PilB from Streptococcus sanguinis is a bimodular type IV pilin with a direct role in adhesion.

Type IV pili (T4P) are functionally versatile filamentous nanomachines, nearly ubiquitous in prokaryotes. They are predominantly polymers of one major pilin but also contain minor pilins whose functions are often poorly defined and likely to be diverse. Here, we show that the minor pilin PilB from the T4P of Streptococcus sanguinis displays an unusual bimodular three-dimensional structure with a bulky von Willebrand factor A-like (vWA) module "grafted" onto a small pilin module via a short loop. Structural modeling suggests that PilB is only compatible with a localization at the tip of T4P. By performing a detailed functional analysis, we found that 1) the vWA module contains a canonical metal ion-dependent adhesion site, preferentially binding Mg2+ and Mn2+, 2) abolishing metal binding has no impact on the structure of PilB or piliation, 3) metal binding is important for S. sanguinis T4P-mediated twitching motility and adhesion to eukaryotic cells, and 4) the vWA module shows an intrinsic binding ability to several host proteins. These findings reveal an elegant yet simple evolutionary tinkering strategy to increase T4P functional versatility by grafting a functional module onto a pilin for presentation by the filaments. This strategy appears to have been extensively used by bacteria, in which modular pilins are widespread and exhibit an astonishing variety of architectures.

Tandem sialoglycan-binding modules in a Streptococcus sanguinis serine-rich repeat adhesin create target dependent avidity effects.

Sialic acid-binding immunoglobulin-like lectins (Siglec)-like domains of streptococcal serine-rich repeat (SRR) adhesins recognize sialylated glycans on human salivary, platelet, and plasma glycoproteins via a YTRY sequence motif. The SRR adhesin from Streptococcus sanguinis strain SK1 has tandem sialoglycan-binding domains and has previously been shown to bind sialoglycans with high affinity. However, both domains contain substitutions within the canonical YTRY motif, making it unclear how they interact with host receptors. To identify how the S. sanguinis strain SK1 SRR adhesin affects interactions with sialylated glycans and glycoproteins, we determined high-resolution crystal structures of the binding domains alone and with purified trisaccharides. These structural studies determined that the ligands still bind at the noncanonical binding motif, but with fewer hydrogen-bonding interactions to the protein than is observed in structures of other Siglec-like adhesins. Complementary biochemical studies identified that each of the two binding domains has a different selectivity profile. Interestingly, the binding of SK1 to platelets and plasma glycoproteins identified that the interaction to some host targets is dominated by the contribution of one binding domain, whereas the binding to other host receptors is mediated by both binding domains. These results provide insight into outstanding questions concerning the roles of tandem domains in targeting host receptors and suggest mechanisms for how pathogens can adapt to the availability of a range of related but nonidentical host receptors. They further suggest that the definition of the YTRY motif should be changed to ϕTRX, a more rigorous description of this sialic acid-recognition motif given recent findings.

Adhesion of streptococci to titanium and zirconia.

The purpose of this study was to evaluate the adherence of streptococci to disks of titanium (commercially pure titanium: CpTi) and zirconia (tetragonal zirconia polycrystals: TZP). CpTi and yttria-stabilized TZP disks with a mirror-polished surface were used as specimens. The arithmetic mean surface roughness (Ra and Sa) and the surface wettability of the experimental specimens were measured. For analyzing the outermost layer of the experimental specimens, X-ray photoelectron spectroscopy (XPS) analysis was performed. Streptococcus sanguinis, S. gordonii, S. oralis, and S. mutans were used as streptococcal bacterial strains. These bacterial cultures were grown for 24 h on CpTi and TZP. The number of bacterial adhesions was estimated using an ATP-bioluminescent assay, and scanning electron microscope (SEM) observation of the adhered bacterial specimens was performed. No significant differences in surface roughness or wettability were found between CpTi and TZP. In XPS analyses, outermost layer of CpTi included Ti0 and Ti4+, and outermost layer of TZP included Zr4+. In the cell adhesion assay, the adherences of S. sanguinis, S. gordonii, and S. oralis to TZP were significantly lower than those to CpTi (p < 0.05); however, significant difference was not observed for S. mutans among the specimens. The adherence to CpTi and TZP of S. mutans was significantly lower than that of S. sanguinis, S. gordonii, and S. oralis. These results were confirmed by SEM. S. sanguinis, S. gordonii, and S. oralis adhered less to TZP than to CpTi, but the adherence of S. mutans was similar to both surfaces. S. mutans was less adherent compare with the other streptococci tested in those specimens.

