Search

Targeting health care associated infections (HCAIs) is a key priority of current health policy. This applies as much to the community as to hospitals, although the latter receive far more media attention. One of the commonest HCAIs in the community is catheter-associated urinary tract infection (CAUTI). Cutting infection rates is a challenge. Many CAUTIs are the result of inappropriate catheterization, often combined with reduced sensitivity of bacteria to antibiotics given prophylactically. The government's rapid reaction task force has recommended use of a silver-alloy coated catheter, but closer surveillance is needed to persuade budget-holders of the cost and clinical benefits of this intervention.

While infection control has increased in profile both clinically and politically, it has also become embedded in clinical governance frameworks of NHS Trusts. Over the last few years the Department of Health has produced a number of documents in an attempt to highlight the increasing risk of healthcare associated infection. Today, the need for education as a key component in health care has been recognized as well as the need for patients and carers to receive information and be trained in, for example, technique of hand decontamination and catheter management following discharge from hospital. The objective of the education programme outlined in this article was to work alongside government guidelines in an attempt to improve education and understanding about infection control and management of infection in the catheterized patient.

Topical photodynamic therapy (PDT) is effective in the treatment of certain non-melanoma skin cancers and is under evaluation in other dermatoses. Its development has been enhanced by a low rate of adverse events and good cosmesis. 5-Aminolaevulinic acid (ALA) is the main agent used, converted within cells into the photosensitizer protoporphyrin IX, with surface illumination then triggering the photodynamic reaction. Despite the relative simplicity of the technique, accurate dosimetry in PDT is complicated by multiple variables in drug formulation, delivery and duration of application, in addition to light-specific parameters. Several non-coherent and coherent light sources are effective in PDT. Optimal disease-specific irradiance, wavelength and total dose characteristics have yet to be established, and are compounded by difficulties comparing light sources. The carcinogenic risk of ALA-PDT appears to be low. Current evidence indicates topical PDT to be effective in actinic keratoses on the face and scalp, Bowen's disease and superficial basal cell carcinomas (BCCs). PDT may prove advantageous where size, site or number of lesions limits the efficacy and/or acceptability of conventional therapies. Topical ALA-PDT alone is a relatively poor option for both nodular BCCs and squamous cell carcinomas. Experience of the modality in other skin diseases remains limited; areas where there is potential benefit include viral warts, acne, psoriasis and cutaneous T-cell lymphoma. A recent British Photodermatology Group workshop considered published evidence on topical PDT in order to establish guidelines to promote the efficacy and safety of this increasingly practised treatment modality.

Streptococcus pneumoniae colonizes the nasopharynx in up to 40% of healthy subjects, and is a leading cause of middle ear infections (otitis media), meningitis and pneumonia. Pneumococci adhere to glycosidic receptors on epithelial cells and to immobilized fibronectin, but the bacterial adhesins mediating these reactions are largely uncharacterized. In this report we describe a novel pneumococcal protein PavA, which binds fibronectin and is associated with pneumococcal adhesion and virulence. The pavA gene, present in 64 independent isolates of S. pneumoniae tested, encodes a 551 amino acid residue polypeptide with 67% identical amino acid sequence to Fbp54 protein in Streptococcus pyogenes. PavA localized to the pneumococcal cell outer surface, as demonstrated by immunoelectron microscopy, despite lack of conventional secretory or cell-surface anchorage signals within the primary sequence. Full-length recombinant PavA polypeptide bound to immobilized human fibronectin in preference to fluid-phase fibronectin, in a heparin-sensitive interaction, and blocked binding of wild-type pneumococcal cells to fibronectin. However, a C-terminally truncated PavA' polypeptide (362 aa residues) failed to bind fibronectin or block pneumococcal cell adhesion. Expression of pavA in Enterococcus faecalis JH2-2 conferred > sixfold increased cell adhesion levels to fibronectin over control JH2-2 cells. Isogenic mutants of S. pneumoniae, either abrogated in PavA expression or producing a 42 kDa C-terminally truncated protein, showed up to 50% reduced binding to immobilized fibronectin. Inactivation of pavA had no effects on growth rate, cell morphology, cell-surface physico-chemical properties, production of pneumolysin, autolysin, or surface proteins PspA and PsaA. Isogenic pavA mutants of encapsulated S. pneumoniae D39 were approximately 104-fold attenuated in virulence in the mouse sepsis model. These results provide evidence that PavA fibronectin-binding protein plays a direct role in the pathogenesis of pneumococcal infections.

This paper describes the management of an outbreak of Shigellosis, reported in the national media, in a village school in Staffordshire. The issues encountered by the outbreak team during the management of this incident have re-affirmed the need for public and environmental health departments to work closely with educational bodies to develop clear and consistent action plans for the management of communicable disease outbreaks. The management of communicable disease in schools, the workplace and other settings should be addressed, possibly as a component of local health improvement plans.

