not differentiate three distinct levels of pathogenicity. Coagulase-negative S. aureus is more virulent than S. epidermidis or Gaffkya, which are more virulent than Micrococcus or Sarcina.     

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.     

Pathogenicity of Staphylococcus lugdunensis, Staphylococcus schleiferi, and three other coagulase-negative staphylococci in a mouse model and possible virulence factors

Staphylococcus lugdunesis and Staphylococcus schleiferi, two newly described species, have been isolated from numerous types of human infections. We compared the pathogenicity of 30 strains of S. lugdunensis, S. schleiferi, Staphylococcus epidermidis, Staphylococcus warneri, and Staphylococcus hominis, using a mouse model in which a foreign body preadhered with the test strain was implanted subcutaneously, followed by injection of the test strain. All five species of staphylococci produced abscesses. Staphylococcus epidermidis, S. schleiferi, and S. lugdunensis yielded species means of 76-91% abscess formation; 80-100% of the infected foreign bodies and tissues were culture positive. These three species were more virulent than S. warneri or S. hominis, which produced abscesses in 54 and 65% of mice, respectively; only 10-48% of the infected samples were culture positive. Transmission electron microscopy of pure cultures of selected strains showed that all species possessed glycocalyx. All species produced a variety of possible virulence factors, such as alpha and delta hemolysins, as well as the aggressins lipase and esterase. The production of exoenzymes did not always correlate with virulence as demonstrated by abscess formation in mice.     

Polymyxin-Coagulase-Mannitol-Agar: I. A Selective Isolation Medium for Coagulase-Positive Staphylococci1

A selective, differential plating medium was developed for the isolation and identification of coagulase-positive and mannitol-fermenting staphylococci. Coagulase produced by growing Staphylococcus aureus caused an opaque zone of fibrin to form around each colony. Several strains of S. aureus produced a visible coagulase reaction by 8 hr, and all strains gave a positive reaction before 12 hr. Mannitol fermentation was usually observed between 12 and 36 hr. Rabbit serum was filtered through Sephadex G-100 to obtain plasmin- and plasminogen-free coagulase-reacting factor (CRF). False-negative reactions, caused by staphylokinase and staphylococcal Müller factor action on plasminogen, were eliminated when this CRF was used. False-positive reactions by lipolytic, coagulase-negative staphylococci were reduced, since gel filtration removed the serum lipoprotein which served as a primary source of opacity. The addition of 75 mug of polymyxin B per ml selectively retarded the growth of S. epidermidis and minimized false-positive reactions caused by citrate-utilizing gram-negative rods. The preparation, characteristics, and use of the medium are presented.     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.