The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Streptococcal Siglec-like adhesins recognize different subsets of human plasma glycoproteins: implications for infective endocarditis.

Streptococcus gordonii and Streptococcus sanguinis are typically found among the normal oral microbiota but can also cause infective endocarditis. These organisms express cell surface serine-rich repeat adhesins containing "Siglec-like" binding regions (SLBRs) that mediate attachment to α2-3-linked sialic acids on human glycoproteins. Two known receptors for the Siglec-like adhesins are the salivary mucin MG2/MUC7 and platelet GPIbα, and the interaction of streptococci with these targets may contribute to oral colonization and endocarditis, respectively. The SLBRs display a surprising diversity of preferences for defined glycans, ranging from highly selective to broader specificity. In this report, we characterize the glycoproteins in human plasma recognized by four SLBRs that prefer different α2-3 sialoglycan structures. We found that the SLBRs recognize a surprisingly small subset of plasma proteins that are extensively O-glycosylated. The preferred plasma protein ligands for a sialyl-T antigen-selective SLBR are proteoglycan 4 (lubricin) and inter-alpha-trypsin inhibitor heavy chain H4. Conversely, the preferred ligand for a 3'sialyllactosamine-selective SLBR is glycocalicin (the extracellular portion of platelet GPIbα). All four SLBRs recognize C1 inhibitor but detect distinctly different glycoforms of this key regulator of the complement and kallikrein protease cascades. The four plasma ligands have potential roles in thrombosis and inflammation, and each has been cited as a biomarker for one or more vascular or other diseases. The combined results suggest that the interaction of Siglec-like adhesins with different subsets of plasma glycoproteins could have a significant impact on the propensity of streptococci to establish endocardial infections.

Buckyballs conjugated with nucleic acid sequences identifies microorganisms in live cell assays.

BACKGROUND: Rapid identification of bacteria can play an important role at the point of care, evaluating the health of the ecosystem, and discovering spatiotemporal distributions of a bacterial community. We introduce a method for rapid identification of bacteria in live cell assays based on cargo delivery of a nucleic acid sequence and demonstrate how a mixed culture can be differentiated using a simple microfluidic system. METHODS: C60 Buckyballs are functionalized with nucleic acid sequences and a fluorescent reporter to show that a diversity of microorganisms can be detected and identified in live cell assays. The nucleic acid complexes include an RNA detector, targeting a species-specific sequence in the 16S rRNA, and a complementary DNA with an attached fluorescent reporter. As a result, each bacterium can be detected and visualized at a specific emission frequency through fluorescence microscopy. RESULTS: The C60 probe complexes can detect and identify a diversity of microorganisms that include gram-position and negative bacteria, yeast, and fungi. More specifically, nucleic-acid probes are designed to identify mixed cultures of Bacillus subtilis and Streptococcus sanguinis, or Bacillus subtilis and Pseudomonas aeruginosa. The efficiency, cross talk, and accuracy for the C60 probe complexes are reported. Finally, to demonstrate that mixed cultures can be separated, a microfluidic system is designed that connects a single source-well to multiple sinks wells, where chemo-attractants are placed in the sink wells. The microfluidic system allows for differentiating a mixed culture. CONCLUSIONS: The technology allows profiling of bacteria composition, at a very low cost, for field studies and point of care.

Effects of Streptococcus sanguinis Bacteriocin on Deformation, Adhesion Ability, and Young's Modulus of Candida albicans.