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major causative agent in the initiation and progression of severe forms of periodontal disease. In order to cause periodontal disease, P. gingivalis must colonize the subgingival region, a process that involves several distinct steps and multiple gene products. The organism must first navigate within the oral fluids in order to reach the hard or soft tissues of the mouth. Retention and growth of bacteria on these surfaces is facilitated by a repertoire of adhesins including fimbriae, hemagglutinins and proteinases. Once established subgingivally, P. gingivalis cells participate in intercellular communication networks with other oral prokaryotic cells and with eukaryotic cells. The establishment of these multiple interactive interfaces can lead to biofilm formation, invasion of root dentin and internalization within gingival epithelial cells. The resulting bacterial and host cellular locations, products and fate contribute to the success of P. gingivalis in colonizing the periodontal region.

The acquisition of transition metal ions by pathogenic bacteria is crucial to their growth and survival within the human host, however, the mechanisms of metal ion homeostasis in streptococci are unknown. The scaCBA operon in the human oral bacterium Streptococcus gordonii encodes the components of an ABC-type transporter for manganese (Mn2+). Production of substrate-binding lipoprotein ScaA was increased approximately fivefold in cells cultured in low Mn2+ medium (< 0.1 microM Mn2+), but not in iron (Fe2+/Fe3+)-limited medium, and was enhanced in the presence of human saliva or serum. mRNA analysis revealed that under low Mn2+ conditions, levels of scaCBA transcript (2.6 kb) were increased > 20-fold. The Mn2+-responsive transcriptional regulator of the sca operon was purified and characterized as a 215-amino-acid residue polypeptide, designated ScaR, with 26% identity to the Corynebacterium diphtheriae diphtheria toxin repressor (DtxR). Inactivation of scaR in S. gordonii DL1 (Challis) resulted in constitutive derepression of sca operon transcription. Expression of tpx, located immediately downstream of scaA and encoding a putative thiol peroxidase, was not subject to ScaR regulation. Purified ScaR protein bound to the scaC promoter region in vitro in the presence of Mn2+ (Kd approximately 80 nM) and, to a lesser extent, in the presence of Ni2+ or Zn2+. The metalloregulator protein binding region was localized by DNA protection analysis to a 46 bp sequence encompassing the -35 and -10 promoter signatures. This sequence was well conserved within the promoters of corresponding virulence-related permease operons in other streptococci. The results identify a new Mn2+-sensing regulator of Mn2+ transport in streptococci, important for Mn2+ homeostasis during infection of the human host.

Streptococcus gordonii is one of the predominant streptococci in the biofilm ecology of the oral cavity. It interacts with other bacteria through receptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specific marker to identify S. gordonii in a two-species in vitro oral biofilm flowcell system. To drive expression of gfp, we isolated and characterized an endogenous S. gordonii promoter, PhppA, which is situated upstream of the chromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromosomal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was constructed and used to demonstrate that PhppA was highly active throughout the growth of bacteria in batch culture. A promoterless 0.8-kb gfp ('gfp) cassette was PCR amplified from pBJ169 and subcloned to replace the cat cassette downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating pMA1. Subsequently, the PhppA-'gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S. gordonii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S. gordonii DL1 aerobically to ensure GFP expression. Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the PhppA-'gfp fusion plasmid stably maintained the fluorescent phenotype. Fluorescent S. gordonii DL1 transformants were used to elucidate the spatial arrangement of S. gordonii DL1 alone in biofilms or with the coadhesion partner Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned in vitro flowcell system. These results show for the first time that GFP expression in oral streptococci can be used as a species-specific marker in model oral biofilms.

Plasmid pGT232 (5.1 kb), an indigenous plasmid of Lactobacillus reuteri 100-23, was determined, on the basis of nucleotide and deduced protein sequence data, to belong to the pC194-pUB110 family of plasmids that replicate via the rolling-circle mechanism. The minimal replicon of pGT232 was located on a 1.7-kb sequence consisting of a double-strand origin of replication and a gene encoding the replication initiation protein, repA. An erythromycin-selectable recombinant plasmid containing this minimal replicon was stably maintained (>97% erythromycin-resistant cells) without antibiotic selection in an L. reuteri population under laboratory growth conditions but was poorly maintained (<33% resistant cells) in the L. reuteri population inhabiting the murine gastrointestinal tract. Stable maintenance (>90% resistant cells) of pGT232-derived plasmids in the lactobacillus population in vivo required an additional 1.0-kb sequence which contained a putative single-strand replication origin (SSO). The SSO of pGT232 is believed to be novel and functions in an orientation-specific manner.

Background/purpose: Complete surgical resection after chemotherapy is the definitive treatment for hepatoblastoma. However, orthotopic liver transplantation (OLT) is now accepted as a treatment modality for patients with unresectable tumours. The aim of this study was to review a single center's experience of OLT for unresectable hepatoblastoma., Methods: A retrospective review of 8 patients with unresectable hepatoblastoma who were referred for liver transplantation was conducted., Results: The patients assessed had an age range of 5 to 105 months at presentation; median, 24 months, (5 boys; 3 girls). Two patients have familial adenomatous polyposis, and one has right hemihypertrophy. All 8 patients had received standard chemotherapy according to SIOP (International Society of Pediatric Oncology) protocols. Extrahepatic metastases were found in 3 patients at diagnosis, but none had detectable metastases at the time of OLT. Four patients continued chemotherapy while awaiting OLT. Three patients received whole grafts, and 5 received reduced grafts. The median follow-up period was 22 months (range, 2 to 78 months). Five patients are alive and well, although 1 patient had a second OLT for biliary cirrhosis secondary to biliary stricture at 6 years. Three patients died: one 26 days post OLT of sepsis and two of disease recurrence at 22 months and 70 months posttransplant. The actuarial survival rate is 88% and 65% at 1 and 5 years, respectively, whereas the overall survival rate is 62.5%., Conclusion: OLT for unresectable hepatoblastoma without extra hepatic metastases is highly successful with a low recurrence rate.

Periodontal disease result from mixed bacterial infections, in which both host resistance barriers and bacterial interactions are important. Approximately ten bacterial species are strongly implicated with various forms of periodontal disease, although species that cannot yet be cultivated are likely also to be relevant. New technologies have shown that pathogenic bacterial species are present in defined complexes within subgingival plaque, thus identifying specific targets for therapeutic intervention. In light of increasing antibiotic resistance amongst oral bacteria, new strategies for control of periodontal bacterial complexes must be developed that inhibit the bacterial factors necessary for colonization and destruction of host tissues.

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.

Human placental trophoblast is critically involved in mediating maternal tolerance of the fetal semiallograft. Genes encoding highly polymorphic major histocompatibility complex (MHC) class I and class II antigens that could provoke maternal immune rejection responses are silenced in trophoblast. However, several MHC class I or class I-related products exhibiting reduced or negligible polymorphism are expressed and assumed to be functionally involved in maintaining pregnancy. The CD1 gene family encodes non-polymorphic MHC class I-like products that have the unusual ability to present non-peptide antigens to T cells. One member, CD1D, is expressed in certain epithelial cells and interacts with a specific T-cell subset that may promote the development of Th2-mediated responses believed to be associated with pregnancy. In this study we examined the expression of CD1D in human trophoblast cell lines and placentally derived trophoblast cells by reverse transcriptase-polymerase chain reaction using CD1D-specific oligonucleotide primers. We have found that CD1D mRNA transcripts are expressed in trophoblast cells and cell lines. We have also identified a novel alternatively spliced CD1D mRNA transcript lacking exon 4. Exon 4-intact and exon 4-deficient CD1D transcripts appear to be differentially expressed in different trophoblast and non-trophoblast cell populations. Our studies suggest that at least one member of the CD1 family is transcribed in human trophoblast.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a stealth role whereby pivotal immune mediators are selectively inactivated. In keeping with its strict metabolic requirements, regulation of gene expression in P. gingivalis can be controlled at the transcriptional level. Finally, although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.

ScaA lipoprotein in Streptococcus gordonii is a member of the LraI family of homologous polypeptides found among streptococci, pneumococci, and enterococci. It is the product of the third gene within the scaCBA operon encoding the components of an ATP-binding cassette (ABC) transporter system. Inactivation of scaC (ATP-binding protein) or scaA (substrate-binding protein) genes resulted in both impaired growth of cells and > 70% inhibition of 54Mn2+ uptake in media containing < 0.5 microM Mn2+. In wild-type and scaC mutant cells, production of ScaA was induced at low concentrations of extracellular Mn2+ (< 0.5 microM) and by the addition of > or = 20 microM Zn2+. Sca permease-mediated uptake of 54Mn2+ was inhibited by Zn2+ but not by Ca2+, Mg2+, Fe2+, or Cu2+. Reduced uptake of 54Mn2+ by sca mutants and by wild-type cells in the presence of Zn2+ was abrogated by the uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting that Mn2+ uptake under these conditions was proton motive force dependent. The frequency of DNA-mediated transformation was reduced > 20-fold in sca mutants. The addition of 0.1 mM Mn2+ to the transformation medium restored only partly the transformability of mutant cells, implying an alternate role for Sca proteins in the transformation process. Cells of sca mutants were unaffected in other binding properties tested and were unaffected in sensitivity to oxidants. The results show that Sca permease is a high-affinity mechanism for the acquisition of Mn2+ and is essential for growth of streptococci under Mn2+-limiting conditions.