In order to study the thallus changes on microscopic morphology and mechanical properties of Candida albicans antagonized by Streptococcus sanguinis bacteriocin, the adhesion ability and Young's modulus of thalli and hypha of Candida albicans were measured by the relative measurement method using atomic force microscope's (AFM) tapping model. The results showed that the average adhesion ability and Young's modulus of thalli were 7.35 ± 0.77 nN and 7.33 ± 1.29 Mpa, respectively; the average adhesion ability and Young's modulus of hypha were 9.82 ± 0.39 nN and 4.04 ± 0.76 Mpa, respectively. After being antagonized by Streptococcus sanguinis bacteriocin, the adhesion ability was decreased along with the increasing of deformation in reaction region and Young's modulus followed the same changes. It could be concluded that the adhesion ability of hypha was greater than thalli, Young's modulus of hypha was less than thalli, and adhesion ability and Young's modulus of Candida albicans were decreased significantly after being antagonized by Streptococcus sanguinis bacteriocin.

Effects of Streptococcus sanguinis Bacteriocin on Cell Surface Hydrophobicity, Membrane Permeability, and Ultrastructure of Candida Thallus.

Candida albicans (C.a) and Candida tropicalis (C.t) were treated with Streptococcus sanguinis bacteriocin (S.s bacteriocin), respectively; the bacteriostatic dynamics of S.s bacteriocin, their effects on cell surface hydrophobicity, leakage of inorganic phosphorus and macromolecular substance, cytosolic calcium concentration, and ultrastructure changes of Candida thallus were detected and analyzed. The results showed that inhibitory effect of S.s bacteriocin on C.a and C.t reached peak level at 24 h, the cell-surface hydrophobicity decreased significantly (P < 0.05) after S.s bacteriocin treatment, and there was leakage of cytoplasmic inorganic phosphorus and macromolecular substance from C.a and C.t; cytosolic calcium concentration decreased greatly. After 24 h treatment by S.s bacteriocin, depressive deformity and defect could be found in the cell surface of C.a and C.t; the thallus displayed irregular forms: C.a was shrunken, there was unclear margins abutting upon cell wall and cell membrane, nucleus disappeared, and cytoplasm was inhomogeneous; likewise, C.t was first plasmolysis, and then the cytoplasm was shrunk, the ultrastructure of cell wall and cell membrane was continuously damaged, and the nucleus was karyolysis. It was illustrated that S.s bacteriocin had similar antifungal effect on C.a and C.t; their cell surface hydrophobicity, membrane permeability, and ultrastructure were changed significantly on exposure to S.s bacteriocin.

Fourier transform infrared microspectroscopy of endocarditis vegetation.

The objectives of this work were to compare the infrared spectra of bacterial endocarditis vegetation with those of native valvular tissue and the infrared spectra of vegetation bacterial masses with those of surrounding vegetation tissue. Streptococcal aortic endocarditis was induced in three rabbits. Vegetation slices were cryo-sectioned for study by Fourier transform infrared (FT-IR) microspectroscopy. Valvular apparatus, vegetation, and bacterial masses within the vegetation were localized on hematoxylin and eosin (H&E) stained contiguous slices. Infrared images of whole vegetations and images of bacterial masses were acquired with apertures set to 80 x 80 and 20 x 20 microm, respectively. Valvular apparatus and vegetation showed different infrared spectra, mainly in the amide I and amide II bands (1674-1518 cm(-1)), and at about 1450, 1400, 1340, 1280, 1240, 1200, 1080, and 1030 cm(-1). Valvular collagen, elastin, and proteoglycans may explain these differences. Bacterial masses and surrounding vegetation showed different infrared patterns, mainly in the amide I and amide II bands and in the 1142-991 cm(-1) carbohydrate spectral range. Bacterial nucleic acids and polysaccharides may partly explain these differences. Study of experimental endocarditis vegetation using FT-IR microspectroscopy distinguishes (1) the vegetation from the valvular tissue, and (2) the bacterial masses from the surrounding tissue. This study demonstrates for the first time that FT-IR microspectroscopy is able to detect bacterial growth in infected tissue. FT-IR microspectroscopy appears to be a useful tool for investigation of the biochemical structure of endocarditis vegetation.

Raman spectroscopic measurement of relative concentrations in mixtures of oral bacteria.