Cell surface proteins SspA and SspB in Streptococcus gordonii and SpaP in Streptococcus mutans are members of the antigen I/II family of polypeptides produced by oral streptococci. These proteins are adhesins and mediate species-specific binding of cells to a variety of host and bacterial receptors. Here we show that antigen I/II polypeptides are involved in the attachment of oral streptococci to collagen and that they also determine the ability of these bacteria to invade human root dentinal tubules. Wild-type S. gordonii DL1 (Challis) cells showed heavy invasion of tubules to a depth of approximately 200 microm, whereas the abilities of cells of isogenic mutant strains OB220 (sspA) and OB219 (sspA sspB) to invade were 50 and >90% reduced, respectively. Likewise, wild-type S. mutans NG8 cells invaded dentinal tubules, whereas cells of isogenic mutant strain 834 (spaP) did not. The invasive abilities of strains OB220 and OB219 were restored by heterologous expression of S. mutans SpaP polypeptide in these strains. The extents of tubule invasion by various wild-type and mutant strains correlated with their levels of adhesion to type I collagen, a major component of dentin. Furthermore, S. gordonii DL1 cells exhibited a growth response to collagen by forming long chains. This was not shown by ssp mutants but was restored by the expression of SpaP in these cells. The production of SspA polypeptide by S. gordonii DL1, but not production of SspB polypeptide by strain OB220 (sspA), was enhanced in the presence of collagen. These results are the first to demonstrate that antigen I/II family polypeptides bind collagen and mediate a morphological growth response of streptococci to collagen. These antigen I/II polypeptide activities are critical for intratubular growth of streptococci and thus for establishment of endodontic infections.

Cytokeratin (CK) expression in untreated, paraffin-treated or dimethylbenzanthracene (DMBA)-treated hamster cheek pouch epithelium was investigated utilizing monoclonal antibodies AE1 or AE3, which react with type I or type II CKs, respectively, and by in situ hybridization utilizing type I CK-specific probes. The latter were isolated from a cDNA library of hamster cheek pouch mRNA and designated CK 13 and CK 10 based on their respective homologies (> 95% amino acids) with murine CK 13 and human CK 10. Treatment of hamster cheek pouch epithelium with DMBA resulted in increased expression of type I CK, detected immunohistochemically with monoclonal AE1, but decreased expression of type II CKs detected with AE3. Despite an overall increase in type I CKs, in situ hybridization demonstrated differential expression of type I CKs with altered distribution of CK 13 mRNA and reduced expression of CK 10 mRNA, providing additional sensitive markers for DMBA-associated changes in CKs. These changes were constant at 2 to 22 weeks in the pre-neoplastic and neoplastic epithelium following the initial application of DMBA.

Strains of the gastrointestinal species Lactobacillus reuteri were electrotransformed with plasmid constructs containing the endo-1,3-1,4-beta-glucanase gene (bglM) of Bacillus macerans. The enzyme was expressed and secreted by the lactobacilli. A plasmid construct containing the bglM gene lacking its promoter was derived and was demonstrated to be useful as a promoter probe vector.

Epidermolysis bullosa simplex with mottled pigmentation (EBS-MP; MIM no 131960) is an autosomal dominant disorder characterized by skin blistering at acral sites, punctate palmo-plantar hyperkeratoses, and mottled pigmentation of the trunk and proximal extremities. Histologically and ultrastructurally, the blistering in EBS-MP closely resembles that found in other EBS subtypes. This is consistent with a disorder of the basal keratinocyte cytoskeleton, in which several groups have found disease-causing mutations within the central rod domains of keratins 5 and 14. We have identified a C --> T transition at base position 71 of K5 causing a P24L substitution in a sporadic case of EBS-MP. Recently, this same mutation was identified in two unrelated families with EBS-MP.

The antigen I/II family of cell-surface-anchored polypeptides in oral streptococci are structurally complex multi-functional adhesins, with multiple ligand-binding sites. Discrete regions within these polypeptides bind human salivary glycoproteins, other microbial cells, and calcium. Sequences within the N-terminal region bind preferentially fluid-phase glycoproteins, while the C-terminal half of the polypeptide contains species-specific adhesion-mediating sequences that bind surface-immobilized glycoproteins. These features may assist streptococcal adhesion to oral surface receptors despite the presence of excess fluid-phase receptors. Immunological studies reveal an array of T-cell and B-cell epitopes presented by antigen I/II polypeptides and suggest the occurrence of natural suppression of human antibodies to the adhesion-mediating sequences. The functional and immunological properties of antigen I/II proteins may account to a major extent for the success of oral streptococci colonizing and surviving within the human host.

Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.

Candida albicans binds to several species of oral streptococci, in particular Streptococcus gordonii, through recognition of a streptococcal cell wall polysaccharide receptor (A. R. Holmes, P. K. Gopal, and H. F. Jenkinson, Infect. Immun. 63:1827-1834, 1995). We now show that isogenic cell surface protein mutants of S. gordonii DL1, unaltered in expression of cell wall polysaccharide, are reduced in ability to support adherence of C. albicans cells in a solid-phase assay. Inactivation of the S. gordonii cshA and cshB genes, encoding high-molecular-mass cell surface polypeptides, and inactivation of the sspA and sspB genes, encoding antigen I/II salivary adhesins, resulted in 40 and 79% reductions, respectively, in adherence of C. albicans cells. Inactivation of the S. gordonii scaA gene encoding a cell surface lipoprotein had no effect on C. albicans adherence. Polyclonal antiserum to streptococcal antigen I/II protein SpaP and antibodies specific to the amino-terminal nonrepetitive (NR) domain of CshA both inhibited adherence of C. albicans to S. gordonii cells. Conversely antibodies to the amino acid repeat block repetitive (R) domain of CshA, or to ScaA, did not inhibit C. albicans adherence. Immobilized recombinant polypeptide fragments of CshA comprising NR domain or R domain sequences both supported adherence of C. albicans cells. Expression of S. gordonii SspB protein on the surface of Enterococcus faecalis conferred on the enterococcal cells the ability to bind C. albicans, and this was ablated by antigen I/II antiserum. Collectively the results suggest that interaction of C. albicans with S. gordonii is mediated by a complement of adhesin-receptor interactions that involves two families of streptococcal multifunctional polypeptide adhesins, bacterial cell wall polysaccharide, and as yet unidentified yeast cell surface components.

Isogenic mutants of Streptococcus gordonii DL1 (Challis) in which the genes encoding high-molecular-mass cell surface polypeptides CshA and/or CshB were inactivated were deficient in binding to four strains of Actinomyces naeslundii and two strains of Streptococcus oralis. Lactose-sensitive interactions of S. gordonii with A. naeslundii ATCC 12104 and PK606 were associated with expression of cshA but not of cshB. Lactose-insensitive interactions of S. gordonii with A. naeslundii T14V and WVU627, and with S. oralis C104 and 34, were dependent on expression of cshA and cshB. S. gordonii DL1 cells bound to immobilized human fibronectin (Fn), but not to soluble Fn, in a dose-dependent manner, and binding was noninhibitable by heparin. S. gordonii cshA and cshB mutants were also deficient in binding to immobilized human Fn. Antibodies to an NH2-terminal nonrepetitive region (amino acid residues 42 to 886) of recombinant CshA inhibited binding of S. gordonii DL1 cells to A. naeslundii T14V and PK606 and to immobilized Fn. Conversely, antibodies to an amino acid repeat block segment of the COOH-terminal domain (amino acid residues 2026 to 2508) were not inhibitory to adherence. Assays using CshA-specific antibodies revealed that surface expression of CshA was reduced in cshB mutants. The results suggest that CshA acts as a multifunctional adhesin in S. gordonii and that major adhesion-mediating sequences are specified within the nonrepetitive NH2-terminal region of the polypeptide.

The highly conserved antigen I/II family of polypeptides produced by oral streptococci are believed to be colonization determinants and may mediate adhesion of bacterial cells to salivary glycoproteins absorbed to cells and tissues in the human oral cavity. Streptococcus gordonii is shown to express, on the cell surface, two antigen I/II polypeptides designated SspA and SspB (formerly Ssp-5) that are the products of tandemly arranged chromosomal genes. The structure and arrangement of these genes is similar in two independently isolated strains, DL1 and M5, of S. gordonii. The mature polypeptide sequences of M5 SspA (1539 amino acid (aa) residues) and SspB (1462 aa residues) are almost wholly conserved (98% identical) in the C-terminal regions (from residues 796 in SspA and 719 in SspB, to the respective C-termini), well-conserved (84%) at the N-terminal regions (residues 1-429), and divergent (only 27% identical residues) within the intervening central regions. Insertional inactivation of the sspA gene in S. gordonii DL1 resulted in reduced binding of cells to salivary agglutinin glycoprotein (SAG), human erythrocytes, and to the oral bacterium Actinomyces naeslundii. Further reductions in streptococcal cell adhesion to SAG and to two strains of A. naeslundii were observed when both sspA and sspB genes were inactivated. The results suggest that both SspA and SspB polypeptides are involved in adhesion of S. gordonii cells to human and bacterial receptors.

Over the past five years, concerns have heightened over the escalating numbers of pathogenic micro-organisms isolated that are resistant to many antibiotics and drugs. This phenomenon poses major problems in the treatment of patients with hospital- or community-acquired infections caused by bacteria, fungi, or parasitic organisms. Microbial cells have acquired resistances to specific antibiotics and drugs by mechanisms that include antibiotic inactivation, target alteration, or drug exclusion. More recently, the importance of another mechanism, that of drug expulsion, has been recognized as contributing significantly to antibiotic and drug resistance in microbes. Drug expulsion, mediated by membrane-associated drug efflux pumps, can protect cells from a range of toxic compounds and therefore may confer single-step multidrug resistance. It is imperative that new drugs be designed or discovered that will poison the pumps or bypass the efflux mechanisms.