Near-infrared Raman spectroscopy has been used for species identification of pure microbial specimens for more than a decade. More recently, this optical method has been extended to the analysis of specimens containing multiple species. In this report, we demonstrate rapid, reagent-free quantitative analysis of a simplified model of oral plaque containing three oral bacteria species, S. mutans, S. sanguis, and S. gordonii, using near-infrared Raman spectroscopy. Raman spectra were acquired from bacterial mixtures in 200 seconds. A prediction model was calibrated by the partial least squares method and validated by additional samples. On a scale from 0 to 1, relative fractions of each species could be predicted with a root mean square error of 0.07. These results suggest that near-infrared Raman spectroscopy is potentially useful in quantification of microbial mixtures in general and oral plaques in particular.

[Purification and characterization of sanguicin--a bacteriocin produced by Streptococcus sanguis].

OBJECTIVE: Streptococcus sanguis plays an important Role in maintaining the periodontal microecological balance. Previous studies have demonstrated that sanguicin--a kind of bacteriocin produced by S. sanguis has a prominent function of inhibiting the growth of putative periodontopathic bacteria (PPB). The aim of this study was to purify sanguicin and investigate its characters. METHODS: The raw extract of sanguicin was obtained from cells of S. sanguis by ultrasonication, salting out and dialysis. Then the diethyl-aminoethyl-sepharose gradual washing was done in conjunction with glucosan gel filtration for in the purfication of sanguicin. The characters of purified sanguicin were determined and its inhibitiory effect on PPB was measured by agar diffusion test. RESULTS: The purifed sanguicin was obtained; it was heat labile and proteinaceous; and by SDS-PAGE, it showed a main band with the relative molecular mass of 65 x 10(3). The purifed sanguicin had a strong inhibitory effect on P. gingivalis, P. intermedia and F. nucleatum in vitro. CONCLUSION: Our methods are useful for the purification of sanguicin. Sanguicin has inhibitory effect on PPB obviously and will have a good prospect in periodontal ecological therapy.

Proteome analysis of oral pathogens.

The oral environment contains diverse communities of micro-organisms including bacteria, fungi, protozoa, and viruses. Studies of oral ecology have led to an appreciation of the complexity of the interactions that oral micro-organisms have with the host in both health and disease. Despite this, diseases such as dental caries and periodontal diseases are still worldwide human ailments, resulting in a high level of morbidity and an economic burden to society. Proteomics offers a new approach to the understanding of holistic changes occurring as oral micro-organisms adapt to environmental change within their habitats in the mouth.

Prevalence of Csh-like fibrillar surface proteins among mitis group oral streptococci.

The prevalence of Csh-like fibrillar surface proteins among oral streptococci was investigated by ELISA and by immunoelectron microscopy using antiserum raised to recombinant fragments of CshA of Streptococcus gordonii DL1. The majority of S. gordonii, Streptococcus sanguis and Streptococcus oralis strains tested elaborated short (ca. 50-80 nm long) surface fibrils and reacted with antiserum to the amino acid repeat region of CshA, demonstrating the widespread nature of Csh-like proteins among these species. In contrast, reactivity with antiserum raised to the adhesion-mediating non-repetitive region of CshA was more restricted. On the basis of the ELISA results, several isolates were selected for immunogold analysis using CshA antisera. Immunogold-negative staining showed a surface distribution of 10 nm gold particles consistent with antibody binding to short fibrils. Long fibrils (>150 nm long), where present, were not significantly labelled with gold. The results suggest that some of the short peritrichous fibrils on many mitis group streptococci comprise Csh-like fibrillar protein. Further, the data are consistent with our hypothesis that the antigenically conserved amino acid repeat region of Csh-like proteins forms a scaffold for cell-distal presentation of the amino-terminal non-repetitive region that, at least in S. gordonii DL1, functions as an adhesin.

Adhesion and surface-aggregation of Candida albicans from saliva on acrylic surfaces with adhering bacteria as studied in a parallel plate flow chamber.

Adhesive interactions between Candida albicans and oral bacteria are generally thought to play a crucial role in the microbial colonization of denture acrylic, which may lead to denture stomatitis. This study investigated the influence of saliva on the adhesive interactions between C. albicans and Streptococcus sanguis or Actinomyces naeslundii on denture acrylic. First, bacteria were allowed to adhere to the acrylic surface from a flowing suspension, and subsequently yeasts were flowed over the acrylic surface. The organisms were assayed in the presence or absence of human whole saliva. All experiments were carried out in a parallel plate flow chamber and enumeration was done in situ with an image analysis system. In the absence of adhering bacteria, adhesion of C. albicans from buffer was more extensive than from saliva. However, in the presence of adhering bacteria, yeast adhesion from saliva was increased with respect to adhesion of yeasts from buffer, indicating that specific salivary components constitute a bridge between bacteria and yeasts. In all cases, yeast aggregates consisting of 3 to 5 yeast cells were observed adhering to the surface. A surface physico-chemical analysis of the microbial cell surfaces prior to and after bathing the microorganisms in saliva, suggests that this bridging is mediated by acid-base interactions since all strains show a major increase in electron-donating surface free energy parameters upon bathing in saliva, with no change in their zeta potentials. The surface physico-chemical analysis furthermore suggests that S. sanguis and A. naeslundii may use a different mechanism for adhesive interactions with C. albicans in saliva.

Whole-cell protein electrophoretic analysis of viridans streptococci: evidence for heterogeneity among Streptococcus mitis biovars.

One hundred reference strains representing all species belonging to the different phylogenetic lineages of the viridans streptococci were examined by means of one-dimensional whole-organism protein electrophoresis. For most of the species examined, multiple strains characterized by DNA-DNA hybridization were included and, wherever described, representatives of different biochemical variants were analysed. Most species were clearly differentiated. The data support the viewpoint that members of the Streptococcus anginosus group constitute a single species and indicate that Streptococcus mitis biovar 2 is a heterogeneous taxon comprising strains from several streptococcal species.

Structures of biologically active muramyl peptides from peptidoglycan of Streptococcus sanguis.

The structures of major muramyl peptides derived from peptidoglycan of the oral pathogen Streptococcus sanguis were determined and the biological activity of the peptides was tested in vitro on human monocytes. The muramyl peptides, produced by muramidase digestion of the purified peptidoglycan, were separated by reversed-phase high-performance liquid chromatography, either in their native form or after reduction with sodium borohydride. Chemical structures of the peptides were elucidated by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, amino acid analysis, post-source decay analysis and Edman sequencing. The study revealed two distinct monomers: N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala) (1), where the Ala-Ala is connected to the epsilon-amino group of lysine, and N-acetylglucosaminyl-N-acetylmuramyl-Ala-iGln-Lys(Ala-Ala)-Ala-Ala (2), where an additional dialanyl residue is attached to the lysine alpha-carboxyl group. Two sets of higher oligomers (di-, tri- and tetramers), related structurally to monomers 1 or 2 were also detected. In these oligomers, the monomeric subunits are linked together by Ala-Ala-Ala bridges. The native muramyl peptides primed human monocytes in vitro for the increased production of the microbicidal superoxide radical.

Streptococcus sanguis expresses a 150-kilodalton two-domain adhesin: characterization of several independent adhesin epitopes.