Cells of the oral bacterium Streptococcus gordonii express three cytoplasmic membrane-bound lipoproteins with apparent molecular masses of 76 to 78 kDa that are the products of three genes (designated hppA, hppG, and hppH). The lipoproteins are immunologically cross-reactive, contain 60% or more identical amino acid residues, and are highly similar to the AmiA, AliA (PlpA), and AliB substrate-binding protein components of an oligopeptide permease in Streptococcus pneumoniae. Insertional inactivation of the hppA or hppH gene resulted in loss of the ability of S. gordonii cells to utilize specific peptides of five to seven amino acid residues for growth. An insertion within the COOH-terminal coding region of hppG that caused apparent truncation of the HppG polypeptide had a similar effect; however, S. gordonii mutants in which HppG polypeptide production was abolished were still able to grow on all oligopeptides tested. Inactivation of hppA gene (but not inactivation of the hppG or hppH gene) caused reduced growth rate of cells in complex medium, slowed the rate of development of competence for transformation, reduced the efficiency of transformation, and increased the resistance of cells to aminopterin. These results suggest that the formation of a solute-binding-protein complex consisting of at least the HppA and the HppH lipopolypeptides is necessary for binding and subsequent uptake of primarily hexa- or heptapeptides by a Hpp (Hexa-heptapeptide permease) system in S. gordonii. In addition, Hpp may play a role in the control of metabolic functions associated with the growth of streptococcal cells on complex nitrogen sources and with the development of competence.

The purpose of this study was to investigate the penetration of smeared and nonsmeared dentine by Streptococcus gordonii. Prepared human roots, grouped as either nonsmeared or smeared, were immersed in a suspension of S. gordonii cells for 3 weeks. The roots were then prepared for scanning electron microscopy and histological analysis. Dentine discs prepared from coronal dentine were grouped similarly. Using a fluid filtration apparatus, the hydraulic conductance (Lp) of each disc was determined before and after incubation with bacterial suspension. Scanning electron microscopy evaluation of the roots following infection with bacteria showed no change in the smear layer (P < 0.0001). Histological sections revealed that bacterial penetration of all the nonsmeared samples had occurred, while nine out of 10 smeared samples showed no bacterial penetration (P < 0.0001). The Lp of the nonsmeared discs was significantly reduced by 42% (P < 0.0001) after bacterial penetration. However, the smeared samples revealed a 1% reduction in Lp which was not significant (P > 0.05). The results suggest that dentinal smear layers are an effective barrier to dentinal tubule invasion by S. gordonii.

Cells of the oral bacterium Streptococcus oralis CN3410 produce lateral tufts of cell-surface fibrils of two lengths. Treatment of cells with trypsin resulted in loss of the tufts and release of longer fibrils intact. SDS-PAGE analysis of trypsin extracts containing fibrils revealed two groups of high molecular mass polypeptides which were denoted group A (molecular mass 227-246 kDa) and group B (molecular mass 175-208 kDa). Antibodies were raised to these two groups of trypsin-extracted polypeptides (TEPs) and to purified fibrils, and the reactivities of the three different antisera were found to be similar both on nitrocellulose blots of cell-surface polypeptides and in ELISA with whole cells. Similar patterns of TEPs were obtained from cells of a spontaneously derived mutant strain, KP34V, which lacked the short fibril components of tufts. Cells of strain KP34V had similar cell-surface hydrophobicity to strain CN3410 cells, and adhered to the same extent to parotid salivary pellicle or human buccal epithelial cells (BECs) as the wild-type cells. Trypsin treatment of strain CN3410 cells abolished their surface hydrophobicity and ability to adhere to BECs, but did not affect streptococcal cell binding to experimental salivary pellicle. Antibodies to TEPs or fibrils had no effect on cell adhesion to BECs or salivary pellicle. The results imply that the short fibril components of tufts are not involved in the cell adhesion properties tested. It is suggested that the TEPs are components of long fibrils, but they are not determinants of streptococcal cell adhesion to pellicle or to epithelial cells.

The yeast Candida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells. C. albicans and Candida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind to Streptococcus gordonii NCTC 7869 while two other Candida species (Candida krusei and Candida kefyr) do not. Adherence of C. albicans was greatest when the yeast had been grown at 30 degrees C to mid-exponential growth phase. For 21 strains of C. albicans there was a positive correlation between the ability to adhere to S. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence of C. albicans to S. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins of C. albicans are co-expressed.

Streptococci are one of the most successful bacterial colonizers of the human body and are major components of oral biofilms. The bacterial cells express multiple cell-surface adhesins that are responsible for the ability of streptococci to adhere to a wide range of substrates which include salivary and serous proteins, epithelial cells and other bacterial cells. Analysis of adherence-defective mutants has indicated the importance of high molecular mass wall-associated polypeptides and of enzymes catalyzing extracellular glucan polysaccharide synthesis to the adherence and accumulation of oral streptococci. The analysis of isogenic mutants of streptococci, generated through insertional inactivation (or allelic exchange), has confirmed the essential roles of specific surface polypeptides both to adhesive processes and to correct assembly of the cell wall layers.

The expression of HLA class I Ag by term human amnion epithelial cells was investigated. In immunostaining and FACS analysis, mAb to monomorphic class I Ag reacted extensively with amnion cells, whereas polymorphic mAb reactivity was more limited and variable. Further studies were conducted on amnion cell preparations containing negligible contaminants. Northern analysis with use of locus-specific probes demonstrated that amnion expresses two class Ib genes, HLA-E and HLA-G. Radio-immunoprecipitation with use of monomorphic mAb identified two fully glycosylated cell surface class I H chains of 44 and 41 kDa; polymorphic mAbs failed to immunoprecipitate the 41-kDa product, although 44-kDa products, typical of class Ia Ag, were identified in some preparations. Class I H chains were isolated from amnion by affinity chromatography. Microsequencing revealed that the first nine residues of the N-terminus of the 41-kDa product aligned perfectly only with HLA-E. Overall, amnion at term appears to express class Ib Ag with limited class Ia Ag. HLA-G is therefore expressed in two extrafetal epithelia: amnion and trophoblast. Identification of the class Ib protein HLA-E in amnion epithelium may have implications for preterm labor that can be associated with infection of the placental membranes.

Candida albicans ATCC 10261 and CA2 bound to cells of the oral bacteria Streptococcus gordonii, Streptococcus oralis, and Streptococcus sanguis when these bacteria were immobilized onto microtiter plate wells, but they did not bind to cells of Streptococcus mutans or Streptococcus salivarius. Cell wall polysaccharide was extracted with alkali from S. gordonii NCTC 7869, the streptococcal species to which C. albicans bound with highest affinity, and was effective in blocking the coaggregation of C. albicans and S. gordonii cells in the fluid phase. When fixed to microtiter plate wells, the S. gordonii polysaccharide was bound by all strains of C. albicans tested. The polysaccharide contained Rha, Glc, GalNAc, GlcNAc, and Gal and was related compositionally to previously characterized cell wall polysaccharides from strains of S. oralis and S. sanguis. The adherence of yeast cells to the immobilized polysaccharide was not inhibitable by a number of saccharides. Antiserum raised to the S. gordonii NCTC 7869 polysaccharide blocked adherence of C. albicans ATCC 10261 to the polysaccharide. The results identify a complex cell wall polysaccharide of S. gordonii as the coaggregation receptor for C. albicans. Adherent interactions of yeast cells with streptococci and other bacteria may be important for colonization of both hard and soft oral surfaces by C. albicans.

The significance of bile salt hydrolase production by lactobacilli in the microecology of the murine intestinal tract has not been extensively studied previously. Assays of bile salt hydrolase (sodium taurocholate as substrate) associated with cell extracts of five Lactobacillus strains of murine origin gave a range of activities (from 915 nmol of cholate released per mg of protein per 30 min to none detected). All of the strains tested colonized the murine gastrointestinal tract equally well. The growth rates of mice were not affected by colonization of their intestinal tracts by lactobacilli whether or not the bacteria produced bile salt hydrolase.

Streptococcus gordonii produces cell-surface lipopolypeptides that have been implicated in the determination of cell adherence and aggregation properties. SarA lipopolypeptide produced by S. gordonii is highly similar to the oligopeptide-binding protein AmiA in Streptococcus pneumoniae and to the OppA and SpoOKA oligopeptide-binding proteins in Bacillus subtilis. Insertional mutagenesis was used to inactivate the genes encoding SarA (76kDa) lipoprotein and a related 78-kDa lipoprotein denoted SarG. SarA- mutants were defective in serum-induced aggregation, competence, growth on complex nitrogen sources, and ability to colonize the oral cavity. Conversely, SarG- mutants were unaltered in the above properties, but were deficient in growth on simple nitrogen sources. It is proposed that SarA plays a central role in environmental sensing of extracellular factors by streptococci leading to modulation of cell-surface composition and growth responses of cells.

Colonization of the oral cavity by Candida albicans involves adherence of yeast cells to oral surfaces. An assay was developed to measure the attachment of C. albicans cells, metabolically labelled with [35S]methionine, to saliva-coated hydroxylapatite (SHA) beads--a model for the tooth surface. Using this assay approximately 1.26 x 10(6) C. albicans cells (50.5% of input cells) attached to the SHA beads (12 mg). Different strains of C. albicans adhered to varying degrees to SHA beads, but in general adherence was promoted by growth of cells at 28 degrees C and by starvation of cells for glucose. Proteins in human whole, or parotid, saliva samples were fractionated by gel filtration (Sephacryl S-200 column) and fractions were adsorbed to hydroxylapatite beads. Fractions that contained proline-rich proteins or statherin promoted attachment of C. albicans ATCC 10261 cells. Neuraminidase treatment of SHA beads, but not of cells, significantly increased yeast cell binding to the SHA beads. Neuraminidase activity in the oral cavity may unmask glycoprotein receptors involved in the adhesion of C. albicans to saliva-coated surfaces.