Streptococcus sanguis binds to saliva-coated hydroxylapatite (sHA), an in vitro model of the enamel pellicle. To learn if more than one adhesin functions during adhesion, 12 reactive monoclonal antibodies (MAbs) were isolated by screening against both adhesive and nonadhesive strains. Two of these MAbs, 1.1 and 1.2, inhibited adhesion in a dose-dependent fashion, although maximum inhibition with either was only 37%. When these two MAbs plus a polyclonal antibody to P1-like adhesin were combined, the inhibition was additive to about 82%. These data indicated that there were at least three distinct, functional adhesion epitopes on the surface of S. sanguis. Western blot analyses of S. sanguis surface macromolecules showed antigens at 36 and 56 (with MAb 1.2), 87 and 150 (with both MAb 1.1 and MAb 1.2), and 100, 130, and 170 kDa (with anti-P1 antibody). The antigens were eluted from gels. Isolated antigens and corresponding antibodies inhibited adhesion similarly. Additivity experiments suggested the distinct epitopes were in three groups: (i) 36/56 kDa, (ii) 87/150 kDa, and (iii) 100/130/170 kDa. The 150-kDa antigen reacting with both MAbs was isolated from gels and digested with trypsin. The digestion revealed a series of tryptic bands. A band at 38 kDa reacted with MAb 1.1 whereas a band at 54 kDa reacted with MAb 1.2 in Western blot analysis, indicating two distinct adhesive epitopes on the 150-kDa antigen. These data strongly suggest that S. sanguis adhesion to sHA is maximized when several adhesin epitopes are coexpressed on surface antigens of different sizes.

comYA, a gene similar to comGA of Bacillus subtilis, is essential for competence-factor-dependent DNA transformation in Streptococcus gordonii.

Tn4001 mutagenesis identified a new competence gene in Streptococcus gordonii Challis designated comYA. A comYA mutant was completely deficient in transformation and exhibited decreased levels of DNA binding and hydrolysis. The deduced 319-amino-acid ComYA protein exhibited 57% similarity and 33% identity to the ComGA transporter protein of Bacillus subtilis and contained the Walker A-box motif conserved in ATP-binding proteins as well as aspartic acid boxes Asp-1 and Asp-2 present in some components of the general secretory pathway of gram-negative bacteria. comYA appeared to be part of a putative operon encompassing a comGB homolog, designated comYB, together with sequences that could encode ComGC- and ComGD-like peptides designated ComYC and ComYD, respectively, as well as other components. The putative ComYC and ComYD peptides had leader sequences similar to the type IV N-methylphenylalanine pilins of gram-negative bacteria, but unlike other examples in this class, including B. subtilis, they contained an alanine at position -1 of the leader instead of the usual glycine residue. Northern analysis identified a single 6.0-kb comYA-containing transcript strictly dependent on exogenous competence factor for expression in ComA1 cells. An identical pattern of expression was seen in wild-type Challis cells grown under conditions of maximal competence but not in cells that were noncompetent.

Lipoteichoic acid from viridans streptococci induces the production of tumor necrosis factor and nitric oxide by murine macrophages.

Bacterial endotoxin or lipopolysaccharide is the major proinflammatory component of gram-negative bacteria, but the components of gram-positive bacteria that trigger the inflammatory cascade are poorly understood. Lipoteichoic acid (LTA) purified from 2 strains of viridans streptococci induced the accumulation of tumor necrosis factor (TNF) mRNA and protein by the murine macrophage cell line RAW 264.7 in a dose- and time-dependent manner. Furthermore, in the presence of recombinant interferon-gamma, LTA from both strains of viridans streptococci provoked the accumulation of inducible nitric oxide (NO) synthase mRNA and the production of NO. Together these observations indicate that LTA can trigger macrophage activation and the production of TNF and NO and suggest that LTA may be an important determinant of the host inflammatory response to gram-positive infection.

Characterization of Streptococcus sanguis isolated from patients with Behçet's disease.

The DNA homology and cell wall sugar constituents of eight Streptococcus sanguis(-like) strains, three isolated from the patients with Behçet's disease (BD114-23, BD113-20, BD118-1), two from patients with Kawasaki disease (MCLS-1, MCLS-2), and three type and reference strains of ATCC (ATCC10556T: S. sanguis, ATCC10557: S. oralis, and ATCC10558T: S. gordonii) were analyzed. Strains BD114-23 and BD118-1 showed high DNA homology to ATCC10556T, and their cell wall constituents were identical. Conversely, BD113-20, MCLS-1, MCLS-2, and ATCC10557 showed little DNA homology to ATCC10556T and ATCC10558T, but showed approximately 50 to 60% homology to each other. The cell wall constituents of BD113-20, MCLS-1, MCLS-2, and ATCC10557, however, were somewhat different, indicating that some of the clinical isolates have different characters from those of the three ATCC strains.