The human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high-molecular-mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41-amino-acid residue leader peptide, (ii) N-terminal 42-878 residues, (iii) residues 879-2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C-terminal anchor domain similar to those present in some other Gram-positive bacterial cell-wall polypeptides. Insertional mutations within cshA reduced both cell-surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.

Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158-166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32-33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces. A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii. Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.

Oral streptococci adhere to human salivary components and coadhere with specific partner oral bacteria. These interactions may favour the ordered development of plaque communities. The primary sequences of several streptococcal polypeptide adhesins are conserved, indicating that similar colonization mechanisms may have evolved. Critical amino acid changes within binding domains of adhesins might account for species- and site-specific adherence and accumulation.

Streptococcus gordonii DL1 (Challis) colonized the oral cavities of BALB/c mice that lacked streptococci, enterococci, and lactobacilli (LF mice) as members of an otherwise complex digestive tract microflora. Conventional mice, in comparison, were refractory to colonization by S. gordonii. Mice that harbored lactobacilli but were free of streptococci and enterococci (EF mice) had a lower incidence of colonization by S. gordonii than LF animals. The LF mouse system should be useful in the study of the molecular mechanisms that enable S. gordonii to inhabit the oral cavity.

beta-N-Acetylglucosaminidase was purified from the spent culture medium of Candida albicans A72 grown in the presence of N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of the protein was determined, two degenerate oligonucleotide probes were constructed, and a 3.9-kb BamHI fragment of DNA that hybridized to both probes was subcloned from a lambda EMBL4 library of C. albicans A72 genomic DNA. This fragment of DNA contained the entire beta-N-acetylglucosaminidase (HEX1) gene, which consisted of an open reading frame coding for a polypeptide precursor of 562 amino acids with a putative 22-amino-acid leader sequence. The deduced HEX1 amino acid sequence showed similarity to hexosaminidases from a variety of organisms. Growth of C. albicans on GlcNAc induced transcription of HEX1, resulting in increased specific beta-N-acetylglucosaminidase activity. HEX1 mRNA (2.35 kb) from GlcNAc-grown cells was approximately 200 bp larger than HEX1 mRNA from cells grown on glucose. This size difference was suggested to result from the use of alternative transcription termination sites. The cloned HEX1 gene introduced into C. albicans SGY-243 on a plasmid also responded to GlcNAc induction.

The effects of four different hyperplastic agents and of the carcinogen DMBA on cytokeratin expression in hamster cheek pouch epithelia were compared. Reversible hyperplasia was produced by the application of either oil of turpentine, vitamin A or TPA. No hyperplastic changes were produced by application of EPP. Apart from the transient appearance of a 45 kDa cytokeratin in one group treated with vitamin A, the immunohistochemical staining patterns and immunoblot profiles of cytokeratins from cheek pouches treated with each of the hyperplastic agents were identical to controls. Following application of DMBA, the cytokeratins stained with increased intensity in the spinous and granular cell layers. This was associated with increased amounts of 42-56 kDa cytokeratins and decreased production of 62-75 kDa cytokeratins. Monoclonal antibody AE1 detected a 45 kDa cytokeratin in extracts of DMBA-treated epithelia that was not detected in untreated epithelial extracts. Monoclonal antibody AE3 detected an additional 54 kDa cytokeratin band in extracts of DMBA-treated epithelia. These cytokeratin changes were present in preneoplastic epithelia and maintained in neoplastic epithelia.

Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.

Cell surface protein SSP-5 in the oral bacterium Streptococcus gordonii M5 binds human salivary agglutinin in a Ca(2+)-dependent reaction (D.R. Demuth, E.E. Golub, and D. Malamud, J. Biol. Chem. 265:7120-7126, 1990). The region of the gene encoding an N-terminal segment of a related polypeptide (SspA) in S. gordonii DL1 (Challis) was isolated following polymerase chain reaction amplification of genomic DNA. The sspA gene in S. gordonii DL1 was insertionally inactivated by homologous recombination of the erythromycin resistance (Emr) determinant ermAM onto the streptococcal chromosome. The SspA polypeptide (apparent molecular mass, 210 kDa) was detected on Western blots (immunoblots) of spheroplast extracts and extracellular culture medium proteins from wild-type strain DL1 but was absent from Emr mutants. One SspA- mutant (designated OB220) was not altered in rate or extent of aggregation by whole saliva or parotid saliva but showed reduced aggregation in the presence of purified salivary agglutinin. Mutant bacteria were unaffected in their ability to adhere to hydroxylapatite beads coated with whole or parotid saliva and were unaltered in cell surface hydrophobicity. However, the SspA- strain OB220 was deficient in binding salivary agglutinin and in binding to six strains of Actinomyces naeslundii. Therefore, expression of SspA polypeptide in S. gordonii is associated with both agglutinin-dependent and agglutinin-independent aggregation and adherence reactions of streptococcal cells.